Number 9 September 2011

VolumeVolume 15 1 -- NumberNumber 91 MaySeptember - September 2011 1997

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Scope

The Atlas of Genetics and Cytogenetics in Oncology and Haematology is a peer reviewed on-line journal in open access, devoted to genes, cytogenetics, and clinical entities in cancer, and cancer-prone diseases. It presents structured review articles ("cards") on genes, leukaemias, solid tumours, cancer-prone diseases, more traditional review articles on these and also on surrounding topics ("deep insights"), case reports in hematology, and educational items in the various related topics for students in Medicine and in Sciences.

Editorial correspondance

Jean-Loup Huret Genetics, Department of Medical Information, University Hospital F-86021 Poitiers, France tel +33 5 49 44 45 46 or +33 5 49 45 47 67 [email protected] or [email protected]

Staff Mohammad Ahmad, Mélanie Arsaban, Marie-Christine Jacquemot-Perbal, Maureen Labarussias, Vanessa Le Berre, Anne Malo, Catherine Morel-Pair, Laurent Rassinoux, Alain Zasadzinski. Philippe Dessen is the Database Director, and Alain Bernheim the Chairman of the on-line version (Gustave Roussy Institute – Villejuif – France).

The Atlas of Genetics and Cytogenetics in Oncology and Haematology (ISSN 1768-3262) is published 12 times a year by ARMGHM, a non profit organisation, and by the INstitute for Scientific and Technical Information of the French National Center for Scientific Research (INIST-CNRS) since 2008.

The Atlas is hosted by INIST-CNRS (http://www.inist.fr)

http://AtlasGeneticsOncology.org

The PDF version of the Atlas of Genetics and Cytogenetics in Oncology and Haematology is a reissue of the original articles published in collaboration with the Institute for Scientific and Technical Information (INstitut de l’Information Scientifique et Technique - INIST) of the French National Center for Scientific Research (CNRS) on its electronic publishing platform I-Revues. Online and PDF versions of the Atlas of Genetics and Cytogenetics in Oncology and Haematology are hosted by INIST-CNRS.

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Editor

Jean-Loup Huret (Poitiers, France) Editorial Board

Sreeparna Banerjee (Ankara, Turkey) Solid Tumours Section Alessandro Beghini (Milan, Italy) Genes Section Anne von Bergh (Rotterdam, The Netherlands) Genes / Leukaemia Sections Judith Bovée (Leiden, The Netherlands) Solid Tumours Section Vasantha Brito-Babapulle (London, UK) Leukaemia Section Charles Buys (Groningen, The Netherlands) Deep Insights Section Anne Marie Capodano (Marseille, France) Solid Tumours Section Fei Chen (Morgantown, West Virginia) Genes / Deep Insights Sections Antonio Cuneo (Ferrara, Italy) Leukaemia Section Paola Dal Cin (Boston, Massachussetts) Genes / Solid Tumours Section Louis Dallaire (Montreal, Canada) Education Section Brigitte Debuire (Villejuif, France) Deep Insights Section François Desangles (Paris, France) Leukaemia / Solid Tumours Sections Enric Domingo-Villanueva (London, UK) Solid Tumours Section Ayse Erson (Ankara, Turkey) Solid Tumours Section Richard Gatti (Los Angeles, California) Cancer-Prone Diseases / Deep Insights Sections Ad Geurts van Kessel (Nijmegen, The Netherlands) Cancer-Prone Diseases Section Oskar Haas (Vienna, Austria) Genes / Leukaemia Sections Anne Hagemeijer (Leuven, Belgium) Deep Insights Section Nyla Heerema (Colombus, Ohio) Leukaemia Section Jim Heighway (Liverpool, UK) Genes / Deep Insights Sections Sakari Knuutila (Helsinki, Finland) Deep Insights Section Lidia Larizza (Milano, Italy) Solid Tumours Section Lisa Lee-Jones (Newcastle, UK) Solid Tumours Section Edmond Ma (Hong Kong, China) Leukaemia Section Roderick McLeod (Braunschweig, Germany) Deep Insights / Education Sections Cristina Mecucci (Perugia, Italy) Genes / Leukaemia Sections Yasmin Mehraein (Homburg, Germany) Cancer-Prone Diseases Section Fredrik Mertens (Lund, Sweden) Solid Tumours Section Konstantin Miller (Hannover, Germany) Education Section Felix Mitelman (Lund, Sweden) Deep Insights Section Hossain Mossafa (Cergy Pontoise, France) Leukaemia Section Stefan Nagel (Braunschweig, Germany) Deep Insights / Education Sections Florence Pedeutour (Nice, France) Genes / Solid Tumours Sections Elizabeth Petty (Ann Harbor, Michigan) Deep Insights Section Susana Raimondi (Memphis, Tennesse) Genes / Leukaemia Section Mariano Rocchi (Bari, Italy) Genes Section Alain Sarasin (Villejuif, France) Cancer-Prone Diseases Section Albert Schinzel (Schwerzenbach, Switzerland) Education Section Clelia Storlazzi (Bari, Italy) Genes Section Sabine Strehl (Vienna, Austria) Genes / Leukaemia Sections Nancy Uhrhammer (Clermont Ferrand, France) Genes / Cancer-Prone Diseases Sections Dan Van Dyke (Rochester, Minnesota) Education Section Roberta Vanni (Montserrato, Italy) Solid Tumours Section Franck Viguié (Paris, France) Leukaemia Section José Luis Vizmanos (Pamplona, Spain) Leukaemia Section Thomas Wan (Hong Kong, China) Genes / Leukaemia Sections

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) Atlas of Genetics and Cytogenetics in Oncology and Haematology

OPEN ACCESS JOURNAL AT INIST-CNRS

Volume 15, Number 9, September 2011

Table of contents

Gene Section

FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) 723 Kunzang Chosdol, Bhawana Dikshit, Subrata Sinha KSR1 (kinase suppressor of ras 1) 727 Mario Fernandez, Robert Lewis PEG10 (paternally expressed 10) 730 Andreas Lux PIK3CD (phosphoinositide-3-kinase, catalytic, delta polypeptide) 738 Emily Burns, Bart Vanhaesebroeck S100A8 (S100 calcium binding protein A8) 742 Claus Kerkhoff, Saeid Ghavami S100A9 (S100 calcium binding protein A9) 754 Claus Kerkhoff, Saeid Ghavami TRIAP1 (TP53 regulated inhibitor of apoptosis 1) 766 Veruska Alves, Roberta Felix, Andre Vettore, Gisele Colleoni

Leukaemia Section t(X;11)(q13;q23) 769 Adriana Zamecnikova t(X;11)(q22;q23) 771 Adriana Zamecnikova t(X;11)(q24;q23) MLL-SEPTIN6 773 Adriana Zamecnikova

Deep Insight Section

S100 Protein Family and Tumorigenesis 776 Geetha Srikrishna, Hudson H Freeze Visualize Dynamic Chromosome 785 Eisuke Gotoh NK cell receptors: evolution and diversity 795 Gwenoline Borhis, Salim I Khakoo Somatostatin (SS), SS receptors and SS analog treatment in tumorigenesis 805 Liliana Steffani, Luca Passafaro, Diego Ferone, Paolo Magni, Massimiliano Ruscica

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) Atlast(11;14)(q 13;q32)of Genetics in multiple myeloma and Cytogenetics Huret JL, Laï JL in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Case Report Section

A new case of translocation t(14;14)(q11;q32) in B lineage ALL 814 Elvira D Rodrigues Pereira Velloso, Priscila Pereira dos Santos Teixeira, Karina Prandi Melillo, Luciana J Rodrigues da Silva, Cristina Alonso Ratis, Daniela Borri, Cristóvão LP Mangueira

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9)

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) Kunzang Chosdol, Bhawana Dikshit, Subrata Sinha Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi- 110029, India (KC, BD, SS)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/FAT1ID40533ch4q35.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI FAT1ID40533ch4q35.txt

Identity Pseudogene FAT tumor suppressor homolog 1 (Drosophila) Other names: CDHF7; CDHR8; FAT; ME5; hFat1 pseudogene 1 (FAT1P1). HGNC (Hugo): FAT1 Other name: dJ697P8.1; sequence accession ID: AL050403; location chromosome: 20p12.2. Location: 4q35.2 Note Protein FAT1 is an ortholog of the Drosophila tumor suppressor gene 'fat'. In Drosophila, it is essential Note for controlling cell proliferation during Known protein coding gene. development. The gene product is a member of the Protein names cadherin superfamily, characterized by the presence Recommended name: protocadherin Fat 1. of cadherin-type repeats. In addition to containing Alternative names: Cadherin-related tumor 34 tandem cadherin-type repeats, the gene product suppressor homolog, Protein fat homolog, Cadherin has five epidermal growth factor (EGF)-like repeats family member 7. and one laminin A-G domain. This gene is Description expressed at high levels in a number of fetal epithelia. Its product probably functions as an 4588 aa (Accession: NP_005236.2). adhesion molecule and/or signaling receptor, and is Expression likely to be important in developmental processes Expressed in epithelial, endothelial and smooth and cell-cell communication. muscle cells. DNA/RNA Localisation Cell membrane; single-pass type I membrane Description protein. FAT1 gene is located on the chromosome 4q35.2 Function (Accession: NC_000004.11). The total length of the gene is 136050 bases (187509746 bp to 187630981 Could function as a cell-adhesion molecule, cell bp from pter) of reverse strand. There are 27 exons. signalling molecule, and have a role in cell An alternate assembly suggested to be starting from migration. 187745931 bp to 187881981 bp from pter. Fat in Drosophila acts via SWH signalling pathway as tumour suppressor gene. Homolog of SWH Transcription pathway molecules are present in human, so there is The length of the transcript is 14773 bps made from a possibility of acting FAT1 as an upstream 27 exons (Accession: NM_005245.3). regulator of SWH pathway in human.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 723

FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) Chosdol K, et al.

Salvador-Warts-Hippo pathway. Mammalian hippo signaling pathway shows homology with Drosophila pathway proteins (depicted in similar color and shape). In Drosophila fat (ft) interacts with core kinase cascade via Expanded (Ex). The core kinase cascade includes kinase Hippo (hpo), adaptor proteins mats and Salvador (Sav) and kinase Warts. The core kinase cascade inhibits phosphorylation of transcriptional co-activator Yorkie (Yki) causing its translocation to nucleus where it binds to transcriptional activator Scalloped (Sd) and modulates gene expression. In mammals, whether FAT1 is involved in hippo pathway regulation is not clear. The effector molecule, phospho-YAP, is reported to interact with p73 in the nucleus and promotes cell death. There is no p73 homolog known to be reported in Drosophila. YAP is also found to interact with other transcription factors and modulate gene expression, thus, the outcome of hippo pathway is context dependent.

Organism Gene Locus Description Similarity to human FAT1 Dog FAT tumor suppressor FAT1 Chr. 16 86.95(n), 91.17(a) (Canis familiaris) homolog 1 (Drosophila) Pig FAT tumor suppressor FAT1 Chr. 17 86(n), 90(a) (Sus scrofa) homolog 1 (Drosophila) Cow FAT tumor suppressor FAT1 Chr. 27 84.18(n) 89.93(a) (Bos Taurus) homolog 1 (Drosophila) Rat FAT tumor suppressor Fat1 Chr. 16q11 82.93(n) 88.16(a) (Rattus norvegicus) homolog 1 (Drosophila) Mouse Chr. 8 FAT tumor suppressor Fat1 82.51(n) 88.14(a) (Mus musculus) (25.00 cM) homolog 1 (Drosophila) Chicken FAT tumor suppressor FAT Chr. 4 76.35(n) 81.43(a) (Gallus gallus) homolog 1 (Drosophila) Zebrafish FAT tumor suppressor fat1 Chr. 1 64.68(n) 64.82(a) (Danio rerio) homolog 1

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 724

FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) Chosdol K, et al.

Fruit fly Chr. 2L (ft) ft - 42.8(n) 42(a), (Drosophila ft and fat2 Chr. 3L fat and fat2 fat2 - 47.99(n) 39.02(a) melanogaster) (fat2) Worm (Caenorhabditis cdh-4 Chr. III Cadherin family 44.19(n) 30.89(a) elegans) African malaria AgaP_AGA mosquito Chr. 3L AGAP011526-PA 48.06(n) 39.5(a) P011526 (Anopheles gambiae) Table. Orthologs for FAT1 gene from other species.

In human, FAT1 expression is highest at the FAT1 expression in more malignant form of breast embryonic stages and diminishes later in adult life. cancer tissues by immunohistochemistry (IHC). In human fetal tissues, high levels of FAT1 There are studies showing LOH and/or deletion of transcripts were found in kidney, lungs, and eye the chromosome 4q34-35 region (which harbors epithelia, and the expression was found to be down FAT gene) in many tumors including gliomas. LOH regulated in the corresponding adult tissues, was found in grade IV gliomas using microsatellite indicating the role of FAT1 in organ development. markers (Hu et al., 2002), though the gene itself has FAT1 also has a role in cell migration (Moeller et not been implicated. Other tumors like small cell al., 2004; Tanoue and Takeichi, 2004) and found to lung carcinoma (Cho et al., 2002), hepatocellular be up-regulated in migrating cells, also crucial for carcinoma (Zhang et al., 2005; Chang et al., 2002) efficient wound healing (Braun et al., 2007). and cervical carcinoma (Backsch et al., 2005) etc In Drosophila, fat is an upstream regulator of the showed alterations/LOH in the chromosomal 4q34- Salvador-Wart-Hippo (SWH) signaling pathway q35 locus and significant association of 4q34-q35 (Cho et al., 2006; Bennett and Harvey, 2006). The region with increased risk of progression of these signalling molecules of SWH pathway are tumors was suggested. Since the FAT gene is conserved in mammals (figure below) but the role located in this region it may have an important role of FAT1 as an apical regulator of SWH pathway in to play in the development and progression of these human has not yet been established. tumors. Homology Astrocytic tumour Paralogs for FAT1 gene: FAT2, FAT3, FAT4. See Note table above. Loss of heterozygosity and altered expression of FAT1 in astrocytic tumors (Chosdol et al., 2009). Mutations Breast cancer Note Note No known mutations. Single nucleotide Increased FAT1 expression contributes to loss of polymorphism (SNPs): gene: FAT1 duct formation, and increased cell migration and (ENSG00000083857). invasion in breast cancer (Kwaepila et al., 2006). Implicated in Oral cancer Note Various cancers Homozygous deletion of FAT in the cell lines and Note in primary oral cancers was studied. Homozygous FAT1, a member of the cadherin gene family, is deletion hot spots were observed in exon 1 (9/20, homologue of Drosophila tumour suppressor gene 45%) and exon 4 (7/20, 35%). The methylation fat. In Drosophila, fat gene is important in status of the FAT CpG island in squamous cell controlling cell proliferation during development carcinomas correlated negatively with its and any defect in the expression of fat would lead expression. Mutations in FAT is suggested as an to tumor development (Bryant et al., 1988). Dunne important factor in the development of oral cancer. et al. (1995) have identified the human homologue Moreover, loss of gene expression was identified in and studied the tissue distribution of FAT other types of squamous cell carcinoma (Nakaya et transcripts in adult and fetal tissues. al., 2007). Loss of heterozygosity and altered expression of Psychiatric disorders FAT1 has been found in human glial tumors Note (Chosdol et al., 2009). Homozygous deletion of FAT1 gene was detected in oral cancer (Nakaya et Bipolar disorder: a positional cloning strategy, al., 2007). Kwaepila et al. (2006) found higher combined with association analysis have provided

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 725

FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) Chosdol K, et al.

evidence that a cadherin gene, FAT, confers zebrafish protocadherin gene, Fat. Gene Expr Patterns. susceptibility to bipolar disorder (Blair et al., 2006). 2005 Apr;5(4):483-90 Magg T, Schreiner D, Solis GP, Bade EG, Hofer HW. Cell migration Processing of the human protocadherin Fat1 and Note translocation of its cytoplasmic domain to the nucleus. Exp FAT1 is known to play role in cell migration. FAT1 Cell Res. 2005 Jul 1;307(1):100-8 knockdown decreases cell migration in vascular Zhang SH, Cong WM, Xian ZH, Wu MC. smooth muscle cells (Hou et al., 2006; Hou and Clinicopathological significance of loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in Sibinga, 2009). FAT1 plays an integrative role in China. World J Gastroenterol. 2005 May 28;11(20):3034-9 regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal Bennett FC, Harvey KF. Fat cadherin modulates organ size in Drosophila via the Salvador/Warts/Hippo signaling dynamics (Moeller et al., 2004). pathway. Curr Biol. 2006 Nov 7;16(21):2101-10 Blair IP, Chetcuti AF, Badenhop RF, Scimone A, Moses References MJ, Adams LJ, Craddock N, Green E, Kirov G, Owen MJ, Kwok JB, Donald JA, Mitchell PB, Schofield PR. Positional Bryant PJ, Huettner B, Held LI Jr, Ryerse J, Szidonya J. cloning, association analysis and expression studies Mutations at the fat locus interfere with cell proliferation provide convergent evidence that the cadherin gene FAT control and epithelial morphogenesis in Drosophila. Dev contains a bipolar disorder susceptibility allele. Mol Biol. 1988 Oct;129(2):541-54 Psychiatry. 2006 Apr;11(4):372-83 Dunne J, Hanby AM, Poulsom R, Jones TA, Sheer D, Chin Cho E, Feng Y, Rauskolb C, Maitra S, Fehon R, Irvine KD. WG, Da SM, Zhao Q, Beverley PC, Owen MJ. Molecular Delineation of a Fat tumor suppressor pathway. Nat Genet. cloning and tissue expression of FAT, the human 2006 Oct;38(10):1142-50 homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion Hou R, Liu L, Anees S, Hiroyasu S, Sibinga NE. The Fat1 molecule. Genomics. 1995 Nov 20;30(2):207-23 cadherin integrates vascular smooth muscle cell growth and migration signals. J Cell Biol. 2006 May 8;173(3):417- Ponassi M, Jacques TS, Ciani L, ffrench Constant C. 29 Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech Dev. 1999 Feb;80(2):207- Katoh Y, Katoh M. Comparative integromics on FAT1, 12 FAT2, FAT3 and FAT4. Int J Mol Med. 2006 Sep;18(3):523-8 Cox B, Hadjantonakis AK, Collins JE, Magee AI. Cloning and expression throughout mouse development of mfat1, a Kwaepila N, Burns G, Leong AS. Immunohistological homologue of the Drosophila tumour suppressor gene fat. localisation of human FAT1 (hFAT) protein in 326 breast Dev Dyn. 2000 Mar;217(3):233-40 cancers. Does this adhesion molecule have a role in pathogenesis? Pathology. 2006 Apr;38(2):125-31 Chang J, Kim NG, Piao Z, Park C, Park KS, Paik YK, Lee WJ, Kim BR, Kim H. Assessment of chromosomal losses Schreiner D, Müller K, Hofer HW. The intracellular domain and gains in hepatocellular carcinoma. Cancer Lett. 2002 of the human protocadherin hFat1 interacts with Homer Aug 28;182(2):193-202 signalling scaffolding proteins. FEBS Lett. 2006 Oct 2;580(22):5295-300 Cho ES, Chang J, Chung KY, Shin DH, Kim YS, Kim SK, Kim SK. Identification of tumor suppressor loci on the long Braun GS, Kretzler M, Heider T, Floege J, Holzman LB, arm of chromosome 4 in primary small cell lung cancers. Kriz W, Moeller MJ. Differentially spliced isoforms of FAT1 Yonsei Med J. 2002 Apr;43(2):145-51 are asymmetrically distributed within migrating cells. J Biol Chem. 2007 Aug 3;282(31):22823-33 Hu J, Jiang C, Ng HK, Pang JC, Tong CY. Chromosome 14q may harbor multiple tumor suppressor genes in Nakaya K, Yamagata HD, Arita N, Nakashiro KI, Nose M, primary glioblastoma multiforme. Chin Med J (Engl). 2002 Miki T, Hamakawa H. Identification of homozygous Aug;115(8):1201-4 deletions of tumor suppressor gene FAT in oral cancer using CGH-array. Oncogene. 2007 Aug 9;26(36):5300-8 Moeller MJ, Soofi A, Braun GS, Li X, Watzl C, Kriz W, Holzman LB. Protocadherin FAT1 binds Ena/VASP Chosdol K, Misra A, Puri S, Srivastava T, Chattopadhyay proteins and is necessary for actin dynamics and cell P, Sarkar C, Mahapatra AK, Sinha S. Frequent loss of polarization. EMBO J. 2004 Oct 1;23(19):3769-79 heterozygosity and altered expression of the candidate tumor suppressor gene 'FAT' in human astrocytic tumors. Tanoue T, Takeichi M. Mammalian Fat1 cadherin BMC Cancer. 2009 Jan 7;9:5 regulates actin dynamics and cell-cell contact. J Cell Biol. 2004 May 24;165(4):517-28 Hou R, Sibinga NE. Atrophin proteins interact with the Fat1 cadherin and regulate migration and orientation in vascular Backsch C, Rudolph B, Kühne-Heid R, Kalscheuer V, smooth muscle cells. J Biol Chem. 2009 Mar Bartsch O, Jansen L, Beer K, Meyer B, Schneider A, Dürst 13;284(11):6955-65 M. A region on human chromosome 4 (q35.1-->qter) induces senescence in cell hybrids and is involved in cervical carcinogenesis. Genes Chromosomes Cancer. This article should be referenced as such: 2005 Jul;43(3):260-72 Chosdol K, Dikshit B, Sinha S. FAT1 (FAT tumor Down M, Power M, Smith SI, Ralston K, Spanevello M, suppressor homolog 1 (Drosophila)). Atlas Genet Burns GF, Boyd AW. Cloning and expression of the large Cytogenet Oncol Haematol. 2011; 15(9):723-726.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 726

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Mini Review

KSR1 (kinase suppressor of ras 1) Mario Fernandez, Robert Lewis Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198, USA (MF, RL)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/KSR1ID41107ch17q11.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI KSR1ID41107ch17q11.txt

Identity Protein Other names: KSR; RSU2 Note HGNC (Hugo): KSR1 Belongs to the KSR family of proteins. Location: 17q11.1 Description Local order: From centromere to telomere on The KSR1 protein is 921 kDa. There have been 15 17q11.2, the KSR1 gene is flanked by: EOE1, phosphorylation sites identified. EVI2A, JJAZ1, KNO3, KRT24, KSR1, LGALS9, Expression LYZL6, MIR144, MIR451 (NCBI). Brain, kidney, lung, pancreas, ovaries, testis, breast. DNA/RNA Localisation Description Sequestered in the cytoplasm in quiescents cells. Upon stimulation, KSR1 protein localizes to the The KSR1 gene was discovered in D. melanogaster plasma membrane. and is highly conserved along the evolutionary tree through H. sapiens. Function Transcription Acts as a scaffold for the Raf/MEK/ERK kinase cascade. Transcription can produce two different mRNA transcripts. The KSR1 is expressed in the thymus, Homology bone marrow, brain, heart, kidney, lung, liver, R. norvegicus KSR1 (91.85%), M. musculus KSR1 pancreas, mammary gland, ovary, testis and muscle. (91.3%), C. familians KSR1 (93.62%), G. gallus Pseudogene KSR1 (76.39%), D. reno KSR1 (67.85%), D. Melanogaster KSR (41.06%), C. elegans KSR1 No known pseudogenes. (35.37%), X. laevis KSR1 (76.06%).

KSR1 contains 17 exons, 154426 base pairs.

The KSR1 protein contains five conserved areas (CA). The CA1 region is unique to KSR1 protein; the CA2 region is a proline- rich region; the CA3 region is implicated in the Ras-induced plasma membrane localization; the CA4 region is a serine/threonine rich region and contains the MAPK docking site (FXFP); and the putative kinase domain (CA5), which contains an amino acid variation in subdomain II that suggests that this protein is catalytically inert. The KSR1 protein is composed of 921 amino acids.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 727

KSR1 (kinase suppressor of ras 1) Fernandez M, Lewis R

Müller J, Ory S, Copeland T, Piwnica-Worms H, Morrison Mutations DK. C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1. Mol Cell. 2001 Nov;8(5):983-93 Note Giblett SM, Lloyd DJ, Light Y, Marais R, Pritchard CA. There have been no prominent germinal or somatic Expression of kinase suppressor of Ras in the normal adult mutations identified. and embryonic mouse. Cell Growth Differ. 2002 Jul;13(7):307-13 Implicated in Nguyen A, Burack WR, Stock JL, Kortum R, Chaika OV, Afkarian M, Muller WJ, Murphy KM, Morrison DK, Lewis Cancer RE, McNeish J, Shaw AS. Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated Note protein kinase activation in vivo. Mol Cell Biol. 2002 KSR1 has not been found to be upregulated or May;22(9):3035-45 downregulated in tumors. However, studies in Roy F, Laberge G, Douziech M, Ferland-McCollough D, KSR1 knockout mice showed that the mice were Therrien M. KSR is a scaffold required for activation of the less susceptible to papilloma virus driven tumors, ERK/MAPK module. Genes Dev. 2002 Feb 15;16(4):427- indicating that KSR1 is required for Ras-mediated 38 oncogenesis. Lozano J, Xing R, Cai Z, Jensen HL, Trempus C, Mark W, Cannon R, Kolesnick R. Deficiency of kinase suppressor of To be noted Ras1 prevents oncogenic ras signaling in mice. Cancer Res. 2003 Jul 15;63(14):4232-8 Note Ory S, Zhou M, Conrads TP, Veenstra TD, Morrison DK. KSR1 is primarily recognized as a scaffold for the Protein phosphatase 2A positively regulates Ras signaling Raf/MEK/ERK kinase cascade. However, there are by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 several published studies that sustain that KSR1 has binding sites. Curr Biol. 2003 Aug 19;13(16):1356-64 catalytic activity. KSR1 is recognized as a Xing HR, Cordon-Cardo C, Deng X, Tong W, pseudokinase, since mammalian KSR1 does not Campodonico L, Fuks Z, Kolesnick R. Pharmacologic inactivation of kinase suppressor of ras-1 abrogates Ras- possess the lysine responsible for ATP orientation mediated pancreatic cancer. Nat Med. 2003 and hydrolysis in the putative kinase domain. This Oct;9(10):1266-8 lysine is present in C. elegans and D. melanogaster, Kortum RL, Lewis RE. The molecular scaffold KSR1 but mutation of this site did not affect activation of regulates the proliferative and oncogenic potential of cells. the Raf/MEK/ERK pathway. A recent publication Mol Cell Biol. 2004 May;24(10):4407-16 (Rajakulendran et al., 2009) showed that KSR1 Laurent MN, Ramirez DM, Alberola-Ila J. Kinase dimerization via its kinase domain to Raf facilitated suppressor of Ras couples Ras to the ERK cascade during the activation of Raf without KSR1 catalytic T cell development. J Immunol. 2004 Jul 15;173(2):986-92 activity. It remains possible that KSR1 proteins Razidlo GL, Kortum RL, Haferbier JL, Lewis RE. have catalytic activity based on a yet to be Phosphorylation regulates KSR1 stability, ERK activation, identified novel mechanism. and cell proliferation. J Biol Chem. 2004 Nov 12;279(46):47808-14 References Yan F, John SK, Wilson G, Jones DS, Washington MK, Polk DB. Kinase suppressor of Ras-1 protects intestinal Kornfeld K, Hom DB, Horvitz HR. The ksr-1 gene encodes epithelium from cytokine-mediated apoptosis during a novel protein kinase involved in Ras-mediated signaling inflammation. J Clin Invest. 2004 Nov;114(9):1272-80 in C. elegans. Cell. 1995 Dec 15;83(6):903-13 Kim M, Yan Y, Kortum RL, Stoeger SM, Sgagias MK, Lee Sundaram M, Han M. The C. elegans ksr-1 gene encodes K, Lewis RE, Cowan KH. Expression of kinase suppressor a novel Raf-related kinase involved in Ras-mediated signal of Ras1 enhances cisplatin-induced extracellular signal- transduction. Cell. 1995 Dec 15;83(6):889-901 regulated kinase activation and cisplatin sensitivity. Cancer Res. 2005 May 15;65(10):3986-92 Therrien M, Chang HC, Solomon NM, Karim FD, Wassarman DA, Rubin GM. KSR, a novel protein kinase Kortum RL, Costanzo DL, Haferbier J, Schreiner SJ, required for RAS signal transduction. Cell. 1995 Dec Razidlo GL, Wu MH, Volle DJ, Mori T, Sakaue H, Chaika 15;83(6):879-88 NV, Chaika OV, Lewis RE. The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates adipogenesis. Mol Therrien M, Michaud NR, Rubin GM, Morrison DK. KSR Cell Biol. 2005 Sep;25(17):7592-604 modulates signal propagation within the MAPK cascade. Genes Dev. 1996 Nov 1;10(21):2684-95 Salerno M, Palmieri D, Bouadis A, Halverson D, Steeg PS. Nm23-H1 metastasis suppressor expression level Michaud NR, Therrien M, Cacace A, Edsall LC, Spiegel S, influences the binding properties, stability, and function of Rubin GM, Morrison DK. KSR stimulates Raf-1 activity in a the kinase suppressor of Ras1 (KSR1) Erk scaffold in kinase-independent manner. Proc Natl Acad Sci U S A. breast carcinoma cells. Mol Cell Biol. 2005 1997 Nov 25;94(24):12792-6 Feb;25(4):1379-88 Müller J, Cacace AM, Lyons WE, McGill CB, Morrison DK. Fusello AM, Mandik-Nayak L, Shih F, Lewis RE, Allen PM, Identification of B-KSR1, a novel brain-specific isoform of Shaw AS. The MAPK scaffold kinase suppressor of Ras is KSR1 that functions in neuronal signaling. Mol Cell Biol. involved in ERK activation by stress and proinflammatory 2000 Aug;20(15):5529-39 cytokines and induction of arthritis. J Immunol. 2006 Nov 1;177(9):6152-8

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 728

KSR1 (kinase suppressor of ras 1) Fernandez M, Lewis R

Kortum RL, Johnson HJ, Costanzo DL, Volle DJ, Razidlo Giurisato E, Lin J, Harding A, Cerutti E, Cella M, Lewis RE, GL, Fusello AM, Shaw AS, Lewis RE. The molecular Colonna M, Shaw AS. The mitogen-activated protein scaffold kinase suppressor of Ras 1 is a modifier of kinase scaffold KSR1 is required for recruitment of RasV12-induced and replicative senescence. Mol Cell Biol. extracellular signal-regulated kinase to the immunological 2006 Mar;26(6):2202-14 synapse. Mol Cell Biol. 2009 Mar;29(6):1554-64 McKay MM, Morrison DK. Caspase-dependent cleavage McKay MM, Ritt DA, Morrison DK. Signaling dynamics of disrupts the ERK cascade scaffolding function of KSR1. J the KSR1 scaffold complex. Proc Natl Acad Sci U S A. Biol Chem. 2007 Sep 7;282(36):26225-34 2009 Jul 7;106(27):11022-7 Ritt DA, Zhou M, Conrads TP, Veenstra TD, Copeland TD, Rajakulendran T, Sahmi M, Lefrançois M, Sicheri F, Morrison DK. CK2 Is a component of the KSR1 scaffold Therrien M. A dimerization-dependent mechanism drives complex that contributes to Raf kinase activation. Curr RAF catalytic activation. Nature. 2009 Sep Biol. 2007 Jan 23;17(2):179-84 24;461(7263):542-5 Chen C, Lewis RE, White MA. IMP modulates KSR1- Razidlo GL, Johnson HJ, Stoeger SM, Cowan KH, Bessho dependent multivalent complex formation to specify T, Lewis RE. KSR1 is required for cell cycle reinitiation ERK1/2 pathway activation and response thresholds. J following DNA damage. J Biol Chem. 2009 Mar Biol Chem. 2008 May 9;283(19):12789-96 13;284(11):6705-15

Casar B, Arozarena I, Sanz-Moreno V, Pinto A, Agudo- This article should be referenced as such: Ibáñez L, Marais R, Lewis RE, Berciano MT, Crespo P. Ras subcellular localization defines extracellular signal- Fernandez M, Lewis R. KSR1 (kinase suppressor of ras 1). regulated kinase 1 and 2 substrate specificity through Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):727- distinct utilization of scaffold proteins. Mol Cell Biol. 2009 729. Mar;29(5):1338-53

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 729

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

PEG10 (paternally expressed 10) Andreas Lux Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany (AL)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/PEG10ID44104ch7q21.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI PEG10ID44104ch7q21.txt

site of exon 1 (Lux et al., 2010) (see also figure 1). Identity PEG10-A, has a length of 6573 bp and PEG10-B of Other names: EDR; HB-1; KIAA1051; MEF3L; 6584 bp. The analysis of four different cell lines Mar2; Mart2; RGAG3 (HepG2, HEK293, HL60 and SH-SY5Y) suggest HGNC (Hugo): PEG10 that variant 1 is the major transcript. How alternative splicing for PEG10 is regulated and why Location: 7q21.3 two alternative splice products exist in parallel is Local order: Located next to the sarcoglycan not known. epsilon gene SGCE in a head-to-head orientation. The transcription start sites of these two genes are Transcription separated by 130 bp. Both genes belong to a The major transcription start site (mTSS) was maternally imprinted gene cluster in humans as determined to be at position 19.519.958 of well as in the syntenic chromosomal regions of reference sequence NT_007933|Hs7_8090 (Lux et several other mammalian species and are expressed al., 2010). This TSS is preceded by a typical from the paternal allele. TATA-box element in the ideal distance of 24-30 nucleotides. It appears as if there is at least one DNA/RNA additional may be cell type dependent but less frequently used TSS further upstream. Several Description studies attempted to analyse the PEG10 promoter The PEG10 gene is comprised of two exons and how PEG10 expression is regulated but the separated by a 6753 bp long intron for transcript results of these studies do not provide a coherent variant 1 (PEG10-A) or by a 6742 bp long intron picture. For example, it was reported that c-MYC for transcript variant 2 (PEG10-B). Transcript upregulates PEG10 expression in pancreatic and variant 2 is the result of alternative splicing where hepatic carcinoma cells as well as in a B- splicing occurs 11 nucleotides after the major splice lymphocyte cell line (Li et al., 2006).

Figure 1. PEG10 splice variants PEG10-A and PEG10-B. Shown is the sequence around the splice junctions for the two PEG10 splice variants. The different start codons, translation initiation sites (TIS), in exon 1 and 2 are in red and underlined.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 730

PEG10 (paternally expressed 10) Lux A

This effect appears to be mediated by c-MYC isolated but none of these transcripts contained the binding to an E-box sequence in the proximal typical polyadenylation signal motif at their 3'-end region of the PEG10 intron, thereby influencing nor any known alternative polyadenylation signal PEG10 promoter activity. In reporter assays, sequence motifs (Lux et al., 2010). If PEG10 analysing just the promoter sequence upstream of transcripts are truly subjected to alternative the mTSS, overexpression of c-MYC showed an polyadenylation then future studies have to address inhibitory effect (Lux et al., 2010). By the question whether this influences PEG10 bioinformatic analysis of the PEG10 promoter expression, mRNA stability, mRNA localisation or region +1 to -220, binding sites for transcription translation and if it might be related to pathological factors like TBP, Sp1 or E2F were identified (Lux processes. Because PEG10 is most likely derived et al., 2010). Binding of E2F members E2F-1 and from a retrotransposon it is interesting to note that E2F-4 to this region was experimentally proven and non-conserved poly(A) sites are associated with it was demonstrated that both factors positively transposable elements to a much greater extent than regulate PEG10 expression (Wang C et al., 2008). conserved ones (Lee et al., 2008). A previous report showed that PEG10 expression is also positively regulated by E2F-2 and E2F-3 in the Protein U2OS osteosarcoma cell line (Müller et al., 2001). These data suggest that PEG10 expression can be Description controled by the E2F/Rb pathway that involves the Transcription of PEG10 results in several protein cyclin D/CDK4 complex, which phosphorylates isoforms due to alternative splicing, alternative pocket proteins like the retinoblastoma protein Rb translation initiation sites, posttranslational and releases E2Fs. Thus, it can be expected that proteolytic cleavage and -1 ribosomal frameshift overexpression of cyclin D and CDK4 does also translation. The most prominent feature of PEG10 increase PEG10 expression, which indeed is the is its -1 ribosomal frameshift translation mechanism case (Wang C et al., 2008). In contrast, the presence that hints at his retroviral/retrotransposon origin. of TGF-beta leads to a dephosphorylation of Rb, The existence of PEG10 was reported by three therefore repressing the expression of E2F target different groups in 2001 (Ono et al., 2001; genes like PEG10 (Wang C et al., 2008). The Shigemoto et al., 2001; Volff et al., 2001). In their repressive activity of TGF-beta on PEG10 search for novelle patternally expressed imprinted expression is in agreement with our own genes for an imprinted region on mouse unpublished results with PEG10 promoter-reporter chromosome 6, syntenic to a human imprinting constructs. Previous results showed that the cluster on chromosome 7q21 containing the PEG10-RF1 protein inhibits TGF-beta3 signalling imprinted SGCE gene, Ono and colleagues in a TGF-beta-specific luciferase reporter assay performed a database search for EST sequences (Lux et al., 2005), may be representing a self- mapping to this region. Three entries were protecting mechanism from down-regulation. identified, HB-1 (GenBank Accession No. Furthermore, it was reported that increased PEG10 AF216076), KIAA1051 (GenBank Accession No. expression is subjected to hormonal regulation by AB028974), and 23915 mRNA (GenBank the male hormon androgen (Jie et al., 2007). In this Accession No. AF038197) that were identical and study, three androgen receptor binding sites (ARE) mapped near SGCE. Sequence analysis of these were identified for PEG10. Two sites were reported clones predicted two open reading frames with for exon 2 and one for the promoter region. homology to Gag and Pol proteins of some Unfortunately, no exact specifications were given vertebrate retrotransposons, respectively. The about these sites. However, using the in the deduced gene was named Paternally Expressed publication given primer sets for a Blast search Gene 10 (PEG10). Similar results were obtained by against the PEG10 sequence reveals that ARE-1 is Volff and colleagues, when they analysed public not located in the promoter but in exon 1. sequence databases for long-terminal-repeat (LTR) Therefore, all three PEG10-specific ARE sites are retrotransposon-like sequences of the Ty3/Gypsy distal to the promoter. retrotransposon family in mammals. They identified Transcript processing. Northern blot analyses KIAA1051, which showed significant similarities have shown that for humans depending on the to the Gag structural core protein of some tissue PEG10 transcripts of different size exist. One Ty3/Gypsy retrotransposons from the Ty3 family, between 6 and 7 kb, corresponding to the major 6.6 including Sushi from the pufferfish Fugu rubripes kb PEG10 transcript, as well as minor sized (42.5% similarities), Skippy, Maggy, and Cft1 from transcripts (Ono et al., 2001; Smallwood et al., different fungi. No significant similarity to other 2003; Lux et al., 2005). The major 6.6 kb PEG10 families of Ty3/Gypsy retrotransposons and transcript is polyadenylated and at the distal end of retroviruses was found and no LTR-like sequences exon 2 there are two canonical polyadenylation flanking KIAA1051 were identified. It was further sequences, AATAAA. In a recent study, minor reported that the KIAA1051 cDNA contains a sized alternatively polyadenylated transcripts were partial pol-like sequence (1.5 kb in length),

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 731

PEG10 (paternally expressed 10) Lux A

Figure 3. Schematic view of the PEG10-RF1b/2 protein. Shown are the locations of different predicted and proven protein domains. which overlaps the gag-like sequence (1 kb in Due to alternative splicing two transcript variants length) over approximately 250 bp. Its conceptual exist, PEG10-A and PEG10-B, leading to several translation product displayed protease and truncated protein isoforms. These isoforms are first, the result reverse transcriptase (RT) regions, including well- of different translation initiation sites in reading conserved first and second of the seven RT domains frame 1 (RF1) (Lux et al., 2010). Second, due to the and showed the highest similarity to the Pol protein fact, whether the reading frames 1 and 2 (RF2) are of again the retrotransposon Sushi (43.7% translated into an RF1 protein or into an RF1/2 similarity). Further evidence for the existence of protein by succesfull -1 frameshift translation, and PEG10 came from the work by Shigemoto and third, in the RF1/2 translation products, shortly colleagues. They analysed the mouse gene Edr, after the frameshift site, there is a retroviral typical which was identified initially by differential functional aspartic protease motif usually for Gag- screening of an embryonal carcinoma cDNA library Pol protein processing leading to proteolytic for genes expressed at a reduced level following cleavage products (Clark et al., 2007). It was retinoic acid induced differentiation (Gorman et al., demonstrated that upstream of the originaly 1985). Their study identified in the Edr gene two predicted ATG translation initiation site (TIS) a open reading frames that overlapped and were set second in frame non-ATG exists. For clarification, appart by a frameshift of one nucleotide. the non-ATG translation site will be named TIS-1a Subsequent analysis demonstrated that both reading and the previous ATG start site TIS-2. This non- frames are translated by -1 frameshifting ATG start codon, a CTG, is 102 nucleotides (Shigemoto et al., 2001; Lux et al., 2005; upstream of the ATG start codon. The alternative Manktelow et al., 2005; Clark et al., 2007). splice event that leads to transcript PEG10-B Translation. In order to perform the -1 frameshift, introduces an additional in frame ATG start codon the reading frame 1 (RF1) - reading frame 2 (RF2) even further upstream of the previous two, which overlap sequence contains a seven nucleotide will be named TIS-1b. Their might be an additional "slippery" sequence with typical consecutive TIS further downstream of TIS-2 (Lux et al., 2010). homopolymeric triplets. The underlined PEG10 Transcript PEG10-A codes for PEG10-RF1 (using "slippery" heptanucleotide sequence G GGA AAC TIS-2), PEG10-RF1a (using TIS-1a), PEG10-RF1/2 TC follows the general pattern of X XXY YYZ and PEG10-RF1a/2. While in theory, transcript where the A- and P-site tRNAs detach from the PEG10-B can lead to all six isoforms, PEG10-RF1, zero frame codons XXY YYZ and re-pair after PEG10-RF1a, PEG10-RF1b (using TIS-1b), shifting back one nucleotide to XXX YYY and PEG10-RF1/2, PEG10-RF1a/2 and PEG10-RF1b/2. restart translation with the codon after the YYY Figure 1 shows in a more schematic way the triplet. Thus, the deduced amino acid sequence of different TIS for transcripts PEG10-A and PEG10- the frameshift site after frameshift translation is B. The deduced amino acid sequences of the GNL. The heptanucleotide "slippery" sequence is different isoforms are listed in figure 2. completely conserved in all species and the Investigation of PEG10 translation in mouse sequence of the downstream pseudoknot is placenta during gestation and human placenta completely conserved in the mammalian species. showed that in vivo both reading frames are except for one nucleotide change in the rodent translated as an RF1 protein and an RF1/2 fusion sequence. A detailed analysis of the PEG10 protein (Clark et al., 2007). The mouse RF1/2 frameshift sequence was done by Manktelow and protein is about 40 kDa larger than the colleagues (2005). corresponding human protein due to an in frame

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 732

PEG10 (paternally expressed 10) Lux A

insertion of approximately 600 nucleotides into the The ELM programme predicted the C-terminal RF2 sequence. Interestingly, the size of RF1 and proline stretch to be a possible binding site for SH3 RF1/2 proteins and the translational frame shift domain containing proteins. As already reported, efficiency varies during gestation. From 9.5 dpc PEG10 contains a retroviral typical aspartyl when PEG10 expression in mice is first detectable, protease consensus sequence, AMIDSGA. In order the 150 kDa frameshift protein is dominant. By to test whether this motif is catalytic active the using an RF1-specific antibody the frameshift aspartate was mutated to an alanine (Clark et al., efficiency was estimated and showed an apparent 2007). This change disrupted the protease activity decrease from 68% at 9.5 dpc to 43% by 21.5 dpc. and proved that the aspartyl protease is responsible At 15.5 dpc an additional protein of 105 kDa was for the cleavage of the full length PEG10 frameshift detected at about equal amounts as the 150 kDa protein in to the RF1 and RF2 parts. Taken the protein. At late gestation, 21.5 dpc, it was present in protease activity into account the previously greater amounts than the 150 kDa PEG10-RF1/2 estimated PEG10 frameshift efficiency of 15-30% protein. Mass spectrometry analysis identified the (Shigemoto et al., 2001; Lux et al., 2005) was 105 kDa protein as a PEG10 product consisting reestimated to be 60% (Clark et al., 2007). primarily of PEG10 RF2 but containing peptides Interacting proteins. Aside from the protease from both reading frames. PEG10 protein analysis motif, for none of the other domains it is known for amniotic membrane showed a similar profile to whether they are functional nor if they bind to other that of placenta. Starting with a low expression at proteins. The only known binding partners for 9.5 dpc and then an increased and continued PEG10 are currently the SIAH1 and SIAH2 expression throughout gestation. The RF1/2 fusion proteins (Okabe et al., 2003) and the TGF-beta type protein again was the dominant band. Surprisingly, I receptor ALK1 (Lux et al., 2005). All three at 10.5 dpc a transient RF1 protein of increased proteins were identified by a yeast two-hybrid mass of about 50 kDa was detected that disappeared screen with the PEG10-RF1 protein and the at later time points and only the slightly smaller 47- interactions were confirmed by co- kDa band identical in size to that in placenta was immunoprecipitation experiments. The exact present again. Furthermore, in this report three RF1 SIAH1/SIAH2 binding region was not determined, protein populations were detected for HepG2 cells but the ELM programme predicted a potential ranging from 47 to 55 kDa. Whether these different SIAH1 binding site (figure 3, PEG10-RF1b amino PEG10 protein masses are the result of post- acids 329-337). Co-immunoprecipitation translational modifications or due to the use of experiments by overexpressing PEG10-RF1 and different TIS or a mixture of both is not clear and several other type I and II receptors of the TGF-beta awaits further investigations. superfamily in COS-1 cells showed that PEG10 In addition, western blot analysis with an RF1- does also interact with other members of this specific antibody of adult mouse heart, spleen and receptor group (Lux et al., 2005). Nevertheless, brain tissue extracts showed a weak, single protein when specifically investigated in the two-hybrid band but of different mass, around 50 kDa, for each assay under stringent conditions none of these tissue (Clark et al., 2007). No RF1/2 proteins were receptors reacted with PEG10-RF1 to activate the detected. The authors concluded based on their reporter system. Thus, the most specific interaction further analysis that these proteins do not represent appears to be with ALK1. Peg10 proteins. Expression Protein domains/motifs. By bioinformatic analyses using different programmes like the Based on data obtained from mice, Peg10 is Simple Modular Architecture Research Tool predominantly expressed during embryonic (SMART), the SUPERFAMILY Sequence Search development, whereas later on expression in most (SCOP domains) and the Eukaryotic Linear Motif tissues ceases or is low except for testis and brain of (ELM) resource for functional site prediction, adult animals (Shigemoto et al., 2001). However, several domains and motifs were predicted. Some significant induction of Peg10 expression was are exemplarily shown schematicaly in figure 3 for detected in hepatocellular carcinomas (HCC) and the 784 amino acid long PEG10-RF1b/2 protein. for the regenerating livers of mice after partial The Zink-finger domain was consistently identified hepatectomy (Tsou et al., 2003). Studies with mice although the size of the domain varies from amino by RNA in situ hybridisation showed a high acid 357-389 or a core region from 370-386 for a expression during embryonic development especially from day 9.5 to 16.5, specifically in bone ZNF-C2HC (CX2CX4HX4C) consensus sequence, which is highly conserved in Gag proteins in most and cartilage forming tissues as well as in extra retroviruses and some retrotransposons. There are embryonic tissues at all stages between E7.5 and two proline rich regions, one at the N-terminus and E17.5. For a detailed description see Shigemoto et one at the C-terminus. Proline-rich regions are al. (2001). In humans, expression of PEG10 in adult recognized as presenting binding motifs to, for tissues was seen in brain, kidney, lung, testis and example, Src homology 2 (SH2) and SH3 domains. only weak to very weak expression in spleen, liver,

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 733

PEG10 (paternally expressed 10) Lux A

colon, small intestine and muscle, but no expression Functional data regarding the different RF1/2 for heart and stomach (Ono et al., 2001). isoforms are lacking too. Furthermore, strong expression was also reported Nevertheless, PEG10-RF1 appears to enhance cell for mouse and human placenta. In humans PEG10 proliferation and blocks apoptosis. Evidence for expression is low at the early hypoxic phase of PEG10's role in cell proliferation were reported by placental growth and increases at 11-12 weeks of Tsou et al. (2003). Induced PEG10 expression was gestation. This high level of expression is found during G2/M phase of regenerating mouse maintained and is significantly increased in term liver and elevated expression of PEG10 was found placenta compared with that in early pregnancy in HCC. The authors state that both, HCC and (Smallwood et al., 2003). The authors hypothesize regenerating mouse livers, represent the two that the gene product might be essential for proliferative states of the otherwise quiescent liver trophoblast differentiation and uterine implantation. tissue. In addition, ectopic expression of PEG10 in Peg10 expression was observed in relation to 293T cells enhanced cell cycle progression. adipocyte differentiation in mice. Peg10 was Complementary data were presented by Okabe and identified as one of the genes expressed early in colleagues (2003). The hepatoma cell line, SNU423 adipogenesis (Hishida et al., 2007). Expression of that has no endogenous PEG10, was stable Peg10 was elevated after the addition of transfected with an expression construct for differentiation inducers in adipocyte differentiable PEG10-RF1 to test the effect of PEG10 on cell 3T3-L1 cells, but not in the non-adipogenic cell line growth. The PEG10 stable transfectant cells NIH-3T3. The knockdown of Peg10 by RNA revealed significant growth promotion compared interference inhibited the differentiation of 3T3-L1 with the parental or mock cells. Under conditions of cells. Moreover, Peg10 siRNA treatment impaired serum starvation (0.1% FBS), the mock cells mitotic clonal expansion (MCE), necessary for rapidly underwent growth arrest, but stable PEG10- adipocyte differentiation, and the crucial expression expressing cells continued to proliferate. In the of C/EBPbeta and C/EBPdelta at the immediate same report, in a yeast two-hybrid screen, the early stage of the differentiation process was apoptosis inducing protein SIAH1 was identified as inhibited by the knock-down. These results indicate a PEG10-RF1 interactor. Overexpression of SIAH1 that Peg10 plays an important role at the immediate increased cell death in different hepatoma cell lines, early stage of adipocyte differentiation. whereas co-expression of PEG10-RF1 in tested Aside from bone and cartilage tissue differentiation SNU423 revealed a partial but significant and adipocyte cell differentiation PEG10 might also protection from apoptosis. Anti-apoptotic activity be involved in neuronal cell differentiation. In a of PEG10 was also reported by Yoshibayashi et al. preliminary experiment with the neuroblastoma cell (2007). The finding that PEG10 enhances cell line SH-SY5Y increasing PEG10 expression was proliferation was further confirmed by PEG10- reported over a period of 14 days after treatment specific siRNA knock-down experiments in a series with all-trans retinoic acid for differentiation (Lux of carcinoma cell lines, i.e. Panc1, HepG2, and et al., 2010). Hep3B, which led to a significantly reduced cell Localisation proliferation (Li et al., 2006; Yoshibayashi et al., 2007). Data regarding PEG10's cellular localisation do Anti-apoptotic activity by PEG10 were not only only exist for PEG10-RF1. Okabe and colleagues seen for HCC/hepatoma but also reported for B-cell (2003) report for the hepatoma cell lines HepG2, acute and chronic lymphocytic leukemia, B-ALL Huh7 and Alexander as well as for hepatocellular and B-CLL. It was observed that B-ALL and B- carcinoma (HCC) tissues nuclear and cytoplasmic CLL CD19+CD34+ B cells expressed elevated staining of PEG10 with a PEG10-RF1-specific levels of PEG10, regulated by the chemokines antibody. In experiments overexpressing PEG10- CXCL13 and CCL19 and that these cells were RF1 in HEK293T cells, only cytoplasmic resistant to TNF-alpha induced apoptosis. localisation was reported (Tsou et al., 2003), which Treatment of the cells with PEG10 antisense was also shown by immunofluorescence analysis constructs reversed this effect (Hu et al., 2004; with a PEG10-RF1-specific antibody for Chunsong et al., 2006; Wang et al., 2007). Kainz endogenous PEG10 in HepG2 and B-CLL cells and colleagues (2007) reported that PEG10 (Lux et al., 2005; Kainz et al., 2007). overexpression is associated with high-risk B-CLL. Function Expression levels in CD19+ B-CLL cells were up to 100-fold higher than in B-cells from healthy The knowledge regarding PEG10's protein function donors and expression levels in B-CLL patient is sparse and all direct evidence that exists so far samples remained stable over time even after was gained by experiments with the PEG10-RF1 chemotherapy. The intensity of intracellular isoform only. No data exists for the recently staining of PEG10 protein corresponded to mRNA identified isoform PEG10-RF1a, most likely the levels. Further analysis of PEG10's anti-apoptotic major RF1 isoform, or the PEG10-RF1b protein. potential showed that short term knock-down (2

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 734

PEG10 (paternally expressed 10) Lux A

days after transfection) of PEG10 in B-CLL cells was not associated with changes in cell survival but Implicated in long term inhibition (4 days after transfection of Various cancers PEG10) led to a significant effect on the induction of apoptosis and late apoptosis/necrosis in B-CLL Note cells. Two major pathological conditions are reported in As described above, during gestation PEG10 is which PEG10 plays a role, hepato cellular highly expressed in the placenta. Mouse placenta is carcinomas and B-cell acute and chronic positive for Peg10 transcripts as well as for proteins lymphocytic leukemia. As already mentioned from reading frame 1 and reading frame 1/2 (Clark above, the presence of PEG10 is linked to et al., 2007). Peg10 knock-out experiments in mice resistance of apoptosis and increased cell growth. In demonstrated that Peg10 and therefore the Peg10 addition, further malignancies in which an over proteins have an important role during placenta expression or prolonged expression of PEG10 was development. Heterozygous knock-outs for the seen are the embryonic kidney malignancy Wilms patternal allele died at 10.5 dpc. Their placentas tumor (Dekel et al., 2006), pancreatic cancer (Li et were severely depleted and the labyrinth layer was al., 2006) and the embryonic form of biliary atresia not developed and the spongiotrophoblast cells (Zhang et al., 2004). were missing (Ono et al., 2006). As discussed by Widespread DNA copy number alterations are well the authors, parthenogenetic embryos die before 9.5 recognized in HCC and concurrent genomic gains d.p.c. and show early embryonic lethality with within the chromosome region 7q21 has been poorly developed extraembryonic tissues. implicated in the progression of HCC. In a study by Morphological defects of the most developed Ip et al. (2007), it was suggested that PEG10 may parthenotes are very similar to those of Peg10-Pat be a potential biomarker in the progressive KO embryos; they lack the diploid trophoblast cells development of HCC. Quantitative PCR and qRT- of the labyrinth layer and the spongiotrophoblast. PCR showed the chromosomal gain of 7q21 as well However, the majority of parthenotes show more as over expression of PEG10 in HCC cell lines and severe phenotypes; suggesting that other genes primary tumors. In addition, qRT-PCR could also contribute to the parthenogenetic demonstrated a significant progressive trend of phenotypes. Nevertheless, the result for mouse increasing PEG10 expression from the putative pre- Peg10 suggests that one of the Peg10 isoforms malignant adjacent livers to early resectable HCC could be critical for parthenogenetic development tumors, and to late inoperable HCCs. The authors in mice and therefore also in man. concluded that genomic gain represents one of the major mechanisms in the induction of PEG10 over Homology expression. This conclusion is further supported by PEG10 orthologous sequences can be found in independent data from Tsuji et al. (2010). Increased several other eutherian species: Pan troglodytes PEG10 expression might also serve as a biomarker (chimpanzee), Papio anubis (baboon), Macaca for nephropathy in peripheral blow cells of type 2 mulatta (Rhesus monkey), Callithrix jacchus diabetes (T2DN) (Guttula et al., 2010). (marmoset), Sus scrofa (pig), Canis familiaris (dog), Felis catus (cat), Bos taurus (bovine), Ovis References aries (sheep), Mus musculus (mouse), Rattus Gorman CM, Lane DP, Watson CJ, Rigby PW. The norvegicus (rat), Rhinolophus ferrumequinum (bat), regulation of gene expression in murine teratocarcinoma Sorex araneus (shrew), Monodelphis domestica cells. Cold Spring Harb Symp Quant Biol. 1985;50:701-6 (opossum) as well as in the metatherian tammar Müller H, Bracken AP, Vernell R, Moroni MC, Christians F, wallaby (Macropus eugenii) (Ono et al., 2001; Grassilli E, Prosperini E, Vigo E, Oliner JD, Helin K. E2Fs Brandt et al., 2005a; Brandt et al., 2005b; Suzuki et regulate the expression of genes involved in differentiation, al., 2007; Clark et al., 2007) and there is a high development, proliferation, and apoptosis. Genes Dev. degree of amino acid conservation. The PEG10 2001 Feb 1;15(3):267-85 sequence is not conserved in reptiles or birds. Ono R, Kobayashi S, Wagatsuma H, Aisaka K, Kohda T, Hence, during evolution the PEG10 gene was Kaneko-Ishino T, Ishino F. A retrotransposon-derived gene, PEG10, is a novel imprinted gene located on human introduced into the therian mammal genome after chromosome 7q21. Genomics. 2001 Apr 15;73(2):232-7 the split of birds about 300 million years (myr) ago and after the split of prototherian mammals Shigemoto K, Brennan J, Walls E, Watson CJ, Stott D, Rigby PW, Reith AD. Identification and characterisation of (monotremes) 166 myr ago, but before the a developmentally regulated mammalian gene that utilises divergence between placental mammals and -1 programmed ribosomal frameshifting. Nucleic Acids marsupials about 148 myr ago. Res. 2001 Oct 1;29(19):4079-88

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 735

PEG10 (paternally expressed 10) Lux A

Volff J, Körting C, Schartl M. Ty3/Gypsy retrotransposon N, Miki H, Kohda T, Ogura A, Yokoyama M, Kaneko-Ishino fossils in mammalian genomes: did they evolve into new T, Ishino F. Deletion of Peg10, an imprinted gene acquired cellular functions? Mol Biol Evol. 2001 Feb;18(2):266-70 from a retrotransposon, causes early embryonic lethality. Nat Genet. 2006 Jan;38(1):101-6 Okabe H, Satoh S, Furukawa Y, Kato T, Hasegawa S, Nakajima Y, Yamaoka Y, Nakamura Y. Involvement of Clark MB, Jänicke M, Gottesbühren U, Kleffmann T, Legge PEG10 in human hepatocellular carcinogenesis through M, Poole ES, Tate WP. Mammalian gene PEG10 interaction with SIAH1. Cancer Res. 2003 Jun expresses two reading frames by high efficiency -1 15;63(12):3043-8 frameshifting in embryonic-associated tissues. J Biol Chem. 2007 Dec 28;282(52):37359-69 Smallwood A, Papageorghiou A, Nicolaides K, Alley MK, Jim A, Nargund G, Ojha K, Campbell S, Banerjee S. Hishida T, Naito K, Osada S, Nishizuka M, Imagawa M. Temporal regulation of the expression of syncytin (HERV- peg10, an imprinted gene, plays a crucial role in adipocyte W), maternally imprinted PEG10, and SGCE in human differentiation. FEBS Lett. 2007 Sep 4;581(22):4272-8 placenta. Biol Reprod. 2003 Jul;69(1):286-93 Ip WK, Lai PB, Wong NL, Sy SM, Beheshti B, Squire JA, Tsou AP, Chuang YC, Su JY, Yang CW, Liao YL, Liu WK, Wong N. Identification of PEG10 as a progression related Chiu JH, Chou CK. Overexpression of a novel imprinted biomarker for hepatocellular carcinoma. Cancer Lett. 2007 gene, PEG10, in human hepatocellular carcinoma and in Jun 8;250(2):284-91 regenerating mouse livers. J Biomed Sci. 2003;10(6 Pt 1):625-35 Jie X, Lang C, Jian Q, Chaoqun L, Dehua Y, Yi S, Yanping J, Luokun X, Qiuping Z, Hui W, Feili G, Boquan J, Youxin Hu C, Xiong J, Zhang L, Huang B, Zhang Q, Li Q, Yang M, J, Jinquan T. Androgen activates PEG10 to promote Wu Y, Wu Q, Shen Q, Gao Q, Zhang K, Sun Z, Liu J, Jin carcinogenesis in hepatic cancer cells. Oncogene. 2007 Y, Tan J. PEG10 activation by co-stimulation of CXCR5 Aug 23;26(39):5741-51 and CCR7 essentially contributes to resistance to apoptosis in CD19+CD34+ B cells from patients with B cell Kainz B, Shehata M, Bilban M, Kienle D, Heintel D, lineage acute and chronic lymphocytic leukemia. Cell Mol Krömer-Holzinger E, Le T, Kröber A, Heller G, Immunol. 2004 Aug;1(4):280-94 Schwarzinger I, Demirtas D, Chott A, Döhner H, Zöchbauer-Müller S, Fonatsch C, Zielinski C, Stilgenbauer Zhang DY, Sabla G, Shivakumar P, Tiao G, Sokol RJ, S, Gaiger A, Wagner O, Jäger U. Overexpression of the Mack C, Shneider BL, Aronow B, Bezerra JA. Coordinate paternally expressed gene 10 (PEG10) from the imprinted expression of regulatory genes differentiates embryonic locus on chromosome 7q21 in high-risk B-cell chronic and perinatal forms of biliary atresia. Hepatology. 2004 lymphocytic leukemia. Int J Cancer. 2007 Nov Apr;39(4):954-62 1;121(9):1984-93 Brandt J, Schrauth S, Veith AM, Froschauer A, Haneke T, Suzuki S, Ono R, Narita T, Pask AJ, Shaw G, Wang C, Schultheis C, Gessler M, Leimeister C, Volff JN. Kohda T, Alsop AE, Marshall Graves JA, Kohara Y, Ishino Transposable elements as a source of genetic innovation: F, Renfree MB, Kaneko-Ishino T. Retrotransposon expression and evolution of a family of retrotransposon- silencing by DNA methylation can drive mammalian derived neogenes in mammals. Gene. 2005 Jan genomic imprinting. PLoS Genet. 2007 Apr 13;3(4):e55 17;345(1):101-11 Wang X, Yuling H, Yanping J, Xinti T, Yaofang Y, Feng Y, Brandt J, Veith AM, Volff JN. A family of neofunctionalized Ruijin X, Li W, Lang C, Jingyi L, Zhiqing T, Jingping O, Ty3/gypsy retrotransposon genes in mammalian genomes. Bing X, Li Q, Chang AE, Sun Z, Youxin J, Jinquan T. Cytogenet Genome Res. 2005;110(1-4):307-17 CCL19 and CXCL13 synergistically regulate interaction between B cell acute lymphocytic leukemia CD23+CD5+ B Lux A, Beil C, Majety M, Barron S, Gallione CJ, Kuhn HM, Cells and CD8+ T cells. J Immunol. 2007 Sep Berg JN, Kioschis P, Marchuk DA, Hafner M. Human 1;179(5):2880-8 retroviral gag- and gag-pol-like proteins interact with the transforming growth factor-beta receptor activin receptor- Yoshibayashi H, Okabe H, Satoh S, Hida K, Kawashima K, like kinase 1. J Biol Chem. 2005 Mar 4;280(9):8482-93 Hamasu S, Nomura A, Hasegawa S, Ikai I, Sakai Y. SIAH1 causes growth arrest and apoptosis in hepatoma cells Manktelow E, Shigemoto K, Brierley I. Characterization of through beta-catenin degradation-dependent and - the frameshift signal of Edr, a mammalian example of independent mechanisms. Oncol Rep. 2007 programmed -1 ribosomal frameshifting. Nucleic Acids Mar;17(3):549-56 Res. 2005;33(5):1553-63 Lee JY, Ji Z, Tian B. Phylogenetic analysis of mRNA Chunsong H, Yuling H, Li W, Jie X, Gang Z, Qiuping Z, polyadenylation sites reveals a role of transposable Qingping G, Kejian Z, Li Q, Chang AE, Youxin J, Jinquan elements in evolution of the 3'-end of genes. Nucleic Acids T. CXC chemokine ligand 13 and CC chemokine ligand 19 Res. 2008 Oct;36(17):5581-90 cooperatively render resistance to apoptosis in B cell lineage acute and chronic lymphocytic leukemia Wang J, Tai LS, Tzang CH, Fong WF, Guan XY, Yang M. CD23+CD5+ B cells. J Immunol. 2006 Nov 1p31, 7q21 and 18q21 chromosomal aberrations and 15;177(10):6713-22 candidate genes in acquired vinblastine resistance of human cervical carcinoma KB cells. Oncol Rep. 2008 Dekel B, Metsuyanim S, Schmidt-Ott KM, Fridman E, May;19(5):1155-64 Jacob-Hirsch J, Simon A, Pinthus J, Mor Y, Barasch J, Amariglio N, Reisner Y, Kaminski N, Rechavi G. Multiple Wang C, Xiao Y, Hu Z, Chen Y, Liu N, Hu G. PEG10 imprinted and stemness genes provide a link between directly regulated by E2Fs might have a role in the normal and tumor progenitor cells of the developing human development of hepatocellular carcinoma. FEBS Lett. 2008 kidney. Cancer Res. 2006 Jun 15;66(12):6040-9 Aug 6;582(18):2793-8 Li CM, Margolin AA, Salas M, Memeo L, Mansukhani M, Guttula SV, Rao AA, Sridhar GR, Chakravarthy MS, Hibshoosh H, Szabolcs M, Klinakis A, Tycko B. PEG10 is Nageshwararo K, Rao PV. Cluster analysis and a c-MYC target gene in cancer cells. Cancer Res. 2006 phylogenetic relationship in biomarker identification of type Jan 15;66(2):665-72 2 diabetes and nephropathy. Int J Diabetes Dev Ctries. 2010 Jan;30(1):52-6 Ono R, Nakamura K, Inoue K, Naruse M, Usami T, Wakisaka-Saito N, Hino T, Suzuki-Migishima R, Ogonuki

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 736

PEG10 (paternally expressed 10) Lux A

Lux H, Flammann H, Hafner M, Lux A. Genetic and hepatocellular carcinoma. Cancer Genet Cytogenet. 2010 molecular analyses of PEG10 reveal new aspects of Apr 15;198(2):118-25 genomic organization, transcription and translation. PLoS One. 2010 Jan 13;5(1):e8686 This article should be referenced as such: Tsuji K, Yasui K, Gen Y, Endo M, Dohi O, Zen K, Lux A. PEG10 (paternally expressed 10). Atlas Genet Mitsuyoshi H, Minami M, Itoh Y, Taniwaki M, Tanaka S, Cytogenet Oncol Haematol. 2011; 15(9):730-737. Arii S, Okanoue T, Yoshikawa T. PEG10 is a probable target for the amplification at 7q21 detected in

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 737

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

PIK3CD (phosphoinositide-3-kinase, catalytic, delta polypeptide) Emily Burns, Bart Vanhaesebroeck Centre of Cell Signalling, Barts Cancer Institute, Queen Mary University of London, London, EC1M 6BQ, UK (EB, BV)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/PIK3CDID46261ch1p36.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI PIK3CDID46261ch1p36.txt

Identity Protein Other names: P110DELTA; PI3K; p110D Description HGNC (Hugo): PIK3CD The PI3K enzymes are a family of activation Location: 1p36.22 dependent lipid kinases, which have been divided Local order: Telomere-SPSB1-SLC25A33- into three classes (Vanhaesebroeck et al., 1999a). TMEM201-PIK3CD-C1orf200-CR604408- PIK3CD encodes p110delta which belongs to the KIAAO00911-centromere. class I PI3Ks alongside p110alpha, p110beta and p110gamma. DNA/RNA The PI3K enzymes are heterodimeric molecules which consist of a catalytic p110 subunit, and a Description constitutively bound p85 regulatory unit of which The PIK3CD gene spans a genomic region of 24 there are five isoforms encoded by three genes coding exons and over 77.17 kb. (PIK3R1, PIK3R2 and PIK3R3) (Vanhaesebroeck and Waterfield, 1999; Geering et al., 2007). Transcription The regulatory subunit has a dual function of The dominant transcript contains two upstream recruiting the p110 catalytic subunit to an activated untranslated exons, -2a and -1, as well as an upstream tyrosine kinase receptor, and inhibiting additional first untranslated exon (-2b) identified in catalytic activity in the absence of this stimulatory the human PIK3CD gene (Kok et al., 2009). interaction (Yu et al., 1998).

Location of PIK3CD on human chromosome 1.

The illustration represents the exon structure of the most prevalent transcript in leukocytes, which contains two untranslated exons upstream of the start site (-2a and -1) and 24 coding exons.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 738

PIK3CD (phosphoinositide-3-kinase, catalytic, delta Burns E, Vanhaesebroeck B polypeptide)

p110delta protein domains. p110delta interacts with its associated p85 regulatory subunit via the adaptor binding domain (ABD). The C Lobe and N Lobe together form the catalytic domain.

Unlike p110alpha, p110delta does not phoshorylate null mouse (Clayton et al., 2002; Jou et al., 2002) its associated p85 regulatory subunit and instead and in a p110delta kinase-dead mouse known as auto-phosphorylates (Vanhaesebroeck et al., 1997; p110deltaD910A/D910A (Okkenhaug et al., 2002). Vanhaesebroeck et al., 1999a). Phenotypic analysis of the p110deltaD910A/D910A Expression mouse revealed an important role for p110delta in immunity; the mice have notable defects in their T p110delta has a restricted expression pattern; it is and B cell responses, as well as several other highly expressed in leukocytes (Seki et al., 1997; immunological abnormalities (Okkenhaug et al., Vanhaesebroeck et al., 1997) and to a lesser extent 2002). In vitro and ex vivo studies suggest that expressed in neurons (Eickholt et al., 2007). In p110delta is the predominant PI3K class IA isoform addition p110delta expression has also been regulating the phenotype and responses of many observed in a number of cancerous tissues leukocyte cell types (Vanhaesebroeck et al., 1999b; including breast cancer and melanoma cells Puri et al., 2004; Papakonstanti et al., 2008). In (Sawyer et al., 2003). mast cells derived from the p110delta kinase-dead A transcription factor binding cluster located mice, ninety percent of the total PI3K lipid kinase immediately upstream of the untranslated exon -2a activity was dependent on p110delta (Ali et al., in the human gene, has been identified and found to 2004). p110delta has been found to be a key player have enhanced promotor activity in leukocytes. in the induction of amongst other things, This promoter region may therefore facilitate the proliferation, chemotaxis, and cytokine/chemokine enhanced expression of p110delta in leukocytes release in response to both physiological stimuli (Kok et al., 2009). and pathological stimuli, in many leukocytes Localisation (Okkenhaug and Vanhaesebroeck, 2003; Dos Santos et al., 2007; Papakonstanti et al., 2008; Dil p110 delta localises to the cytosol and is recruited et al., 2009; Low et al., 2010). to the periplasmic region upon stimulation of upstream activators. Homology Function p110delta is most homologous to p110beta (Clayton et al., 2001). Cell signalling and lipid kinase activity. PI3K signalling has been found to play a crucial role in the regulation of numerous cellular processes Mutations including proliferation, metabolism (Foukas et al., Note 2006; Engelman et al., 2006), and migration No prevalent p110delta mutations have been (Papakonstanti et al., 2007). observed in primary samples or cell lines. Active p110delta initiates signalling cascades by phosphorylating phosphoinositide (PI) lipids such Implicated in as PIP2 producing pools of PIP3 in the periplasmic region (Vanhaesebroeck et al., 1997). A wide range Haematological malignancy of effector proteins recognise and interact with the Note PI species produced, and the resulting alteration in Constitutive phosphorylation of Akt (indicating location and activities of these effectors initiates a constitutive activity) has been observed in many number of signalling cascades. The classical haematological malignancies, and appears to be effector of PI3K signalling is the serine/threonin responsible for or contributing to growth factor- kinase AKT (aka protein kinase B), which contains independent proliferation (Min et al., 2003, Ikeda et a pleckstrin homology (PH) domain that interacts al., 2010). p110delta appears to be upstream of the with PIP (Franke et al., 1997). The affinity of Akt 3 constitutively active Akt in these transformed cells; for PIP facilitates activation of Akt's protein kinase 3 p110delta-selective inhibitors significantly reduce activity, resulting in activity-modifying the abberent Akt activation and also results in a phosphorylation of a range of downstream Akt reduction in proliferation (Billottet et al., 2006; targets. Ikeda et al., 2010; Herman et al., 2010; Lannutti et Roles of p110delta in immune function. The role al., 2011). of p110delta in vivo has been studied in a p110delta

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 739

PIK3CD (phosphoinositide-3-kinase, catalytic, delta Burns E, Vanhaesebroeck B polypeptide)

Despite the dominance of p110delta in the regulation of proliferation in leukocytes and the References abberant activity of it's major downstream target Franke TF, Kaplan DR, Cantley LC, Toker A. Direct Akt, no mutations or amplifications of p110delta regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 1997 Jan have been observed in these or other 31;275(5300):665-8 haematological malignancies. A series of clinical trials are now underway to Seki N, Nimura Y, Ohira M, Saito T, Ichimiya S, Nomura N, Nakagawara A. Identification and chromosome assignment assess the effects of selective inhibition of of a human gene encoding a novel phosphatidylinositol-3 p110delta on refractory haematological kinase. DNA Res. 1997 Oct 31;4(5):355-8 malignancies, and preliminary results are promising Vanhaesebroeck B, Welham MJ, Kotani K, Stein R, Warne (Furman, 2010). PH, Zvelebil MJ, Higashi K, Volinia S, Downward J, Disease Waterfield MD. P110delta, a novel phosphoinositide 3- kinase in leukocytes. Proc Natl Acad Sci U S A. 1997 Apr CLL, T-ALL, B-ALL, DLBCL, AML, and multiple 29;94(9):4330-5 myeloma. Yu J, Zhang Y, McIlroy J, Rordorf-Nikolic T, Orr GA, Allergic disease (Ali et al., 2004; Lee et al., Backer JM. Regulation of the p85/p110 2006; Medina-Tato et al., 2007; Park et al., 2008) phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory Note subunit. Mol Cell Biol. 1998 Mar;18(3):1379-87 D910A/D910A Mast cells derived from p110delta mice Vanhaesebroeck B, Higashi K, Raven C, Welham M, have a degranulation defect, as do wild-type mast Anderson S, Brennan P, Ward SG, Waterfield MD. cells treated with a p110delta-selective inhibitor. Autophosphorylation of p110delta phosphoinositide 3- Specifically cells lacking active p110delta showed a kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo. EMBO J. 1999a Mar 1;18(5):1292-302 45-55% reduction in degranulation induced by an antigen-IgE complex, compared to the Vanhaesebroeck B, Jones GE, Allen WE, Zicha D, degranulation observed in WT mast cells (Ali et al., Hooshmand-Rad R, Sawyer C, Wells C, Waterfield MD, Ridley AJ. Distinct PI(3)Ks mediate mitogenic signalling 2004). Furthermore inactivation of p110delta has and cell migration in macrophages. Nat Cell Biol. 1999b been shown to attenuate anaphylactic responses in May;1(1):69-71 mice (Ali et al., 2004). p110delta-selective Vanhaesebroeck B, Waterfield MD. Signaling by distinct inhibitors have been shown to attenuate OVA- classes of phosphoinositide 3-kinases. Exp Cell Res. 1999 induced influx into lungs of total leukocytes, Nov 25;253(1):239-54 eosinophils, neutrophils, and lymphocytes, as well Clayton E, McAdam S, Coadwell J, Chantry D, Turner M. as reducing the production of a number of pro- Structural organization of the mouse phosphatidylinositol inflammatory mediators (Lee et al., 2006). 3-kinase p110d gene. Biochem Biophys Res Commun. 2001 Feb 9;280(5):1328-32 Disease Asthma, allergic rhinitis. Clayton E, Bardi G, Bell SE, Chantry D, Downes CP, Gray A, Humphries LA, Rawlings D, Reynolds H, Vigorito E, Chronic inflammatory conditions Turner M. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and Note activation. J Exp Med. 2002 Sep 16;196(6):753-63 p110delta has been identified as a major regulator Jou ST, Carpino N, Takahashi Y, Piekorz R, Chao JR, of inflammatory responses in a wide range of Carpino N, Wang D, Ihle JN. Essential, nonredundant role leukocytes (including macrophages, neutrophils, for the phosphoinositide 3-kinase p110delta in signaling by mast cells as well as the adaptive immune cells). It the B-cell receptor complex. Mol Cell Biol. 2002 it is considered an excellent potential therapeutic Dec;22(24):8580-91 target for reducing inflammation both in the acute Okkenhaug K, Bilancio A, Farjot G, Priddle H, Sancho S, allergic context but also in the context of chronic Peskett E, Pearce W, Meek SE, Salpekar A, Waterfield MD, Smith AJ, Vanhaesebroeck B. Impaired B and T cell inflammatory disorders (Ji et al., 2007; Rommel et antigen receptor signaling in p110delta PI 3-kinase mutant al., 2007). The importance of p110delta in the mice. Science. 2002 Aug 9;297(5583):1031-4 regulation of inflammatory responses is highlighted Min YH, Eom JI, Cheong JW, Maeng HO, Kim JY, Jeung by the development of spontaneous chronic colonic HK, Lee ST, Lee MH, Hahn JS, Ko YW. Constitutive D910A/D910A inflammation in the p110delta mice, phosphorylation of Akt/PKB protein in acute myeloid which has been attributed to a failure to repress leukemia: its significance as a prognostic variable. inflammatory responses to commensal bacteria in Leukemia. 2003 May;17(5):995-7 the gut (Uno et al., 2010). Thus p110delta-selective Okkenhaug K, Vanhaesebroeck B. PI3K in lymphocyte drugs are currently under investigation as a development, differentiation and activation. Nat Rev Immunol. 2003 Apr;3(4):317-30 treatment for rheumatoid arthritis, asthma and other inflammatory conditions. Sawyer C, Sturge J, Bennett DC, O'Hare MJ, Allen WE, Bain J, Jones GE, Vanhaesebroeck B. Regulation of Disease breast cancer cell chemotaxis by the phosphoinositide 3- Rheumatoid arthritis, inflammatory bowel disease kinase p110delta. Cancer Res. 2003 Apr 1;63(7):1667-75 (IBD). Ali K, Bilancio A, Thomas M, Pearce W, Gilfillan AM, Tkaczyk C, Kuehn N, Gray A, Giddings J, Peskett E, Fox

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 740

PIK3CD (phosphoinositide-3-kinase, catalytic, delta Burns E, Vanhaesebroeck B polypeptide)

R, Bruce I, Walker C, Sawyer C, Okkenhaug K, Finan P, Papakonstanti EA, Zwaenepoel O, Bilancio A, Burns E, Vanhaesebroeck B. Essential role for the p110delta Nock GE, Houseman B, Shokat K, Ridley AJ, phosphoinositide 3-kinase in the allergic response. Nature. Vanhaesebroeck B. Distinct roles of class IA PI3K isoforms 2004 Oct 21;431(7011):1007-11 in primary and immortalised macrophages. J Cell Sci. 2008 Dec 15;121(Pt 24):4124-33 Puri KD, Doggett TA, Douangpanya J, Hou Y, Tino WT, Wilson T, Graf T, Clayton E, Turner M, Hayflick JS, Park SJ, Min KH, Lee YC. Phosphoinositide 3-kinase delta Diacovo TG. Mechanisms and implications of inhibitor as a novel therapeutic agent in asthma. phosphoinositide 3-kinase delta in promoting neutrophil Respirology. 2008 Nov;13(6):764-71 trafficking into inflamed tissue. Blood. 2004 May 1;103(9):3448-56 Dil N, Marshall AJ. Role of phosphoinositide 3-kinase p110 delta in TLR4- and TLR9-mediated B cell cytokine Billottet C, Grandage VL, Gale RE, Quattropani A, Rommel production and differentiation. Mol Immunol. 2009 C, Vanhaesebroeck B, Khwaja A. A selective inhibitor of Jun;46(10):1970-8 the p110delta isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic Kok K, Nock GE, Verrall EA, Mitchell MP, Hommes DW, effects of VP16. Oncogene. 2006 Oct 26;25(50):6648-59 Peppelenbosch MP, Vanhaesebroeck B. Regulation of p110delta PI 3-kinase gene expression. PLoS One. Engelman JA, Luo J, Cantley LC. The evolution of 2009;4(4):e5145 phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006 Aug;7(8):606-19 Furman RR. New agents in early clinical trials for CLL therapy. Clin Adv Hematol Oncol. 2010 Jul;8(7):475-6 Foukas LC, Claret M, Pearce W, Okkenhaug K, Meek S, Peskett E, Sancho S, Smith AJ, Withers DJ, Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao Vanhaesebroeck B. Critical role for the p110alpha W, Flynn JM, Jones J, Andritsos L, Puri KD, Lannutti BJ, phosphoinositide-3-OH kinase in growth and metabolic Giese NA, Zhang X, Wei L, Byrd JC, Johnson AJ. regulation. Nature. 2006 May 18;441(7091):366-70 Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic Lee KS, Lee HK, Hayflick JS, Lee YC, Puri KD. Inhibition leukemia by antagonizing intrinsic and extrinsic cellular of phosphoinositide 3-kinase delta attenuates allergic survival signals. Blood. 2010 Sep 23;116(12):2078-88 airway inflammation and hyperresponsiveness in murine asthma model. FASEB J. 2006 Mar;20(3):455-65 Ikeda H, Hideshima T, Fulciniti M, Perrone G, Miura N, Yasui H, Okawa Y, Kiziltepe T, Santo L, Vallet S, Cristea Dos Santos S, Delattre AI, De Longueville F, Bult H, Raes D, Calabrese E, Gorgun G, Raje NS, Richardson P, M. Gene expression profiling of LPS-stimulated murine Munshi NC, Lannutti BJ, Puri KD, Giese NA, Anderson KC. macrophages and role of the NF-kappaB and PI3K/mTOR PI3K/p110{delta} is a novel therapeutic target in multiple signaling pathways. Ann N Y Acad Sci. 2007 Jan;1096:70- myeloma. Blood. 2010 Sep 2;116(9):1460-8 7 Low PC, Misaki R, Schroder K, Stanley AC, Sweet MJ, Eickholt BJ, Ahmed AI, Davies M, Papakonstanti EA, Teasdale RD, Vanhaesebroeck B, Meunier FA, Taguchi T, Pearce W, Starkey ML, Bilancio A, Need AC, Smith AJ, Stow JL. Phosphoinositide 3-kinase δ regulates membrane Hall SM, Hamers FP, Giese KP, Bradbury EJ, fission of Golgi carriers for selective cytokine secretion. J Vanhaesebroeck B. Control of axonal growth and Cell Biol. 2010 Sep 20;190(6):1053-65 regeneration of sensory neurons by the p110delta PI 3- kinase. PLoS One. 2007 Sep 11;2(9):e869 Uno JK, Rao KN, Matsuoka K, Sheikh SZ, Kobayashi T, Li F, Steinbach EC, Sepulveda AR, Vanhaesebroeck B, Geering B, Cutillas PR, Nock G, Gharbi SI, Sartor RB, Plevy SE. Altered macrophage function Vanhaesebroeck B. Class IA phosphoinositide 3-kinases contributes to colitis in mice defective in the are obligate p85-p110 heterodimers. Proc Natl Acad Sci U phosphoinositide-3 kinase subunit p110δ. S A. 2007 May 8;104(19):7809-14 Gastroenterology. 2010 Nov;139(5):1642-53, 1653.e1-6 Ji H, Rintelen F, Waltzinger C, Bertschy Meier D, Bilancio Lannutti BJ, Meadows SA, Herman SE, Kashishian A, A, Pearce W, Hirsch E, Wymann MP, Rückle T, Camps M, Steiner B, Johnson AJ, Byrd JC, Tyner JW, Loriaux MM, Vanhaesebroeck B, Okkenhaug K, Rommel C. Inactivation Deininger M, Druker BJ, Puri KD, Ulrich RG, Giese NA. of PI3Kgamma and PI3Kdelta distorts T-cell development CAL-101, a p110delta selective phosphatidylinositol-3- and causes multiple organ inflammation. Blood. 2007 Oct kinase inhibitor for the treatment of B-cell malignancies, 15;110(8):2940-7 inhibits PI3K signaling and cellular viability. Blood. 2011 Jan 13;117(2):591-4 Medina-Tato DA, Ward SG, Watson ML. Phosphoinositide 3-kinase signalling in lung disease: leucocytes and This article should be referenced as such: beyond. Immunology. 2007 Aug;121(4):448-61 Burns E, Vanhaesebroeck B. PIK3CD (phosphoinositide-3- Papakonstanti EA, Ridley AJ, Vanhaesebroeck B. The kinase, catalytic, delta polypeptide). Atlas Genet Cytogenet p110delta isoform of PI 3-kinase negatively controls RhoA Oncol Haematol. 2011; 15(9):738-741. and PTEN. EMBO J. 2007 Jul 11;26(13):3050-61 Rommel C, Camps M, Ji H. PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond? Nat Rev Immunol. 2007 Mar;7(3):191-201

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 741

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

S100A8 (S100 calcium binding protein A8) Claus Kerkhoff, Saeid Ghavami Dept VAC / IMCI, Helmholtz Centre for Infection Research, Inhoffenstr 7, D-38124 Braunschweig, Germany (CK), Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada (SG)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/S100A8ID46446ch1q21.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI S100A8ID46446ch1q21.txt

proteins that fulfil important functions in terminal Identity differentiation in the human epidermis, including Other names: 60B8AG; CAGA; CFAG; CGLA; filaggrin, loricrin and others. In addition, linkage CP-10; L1Ag; MA387; MIF; MRP8; NIF; P8 analyses have identified a psoriasis susceptibility HGNC (Hugo): S100A8 region, the PSORS4 locus, that is close to the S100 gene cluster (Hardas et al., 1996; Semprini et al., Location: 1q21.3 2002). These data are important indications for the Local order: Distal to LOC645900, proximal to involvement of S100 genes in inflammatory as well S100A7A (S100 calcium binding protein A7A). as neoplastic disorders. It has been speculated that the rearrangements result in a deregulated DNA/RNA expression of S100 genes associated with neoplasia. Note Description S100A8 belongs to the S100/calgranulin family of The S100 gene structure has been structurally small non-ubiquitous cytoplasmic Ca2+-binding conserved during evolution. Similar to most S100 proteins of EF-hand type. The proteins were genes S100A8 consists of three exons that are referred to "S100" because of their solubility in separated by two introns. saturated ammonium sulphate solution. Sixteen of Transcription 21 members are localised in a cluster on human chromosome 1q21. In the S100A8 gene, exon 1 encodes the The clustered organization of these S100 genes is untranslated region. The protein is encoded by conserved during evolution (Ridinger et al., 1998). sequences in exon 2 and exon3, encoding a N- A comparison between man and mouse has shown terminal and a C-terminal EF-hand motif, that during evolution, the colinearity of the S100 respectively. gene cluster has been destroyed by some inversions. The sequence of human S100A8 cDNA has an open However, the colocalization of the myeloid reading frame of 279 nucleotides predicting a expressed S100 genes such as S100A8, S100A9, protein of 93 amino acids. and S100A12 is conserved. It has been speculated, S100A8 expression appears to be restricted to a that the structural integrity of that part of the locus specific stage of myeloid differentiation. The is necessary for the coordinated expression of these protein is present in circulating neutrophils and genes (Nacken et al., 2001). monocytes, but not in resting tissue macrophages. Remarkably, the S100 gene cluster is located in In peripheral blood monocytes it is down regulated close proximity to a region which has been during maturation to macrophages. Despite a frequently rearranged in human cancer (Carlsson et number of distinct regulatory regions are located al., 2005) and to the epidermal differentiation upstream of the transcription initiation site, the complex (EDC) (Mischke et al., 1996). EDC is a corresponding nuclear factors as well as the cluster of genes on chromosome 1q21 encoding underlying molecular mechanisms still remain

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 742

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

unclear. Transcription factors such as PU.1 (Henkel Expression et al., 2002), C/EBP-alpha and C/EBP-beta S100A8 is mainly expressed in cells of the myeloid (Kuruto-Niwa et al., 1998) have been shown to lineage, however, its gene expression is induced in drive S100 gene expression within the myeloid epithelial cells in response to stress, in specific lineage. conditions such as wound healing, UV exposition, Beside its expression in myeloid cells S100A8 is abundant in psoriasis keratinocytes, differentially expressed in epithelia under specific conditions. Its expressed in several cancers. expression is transiently induced in keratinocytes after epidermal injury and UVB irradiation, and the Localisation protein is expressed at extremely high levels in Mostly cytoplasmic, but also at membranes and psoriatic keratinocytes. Furthermore, its expression cytoskeleton. is induced by pro-inflammatory cytokines such as In resting phagocytes the S100A8/A9 protein TNFalpha and IL1beta. Recently, a complex of complex is mainly located in the cytosol. Upon Poly (ADP-ribose) polymerase (PARP-1) and cellular activation the protein complex is either Ku70/Ku80 has been demonstrated to drive the translocated to cytoskeleton and plasma membrane stress response-specific S100 gene expression or released into the extracellular environment. (Grote et al., 2006). The stress response-induced The translocation pathways occur upon the expression of the S100 proteins points to an elevation of the intracellular calcium level (Roth et important role in skin pathology. al., 1993). At a later time point, the S100A8/A9 Pseudogene heterodimers can be detected on the surface of monocytes (Bhardwaj et al., 1992). The mechanism Not known. by which the S100A8/A9 heterodimer penetrates the plasma membrane remains unclear since the Protein S100 proteins lack a transmembrane signaling Description region. The secretion pathway relies on the activation of The sequence of human S100A8 cDNA has an open protein kinase C. This pathway differs from the reading frame of 279 nucleotides predicting a classical as well as the alternative secretion protein of 93 amino acids and a calculated Mr of pathways of cytokines (Moqbel and Coughlin, 10835 Da. The theoretical isoelectric point is 6.6. 2006). It has been demonstrated that this novel S100A8 is composed of two helix-loop-helix EF- secretion pathway is energy-consuming and hand motifs. The C-terminal EF-hand contains a 2+ depends on an intact microtubule network (Murao canonical Ca -binding loop of 12 amino acids. et al., 1990; Rammes et al., 1997). Conversely, the N-terminal EF-hand contains a 2+ 2+ Recent investigations give evidence that interaction Ca -binding loop of 14 residues that binds Ca of S100A8/A9 with annexin-6 is involved in mostly through main-chain carbonyl groups that surface expression and release of S100A8/A9 which is specific to S100 proteins. Consequently, 2+ (Bode et al., 2008). Annexins are another class of S100 proteins have a weaker Ca affinity than 2+ 2+ Ca -regulated proteins. They are characterized by typical Ca sensors such as calmodulin (Donato, the unique architecture of their Ca2+-binding sites, 2003). which enables them to peripherally dock onto In vivo and in vitro experiments have shown that negatively charged membrane surfaces in their S100 proteins form homo-, hetero- and oligomeric Ca2+-bound conformation. This property links assemblies (Hunter and Chazin, 1998; Osterloh et annexins to many membrane-related events such as al., 1998; Pröpper et al., 1999; Moroz et al., 2003). certain exocytic and endocytic transport steps. This Together with their specific cell- and tissue- is an interesting finding since S100A8 and S100A9 expression patterns, the structural variations, and are expressed in cancerous cells of secretory tissues the different metal ion binding properties (Ca2+, 2+ 2+ as breast and prostate. Cells originating from such Zn and Cu ) the S100 protein complexes might glandular tissues are rich in membrane structures, be functionally diversified. S100A8 preferentially suggesting that membrane-associated molecular interacts with S100A9. targets for the S100A8/A9 proteins could be It is worthwhile mentioning that the murine analogs potentially found in these cells. display a stronger tendency to form homodimeric Recent investigations also demonstrated the protein complexes. In view of the formation of association of S100A8/A9 with cholesterol- different tertiary structures with putative distinct enriched membrane microdomains (lipid rafts) functions it is tempting to speculate that S100A8 (Nacken et al., 2004). This observation is in and/or A9 have different functions in mouse and agreement with the enhancing effect of S100A8/A9 man. on NADPH oxidase since the formation of the oxidase complex takes place at lipid rafts.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 743

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

Function In view of the stress response-induced expression of the two S100 proteins in keratinocytes these Intra- as well as extracellular roles have been findings have great implications for tissue proposed for the S100 proteins. remodeling and repair. For example, keratinocytes Intracellular activities of S100A8/A9 acquire an activated state after cutaneous wounding In the intracellular milieu, S100 proteins are in which proliferation is favored over considered as calcium sensors changing their differentiation in order to replenish the lost material conformation in response to calcium influx and and rapidly close the site of injury. Thus, it is likely then mediating calcium signals by binding to other to hypothesize that S100A8/A9-mediated growth intracellular proteins. In a mouse knock-out model reduction is required for the upcoming cell fate chemokine-induced down regulation of the 2+ decision of damaged cells, i.e. for a survival phase cytosolic Ca -level was detected (Nacken et al., to be followed by differentiation, proliferation, or 2005). apoptosis. These data have also an impact on After calcium binding, the S100A8/A9 protein tumorigenesis since S100 gene expression is complex binds specifically polyunsaturated fatty associated with neoplastic disorders. acids. S100A8/A9 represents the exclusive In migrating monocytes the S100A8/A9 complex arachidonic acid-binding capacity in the neutrophil has been found to be associated with cytoskeletal cytosol (Kerkhoff et al., 1999), and participates in tubulin and to modulate transendothelial migration NADPH oxidase activation by transferring (Vogl et al., 2004). Investigations using two arachidonic acid to membrane-bound gp91phox different mouse knock-out models demonstrated no during interactions with two cytosolic oxidase obvious phenotype (Manitz et al., 2003; Hobbs et activation factors, p67phox and Rac-2. The al., 2003). However, reduced migration of S100A9- functional relevance of S100A8/A9 in the deficient neutrophils and decreased surface phagocyte NADPH oxidase activation was expression of CD11b, which belongs to the integrin demonstrated by the impairment of NADPH family, were observed upon in vitro stimulation. oxidase activity in neutrophil-like NB4 cells, after Extracellular activities of S100A8/A9 specifically blocking S100A9 expression, and The S100/calgranulins display antimicrobial employing bone marrow-derived PMNs from -/- activity by depriving bacterial pathogens of S100A9 mice (Kerkhoff et al., 2005). essential trace metals such as Zn2+ and Mn2+ In accordance to their role in myeloid cells, (Steinbakk et al., 1990; Murthy et al., 1993; S100A8/A9 enhances epithelial NADPH oxidases Clohessy and Golden, 1995; Sohnle et al., 2000). In (Benedyk et al., 2007). As a consequence of the context of inflammation, it has been proposed enhanced ROS levels, NF-kB activation and that S100A8/A9 is massively released when subsequently TNF-alpha and IL-8 mRNA levels are neutrophils die to provide a growth-inhibitory type increased in S100A8/A9-HaCaT keratinocytes, of host defense that is adjunctive to the usual consistent with the view that NF-kB is a redox- microbicidal functions by binding metals other than sensitive transcription factor. Further consequences Ca2+ (Corbin et al., 2008). of S100A8/A9-mediated NF-kB activation are In addition, S100/calgranulins serve as leukocyte reduced cell growth, increased expression of chemoattractants (Lackmann et al., 1992; differentiation markers, and enhanced PARP Lackmann et al., 1993; Kocher et al., 1996; Lim et cleavage as an indicator of increased cell death al., 2008). Murine S100A8 has potent chemotactic (Voss et al., 2011). activity for neutrophils and monocytes in vitro and

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 744

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

in vivo (Lackmann et al., 1992). In contrast, human receptor for advanced glycation end products S100A8 displays only weak leukocyte chemotactic (RAGE) (Srikrishna and Freeze, 2009). activity in vitro and in vivo (Lackmann et al., The S100/calgranulins display cytokine-like 1993). Detailed analysis revealed that the hinge functions, including activation of the receptor for region contributes to the chemotactic activity of advanced glycation endproducts (RAGE) (Hofmann murine, but not human S100A8. These data et al., 1999; Herold et al., 2007). RAGE is a questioned whether the proteins are orthologs since member of the immunoglobulin superfamily and there is a high degree of homology between murine present on numerous cell types. It has been shown and human S100A8 but a functional divergence. to play crucial roles in a variety of Intriguingly, human S100A12 is chemotactic and pathophysiological situations, such as wound the hinge region of human S100A12 has been healing, atherosclerotic lesion development, tumor implicated herein (Yang et al., 2001). Thus, the growth and metastasis, systemic amyloidosis, and functional and sequence divergence suggested Alzheimer disease (Bierhaus et al., 2005). complex evolution of the S100 family in mammals. RAGE/S100 interaction has been considered a very The putative pro-inflammatory functions of attractive model to explain how RAGE and its S100A8 and S100A9 have recently been proinflammatory ligand contribute to the investigated in two different mouse knock-out pathophysiology of several inflammatory diseases. models. S100A9 deficiency did not result in an Beside the above mentioned receptors a number of obvious phenotype (Manitz et al., 2003; Hobbs et other cell surface binding sites specific for al., 2003). However, reduced migration of S100A9- S100A8/A9 have been reported, such as novel deficient neutrophils and decreased surface carboxylated glycans (Srikrishna et al., 2001), expression of CD11b, which belongs to the integrin heparan sulfate glycosaminoglycans (Robinson et family, were observed upon in vitro stimulation. In al., 2002), beta2-integrin (Newton and Hogg, addition, chemokine-induced down regulation of 1998), and the fatty acid transporter FAT/CD36 the cytosolic Ca2+-level was detected. Obviously, (Kerkhoff et al., 2001). Therefore, the cell surface these in vitro effects are compensated by alternative receptor of S100A8/A9 is still in debate. pathways in vivo. Interestingly, the growth-stimulatory activity of Remarkably, cancer cells utilize S100A8 and S100A8/A9 has been demonstrated to be mediated S100A9 as guidance for the adhesion and invasion by binding to the receptor of advanced glycation of disseminating malignant cells (Hiratsuka et al., end products (RAGE) (Ghavami et al., 2008b; 2006). In the context of malignancy it was reported Turovskaya et al., 2008; Gebhardt et al., 2008). It is that S100A8/A9 attracts Mac-1+ myeloid cells to likely to speculate that the selective up-regulation the lung tissue. Recruited Mac-1+ myeloid cells in of S100 proteins may be of importance for survival lung in turn produce S100A8/A9 in response to and proliferation of metastasizing cancer cells. primary malignant cells in a so called S100A8/A9 complexes that are secreted from "premetastatic phase". This phase shows the general phorbolester-stimulated neutrophil-like HL-60 cells characteristics of an inflammation state which have been shown to carry the eicosanoid precursor facilitates the micro-environmental changes arachidonic acid (Kerkhoff et al., 1999). The required for the migration and implantation of S100A8/A9-arachidonic acid complex is primary tumor cells to lung tissue. After preparation recognized by the fatty acid transporter FAT/CD36, of the target tissue for accepting the malignant cells, and the fatty acid is rapidly taken up (Kerkhoff et tumor cells mimic Mac-1+ myeloid cells in response al., 2001). Endothelial cells as well as neutrophils to S100A8/A9 chemotactic signaling and migrate to themselves utilize both endogenous and exogenous lung. So, it seems that tumor cells and Mac-1+ arachidonic acid for transcellular production of myeloid cells utilize a common pathway for eicosanoids (Sala et al., 1999). Therefore, the migration to lung which involves the activation of secreted S100A8/A9-AA complex may serve as a mitogen-activated protein kinase pathway transport protein to move AA to its target cells. (Hiratsuka et al., 2006). These findings suggest This may represent a mechanism by which AA- S100A8/A9 as an attractive target for the derived eicosanoids are synthesized in a development of strategies counteracting tumor cooperative manner between different cell species metastasizing to certain organs. due to environmental cues. S100A8 and S100A9 have been identified as S100A8/A9 displays apoptosis-inducing activity important endogenous damage-associated against various tumor cells (Yui et al., 1995; Yui et molecular pattern (DAMP) proteins. Although al., 2002; Ghavami et al., 2004; Ghavami et al., receptors for S100A8/A9 are still largely 2008a; Kerkhoff and Ghavami, 2009; Ghavami et uncharacterized, more recent findings support the al., 2009; Ghavami et al., 2010). It was speculated notion that they function as potent ligands of that this activity was due to the ability to bind pattern-recognition receptors, such as the toll-like divalent metal ions including Zn2+, Mn2+ and Cu2+ receptor 4 (TLR4) (Vogl et al., 2007) and the at sites that are distinct from Ca2+-binding sites.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 745

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

However, a number of recent reports now indicate observed in the squamous epithelia under normal, that S100A8/A9 exerts its activity by both chelation inflammatory and cancerous conditions. of trace metal ions such as Zn2+ and cell surface Immunohistochemical investigations have shown receptor mediated pathways. that the S100 proteins are over expressed in skin Although a number of receptors have been shown cancers, pulmonary adenocarcinoma, pancreatic to bind S100A8/A9, the nature of the receptor adenocarcinoma, bladder cancers, ductal carcinoma involved in S100A8/A9-induced cell death remains of the breast, and prostate adenocarcinoma. In to be elucidated. Experiments with certain cell lines contrast, S100A8 and S100A9 are down-regulated either deficient for or over expressing components in esophageal squamous cell carcinomas. of the death signaling machinery as well as RAGE Furthermore, plasma levels of S100A8/A9 are gene silencing and blocking RAGE-specific elevated in patients suffering from various cancers. antibody approaches excluded both RAGE and the Insofar, S100A8 and S100A9 might represent novel classical death receptor to be involved in diagnostic markers for some carcinomas. S100A8/A9-induced cell death, even though S100A8 and S100A9 have been suggested to have S100A8/A9 can specifically bind to cancer cells potential roles in carcinogenesis and tumor and RAGE mediates the growth-promoting activity progression. However, the biological role of obvious at low micromolar concentrations of S100A8/A9 remains to be elucidated. It is S100A8/A9. Clearly, investigations to identify the conceivable that S100A8 and S100A9 modulate receptor involved in S100A8/A9-induced cell death signal pathways to directly promote invasion, are critical. migration and metastasis, probably via activation of Homology NF-kB, Akt or MAP kinases. In the last decade the concept of the functional Overall, the S100 proteins share significant relationship between inflammation and cancer has sequential homology in the EF-hand motifs, but are been developed that is based on numerous findings, least conserved in the hinge region. This region is ranging from epidemiological studies to molecular proposed to provide for specific interaction with analyses of mouse models (Coussens and Werb, target proteins (Groves et al., 1998; Zimmer et al., 2002). In this concept, the generation of an 2003; Santamaria-Kisiel et al., 2006; Fernandez- inflammatory microenvironment supports Fernandez et al., 2008; van Dieck et al., 2009). The tumorigenesis by promoting cancer cell survival, availability of high-resolution S100-target proliferation, migration, and invasion. Although it structures has highlighted important structural is clear that inflammation alone does not cause features that contribute to S100 protein functional cancer, it is evident that an environment that is rich specificity (Bhattacharya et al., 2003). in inflammatory cells, growth factors, activated The functional diversification of S100 proteins is stroma, and DNA-damage-promoting agents achieved by their specific cell- and tissue- certainly potentiates and/or promotes neoplastic expression patterns, structural variations, different 2+ 2+ 2+ risk. In addition, many cancers arise from sites of metal ion binding properties (Ca , Zn and Cu ) infection, chronic irritation and inflammation. as well as their ability to form homo-, hetero- and Recent data have expanded our knowledge oligomeric assemblies (Hunter and Chazin, 1998; demonstrating that specific soluble factors released Osterloh et al., 1998; Pröpper et al., 1999; from primary tumors induce the S100A8 and Tarabykina et al., 2001; Moroz et al., 2003; Fritz et S100A9 gene expression in the target tissue. After al., 2010). Although the function of S100 proteins secretion S100A8 and S100A9 might display in cancer cells in most cases is still unknown, the chemokine- and cytokine-like properties that specific expression patterns of these proteins are a promote invasion, migration and metastasis. These valuable diagnostic tool. data indicate that tumor cells are able to reprogram some of the signaling molecules of the innate Implicated in immune system. These insights are fostering new anti-inflammatory therapeutic approaches to cancer General note development. Note Skin cancer Comparative and functional genomics have revealed that a number of S100 proteins are found Note to be differentially expressed in cancer cells. The expression of S100A8 and S100A9 in Several of these have been associated with tumor epithelial cells was first detected in the squamous development, cancer invasion or metastasis in epithelia (Gabrielsen et al., 1986). Normal S100A8 recent studies (for review see Salama et al., 2008). and S100A9 are expressed at minimal levels in the S100A8 and S100A9 are abundant in cells of the epidermis. However, their expression is induced in myeloid lineage, are released from activated inflammatory and cancerous conditions, and pro- phagocytes and display intra- and extracellular inflammatory cytokines such as TNF-alpha and IL1 functions. Their expression is ubiquitously beta are involved herein.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 746

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

Gene expression analysis in a mouse model of this study correlates S100A9 expression with chemically induced skin carcinogenesis identified a inflammation and other clinical features (Su et al., large set of novel tumor-associated genes including 2010). S100A8 (Hummerich et al., 2006). The data was Primary tumors influence the environment in the confirmed by in situ hybridization and lungs before metastasis. They release specific immunofluorescence analysis on mouse tumor soluble factors that prepare the premetastatic niche sections, in mouse keratinocyte cell lines that form for the engraftment of tumor cells. In several tumors in vivo, and in human skin tumor studies it has been shown that tumor cells induce specimens. both expression and secretion of S100A8 and However, conflicting results have been published S100A9 in the target organ that display a promoting concerning S100 expression in skin cancer. For role in cancer cell survival, proliferation, migration, instance, esophageal squamous cell carcinoma and invasion (Hiratsuka et al., 2002; Hiratsuka et (ESCC) is one of the most common cancers al., 2006; Hiratsuka et al., 2008; Saha et al., 2010). worldwide. DNA microarray data analysis revealed Microarray analysis of lungs from tumor-bearing that S100A8 and S100A9 were significantly down and non-bearing mice revealed the strong up- regulated in human ESCC versus the normal regulation of a number of genes including S100A8 counterparts (Zhi et al., 2003). Interestingly, among and S100A9 (Hiratsuka et al., 2002). Their the 42 genes either up regulated or down regulated expression in Mac 1+-myeloid cells and endothelial in tumors, as compared to normal esophageal cells was induced by factors such as vascular squamous epithelia, nine of the altered expression endothelial growth factor A (VEGF-A), tumor genes were related to arachidonic acid (AA) necrosis factor-alpha (TNF-alpha) and transforming metabolism, suggesting that AA metabolism growth factor-beta (TGF-beta), both in vitro and in pathway and its altered expression may contribute vivo (Hiratsuka et al., 2006). Remarkably, anti- to esophageal squamous cell carcinogenesis. S100A8 neutralizing antibody treatment blocked Similar data were obtained by Ji et al. (2004). They metastasis. S100A8 and S100A9 were shown to investigated the differential expression of the S100 induce the expression of serum amyloid A (SAA) gene family at the RNA level in human ESCC. that attracted Mac 1+-myeloid cells in the Eleven out of 16 S100 genes were significantly premetastatic lung (Hiratsuka et al., 2008). These down regulated in ESCC versus the normal studies demonstrated that lung cancer cells utilize counterparts. Only the S100A7 gene was found to S100A8 and S100A9 as guidance for the adhesion be markedly up regulated. Another study and invasion of disseminating malignant cells. demonstrated that poorly differentiated ESCC Pancreatic adenocarcinoma displayed a stronger decrease in S100A8 and S100A9 expression than well and moderately Note differentiated tumors, with a correlation between Patients with ductal adenocarcinoma of the protein level and histopathological grading (Kong pancreas have a dismal prognosis. Thus, there is an et al., 2004). These findings suggest that decreased urgent need for early detection markers and the expression of S100A8 and S100A9 might play an development of immunotherapeutical approaches important role in the ESCC pathogenesis, being concentrating on the induction and enhancement of particularly associated with poor differentiation of immune responses against tumors. Proteomic tumor cells. analyses of pancreatic adenocarcinoma, normal adjacent tissues, pancreatitis, and normal pancreatic Lung adenocarcinomas tissues revealed a number of differentially Note expressed genes (Shen et al., 2004). S100A8 was S100A9 over expression has been detected in found to be specifically over expressed in tumors various carcinomas of glandular cell origin, and its compared with normal and pancreatitis tissues. expression has been associated with poor tumor These data are in accordance with another study differentiation. Similarly, S100A9 (Sheikh et al., 2007). Strong expression of S100A8 immunopositivity was also detected in pulmonary and S100A9 was found in tumor-associated stroma adenocarcinoma cell lines and resected pulmonary but not in benign or malignant epithelia. Further adenocarcinoma (Arai et al., 2001). Examination of analyses identified stromal CD14+ CD68- the relation of S100A9 expression to tumor monocytes/macrophages as source for S100 differentiation showed that the expression rate in expression. Interestingly, the number of S100A8- pulmonary adenocarcinoma showed higher positive cells in the tumor microenvironment correlation in poorly differentiated carcinomas. negatively correlated with the expression of the Another study confirmed these data (Su et al., tumor suppressor protein, Smad4. The number of 2010). Immunohistochemical staining of both S100 S100A9-positive cells was not altered in Smad4- proteins showed a significant up-regulation in lung negative or Smad4-positive tumors. cancer tissue, and quantitative PCR revealed A similar correlation was found in colorectal cancer significantly higher levels of S100A8 and S100A9 tumors (Ang et al., 2010). The number of stromal mRNA transcripts in lung cancer tissues. Moreover, S100A8- and S100A9-positive cells was associated

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 747

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

with the presence or absence of Smad4. Smad4- bladder cancer (NMIBC) (Ha et al., 2010). S100A8 negative tumors showed enhanced numbers of expression was evaluated in a total of 103 primary S100A8/A9 stroma cells, and the corresponding NMIBC samples by quantitative PCR. The mRNA patients had a poor survival prognosis. expression levels of S100A8 were significantly Investigation of the underlying molecular related to the progression of NMIBC, suggesting mechanisms revealed that both migration and that S100A8 might be a useful prognostic marker proliferation was enhanced in response to for disease progression of NMIBC. exogenous S100A8 and S100A9, irrespective of Breast cancers Smad4-presence. However, depletion of Smad4 resulted in loss of responsiveness to exogenous Note S100A8, but not S100A9. Vice versa, Smad4 S100A8 and S100A9 are expressed in breast expression in Smad4-negative cells enhanced the cancers (Cross et al., 2005), especially in invasive responsive-ness to S100A8 and S100A9. Further breast carcinoma (Arai et al., 2004). By analyses give evidence that similar to TGF-beta, immunohistochemical analyses a strong S100A9 S100A8 and S100A9 induce the phosphorylation of immunoreactivity has been demonstrated in both Smad2 and Smad3 that was blocked by a invasive as well as non-invasive ductal carcinoma. RAGE-specific antibody. These data point to a No immunopositive reaction was observed in functional relationship between inflammation and invasive lobular carcinomas, and no significant tumorigenesis. differences were detected in the number of myelomonocytic cells expressing S100A9. These Bladder cancers data give evidence that S100A9 in glandular Note epithelial cells is newly expressed under cancerous Gene expression profiles revealed that thirteen conditions and is over-expressed in poorly members of the S100 gene family were differentiated adenocarcinoma (Arai et al., 2004). differentially expressed in human bladder cancers. Further analyses target on the relationship between S100A8 and S100A9 were found to be over S100A8/A9 expression and pathological parameters expressed (Yao et al., 2007). that reflect the aggressiveness of carcinoma. The Another study investigated S100A9 expression and immunopositivity for S100A8/A9 correlated with DNA methylation in urothelial cancer cell lines and poor tumor differentiation, mitotic activity, cancer tissue (Dokun et al., 2008). Expression of HER2/neu over expression, poor pT categories, S100A9 was found to be generally elevated in the node metastasis, and poor pStage, but not with tumor tissues but S100A9 was weakly expressed in vessel invasion. These data may indicate that most cancer cell lines. The S100A9 promoter S100A8 and S100A9 over expression should be contains 6 CpG sites, and its methylation state was considered marker of poor prognosis in invasive unrelated to the variable expression. It has been breast ductal carcinoma (Arai et al., 2008). hypothesized that over expression of genes is the By analyses of ductal carcinoma in situ and consequence of DNA hypomethylation, however, invasive ductal carcinoma of the breast S100A9 has DNA methylation and gene expression are less been demonstrated to be most abundantly expressed strictly related for those genes having promoters in the invasive tumor (Seth et al., 2003). Therefore, within CpG-islands. Alternatively, the increased the expression of S100A8 and S100A9 has been S100A9 gene expression may be related to that of correlated with the degree of noninvasive / invasive other immune-related genes in the carcinoma cell behavior. There are conflicting data concerning this cultures. This is sustained by the facts that S100A9 correlation. For instance, non-invasive MCF-7 is secreted by epithelial and other cell types to breast cancer cells do not express S100A9, and its modulate inflammatory reactions as well as to gene expression is induced by cytokine oncostatin promote cancer proliferation and metastasis. M through the STAT3 signaling cascade (Li et al., Two recent studies propose S100A8 and S100A9 2004). However, non-invasive MDA-MB-468 cells gene expression as prognostic value for bladder are abundant for both S100 proteins (Bode et al., cancer (Minami et al., 2010; Ha et al., 2010). By 2008) and invasive breast cancer cells MDA-MB- proteomic analysis of pre- and postoperative sera 231 show low transcript level of S100A9 (Nagaraja from bladder cancer patients S100A8 and S100A9 et al., 2006). were identified as tumor-associated proteins Thyroid carcinoma (Minami et al., 2010). Interestingly, S100A8 expression was associated with bladder wall muscle Note invasion of the tumor and cancer-specific survival Similar to other carcinomas of glandular cell origin, while S100A9 expression was associated with the expression of S100A8 and S100A9 is significantly tumor grade. In addition, the expression of both linked to dedifferentiation of thyroid carcinoma (Ito proteins S100A8/A9 was correlated with et al., 2005; Ito et al., 2009). S100A8 and S100A9 recurrence-free survival. immunreactivity was found in all undifferentiated In another study it was evaluated whether S100A8 carcinomas examined, while papillary carcinoma, is a prognostic value for non-muscle-invasive follicular carcinoma, follicular adenoma and

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 748

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

medullary carcinoma and normal follicules were Bhardwaj RS, Zotz C, Zwadlo-Klarwasser G, Roth J, negative for both proteins. Further analyses Goebeler M, Mahnke K, Falk M, Meinardus-Hager G, Sorg C. The calcium-binding proteins MRP8 and MRP14 form a revealed that S100A9 is a useful marker for membrane-associated heterodimer in a subset of discriminating intrathyroid epithelial tumor from monocytes/macrophages present in acute but absent in squamous cell carcinoma or undifferentiated chronic inflammatory lesions. Eur J Immunol. 1992 carcinoma with squamoid component (Ito et al., Jul;22(7):1891-7 2006). Lackmann M, Cornish CJ, Simpson RJ, Moritz RL, Geczy CL. Purification and structural analysis of a murine Prostate cancer chemotactic cytokine (CP-10) with sequence homology to Note S100 proteins. J Biol Chem. 1992 Apr 15;267(11):7499- 504 Increased levels of S100A8, S100A9, and RAGE have been reported in prostatic intra epithelial Kuwayama A, Kuruto R, Horie N, Takeishi K, Nozawa R. Appearance of nuclear factors that interact with genes for neoplasia and preferentially in high-grade myeloid calcium binding proteins (MRP-8 and MRP-14) in adenocarcinomas, whereas benign tissue was differentiated HL-60 cells. Blood. 1993 Jun 1;81(11):3116- negative or showed weak expression of the 21 proteins. The three proteins showed a strong Lackmann M, Rajasekariah P, Iismaa SE, Jones G, overlap in the expression pattern. S100A9 serum Cornish CJ, Hu S, Simpson RJ, Moritz RL, Geczy CL. level was significantly elevated in cancer patients Identification of a chemotactic domain of the pro- compared with benign prostatic hyperplasia patients inflammatory S100 protein CP-10. J Immunol. 1993 Apr 1;150(7):2981-91 or healthy individuals. Therefore, S100A8 and S100A9 might represent novel diagnostic markers Miyasaki KT, Bodeau AL, Murthy AR, Lehrer RI. In vitro antimicrobial activity of the human neutrophil cytosolic S- for prostate cancer and benign prostate hyperplasia 100 protein complex, calprotectin, against (Hermani et al., 2005). Capnocytophaga sputigena. J Dent Res. 1993 In further analyses it has been demonstrated that Feb;72(2):517-23 S100A8 and S100A9 are secreted by prostate Murthy AR, Lehrer RI, Harwig SS, Miyasaki KT. In vitro cancer cells, and extracellular S100A8/A9 candidastatic properties of the human neutrophil stimulates migration of benign prostatic cells in calprotectin complex. J Immunol. 1993 Dec vitro by activation of NF-kB and increased 1;151(11):6291-301 phosphorylation of p38 and p44/p42 MAP kinases. Roth J, Burwinkel F, van den Bos C, Goebeler M, Vollmer Immunofluorescence analyses give evidence for a E, Sorg C. MRP8 and MRP14, S-100-like proteins associated with myeloid differentiation, are translocated to RAGE-mediated response (Hermani et al., 2006). plasma membrane and intermediate filaments in a The significance of being diagnostic markers for calcium-dependent manner. Blood. 1993 Sep prostate cancer has been questioned by Ludwig et 15;82(6):1875-83 al. (2007). Their re-evaluation study has shown that Clohessy PA, Golden BE. Calprotectin-mediated zinc S100A8/A9 did not improve the differentiation chelation as a biostatic mechanism in host defence. Scand between patients with and without prostate cancer. J Immunol. 1995 Nov;42(5):551-6 The data give no evidence for the replacement of Yui S, Mikami M, Yamazaki M. Induction of apoptotic cell the established marker PSA by S100A8/A9. death in mouse lymphoma and human leukemia cell lines by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells. J Leukoc Biol. References 1995 Dec;58(6):650-8 Gabrielsen TO, Dale I, Brandtzaeg P, Hoel PS, Fagerhol Hardas BD, Zhao X, Zhang J, Longqing X, Stoll S, Elder MK, Larsen TE, Thune PO. Epidermal and dermal JT. Assignment of psoriasin to human chromosomal band distribution of a myelomonocytic antigen (L1) shared by 1q21: coordinate overexpression of clustered genes in epithelial cells in various inflammatory skin diseases. J Am psoriasis. J Invest Dermatol. 1996 Apr;106(4):753-8 Acad Dermatol. 1986 Aug;15(2 Pt 1):173-9 Kocher M, Kenny PA, Farram E, Abdul Majid KB, Finlay- Gimbrone MA Jr, Bevilacqua MP, Cybulsky MI. Jones JJ, Geczy C L. Functional chemotactic factor CP-10 Endothelial-dependent mechanisms of leukocyte adhesion and MRP-14 are abundant in murine abscesses. Infect in inflammation and atherosclerosis. Ann N Y Acad Sci. Immun. 1996 Apr;64(4):1342-50 1990;598:77-85 Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. Murao S, Collart F, Huberman E. A protein complex Genes encoding structural proteins of epidermal expressed during terminal differentiation of cornification and S100 calcium-binding proteins form a monomyelocytic cells is an inhibitor of cell growth. Cell gene complex ("epidermal differentiation complex") on Growth Differ. 1990 Oct;1(10):447-54 human chromosome 1q21. J Invest Dermatol. 1996 Steinbakk M, Naess-Andresen CF, Lingaas E, Dale I, May;106(5):989-92 Brandtzaeg P, Fagerhol MK. Antimicrobial actions of Rammes A, Roth J, Goebeler M, Klempt M, Hartmann M, calcium binding leucocyte L1 protein, calprotectin. Lancet. Sorg C. Myeloid-related protein (MRP) 8 and MRP14, 1990 Sep 29;336(8718):763-5 calcium-binding proteins of the S100 family, are secreted Sohnle PG, Collins-Lech C, Wiessner JH. Antimicrobial by activated monocytes via a novel, tubulin-dependent activity of an abundant calcium-binding protein in the pathway. J Biol Chem. 1997 Apr 4;272(14):9496-502 cytoplasm of human neutrophils. J Infect Dis. 1991 Groves P, Finn BE, Kuźnicki J, Forsén S. A model for Jan;163(1):187-92 target protein binding to calcium-activated S100 dimers. FEBS Lett. 1998 Jan 16;421(3):175-9

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 749

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

Hunter MJ, Chazin WJ. High level expression and dimer Hiratsuka S, Maru Y, Okada A, Seiki M, Noda T, Shibuya characterization of the S100 EF-hand proteins, migration M. Involvement of Flt-1 tyrosine kinase (vascular inhibitory factor-related proteins 8 and 14. J Biol Chem. endothelial growth factor receptor-1) in pathological 1998 May 15;273(20):12427-35 angiogenesis. Cancer Res. 2001 Feb 1;61(3):1207-13 Klempt M, Melkonyan H, Hofmann HA, Eue I, Sorg C. The Kerkhoff C, Sorg C, Tandon NN, Nacken W. Interaction of transcription factors c-myb and C/EBP alpha regulate the S100A8/S100A9-arachidonic acid complexes with the monocytic/myeloic gene MRP14. Immunobiology. 1998 scavenger receptor CD36 may facilitate fatty acid uptake Jul;199(1):148-51 by endothelial cells. Biochemistry. 2001 Jan 9;40(1):241-8 Kuruto-Niwa R, Nakamura M, Takeishi K, Nozawa R. Nacken W, Lekstrom-Himes JA, Sorg C, Manitz MP. Transcriptional regulation by C/EBP alpha and -beta in the Molecular analysis of the mouse S100A9 gene and expression of the gene for the MRP14 myeloid calcium evidence that the myeloid specific transcription factor binding protein. Cell Struct Funct. 1998 Jun;23(3):109-18 C/EBPepsilon is not required for the regulation of the S100A9/A8 gene expression in neutrophils. J Cell Loomans HJ, Hahn BL, Li QQ, Phadnis SH, Sohnle PG. Biochem. 2001;80(4):606-16 Histidine-based zinc-binding sequences and the antimicrobial activity of calprotectin. J Infect Dis. 1998 Srikrishna G, Panneerselvam K, Westphal V, Abraham V, Mar;177(3):812-4 Varki A, Freeze HH. Two proteins modulating transendothelial migration of leukocytes recognize novel Melkonyan H, Hofmann HA, Nacken W, Sorg C, Klempt M. carboxylated glycans on endothelial cells. J Immunol. 2001 The gene encoding the myeloid-related protein 14 Apr 1;166(7):4678-88 (MRP14), a calcium-binding protein expressed in granulocytes and monocytes, contains a potent enhancer Tarabykina S, Scott DJ, Herzyk P, Hill TJ, Tame JR, element in the first intron. J Biol Chem. 1998 Oct Kriajevska M, Lafitte D, Derrick PJ, Dodson GG, Maitland 9;273(41):27026-32 NJ, Lukanidin EM, Bronstein IB. The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in Newton RA, Hogg N. The human S100 protein MRP-14 is vivo and in vitro studies. J Biol Chem. 2001 Jun a novel activator of the beta 2 integrin Mac-1 on 29;276(26):24212-22 neutrophils. J Immunol. 1998 Feb 1;160(3):1427-35 Yang Z, Tao T, Raftery MJ, Youssef P, Di Girolamo N, Osterloh D, Ivanenkov VV, Gerke V. Hydrophobic residues Geczy CL. Proinflammatory properties of the human S100 in the C-terminal region of S100A1 are essential for target protein S100A12. J Leukoc Biol. 2001 Jun;69(6):986-94 protein binding but not for dimerization. Cell Calcium. 1998 Aug;24(2):137-51 Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002 Dec 19-26;420(6917):860-7 Ridinger K, Ilg EC, Niggli FK, Heizmann CW, Schäfer BW. Clustered organization of S100 genes in human and Henkel GW, McKercher SR, Maki RA. Identification of mouse. Biochim Biophys Acta. 1998 Dec 10;1448(2):254- three genes up-regulated in PU.1 rescued monocytic 63 precursor cells. Int Immunol. 2002 Jul;14(7):723-32 Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, Avila Kerkhoff C, Hofmann HA, Vormoor J, Melkonyan H, Roth C, Kambham N, Bierhaus A, Nawroth P, Neurath MF, J, Sorg C, Klempt M. Binding of two nuclear complexes to Slattery T, Beach D, McClary J, Nagashima M, Morser J, a novel regulatory element within the human S100A9 Stern D, Schmidt AM. RAGE mediates a novel promoter drives the S100A9 gene expression. J Biol proinflammatory axis: a central cell surface receptor for Chem. 2002 Nov 1;277(44):41879-87 S100/calgranulin polypeptides. Cell. 1999 Jun 25;97(7):889-901 Robinson MJ, Tessier P, Poulsom R, Hogg N. The S100 family heterodimer, MRP-8/14, binds with high affinity to Kerkhoff C, Klempt M, Kaever V, Sorg C. The two calcium- heparin and heparan sulfate glycosaminoglycans on binding proteins, S100A8 and S100A9, are involved in the endothelial cells. J Biol Chem. 2002 Feb 1;277(5):3658-65 metabolism of arachidonic acid in human neutrophils. J Biol Chem. 1999 Nov 12;274(46):32672-9 Semprini S, Capon F, Tacconelli A, Giardina E, Orecchia A, Mingarelli R, Gobello T, Zambruno G, Botta A, Fabrizi Klempt M, Melkonyan H, Hofmann HA, Sorg C. G, Novelli G. Evidence for differential S100 gene over- Identification of epithelial and myeloid-specific DNA expression in psoriatic patients from genetically elements regulating MRP14 gene transcription. J Cell heterogeneous pedigrees. Hum Genet. 2002 Oct;111(4- Biochem. 1999 Apr 1;73(1):49-55 5):310-3 Pröpper C, Huang X, Roth J, Sorg C, Nacken W. Analysis Yui S, Nakatani Y, Hunter MJ, Chazin WJ, Yamazaki M. of the MRP8-MRP14 protein-protein interaction by the two- Implication of extracellular zinc exclusion by recombinant hybrid system suggests a prominent role of the C-terminal human calprotectin (MRP8 and MRP14) from target cells domain of S100 proteins in dimer formation. J Biol Chem. in its apoptosis-inducing activity. Mediators Inflamm. 2002 1999 Jan 1;274(1):183-8 Jun;11(3):165-72 Sala A, Zarini S, Folco G, Murphy RC, Henson PM. Bhattacharya S, Large E, Heizmann CW, Hemmings B, Differential metabolism of exogenous and endogenous Chazin WJ. Structure of the Ca2+/S100B/NDR kinase arachidonic acid in human neutrophils. J Biol Chem. 1999 peptide complex: insights into S100 target specificity and Oct 1;274(40):28264-9 activation of the kinase. Biochemistry. 2003 Dec 16;42(49):14416-26 Sohnle PG, Hunter MJ, Hahn B, Chazin WJ. Zinc- reversible antimicrobial activity of recombinant calprotectin Donato R. Intracellular and extracellular roles of S100 (migration inhibitory factor-related proteins 8 and 14). J proteins. Microsc Res Tech. 2003 Apr 15;60(6):540-51 Infect Dis. 2000 Oct;182(4):1272-5 Hobbs JA, May R, Tanousis K, McNeill E, Mathies M, Arai K, Teratani T, Nozawa R, Yamada T. Gebhardt C, Henderson R, Robinson MJ, Hogg N. Myeloid Immunohistochemical investigation of S100A9 expression cell function in MRP-14 (S100A9) null mice. Mol Cell Biol. in pulmonary adenocarcinoma: S100A9 expression is 2003 Apr;23(7):2564-76 associated with tumor differentiation. Oncol Rep. 2001 May-Jun;8(3):591-6 Manitz MP, Horst B, Seeliger S, Strey A, Skryabin BV, Gunzer M, Frings W, Schönlau F, Roth J, Sorg C, Nacken

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 750

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

W. Loss of S100A9 (MRP14) results in reduced human keratinocytes. J Immunol. 2005 Mar interleukin-8-induced CD11b surface expression, a 15;174(6):3695-702 polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. Mol Cell Biol. Carlsson H, Petersson S, Enerbäck C. Cluster analysis of 2003 Feb;23(3):1034-43 S100 gene expression and genes correlating to psoriasin (S100A7) expression at different stages of breast cancer Moroz OV, Dodson GG, Wilson KS, Lukanidin E, Bronstein development. Int J Oncol. 2005 Dec;27(6):1473-81 IB. Multiple structural states of S100A12: A key to its functional diversity. Microsc Res Tech. 2003 Apr Cross SS, Hamdy FC, Deloulme JC, Rehman I. 15;60(6):581-92 Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: S100A6, Seth A, Kitching R, Landberg G, Xu J, Zubovits J, Burger S100A8, S100A9 and S100A11 are all overexpressed in AM. Gene expression profiling of ductal carcinomas in situ common cancers. Histopathology. 2005 Mar;46(3):256-69 and invasive breast tumors. Anticancer Res. 2003 May- Jun;23(3A):2043-51 Hermani A, Hess J, De Servi B, Medunjanin S, Grobholz R, Trojan L, Angel P, Mayer D. Calcium-binding proteins Zhi H, Zhang J, Hu G, Lu J, Wang X, Zhou C, Wu M, Liu Z. S100A8 and S100A9 as novel diagnostic markers in The deregulation of arachidonic acid metabolism-related human prostate cancer. Clin Cancer Res. 2005 Jul genes in human esophageal squamous cell carcinoma. Int 15;11(14):5146-52 J Cancer. 2003 Sep 1;106(3):327-33 Ito Y, Arai K, Ryushi, Nozawa, Yoshida H, Tomoda C, Zimmer DB, Wright Sadosky P, Weber DJ. Molecular Uruno T, Miya A, Kobayashi K, Matsuzuka F, Kuma K, mechanisms of S100-target protein interactions. Microsc Kakudo K, Miyauchi A. S100A9 expression is significantly Res Tech. 2003 Apr 15;60(6):552-9 linked to dedifferentiation of thyroid carcinoma. Pathol Res Pract. 2005;201(8-9):551-6 Arai K, Teratani T, Kuruto-Niwa R, Yamada T, Nozawa R. S100A9 expression in invasive ductal carcinoma of the Kerkhoff C, Nacken W, Benedyk M, Dagher MC, Sopalla breast: S100A9 expression in adenocarcinoma is closely C, Doussiere J. The arachidonic acid-binding protein associated with poor tumour differentiation. Eur J Cancer. S100A8/A9 promotes NADPH oxidase activation by 2004 May;40(8):1179-87 interaction with p67phox and Rac-2. FASEB J. 2005 Mar;19(3):467-9 Ghavami S, Kerkhoff C, Los M, Hashemi M, Sorg C, Karami-Tehrani F. Mechanism of apoptosis induced by Lominadze G, Rane MJ, Merchant M, Cai J, Ward RA, S100A8/A9 in colon cancer cell lines: the role of ROS and McLeish KR. Myeloid-related protein-14 is a p38 MAPK the effect of metal ions. J Leukoc Biol. 2004 Jul;76(1):169- substrate in human neutrophils. J Immunol. 2005 Jun 75 1;174(11):7257-67 Ji J, Zhao L, Wang X, Zhou C, Ding F, Su L, Zhang C, Mao Nacken W, Mooren FC, Manitz MP, Bode G, Sorg C, X, Wu M, Liu Z. Differential expression of S100 gene Kerkhoff C. S100A9 deficiency alters adenosine-5'- family in human esophageal squamous cell carcinoma. J triphosphate induced calcium signalling but does not Cancer Res Clin Oncol. 2004 Aug;130(8):480-6 generally interfere with calcium and zinc homeostasis in murine neutrophils. Int J Biochem Cell Biol. 2005 Kong JP, Ding F, Zhou CN, Wang XQ, Miao XP, Wu M, Liu Jun;37(6):1241-53 ZH. Loss of myeloid-related proteins 8 and myeloid-related proteins 14 expression in human esophageal squamous Grote J, König S, Ackermann D, Sopalla C, Benedyk M, cell carcinoma correlates with poor differentiation. World J Los M, Kerkhoff C. Identification of poly(ADP- Gastroenterol. 2004 Apr 15;10(8):1093-7 ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression. BMC Mol Biol. Li C, Zhang F, Lin M, Liu J. Induction of S100A9 gene 2006 Dec 22;7:48 expression by cytokine oncostatin M in breast cancer cells through the STAT3 signaling cascade. Breast Cancer Res Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer Treat. 2004 Sep;87(2):123-34 D. S100A8 and S100A9 activate MAP kinase and NF- kappaB signaling pathways and trigger translocation of Nacken W, Sorg C, Kerkhoff C. The myeloid expressed RAGE in human prostate cancer cells. Exp Cell Res. 2006 EF-hand proteins display a diverse pattern of lipid raft Jan 15;312(2):184-97 association. FEBS Lett. 2004 Aug 13;572(1-3):289-93 Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour- Shen J, Person MD, Zhu J, Abbruzzese JL, Li D. Protein mediated upregulation of chemoattractants and expression profiles in pancreatic adenocarcinoma recruitment of myeloid cells predetermines lung compared with normal pancreatic tissue and tissue metastasis. Nat Cell Biol. 2006 Dec;8(12):1369-75 affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry. Cancer Res. Hummerich L, Müller R, Hess J, Kokocinski F, Hahn M, 2004 Dec 15;64(24):9018-26 Fürstenberger G, Mauch C, Lichter P, Angel P. Identification of novel tumour-associated genes Vogl T, Ludwig S, Goebeler M, Strey A, Thorey IS, differentially expressed in the process of squamous cell Reichelt R, Foell D, Gerke V, Manitz MP, Nacken W, cancer development. Oncogene. 2006 Jan 5;25(1):111-21 Werner S, Sorg C, Roth J. MRP8 and MRP14 control microtubule reorganization during transendothelial Ito Y, Miyauchi A, Arai K, Nozawa R, Miya A, Kobayashi K, migration of phagocytes. Blood. 2004 Dec Nakamura Y, Kakudo K. Usefulness of S100A9 for 15;104(13):4260-8 diagnosis of intrathyroid epithelial thymoma (ITET)/carcinoma showing thymus-like differentiation Bierhaus A, Humpert PM, Stern DM, Arnold B, Nawroth (CASTLE). Pathology. 2006 Dec;38(6):541-4 PP. Advanced glycation end product receptor-mediated cellular dysfunction. Ann N Y Acad Sci. 2005 Moqbel R, Coughlin JJ. Differential secretion of cytokines. Jun;1043:676-80 Sci STKE. 2006 Jun 6;2006(338):pe26 Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Nagaraja GM, Othman M, Fox BP, Alsaber R, Pellegrino Morel F. IL-22 inhibits epidermal differentiation and CM, Zeng Y, Khanna R, Tamburini P, Swaroop A, Kandpal induces proinflammatory gene expression and migration of RP. Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells:

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 751

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

comprehensive profiles by representational difference novel, RAGE-independent pathway that involves selective analysis, microarrays and proteomics. Oncogene. 2006 release of Smac/DIABLO and Omi/HtrA2. Biochim Biophys Apr 13;25(16):2328-38 Acta. 2008 Feb;1783(2):297-311 Santamaria-Kisiel L, Rintala-Dempsey AC, Shaw GS. Ghavami S, Rashedi I, Dattilo BM, Eshraghi M, Chazin Calcium-dependent and -independent interactions of the WJ, Hashemi M, Wesselborg S, Kerkhoff C, Los M. S100 protein family. Biochem J. 2006 Jun 1;396(2):201-14 S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent Benedyk M, Sopalla C, Nacken W, Bode G, Melkonyan H, pathway. J Leukoc Biol. 2008 Jun;83(6):1484-92 Banfi B, Kerkhoff C. HaCaT keratinocytes overexpressing the S100 proteins S100A8 and S100A9 show increased Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, NADPH oxidase and NF-kappaB activities. J Invest Ishibashi S, Miyake K, Shibuya M, Akira S, Aburatani H, Dermatol. 2007 Aug;127(8):2001-11 Maru Y. The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nat Cell Biol. Herold K, Moser B, Chen Y, Zeng S, Yan SF, Ramasamy 2008 Nov;10(11):1349-55 R, Emond J, Clynes R, Schmidt AM. Receptor for advanced glycation end products (RAGE) in a dash to the Lim SY, Raftery M, Cai H, Hsu K, Yan WX, Hseih HL, rescue: inflammatory signals gone awry in the primal Watts RN, Richardson D, Thomas S, Perry M, Geczy CL. response to stress. J Leukoc Biol. 2007 Aug;82(2):204-12 S-nitrosylated S100A8: novel anti-inflammatory properties. J Immunol. 2008 Oct 15;181(8):5627-36 Ludwig S, Stephan C, Lein M, Loening SA, Jung K. S100A8, S100A9, and the S100A8/A9 complex in Salama I, Malone PS, Mihaimeed F, Jones JL. A review of circulating blood are not associated with prostate cancer the S100 proteins in cancer. Eur J Surg Oncol. 2008 risk-A re-evaluation study. Prostate. 2007 Sep Apr;34(4):357-64 1;67(12):1301-7 Turovskaya O, Foell D, Sinha P, Vogl T, Newlin R, Nayak Sheikh AA, Vimalachandran D, Thompson CC, Jenkins J, Nguyen M, Olsson A, Nawroth PP, Bierhaus A, Varki N, RE, Nedjadi T, Shekouh A, Campbell F, Dodson A, Prime Kronenberg M, Freeze HH, Srikrishna G. RAGE, W, Crnogorac-Jurcevic T, Lemoine NR, Costello E. The carboxylated glycans and S100A8/A9 play essential roles expression of S100A8 in pancreatic cancer-associated in colitis-associated carcinogenesis. Carcinogenesis. 2008 monocytes is associated with the Smad4 status of Oct;29(10):2035-43 pancreatic cancer cells. Proteomics. 2007 Jun;7(11):1929- 40 Ghavami S, Chitayat S, Hashemi M, Eshraghi M, Chazin WJ, Halayko AJ, Kerkhoff C. S100A8/A9: a Janus-faced Vogl T, Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van molecule in cancer therapy and tumorgenesis. Eur J Zoelen MA, Nacken W, Foell D, van der Poll T, Sorg C, Pharmacol. 2009 Dec 25;625(1-3):73-83 Roth J. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced Ito Y, Arai K, Nozawa R, Yoshida H, Hirokawa M, shock. Nat Med. 2007 Sep;13(9):1042-9 Fukushima M, Inoue H, Tomoda C, Kihara M, Higashiyama T, Takamura Y, Miya A, Kobayashi K, Yao R, Lopez-Beltran A, Maclennan GT, Montironi R, Eble Matsuzuka F, Miyauchi A. S100A8 and S100A9 JN, Cheng L. Expression of S100 protein family members expression is a crucial factor for dedifferentiation in thyroid in the pathogenesis of bladder tumors. Anticancer Res. carcinoma. Anticancer Res. 2009 Oct;29(10):4157-61 2007 Sep-Oct;27(5A):3051-8 Kerkhoff C, Ghavami S.. Innate immunity molecules Arai K, Takano S, Teratani T, Ito Y, Yamada T, Nozawa R. S100A8/A9 involved in stress response and cancer S100A8 and S100A9 overexpression is associated with biology. Anti-Inflammatory and Anti-Allergy Agents in poor pathological parameters in invasive ductal carcinoma Medicinal Chemistry, 2009; (8)4:279-281. of the breast. Curr Cancer Drug Targets. 2008 Jun;8(4):243-52 Srikrishna G, Freeze HH.. Endogenous damage- associated molecular pattern molecules at the crossroads Bode G, Lüken A, Kerkhoff C, Roth J, Ludwig S, Nacken of inflammation and cancer. Neoplasia. 2009 W. Interaction between S100A8/A9 and annexin A6 is Jul;11(7):615-28. (REVIEW) involved in the calcium-induced cell surface exposition of S100A8/A9. J Biol Chem. 2008 Nov 14;283(46):31776-84 van Dieck J, Fernandez-Fernandez MR, Veprintsev DB, Fersht AR.. Modulation of the oligomerization state of p53 Corbin BD, Seeley EH, Raab A, Feldmann J, Miller MR, by differential binding of proteins of the S100 family to p53 Torres VJ, Anderson KL, Dattilo BM, Dunman PM, Gerads monomers and tetramers. J Biol Chem. 2009 May R, Caprioli RM, Nacken W, Chazin WJ, Skaar EP. Metal 15;284(20):13804-11. Epub 2009 Mar 18. chelation and inhibition of bacterial growth in tissue abscesses. Science. 2008 Feb 15;319(5865):962-5 Ang CW, Nedjadi T, Sheikh AA, Tweedle EM, Tonack S, Honap S, Jenkins RE, Park BK, Schwarte-Waldhoff I, Dokun OY, Florl AR, Seifert HH, Wolff I, Schulz WA. Khattak I, Azadeh B, Dodson A, Kalirai H, Neoptolemos Relationship of SNCG, S100A4, S100A9 and LCN2 gene JP, Rooney PS, Costello E.. Smad4 loss is associated with expression and DNA methylation in bladder cancer. Int J fewer S100A8-positive monocytes in colorectal tumors and Cancer. 2008 Dec 15;123(12):2798-807 attenuated response to S100A8 in colorectal and pancreatic cancer cells. Carcinogenesis. 2010 Fernandez-Fernandez MR, Rutherford TJ, Fersht AR. Sep;31(9):1541-51. Epub 2010 Jul 9. Members of the S100 family bind p53 in two distinct ways. Protein Sci. 2008 Oct;17(10):1663-70 Fritz G, Botelho HM, Morozova-Roche LA, Gomes CM.. Natural and amyloid self-assembly of S100 proteins: Gebhardt C, Riehl A, Durchdewald M, Németh J, structural basis of functional diversity. FEBS J. 2010 Fürstenberger G, Müller-Decker K, Enk A, Arnold B, Nov;277(22):4578-90. doi: 10.1111/j.1742- Bierhaus A, Nawroth PP, Hess J, Angel P. RAGE signaling 4658.2010.07887.x. (REVIEW) sustains inflammation and promotes tumor development. J Exp Med. 2008 Feb 18;205(2):275-85 Ghavami S, Eshragi M, Ande SR, Chazin WJ, Klonisch T, Halayko AJ, McNeill KD, Hashemi M, Kerkhoff C, Los M.. Ghavami S, Kerkhoff C, Chazin WJ, Kadkhoda K, Xiao W, S100A8/A9 induces autophagy and apoptosis via ROS- Zuse A, Hashemi M, Eshraghi M, Schulze-Osthoff K, mediated cross-talk between mitochondria and lysosomes Klonisch T, Los M. S100A8/9 induces cell death via a

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 752

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S

that involves BNIP3. Cell Res. 2010 Mar;20(3):314-31. Su YJ, Xu F, Yu JP, Yue DS, Ren XB, Wang CL.. Up- Epub 2009 Nov 24. regulation of the expression of S100A8 and S100A9 in lung adenocarcinoma and its correlation with inflammation Ha YS, Kim MJ, Yoon HY, Kang HW, Kim YJ, Yun SJ, Lee and other clinical features. Chin Med J (Engl). 2010 SC, Kim WJ.. mRNA Expression of S100A8 as a Aug;123(16):2215-20. Prognostic Marker for Progression of Non-Muscle-Invasive Bladder Cancer. Korean J Urol. 2010 Jan;51(1):15-20. Voss A, Bode G, Sopalla C, Benedyk M, Varga G, Bohm Epub 2010 Jan 21. M, Nacken W, Kerkhoff C.. Expression of S100A8/A9 in HaCaT keratinocytes alters the rate of cell proliferation and Minami S, Sato Y, Matsumoto T, Kageyama T, Kawashima differentiation. FEBS Lett. 2011 Jan 21;585(2):440-6. Epub Y, Yoshio K, Ishii J, Matsumoto K, Nagashio R, Okayasu 2010 Dec 28. I.. Proteomic study of sera from patients with bladder cancer: usefulness of S100A8 and S100A9 proteins. This article should be referenced as such: Cancer Genomics Proteomics. 2010 Jul-Aug;7(4):181-9. Kerkhoff C, Ghavami S. S100A8 (S100 calcium binding Saha A, Lee YC, Zhang Z, Chandra G, Su SB, Mukherjee protein A8). Atlas Genet Cytogenet Oncol Haematol. 2011; AB.. Lack of an endogenous anti-inflammatory protein in 15(9):742-753. mice enhances colonization of B16F10 melanoma cells in the lungs. J Biol Chem. 2010 Apr 2;285(14):10822-31. Epub 2010 Jan 29.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 753

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

S100A9 (S100 calcium binding protein A9) Claus Kerkhoff, Saeid Ghavami Dept VAC / IMCI, Helmholtz Centre for Infection Research, Inhoffenstr 7, D-38124 Braunschweig, Germany (CK), Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada (SG)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/S100A9ID45569ch1q21.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI S100A9ID45569ch1q21.txt

cluster of genes on chromosome 1q21 encoding Identity proteins that fulfil important functions in terminal Other names: 60B8AG; CAGB; CFAG; CGLB; differentiation in the human epidermis, including L1AG; LIAG; MAC387; MIF; MRP14; NIF; P14 filaggrin, loricrin and others. In addition, linkage HGNC (Hugo): S100A9 analyses have identified a psoriasis susceptibility region, the PSORS4 locus, that is close to the S100 Location: 1q21.3 gene cluster (Hardas et al., 1996; Semprini et al., Local order: Distal to PGLYRP4 peptidoglycan 2002). These data are important indications for the recognition protein 4, proximal to S100A12 (S100 involvement of S100 genes in inflammatory as well calcium binding protein A12). as neoplastic disorders. It has been speculated that the rearrangements result in a deregulated DNA/RNA expression of S100 genes associated with neoplasia. Note Description S100A9 belongs to the S100/calgranulin family of The S100 gene structure has been structurally small non-ubiquitous cytoplasmic Ca2+-binding conserved during evolution. Similar to most S100 proteins of EF-hand type. The proteins were genes S100A9 consists of three exons that are referred to "S100" because of their solubility in separated by two introns. saturated ammonium sulphate solution. Sixteen of Transcription 21 members are localised in a cluster on human chromosome 1q21. In the S100A9 gene, exon 1 encodes the The clustered organization of these S100 genes is untranslated region. The protein is encoded by conserved during evolution (Ridinger et al., 1998). sequences in exon 2 and exon 3, encoding a N- A comparison between man and mouse has shown terminal and a C-terminal EF-hand motif, that during evolution, the colinearity of the S100 respectively. gene cluster has been destroyed by some inversions. The sequence of human S100A9 cDNA has an open However, the colocalization of the myeloid reading frame of 352 nucleotides. expressed S100 genes such as S100A8, S100A9, S100A9 expression appears to be restricted to a and S100A12 is conserved. It has been speculated, specific stage of myeloid differentiation. The that the structural integrity of that part of the locus protein is present in circulating neutrophils and is necessary for the coordinated expression of these monocytes, but not in resting tissue macrophages. genes (Nacken et al., 2001). In peripheral blood monocytes it is down regulated Remarkably, the S100 gene cluster is located in during maturation to macrophages. Despite a close proximity to a region which has been number of distinct regulatory regions are located frequently rearranged in human cancer (Carlsson et upstream of the transcription initiation site, the al., 2005) and to the epidermal differentiation corresponding nuclear factors as well as the complex (EDC) (Mischke et al., 1996). EDC is a underlying molecular mechanisms still remain

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 754

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

unclear. Transcription factors such as PU.1 (Henkel Ku70/Ku80 has been demonstrated to drive the et al., 2002), C/EBP-alpha and C/EBP-beta stress response-specific S100 gene expression (Kuruto-Niwa et al., 1998) have been shown to (Grote et al., 2006). The stress response-induced drive S100 gene expression within the myeloid expression of the S100 proteins points to an lineage. important role in skin pathology. For example, during differentiation of HL-60 cells In breast cancer cells S100A9 gene expression is into monocyte-like cells two still not identified induced by the cytokine oncostatin M (OM) factors were found to bind to the upstream regions through the STAT3-signaling cascade (Li et al., of S100A9 gene; one adjacent to the TATA box 2004). This finding is in accordance with another and another in the region between -400 and -150 study showing that IL-22 up-regulates the (Kuwayama et al., 1993). Another study revealed a expression of S100A7, S100A8, and S100A9 in CCAAT/enhancer binding protein (C/EBP)-binding keratinocytes. IL-22 has been demonstrated to motif located at position -81 upstream of the induce STAT3 activation in keratinocytes (Boniface S100A9 gene. Both C/EBP-alpha and -beta bind to et al., 2005). this motif in a myeloid/monocytic differentiation- Pseudogene dependent manner (Kuruto-Niwa et al., 1998). C/EBP was shown to be alone sufficient to drive Not known. S100A9 expression in otherwise negative cells. C/EBP up-regulation is antagonized by myb, a Protein transcription factor active in differentiated myeloid/monocytic cells (Klempt et al., 1998). The Description presence of distinct epithelial and myeloid-specific The sequence of human S100A9 cDNA has an open regulatory regions upstream of the transcription reading frame of 352 nucleotides predicting a initiation site has been demonstrated by detailed protein of 114 amino acids and a calculated Mr of deletion analysis (Klempt et al., 1999). Besides the 13242 Da. very specific action of particular upstream DNA Beside the full-length form of S100A9 there is a elements, the S100A9 gene contains a potent truncated isoform of S100A9 resulting from enhancer, which is harbored within positions 153 to alternative translation. 361 of its first intron (Melkonyan et al., 1998). The The full-length form of S100A9 lacks the first Met, functional relevance of this enhancer in S100A9 and Thr at position 2 is acetylated leading to a expression is supported by its conservation in calculated molecular mass of 13154 Da. human and murine S100A9 genes at almost The N-truncated isoform starts with Met at position identical positions. 5. Posttranslational removing of Met at position 5 Promoter analyses revealed a regulatory element and consequent acetylation of Ser at position 6 within the S100A9 promoter referred to as MRP leads to a calculated molecular mass of 12690 Da. regulatory element (MRE) that drives the S100A9 The theoretical isoelectric point of the full length gene expression in a cell-specific and form is 5.7 and for the truncated form is 5.5, differentiation-dependent manner. This regulatory respectively. region is located at position -400 to -374 bp, and S100A9 is composed of two helix-loop-helix EF- two distinct nuclear complexes were demonstrated hand motifs. The C-terminal EF-hand contains a 2+ to bind to this region. Interestingly, the formation of canonical Ca -binding loop of 12 amino acids. the nuclear protein complexes closely correlates Conversely, the N-terminal EF-hand contains a 2+ 2+ with the myeloid-specific expression of the S100A9 Ca -binding loop of 14 residues that binds Ca gene and, were therefore referred to as MRE- mostly through main-chain carbonyl groups that binding complex A (MbcA) and MbcB, which is specific to S100 proteins. Consequently, 2+ respectively. Analysis of one of the two nuclear S100 proteins have a weaker Ca affinity than 2+ complexes revealed a heterocomplex consisting of typical Ca sensors such as calmodulin (Donato, transcriptional intermediary factor 1 beta (TIF1 2003). beta) and a yet unidentified protein with homology An important posttranslational modification of to KRAB domain-containing (Kruppel-related) zinc S100A9 represents the phosphorylation of finger proteins (ZFP) (Kerkhoff et al., 2002). threonine at position 113. It can be phosphorylated Beside its expression in myeloid cells S100A9 is upon PMN activation, and phosphorylation of this 2+ expressed in epithelia under specific conditions. Its residue is specifically regulated by the Ca - expression is transiently induced in keratinocytes ionophore, ionomycin. Recent studies give after epidermal injury and UVB irradiation, and the evidence for S100A9 being a p38 MAPK substrate protein is expressed at extremely high levels in in human neutrophils (Lominadze et al., 2005). This psoriatic keratinocytes. Furthermore, its expression phosphorylation is involved in translocation and is induced by pro-inflammatory cytokines such as functional events. TNF alpha and IL1 beta. Recently, a complex of In vivo and in vitro experiments have shown that Poly (ADP-ribose) polymerase (PARP-1) and S100 proteins form homo-, hetero- and oligomeric

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 755

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

Gene: Box = exon (light blue = 5'UTR, yellow = CDS, red = 3'UTR); Line = intron. Protein: Upper boxes, alternating colours: exons (coding part only). Lower boxes: protein domains. Green box = not structure; blue box = helix; violett box = calcium-binding domain. assemblies (Hunter and Chazin, 1998; Osterloh et The secretion pathway relies on the activation of al., 1998; Pröpper et al., 1999; Moroz et al., 2003). protein kinase C. This pathway differs from the Together with their specific cell- and tissue- classical as well as the alternative secretion expression patterns, the structural variations, and pathways of cytokines (Moqbel and Coughlin, the different metal ion binding properties (Ca2+, 2006). It has been demonstrated that this novel Zn2+ and Cu2+) the S100 protein complexes might secretion pathway is energy-consuming and be functionally diversified. S100A9 preferentially depends on an intact microtubule network (Murao interacts with S100A8. et al., 1990; Rammes et al., 1997). It is worthwhile mentioning that the murine analogs Recent investigations give evidence that interaction display a stronger tendency to form homodimeric of S100A8/A9 with annexin-6 is involved in protein complexes. In view of the formation of surface expression and release of S100A8/A9 different tertiary structures with putative distinct (Bode et al., 2008). Annexins are another class of functions it is tempting to speculate that S100A8 Ca2+-regulated proteins. They are characterized by and/or A9 have different functions in mouse and the unique architecture of their Ca2+-binding sites, man. which enables them to peripherally dock onto Expression negatively charged membrane surfaces in their Ca2+-bound conformation. This property links S100A9 is mainly expressed in cells of the myeloid annexins to many membrane-related events such as lineage, however, its gene expression is induced in certain exocytic and endocytic transport steps. This epithelial cells in response to stress, in specific is an interesting finding since S100A8 and S100A9 conditions such as wound healing, UV exposition, are expressed in cancerous cells of secretory tissues abundant in psoriais keratinocytes, differentially as breast and prostate. Cells originating from such expressed in several cancers. glandular tissues are rich in membrane structures, Localisation suggesting that membrane-associated molecular targets for the S100A8/A9 proteins could be Mostly cytoplasmic, but also at membranes and potentially found in these cells. cytoskeleton. Recent investigations also demonstrated the In resting phagocytes the S100A8/A9 protein association of S100A8/A9 with cholesterol- complex is mainly located in the cytosol. Upon enriched membrane microdomains (lipid rafts) cellular activation the protein complex is either (Nacken et al., 2004). This observation is in translocated to cytoskeleton and plasma membrane agreement with the enhancing effect of S100A8/A9 or released into the extracellular environment. on NADPH oxidase since the formation of the The translocation pathways occur upon the oxidase complex takes place at lipid rafts. elevation of the intracellular calcium level (Roth et al., 1993). At a later time point, the S100A8/A9 Function heterodimers can be detected on the surface of Intra- as well as extracellular roles have been monocytes (Bhardwaj et al., 1992). The mechanism proposed for the S100 proteins. by which the S100A8/A9 heterodimer penetrates Intracellular activities of S100A8/A9 the plasma membrane remains unclear since the In the intracellular milieu, S100 proteins are S100 proteins lack a transmembrane signaling considered as calcium sensors changing their region.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 756

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

conformation in response to calcium influx and Extracellular activities of S100A8/A9 then mediating calcium signals by binding to other The S100/calgranulins display antimicrobial intracellular proteins. In a mouse knock-out model activity by depriving bacterial pathogens of chemokine-induced down regulation of the essential trace metals such as Zn2+ and Mn2+ cytosolic Ca2+-level was detected (Nacken et al., (Steinbakk et al., 1990; Murthy et al., 1993; 2005). Clohessy and Golden, 1995; Sohnle et al., 2000). In After calcium binding, the S100A8/A9 protein the context of inflammation, it has been proposed complex binds specifically polyunsaturated fatty that S100A8/A9 is massively released when acids. S100A8/A9 represents the exclusive neutrophils die to provide a growth-inhibitory type arachidonic acid-binding capacity in the neutrophil of host defense that is adjunctive to the usual cytosol (Kerkhoff et al., 1999), and participates in microbicidal functions by binding metals other than NADPH oxidase activation by transferring Ca2+ (Corbin et al., 2008). arachidonic acid to membrane-bound gp91phox In addition, S100/calgranulins serve as leukocyte during interactions with two cytosolic oxidase chemoattractants (Lackmann et al., 1992; activation factors, p67phox and Rac-2. The Lackmann et al., 1993; Kocher et al., 1996; Lim et functional relevance of S100A8/A9 in the al., 2008). Murine S100A8 has potent chemotactic phagocyte NADPH oxidase activation was activity for neutrophils and monocytes in vitro and demonstrated by the impairment of NADPH in vivo (Lackmann et al., 1992). In contrast, human oxidase activity in neutrophil-like NB4 cells, after S100A8 displays only weak leukocyte chemotactic specifically blocking S100A9 expression, and activity in vitro and in vivo (Lackmann et al., employing bone marrow-derived PMNs from 1993). Detailed analysis revealed that the hinge S100A9-/- mice (Kerkhoff et al., 2005). region contributes to the chemotactic activity of In accordance to their role in myeloid cells, murine, but not human S100A8. These data S100A8/A9 enhances epithelial NADPH oxidases questioned whether the proteins are orthologs since (Benedyk et al., 2007). As a consequence of there is a high degree of homology between murine enhanced ROS levels, NF-kB activation and and human S100A8 but a functional divergence. subsequently TNF-alpha and IL-8 mRNA levels are Intriguingly, human S100A12 is chemotactic and increased in S100A8/A9-HaCaT keratinocytes, the hinge region of human S100A12 has been consistent with the view that NF-kB is a redox- implicated herein (Yang et al., 2001). Thus, the sensitive transcription factor. Further consequences functional and sequence divergence suggested of S100A8/A9-mediated NF-kB activation are complex evolution of the S100 family in mammals. reduced cell growth, increased expression of The putative pro-inflammatory functions of differentiation markers, and enhanced PARP S100A8 and S100A9 have recently been cleavage as an indicator of increased cell death investigated in two different mouse knock-out (Voss et al., 2011). models. S100A9 deficiency did not result in an In view of the stress response-induced expression obvious phenotype (Manitz et al., 2003; Hobbs et of the two S100 proteins in keratinocytes these al., 2003). However, reduced migration of S100A9- findings have great implications for tissue deficient neutrophils and decreased surface remodeling and repair. For example, keratinocytes expression of CD11b, which belongs to the integrin acquire an activated state after cutaneous wounding family, were observed upon in vitro stimulation. In in which proliferation is favored over addition, chemokine-induced down regulation of differentiation in order to replenish the lost material the cytosolic Ca2+-level was detected. Obviously, and rapidly close the site of injury. Thus, it is likely these in vitro effects are compensated by alternative to hypothesize that S100A8/A9-mediated growth pathways in vivo. reduction is required for the upcoming cell fate Remarkably, cancer cells utilize S100A8 and decision of damaged cells, i.e. for a survival phase S100A9 as guidance for the adhesion and invasion to be followed by differentiation, proliferation, or of disseminating malignant cells (Hiratsuka et al., apoptosis. These data have also an impact on 2006). In the context of malignancy it was reported tumorigenesis since S100 gene expression is that S100A8/A9 attracts Mac-1+ myeloid cells to associated with neoplastic disorders. the lung tissue. Recruited Mac-1+ myeloid cells in In migrating monocytes the S100A8/A9 complex lung in turn produce S100A8/A9 in response to has been found to be associated with cytoskeletal primary malignant cells in a so called tubulin and to modulate transendothelial migration "premetastatic phase". This phase shows the general (Vogl et al., 2004). Investigations using two characteristics of an inflammation state which different mouse knock-out models demonstrated no facilitates the micro-environmental changes obvious phenotype (Manitz et al., 2003; Hobbs et required for the migration and implantation of al., 2003). However, reduced migration of S100A9- primary tumor cells to lung tissue. After preparation deficient neutrophils and decreased surface of the target tissue for accepting the malignant cells, expression of CD11b, which belongs to the integrin tumor cells mimic Mac-1+ myeloid cells in response family, were observed upon in vitro stimulation. to S100A8/A9 chemotactic signaling and migrate to

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 757

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

lung. So, it seems that tumor cells and Mac-1+ eicosanoids (Sala et al., 1999). Therefore, the myeloid cells utilize a common pathway for secreted S100A8/A9-AA complex may serve as a migration to lung which involves the activation of transport protein to move AA to its target cells. mitogen-activated protein kinase pathway This may represent a mechanism by which AA- (Hiratsuka et al., 2006). These findings suggest derived eicosanoids are synthesized in a S100A8/A9 as an attractive target for the cooperative manner between different cell species development of strategies counteracting tumor due to environmental cues. metastasizing to certain organs. S100A8/A9 displays apoptosis-inducing activity S100A8 and S100A9 have been identified as against various tumor cells (Yui et al., 1995; Yui et important endogenous damage-associated al., 2002; Ghavami et al., 2004; Ghavami et al., molecular pattern (DAMP) proteins. Although 2008a; Kerkhoff and Ghavami, 2009; Ghavami et receptors for S100A8/A9 are still largely al., 2009; Ghavami et al., 2010). It was speculated uncharacterized, more recent findings support the that this activity was due to the ability to bind notion that they function as potent ligands of divalent metal ions including Zn2+, Mn2+ and Cu2+ pattern-recognition receptors, such as the toll-like at sites that are distinct from Ca2+-binding sites. receptor 4 (TLR4) (Vogl et al., 2007) and the However, a number of recent reports now indicate receptor for advanced glycation end products that S100A8/A9 exerts its activity by both chelation (RAGE) (Srikrishna and Freeze, 2009). of trace metal ions such as Zn2+ and cell surface The S100/calgranulins display cytokine-like receptor mediated pathways. functions, including activation of the receptor for Although a number of receptors have been shown advanced glycation endproducts (RAGE) (Hofmann to bind S100A8/A9, the nature of the receptor et al., 1999; Herold et al., 2007). RAGE is a involved in S100A8/A9-induced cell death remains member of the immunoglobulin superfamily and to be elucidated. Experiments with certain cell lines present on numerous cell types. It has been shown either deficient for or over expressing components to play crucial roles in a variety of of the death signaling machinery as well as RAGE pathophysiological situations, such as wound gene silencing and blocking RAGE-specific healing, atherosclerotic lesion development, tumor antibody approaches excluded both RAGE and the growth and metastasis, systemic amyloidosis, and classical death receptor to be involved in Alzheimer disease (Bierhaus et al., 2005). S100A8/A9-induced cell death, even though RAGE/S100 interaction has been considered a very S100A8/A9 can specifically bind to cancer cells attractive model to explain how RAGE and its and RAGE mediates the growth-promoting activity proinflammatory ligand contribute to the obvious at low micromolar concentrations of pathophysiology of several inflammatory diseases. S100A8/A9. Clearly, investigations to identify the Beside the above mentioned receptors a number of receptor involved in S100A8/A9-induced cell death other cell surface binding sites specific for are critical. S100A8/A9 have been reported, such as novel Homology carboxylated glycans (Srikrishna et al., 2001), heparan sulfate glycosaminoglycans (Robinson et Overall, the S100 proteins share significant al., 2002), beta2-integrin (Newton and Hogg, sequential homology in the EF-hand motifs, but are 1998), and the fatty acid transporter FAT/CD36 least conserved in the hinge region. This region is (Kerkhoff et al., 2001). Therefore, the cell surface proposed to provide for specific interaction with receptor of S100A8/A9 is still in debate. target proteins (Groves et al., 1998; Zimmer et al., Interestingly, the growth-stimulatory activity of 2003; Santamaria-Kisiel et al., 2006; Fernandez- S100A8/A9 has been demonstrated to be mediated Fernandez et al., 2008; van Dieck et al., 2009). The by binding to the receptor of advanced glycation availability of high-resolution S100-target end products (RAGE) (Ghavami et al., 2008b; structures has highlighted important structural Turovskaya et al., 2008; Gebhardt et al., 2008). It is features that contribute to S100 protein functional likely to speculate that the selective up-regulation specificity (Bhattacharya et al., 2003). of S100 proteins may be of importance for survival The functional diversification of S100 proteins is and proliferation of metastasizing cancer cells. achieved by their specific cell- and tissue- expression patterns, structural variations, different S100A8/A9 complexes that are secreted from 2+ 2+ 2+ phorbolester-stimulated neutrophil-like HL-60 cells metal ion binding properties (Ca , Zn and Cu ) have been shown to carry the eicosanoid precursor as well as their ability to form homo-, hetero- and arachidonic acid (Kerkhoff et al., 1999). The oligomeric assemblies (Hunter and Chazin, 1998; S100A8/A9-arachidonic acid complex is Osterloh et al., 1998; Pröpper et al., 1999; recognized by the fatty acid transporter FAT/CD36, Tarabykina et al., 2001; Moroz et al., 2003; Fritz et and the fatty acid is rapidly taken up (Kerkhoff et al., 2010). Although the function of S100 proteins al., 2001). Endothelial cells as well as neutrophils in cancer cells in most cases is still unknown, the themselves utilize both endogenous and exogenous specific expression patterns of these proteins are a arachidonic acid for transcellular production of valuable diagnostic tool.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 758

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

immune system. These insights are fostering new Implicated in anti-inflammatory therapeutic approaches to cancer General note development. Note Skin cancer Comparative and functional genomics have Note revealed that a number of S100 proteins are found The expression of S100A8 and S100A9 in to be differentially expressed in cancer cells. epithelial cells was first detected in the squamous Several of these have been associated with tumor epithelia (Gabrielsen et al., 1986). Normal S100A8 development, cancer invasion or metastasis in and S100A9 are expressed at minimal levels in the recent studies (for review see Salama et al., 2008). epidermis. However, their expression is induced in S100A8 and S100A9 are abundant in cells of the inflammatory and cancerous conditions, and pro- myeloid lineage, are released from activated inflammatory cytokines such as TNF-alpha and IL1 phagocytes and display intra- and extracellular beta are involved herein. functions. Their expression is ubiquitously Gene expression analysis in a mouse model of observed in the squamous epithelia under normal, chemically induced skin carcinogenesis identified a inflammatory and cancerous conditions. large set of novel tumor-associated genes including Immunohistochemical investigations have shown S100A8 (Hummerich et al., 2006). The data was that the S100 proteins are over expressed in skin confirmed by in situ hybridization and cancers, pulmonary adenocarcinoma, pancreatic immunofluorescence analysis on mouse tumor adenocarcinoma, bladder cancers, ductal carcinoma sections, in mouse keratinocyte cell lines that form of the breast, and prostate adenocarcinoma. In tumors in vivo, and in human skin tumor contrast, S100A8 and S100A9 are down-regulated specimens. in esophageal squamous cell carcinomas. However, conflicting results have been published Furthermore, plasma levels of S100A8/A9 are concerning S100 expression in skin cancer. For elevated in patients suffering from various cancers. instance, esophageal squamous cell carcinoma Insofar, S100A8 and S100A9 might represent novel (ESCC) is one of the most common cancers diagnostic markers for some carcinomas. worldwide. DNA microarray data analysis revealed S100A8 and S100A9 have been suggested to have that S100A8 and S100A9 were significantly down potential roles in carcinogenesis and tumor regulated in human ESCC versus the normal progression. However, the biological role of counterparts (Zhi et al., 2003). Interestingly, among S100A8/A9 remains to be elucidated. It is the 42 genes either up regulated or down regulated conceivable that S100A8 and S100A9 modulate in tumors, as compared to normal esophageal signal pathways to directly promote invasion, squamous epithelia, nine of the altered expression migration and metastasis, probably via activation of genes were related to arachidonic acid (AA) NF-kB, Akt or MAP kinases. metabolism, suggesting that AA metabolism In the last decade the concept of the functional pathway and its altered expression may contribute relationship between inflammation and cancer has to esophageal squamous cell carcinogenesis. been developed that is based on numerous findings, Similar data were obtained by Ji et al. (2004). They ranging from epidemiological studies to molecular investigated the differential expression of the S100 analyses of mouse models (Coussens and Werb, gene family at the RNA level in human ESCC. 2002). In this concept, the generation of an Eleven out of 16 S100 genes were significantly inflammatory microenvironment supports down regulated in ESCC versus the normal tumorigenesis by promoting cancer cell survival, counterparts. Only the S100A7 gene was found to proliferation, migration, and invasion. Although it be markedly up regulated. Another study is clear that inflammation alone does not cause demonstrated that poorly differentiated ESCC cancer, it is evident that an environment that is rich displayed a stronger decrease in S100A8 and in inflammatory cells, growth factors, activated S100A9 expression than well and moderately stroma, and DNA-damage-promoting agents differentiated tumors, with a correlation between certainly potentiates and/or promotes neoplastic protein level and histopathological grading (Kong risk. In addition, many cancers arise from sites of et al., 2004). These findings suggest that decreased infection, chronic irritation and inflammation. expression of S100A8 and S100A9 might play an Recent data have expanded our knowledge important role in the ESCC pathogenesis, being demonstrating that specific soluble factors released particularly associated with poor differentiation of from primary tumors induce the S100A8 and tumor cells. S100A9 gene expression in the target tissue. After secretion S100A8 and S100A9 might display Lung adenocarcinomas chemokine- and cytokine-like properties that Note promote invasion, migration and metastasis. These S100A9 over expression has been detected in data indicate that tumor cells are able to reprogram various carcinomas of glandular cell origin, and its some of the signaling molecules of the innate expression has been associated with poor tumor

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 759

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

differentiation. Similarly, S100A9 These data are in accordance with another study immunopositivity was also detected in pulmonary (Sheikh et al., 2007). Strong expression of S100A8 adenocarcinoma cell lines and resected pulmonary and S100A9 was found in tumor-associated stroma adenocarcinoma (Arai et al., 2001). Examination of but not in benign or malignant epithelia. Further the relation of S100A9 expression to tumor analyses identified stromal CD14+ CD68- differentiation showed that the expression rate in monocytes/macrophages as source for S100 pulmonary adenocarcinoma showed higher expression. Interestingly, the number of S100A8- correlation in poorly differentiated carcinomas. positive cells in the tumor microenvironment Another study confirmed these data (Su et al., negatively correlated with the expression of the 2010). Immunohistochemical staining of both S100 tumor suppressor protein, Smad4. The number of proteins showed a significant up-regulation in lung S100A9-positive cells was not altered in Smad4- cancer tissue, and quantitative PCR revealed negative or Smad4-positive tumors. significantly higher levels of S100A8 and S100A9 A similar correlation was found in colorectal cancer mRNA transcripts in lung cancer tissues. Moreover, tumors (Ang et al., 2010). The number of stromal this study correlates S100A9 expression with S100A8- and S100A9-positive cells was associated inflammation and other clinical features (Su et al., with the presence or absence of Smad4. Smad4- 2010). negative tumors showed enhanced numbers of Primary tumors influence the environment in the S100A8/A9 stroma cells, and the corresponding lungs before metastasis. They release specific patients had a poor survival prognosis. soluble factors that prepare the premetastatic niche Investigation of the underlying molecular for the engraftment of tumor cells. In several mechanisms revealed that both migration and studies it has been shown that tumor cells induce proliferation was enhanced in response to both expression and secretion of S100A8 and exogenous S100A8 and S100A9, irrespective of S100A9 in the target organ that display a promoting Smad4-presence. However, depletion of Smad4 role in cancer cell survival, proliferation, migration, resulted in loss of responsiveness to exogenous and invasion (Hiratsuka et al., 2002; Hiratsuka et S100A8, but not S100A9. Vice versa, Smad4 al., 2006; Hiratsuka et al., 2008; Saha et al., 2010). expression in Smad4-negative cells enhanced the Microarray analysis of lungs from tumor-bearing responsive-ness to S100A8 and S100A9. Further and non-bearing mice revealed the strong up- analyses give evidence that similar to TGF-beta, regulation of a number of genes including S100A8 S100A8 and S100A9 induce the phosphorylation of and S100A9 (Hiratsuka et al. 2002). Their both Smad2 and Smad3 that was blocked by a expression in Mac 1+-myeloid cells and endothelial RAGE-specific antibody. These data point to a cells was induced by factors such as vascular functional relationship between inflammation and endothelial growth factor A (VEGF-A), tumor tumorigenesis. necrosis factor-alpha (TNF-alpha) and transforming Bladder cancers growth factor-beta (TGF-beta), both in vitro and in vivo (Hiratsuka et al., 2006). Remarkably, anti- Note S100A8 neutralizing antibody treatment blocked Gene expression profiles revealed that thirteen metastasis. S100A8 and S100A9 were shown to members of the S100 gene family were induce the expression of serum amyloid A (SAA) differentially expressed in human bladder cancers. that attracted Mac 1+-myeloid cells in the S100A8 and S100A9 were found to be over premetastatic lung (Hiratsuka et al., 2008). These expressed (Yao et al., 2007). studies demonstrated that lung cancer cells utilize Another study investigated S100A9 expression and S100A8 and S100A9 as guidance for the adhesion DNA methylation in urothelial cancer cell lines and and invasion of disseminating malignant cells. cancer tissue (Dokun et al., 2008). Expression of S100A9 was found to be generally elevated in the Pancreatic adenocarcinoma tumor tissues but S100A9 was weakly expressed in Note most cancer cell lines. The S100A9 promoter Patients with ductal adenocarcinoma of the contains 6 CpG sites, and its methylation state was pancreas have a dismal prognosis. Thus, there is an unrelated to the variable expression. It has been urgent need for early detection markers and the hypothesized that over expression of genes is the development of immunotherapeutical approaches consequence of DNA hypomethylation, however, concentrating on the induction and enhancement of DNA methylation and gene expression are less immune responses against tumors. Proteomic strictly related for those genes having promoters analyses of pancreatic adenocarcinoma, normal within CpG-islands. Alternatively, the increased adjacent tissues, pancreatitis, and normal pancreatic S100A9 gene expression may be related to that of tissues revealed a number of differentially other immune-related genes in the carcinoma cell expressed genes (Shen et al., 2004). S100A8 was cultures. This is sustained by the facts that S100A9 found to be specifically over expressed in tumors is secreted by epithelial and other cell types to compared with normal and pancreatitis tissues. modulate inflammatory reactions as well as to promote cancer proliferation and metastasis.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 760

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

Two recent studies propose S100A8 and S100A9 2004). However, non-invasive MDA-MB-468 cells gene expression as prognostic value for bladder are abundant for both S100 proteins (Bode et al., cancer (Minami et al., 2010; Ha et al., 2010). By 2008) and invasive breast cancer cells MDA-MB- proteomic analysis of pre- and postoperative sera 231 show low transcript level of S100A9 (Nagaraja from bladder cancer patients S100A8 and S100A9 et al., 2006). were identified as tumor-associated proteins Thyroid carcinoma (Minami et al., 2010). Interestingly, S100A8 expression was associated with bladder wall muscle Note invasion of the tumor and cancer-specific survival Similar to other carcinomas of glandular cell origin, while S100A9 expression was associated with the expression of S100A8 and S100A9 is significantly tumor grade. In addition, the expression of both linked to dedifferentiation of thyroid carcinoma (Ito proteins S100A8/A9 was correlated with et al., 2005; Ito et al., 2009). S100A8 and S100A9 recurrence-free survival. immunreactivity was found in all undifferentiated In another study it was evaluated whether S100A8 carcinomas examined, while papillary carcinoma, is a prognostic value for non-muscle-invasive follicular carcinoma, follicular adenoma and bladder cancer (NMIBC) (Ha et al., 2010). S100A8 medullary carcinoma and normal follicules were expression was evaluated in a total of 103 primary negative for both proteins. Further analyses NMIBC samples by quantitative PCR. The mRNA revealed that S100A9 is a useful marker for expression levels of S100A8 were significantly discriminating intrathyroid epithelial tumor from related to the progression of NMIBC, suggesting squamous cell carcinoma or undifferentiated that S100A8 might be a useful prognostic marker carcinoma with squamoid component (Ito et al., for disease progression of NMIBC. 2006). Breast cancers Prostate cancer Note Note S100A8 and S100A9 are expressed in breast Increased levels of S100A8, S100A9, and RAGE cancers (Cross et al., 2005), especially in invasive have been reported in prostatic intra epithelial breast carcinoma (Arai et al., 2004). By neoplasia and preferentially in high-grade immunohistochemical analyses a strong S100A9 adenocarcinomas, whereas benign tissue was immunoreactivity has been demonstrated in negative or showed weak expression of the invasive as well as non-invasive ductal carcinoma. proteins. The three proteins showed a strong No immunopositive reaction was observed in overlap in the expression pattern. S100A9 serum invasive lobular carcinomas, and no significant level was significantly elevated in cancer patients differences were detected in the number of compared with benign prostatic hyperplasia patients myelomonocytic cells expressing S100A9. These or healthy individuals. Therefore, S100A8 and data give evidence that S100A9 in glandular S100A9 might represent novel diagnostic markers epithelial cells is newly expressed under cancerous for prostate cancer and benign prostate hyperplasia conditions and is over-expressed in poorly (Hermani et al., 2005). differentiated adenocarcinoma (Arai et al., 2004). In further analyses it has been demonstrated that Further analyses target on the relationship between S100A8 and S100A9 are secreted by prostate S100A8/A9 expression and pathological parameters cancer cells, and extracellular S100A8/A9 that reflect the aggressiveness of carcinoma. The stimulates migration of benign prostatic cells in immunopositivity for S100A8/A9 correlated with vitro by activation of NF-kB and increased poor tumor differentiation, mitotic activity, phosphorylation of p38 and p44/p42 MAP kinases. HER2/neu over expression, poor pT categories, Immunofluorescence analyses give evidence for a node metastasis, and poor pStage, but not with RAGE-mediated response (Hermani et al., 2006). vessel invasion. These data may indicate that The significance of being diagnostic markers for S100A8 and S100A9 over expression should be prostate cancer has been questioned by Ludwig et considered marker of poor prognosis in invasive al. (2007). Their re-evaluation study has shown that breast ductal carcinoma (Arai et al., 2008). S100A8/A9 did not improve the differentiation By analyses of ductal carcinoma in situ and between patients with and without prostate cancer. invasive ductal carcinoma of the breast S100A9 has The data give no evidence for the replacement of been demonstrated to be most abundantly expressed the established marker PSA by S100A8/A9. in the invasive tumor (Seth et al., 2003). Therefore, the expression of S100A8 and S100A9 has been References correlated with the degree of noninvasive / invasive Gabrielsen TO, Dale I, Brandtzaeg P, Hoel PS, Fagerhol behavior. There are conflicting data concerning this MK, Larsen TE, Thune PO. Epidermal and dermal correlation. For instance, non-invasive MCF-7 distribution of a myelomonocytic antigen (L1) shared by breast cancer cells do not express S100A9, and its epithelial cells in various inflammatory skin diseases. J Am Acad Dermatol. 1986 Aug;15(2 Pt 1):173-9 gene expression is induced by cytokine oncostatin M through the STAT3 signaling cascade (Li et al.,

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 761

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

Gimbrone MA Jr, Bevilacqua MP, Cybulsky MI. Kocher M, Kenny PA, Farram E, Abdul Majid KB, Finlay- Endothelial-dependent mechanisms of leukocyte adhesion Jones JJ, Geczy C L. Functional chemotactic factor CP-10 in inflammation and atherosclerosis. Ann N Y Acad Sci. and MRP-14 are abundant in murine abscesses. Infect 1990;598:77-85 Immun. 1996 Apr;64(4):1342-50 Murao S, Collart F, Huberman E. A protein complex Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. expressed during terminal differentiation of Genes encoding structural proteins of epidermal monomyelocytic cells is an inhibitor of cell growth. Cell cornification and S100 calcium-binding proteins form a Growth Differ. 1990 Oct;1(10):447-54 gene complex ("epidermal differentiation complex") on human chromosome 1q21. J Invest Dermatol. 1996 Steinbakk M, Naess-Andresen CF, Lingaas E, Dale I, May;106(5):989-92 Brandtzaeg P, Fagerhol MK. Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet. Rammes A, Roth J, Goebeler M, Klempt M, Hartmann M, 1990 Sep 29;336(8718):763-5 Sorg C. Myeloid-related protein (MRP) 8 and MRP14, calcium-binding proteins of the S100 family, are secreted Sohnle PG, Collins-Lech C, Wiessner JH. Antimicrobial by activated monocytes via a novel, tubulin-dependent activity of an abundant calcium-binding protein in the pathway. J Biol Chem. 1997 Apr 4;272(14):9496-502 cytoplasm of human neutrophils. J Infect Dis. 1991 Jan;163(1):187-92 Groves P, Finn BE, Kuźnicki J, Forsén S. A model for target protein binding to calcium-activated S100 dimers. Bhardwaj RS, Zotz C, Zwadlo-Klarwasser G, Roth J, FEBS Lett. 1998 Jan 16;421(3):175-9 Goebeler M, Mahnke K, Falk M, Meinardus-Hager G, Sorg C. The calcium-binding proteins MRP8 and MRP14 form a Hunter MJ, Chazin WJ. High level expression and dimer membrane-associated heterodimer in a subset of characterization of the S100 EF-hand proteins, migration monocytes/macrophages present in acute but absent in inhibitory factor-related proteins 8 and 14. J Biol Chem. chronic inflammatory lesions. Eur J Immunol. 1992 1998 May 15;273(20):12427-35 Jul;22(7):1891-7 Klempt M, Melkonyan H, Hofmann HA, Eue I, Sorg C. The Lackmann M, Cornish CJ, Simpson RJ, Moritz RL, Geczy transcription factors c-myb and C/EBP alpha regulate the CL. Purification and structural analysis of a murine monocytic/myeloic gene MRP14. Immunobiology. 1998 chemotactic cytokine (CP-10) with sequence homology to Jul;199(1):148-51 S100 proteins. J Biol Chem. 1992 Apr 15;267(11):7499- 504 Kuruto-Niwa R, Nakamura M, Takeishi K, Nozawa R. Transcriptional regulation by C/EBP alpha and -beta in the Kuwayama A, Kuruto R, Horie N, Takeishi K, Nozawa R. expression of the gene for the MRP14 myeloid calcium Appearance of nuclear factors that interact with genes for binding protein. Cell Struct Funct. 1998 Jun;23(3):109-18 myeloid calcium binding proteins (MRP-8 and MRP-14) in differentiated HL-60 cells. Blood. 1993 Jun 1;81(11):3116- Loomans HJ, Hahn BL, Li QQ, Phadnis SH, Sohnle PG. 21 Histidine-based zinc-binding sequences and the antimicrobial activity of calprotectin. J Infect Dis. 1998 Lackmann M, Rajasekariah P, Iismaa SE, Jones G, Mar;177(3):812-4 Cornish CJ, Hu S, Simpson RJ, Moritz RL, Geczy CL. Identification of a chemotactic domain of the pro- Melkonyan H, Hofmann HA, Nacken W, Sorg C, Klempt M. inflammatory S100 protein CP-10. J Immunol. 1993 Apr The gene encoding the myeloid-related protein 14 1;150(7):2981-91 (MRP14), a calcium-binding protein expressed in granulocytes and monocytes, contains a potent enhancer Miyasaki KT, Bodeau AL, Murthy AR, Lehrer RI. In vitro element in the first intron. J Biol Chem. 1998 Oct antimicrobial activity of the human neutrophil cytosolic S- 9;273(41):27026-32 100 protein complex, calprotectin, against Capnocytophaga sputigena. J Dent Res. 1993 Newton RA, Hogg N. The human S100 protein MRP-14 is Feb;72(2):517-23 a novel activator of the beta 2 integrin Mac-1 on neutrophils. J Immunol. 1998 Feb 1;160(3):1427-35 Murthy AR, Lehrer RI, Harwig SS, Miyasaki KT. In vitro candidastatic properties of the human neutrophil Osterloh D, Ivanenkov VV, Gerke V. Hydrophobic residues calprotectin complex. J Immunol. 1993 Dec in the C-terminal region of S100A1 are essential for target 1;151(11):6291-301 protein binding but not for dimerization. Cell Calcium. 1998 Aug;24(2):137-51 Roth J, Burwinkel F, van den Bos C, Goebeler M, Vollmer E, Sorg C. MRP8 and MRP14, S-100-like proteins Ridinger K, Ilg EC, Niggli FK, Heizmann CW, Schäfer BW. associated with myeloid differentiation, are translocated to Clustered organization of S100 genes in human and plasma membrane and intermediate filaments in a mouse. Biochim Biophys Acta. 1998 Dec 10;1448(2):254- calcium-dependent manner. Blood. 1993 Sep 63 15;82(6):1875-83 Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, Avila Clohessy PA, Golden BE. Calprotectin-mediated zinc C, Kambham N, Bierhaus A, Nawroth P, Neurath MF, chelation as a biostatic mechanism in host defence. Scand Slattery T, Beach D, McClary J, Nagashima M, Morser J, J Immunol. 1995 Nov;42(5):551-6 Stern D, Schmidt AM. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for Yui S, Mikami M, Yamazaki M. Induction of apoptotic cell S100/calgranulin polypeptides. Cell. 1999 Jun death in mouse lymphoma and human leukemia cell lines 25;97(7):889-901 by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells. J Leukoc Biol. Kerkhoff C, Klempt M, Kaever V, Sorg C. The two calcium- 1995 Dec;58(6):650-8 binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils. J Hardas BD, Zhao X, Zhang J, Longqing X, Stoll S, Elder Biol Chem. 1999 Nov 12;274(46):32672-9 JT. Assignment of psoriasin to human chromosomal band 1q21: coordinate overexpression of clustered genes in Klempt M, Melkonyan H, Hofmann HA, Sorg C. psoriasis. J Invest Dermatol. 1996 Apr;106(4):753-8 Identification of epithelial and myeloid-specific DNA elements regulating MRP14 gene transcription. J Cell Biochem. 1999 Apr 1;73(1):49-55

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 762

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

Pröpper C, Huang X, Roth J, Sorg C, Nacken W. Analysis Yui S, Nakatani Y, Hunter MJ, Chazin WJ, Yamazaki M. of the MRP8-MRP14 protein-protein interaction by the two- Implication of extracellular zinc exclusion by recombinant hybrid system suggests a prominent role of the C-terminal human calprotectin (MRP8 and MRP14) from target cells domain of S100 proteins in dimer formation. J Biol Chem. in its apoptosis-inducing activity. Mediators Inflamm. 2002 1999 Jan 1;274(1):183-8 Jun;11(3):165-72 Sala A, Zarini S, Folco G, Murphy RC, Henson PM. Bhattacharya S, Large E, Heizmann CW, Hemmings B, Differential metabolism of exogenous and endogenous Chazin WJ. Structure of the Ca2+/S100B/NDR kinase arachidonic acid in human neutrophils. J Biol Chem. 1999 peptide complex: insights into S100 target specificity and Oct 1;274(40):28264-9 activation of the kinase. Biochemistry. 2003 Dec 16;42(49):14416-26 Sohnle PG, Hunter MJ, Hahn B, Chazin WJ. Zinc- reversible antimicrobial activity of recombinant calprotectin Donato R. Intracellular and extracellular roles of S100 (migration inhibitory factor-related proteins 8 and 14). J proteins. Microsc Res Tech. 2003 Apr 15;60(6):540-51 Infect Dis. 2000 Oct;182(4):1272-5 Hobbs JA, May R, Tanousis K, McNeill E, Mathies M, Arai K, Teratani T, Nozawa R, Yamada T. Gebhardt C, Henderson R, Robinson MJ, Hogg N. Myeloid Immunohistochemical investigation of S100A9 expression cell function in MRP-14 (S100A9) null mice. Mol Cell Biol. in pulmonary adenocarcinoma: S100A9 expression is 2003 Apr;23(7):2564-76 associated with tumor differentiation. Oncol Rep. 2001 May-Jun;8(3):591-6 Manitz MP, Horst B, Seeliger S, Strey A, Skryabin BV, Gunzer M, Frings W, Schönlau F, Roth J, Sorg C, Nacken Hiratsuka S, Maru Y, Okada A, Seiki M, Noda T, Shibuya W. Loss of S100A9 (MRP14) results in reduced M. Involvement of Flt-1 tyrosine kinase (vascular interleukin-8-induced CD11b surface expression, a endothelial growth factor receptor-1) in pathological polarized microfilament system, and diminished angiogenesis. Cancer Res. 2001 Feb 1;61(3):1207-13 responsiveness to chemoattractants in vitro. Mol Cell Biol. 2003 Feb;23(3):1034-43 Kerkhoff C, Sorg C, Tandon NN, Nacken W. Interaction of S100A8/S100A9-arachidonic acid complexes with the Moroz OV, Dodson GG, Wilson KS, Lukanidin E, Bronstein scavenger receptor CD36 may facilitate fatty acid uptake IB. Multiple structural states of S100A12: A key to its by endothelial cells. Biochemistry. 2001 Jan 9;40(1):241-8 functional diversity. Microsc Res Tech. 2003 Apr 15;60(6):581-92 Nacken W, Lekstrom-Himes JA, Sorg C, Manitz MP. Molecular analysis of the mouse S100A9 gene and Seth A, Kitching R, Landberg G, Xu J, Zubovits J, Burger evidence that the myeloid specific transcription factor AM. Gene expression profiling of ductal carcinomas in situ C/EBPepsilon is not required for the regulation of the and invasive breast tumors. Anticancer Res. 2003 May- S100A9/A8 gene expression in neutrophils. J Cell Jun;23(3A):2043-51 Biochem. 2001;80(4):606-16 Zhi H, Zhang J, Hu G, Lu J, Wang X, Zhou C, Wu M, Liu Z. Srikrishna G, Panneerselvam K, Westphal V, Abraham V, The deregulation of arachidonic acid metabolism-related Varki A, Freeze HH. Two proteins modulating genes in human esophageal squamous cell carcinoma. Int transendothelial migration of leukocytes recognize novel J Cancer. 2003 Sep 1;106(3):327-33 carboxylated glycans on endothelial cells. J Immunol. 2001 Apr 1;166(7):4678-88 Zimmer DB, Wright Sadosky P, Weber DJ. Molecular mechanisms of S100-target protein interactions. Microsc Tarabykina S, Scott DJ, Herzyk P, Hill TJ, Tame JR, Res Tech. 2003 Apr 15;60(6):552-9 Kriajevska M, Lafitte D, Derrick PJ, Dodson GG, Maitland NJ, Lukanidin EM, Bronstein IB. The dimerization interface Arai K, Teratani T, Kuruto-Niwa R, Yamada T, Nozawa R. of the metastasis-associated protein S100A4 (Mts1): in S100A9 expression in invasive ductal carcinoma of the vivo and in vitro studies. J Biol Chem. 2001 Jun breast: S100A9 expression in adenocarcinoma is closely 29;276(26):24212-22 associated with poor tumour differentiation. Eur J Cancer. 2004 May;40(8):1179-87 Yang Z, Tao T, Raftery MJ, Youssef P, Di Girolamo N, Geczy CL. Proinflammatory properties of the human S100 Ghavami S, Kerkhoff C, Los M, Hashemi M, Sorg C, protein S100A12. J Leukoc Biol. 2001 Jun;69(6):986-94 Karami-Tehrani F. Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and Coussens LM, Werb Z. Inflammation and cancer. Nature. the effect of metal ions. J Leukoc Biol. 2004 Jul;76(1):169- 2002 Dec 19-26;420(6917):860-7 75 Henkel GW, McKercher SR, Maki RA. Identification of Ji J, Zhao L, Wang X, Zhou C, Ding F, Su L, Zhang C, Mao three genes up-regulated in PU.1 rescued monocytic X, Wu M, Liu Z. Differential expression of S100 gene precursor cells. Int Immunol. 2002 Jul;14(7):723-32 family in human esophageal squamous cell carcinoma. J Cancer Res Clin Oncol. 2004 Aug;130(8):480-6 Kerkhoff C, Hofmann HA, Vormoor J, Melkonyan H, Roth J, Sorg C, Klempt M. Binding of two nuclear complexes to Kong JP, Ding F, Zhou CN, Wang XQ, Miao XP, Wu M, Liu a novel regulatory element within the human S100A9 ZH. Loss of myeloid-related proteins 8 and myeloid-related promoter drives the S100A9 gene expression. J Biol proteins 14 expression in human esophageal squamous Chem. 2002 Nov 1;277(44):41879-87 cell carcinoma correlates with poor differentiation. World J Gastroenterol. 2004 Apr 15;10(8):1093-7 Robinson MJ, Tessier P, Poulsom R, Hogg N. The S100 family heterodimer, MRP-8/14, binds with high affinity to Li C, Zhang F, Lin M, Liu J. Induction of S100A9 gene heparin and heparan sulfate glycosaminoglycans on expression by cytokine oncostatin M in breast cancer cells endothelial cells. J Biol Chem. 2002 Feb 1;277(5):3658-65 through the STAT3 signaling cascade. Breast Cancer Res Treat. 2004 Sep;87(2):123-34 Semprini S, Capon F, Tacconelli A, Giardina E, Orecchia A, Mingarelli R, Gobello T, Zambruno G, Botta A, Fabrizi Nacken W, Sorg C, Kerkhoff C. The myeloid expressed G, Novelli G. Evidence for differential S100 gene over- EF-hand proteins display a diverse pattern of lipid raft expression in psoriatic patients from genetically association. FEBS Lett. 2004 Aug 13;572(1-3):289-93 heterogeneous pedigrees. Hum Genet. 2002 Oct;111(4- 5):310-3 Shen J, Person MD, Zhu J, Abbruzzese JL, Li D. Protein expression profiles in pancreatic adenocarcinoma

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 763

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

compared with normal pancreatic tissue and tissue Hummerich L, Müller R, Hess J, Kokocinski F, Hahn M, affected by pancreatitis as detected by two-dimensional Fürstenberger G, Mauch C, Lichter P, Angel P. gel electrophoresis and mass spectrometry. Cancer Res. Identification of novel tumour-associated genes 2004 Dec 15;64(24):9018-26 differentially expressed in the process of squamous cell cancer development. Oncogene. 2006 Jan 5;25(1):111-21 Vogl T, Ludwig S, Goebeler M, Strey A, Thorey IS, Reichelt R, Foell D, Gerke V, Manitz MP, Nacken W, Ito Y, Miyauchi A, Arai K, Nozawa R, Miya A, Kobayashi K, Werner S, Sorg C, Roth J. MRP8 and MRP14 control Nakamura Y, Kakudo K. Usefulness of S100A9 for microtubule reorganization during transendothelial diagnosis of intrathyroid epithelial thymoma migration of phagocytes. Blood. 2004 Dec (ITET)/carcinoma showing thymus-like differentiation 15;104(13):4260-8 (CASTLE). Pathology. 2006 Dec;38(6):541-4 Bierhaus A, Humpert PM, Stern DM, Arnold B, Nawroth Moqbel R, Coughlin JJ. Differential secretion of cytokines. PP. Advanced glycation end product receptor-mediated Sci STKE. 2006 Jun 6;2006(338):pe26 cellular dysfunction. Ann N Y Acad Sci. 2005 Jun;1043:676-80 Nagaraja GM, Othman M, Fox BP, Alsaber R, Pellegrino CM, Zeng Y, Khanna R, Tamburini P, Swaroop A, Kandpal Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, RP. Gene expression signatures and biomarkers of Morel F. IL-22 inhibits epidermal differentiation and noninvasive and invasive breast cancer cells: induces proinflammatory gene expression and migration of comprehensive profiles by representational difference human keratinocytes. J Immunol. 2005 Mar analysis, microarrays and proteomics. Oncogene. 2006 15;174(6):3695-702 Apr 13;25(16):2328-38 Carlsson H, Petersson S, Enerbäck C. Cluster analysis of Santamaria-Kisiel L, Rintala-Dempsey AC, Shaw GS. S100 gene expression and genes correlating to psoriasin Calcium-dependent and -independent interactions of the (S100A7) expression at different stages of breast cancer S100 protein family. Biochem J. 2006 Jun 1;396(2):201-14 development. Int J Oncol. 2005 Dec;27(6):1473-81 Benedyk M, Sopalla C, Nacken W, Bode G, Melkonyan H, Cross SS, Hamdy FC, Deloulme JC, Rehman I. Banfi B, Kerkhoff C. HaCaT keratinocytes overexpressing Expression of S100 proteins in normal human tissues and the S100 proteins S100A8 and S100A9 show increased common cancers using tissue microarrays: S100A6, NADPH oxidase and NF-kappaB activities. J Invest S100A8, S100A9 and S100A11 are all overexpressed in Dermatol. 2007 Aug;127(8):2001-11 common cancers. Histopathology. 2005 Mar;46(3):256-69 Herold K, Moser B, Chen Y, Zeng S, Yan SF, Ramasamy Hermani A, Hess J, De Servi B, Medunjanin S, Grobholz R, Emond J, Clynes R, Schmidt AM. Receptor for R, Trojan L, Angel P, Mayer D. Calcium-binding proteins advanced glycation end products (RAGE) in a dash to the S100A8 and S100A9 as novel diagnostic markers in rescue: inflammatory signals gone awry in the primal human prostate cancer. Clin Cancer Res. 2005 Jul response to stress. J Leukoc Biol. 2007 Aug;82(2):204-12 15;11(14):5146-52 Ludwig S, Stephan C, Lein M, Loening SA, Jung K. Ito Y, Arai K, Ryushi, Nozawa, Yoshida H, Tomoda C, S100A8, S100A9, and the S100A8/A9 complex in Uruno T, Miya A, Kobayashi K, Matsuzuka F, Kuma K, circulating blood are not associated with prostate cancer Kakudo K, Miyauchi A. S100A9 expression is significantly risk-A re-evaluation study. Prostate. 2007 Sep linked to dedifferentiation of thyroid carcinoma. Pathol Res 1;67(12):1301-7 Pract. 2005;201(8-9):551-6 Sheikh AA, Vimalachandran D, Thompson CC, Jenkins Kerkhoff C, Nacken W, Benedyk M, Dagher MC, Sopalla RE, Nedjadi T, Shekouh A, Campbell F, Dodson A, Prime C, Doussiere J. The arachidonic acid-binding protein W, Crnogorac-Jurcevic T, Lemoine NR, Costello E. The S100A8/A9 promotes NADPH oxidase activation by expression of S100A8 in pancreatic cancer-associated interaction with p67phox and Rac-2. FASEB J. 2005 monocytes is associated with the Smad4 status of Mar;19(3):467-9 pancreatic cancer cells. Proteomics. 2007 Jun;7(11):1929- 40 Lominadze G, Rane MJ, Merchant M, Cai J, Ward RA, McLeish KR. Myeloid-related protein-14 is a p38 MAPK Vogl T, Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van substrate in human neutrophils. J Immunol. 2005 Jun Zoelen MA, Nacken W, Foell D, van der Poll T, Sorg C, 1;174(11):7257-67 Roth J. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced Nacken W, Mooren FC, Manitz MP, Bode G, Sorg C, shock. Nat Med. 2007 Sep;13(9):1042-9 Kerkhoff C. S100A9 deficiency alters adenosine-5'- triphosphate induced calcium signalling but does not Yao R, Lopez-Beltran A, Maclennan GT, Montironi R, Eble generally interfere with calcium and zinc homeostasis in JN, Cheng L. Expression of S100 protein family members murine neutrophils. Int J Biochem Cell Biol. 2005 in the pathogenesis of bladder tumors. Anticancer Res. Jun;37(6):1241-53 2007 Sep-Oct;27(5A):3051-8 Grote J, König S, Ackermann D, Sopalla C, Benedyk M, Arai K, Takano S, Teratani T, Ito Y, Yamada T, Nozawa R. Los M, Kerkhoff C. Identification of poly(ADP- S100A8 and S100A9 overexpression is associated with ribose)polymerase-1 and Ku70/Ku80 as transcriptional poor pathological parameters in invasive ductal carcinoma regulators of S100A9 gene expression. BMC Mol Biol. of the breast. Curr Cancer Drug Targets. 2008 2006 Dec 22;7:48 Jun;8(4):243-52 Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer Bode G, Lüken A, Kerkhoff C, Roth J, Ludwig S, Nacken D. S100A8 and S100A9 activate MAP kinase and NF- W. Interaction between S100A8/A9 and annexin A6 is kappaB signaling pathways and trigger translocation of involved in the calcium-induced cell surface exposition of RAGE in human prostate cancer cells. Exp Cell Res. 2006 S100A8/A9. J Biol Chem. 2008 Nov 14;283(46):31776-84 Jan 15;312(2):184-97 Corbin BD, Seeley EH, Raab A, Feldmann J, Miller MR, Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour- Torres VJ, Anderson KL, Dattilo BM, Dunman PM, Gerads mediated upregulation of chemoattractants and R, Caprioli RM, Nacken W, Chazin WJ, Skaar EP. Metal recruitment of myeloid cells predetermines lung chelation and inhibition of bacterial growth in tissue metastasis. Nat Cell Biol. 2006 Dec;8(12):1369-75 abscesses. Science. 2008 Feb 15;319(5865):962-5

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 764

S100A9 (S100 calcium binding protein A9) Kerkhoff C, Ghavami S

Dokun OY, Florl AR, Seifert HH, Wolff I, Schulz WA. of inflammation and cancer. Neoplasia. 2009 Relationship of SNCG, S100A4, S100A9 and LCN2 gene Jul;11(7):615-28. (REVIEW) expression and DNA methylation in bladder cancer. Int J Cancer. 2008 Dec 15;123(12):2798-807 van Dieck J, Fernandez-Fernandez MR, Veprintsev DB, Fersht AR.. Modulation of the oligomerization state of p53 Fernandez-Fernandez MR, Rutherford TJ, Fersht AR. by differential binding of proteins of the S100 family to p53 Members of the S100 family bind p53 in two distinct ways. monomers and tetramers. J Biol Chem. 2009 May Protein Sci. 2008 Oct;17(10):1663-70 15;284(20):13804-11. Epub 2009 Mar 18. Gebhardt C, Riehl A, Durchdewald M, Németh J, Ang CW, Nedjadi T, Sheikh AA, Tweedle EM, Tonack S, Fürstenberger G, Müller-Decker K, Enk A, Arnold B, Honap S, Jenkins RE, Park BK, Schwarte-Waldhoff I, Bierhaus A, Nawroth PP, Hess J, Angel P. RAGE signaling Khattak I, Azadeh B, Dodson A, Kalirai H, Neoptolemos sustains inflammation and promotes tumor development. J JP, Rooney PS, Costello E.. Smad4 loss is associated with Exp Med. 2008 Feb 18;205(2):275-85 fewer S100A8-positive monocytes in colorectal tumors and attenuated response to S100A8 in colorectal and Ghavami S, Kerkhoff C, Chazin WJ, Kadkhoda K, Xiao W, pancreatic cancer cells. Carcinogenesis. 2010 Zuse A, Hashemi M, Eshraghi M, Schulze-Osthoff K, Sep;31(9):1541-51. Epub 2010 Jul 9. Klonisch T, Los M. S100A8/9 induces cell death via a novel, RAGE-independent pathway that involves selective Fritz G, Botelho HM, Morozova-Roche LA, Gomes CM.. release of Smac/DIABLO and Omi/HtrA2. Biochim Biophys Natural and amyloid self-assembly of S100 proteins: Acta. 2008 Feb;1783(2):297-311 structural basis of functional diversity. FEBS J. 2010 Nov;277(22):4578-90. doi: 10.1111/j.1742- Ghavami S, Rashedi I, Dattilo BM, Eshraghi M, Chazin 4658.2010.07887.x. (REVIEW) WJ, Hashemi M, Wesselborg S, Kerkhoff C, Los M. S100A8/A9 at low concentration promotes tumor cell Ghavami S, Eshragi M, Ande SR, Chazin WJ, Klonisch T, growth via RAGE ligation and MAP kinase-dependent Halayko AJ, McNeill KD, Hashemi M, Kerkhoff C, Los M.. pathway. J Leukoc Biol. 2008 Jun;83(6):1484-92 S100A8/A9 induces autophagy and apoptosis via ROS- mediated cross-talk between mitochondria and lysosomes Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, that involves BNIP3. Cell Res. 2010 Mar;20(3):314-31. Ishibashi S, Miyake K, Shibuya M, Akira S, Aburatani H, Epub 2009 Nov 24. Maru Y. The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nat Cell Biol. Ha YS, Kim MJ, Yoon HY, Kang HW, Kim YJ, Yun SJ, Lee 2008 Nov;10(11):1349-55 SC, Kim WJ.. mRNA Expression of S100A8 as a Prognostic Marker for Progression of Non-Muscle-Invasive Lim SY, Raftery M, Cai H, Hsu K, Yan WX, Hseih HL, Bladder Cancer. Korean J Urol. 2010 Jan;51(1):15-20. Watts RN, Richardson D, Thomas S, Perry M, Geczy CL. Epub 2010 Jan 21. S-nitrosylated S100A8: novel anti-inflammatory properties. J Immunol. 2008 Oct 15;181(8):5627-36 Minami S, Sato Y, Matsumoto T, Kageyama T, Kawashima Y, Yoshio K, Ishii J, Matsumoto K, Nagashio R, Okayasu Salama I, Malone PS, Mihaimeed F, Jones JL. A review of I.. Proteomic study of sera from patients with bladder the S100 proteins in cancer. Eur J Surg Oncol. 2008 cancer: usefulness of S100A8 and S100A9 proteins. Apr;34(4):357-64 Cancer Genomics Proteomics. 2010 Jul-Aug;7(4):181-9. Turovskaya O, Foell D, Sinha P, Vogl T, Newlin R, Nayak Saha A, Lee YC, Zhang Z, Chandra G, Su SB, Mukherjee J, Nguyen M, Olsson A, Nawroth PP, Bierhaus A, Varki N, AB.. Lack of an endogenous anti-inflammatory protein in Kronenberg M, Freeze HH, Srikrishna G. RAGE, mice enhances colonization of B16F10 melanoma cells in carboxylated glycans and S100A8/A9 play essential roles the lungs. J Biol Chem. 2010 Apr 2;285(14):10822-31. in colitis-associated carcinogenesis. Carcinogenesis. 2008 Epub 2010 Jan 29. Oct;29(10):2035-43 Su YJ, Xu F, Yu JP, Yue DS, Ren XB, Wang CL.. Up- Ghavami S, Chitayat S, Hashemi M, Eshraghi M, Chazin regulation of the expression of S100A8 and S100A9 in WJ, Halayko AJ, Kerkhoff C. S100A8/A9: a Janus-faced lung adenocarcinoma and its correlation with inflammation molecule in cancer therapy and tumorgenesis. Eur J and other clinical features. Chin Med J (Engl). 2010 Pharmacol. 2009 Dec 25;625(1-3):73-83 Aug;123(16):2215-20. Ito Y, Arai K, Nozawa R, Yoshida H, Hirokawa M, Voss A, Bode G, Sopalla C, Benedyk M, Varga G, Bohm Fukushima M, Inoue H, Tomoda C, Kihara M, M, Nacken W, Kerkhoff C.. Expression of S100A8/A9 in Higashiyama T, Takamura Y, Miya A, Kobayashi K, HaCaT keratinocytes alters the rate of cell proliferation and Matsuzuka F, Miyauchi A. S100A8 and S100A9 differentiation. FEBS Lett. 2011 Jan 21;585(2):440-6. Epub expression is a crucial factor for dedifferentiation in thyroid 2010 Dec 28. carcinoma. Anticancer Res. 2009 Oct;29(10):4157-61 Kerkhoff C, Ghavami S.. Innate immunity molecules This article should be referenced as such: S100A8/A9 involved in stress response and cancer Kerkhoff C, Ghavami S. S100A9 (S100 calcium binding biology. Anti-Inflammatory and Anti-Allergy Agents in protein A9). Atlas Genet Cytogenet Oncol Haematol. 2011; Medicinal Chemistry, 2009; (8)4:279-281. 15(9):754-765. Srikrishna G, Freeze HH.. Endogenous damage- associated molecular pattern molecules at the crossroads

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 765

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Mini Review

TRIAP1 (TP53 regulated inhibitor of apoptosis 1) Veruska Alves, Roberta Felix, Andre Vettore, Gisele Colleoni Universidade Federal de Sao Paulo - UNIFESP, Laboratory of Cancer Molecular Biology, Sao Paulo, Brazil (VA, RF, AV, GC)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/TRIAP1ID44577ch12q24.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI TRIAP1ID44577ch12q24.txt

Identity Transcription Other names: HSPC132; p53CSV; P53CSV; WF- This gene contains 2 introns which transcription 1 gives 3 different mRNAs, 2 alternatively spliced variants and 1 unspliced form that encodes good HGNC (Hugo): TRIAP1 proteins (see figure 1). Location: 12q24.31 Protein DNA/RNA Note Description The P53CSV protein is involved in programmed 2452 bases, starts at 119366147 and ends at cell death. It contains a p53-binding site and it is 119368598 bp from promoter with minus strand induced when cells are at low genotoxic stress. It is orientation. probably involved in cell survival by interaction between Apaf-1 (apoptosis protease activating factor 1) and heat shock protein 70 (Hsp70) with subsequent inhibition of caspase-9 activation.

Figure 1.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 766

TRIAP1 (TP53 regulated inhibitor of apoptosis 1) Alves V, et al.

Description Mutations This protein contains 76 amino acids and has 8786 Note (Da) of weight. There are two identified alterations until now. One of them is located at position 270 of mRNA and the allele G (guanine) is switched to the allele C

Figure 2. (cytosine) at position 77 of the amino acid sequence protein. The other one is a synonymous alteration Localisation localized at position 160 of mRNA involving the The protein is localized in cytoplasm and protein residue Leucine. The allele C (cytosine) is perinuclear region. switched to the allele T (thymine) at position 40 of Function the amino acid sequence protein (NCBI). P53CSV is a novel p53-target gene. This gene can Implicated in modulate apoptotic pathways by interaction with heat shock protein 70 (HSP70), preventing the Multiple myeloma induction of apoptosis. When cells are submitted to Note low levels of genotoxic stress, it is an important Felix et al. (2009), described that P53CSV gene player in P53-mediated cell survivor pathway (Park was upregulated in multiple myeloma SAGE (serial and Nakamura, 2005; Felix et al., 2009). analysis of gene expression) library when compared P53CSV can inhibit apoptosis through interaction to normal/reactive plasma cells. with APAF1 and HSP70 complex.

Figure 3. Hypothetical illustration about TRIAP1 (P53CSV) involvement in the p53-dependent cell survival pathway. The TRIAP1 mediates cell survival at low level of genotoxic stress by inhibiting activation of the complex APAF-1/caspase-9/cytochrome C preventing the apoptosis induction.

Figure 4. Hypothetical P53CSV mechanism of action in interaction with heat shock protein 70 in normal and tumor cells (Felix et al., 2009).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 767

TRIAP1 (TP53 regulated inhibitor of apoptosis 1) Alves V, et al.

They suggested that the interaction between P53CSV/Hsp70 should be evaluated as a potential References target for multiple myeloma patients. Real time Park WR, Nakamura Y. p53CSV, a novel p53-inducible quantitative PCR analyses confirmed upregulation gene involved in the p53-dependent cell-survival pathway. of P53CSV in 90% of multiple myeloma plasma Cancer Res. 2005 Feb 15;65(4):1197-206 samples cells. Staib F, Robles AI, Varticovski L, Wang XW, Zeeberg BR, Sirotin M, Zhurkin VB, Hofseth LJ, Hussain SP, Weinstein Inflammatory stress JN, Galle PR, Harris CC. The p53 tumor suppressor network is a key responder to microenvironmental Note components of chronic inflammatory stress. Cancer Res. Staib et al. (2005) reported P53CSV expression in 2005 Nov 15;65(22):10255-64 colon carcinoma cells in the course of inflammatory Yu K, Ganesan K, Tan LK, Laban M, Wu J, Zhao XD, Li H, responses associated with four microenvironmental Leung CH, Zhu Y, Wei CL, Hooi SC, Miller L, Tan P. A components: nitric oxide, hydrogen peroxide, DNA precisely regulated gene expression cassette potently replication arrest, and hypoxia. modulates metastasis and survival in multiple solid cancers. PLoS Genet. 2008 Jul 18;4(7):e1000129 Solid cancers Felix RS, Colleoni GW, Caballero OL, Yamamoto M, Note Almeida MS, Andrade VC, Chauffaille Mde L, Silva WA Jr, Yu Kun et al. (2008), using a genome-wide Begnami MD, Soares FA, Simpson AJ, Zago MA, Vettore AL. SAGE analysis highlights the importance of p53csv, computational strategy identified genes exhibiting ddx5, mapkapk2 and ranbp2 to multiple myeloma precise transcriptional control in solid tumors and tumorigenesis. Cancer Lett. 2009 Jun 8;278(1):41-8 evaluated if they linked to multiple cancer-related pathways such as metastatic and invasive potential. This article should be referenced as such: siRNA knockdown of five genes supports the Alves V, Felix R, Vettore A, Colleoni G. TRIAP1 (TP53 existence of precisely controlled genes in solid regulated inhibitor of apoptosis 1). Atlas Genet Cytogenet tumors, including P53CSV. Oncol Haematol. 2011; 15(9):766-768.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 768

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Leukaemia Section Mini Review t(X;11)(q13;q23) Adriana Zamecnikova Kuwait Cancer Control Center, Laboratory of Cancer Genetics, Department of Hematology, Shuwaikh, 70653 Kuwait (AZ)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0X11ID1127.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0X11ID1127.txt

This article is an update of : Bojesen SE. t(X;11)(q13;q23). Atlas Genet Cytogenet Oncol Haematol 2001;5(4)

Identity Phenotype/cell stem origin Suggested involvement of a pluripotent stem cell or a myeloid progenitor cell; very rarely in lymphoid lineage. Etiology No known prior exposure; case of AML M2 developed in a 6 years old male previously treated by chemotherapy and radiotherapy for acute t(X;11)(q13;q23) G- banding - Courtesy Melanie Zenger lymphoblastic leukemia (Harrison et al., 1998). and Claudia Haferlach. Epidemiology Clinics and pathology 6 cases to date, children aged 6 months to 5 years, male predominance; sex ratio 4M/2F. Disease Clinics Described in infants and young children; 4 cases of From the known data: WBC: 21.6 to 91x109/L, case acute myeloid leukemia (AML) (Pui et al., 1987; with a complex t(X;11) associated with fever, Raimondi et al., 1989; Pui et al., 1989; Harrison et enlargement of the liver, spleen and parotid glands, al., 1998) and one case of acute lymphoblastic blood in the stool (Karpas et al., 1977); mediastinal leukemia (ALL) (Smith et al., 1973). With one mass, dyspnoea, no hepatosplenomegaly, WBC: exception, the FAB types in cases of AML were 5x109/L in T-ALL (Smith et al., 1973). M4. Peripheral leucocytes at diagnosis of this ALL case were cultured and are presently known as the Prognosis KARPAS-45 cell line (Karpas et al., 1977). In Survival: poor prognosis; 3 patients died within a addition, MLL/AFX1 fusion was confirmed in an year after diagnosis, and one patient died after 24.5 AML case with highly complex change originally months. published involving the Xq22 locus (Nacheva et al., 1982; Parry et al., 1994; Borkhardtet al., 1997). Genetics Note This translocation has also been found in 2 cases of Note CLL (Bentz et al., 1995; Kalla et al., 2005). In one Breakpoints difficult to ascertain in suboptimal case a t(X;11)(q13;q23) was cloned revealing the preparations. involvement of BRWD3 gene recently located on Xq21.1 (Kalla et al., 2005).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 769 t(X;11)(q13;q23) Zamecnikova A

code for the following domains: AT-hook + DNA Cytogenetics methyltransferase (from MLL) + part, aa 147-187 Probes of the DNA-binding domain (from AFX1). Pooled cDNA FISH-probes from AFX1: AFX 12, References 115, 106, 108, 114. Smith JL, Clein GP, Barker CR, Collins RD. Additional anomalies Characterisation of malignant mediastinal lymphoid Part of a highly complex change in one case; in neoplasm (Sternberg sarcoma) as thymic in origin. Lancet. KARPAS 45: Hypotetraploidy. -Y, -3, +6, -14, -18 1973 Jan 13;1(7794):74-7 t(1;5)(q21;q12.2)x2, del4(4)(q22), del(16)(q22). Karpas A, Hayhoe FGJ, Greenberger JS, Barker CR, Cawley JC, Lowenthal RM, Moloney WC.. The Establishment and Cytological, Cytochemical and Genes involved and Immunological Characterisation of Human Haemic Cell Lines: Evidence for Heterogeneity. Leukaemia Res 1977; 1 proteins : 35-49. Note Nacheva E, Fischer P, Haas O, Manolova Y, Manolov G, Cloning and characterization of AFX the gene that Levan A.. Acute myelogenous leukemia in a child with primary involvement of chromosomes 11 and X. Hereditas. fuses to MLL in one case of AML and in the 1982;97(2):273-88. leukemic cell line. Pui CH, Raimondi SC, Murphy SB, Ribeiro RC, Kalwinsky AFX1 (All-1 fusion partner on DK, Dahl GV, Crist WM, Williams DL.. An analysis of leukemic cell chromosomal features in infants. Blood. 1987 chromosome X, MLLT7) May;69(5):1289-93. Location Pui CH, Behm FG, Raimondi SC, Dodge RK, George SL, Xq13 Rivera GK, Mirro J Jr, Kalwinsky DK, Dahl GV, Murphy DNA/RNA SB.. Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia. N Engl J Med. 1989 Jul AFX consists of two exons and encodes for a 20;321(3):136-42. protein of 501 amino acids. Raimondi SC, Kalwinsky DK, Hayashi Y, Behm FG, Mirro J Protein Jr, Williams DL.. Cytogenetics of childhood acute Transcription factor; high degree of homology nonlymphocytic leukemia. Cancer Genet Cytogenet. 1989 between AFXI and the forkhead protein family and Jul 1;40(1):13-27. highly homologous to the human FKHR protein. Parry P, Wei Y, Evans G.. Cloning and characterization of the t(X;11) breakpoint from a leukemic cell line identify a MLL (Mixed lineage leukemia gene, new member of the forkhead gene family. Genes ALL1, HRX, and Hrtx) Chromosomes Cancer. 1994 Oct;11(2):79-84. Location Bentz M, Huck K, du Manoir S, Joos S, Werner CA, Fischer K, Dohner H, Lichter P.. Comparative genomic 11q23 hybridization in chronic B-cell leukemias shows a high DNA/RNA incidence of chromosomal gains and losses. Blood. 1995 Jun 15;85(12):3610-8. The Mixed-Lineage Leukemia gene consists of at least 36 exons, encoding a 3969 amino-acid nuclear Borkhardt A, Repp R, Haas OA, Leis T, Harbott J, Kreuder J, Hammermann J, Henn T, Lampert F.. Cloning and protein with a molecular weight of nearly 430 kDa. characterization of AFX, the gene that fuses to MLL in Protein acute leukemias with a t(X;11)(q13;q23). Oncogene. 1997 Multidomain molecule; shares homology with the Jan 16;14(2):195-202. Drosophila trithorax protein; function as a positive Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage- regulator of gene expression in embryonic Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM.. Ten novel 11q23 chromosomal partner sites. development and hematopoiesis. European 11q23 Workshop participants. Leukemia. 1998 May;12(5):811-22. Result of the chromosomal Kalla C, Nentwich H, Schlotter M, Mertens D, Wildenberger K, Dohner H, Stilgenbauer S, Lichter P.. anomaly Translocation t(X;11)(q13;q23) in B-cell chronic lymphocytic leukemia disrupts two novel genes. Genes Hybrid gene Chromosomes Cancer. 2005 Feb;42(2):128-43. Note This article should be referenced as such: 5' MLL - AFX 3' as well as the 5' AFX - MLL 3'. Zamecnikova A. t(X;11)(q13;q23). Atlas Genet Cytogenet Fusion protein Oncol Haematol. 2011; 15(9):769-770. Note Chimeric proteins that contain the N-terminus of MLL; hybrid transcript MLL-AFX1 contains the

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 770

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Leukaemia Section Mini Review t(X;11)(q22;q23) Adriana Zamecnikova Kuwait Cancer Control Center, Laboratory of Cancer Genetics, Department of Hematology, Shuwaikh, 70653 Kuwait (AZ)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0X11q22q23ID1573.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0X11q22q23ID1573.txt

Identity

Partial karyotypes showing the chromosomal translocation t(X;11)(q22;q23).

monosomy 22 (Soszynska et al., 2008). Of note, the Clinics and pathology fourth AML (FAB type M2) case reported by Slater in a 10 months old male was shown to involve the Disease SEPTIN6 gene located on Xq24 (Slater et al., The chromosomal translocation t(X;11)(q22;q23) 2002). occurs very rarely, with only three cases of infants Phenotype/cell stem origin young children having been described in the literature; 2 AML cases: a 3 years old male, Suggested involvement of a pluripotent stem cell or diagnosed with AML-M2 (Harrison et al., 1998) a myeloid progenitor cell; myeloid lineage. and 2 years old female diagnosed with acute Etiology megakaryoblastic leukemia (FAB type M7) No known prior exposure. (Ribeiro et al., 1993). The one ALL case described in a 4 years old male had a complex karyotype with Epidemiology chromosomal translocation t(11;14)(q13;q32), and Only 3 cases to date, sex ratio 2M/1F.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 771 t(X;11)(q22;q23) Zamecnikova A

Prognosis Protein From the known data, the 3 years old male, The MLL protein is a multi-domain molecule with diagnosed with AML-M2 remained alive in regions of homology to diverse proteins; a major complete remission at 97 months; the ALL patient regulator of class I homeobox (HOX) gene was in complete remission after 39 months. expression. Cytogenetics Result of the chromosomal Note anomaly Breakpoints difficult to ascertain; cytogenetic Hybrid gene appearance may be similar to t(X;11)(q13;q23) involving the AFX gene that fuses to MLL in acute Note leukemias. 5' MLL - PARTNER GENE 3'. MLL translocation breakpoints cluster within an Cytogenetics morphological 8.3-kb region spanning exons 5-11; genomic t(X;11)(q22;q23). breakpoint junction usually created on the der(11) Additional anomalies chromosome. Sole abnormality in AML-M2 case, part of a Fusion protein hyperploid karyotype associated with +6, +8, +19, Oncogenesis +21, +21 in a child with acute megakaryoblastic Expression of a chimeric protein with actively leukemia and complex karyotype in ALL case transforming properties; altered patterns of MLL associated with t(11;14)(q13;q32), and monosomy activity in hematopoietic stem cells resulting in 22, indicating that the t(X;11)(q22;q23) is likely to blockage of hematopoietic maturation. be a secondary anomaly to t(11;14)(q13;q32) in ALL. References Ribeiro RC, Oliveira MS, Fairclough D, Hurwitz C, Mirro J, Genes involved and Behm FG, Head D, Silva ML, Raimondi SC, Crist WM. Acute megakaryoblastic leukemia in children and proteins adolescents: a retrospective analysis of 24 cases. Leuk Lymphoma. 1993 Jul;10(4-5):299-306 Note The gene in Xq22 is yet unknown, it is therefore Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage- Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker uncertain whether this translocation involve a new LM. Ten novel 11q23 chromosomal partner sites. MLL partner. European 11q23 Workshop participants. Leukemia. 1998 MLL May;12(5):811-22 Slater DJ, Hilgenfeld E, Rappaport EF, Shah N, Meek RG, Location Williams WR, Lovett BD, Osheroff N, Autar RS, Ried T, 11q23 Felix CA. MLL-SEPTIN6 fusion recurs in novel translocation of chromosomes 3, X, and 11 in infant acute Note myelomonocytic leukaemia and in t(X;11) in infant acute The MLL gene is frequently disrupted by a variety myeloid leukaemia, and MLL genomic breakpoint in of chromosomal rearrangements that occur in acute complex MLL-SEPTIN6 rearrangement is a DNA myeloblastic leukemia (AML) and in acute topoisomerase II cleavage site. Oncogene. 2002 Jul 11;21(30):4706-14 lymphoblastic leukemia (ALL), with a peak incidence in infant leukemia as well as in Soszynska K, Mucha B, Debski R, Skonieczka K, secondary, topoisomerase II inhibitor-related Duszenko E, Koltan A, Wysocki M, Haus O. The application of conventional cytogenetics, FISH, and RT- leukemia. PCR to detect genetic changes in 70 children with ALL. DNA/RNA Ann Hematol. 2008 Dec;87(12):991-1002 The MLL genomic structure consists of 36 exons This article should be referenced as such: distributed over 100 kb, the mRNA of ~11.9 kb encodes a 3969 amino-acid nuclear protein with a Zamecnikova A. t(X;11)(q22;q23). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):771-772. molecular weight of of 430 kDa.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 772

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Leukaemia Section Mini Review t(X;11)(q24;q23) MLL-SEPTIN6 Adriana Zamecnikova Kuwait Cancer Control Center, Laboratory of Cancer Genetics, Department of Hematology, Shuwaikh, 70653 Kuwait (AZ)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0X11q24q23ID1219.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0X11q24q23ID1219.txt

Notably, in 2 of the patients bilateral and right Clinics and pathology exophthalmus was described. Peripheral blood leukocytosis (WBC 13.4x109/L to 608x109/L; mean Disease 223x109/L), anemia and thrombocytopenia were All the described cases were diagnosed as having reported in the majority of patients. acute myeloid leukemia (AML), classified as FAB- Prognosis M2 (5 cases), M4 (4 cases), M1 (1 case) and M5 (1 case), indicating that AML with the MLL-SEPTIN6 From the 4 patients treated with chemotherapy one fusion gene have a tendency to differentiate into the is alive (13+ months), 3 patients died 1 to 8 months myeloid lineage. All the patients were infants and from diagnosis; 8 patients received bone marrow young children aged 0 to 29 months, suggesting transplantation, among them 2 of the patients died that AML with t(X;11)(q24;q23) is a subgroup of after 9 and 11 months, 6 patients are alive (one infant leukemia. months to 7 years) indicating the prognosis is rather poor. Phenotype/cell stem origin Suggested involvement of a pluripotent stem cell or Cytogenetics a myeloid progenitor cell. Cytogenetics morphological Etiology Chromosomal rearrangements of 11q23 and Xq24 No known prior exposure; putative association with resulting in MLL-SEPT6 fusions are often complex in utero exposure to recurrent genetic insults. and sometimes cryptic associated with 11q Epidemiology insertions. In addition, molecular detection of MLL-SEPTIN6 transcripts in cases with normal Involvement of the SEPTIN6 gene on Xq24 in cytogenetics and in patients with chromosomal MLL rearrangements occurs very rarely, with only Xq22 breakpoints indicates the difficulty in precise 13 cases (7 males, 6 females) having been chromosomal breakpoint definition. documented in the literature. In addition, 3 AML cases with chromosomal translocation Additional anomalies t(X;11)(q24;q23) (3 males aged 0 to 6 years), which +6 (2 cases), del(11)(q13), i(10)(q10), add(X)(p11) also potentially could be found to involve MLL and described in single cases. SEPTIN6 genes have been described confirming the recurrent nature of this translocation. Variants Clinics At least four different types of chromosomal rearrangements have been described that can Hepatosplenomegaly (3 cases), massive and diffuse generate the MLL-SEPT6 fusion. adenopathy (2 cases), lympadenophaty (2 cases), CNS involvement in 2 cases as well as chloroma, Genes involved and scalp nodules, mucosal and cutaneous pallor, bluish cutaneous nodules and petecchiae were described. proteins

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 773 t(X;11)(q24;q23) MLL-SEPTIN6 Zamecnikova A

Note Protein MLL and SEPTIN6 reside on their respective 434 amino acids; 49717 Da. chromosome loci in reverse orientation, that is, the SEPT6 is a GTP-binding protein with a central orientation of the MLL gene is centromere-to- conserved ATP-GTP binding motif, a lysin rich telomere and the orientation of the SEPTIN6 gene region, a variable N-terminal extension domain and is reversed, telomere to centromere at Xq24. This a C-terminal coiled coil. May function in may explain why the MLL/SEPTIN6/Xq24 heteropolymeric complexes; roles in GTPase rearrangement is often associated with complex signaling, cell division, cytokinesis, cytoskeletal translocations and with 11q insertions. filament formation, cell polarity, and oncogenesis. MLL (Mixed lineage leukemia gene, Septins, a family of conserved GTP-binding proteins, are characteristically found in the ALL1, HRX, and HRTX) heteropolymeric filaments and associate with Location cellular membranes, microtubules and actin 11q23 filaments which are assembled from asymmetrical DNA/RNA heterotrimers, composed of SEPT2, SEPT6 and The MLL genomic structure consists of at least 36 SEPT7 that associate head-to-head to form a exons spanning a region of ~89 kb. The mRNA of hexameric unit. Mammalian septins localize in the ~11.9 kb encodes a massive nuclear protein of 3969 cytoplasm and assemble into heteromeric amino acids with a molecular weight of nearly 430 complexes composed of three or more septin kDa. subunits. Protein Result of the chromosomal Multi-domain protein characteristic of several domains with assigned activities including an N anomaly terminus with DNA binding motifs; AT-hook motifs, 4 cysteine-rich zinc fingers, a Hybrid gene transactivation domain, and a highly conserved C- Note terminal domain with histone methyltransferase 5' MLL - SEPTIN6 3' activity. Nuclear protein; a major regulator of class The MLL genomic breakpoints in MLL-SEPT6 I homeobox (HOX) gene expression; functions as a AML patients in all cases occurred in the MLL 8.3 positive regulator of gene expression in early kb breakpoint cluster region (BCR) and seem to embryonic development and hematopoiesis occur preferentially in the telomeric half (between regulation. introns 7 and 11) of the MLL BCR. In the majority SEPTIN6 of reported cases 5' MLL sequences joined in-frame with SEPTIN6 downstream of SEPT6 exon 1. In Location rare cases, out-of-frame fusion between MLL exon Xq24 7 and SEPT6 exon 2, with splicing of MLL exon 6 DNA/RNA have been described. The SEPT6 gene, belongs to the evolutionarily The breakpoint junctions in the SEPT6 intron 1 conserved family of genes of septins consisting of mapped to the vicinity of GC-rich low-complexity 12 exons. Four types of transcripts: 2.3 kb, 2.7 kb, repeats, Alu repeats, and a topoisomerase II 3.1 kb and 4.6 kb coding for three isoforms. SEPT6 recognition sequence raising the possibility that the is ubiquitously expressed in tissues; in the human, non-homologous DNA end-joining pathway may be several alternatively spliced SEPTIN6 transcripts involved in the in the generation of MLL-SEPT6 are differentially expressed in adult and fetal rearrangements in infant acute myeloid leukemia tissues. and a putative association with in utero exposure to topoisomerase II inhibitors has been hypothesized.

Schematic representation of MLL-SEPTIN6 fusion protein.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 774 t(X;11)(q24;q23) MLL-SEPTIN6 Zamecnikova A

Transcript European 11q23 Workshop participants. Leukemia. 1998 May;12(5):811-22 5'-MLL/SEPTIN6-3' chimeric transcript. Nakata Y, Mori T, Yamazaki T, Suzuki T, Okazaki T, Fusion protein Kurosawa Y, Kinoshita A, Ohyashiki K, Nakazawa S. Acute myeloid leukemia with hypergranular cytoplasm Note accompanied by t(X;11)(q24;q23) and rearrangement of The MLL-SEPT6 chimeric protein consists of the the MLL gene. Leuk Res. 1999 Jan;23(1):85-8 AT-hook DNA-binding, the DNA Borkhardt A, Teigler-Schlegel A, Fuchs U, Keller C, König methyltransferase, the and repression domains of M, Harbott J, Haas OA. An ins(X;11)(q24;q23) fuses the MLL and almost the entire open reading frame of MLL and the Septin 6/KIAA0128 gene in an infant with SEPT6 including the central conserved ATP-GTP AML-M2. Genes Chromosomes Cancer. 2001 binding motif. Sep;32(1):82-8 Expression / Localisation Ono R, Taki T, Taketani T, Kawaguchi H, Taniwaki M, Okamura T, Kawa K, Hanada R, Kobayashi M, Hayashi Y. MLL fusion genes express in- frame chimeric SEPTIN6, a human homologue to mouse Septin6, is fused proteins residing in the nucleus. to MLL in infant acute myeloid leukemia with complex chromosomal abnormalities involving 11q23 and Xq24. Oncogenesis Cancer Res. 2002 Jan 15;62(2):333-7 MLL is fused with a partner gene in MLL-related Slater DJ, Hilgenfeld E, Rappaport EF, Shah N, Meek RG, leukemias leading to the aberrant activation of Williams WR, Lovett BD, Osheroff N, Autar RS, Ried T, target genes, including HOX genes. The phenotype Felix CA. MLL-SEPTIN6 fusion recurs in novel depends on the fusion partner, indicating that each translocation of chromosomes 3, X, and 11 in infant acute fusion partner is critical for the leukemogenesis. myelomonocytic leukaemia and in t(X;11) in infant acute myeloid leukaemia, and MLL genomic breakpoint in Among partner genes, septins are the protein family complex MLL-SEPTIN6 rearrangement is a DNA most frequently involved in rearrangements with topoisomerase II cleavage site. Oncogene. 2002 Jul MLL, suggesting that SEPTIN family members are 11;21(30):4706-14 particularly vulnerable to form MLL translocations. Fu JF, Liang DC, Yang CP, Hsu JJ, Shih LY. Molecular MLL fusions with several different SEPTIN family analysis of t(X;11)(q24;q23) in an infant with AML-M4. members (SEPT2, SEPT5, SEPT9, and SEPT11) Genes Chromosomes Cancer. 2003 Nov;38(3):253-9 are preferentially associated with myeoloblastic Kim HJ, Ki CS, Park Q, Koo HH, Yoo KH, Kim EJ, Kim SH. rather than lymphoblastic leukemogenesis MLL/SEPTIN6 chimeric transcript from inv suggesting an important common pathway to ins(X;11)(q24;q23q13) in acute monocytic leukemia: report leukaemogenesis in AML with these translocations. of a case and review of the literature. Genes Chromosomes Cancer. 2003 Sep;38(1):8-12 The observation that overexpression of SEPT6 itself does not lead to the myeloid immortalization Ono R, Nakajima H, Ozaki K, Kumagai H, Kawashima T, Taki T, Kitamura T, Hayashi Y, Nosaka T. Dimerization of of murine hematopoietic progenitors in vitro, MLL fusion proteins and FLT3 activation synergize to whereas the overexpression of MLL-SEPT6 does induce multiple-lineage leukemogenesis. J Clin Invest. indicate that the fusion partner-mediated 2005 Apr;115(4):919-29 homooligomerization of MLL-SEPT6 through its Kadkol SS, Bruno A, Oh S, Schmidt ML, Lindgren V. MLL- intact GTP-binding domain and coiled-coil region SEPT6 fusion transcript with a novel sequence in an infant in the nucleus is essential to immortalize with acute myeloid leukemia. Cancer Genet Cytogenet. hematopoietic progenitors. However, MLL-SEPT6 2006 Jul 15;168(2):162-7 rearrangment induced lethal myeloproliferative Strehl S, König M, Meyer C, Schneider B, Harbott J, Jäger disease with long latency in mice, but not acute U, von Bergh AR, Loncarevic IF, Jarosova M, Schmidt HH, Moore SD, Marschalek R, Haas OA. Molecular dissection leukemia in experimental models. These findings of t(11;17) in acute myeloid leukemia reveals a variety of suggest that secondary genotoxic effects on DNA gene fusions with heterogeneous fusion transcripts and repair and/or cell-cycle regulation are required for multiple splice variants. Genes Chromosomes Cancer. oncogenesis in MLL-SEPT6 associated leukemias. 2006 Nov;45(11):1041-9 Cerveira N, Micci F, Santos J, Pinheiro M, Correia C, References Lisboa S, Bizarro S, Norton L, Glomstein A, Asberg AE, Heim S, Teixeira MR. Molecular characterization of the Köller U, Haas OA, Ludwig WD, Bartram CR, Harbott J, MLL-SEPT6 fusion gene in acute myeloid leukemia: Panzer-Grümayer R, Hansen-Hagge T, Ritter J, Creutzig identification of novel fusion transcripts and cloning of U, Knapp W. Phenotypic and genotypic heterogeneity in genomic breakpoint junctions. Haematologica. 2008 infant acute leukemia. II. Acute nonlymphoblastic Jul;93(7):1076-80 leukemia. Leukemia. 1989 Oct;3(10):708-14 This article should be referenced as such: Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage- Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker Zamecnikova A. t(X;11)(q24;q23) MLL-SEPTIN6. Atlas LM. Ten novel 11q23 chromosomal partner sites. Genet Cytogenet Oncol Haematol. 2011; 15(9):773-775.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 775

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Deep Insight Section

S100 Protein Family and Tumorigenesis Geetha Srikrishna, Hudson H Freeze Sanford-Burnham Medical Research Institute, 10905 Road to the Cure, San Diego, CA 92121, USA (GS, HHF)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Deep/S100ProtFamilyTumorID20092.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI S100ProtFamilyTumorID20092.txt

1. Introduction S100 proteins are a family of 25 homologous intracellular calcium-binding proteins characterized by EF hand motifs, low molecular weights (9-13 kDa), ability to form homodimers, heterodimers and oligomeric assemblies, and are characterized by tissue and cell-specific expression (Donato, 2001; Heizmann et al., 2002; Marenholz et al., 2004; Roth et al., 2003) (Figure 1). They are solely present in vertebrates (Donato, 2001). While human S100B, S100P, S100Z and S100G are located at 21q22, 4p16, 5q14 and Xp22 respectively, 21 of the human S100 genes (S100A1-S100A18, trichohyalin, filaggrin and repetin) are clustered at the chromosomal region 1q21, a region that is frequently deleted, translocated or duplicated in epithelial tumors and tumors of soft tissues (Craig et al., 1994; Donato, 2001; Gebhardt et al., 2006; Figure 1. Dimer structure of S100 proteins. S100 Heizmann et al., 2002) (Figure 2). There is growing proteins form a large multi-gene family of low molecular evidence that expression of S100 proteins is altered weight proteins that are characterized by calcium-binding in many tumors, often in association with tumor EF hand motifs and exhibit remarkable tissue and cell- progression, and they are therefore potentially specific expression. They exist as homo and heterodimers, and oligomers. Each monomer consists of two EF-hands important tumor biomarkers and therapeutic targets. connected by a hinge region. (Reproduced from Heizmann However, their precise roles in tumor progression CW, Fritz G, Schafer BW. Frontiers in Bioscience. 2002 are not completely understood. May 1;7:d1356-68).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 776

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

causes conformational changes that exposes binding sites for target proteins. Intracellular functions of S100 proteins have been extensively studied. These include calcium homeostasis, cell cycle regulation, cell growth and migration, cytoskeletal interactions, membrane trafficking, protein phosphorylation, and regulation of transcriptional factors among others (Donato, 2001; Heizmann et al., 2002; Marenholz et al., 2004; Roth et al., 2003). S100 proteins can also be released into extracellular space in response to stimuli, or during cell damage, and they promote responses including neuronal survival and extension (S100B), apoptosis (S100A4 and S100A6), inflammation (S100B, S100A8/A9, S100A11 and S100A12), autoimmunity (S100A8/A9), chemotaxis (S100A8/A9) and cell proliferation and survival (S100P, S100A7), thus effectively functioning as paracrine and autocrine mediators. S100B, S100P, S100A4, S100A6, S100A8/A9, S100A11 and S100A12 are known to act via interaction with cell surface receptors, primarily the Receptor for Advanced Glycation End Products (RAGE) (Donato, 2007; Leclerc et al.,

Figure 2. The S100 gene cluster on human 2009), while S100A8/A9 also bind Toll-like chromosome 1q21. Most human S100 genes are located receptors or TLRs (Vogl et al., 2007). Multimeric in the epidermal differentiation complex on chromosome forms of S100 proteins appear to be necessary for 1q21, a region prone to rearrangements. Genes located in the cluster region are indicated, as well as two commonly the extracellular functions of S100 proteins used genomic markers (D1S1664 and D1S2346). p and q (Donato, 2007; Leukert et al., 2006). Multimeric indicate the short and the long arm of the chromosome, assemblies have been reported for S100A12, respectively. Human S100B, S100P, S100Z and S100G are S100A4, S100B and S100A8/A9 (Fritz et al., located on chromosomes 21q22, 4p16, 5q14 and Xp22 respectively. (Reproduced from Heizmann CW, Fritz G, 2010). Elevated S100 protein levels are associated Schafer BW. Frontiers in Bioscience. 2002 May 1;7:d1356- with chronic inflammation, neurodegeneration, 68). cardiomyopathies, atherosclerosis and cancer 2. Structure and functions of (Pietzsch, 2010). S100 proteins 3. S100 proteins and S100 proteins have two distinct EF-hand (helix- tumorigenesis loop-helix motif) calcium-binding domains A number of S100 proteins are up-regulated in connected by a hinge region (Fritz et al., 2010). The tumors (Salama et al., 2008). With the identification canonical C-terminal calcium-binding EF- hand is of binding proteins and signaling pathways for at common to all EF-hand proteins, while the N- least a few of its family members, S100 proteins terminal EF-hand is non-canonical. There is promise to offer both functional biomarkers and variable degree of sequence identity among S100 therapeutic targets in cancers, and the case for proteins. S100 proteins exist as anti-parallel hetero considering their importance is evolving rapidly and homodimers within cells. Calcium binding (summarized in Table 1). Table 1. Expression of S100 proteins in tumors*. Tumors in which S100 Tumors in which Known functions / interactions / mechanism expression is proteins expression is up-regulated of action down-regulated Renal, clear cell and papillary S100A1 Known RAGE ligand. Endometriod subtype of ovarian and endometrial Lung, non-small cell Pancreatic Oral squamous cell Promotes p53 transcriptional activity and S100A2 Gastric Prostate reduces expression of Cox-2. Thyroid, papillary and anaplastic

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 777

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

Breast Colorectal Gastric Promotes tumor migration, invasion and Prostate angiogenesis. Regulates matrix S100A4 Lung, non-small cell metalloproteinases and interacts with p53 and Ovarian inhibits p53 phosphorylation. Pancreatic Melanoma Colorectal Pancreatic Gastric S100A6 Known RAGE ligand. Hepatocellular Lung Melanoma Breast, ER negative invasive, DCIS S100A7 Possible interaction with Jab-1. Bladder Skin Gastric Colon Interaction with RAGE and TLR4; promote Pancreatic tumor proliferation, and migration, Bladder accumulation of myeloid derived suppressor S100A8/A9 Ovarian cells, activation of protumorigenic genes, and Thyroid formation of premetastatic niches in distal Breast organs. Skin Uterine, smooth muscle Bladder Lymphoma, anaplastic S100A11 Esophageal, large cell squamous cell Pancreatic Melanoma (biomarker) Interacts with p53 and down-regulates p53- S100B Astrocytoma, anaplastic mediated apoptosis in melanoma. Well known Glioblastomas RAGE ligand. Ovarian Pancreatic Breast Activation of RAGE dependent signaling S100P Gastric pathways. Colorectal Prostate Lung * Relevant references are provided in the text.

3.1. S100A1 2009). Besides cardiac and skeletal muscle, S100A1 is predominantly expressed in the heart, expression of S100A1 is low in most normal and to a lesser extent in the skeletal muscle tissues, but up-regulated in cancers of the kidneys, (Heizmann et al., 2007; Leclerc et al., 2009). skin and ovary. S100A1 expression helps to S100A1 is a key modulator of calcium homeostasis differentiate subtypes of renal carcinoma. S100A1 in the heart and targets several key regulators of protein is expressed in renal oncocytomas, and in sarcoplasmic reticulum including Ca2+ ATPase, clear cell and papillary renal cell carcinomas but ryanodine receptors and other targets, thereby not in chromophobe renal cell carcinomas (Cossu- enhancing cardiomyocyte performance. Rocca et al., 2009; Li et al., 2007). In addition, Dysregulation of cardiomyocyte S100A1 protein, S100A1 is a specific and sensitive and diminished levels following myocardial immunohistochemical marker to differentiate infarction contributes to cardiac hypertrophy and nephrogenic adenoma from prostatic heart failure. Extracellularly, S100A1 exists as both adenocarcinoma where it is not expressed (Cossu- a homodimer and heterodimer with S100B, S100A4 Rocca et al., 2009). S100A1 messenger RNA and and S100P and interacts with RAGE (Leclerc et al., protein are up-regulated in ovarian tumors and

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 778

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

ovarian cancer metastasis compared with normal Maelandsmo, 2010), is normally found in the ovarian tissues. In the endometrioid subtype of nervous system and believed to be involved in ovarian and endometrial cancers, there is a negative neuritogenesis. S100A4 is also expressed at low correlation between relapse-free survival and levels in normal human fibroblasts, monocytes, S100A1 expression, suggesting that S100A1 is a macrophages, T cells, neutrophils, and endothelial marker for poor prognosis of endometrioid subtypes cells. Although its expression is low in normal of cancer (DeRycke et al., 2009). tissues, it is highly expressed in many tumors such 3.2. S100A2 as breast, colorectal, gastric, prostate and non-small S100A2 protein is present in many organs or cell lung cancer, ovarian and pancreatic cancers, tissues, with high expression in lung and kidney. and malignant melanoma (Boye and Maelandsmo, Unlike most other S100 proteins, S100A2 is 2010; Sherbet, 2009). Expression is a significant markedly down-regulated in many tumors predictor of patient survival and metastatic disease. suggesting that it acts as a tumor suppressor gene. It S100A4 was first cloned from highly metastatic promotes transcriptional activity of p53 (Mueller et breast cancer cells, and since then its involvement al., 2005) and reduces expression of Cox-2 (Tsai et in cancer metastasis has been substantiated by al., 2006). On the other hand, S100A2 is also up- several studies (Boye and Maelandsmo, 2010; regulated in some tumors (Salama et al., 2008; Sherbet, 2009; Tarabykina et al., 2007). Studies Wolf et al., 2010). show that extracellular, intracellular, tumor-derived, Loss of S100A2 expression has been associated and stroma-derived S100A4 all contribute to the with poor prognosis and shorter survival. S100A2 metastatic process, and influence several steps in expression decreases in epithelial cells from normal the metastatic cascade, including migration, to tumor stages, with the decrease more pronounced invasion, and angiogenesis. Much of this metastatic in glandular than in squamous epithelial tissue potential has been linked to the ability of S100A4 (Nagy et al., 2002). S100A2 expression is reduced to regulate matrix metalloproteinases, modulate cell in early-stage oral squamous cell carcinoma and is motility, promote angiogenesis and epithelial significantly associated with tumor recurrence and mesenchymal transition, and the association of metastasis (Suzuki et al., 2005; Tsai et al., 2005). S100A4 expression with reduced expression of Patients with S100A2 positive laryngeal squamous tumor suppressor genes such as p53 (Garrett et al., cell carcinoma had a better relapse-free overall 2006; Salama et al., 2008). S100A4 interacts with survival than patients with S100A2-negative tumors p53 and inhibits p53-mediated tumor suppression (Almadori et al., 2009). Benign prostate hyperplasia by inhibiting its phosphorylation (Grigorian et al., and prostatitis are characterized by higher levels of 2001). The extensive association of S100A4 with S100A2 than low-grade cancer, and expression is tumor progression and metastasis has positioned it lost in high-grade and metastatic cancer specimens to be a target for novel therapeutic strategies. (Gupta et al., 2003). 3.4. S100A6 S100A2 is up-regulated in some tumors such as S100A6 is highly expressed in various organs, and non-small cell lung carcinoma (Smith et al., 2004), on fibroblasts, epithelial and other cells (Leclerc et with higher expression found in early-stage al., 2009; Lesniak et al., 2009). S100A6 is carcinomas, associated with reduced overall predominantly cytoplasmic protein but can survival, and higher propensity to metastasis translocate in the presence of Ca2+ to plasma (Bartling et al., 2007; Bulk et al., 2009; Feng et al., membrane and the nuclear envelope. S100A6 2001; Wang et al., 2005; Zech et al., 2006). In expression can be up-regulated by platelet-derived pancreatic cancer tissues and cell lines, S100A2 growth factor, epidermal growth factor, tumor expression is significantly higher than in normal necrosis factor, retinoic acid and estrogen, and upon pancreatic tissues, and higher expression co-related stress conditions, and at the transcriptional level by with poor disease outcome (Biankin et al., 2009; NF-κB. S100A6 interacts with many proteins Ohuchida et al., 2007). S100A2 is also over- including CacyBP/SIP, annexins II and XI, expressed in gastric tumors compared to normal tropomycin and RAGE (Leclerc et al., 2009; gastric mucosa (Lee et al., 2006). Varied expression Lesniak et al., 2009). S100A6 is overexpressed in has been reported with oesophageal squamous many cancers including colorectal, pancreatic, carcinoma (Cao et al., 2009; Imazawa et al., 2005; gastric, hepatocellular and lung cancers, and Ji et al., 2004). In thyroid carcinomas, S100A2 is melanoma. Expression in melanoma, pancreatic and absent in follicular adenomas and carcinomas, but colorectal cancers has been shown to correlate with up-regulated in papillary and anaplastic carcinomas, tumor growth and metastatic progression providing a possible marker for distinguishing these suggesting a potential role for S100A6 in the two types of thyroid carcinoma (Ito et al., 2005). development of malignancy (Lesniak et al., 2009; 3.3. S100A4 Salama et al., 2008). It is however down-regulated S100A4, also called Metastatin due to its well- in prostate cancer and medulloblastoma. established association with tumor metastasis (Tarabykina et al., 2007; Sherbet, 2009; Boye and

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 779

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

3.5. S100A7 S100A8/A9 exhibit concentration-dependent S100A7 was first identified in inflamed psoriatic dichotomy of function in tumors. At high skin, hence is also called psoriasin (Watson et al., concentrations S100A8/A9 exert apoptotic effects 1998). It is released from keratinocytes around on tumor cells (Ghavami et al., 2004), while at low wounds and believed to exert cytokine and anti concentrations promote tumor cell growth bacterial effects, and is also chemotactic for (Ghavami et al., 2008; Turovskaya et al., 2008). granulocyes, monocytes and lymphocytes (Eckert et S100A8/A9 also stimulate tumor cell migration at al., 2004). Contrary to other S100 proteins, calcium low concentrations (Ang et al., 2010; Hermani et binding does not induce large conformational al., 2006; Hiratsuka et al., 2006; Moon et al., 2008; changes in S100A7 (Streicher et al., 2010). S100A7 Saha et al., 2010). S100A8/A9 regulate the is up regulated in breast, bladder and skin cancers accumulation of MDSC (Cheng et al., 2008; Sinha (Salama et al., 2008). Increasing evidence show that et al., 2008), which are immature myeloid cells that S100A7 is up-regulated in ductal carcinoma in situ expand during inflammation and in tumors, and are and ER negative invasive breast cancer and potent suppressors of T-cell mediated immune expression correlates with aggressive phenotype responses (Dolcetti et al., 2008; Gabrilovich and and patient survival (Emberley et al., 2004b). Nagaraj, 2009; Ostrand-Rosenberg and Sinha, S100A7 interacts with Jab1 (c-jun activation 2009). Tumor derived factors promote sustained domain binding protein 1) a protein with highly STAT3 dependent upregulation of S100A9 in recognizable role in tumorigenesis to elicit myeloid precursors which results in inhibition of functional effects in breast tumor progression differentiation to DC and accumulation of MDSC (Emberley et al., 2004a). (Cheng et al., 2008). S100A8/A9 are not only 3.6. S100A8/A9 synthesized and secreted by MDSC, but they also S100A8 and S100A9 are expressed predominantly have binding sites for S100A8/A9, and activate by myeloid cells, including granulocytes, intracellular signaling that promote their migration monocytes, myeloid derived suppressor cells (Sinha, et al., 2008), suggesting that S100A8/A9 (MDSC) and other immature cells of myeloid support an autocrine feedback loop that sustains lineage (Cheng et al., 2008; Goyette and Geczy, accumulation of MDSC in tumors (Ostrand- 2010; Roth et al., 2003; Sinha et al., 2008). Rosenberg, 2008). They also bind to tumor cells Although the proteins are products of distinct and activate MAPK and NF-κB signaling pathways genes, they are often co-expressed and function and specific downstream genes that promote mainly as heterodimer of S100A8/A9 tumorigenesis (Ghavami et al., 2009; Ichikawa et (calprotectin). Expression is down-regulated during al., 2011). S100A8/A9 are involved in early macrophage and dendritic cell differentiation metastatic processes. Expression of S100A8/A9 in (Cheng et al., 2008; Lagasse and Clerc, 1988; myeloid and endothelial cells in premetastatic Odink et al., 1987), but can be induced in epithelial organs in response to soluble factors such as VEGF, cells, osteoclasts and keratinocytes (Gebhardt et al., TGFβ and TNFα expressed by distal primary 2006). S100A8/A9 released into the extracellular tumors promotes homing of tumor cells to medium in response to cell damage or activation premetastatic niches (Hiratsuka et al., 2006). Recent become danger signals (Damage Associated studies argue for prominent roles for two pattern Molecular Pattern molecules or DAMP), which recognition receptors, TLR4 and RAGE, in alert the host of danger by triggering immune S100A8/A9 mediated pathological effects responses and activating repair mechanisms (Gebhardt et al., 2008; Loser et al., 2010; Sinha et through interaction with pattern recognition al., 2008; Turovskaya et al., 2008; Vogl et al., receptors (Donato, 2007; Ehrchen et al., 2009; Foell 2007). et al., 2007; Leclerc et al., 2009; Srikrishna and 3.7. S100A11 Freeze, 2009). Elevated S100A8/A9 is the hallmark S100A11, also called S100C or calgizzarin, is of inflammatory conditions such as rheumatoid expressed in many tissues including the placenta, arthritis, inflammatory bowel disease, multiple heart, lung, and kidney (He et al., 2009; Inada et al., sclerosis, cystic fibrosis and psoriasis (Foell et al., 1999; Salama et al., 2008). Expression levels are 2007; Roth et al., 2001; Roth et al., 2003). Critical low in skeletal muscle and liver (Inada et al., 1999). roles for these proteins in endotoxin-induced S100A11 is overexpressed in uterine smooth lethality and systemic autoimmunity have recently muscle tumors (Kanamori et al., 2004), anaplastic been recognized (Loser et al., 2010; Vogl et al., large cell lymphomas (Rust et al., 2005), and 2007). In addition to expression within pancreatic tumors (Ohuchida et al., 2006b), while inflammatory milieu, strong up-regulation of these significantly down-regulated in esophageal proteins has also been observed in many tumors, squamous cell (Ji et al., 2004) and bladder tumors including gastric, colon, pancreatic, bladder, (Memon et al., 2005). In bladder carcinoma, down- ovarian, thyroid, breast and skin cancers (Gebhardt regulation of S100A11 is associated with poor et al., 2006; Salama et al., 2008). prognosis and decreased survival, suggesting that S100A11 functions as a tumor suppressor (Memon

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 780

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

et al., 2005). In pancreatic cancer, S100A11 is over- studies show correlation between S100P and expressed in early stages and down-regulated in estrogen receptor expression, and with high-risk advanced tumors, again supporting a tumor- lesions and decreased survival (Schor et al., 2006). suppressor role and suggesting that S100A11 S100P has been shown to induce breast cancer expression could be valuable in detecting early metastasis (Wang et al., 2006). S100P is also up- pancreatic tumors (Ohuchida et al., 2006b). On the regulated in colon cancer cells (Bertram et al., other hand, S100A11 expression in prostate cancer 1998; Fuentes et al., 2007), and in flat adenomas in is associated with advanced disease (Rehman et al., the colon (Kita et al., 2006). S100P can be secreted, 2004), showing that S100A11 plays opposing roles and interacts with RAGE on pancreatic and colon depending on the tumor involved. tumor cells (Arumugam et al., 2005; Fuentes et al., 3.8. S100B 2007), activating NF-κB and MAPK pathways S100B is one of the best-studied proteins of the (Fuentes et al., 2007). Efforts are therefore S100 family and its interaction with RAGE has underway to develop small molecule inhibitors that been well characterized (Donato, 2007; Donato et block the interaction of S100P and RAGE. al., 2008; Leclerc et al., 2009). S100B is particularly abundant in the brain and is highly Summary expressed by astrocytes, oligodendrocytes and There is growing evidence that many S100 proteins Schwann cells. It is considered as an intracellular are altered in human tumors. Future studies are regulator and extracellular signal, exerting likely to reveal molecular mechanisms that define concentration-dependent trophic as well as toxic the multiple and specific roles that S100 proteins effects on neurons (Donato et al., 2008). It also play in tumor progression and metastasis, providing activates microglia, and may have a role in the novel therapeutic targets and biomarkers. pathogenesis of neurodegenerative disorders. S100B is over-expressed in anaplastic astrocytomas References and glioblastomas (Camby et al., 1999), and Odink K, Cerletti N, Brüggen J, Clerc RG, Tarcsay L, melanomas (Salama et al., 2008). S100B interacts Zwadlo G, Gerhards G, Schlegel R, Sorg C. Two calcium- with p53 in melanomas and down-regulates p53 binding proteins in infiltrate macrophages of rheumatoid mediated apopotosis (Lin et al., 2010). S100B is the arthritis. Nature. 1987 Nov 5-11;330(6143):80-2 best-studied biomarker for melanoma (Gogas et al., Lagasse E, Clerc RG. Cloning and expression of two 2009; Salama et al., 2008). Serum levels of S100B human genes encoding calcium-binding proteins that are increases in a stage-dependent manner in patients regulated during myeloid differentiation. Mol Cell Biol. 1988 Jun;8(6):2402-10 with melanoma, reflecting tumor load, with decline during therapy and remission, and correlate well Matsuoka N, Maeda N, Ohkubo Y, Yamaguchi I. Differential effects of physostigmine and pilocarpine on the with overall survival. Elevated levels following spatial memory deficits produced by two septo- therapy has shown to correlate with melanoma hippocampal deafferentations in rats. Brain Res. 1991 Sep recurrence. S100B is thus the first of the S100 20;559(2):233-40 proteins to be validated in a clinical setting. Craig RW, Jabs EW, Zhou P, Kozopas KM, Hawkins AL, 3.9. S100P Rochelle JM, Seldin MF, Griffin CA. Human and mouse S100P was first purified from placenta, but it is also chromosomal mapping of the myeloid cell leukemia-1 expressed in many normal tissues including the GI gene: MCL1 maps to human chromosome 1q21, a region that is frequently altered in preneoplastic and neoplastic tract, and in prostate and leukocytes (Arumugam disease. Genomics. 1994 Sep 15;23(2):457-63 and Logsdon, 2010; Leclerc et al., 2009). S100P is also expressed in many tumors, including ovarian, Bertram J, Palfner K, Hiddemann W, Kneba M. Elevated expression of S100P, CAPL and MAGE 3 in doxorubicin- pancreatic, breast, gastric, colorectal, prostate, and resistant cell lines: comparison of mRNA differential lung carcinomas, and has been shown to be display reverse transcription-polymerase chain reaction associated with poor clinical outcomes (Arumugam and subtractive suppressive hybridization for the analysis and Logsdon, 2010; Leclerc et al., 2009). Recent of differential gene expression. Anticancer Drugs. 1998 Apr;9(4):311-7 studies implicate DNA hypomethylation, bone morphogenic protein and non-steroidal anti- Watson PH, Leygue ER, Murphy LC. Psoriasin (S100A7). inflammatory drugs in the regulation of S100P Int J Biochem Cell Biol. 1998 May;30(5):567-71 expression in tumors (Hamada et al., 2009; Namba Camby I, Nagy N, Lopes MB, Schäfer BW, Maurage CA, et al., 2009; Sato et al., 2004). S100P is expressed Ruchoux MM, Murmann P, Pochet R, Heizmann CW, Brotchi J, Salmon I, Kiss R, Decaestecker C. in pancreatic cancer cells, but not in pancreatic Supratentorial pilocytic astrocytomas, astrocytomas, inflammation, and is also elevated early in anaplastic astrocytomas and glioblastomas are preneoplastic cells, suggesting that S100P may be a characterized by a differential expression of S100 proteins. useful biomarker for pancreatic cancer (Logsdon et Brain Pathol. 1999 Jan;9(1):1-19 al., 2003; Ohuchida et al., 2006a). S100P is absent Inada H, Naka M, Tanaka T, Davey GE, Heizmann CW. in normal breast tissue but detected in hyperplasia, Human S100A11 exhibits differential steady-state RNA levels in various tissues and a distinct subcellular as well as in situ and invasive ductal carcinoma localization. Biochem Biophys Res Commun. 1999 Sep (Guerreiro Da Silva et al., 2000). Immunochemical 16;263(1):135-8

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 781

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

Guerreiro Da Silva ID, Hu YF, Russo IH, Ao X, Salicioni Kanamori T, Takakura K, Mandai M, Kariya M, Fukuhara AM, Yang X, Russo J. S100P calcium-binding protein K, Sakaguchi M, Huh NH, Saito K, Sakurai T, Fujita J, Fujii overexpression is associated with immortalization of S. Increased expression of calcium-binding protein S100 in human breast epithelial cells in vitro and early stages of human uterine smooth muscle tumours. Mol Hum Reprod. breast cancer development in vivo. Int J Oncol. 2000 2004 Oct;10(10):735-42 Feb;16(2):231-40 Marenholz I, Heizmann CW, Fritz G. S100 proteins in Donato R. S100: a multigenic family of calcium-modulated mouse and man: from evolution to function and pathology proteins of the EF-hand type with intracellular and (including an update of the nomenclature). Biochem extracellular functional roles. Int J Biochem Cell Biol. 2001 Biophys Res Commun. 2004 Oct 1;322(4):1111-22 Jul;33(7):637-68 Rehman I, Azzouzi AR, Cross SS, Deloulme JC, Catto JW, Feng G, Xu X, Youssef EM, Lotan R. Diminished Wylde N, Larre S, Champigneuille J, Hamdy FC. expression of S100A2, a putative tumor suppressor, at Dysregulated expression of S100A11 (calgizzarin) in early stage of human lung carcinogenesis. Cancer Res. prostate cancer and precursor lesions. Hum Pathol. 2004 2001 Nov 1;61(21):7999-8004 Nov;35(11):1385-91 Grigorian M, Andresen S, Tulchinsky E, Kriajevska M, Sato N, Fukushima N, Matsubayashi H, Goggins M. Carlberg C, Kruse C, Cohn M, Ambartsumian N, Identification of maspin and S100P as novel Christensen A, Selivanova G, Lukanidin E. Tumor hypomethylation targets in pancreatic cancer using global suppressor p53 protein is a new target for the metastasis- gene expression profiling. Oncogene. 2004 Feb associated Mts1/S100A4 protein: functional consequences 26;23(8):1531-8 of their interaction. J Biol Chem. 2001 Jun 22;276(25):22699-708 Smith SL, Gugger M, Hoban P, Ratschiller D, Watson SG, Field JK, Betticher DC, Heighway J. S100A2 is strongly Roth J, Goebeler M, Sorg C. S100A8 and S100A9 in expressed in airway basal cells, preneoplastic bronchial inflammatory diseases. Lancet. 2001 Mar lesions and primary non-small cell lung carcinomas. Br J 31;357(9261):1041 Cancer. 2004 Oct 18;91(8):1515-24 Heizmann CW, Fritz G, Schäfer BW. S100 proteins: Arumugam T, Simeone DM, Van Golen K, Logsdon CD. structure, functions and pathology. Front Biosci. 2002 May S100P promotes pancreatic cancer growth, survival, and 1;7:d1356-68 invasion. Clin Cancer Res. 2005 Aug 1;11(15):5356-64 Nagy N, Hoyaux D, Gielen I, Schäfer BW, Pochet R, Imazawa M, Hibi K, Fujitake S, Kodera Y, Ito K, Akiyama Heizmann CW, Kiss R, Salmon I, Decaestecker C. The S, Nakao A. S100A2 overexpression is frequently Ca2+-binding S100A2 protein is differentially expressed in observed in esophageal squamous cell carcinoma. epithelial tissue of glandular or squamous origin. Histol Anticancer Res. 2005 Mar-Apr;25(2B):1247-50 Histopathol. 2002 Jan;17(1):123-30 Ito Y, Yoshida H, Tomoda C, Uruno T, Miya A, Kobayashi Gupta S, Hussain T, MacLennan GT, Fu P, Patel J, K, Matsuzuka F, Kakudo K, Kuma K, Miyauchi A. Mukhtar H. Differential expression of S100A2 and S100A4 Expression of S100A2 and S100A6 in thyroid carcinomas. during progression of human prostate adenocarcinoma. J Histopathology. 2005 May;46(5):569-75 Clin Oncol. 2003 Jan 1;21(1):106-12 Memon AA, Sorensen BS, Meldgaard P, Fokdal L, Logsdon CD, Simeone DM, Binkley C, Arumugam T, Thykjaer T, Nexo E. Down-regulation of S100C is Greenson JK, Giordano TJ, Misek DE, Kuick R, Hanash S. associated with bladder cancer progression and poor Molecular profiling of pancreatic adenocarcinoma and survival. Clin Cancer Res. 2005 Jan 15;11(2 Pt 1):606-11 chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer. Cancer Res. 2003 May Mueller A, Schäfer BW, Ferrari S, Weibel M, Makek M, 15;63(10):2649-57 Höchli M, Heizmann CW. The calcium-binding protein S100A2 interacts with p53 and modulates its Roth J, Vogl T, Sorg C, Sunderkötter C. Phagocyte- transcriptional activity. J Biol Chem. 2005 Aug specific S100 proteins: a novel group of proinflammatory 12;280(32):29186-93 molecules. Trends Immunol. 2003 Apr;24(4):155-8 Rust R, Visser L, van der Leij J, Harms G, Blokzijl T, Eckert RL, Broome AM, Ruse M, Robinson N, Ryan D, Lee Deloulme JC, van der Vlies P, Kamps W, Kok K, Lim M, K. S100 proteins in the epidermis. J Invest Dermatol. 2004 Poppema S, van den Berg A. High expression of calcium- Jul;123(1):23-33 binding proteins, S100A10, S100A11 and CALM2 in anaplastic large cell lymphoma. Br J Haematol. 2005 Emberley ED, Alowami S, Snell L, Murphy LC, Watson PH. Dec;131(5):596-608 S100A7 (psoriasin) expression is associated with aggressive features and alteration of Jab1 in ductal Suzuki F, Oridate N, Homma A, Nakamaru Y, Nagahashi carcinoma in situ of the breast. Breast Cancer Res. T, Yagi K, Yamaguchi S, Furuta Y, Fukuda S. S100A2 2004;6(4):R308-15 expression as a predictive marker for late cervical metastasis in stage I and II invasive squamous cell Emberley ED, Murphy LC, Watson PH. S100A7 and the carcinoma of the oral cavity. Oncol Rep. 2005 progression of breast cancer. Breast Cancer Res. Dec;14(6):1493-8 2004;6(4):153-9 Tsai ST, Jin YT, Tsai WC, Wang ST, Lin YC, Chang MT, Ghavami S, Kerkhoff C, Los M, Hashemi M, Sorg C, Wu LW. S100A2, a potential marker for early recurrence in Karami-Tehrani F. Mechanism of apoptosis induced by early-stage oral cancer. Oral Oncol. 2005 Apr;41(4):349-57 S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions. J Leukoc Biol. 2004 Jul;76(1):169- Wang H, Zhang Z, Li R, Ang KK, Zhang H, Caraway NP, 75 Katz RL, Jiang F. Overexpression of S100A2 protein as a prognostic marker for patients with stage I non small cell Ji J, Zhao L, Wang X, Zhou C, Ding F, Su L, Zhang C, Mao lung cancer. Int J Cancer. 2005 Aug 20;116(2):285-90 X, Wu M, Liu Z. Differential expression of S100 gene family in human esophageal squamous cell carcinoma. J Garrett SC, Varney KM, Weber DJ, Bresnick AR. S100A4, Cancer Res Clin Oncol. 2004 Aug;130(8):480-6 a mediator of metastasis. J Biol Chem. 2006 Jan 13;281(2):677-80

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 782

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

Gebhardt C, Németh J, Angel P, Hess J. S100A8 and S100P in colon cancer stimulates growth, migration, and S100A9 in inflammation and cancer. Biochem Pharmacol. cell signaling pathways. Dis Colon Rectum. 2007 2006 Nov 30;72(11):1622-31 Aug;50(8):1230-40 Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer Heizmann CW, Ackermann GE, Galichet A. Pathologies D. S100A8 and S100A9 activate MAP kinase and NF- involving the S100 proteins and RAGE. Subcell Biochem. kappaB signaling pathways and trigger translocation of 2007;45:93-138 RAGE in human prostate cancer cells. Exp Cell Res. 2006 Jan 15;312(2):184-97 Li G, Barthelemy A, Feng G, Gentil-Perret A, Peoc'h M, Genin C, Tostain J. S100A1: a powerful marker to Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour- differentiate chromophobe renal cell carcinoma from renal mediated upregulation of chemoattractants and oncocytoma. Histopathology. 2007 Apr;50(5):642-7 recruitment of myeloid cells predetermines lung metastasis. Nat Cell Biol. 2006 Dec;8(12):1369-75 Tarabykina S, Griffiths TR, Tulchinsky E, Mellon JK, Bronstein IB, Kriajevska M. Metastasis-associated protein Kita H, Hikichi Y, Hikami K, Tsuneyama K, Cui ZG, Osawa S100A4: spotlight on its role in cell migration. Curr Cancer H, Ohnishi H, Mutoh H, Hoshino H, Bowlus CL, Yamamoto Drug Targets. 2007 May;7(3):217-28 H, Sugano K. Differential gene expression between flat adenoma and normal mucosa in the colon in a microarray Vogl T, Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van analysis. J Gastroenterol. 2006 Nov;41(11):1053-63 Zoelen MA, Nacken W, Foell D, van der Poll T, Sorg C, Roth J. Mrp8 and Mrp14 are endogenous activators of Lee OJ, Hong SM, Razvi MH, Peng D, Powell SM, Smoklin Toll-like receptor 4, promoting lethal, endotoxin-induced M, Moskaluk CA, El-Rifai W. Expression of calcium-binding shock. Nat Med. 2007 Sep;13(9):1042-9 proteins S100A2 and S100A4 in Barrett's adenocarcinomas. Neoplasia. 2006 Oct;8(10):843-50 Cheng P, Corzo CA, Luetteke N, Yu B, Nagaraj S, Bui MM, Ortiz M, Nacken W, Sorg C, Vogl T, Roth J, Gabrilovich DI. Leukert N, Vogl T, Strupat K, Reichelt R, Sorg C, Roth J. Inhibition of dendritic cell differentiation and accumulation Calcium-dependent tetramer formation of S100A8 and of myeloid-derived suppressor cells in cancer is regulated S100A9 is essential for biological activity. J Mol Biol. 2006 by S100A9 protein. J Exp Med. 2008 Sep Jun 16;359(4):961-72 29;205(10):2235-49 Ohuchida K, Mizumoto K, Egami T, Yamaguchi H, Fujii K, Dolcetti L, Marigo I, Mantelli B, Peranzoni E, Zanovello P, Konomi H, Nagai E, Yamaguchi K, Tsuneyoshi M, Tanaka Bronte V. Myeloid-derived suppressor cell role in tumor- M. S100P is an early developmental marker of pancreatic related inflammation. Cancer Lett. 2008 Aug carcinogenesis. Clin Cancer Res. 2006 Sep 28;267(2):216-25 15;12(18):5411-6 Gebhardt C, Riehl A, Durchdewald M, Németh J, Ohuchida K, Mizumoto K, Ohhashi S, Yamaguchi H, Fürstenberger G, Müller-Decker K, Enk A, Arnold B, Konomi H, Nagai E, Yamaguchi K, Tsuneyoshi M, Tanaka Bierhaus A, Nawroth PP, Hess J, Angel P. RAGE signaling M. S100A11, a putative tumor suppressor gene, is sustains inflammation and promotes tumor development. J overexpressed in pancreatic carcinogenesis. Clin Cancer Exp Med. 2008 Feb 18;205(2):275-85 Res. 2006 Sep 15;12(18):5417-22 Ghavami S, Rashedi I, Dattilo BM, Eshraghi M, Chazin Schor AP, Carvalho FM, Kemp C, Silva ID, Russo J. WJ, Hashemi M, Wesselborg S, Kerkhoff C, Los M. S100P calcium-binding protein expression is associated S100A8/A9 at low concentration promotes tumor cell with high-risk proliferative lesions of the breast. Oncol Rep. growth via RAGE ligation and MAP kinase-dependent 2006 Jan;15(1):3-6 pathway. J Leukoc Biol. 2008 Jun;83(6):1484-92 Tsai WC, Tsai ST, Jin YT, Wu LW. Cyclooxygenase-2 is Moon A, Yong HY, Song JI, Cukovic D, Salagrama S, involved in S100A2-mediated tumor suppression in Kaplan D, Putt D, Kim H, Dombkowski A, Kim HR. Global squamous cell carcinoma. Mol Cancer Res. 2006 gene expression profiling unveils S100A8/A9 as candidate Aug;4(8):539-47 markers in H-ras-mediated human breast epithelial cell invasion. Mol Cancer Res. 2008 Oct;6(10):1544-53 Wang G, Platt-Higgins A, Carroll J, de Silva Rudland S, Winstanley J, Barraclough R, Rudland PS. Induction of Ostrand-Rosenberg S. Cancer and complement. Nat metastasis by S100P in a rat mammary model and its Biotechnol. 2008 Dec;26(12):1348-9 association with poor survival of breast cancer patients. Cancer Res. 2006 Jan 15;66(2):1199-207 Salama I, Malone PS, Mihaimeed F, Jones JL. A review of the S100 proteins in cancer. Eur J Surg Oncol. 2008 Zech VF, Dlaska M, Tzankov A, Hilbe W. Prognostic and Apr;34(4):357-64 diagnostic relevance of hnRNP A2/B1, hnRNP B1 and S100 A2 in non-small cell lung cancer. Cancer Detect Sinha P, Okoro C, Foell D, Freeze HH, Ostrand- Prev. 2006;30(5):395-402 Rosenberg S, Srikrishna G. Proinflammatory S100 proteins regulate the accumulation of myeloid-derived Bartling B, Rehbein G, Schmitt WD, Hofmann HS, Silber suppressor cells. J Immunol. 2008 Oct 1;181(7):4666-75 RE, Simm A. S100A2-S100P expression profile and diagnosis of non-small cell lung carcinoma: impairment by Turovskaya O, Foell D, Sinha P, Vogl T, Newlin R, Nayak advanced tumour stages and neoadjuvant chemotherapy. J, Nguyen M, Olsson A, Nawroth PP, Bierhaus A, Varki N, Eur J Cancer. 2007 Sep;43(13):1935-43 Kronenberg M, Freeze HH, Srikrishna G. RAGE, carboxylated glycans and S100A8/A9 play essential roles Donato R. RAGE: a single receptor for several ligands and in colitis-associated carcinogenesis. Carcinogenesis. 2008 different cellular responses: the case of certain S100 Oct;29(10):2035-43 proteins. Curr Mol Med. 2007 Dec;7(8):711-24 Almadori G, Bussu F, Galli J, Rigante M, Lauriola L, Foell D, Wittkowski H, Vogl T, Roth J. S100 proteins Michetti F, Maggiano N, Schafer BW, Heizmann CW, expressed in phagocytes: a novel group of damage- Ranelletti FO, Paludetti G. Diminished expression of associated molecular pattern molecules. J Leukoc Biol. S100A2, a putative tumour suppressor, is an independent 2007 Jan;81(1):28-37 predictive factor of neck node relapse in laryngeal squamous cell carcinoma. J Otolaryngol Head Neck Surg. Fuentes MK, Nigavekar SS, Arumugam T, Logsdon CD, 2009 Feb;38(1):16-22 Schmidt AM, Park JC, Huang EH. RAGE activation by

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 783

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH

Biankin AV, Kench JG, Colvin EK, Segara D, Scarlett CJ, tumorigenic effects. J Biol Chem. 2009 Feb Nguyen NQ, Chang DK, Morey AL, Lee CS, Pinese M, 13;284(7):4158-67 Kuo SC, Susanto JM, Cosman PH, Lindeman GJ, Visvader JE, Nguyen TV, Merrett ND, Warusavitarne J, Ostrand-Rosenberg S, Sinha P. Myeloid-derived Musgrove EA, Henshall SM, Sutherland RL. Expression of suppressor cells: linking inflammation and cancer. J S100A2 calcium-binding protein predicts response to Immunol. 2009 Apr 15;182(8):4499-506 pancreatectomy for pancreatic cancer. Gastroenterology. Sherbet GV. Metastasis promoter S100A4 is a potentially 2009 Aug;137(2):558-68, 568.e1-11 valuable molecular target for cancer therapy. Cancer Lett. Bulk E, Sargin B, Krug U, Hascher A, Jun Y, Knop M, 2009 Jul 18;280(1):15-30 Kerkhoff C, Gerke V, Liersch R, Mesters RM, Hotfilder M, Srikrishna G, Freeze HH. Endogenous damage-associated Marra A, Koschmieder S, Dugas M, Berdel WE, Serve H, molecular pattern molecules at the crossroads of Müller-Tidow C. S100A2 induces metastasis in non-small inflammation and cancer. Neoplasia. 2009 Jul;11(7):615- cell lung cancer. Clin Cancer Res. 2009 Jan 1;15(1):22-9 28 Cao LY, Yin Y, Li H, Jiang Y, Zhang HF. Expression and Ang CW, Nedjadi T, Sheikh AA, Tweedle EM, Tonack S, clinical significance of S100A2 and p63 in esophageal Honap S, Jenkins RE, Park BK, Schwarte-Waldhoff I, carcinoma. World J Gastroenterol. 2009 Sep Khattak I, Azadeh B, Dodson A, Kalirai H, Neoptolemos 7;15(33):4183-8 JP, Rooney PS, Costello E. Smad4 loss is associated with Cossu-Rocca P, Contini M, Brunelli M, Festa A, Pili F, fewer S100A8-positive monocytes in colorectal tumors and Gobbo S, Eccher A, Mura A, Massarelli G, Martignoni G. attenuated response to S100A8 in colorectal and S-100A1 is a reliable marker in distinguishing nephrogenic pancreatic cancer cells. Carcinogenesis. 2010 adenoma from prostatic adenocarcinoma. Am J Surg Sep;31(9):1541-51 Pathol. 2009 Jul;33(7):1031-6 Arumugam T, Logsdon CD. S100P: a novel therapeutic DeRycke MS, Andersen JD, Harrington KM, Pambuccian target for cancer. Amino Acids. 2011 Oct;41(4):893-9 SE, Kalloger SE, Boylan KL, Argenta PA, Skubitz AP. Boye K, Maelandsmo GM. S100A4 and metastasis: a S100A1 expression in ovarian and endometrial small actor playing many roles. Am J Pathol. 2010 endometrioid carcinomas is a prognostic indicator of Feb;176(2):528-35 relapse-free survival. Am J Clin Pathol. 2009 Dec;132(6):846-56 Fritz G, Botelho HM, Morozova-Roche LA, Gomes CM. Natural and amyloid self-assembly of S100 proteins: Donato R, Sorci G, Riuzzi F, Arcuri C, Bianchi R, Brozzi F, structural basis of functional diversity. FEBS J. 2010 Tubaro C, Giambanco I. S100B's double life: intracellular Nov;277(22):4578-90 regulator and extracellular signal. Biochim Biophys Acta. 2009 Jun;1793(6):1008-22 Goyette J, Geczy CL. Inflammation-associated S100 proteins: new mechanisms that regulate function. Amino Ehrchen JM, Sunderkötter C, Foell D, Vogl T, Roth J. The Acids. 2011 Oct;41(4):821-42 endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, Lin J, Yang Q, Wilder PT, Carrier F, Weber DJ. The and cancer. J Leukoc Biol. 2009 Sep;86(3):557-66 calcium-binding protein S100B down-regulates p53 and apoptosis in malignant melanoma. J Biol Chem. 2010 Aug Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor 27;285(35):27487-98 cells as regulators of the immune system. Nat Rev Immunol. 2009 Mar;9(3):162-74 Loser K, Vogl T, Voskort M, Lueken A, Kupas V, Nacken W, Klenner L, Kuhn A, Foell D, Sorokin L, Luger TA, Roth Ghavami S, Chitayat S, Hashemi M, Eshraghi M, Chazin J, Beissert S. The Toll-like receptor 4 ligands Mrp8 and WJ, Halayko AJ, Kerkhoff C. S100A8/A9: a Janus-faced Mrp14 are crucial in the development of autoreactive molecule in cancer therapy and tumorgenesis. Eur J CD8+ T cells. Nat Med. 2010 Jun;16(6):713-7 Pharmacol. 2009 Dec 25;625(1-3):73-83 Pietzsch J. S100 proteins in health and disease. Amino Gogas H, Eggermont AM, Hauschild A, Hersey P, Mohr P, Acids. 2011 Oct;41(4):755-60 Schadendorf D, Spatz A, Dummer R. Biomarkers in melanoma. Ann Oncol. 2009 Aug;20 Suppl 6:vi8-13 Saha A, Lee YC, Zhang Z, Chandra G, Su SB, Mukherjee AB. Lack of an endogenous anti-inflammatory protein in Hamada S, Satoh K, Hirota M, Fujibuchi W, Kanno A, mice enhances colonization of B16F10 melanoma cells in Umino J, Ito H, Satoh A, Kikuta K, Kume K, Masamune A, the lungs. J Biol Chem. 2010 Apr 2;285(14):10822-31 Shimosegawa T. Expression of the calcium-binding protein S100P is regulated by bone morphogenetic protein in Streicher WW, Lopez MM, Makhatadze GI. Modulation of pancreatic duct epithelial cell lines. Cancer Sci. 2009 quaternary structure of S100 proteins by calcium ions. Jan;100(1):103-10 Biophys Chem. 2010 Oct;151(3):181-6 He H, Li J, Weng S, Li M, Yu Y. S100A11: diverse function Wolf S, Haase-Kohn C, Pietzsch J. S100A2 in and pathology corresponding to different target proteins. cancerogenesis: a friend or a foe? Amino Acids. 2011 Cell Biochem Biophys. 2009;55(3):117-26 Oct;41(4):849-61 Leclerc E, Fritz G, Vetter SW, Heizmann CW. Binding of Ichikawa M, Williams R, Wang L, Vogl T, Srikrishna G. S100 proteins to RAGE: an update. Biochim Biophys Acta. S100A8/A9 activate key genes and pathways in colon 2009 Jun;1793(6):993-1007 tumor progression. Mol Cancer Res. 2011 Feb;9(2):133-48

Leśniak W, Słomnicki ŁP, Filipek A. S100A6 - new facts This article should be referenced as such: and features. Biochem Biophys Res Commun. 2009 Dec 25;390(4):1087-92 Srikrishna G, Freeze HH. S100 Protein Family and Tumorigenesis. Atlas Genet Cytogenet Oncol Haematol. Namba T, Homan T, Nishimura T, Mima S, Hoshino T, 2011; 15(9):776-784. Mizushima T. Up-regulation of S100P expression by non- steroidal anti-inflammatory drugs and its role in anti-

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 784

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Deep Insight Section

Visualize Dynamic Chromosome Eisuke Gotoh Division of Genetic Resources, National Institute of Infectious Diseases Japan 1-23-1, Toyama, Shin- juku-ku, Tokyo, 162-8640, Japan (EG)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Deep/VisuDynChID20093.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI VisuDynChID20093.txt

Key words: technique was introduced in the 1970's as a useful chromosome condensation/compaction, technique that allows the interphase nuclei to be chromosome structure, DNA replication, cell cycle, visualized as condensed mitotic chromosome mitosis, S-phase, premature chromosome (Johnson and Rao, 1970; Johnson et al., 1970; condensation (PCC), prematurely condensed Sperling and Rao, 1974). Since then, a lot of studies chromosomes (PCCs), calyculin A, beads loading including DNA replication and chromosome method packaging have been archived using the PCC method (Hittelman and Rao, 1976; Rao et al., 1977; A most miracle mysterious and profound event in Hanks and Rao, 1980; Mullinger and Johnson, eukaryote cell is how DNA folds to chromosomes. 1980; Lau and Arrighi, 1981; Mullinger and In human diploid cell (2n), for example, the total Johnson, 1983). These studies seem to teach that DNA (~6x109 nucleotide base pairs, a meter of the different DNA packaging appearance in length when fully relaxed) is packed to 46 different sub-phase of S-phase suggest that the chromosomes (22 pairs of autosomes and 1 pair of degree of chromosome condensation might be sex chromosomes) and contained in nuclei size of tightly coupled with the progressing of DNA ~5 μm in diameter (Alberts et al., 1989). It is quite replication. However, the limited available difficult to imagine how such long length thin methodologies at that time did not allow the precise fibrous linear molecule is folded in small sized mechanism to be cleared. chromosomes without entangling in a narrow More recently, accumulated evidences have further nucleus space. Very earlier, the concept about the concrete that eukaryote DNA chromosome architecture formation during cell replication/transcription is involved in compaction cycling was conceived as follows: (1) chromosomes of chromosomes (Zink et al., 1998; Manders et al., are diffused over nucleus as decondensed form in 1999; Samaniego et al., 2002, Pflumm, 2002). G1-phase (Gap 1 phase), (2) DNA synthesis starts Molecular genetic studies have also provided and chromosome replicates in S-phase (Synthesis of supporting evidence for the idea that mutation (in DNA phase), (3) DNA synthesis finished, the genes as HIRA/Tuple1, XCDT1, cdt1, Orc2, Orc3, resulted chromosomes are duplicated and ready for Orc5, MCM2, MCM4, MCM10, RECQL4, cell division in G2-phase (Gap 2 phase) and then required for DNA replication) showed abnormal (4) chromosomes condense: separation/segregation phenotype in chromosome condensation (Loupart et and cell division occurs in M-phase (Mitotic phase). al., 2000; Maiorano et al., 2000; Nishitani et al., This traditional concept seems to tell that DNA 2000; Pflumm and Botchan, 2001; Christensen and replication and chromosome condensation are Tye, 2003; McHugh and Heck, 2003; Prasanth et independent events that proceed in S- and M- cell al., 2004), inherited diseases (D'Antoni et al., 2004; cycle stage, respectively. Sangrithi et al., 2005), genomic instability or prone Recently, number of accumulated evidences to cancer (Tatsumi et al., 2006; Pruitt et al., 2007; suggests a close relationship between DNA Shima et al., 2007), or aberrant replication timing replication and chromosome condensation. causes abnormal chromosome condensation Premature chromosome condensation (PCC) (Loupart et al., 2000; Marheineke and Hyrien,

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 785

Visualize Dynamic Chromosome Gotoh E

2001; Pliss et al., 2009). For more detailed 1974; Draetta and Beach, 1988; Dunphy et al., knowledge about the DNA replication, see also the 1988; Kumagai and Dunphy, 1992), Polo and Rho following excellent reviews; Bell and Dutta, 2002 in cytokinesis (Burkard et al., 2009; Wolfe et al., and Masai et al., 2010. 2009; Li et al., 2010) and many other proteins. In the present article, using drug-induced PCC Chromosome dynamic consists of such number of technique and direct Cy3-dUTP fluorescent various elements. Regarding these dynamics, replicating DNA by beads loading method, we numerous visualizing studies reported or in demonstrate the dynamics of chromosome progression are achieved through the mitosis structure, formation and transition during the S- events, relatively easy to observe under microscope. phase progression in which tight-coupled relation However, visualizing approaches in chromosome between DNA replication and chromosome dynamics, coupled with DNA replication, is still condensation /compaction. Possible hypothetical limited. This restraint is due to difficulties as to chromosome condensation/compaction model observe the chromosomes in the S-phase: involving the role of DNA replication will be chromosomes are usually invisible at this stage, suggested. since they are decondensed. In the present review, we simply focus on visualizing the chromosome Chromosome dynamics: DNA replication, dynamics coupled with DNA replication during the condensation, decondensation, cohesion, S-phase progression and we show how replicating separation, segregation, cytokinesis etc. DNA is folded into higher order chromosomes. Under a quite stringent and higher ordered mechanism, chromosomes condense during mitosis Tools to visualize the dynamic chromosomes within a very short lapse of time in the mitotic Drug-induced premature chromosome phase. Mitotic phase is further divided into several condensation (PCC) method subphases (preprophase, prophase, prometaphase, Cytogenetic analysis studies are usually performed metaphase, anaphase and telophase), followed by on chromosomes. As condensed in mitosis, cytokinesis. In the course of Mitotic phase, number chromosomes are usually visible, but as they of sequential drastic conformational transactions are decondensed in the interphase, they are invisible proceeded as following: (1) chromatins condense to (Manders et al., 1996; Gotoh and Durante, 2006). well-defined visible chromosomes under the Therefore, it is practically difficult or even microscope (2) mitotic spindle assemble begin, impossible to analyze the dynamics of chromosome nuclear envelope breakdown into membrane condensation during the interphase by conventional vesicles, centriole and mitotic spindle formation chromosome methods such as colcemid block. followed by spindle attaches to chromosome Premature chromosome condensation (PCC) is a centromeres (3) kinetocore microtubules align the useful and a unique technique that allows the chromosomes at metaphase plate (4) chromosome interphase nuclei to be visualized as a condensed separation segregates to spindle poles (5) separated form of mitotic chromosome (Johnson and Rao, daughter chromatids reach the poles followed by 1970; Rao and Johnson, 1970). Conventional PCC the nuclear envelope re-forms (6) formation of has been carried out by cell fusion using either contractile ring and cleavage furrows which fusogenic viruses (i.e. Sendai virus) (Johnson and constrict the cell center, cytokinesis, cell dividing Rao, 1970) or polyethylene glycol (PEG) (Pantelias into two daughter cells, chromosome and Maillie, 1983) (cell fusion-mediated PCC). But decondensation in divided cells and finally re- these protocols are usually technically demanding entering the cells in the G1 phase (Alberts et al., and keenly depend on the activity of the virus or 1989). The detailed of the whole mechanism is still PEG. Virus-mediated PCC might be also almost unclear. However, number of molecules problematic because of infectious viruses use. which involved in the mitotic events have been Moreover, resulting chromosomes are mixture of identified such as SMC proteins, including those inducer and recipient cells (Gotoh and condensin (chromosome condensation), cohesion Durante, 2006). Due to these restrictions, (chromosome cohesion of replicated chromosomes) conventional PCC has been used in limited (Swedlow and Hirano, 2003), NuMA protein for institutions. These drawbacks of the conventional spindle pole formation (Chang et al., 2009; Haren et PCC technique have been recently overcome with a al., 2009; Silk et al., 2009; Torres et al., 2010), much easier and more rapid technique using nuclear lamins (Moir et al., 2000), aurora kinases in calyculin A or okadaic acid, specific inhibitors of centromere function (Tanno et al., 2006; Meyer et protein phosphatases (drug-induced PCC technique) al., 2010; Tanno et al., 2010), shugoshin and (Gotoh et al., 1995; Gotoh and Asakawa, 1996; protein protein phosphatase 2A in chromosome Asakawa and Gotoh, 1997; Durante et al., 1998; cohesion (Kitajima et al., 2006; Tanno et al., 2010), Gotoh and Durante, 2006). Drug-induced PCC is cdk1 in chromosome condensation, chromosome becoming now 'popular' and has been used in a bi-orientation (Tsukahara et al., 2010), cyclin B, wide range of cytogenetic applications (Gotoh and cdc2, cdc25 in chromosome condensation (Masui, Asakawa, 1996; Asakawa and Gotoh, 1997; Gotoh

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 786

Visualize Dynamic Chromosome Gotoh E

et al., 1999; Ito et al., 2002; Terzoudi et al., 2003; Fluorochrome choice is strictly dependent on the El Achkar et al., 2005; Gotoh and Tanno, 2005; light source (laser) equipped on the microscope Gotoh et al., 2005; Srebniak et al., 2005; Terzoudi available in the individual institute. BrdU (Bromo- et al., 2005; Deckbar et al., 2007; Gotoh, 2007; deoxy-Uridine) is commonly used for labeling Beucher et al., 2009; van Harn et al., 2010). Thus, replicating DNA and gives substantial high quality drug-induced PCC technique is suitable to visualize signals. But combined to laser confocal microscope, dynamic chromosomes particularly in interphase Cy3-dUTP gives much more fine signals. nuclei. This technique will be also useful and Glass beads for beads loading methods. Various applicable in many fields of cytogenetic approaches particle size and various surface treatment glass including traditional chromosome analysis study, beads are provided from the company. DNA because the technique is very simple and much labeling efficiency using beads loading method may easier even than the conventional colcemid be varies in different cell lines, cell conditions, blocking method (Gotoh, 2009). beads size and surface treatment. The optimum Beads loading method choice of beads for individual cell lines should be Cytogenetically visualization of the replicating determined prior to the experiment. DNA is certainly a most direct approach to identify Microscope. Confocal laser microscope is ideal for the DNA replication dynamics. In the very earlier visualize dynamic chromosome coupled with DNA studies, the fibre autoradiography of DNA had been replication, although the conventional microscope labeled with 3H-thymidine (Fakan and Hancock, may substantially work. 1974; Edenberg and Huberman, 1975; Hand, 1978). The spatial resolution of fibre autoradiography is, Visualize the dynamics of chromosome structure however, limited because the location of the silver formation coupled with DNA replication during grains, developed in photosensitive emulsion layer S-phase and covered the specimens, do not correctly reflect Many studies, for visualizing the dynamics of the actual regions of the foci incorporating the 3H- chromosome condensation during cell division in thymidine and the size of grains are not enough tiny mitosis, have been achieved and well documented. to determine the precise location of replicating However, the visualizing study on the relationship regions. More precise localization and measure the between chromosome condensation and DNA replication foci were then done using thymidine replication is still limited. Several studies tried to analog BrdU (Bromodeoxy Uridine) labeling and define fairly well the replication foci distribution in its antibodies (Nakamura et al., 1986; Mills et al., interphase nuclei (Nakamura et al., 1986), but little 1989; Nakayasu and Berezney, 1989). However, the is yet known about how replicating DNA is folded resolution is still limited presumably because it is to higher order chromosomes (since chromosomes based on accessibility problems or size of are invisible in interphase stage as they immunocomplex (antigen/antibodies). Recently, the decondensed). replication regions and chromosome formation in To visualize the chromosome compaction dynamics living cells were visualized using Cy5-dUTP coupled with DNA replication, more precisely in S- directly labeled fluorescent DNA (Manders et al., phase nucleus, the drug-induced PCC method was 1999) by beads loading methods (McNeil and used (Gotoh et al., 1995; Asakawa and Gotoh, Warder, 1987). The procedure facilitate the 1997; Johnson et al., 1999; Ito et al., 2002). The analogues (Cy3-dUTP or Cy5-dUTP) to be cells were unsynchronized because cell incorporated in the cell nucleus in a very short time synchronization using DNA synthesis inhibitor whereby transiently permeabilizes the cell such as thymidine may give some bias in DNA membranes. This method allows the replicating replication and consequently all phases of DNA to be Cy3 fluorescently imaged within very replication can be observed. Individual substage of short lapse of time. The obtaining fluorescence S-phase can be easily identified by typical signal reflects the real incorporated site of analogue diagnostic appearances seen in different phases of replicating DNA with a very fine signal resolution. S-PCCs (Mullinger and Johnson, 1983; Gollin et Combined with the beads loading method and drug- al., 1984; Hameister and Sperling, 1984; Savage et induced PCC, dynamic study of chromosome al., 1984; Gotoh et al., 1995; Gotoh and Durante, condensation, involving DNA replication, has been 2006). A drastic conformational change of realized (Gotoh, 2007). chromosome structure formation along with the proceed of DNA replication, as shown in Fig. 1 Chemicals and Instruments (reproduced from Chromosoma. Gotoh, 2007; Calyculin A to induce PCC in interphase nuclei; 116(5):453-462), is clearly revealed in PCCs purchased from Wako Chemicals (Osaka, Japan), following Cy3-dUTP loading. Cy3-dUTP loading dissolved in 100% DMSO, 100 μM of stock procedure takes 10 minutes followed by 10 minutes solution was stored at -20°C. of PCC induction and fixation (for details, see Cy3- or Cy5-dUTP or other fluorochrome Materials and Methods in Chromosoma. Gotoh, conjugated dUTP for labelling replicating DNA. 2007; 116(5):453-462). Accordingly, only

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 787

Visualize Dynamic Chromosome Gotoh E

replicated DNA in this short lapse will be were mostly condensed like mitotic chromosomes. fluoresced. Thus, the observed S-PCCs in the Cy3-dUTP incorporated regions were recognized as present study reflected the replication stages at most band arrays inserted in the condensed chromosome 20 minutes before the cell fixation. (i) In early S- (Fig. 1K, indicated by arrows). The similar phase, PCCs showed a cloudy spreading mass of appearance of replication foci along longitudinally thin fibres like a 'nebula', where numerous fine on chromosomes were previously reported on granular foci homogeneously distributed on overall metaphase of kangaroo-rat kidney PtK1 cells (Ma et the fibres (Fig. 1I), showing 'beads on a string' or al., 1998). The size of foci is still up and their 'particles on a string': these structures are observed number is still down to the point that they could be under an electron microscope (Olins and Olins, easily scored. (iv) In the very late S-phase, the 1974; Thoma et al., 1979). (ii) In the middle of S- number of foci is further reduced and phase, typical 'pulverized' PCCs were recognized; predominantly they are localized at centromeric or the size of foci was increased while the number of telomeric regions (Fig. 1L, indicated by arrows). foci, unevenly distributed on chromosomes, was These regions are actually known as satellite decreased. As shown in Fig. 1J, the foci become heterochromatic DNA regions where DNA brighter. (iii) In the late S-phase, chromosomes replicates at very late S (O'Keefe et al., 1992).

Figure 1: (1) DNA replication regions on prematurely condensed chromosomes (PCCs) of different substages of S-phase. Ten minutes after Cy3-dUTP loading, cells were condensed prematurely using 50 nM of calyculin A (Gotoh et al., 1995). From left to right column, (A,B,C) early S-phase PCCs, (D,E,F) middle S-PCCs, (G,H,I) late S-PCCs and (J,K,L) very late S-PCCs. (A,D,G,J) DAPI counterstained DNA, (B,E,H,K) Cy3-dUTP labelled DNA replication region and (C,F,I,L) Merged image of DAPI and Cy3. (L) Centromeric region (arrow) or telomeric region (arrowhead) replicates in very-late S-phase are indicated. (I,L) Late S- and very late S-PCCs already condensed like as mitotic chromosomes, but these PCCs were actually S-phase chromosomes because they incorporated Cy3-dUTP. G2/M chromosomes are easily distinguished from late or very late S chromosomes as G2/M chromosomes do not incorporate Cy3-dUTP (data not shown). Inset in (C) is higher magnification of the boxed portion. Scale bar, 10 μm. (2) DNA replication regions seen on prominent fibre of PCCs. (M) early-S-phase and (N) middle S-phase. Replication foci are clearly seen as 'beads on a string' structure, some of these are indicated by arrowhead. Scale bar, 10 μm. (Figure reproduced from Figure 2 of Chromosoma 2007; 116(5):453-462. By Gotoh).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 788

Visualize Dynamic Chromosome Gotoh E

Chromosome condensation/compaction coupled nuclear order of these stable chromosomal units with DNA replication (Sadoni et al., 2004). (ix) Chromatin domains with In the present review, the dynamics of the dimension of replication foci may be chromosomal conformation change, which is tightly fundamental units of chromosomal architecture coupled with DNA replication during S-phase, was (Berezney et al., 2000). (x) DNA replication occurs clearly seen on PCCs of different sub S-phase using at fixed sites and replicated DNA move through drug-induced PCC method (Gotoh and Durante, replication center (Berezney and Coffey, 1975; 2006) and on Cy3-dUTP direct labeling method Pardoll et al., 1980; Hozak et al., 1993). (xi) DNA (McNeil and Warder, 1987). Drug-induced PCC replication contributes to a longitudinal contraction would be, therefore, a useful tool that provides new of the chromosome axis (Hearst et al., 1998). (xii) insights of the dynamics of chromosome formation Functional replication origins are a critical and DNA replication. requirement for longitudinal condensation of the As described in the previous section, number of chromosome axis (Pflumm and Botchan, 2001). accumulated evidences suggested the role of DNA The results presented in this review and previous replication in chromosome findings strongly suggest that DNA replication, condensation/compaction (Pflumm, 2002). As nuclear organization and chromosome condensation previously reported, evidence and results of this are mutually integrated to construct a higher study show that: (i) The different appearance of ordered structure of eukaryote chromosomes. condensation in different sub-phase of S-PCCs is thought to be depended on the different degrees of A hypothetical chromosome compaction model chromosome conformation at the time of PCC coupled with DNA replication induction (Johnson and Rao, 1970; Rao, 1977; Rao Number of models for eukaryote chromosome et al., 1977). In the late or very late S phase, architecture have been proposed (Marsden and particularly, chromosome conformation already Laemmli, 1979; Woodcock et al., 1984; Woodcock changes like mitotic chromosomes (Fig. 1F). (ii) and Dimitrov, 2001; Swedlow and Hirano, 2003; Chromosomes condense asynchronous and the Kireeva et al., 2004), but they are controversial and different degree of condensation depend on the time many aspects are still unclear. In addition, these of chromatin replication (Kuroiwa, 1971). (iii) models do not take account of the involvement of Chromosomes are not fully diffused nor DNA replication/transcription in chromosome nonrandomely positioned in the nucleus, but are packaging. DNA/RNA polymerase are known to be separately compartmentalized in interphase nuclei tightly immobilized to the replication/transcription (Cremer et al., 1993; Ferreira et al., 1997; Berezney factories (Cook, 1999; Frouin et al., 2003). In the et al., 2000). These chromosomes, occupying the proposed model, DNA polymerase is thought to be 'territory', do not intermingle (Hadlaczky et al., a 'reel in DNA template and extrude replicated 1986; Cremer et al., 1993; Swedlow and Hirano, DNA' (Hozak et al., 1996; Cook, 1999) rather than 2003; Cremer et al., 2006; Heard and Bickmore, an enzyme track along DNA template, which is 2007). (iv) Late replication foci were prealigned proposed in many conventional models. In the during interphase. They moved subtly to generate context of Cook's model, some kinds of mechanical recognizable chromosomes presumably due to tension force should be generated in the DNA shortening of the longitudinal chromosome axis template along with DNA replication goes on (Manders et al., 1999). (v) The gross structure of an because the factory is not freely suspended in the interphase chromosome territories is directly related nucleus but attached to nucleoskeleton. to that of the prophase chromosomes (Manders et Consequently, this force may pull and aggregate the al., 1999). (vi) The structure of mitotic replication foci of both sides as to release the chromosomes and the nuclear chromosome tension in DNA strands, which may result in the territories are closely related (Manders et al., 1999) formation of the chromosomes as seen in mitosis. and the different bands of mitotic chromosomes are Based on the above mechanism and the observed presented as distinct domains regarded findings obtained from chromosome structure subchromosomal foci within chromosome dynamics coupled with DNA replication, Fig. 2 territories (Zink et al., 1999). (vii) During the cell shows a hypothetical model for the relationship cycling, the global chromosome territories are between DNA replication and chromosomal conserved. Although some conflicts still remains, conformation changes, and it shows too how the several studies reported that chromosome territories interphase chromatin is constructed into are transmitted through mitosis (Manders et al., chromosomes (Figure reproduced from 1999; Gerlich et al., 2003; Gerlich and Ellenberg, Chromosoma. Gotoh, 2007; 116(5): 453-462). 2003) whereas others reported that positional During the S-phase, chromosomal conformation relations of chromosome territories are lost either at changes and the chromosome formation would be mitosis (Walter et al., 2003) or at early G1 (Essers mostly completed at the end of DNA replication et al., 2005). (viii) The spatio-temporal organization (Fig. 2A,B,C). From G2 to prophase, chromosomes of DNA replication is determined by the specific are still more elastic, less condensed, folded only

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 789

Visualize Dynamic Chromosome Gotoh E

several times and prealigned in interphase nuclei Entering in mitosis, these chromosomes would (Manders et al., 1999). At these phases, the condense even more as shortening the longitudinal chromosomes would be observed as chromosome axis to form solid and rod shape appearance of territories (Cremer et al., 1993; Berezney et al., recognizable mitotic chromosomes (Manders et al., 2000) (Fig. 2D). 1999) (Fig. 2E).

Figure 2: A hypothetical two-dimensional model for chromosome conformational change involving DNA replication based on the models proposed by Cook (Cook, 1995) or Pflumm (Pflumm, 2002). (A) Early S-phase. DNA replication starts at multiple origins and proceeds bi-directionally. Early S-PCCs are seen as 'beads on a string' appearance. (B) Middle S-phase. As DNA replication proceeds, replicated DNA pass through replication factory and some tension are generated. The generated tension may pull back the replication factories close together so as to release the tension. Replication factories may in turn fuse together and chromosomes compact. Middle S-PCCs are seen as well known 'pulverized chromosomes' appearance. (C) Late S-phase. Most of DNA finished replication and conformation was changed. Late S-PCCs are seen as 'tandem band arrayed structured chromosomes' like as mitotic chromosomes. (D) G2 to prophase. After finishing of DNA replication, chromosome conformation changed like as mitotic chromosomes, but still so elastic that packed in nucleus. Before fixation, each chromosome occupies individual chromosome territory (CT) in interphase nucleus, thus observed as compartment regions (colorized). (E) Mitosis. After prophase, chromosomes further shortening in longitudinal axis of chromosomes, consequently a straight rod shaped recognizable chromosome formed as usually seen by cytologists under a microscope. For simplicity, the model is shown as two- dimensional and the scaling is arbitrary. The model intends not to depict actual events of chromosome conformation change but to help imagine how DNA replication is involved in chromosomal conformation. As the real chromosomes condense as three- dimensionally, other elements such as coiling and helical winding should be considered together to construct a stereoscopic hierarchical structure of eukaryote chromosomes (Woodcock and Dimitrov, 2001; Swedlow and Hirano, 2003). (Figure reproduced from Figure 3 of Chromosoma 2007; 116(5):453-462. By Gotoh).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 790

Visualize Dynamic Chromosome Gotoh E

Summary and Conclusion Olins AL, Olins DE. Spheroid chromatin units (v bodies). A basic and principle question of cell biology is: Science. 1974 Jan 25;183(4122):330-2 how DNA folds to chromosome? Numbers of Sperling K, Rao PN. The phenomenon of premature evidence have suggested the involvement of DNA chromosome condensation: its relevance to basic and replication in chromosome structure formation. To applied research. Humangenetik. 1974;23(4):235-58 visualize the dynamics of chromosome structure Berezney R, Coffey DS. Nuclear protein matrix: formation coupled with DNA replication, Cy3- association with newly synthesized DNA. Science. 1975 Jul 25;189(4199):291-3 dUTP direct-labeled active replicating DNA was observed in prematurely condensed chromosomes Edenberg HJ, Huberman JA. Eukaryotic chromosome (PCCs) utilized with drug-induced premature replication. Annu Rev Genet. 1975;9:245-84 chromosome condensation technique, which Hittelman WN, Rao PN. Premature chromosome facilitates the visualization of interphase chromatin condensation. Conformational changes of chromatin associated with phytohemagglutinin stimulation of as well as the condensed chromosome form. S- peripheral lymphocytes. Exp Cell Res. 1976 phase PCCs revealed clearly the drastic dynamic Jul;100(2):219-22 transition of chromosome formation during S-phase Rao PN.. Premature chromosome condensation and the along with the progress of DNA replication: from a fine structure of chromosomes. In Molecular Structure of 'cloudy nebula' structure in early S-phase to Human Chromosomes (ed Yunis JJ) Volume 1977. numerous number of 'beads on a string' in middle S- Academic Press, New York, p 205-231. phase and finally to 'striped arrays of banding Rao PN, Wilson B, Puck TT.. Premature chromosome structured chromosome' in the late S-phase as usual condensation and cell cycle analysis. J Cell Physiol. 1977 observed in mitotic chromosomes. The drug- Apr;91(1):131-41. induced PCC is clearly provided a new insight that Hand R.. Eucaryotic DNA: organization of the genome for the eukaryote DNA replication is tightly coupled replication. Cell. 1978 Oct;15(2):317-25. with the dynamics of chromosome Marsden MP, Laemmli UK.. Metaphase chromosome condensation/compaction for the construction of structure: evidence for a radial loop model. Cell. 1979 eukaryote higher ordered chromosome structure. Aug;17(4):849-58. Based on these findings, a hypothetical model for Thoma F, Koller T, Klug A.. Involvement of histone H1 in chromosome compaction involved the role of DNA the organization of the nucleosome and of the salt- replication is proposed. In this model, dependent superstructures of chromatin. J Cell Biol. 1979 conformational change is simply illustrated as two- Nov;83(2 Pt 1):403-27. dimensional but the real architecture is a three- Hanks SK, Rao PN.. Initiation of DNA synthesis in the dimensionally chromosome constructs, with much prematurely condensed chromosomes of G1 cells. J Cell Biol. 1980 Oct;87(1):285-91. more complex fashion. It is mostly unclear how DNA replication/transcription conducts to make up Mullinger AM, Johnson RT.. Packing DNA into a three-dimensional hierarchical structure of chromosomes. J Cell Sci. 1980 Dec;46:61-86. chromosomes coupled with Pardoll DM, Vogelstein B, Coffey DS.. A fixed site of DNA twisting/folding/winding or other factors. It should replication in eucaryotic cells. Cell. 1980 Feb;19(2):527-36. be a most principle challenge in cell science. Lau YF, Arrighi FE.. Studies of mammalian chromosome replication. II. Evidence for the existence of defined chromosome replicating units. Chromosoma. References 1981;83(5):721-41. Johnson RT, Rao PN. Mammalian cell fusion: induction of Mullinger AM, Johnson RT.. Units of chromosome premature chromosome condensation in interphase nuclei. replication and packing. J Cell Sci. 1983 Nov;64:179-93. Nature. 1970 May 23;226(5247):717-22 Pantelias GE, Maillie HD.. A simple method for premature Johnson RT, Rao PN, Hughes HD. Mammalian cell fusion. chromosome condensation induction in primary human 3. A HeLa cell inducer of premature chromosome and rodent cells using polyethylene glycol. Somatic Cell condensation active in cells from a variety of animal Genet. 1983 Sep;9(5):533-47. species. J Cell Physiol. 1970 Oct;76(2):151-7 Gollin SM, Wray W, Hanks SK, Hittelman WN, Rao PN.. Rao PN, Johnson RT. Mammalian cell fusion: studies on The ultrastructural organization of prematurely condensed the regulation of DNA synthesis and mitosis. Nature. 1970 chromosomes. J Cell Sci Suppl. 1984;1:203-21. Jan 10;225(5228):159-64 Hameister H, Sperling K.. Description of a chromosome Kuroiwa T. Asynchronous condensation of chromosomes replication unit in individual prematurely condensed human from early prophase to late prophase as revealed by S-phase chromosomes. Chromosoma. 1984;90(5):389-93. electron microscopic autoradiography. Exp Cell Res. 1971 Nov;69(1):97-105 Savage JR, Prasad R, Papworth DG.. Subdivision of S- phase and its use for comparative purposes in cultured Fakan S, Hancock R. Localization of newly-synthesized human cells. J Theor Biol. 1984 Nov 21;111(2):355-67. DNA in a mammalian cell as visualized by high resolution autoradiography. Exp Cell Res. 1974 Jan;83(1):95-102 Woodcock CL, Frado LL, Rattner JB.. The higher-order structure of chromatin: evidence for a helical ribbon Masui Y. A cytostatic factor in amphibian oocytes: its arrangement. J Cell Biol. 1984 Jul;99(1 Pt 1):42-52. extraction and partial characterization. J Exp Zool. 1974 Jan;187(1):141-7 Hadlaczky G, Went M, Ringertz NR.. Direct evidence for the non-random localization of mammalian chromosomes

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 791

Visualize Dynamic Chromosome Gotoh E

in the interphase nucleus. Exp Cell Res. 1986 phosphorylation and subunit rearrangement. Cell. 1988 Jul Nov;167(1):1-15. 1;54(1):17-26. Nakamura H, Morita T, Sato C.. Structural organizations of Durante M, Furusawa Y, Gotoh E.. A simple method for replicon domains during DNA synthetic phase in the simultaneous interphase-metaphase chromosome analysis mammalian nucleus. Exp Cell Res. 1986 Aug;165(2):291- in biodosimetry. Int J Radiat Biol. 1998 Oct;74(4):457-62; 7. Hearst JE, Kauffman L, McClain WM.. A simple McNeil PL, Warder E.. Glass beads load macromolecules mechanism for the avoidance of entanglement during into living cells. J Cell Sci. 1987 Dec;88 ( Pt 5):669-78. chromosome replication. Trends Genet. 1998 Jun;14(6):244-7. (REVIEW) Dunphy WG, Brizuela L, Beach D, Newport J.. The Xenopus cdc2 protein is a component of MPF, a Ma H, Samarabandu J, Devdhar RS, Acharya R, Cheng cytoplasmic regulator of mitosis. Cell. 1988 Jul PC, Meng C, Berezney R.. Spatial and temporal dynamics 29;54(3):423-31. of DNA replication sites in mammalian cells. J Cell Biol. 1998 Dec 14;143(6):1415-25. Mills AD, Blow JJ, White JG, Amos WB, Wilcock D, Laskey RA.. Replication occurs at discrete foci spaced throughout Zink D, Cremer T, Saffrich R, Fischer R, Trendelenburg nuclei replicating in vitro. J Cell Sci. 1989 Nov;94 ( Pt MF, Ansorge W, Stelzer EH.. Structure and dynamics of 3):471-7. human interphase chromosome territories in vivo. Hum Genet. 1998 Feb;102(2):241-51. Nakayasu H, Berezney R.. Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol. 1989 Jan;108(1):1- Cook PR.. The organization of replication and 11. transcription. Science. 1999 Jun 11;284(5421):1790-5. (REVIEW) O'Keefe RT, Henderson SC, Spector DL.. Dynamic organization of DNA replication in mammalian cell nuclei: Gotoh E, Kawata T, Durante M.. Chromatid break rejoining spatially and temporally defined replication of and exchange aberration formation following gamma-ray chromosome-specific alpha-satellite DNA sequences. J exposure: analysis in G2 human fibroblasts by chemically Cell Biol. 1992 Mar;116(5):1095-110. induced premature chromosome condensation. Int J Radiat Biol. 1999 Sep;75(9):1129-35. Kumagai A, Dunphy WG.. Regulation of the cdc25 protein during the cell cycle in Xenopus extracts. Cell. 1992 Jul Johnson RT, Gotoh E, Mullinger AM, Ryan AJ, Shiloh Y, 10;70(1):139-51. Ziv Y, Squires S.. Targeting double-strand breaks to replicating DNA identifies a subpathway of DSB repair that Cremer T, Kurz A, Zirbel R, Dietzel S, Rinke B, Schrock E, is defective in ataxia-telangiectasia cells. Biochem Biophys Speicher MR, Mathieu U, Jauch A, Emmerich P, Res Commun. 1999 Aug 2;261(2):317-25. Scherthan H, Ried T, Cremer C, Lichter P.. Role of chromosome territories in the functional Manders EM, Kimura H, Cook PR.. Direct imaging of DNA compartmentalization of the cell nucleus. Cold Spring Harb in living cells reveals the dynamics of chromosome Symp Quant Biol. 1993;58:777-92. formation. J Cell Biol. 1999 Mar 8;144(5):813-21. Hozak P, Hassan AB, Jackson DA, Cook PR.. Zink D, Bornfleth H, Visser A, Cremer C, Cremer T.. Visualization of replication factories attached to Organization of early and late replicating DNA in human nucleoskeleton. Cell. 1993 Apr 23;73(2):361-73. chromosome territories. Exp Cell Res. 1999 Feb 25;247(1):176-88. Cook PR.. A chromomeric model for nuclear and chromosome structure. J Cell Sci. 1995 Sep;108 ( Pt Berezney R, Dubey DD, Huberman JA.. Heterogeneity of 9):2927-35. (REVIEW) eukaryotic replicons, replicon clusters, and replication foci. Chromosoma. 2000 Mar;108(8):471-84. (REVIEW) Gotoh E, Asakawa Y, Kosaka H.. Inhibition of protein serine/threonine phophatases directly induces premature Loupart ML, Krause SA, Heck MS.. Aberrant replication chromosome condensation in mammalian somatic cells. timing induces defective chromosome condensation in Biomedical Research (Tokyo).1995; 16(1):63-68. Drosophila ORC2 mutants. Curr Biol. 2000 Dec 14- 28;10(24):1547-56. Gotoh E, Asakawa Y.. Detection and evaluation of chromosomal aberrations induced by high doses of Maiorano D, Moreau J, Mechali M.. XCDT1 is required for gamma-irradiation using immunogold-silver painting of the assembly of pre-replicative complexes in Xenopus prematurely condensed chromosomes. Int J Radiat Biol. laevis. Nature. 2000 Apr 6;404(6778):622-5. 1996 Nov;70(5):517-20. Moir RD, Yoon M, Khuon S, Goldman RD.. Nuclear lamins Hozak P, Jackson DA, Cook PR.. The role of nuclear A and B1: different pathways of assembly during nuclear structure in DNA replication. In Eukaryotic DNA envelope formation in living cells. J Cell Biol. 2000 Dec Replication: Frontiers in Molecular Biology (ed Blow JJ) 11;151(6):1155-68. Volume 1996.Oxford University Press, Oxford, p 124-142. Nishitani H, Lygerou Z, Nishimoto T, Nurse P.. The Cdt1 Manders EM, Stap J, Strackee J, van Driel R, Aten JA.. protein is required to license DNA for replication in fission Dynamic behavior of DNA replication domains. Exp Cell yeast. Nature. 2000 Apr 6;404(6778):625-8. Res. 1996 Aug 1;226(2):328-35. Marheineke K, Hyrien O.. Aphidicolin triggers a block to Asakawa Y, Gotoh E.. A method for detecting sister replication origin firing in Xenopus egg extracts. J Biol chromatid exchanges using prematurely condensed Chem. 2001 May 18;276(20):17092-100. chromosomes and immunogold-silver staining. Mutagenesis. 1997 May;12(3):175-7. Pflumm MF, Botchan MR.. Orc mutants arrest in metaphase with abnormally condensed chromosomes. Ferreira J, Paolella G, Ramos C, Lamond AI.. Spatial Development. 2001 May;128(9):1697-707. organization of large-scale chromatin domains in the nucleus: a magnified view of single chromosome Woodcock CL, Dimitrov S.. Higher-order structure of territories. J Cell Biol. 1997 Dec 29;139(7):1597-610. chromatin and chromosomes. Curr Opin Genet Dev. 2001 Apr;11(2):130-5. (REVIEW) Draetta G, Beach D.. Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 792

Visualize Dynamic Chromosome Gotoh E

Bell SP, Dutta A.. DNA replication in eukaryotic cells. Annu dynamics of PCNA in DNA replication and repair. Mol Cell Rev Biochem. 2002;71:333-74. (REVIEW) Biol. 2005 Nov;25(21):9350-9. Ito S, Gotoh E, Ozawa S, Yanagi K.. Epstein-Barr virus Gotoh E, Tanno Y.. Simple biodosimetry method for cases nuclear antigen-1 is highly colocalized with interphase of high-dose radiation exposure using the ratio of the chromatin and its newly replicated regions in particular. J longest/shortest length of Giemsa-stained drug-induced Gen Virol. 2002 Oct;83(Pt 10):2377-83. prematurely condensed chromosomes (PCC). Int J Radiat Biol. 2005 May;81(5):379-85. Pflumm MF.. The role of DNA replication in chromosome condensation. Bioessays. 2002 May;24(5):411-8. Gotoh E, Tanno Y, Takakura K.. Simple biodosimetry (REVIEW) method for use in cases of high-dose radiation exposure that scores the chromosome number of Giemsa-stained Samaniego R, de la Torre C, Moreno Diaz de la Espina S.. drug-induced prematurely condensed chromosomes Dynamics of replication foci and nuclear matrix during S (PCC). Int J Radiat Biol. 2005 Jan;81(1):33-40. phase in Allium cepa L. cells. Planta. 2002 Jun;215(2):195- 204. Sangrithi MN, Bernal JA, Madine M, Philpott A, Lee J, Dunphy WG, Venkitaraman AR.. Initiation of DNA Christensen TW, Tye BK.. Drosophila MCM10 interacts replication requires the RECQL4 protein mutated in with members of the prereplication complex and is Rothmund-Thomson syndrome. Cell. 2005 Jun required for proper chromosome condensation. Mol Biol 17;121(6):887-98. Cell. 2003 Jun;14(6):2206-15. Srebniak MI, Trapp GG, Wawrzkiewicz AK, Kaz'mierczak Frouin I, Montecucco A, Spadari S, Maga G.. DNA W, Wiczkowski AK.. The usefulness of calyculin a for replication: a complex matter. EMBO Rep. 2003 cytogenetic prenatal diagnosis. J Histochem Cytochem. Jul;4(7):666-70. (REVIEW) 2005 Mar;53(3):391-4. Gerlich D, Beaudouin J, Kalbfuss B, Daigle N, Eils R, Terzoudi GI, Manola KN, Pantelias GE, Iliakis G.. Ellenberg J.. Global chromosome positions are transmitted Checkpoint abrogation in G2 compromises repair of through mitosis in mammalian cells. Cell. 2003 Mar chromosomal breaks in ataxia telangiectasia cells. Cancer 21;112(6):751-64. Res. 2005 Dec 15;65(24):11292-6. Gerlich D, Ellenberg J.. Dynamics of chromosome Cremer T, Cremer M, Dietzel S, Muller S, Solovei I, Fakan positioning during the cell cycle. Curr Opin Cell Biol. 2003 S.. Chromosome territories--a functional nuclear Dec;15(6):664-71. (REVIEW) landscape. Curr Opin Cell Biol. 2006 Jun;18(3):307-16. McHugh B, Heck MM.. Regulation of chromosome (REVIEW) condensation and segregation. Curr Opin Genet Dev. 2003 Gotoh E, Durante M.. Chromosome condensation outside Apr;13(2):185-90. (REVIEW) of mitosis: mechanisms and new tools. J Cell Physiol. Swedlow JR, Hirano T.. The making of the mitotic 2006 Nov;209(2):297-304. (REVIEW) chromosome: modern insights into classical questions. Mol Kitajima TS, Sakuno T, Ishiguro K, Iemura S, Natsume T, Cell. 2003 Mar;11(3):557-69. (REVIEW) Kawashima SA, Watanabe Y.. Shugoshin collaborates with Terzoudi GI, Malik SI, Pantelias GE, Margaritis K, Manola protein phosphatase 2A to protect cohesin. Nature. 2006 K, Makropoulos W.. A new cytogenetic approach for the May 4;441(7089):46-52. evaluation of mutagenic potential of chemicals that induce Tanno Y, Kobayashi K, Tatsuka M, Gotoh E, Takakura K.. cell cycle arrest in the G2 phase. Mutagenesis. 2003 Mitotic arrest caused by an X-ray microbeam in a single Nov;18(6):539-43. cell expressing EGFP-aurora kinase B. Radiat Prot Walter J, Schermelleh L, Cremer M, Tashiro S, Cremer T.. Dosimetry. 2006;122(1-4):301-6. Chromosome order in HeLa cells changes during mitosis Tatsumi Y, Sugimoto N, Yugawa T, Narisawa-Saito M, and early G1, but is stably maintained during subsequent Kiyono T, Fujita M.. Deregulation of Cdt1 induces interphase stages. J Cell Biol. 2003 Mar 3;160(5):685-97. chromosomal damage without rereplication and leads to D'Antoni S, Mattina T, Di Mare P, Federico C, Motta S, chromosomal instability. J Cell Sci. 2006 Aug 1;119(Pt Saccone S.. Altered replication timing of the HIRA/Tuple1 15):3128-40. locus in the DiGeorge and Velocardiofacial syndromes. Deckbar D, Birraux J, Krempler A, Tchouandong L, Gene. 2004 May 26;333:111-9. Beucher A, Walker S, Stiff T, Jeggo P, Lobrich M.. Kireeva N, Lakonishok M, Kireev I, Hirano T, Belmont AS.. Chromosome breakage after G2 checkpoint release. J Cell Visualization of early chromosome condensation: a Biol. 2007 Mar 12;176(6):749-55. hierarchical folding, axial glue model of chromosome Heard E, Bickmore W.. The ins and outs of gene regulation structure. J Cell Biol. 2004 Sep 13;166(6):775-85. and chromosome territory organisation. Curr Opin Cell Prasanth SG, Prasanth KV, Siddiqui K, Spector DL, Biol. 2007 Jun;19(3):311-6. (REVIEW) Stillman B.. Human Orc2 localizes to centrosomes, Gotoh E.. Visualizing the dynamics of chromosome centromeres and heterochromatin during chromosome structure formation coupled with DNA replication. inheritance. EMBO J. 2004 Jul 7;23(13):2651-63. Chromosoma. 2007 Oct;116(5):453-62. Sadoni N, Cardoso MC, Stelzer EH, Leonhardt H, Zink D.. Pruitt SC, Bailey KJ, Freeland A. Reduced Mcm2 Stable chromosomal units determine the spatial and expression results in severe stem/progenitor cell deficiency temporal organization of DNA replication. J Cell Sci. 2004 and cancer. Stem Cells. 2007 Dec;25(12):3121-32. Oct 15;117(Pt 22):5353-65. Shima N, Alcaraz A, Liachko I, Buske TR, Andrews CA, El Achkar E, Gerbault-Seureau M, Muleris M, Dutrillaux B, Munroe RJ, Hartford SA, Tye BK, Schimenti JC.. A viable Debatisse M.. Premature condensation induces breaks at allele of Mcm4 causes chromosome instability and the interface of early and late replicating chromosome mammary adenocarcinomas in mice. Nat Genet. 2007 bands bearing common fragile sites. Proc Natl Acad Sci U Jan;39(1):93-8. S A. 2005 Dec 13;102(50):18069-74. Beucher A, Birraux J, Tchouandong L, Barton O, Shibata Essers J, Theil AF, Baldeyron C, van Cappellen WA, A, Conrad S, Goodarzi AA, Krempler A, Jeggo PA, Lobrich Houtsmuller AB, Kanaar R, Vermeulen W.. Nuclear

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 793

Visualize Dynamic Chromosome Gotoh E

M.. ATM and Artemis promote homologous recombination Masai H, Matsumoto S, You Z, Yoshizawa-Sugata N, Oda of radiation-induced DNA double-strand breaks in G2. M.. Eukaryotic chromosome DNA replication: where, when, EMBO J. 2009 Nov 4;28(21):3413-27. and how? Annu Rev Biochem. 2010;79:89-130. (REVIEW) Burkard ME, Maciejowski J, Rodriguez-Bravo V, Repka M, Meyer H, Drozdowska A, Dobrynin G.. A role for Lowery DM, Clauser KR, Zhang C, Shokat KM, Carr SA, Cdc48/p97 and Aurora B in controlling chromatin Yaffe MB, Jallepalli PV.. Plk1 self-organization and priming condensation during exit from mitosis. Biochem Cell Biol. phosphorylation of HsCYK-4 at the spindle midzone 2010 Feb;88(1):23-8. (REVIEW) regulate the onset of division in human cells. PLoS Biol. 2009 May 5;7(5):e1000111. Tanno Y, Kitajima TS, Honda T, Ando Y, Ishiguro K, Watanabe Y.. Phosphorylation of mammalian Sgo2 by Chang P, Coughlin M, Mitchison TJ.. Interaction between Aurora B recruits PP2A and MCAK to centromeres. Genes Poly(ADP-ribose) and NuMA contributes to mitotic spindle Dev. 2010 Oct 1;24(19):2169-79. pole assembly. Mol Biol Cell. 2009 Nov;20(21):4575-85. Torres JZ, Ban KH, Jackson PK.. A specific form of Haren L, Gnadt N, Wright M, Merdes A.. NuMA is required phospho protein phosphatase 2 regulates anaphase- for proper spindle assembly and chromosome alignment in promoting complex/cyclosome association with spindle prometaphase. BMC Res Notes. 2009 Apr 28;2:64. poles. Mol Biol Cell. 2010 Mar;21(6):897-904. Gotoh E.. Drug-induced premature chromosome Tsukahara T, Tanno Y, Watanabe Y.. Phosphorylation of condensation (PCC) protocols: cytogenetic approaches in the CPC by Cdk1 promotes chromosome bi-orientation. mitotic chromosome and interphase chromatin. Methods Nature. 2010 Oct 7;467(7316):719-23. Mol Biol. 2009;523:83-92. van Harn T, Foijer F, van Vugt M, Banerjee R, Yang F, Pliss A, Malyavantham K, Bhattacharya S, Zeitz M, Oostra A, Joenje H, te Riele H.. Loss of Rb proteins Berezney R.. Chromatin dynamics is correlated with causes genomic instability in the absence of mitogenic replication timing. Chromosoma. 2009 Aug;118(4):459-70. signaling. Genes Dev. 2010 Jul 1;24(13):1377-88. Silk AD, Holland AJ, Cleveland DW.. Requirements for Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD.. NuMA in maintenance and establishment of mammalian Molecular Biology of The Cell. Garland Publishing Inc. spindle poles. J Cell Biol. 2009 Mar 9;184(5):677-90. New York 1219 pp.

Wolfe BA, Takaki T, Petronczki M, Glotzer M.. Polo-like This article should be referenced as such: kinase 1 directs assembly of the HsCyk-4 RhoGAP/Ect2 RhoGEF complex to initiate cleavage furrow formation. Gotoh E. Visualize Dynamic Chromosome. Atlas Genet PLoS Biol. 2009 May 5;7(5):e1000110. Cytogenet Oncol Haematol. 2011; 15(9):785-794. Li J, Wang J, Jiao H, Liao J, Xu X.. Cytokinesis and cancer: Polo loves ROCK'n' Rho(A). J Genet Genomics. 2010 Mar;37(3):159-72. (REVIEW)

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 794

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Deep Insight Section

NK cell receptors: evolution and diversity Gwenoline Borhis, Salim I Khakoo Department of Hepatology, Division of Medicine, Imperial College London, UK (GB, SIK)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Deep/NKCellRecEvoDivID20095.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI NKCellRecEvoDivID20095.txt

shifts the balance towards NK cell activation and Summary target cell destruction by removing this inhibitory Natural Killer cell functions are regulated by signal. Thus in health NK cells are tolerant towards combinations of activating and inhibitory receptors, host cells, but in disease this tolerance can be derived from a number of different gene families. readily broken. This review focuses on receptors for MHC class I, which include the killer cell immunoglobulin-like MHC class I receptors on NK receptors (KIR) and the CD94:NKG2 family of cells receptors. In particular the KIR are diverse and MHC class I receptors on NK cells can be either rapidly co-evolving with their classical MHC class inhibitory or activating. The inhibitory receptors for I ligands. Thus NK cells are part of the innate MHC class I regulate NK cell function by immune system that are continuing to adapt to the generating a tonic inhibitory signal as hypothesized challenges of pathogens. in the "missing-self" model (Ljunggren and Karre, Introduction 1990). The role of the activating receptors for MHC class I appears less clear, but genetic studies have NK cells are an important component of the innate implicated them in recognition of virally infected immune system, which participate in the early cells. immune defence against intracellular pathogens and Several inhibitory receptors have been identified, tumour transformation. They were originally but there are two main families involved in NK defined by their ability to spontaneously eliminate regulation by MHC class I: the Killer cell rare cells lacking expression of class I Major Immunoglobulin-like Receptors (KIR) and the C- Histocompatibility Complex (MHC class I) self type lectin-like CD94/NKG2A heterodimers. KIR molecules, a process commonly referred to as interact with the classical MHC class Ia (HLA-A, - "missing self" recognition (Biron et al., 1999; Purdy B and -C) while CD94/NKG2A recognizes the non- and Campbell, 2009). classical MHC class Ib, HLA-E. Both synergize Upon activation, NK cells can mediate direct and permit NK cells to sense and respond to cytotoxicity or secrete cytokines and chemokine changes in MHC class I expression. These receptors that modulate subsequent steps in the adaptive are expressed in a combinatorial fashion on NK immune response. These functions are regulated by cells to generate an NK cell repertoire. The the combination of signals from activating and importance of this is gradually being realised. inhibitory receptors (Lanier, 1998). The MHC class Expression of an MHC class I inhibitory receptor I receptors are particularly important for NK cells appears to confer additional reactivity on these NK to discriminate "self" (healthy cells) from "altered- cells, a phenomenon originally termed "licensing" self" (infected- and transformed-cells) or "missing (Kim et al., 2005). Thus NK cells without self". MHC class I receptor:ligand interactions can inhibitory receptors for MHC class I are thought to induce inhibitory signals that counteract activating be relatively hypofunctional, although in specific receptor signals and lead to NK cell inhibition. In scenarios, these cells can become important for contrast down-regulation or loss of MHC class I viral eradication as shown by studies in murine expression, during viral infection or carcinogenesis,

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 795

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

CMV infection (Orr et al., 2010). Furthermore in activation leads to the suppression of activating disease states in which a specific inhibitory receptor receptor signals (Long, 2008). Activating KIR may be beneficial, such as KIR2DL3 in hepatitis C possess a positively charged residue (usually virus infection, then individuals with more arginine) in the transmembrane region, which beneficial repertoires may be more likely to clear facilitate the association with accessory molecules, infection (Alter et al., 2010). DAP12 or FcεRIγ (KIR2DL4) and NK cell In addition to these key receptors, members of the activation (cytotoxicity and/or cytokine production) Ig-Like Transcripts (ILTs) family, which are (MacFarlane and Campbell, 2006). The exception, genetically related to KIR (Wilson et al., 2000), can KIR2DL4 contains both ITIMs and a positively also recognize MHC class I. For example, LILRB1 charged residue (lysine), which facilitates the (LIR-1, ILT-2), which binds a broad range of MHC association with FcεRIγ and the induction of the class I molecules including HLA-G (Vitale et al., activating signals (Kikuchi-Maki et al., 2005). 1999), is able to inhibit the NK cell line NK92 The KIR genes can be divided into six lineages (Kirwan and Burshtyn, 2005). It is also expressed in based on phylogenetic analysis. This has allowed a variegated fashion on NK cells (Davidson et al., "tracking" of the KIR across species and given 2010). However it does not appear to play a part in insights into the evolution of the KIR gene families. NK cell education and it has yet to be demonstrated Lineage I KIR have two extracellular domains in that this gene family play a major role in inhibiting the D0D2 conformation; lineage II KIR are specific NK cells in vivo (Yawata et al., 2008). for MHC-A and -B; lineage III KIR bind HLA-C; CD94/NKG2A and KIR molecules have adopted lineage V KIR are related to the human KIR two different recognition strategies. To a large framework gene KIR3DL3; and lineage IV and VI extent CD94/NKG2A ignore MHC class I diversity KIR are expansions specific to the rhesus macaque by recognizing HLA-E. This non-polymorphic and new world monkeys respectively. MHC class I molecule binds leader peptide sequences derived from classical MHC-A, -B and - KIR locus and diversity C molecules and also from HLA-G (Llano et al., Genotyping of individuals for specific KIR genes 1998). In contrast the KIR family embrace the demonstrated an unexpected diversity in gene diversity of MHC class I through direct recognition content amongst the population (Uhrberg et al., of polymorphic determinants. This strategy leads to 1997). The genotype of these individuals correlated a highly variable and polymorphic KIR system with with expression of KIR genes thus demonstrating diversity comparable to that of MHC class I that this genetic diversity would be important for (Valiante et al., 1997a). NK cell function. This seminal study started a detailed investigation into KIR genetics. KIR structure and signalling Sequencing of two KIR haplotypes from a single function individual showed that the KIR are encoded by a The KIR family (assigned the designation of compact family of genes which occupy about 150 CD158) is a member of the immunoglobulin kb of the Leukocyte Receptor Complex (LRC) on superfamily that comprises 15 expressed receptors chromosome 19q13.4 (Wilson et al., 2000; Wende (KIRDL1-5B, KIR3DL1-3, KIR2DS1-5 and et al., 1999). The locus is flanked by the LILR and KIR3DS1): which can be either inhibitory or the FCAR genes and contains up to 17 KIR genes activating (Table 1). All KIR are type I and pseudogenes (Kelley et al., 2005). Because the transmembrane glycoproteins formed from either KIR genes have high sequence homology to each two (KIR2D) or three (KIR3D) extracellular Ig-like other (90-95%) and are closely distributed within domains, a stem region, a transmembrane region the LRC, they have been proposed to evolve by and a cytoplasmic tail. Depending on the length of non-allelic homologous recombination (Carrington the cytoplasmic tail KIR can be subdivided into and Cullen, 2004). This mechanism could explain long-tailed and short-tailed receptors. In general the expansion and contraction of the KIR locus and these structural characteristics correlate with their provide a basis for the substantial diversity function. Long-tailed KIR are generally inhibitory observed (Hsu et al., 2002a; Hsu et al., 2002b; and short-tailed KIR are activating (Vilches and Shilling et al., 2002; Uhrberg et al., 2002; Whang et Parham, 2002). An exception to this rule is the al., 2005; Martin et al., 2003). receptor KIR2DL4 which has a long Haplotypic diversity intracytoplasmic tail but stimulates potent cytokine The number of putatively expressed KIR genes production, although only minimal cytotoxicity usually ranges from 7 to 12, depending primarily on (Rajagopalan et al., 2006). the presence or absence of activating KIR loci Inhibitory KIR contain one or two Immunoreceptor (Wilson et al., 2000; Uhrberg et al, 2002; Witt et Tyrosine-based Inhibitory Motifs (ITIMs; al., 1999). This variation in gene content is one V/I/LxYxxL/V), which are require for NK cell component of KIR diversity. Despite this extreme inhibition via recruitment of the protein tyrosine variability some systematic features are conserved phosphatases SHP-1 and SHP-2. SHP-1/2 in the organisation of the KIR locus. Four KIR genes, KIR3DL3, KIR3DP1, KIR2DL4 and

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 796

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

KIR3DL2, are found in all individuals and have sections a centromeric section (KIR3DL3- been named framework loci (Bashirova et al., KIR3DP1) and a telomeric section (KIR2DL4- 2006). KIR3DL3 and KIR3DL2 define the ends of KIR3DL2) which appear to have diversified the KIR-gene region and KIR3DP1-KIR2DL4, the independently (Pyo et al., 2010). Sequence analysis middle. Regions of genetic variability are located of a number of KIR haplotypes shows that allelic between KIR3DL3 and KIR3DP1, and between diversity of the centromeric section is predominant KIR2DL4 and KIR3DL2 (Wilson et al, 2000; Martin in the A haplotypes, but it is in the telomeric et al., 2000). section of the B haplotypes that allelic diversity is Two distinct forms of haplotype, termed A and B, most noticeable. can be distinguished on the basis of gene content. The distribution of A and B haplotypes varies Haplotype A has a fixed gene content (KIR2DL1, widely between distinct ethnic groups. The A and B KIR2DL3, KIR2DL4, KIR2DS4, KIR3DL1, haplotype frequencies are relatively even in KIR3DL2, KIR3DP1 and KIR3DL3) (Uhrberg et al., Caucasian populations (Uhrberg et al., 1997; Hsu et 1997) and fewer genes than B haplotype but the al., 2002b). However the A haplotype dominates in most functionally relevant distinction between these the Korean, Japanese and Han Chinese populations two haplotypes is the number of activating with an approximate 75% frequency (Whang et al., receptors. Haplotype A contains only a single 2005; Yawata et al., 2002b; Jiang et al., 2005) as activating KIR gene, KIR2DS4, whereas haplotype compared to the Australian Aborigines, who have a B contains various combinations of KIR2DS1, very low frequency of the A haplotypes of about KIR2DS2, KIR2DS3, KIR2DS5, KIR3DS1 and 13% (Toneva et al., 2001). These differences may KIR2DS4. Furthermore, the KIR2DS4 gene has a reflect both founder effects and selection by null allele with a population frequency of about pathogens and may account for some variation in 84% (Maxwell et al., 2002), thus some homozygous worldwide disease susceptibility. A haplotype individuals don't express any Allelic polymorphism activating KIR (Hsu et al., 2002b). Although A Point mutation and homologous recombination haplotypes are fixed in term of the number and type generate allelic polymorphism (Table 1) (Norman of genes present, they show extensive allelic et al., 2009; Shilling et al., 1998). This allelic variation at several of the genes. In contrast to A polymorphism gives an additional dimension to haplotype, B haplotype displays a much greater KIR diversity in that unrelated individuals are variety of gene contents. Based on segregation unlikely to have identical KIR alleles, similar to the analysis, more than 20 different B haplotypes have situation for MHC diversity (Gardiner et al., 2001). been described (Hsu et al., 2002a; Yawata et al., Allelic polymorphism has been described for all the 2002a). These haplotypes contain various inhibitory KIR genes and names for alleles at combinations of KIR genes, including several several of the most polymorphic loci have been activating KIR but there is a very high linkage specified based on nomenclature used for HLA loci disequilibrium (LD) between many pairs of genes, (Shilling et al., 2002). This polymorphism as KIR2DL1/KIR2DL3 or KIR3DL1/KIR3DL2 significantly influences their ligand affinities and alleles (Uhrberg et al., 1997; Shilling et al., 2002; levels of cell surface expression. For example, Witt et al., 1999; Norman et al., 2002; Norman et distinct alleles of KIR3DL1, one of the most al., 2001; Toneva et al., 2001; Crum et al., 2000). polymorphic KIR genes encode molecules that However despite the broad categorizations, there appear to be expressed at different levels on the are several exceptions to these simple rules. For surface of NK cells (Gardiner et al., 2001; Yawata instance despite the fact that KIR3DL1 (inhibitory) et al., 2006; Pando et al., 2003). Moreover this and KIR3DS1 (activating) segregate as alleles of a allelic variability can occur at positions encoding single locus in the vast majority of individuals, residues that affect interaction with HLA class I haplotypes have been described in which they occur (Boyington et al., 2000; Fan et al., 2001) and on the same chromosome (Martin et al., 2008; influences both the binding affinity and the Norman et al., 2009). Similarly some KIR inhibitory capacity. Similarly the genes KIR2DL2 haplotypes have fewer than expected KIR. For and KIR2DL3 also segregate as alleles of a single instance a recently described haplotype contains locus and although they have broadly similar MHC only 3 KIR genes KIR3DL3, KIR2DS1 and class I specificity, bind their ligands with KIR3DL2 (Traherne et al., 2010). Thus the rules for substantially different avidities (Moesta et al., the KIR locus appear unusually flexible, perhaps 2008). The synergy of haplotype variability and, due to the combination of a high sequence allelic polymorphism has generated substantial homology between the genes, and an overlap in diversity across both individual populations, but function of these receptors with the well conserved also across different ethnic groups (Rajalingam et CD94:NKG2 family of receptors. Furthermore al., 2001). This diversity is likely driven by both some genes (KIR2DS3 and KIR2DS5) appear to encounters with pathogens, but also by reproductive occur in two different chromosomal locations. This fitness. has led to the splitting of the locus into two separate

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 797

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

Table 1: KIR nomenclature, lineages and ligands(IPD - KIR Database; Cadavid and Lun, 2009) No. of No. of Gene name CD nomenclature Lineage Ligand(s) Function alleles protein KIR2DL1 CD158a 43 24 III HLA-C2 inhibitory KIR2DL2 CD158b1 29 12 III HLA-C1 (weakly HLA-C2) inhibitory KIR2DL3 CD158b2 33 17 III HLA-C1 (weakly HLA-C2) inhibitory KIR2DL5A* CD158f I Unknown inhibitory 45 18 KIR2DL5B* I Unknown inhibitory

KIR3DL1 CD158e1 74 58 II HLA-BBw4 and HLA-ABw4 inhibitory Certain HLA-A3 and HLA- KIR3DL2 CD158k 84 62 II inhibitory A*11 KIR3DL3 CD158z 107 56 V Unknown inhibitory KIR2DL4 CD158d 47 22 I HLA-G activating KIR2DS1 CD158h 15 7 III HLA-C2A activating Potentially HLA-C1 KIR2DS2 CD158j 22 8 III activating (binding not detectable) KIR2DS3 14 5 III Potentially HLA-C1 activating

KIR2DS4 CD158i 30 13 III HLA-Cw4 and HLA-11 activating KIR2DS5 CD158g 15 10 III Unknown activating Potentially HLA-BBw4 KIR3DS1 CD158e2 16 12 II activating (binding not detectable) KIR2DP1 22 0 III / pseudogene

KIR3DP1 CD158c 23 0 V / pseudogene *KIR2DL5 gene is duplicated and encoded by two separate loci within the LRC gene cluster.

Given the high sequence homology between the KIR recognition and peptide extracellular domains of some activating and selectivity inhibitory KIR (~99%), several activating KIR have Individual KIR recognize distinct subsets of the been reported to bind the same HLA molecules as classical human MHC class I allotypes. This their inhibitory counterparts, although with binding specificity is determined both by residues significantly weaker affinity (Biassoni et al., 1997; of the MHC class I and those of the peptide bound Valés-Gómez et al., 1998; Stewart et al., 2005). by the MHC class I molecule. Inhibitory KIR are Due to their low affinities, the activating KIR-HLA able to recognize all the known HLA-C allotypes binding specificity is quite uncertain. Moreover the (C1 and C2 subgroup) and some subsets of HLA-A KIR-HLA affinities can be enhanced by specific and HLA-B allotypes. KIR2DL1 binds HLA-C2 peptides presented in the HLA molecules, as has allotypes, which all have a lysine at position 80 been shown for KIR2DS1 interactions with (Colonna et al., 1993). KIR2DL2 and KIR2DL3, Epstein-Barr virus-infected cells (Stewart et al., which segregate as alleles of the same locus, bind 2005). One potential model is that these receptors mainly HLA-C1 allotypes (with an asparagine at may bind specific viral peptides that have yet to be position 80), some HLA-C2 allotypes and a few determined. HLA-B allotypes which have an asparagine at In addition to the MHC class I heavy chain, all position 80 and also a valine at position 76 (Moesta inhibitory KIR tested to date have some degree of et al., 2008; Wagtmann et al., 1995; Pende et al., peptide selectivity (Boyington et al., 2000; Malnati 2009). KIR3DL1 recognize the "Bw4" motif et al., 1995; Rajagopalan and Long, 1997; present in 40% of the known HLA-B allotypes and Hansasuta et al., 2004). This appears to have a in some HLA-A allotypes, with a higher affinity for functional relevance in that NK cells expressing the Bw4 motifs containing an isoleucine at position KIR2DL3 are exquisitely sensitive to the peptide 80 (Cella et al., 1994; Gumperz et al., 1995). bound by MHC class I. This is because peptides KIR3DL2 is only known to bind HLA-A3 and that stabilise MHC class I, but bind KIR weakly can HLA-A11 allotypes whilst ligands for KIR2DL5 antagonize the inhibition due to MHC class and KIR3DL3 have not yet been identified. I:peptide complexes that bind KIR strongly (Fadda et al., 2010). This process appears to be more

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 798

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

efficient than MHC class I downregulation in Studies in other species have revealed the activating NK cells, and may be important for uniqueness of KIR in the simian primates. KIR recognition of infected targets (Rajagopalan and genes have been found in species as diverse as Long, 2010). cattle, horses, dogs and pinnipeds (Parham et al., 2010). These are thought to have derived from KIR evolution duplication of an ancestral KIR3D gene over 100 A comparison of the KIR genes in human and million years ago. This resulted in two genes: chimpanzees revealed unexpectedly rapid evolution KIR3DL and KIR3DX (Guethlein et al., 2007). The of the KIR locus, in many ways exceeding the pace KIR3DL gene is thought to have spawned the KIR of their MHC class I ligand (Khakoo et al., 2000). genes of the primates, and the KIR3DX gene given This contrasts with the high conservation of the rise to the multigene KIR family in cattle CD94:NKG2A system (Shum et al., 2002). Work in (Sambrook et al., 2006). KIR3DX is retained in the higher primates has revealed that in these humans, however it is a non-functional pseudogene species the KIR genes have expanded substantially. in the LRC amongst the LILR gene. The adoption Mice, which are the most frequently used of different solutions to the issue of NK cell immunological model for the immune system of receptor variability is further illustrated by the man and his response to disease, do not have KIR expansion of the NKG2 family of genes in the as regulators of NK cell activity (Figure 1). Instead prosimians (Averdam et al., 2009) and the they have an expansion of the C-type lectin-like observation that the pinnipeds (seals and sea lions) receptors, the Ly49 genes which also bind classical seem to cope with having only one functional KIR MHC class I molecules. These genes are related to and one functional Ly49 gene (Hammond et al., the NKG2A family of receptors, and both these 2009). gene families in addition to the CD94 gene are Genetic studies implicating specific combinations found on murine chromosome 6 in a region of KIR in infectious diseases imply that pathogens designated the natural killer cell complex (NKC) are a major driving force in KIR evolution. This (Vance et al., 1998). In mice CD94:NKG2A binds follows naturally from the observation that natural the non-classical MHC class I molecule Qa-1, killer cells are important in clearing viral infections. which also binds MHC class I leader sequences. Pathogens can drive KIR selection both by a direct Thus comparison of humans and rodents has effect on specific KIR genes and also via an indirect revealed two distinct evolutionary pathways for NK selective pressure through driving the evolution of cell receptors: one leading to diversification of KIR MHC Class I. This is well illustrated by the co- and the other to diversification of Ly49. Both evolution of KIR and the MHC-C locus in the great species have inhibitory NK cell receptors for apes. The KIR can be divided into lineages based classical class I molecules and both for a non- on sequence homologies. The lineage III KIR have classical MHC class I molecule, although NKG2A MHC-C ligands. The most divergent species from in mouse and human are not strictly orthologous. man with an MHC-C allele is the orangutan Remnants of non-functional genes can be found in (Adams et al., 1999). In this species this locus is the alternate species: the KIR are represented by a present in only about half the individuals. gene on murine chromosome X and Ly49 is a Nevertheless, in the orangutan and man's more pseudogene in humans (Kelley et al., 2005) (Figure closely related ancestors 1).

Figure 1

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 799

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

(the gorilla, the common chimpanzee and the functional relationship and which may be fine tuned pygmy chimpanzee) lineage III KIR have by the affinity of the KIR for its MHC class I ligand expanded, implying that MHC and the KIR are co- (Single et al., 2007). Thus in the Yucpa population evolving (Abi-Rached et al., 2010b). Similarly in there is a relatively high frequency of the "strong the old world monkeys the expansion of MHC-A/B educating" HLA-C*07 allele, and correspondingly locus has led to expansion of lineage II KIR, and in higher frequencies of KIR2DL3 alleles with low the hoolock gibbon loss of MHC-G corresponds to avidities for HLA-C (Gendzekhadze et al., 2009). loss of the lineage I gene KIR2DL4, which has This implies that evolutionary pressures have been shown to be relatively conserved amongst combined to ensure that inhibitory signals to NK higher primates (Abi-Rached et al., 2010a; Parham cells can be easily overcome and so NK cells can be et al., 2010). readily activated in response to pathogens. Whilst humans and chimpanzees share MHC-A, -B and -C loci, they share relatively few KIR genes. Impact of KIR diversity on Human These include the framework genes KIR3DL3, health and disease KIR2DL4 and KIR3DL2 (which in the chimpanzee The impact of KIR diversity on human health is is a chimera of human KIR3DL1 and KIR3DL2 well illustrated by disease association studies. called Pt-KIR3DL1/2), and the genes KIR2DL5 Whilst infection is thought to be the major driving and KIR2DS4 (Khakoo et al., 2000). Humans have force for the evolution of KIR, there is substantial retained lineage III inhibitory KIR which bind evidence that KIR diversity impacts a number of strongly to HLA-C, however the activating KIR for pregnancy associated disorders, including pre- HLA-C are low avidity. Conversely the common eclampsia and recurrent spontaneous abortion chimpanzee has retained high avidity activating and (Moffett-King, 2002). During placentation the inhibitory for both group 1 and group 2 HLA-C trophoblast burrows into the placenta, and natural allotypes. Furthermore, although the lineage II killer cells appear to be important for this process. human KIR bind both HLA-A and -B allotypes, the Analysis of maternal and foetal KIR and MHC most relevant interaction appears to be that of class I genotypes demonstrate that if these result in KIR3DL1 with HLA-B allotypes with the Bw4 greater foetal NK cell activation then pregnancy is serological motif. Although this KIR does bind more likely to be successful, due to improved HLA-A allotypes with the same serological motif. placentation. Thus in cases where the KIR The binding of other KIR to HLA-A allotypes is haplotype of the fetus has a preponderance of less well documented. KIR3DL2 binds HLA-A3 activating receptors, such as a type B KIR and HLA-A11, although the avidity of this haplotype, then there I a lower probability of pre- interaction is not well studied (Dohring et al., 1996; eclampsia (Hiby et al., 2004). Conversely if the Pende et al., 1996; Valiante et al., 1997b). It has fetus has only one activating KIR, as is found in a also been shown to bind tetramers of HLA-B27 group A KIR haplotype and the mother has a strong homodimers (Kollnberger et al., 2007). KIR2DS4 inhibitory MHC class I type for example two group also binds HLA-A11 (Graef et al., 2009). However 2 HLA-C alleles then there is a greater risk of pre- the functional relevance of these interactions with eclampsia, foetal growth retardation and recurrent HLA-A is not as well documented as for HLA-B: spontaneous abortion (Hiby et al., 2010). This and KIR. Conversely Pt-KIR3DL2, binds both likely drives the evolution of the KIR locus towards MHC-A and -B allotypes, and can demonstrably a preponderance of activating receptors. inhibit chimpanzee NK cells in a manner not Early studies in infectious disease would concur restricted by the Bw4 serological motif, even with this evolutionary direction. In HIV infection though this motif is present on a number of the activating receptor KIR3DS1 and its HLA-B chimpanzee MHC-B allotypes (Khakoo et al., ligands Bw4 with isoleucine at position 80 (Bw480I) 2002). Thus even where the most simple motifs for are associated with a slower progression to AIDS KIR binding on MHC appear relatively conserved (Martin et al., 2002). This begs the question as to between species there is substantial evidence for a the persistence of the "inhibitory" A haplotypes in more rapid evolution at the KIR locus. the human population. Further studies of HIV Within different human populations there is a great infection have revealed a second model in which a diversity in the frequencies of individual KIR genes hierarchy of inhibitory interactions between the (Gonzalez-Galarza et al., 2011). The essential role inhibitory receptor KIR3DL1 and its HLA-BBw4 for unequal crossing over in generating this ligands influences the progression to AIDS (Martin diversity is illustrated by in depth study of the et al., 2007). Interestingly in this genetic analysis KIR3DL1/KIR3DS1 locus in which it has been the most protective allele KIR3DL1*004 is one demonstrated that this process can account for both which is not expressed on the cell surface (Pando et duplication and deletion within the KIR gene al., 2003). This is a feature of other KIR alleles, complex (Norman et al., 2009). Further selection including KIR2DL2*004 and a number of alleles of may occur on the basis of the interaction of KIR KIR2DS3 (VandenBussche et al., 2006; with its MHC class I ligands, to maintain a VandenBussche et al., 2009). This implies that care

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 800

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

must be taken with the interpretation of these Colonna M, Borsellino G, Falco M, Ferrara GB, Strominger genetic analyses as the presence of the receptor and JL. HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer its ligand does not necessarily mean that there is a cells. Proc Natl Acad Sci U S A. 1993 Dec simple functional relationship. 15;90(24):12000-4 The importance of inhibitory receptor:ligand Cella M, Longo A, Ferrara GB, Strominger JL, Colonna M. interactions is probably best illustrated by NK3-specific natural killer cells are selectively inhibited by consideration of hepatitis C virus (HCV) infection. Bw4-positive HLA alleles with isoleucine 80. J Exp Med. This is a positive stranded RNA virus that has 1994 Oct 1;180(4):1235-42 relatively little specific effects on MHC class I Gumperz JE, Litwin V, Phillips JH, Lanier LL, Parham P. expression, and so may act as a more general The Bw4 public epitope of HLA-B molecules confers template for understanding the role of NK cells in reactivity with natural killer cell clones that express NKB1, a putative HLA receptor. J Exp Med. 1995 Mar viral infection. Genetic analysis has shown that 1;181(3):1133-44 weaker inhibitory interactions are associated with a more beneficial outcome of HCV infection. It was Malnati MS, Peruzzi M, Parker KC, Biddison WE, Ciccone E, Moretta A, Long EO. Peptide specificity in the originally shown that KIR2DL3 and its group 1 recognition of MHC class I by natural killer cell clones. HLA-C ligands were associated with spontaneous Science. 1995 Feb 17;267(5200):1016-8 resolution of HCV infection, which in the vast Wagtmann N, Rajagopalan S, Winter CC, Peruzzi M, Long majority of individuals leads to chronic infection EO. Killer cell inhibitory receptors specific for HLA-C and (Khakoo et al., 2004). Binding analysis reveals that HLA-B identified by direct binding and by functional KIR2DL3 is a weaker binder to HLA-C than its transfer. Immunity. 1995 Dec;3(6):801-9 allele KIR2DL2, which has a similar MHC class I Döhring C, Scheidegger D, Samaridis J, Cella M, Colonna specificity (Winter et al., 1998). It is therefore M. A human killer inhibitory receptor specific for HLA-A1,2. thought that a weaker inhibitory receptor:ligand J Immunol. 1996 May 1;156(9):3098-101 interaction can be more easily perturbed than a Pende D, Biassoni R, Cantoni C, Verdiani S, Falco M, di strong one and hence is more likely to lead to NK Donato C, Accame L, Bottino C, Moretta A, Moretta L. The natural killer cell receptor specific for HLA-A allotypes: a cell activation. This protection has been observed in novel member of the p58/p70 family of inhibitory receptors other HCV exposed cohorts and disease settings, that is characterized by three immunoglobulin-like domains including in the clinically important treatment and is expressed as a 140-kD disulphide-linked dimer. J setting (Romero et al., 2008; Knapp et al. 2010; Exp Med. 1996 Aug 1;184(2):505-18 Vidal-Castiñeira et al., 2010). Furthermore, similar Biassoni R, Pessino A, Malaspina A, Cantoni C, Bottino C, to HIV, it can be mapped to the allelic level, but in Sivori S, Moretta L, Moretta A. Role of amino acid position this case at the HLA-C locus. Thus the common 70 in the binding affinity of p50.1 and p58.1 receptors for HLA-Cw4 molecules. Eur J Immunol. 1997 group 1 HLA-C*07 alleles are not protective Dec;27(12):3095-9 whereas most other group 1 HLA-C alleles are protective in combination with KIR2DL3 (Knapp et Rajagopalan S, Long EO. The direct binding of a p58 killer cell inhibitory receptor to human histocompatibility al., 2010). Thus it appears that there is a balancing leukocyte antigen (HLA)-Cw4 exhibits peptide selectivity. J selection on the KIR "A" and "B" haplotypes in Exp Med. 1997 Apr 21;185(8):1523-8 humans which has permitted maintenance of both Valiante NM, Lienert K, Shilling HG, Smits BJ, Parham P. in the extant human populations (Gendzekhadze et Killer cell receptors: keeping pace with MHC class I al., 2009). evolution. Immunol Rev. 1997 Feb;155:155-64 Conclusion Valiante NM, Uhrberg M, Shilling HG, Lienert-Weidenbach K, Arnett KL, D'Andrea A, Phillips JH, Lanier LL, Parham By virtue of the expression of KIR, natural killer P. Functionally and structurally distinct NK cell receptor cells are a branch of the innate immune system that repertoires in the peripheral blood of two human donors. are undergoing constant evolution in response to Immunity. 1997 Dec;7(6):739-51 both pathogens and the challenge of successful Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert- reproduction. The KIR have diversified in response Weidenbach K, Corliss B, Tyan D, Lanier LL, Parham P. Human diversity in killer cell inhibitory receptor genes. to MHC driven selective pressures, following Immunity. 1997 Dec;7(6):753-63 exposure to pathogens. Due to the independent segregation of KIR and their MHC class I ligands, Lanier LL. NK cell receptors. Annu Rev Immunol. 1998;16:359-93 in any given individual some KIR may be redundant. Nevertheless, on a population level, by Llano M, Lee N, Navarro F, García P, Albar JP, Geraghty fine tuning natural killer activity these receptors are DE, López-Botet M. HLA-E-bound peptides influence recognition by inhibitory and triggering CD94/NKG2 key regulators of the innate immune response to receptors: preferential response to an HLA-G-derived pathogens. nonamer. Eur J Immunol. 1998 Sep;28(9):2854-63 Shilling HG, Lienert-Weidenbach K, Valiante NM, Uhrberg References M, Parham P. Evidence for recombination as a mechanism for KIR diversification. Immunogenetics. 1998 Nov- Ljunggren HG, Kärre K. In search of the 'missing self': Dec;48(6):413-6 MHC molecules and NK cell recognition. Immunol Today. 1990 Jul;11(7):237-44 Valés-Gómez M, Reyburn HT, Erskine RA, Strominger J. Differential binding to HLA-C of p50-activating and p58-

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 801

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

inhibitory natural killer cell receptors. Proc Natl Acad Sci U immunoglobulin-like receptor sequences in three ethnic S A. 1998 Nov 24;95(24):14326-31 groups. Immunogenetics. 2001;52(3-4):195-205 Vance RE, Kraft JR, Altman JD, Jensen PE, Raulet DH. Rajalingam R, Gardiner CM, Canavez F, Vilches C, Mouse CD94/NKG2A is a natural killer cell receptor for the Parham P. Identification of seventeen novel KIR variants: nonclassical major histocompatibility complex (MHC) class fourteen of them from two non-Caucasian donors. Tissue I molecule Qa-1(b). J Exp Med. 1998 Nov Antigens. 2001 Jan;57(1):22-31 16;188(10):1841-8 Toneva M, Lepage V, Lafay G, Dulphy N, Busson M, Winter CC, Gumperz JE, Parham P, Long EO, Wagtmann Lester S, Vu-Trien A, Michaylova A, Naumova E, N. Direct binding and functional transfer of NK cell McCluskey J, Charron D. Genomic diversity of natural killer inhibitory receptors reveal novel patterns of HLA-C allotype cell receptor genes in three populations. Tissue Antigens. recognition. J Immunol. 1998 Jul 15;161(2):571-7 2001 Apr;57(4):358-62 Adams EJ, Thomson G, Parham P. Evidence for an HLA- Hsu KC, Chida S, Geraghty DE, Dupont B. The killer cell C-like locus in the orangutan Pongo pygmaeus. immunoglobulin-like receptor (KIR) genomic region: gene- Immunogenetics. 1999 Sep;49(10):865-71 order, haplotypes and allelic polymorphism. Immunol Rev. 2002 Dec;190:40-52 Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar- Mather TP. Natural killer cells in antiviral defense: function Hsu KC, Liu XR, Selvakumar A, Mickelson E, O'Reilly RJ, and regulation by innate cytokines. Annu Rev Immunol. Dupont B. Killer Ig-like receptor haplotype analysis by 1999;17:189-220 gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with Vitale M, Castriconi R, Parolini S, Pende D, Hsu ML, multiple subsets. J Immunol. 2002 Nov 1;169(9):5118-29 Moretta L, Cosman D, Moretta A. The leukocyte Ig-like receptor (LIR)-1 for the cytomegalovirus UL18 protein Khakoo SI, Geller R, Shin S, Jenkins JA, Parham P. The displays a broad specificity for different HLA class I alleles: D0 domain of KIR3D acts as a major histocompatibility analysis of LIR-1 + NK cell clones. Int Immunol. 1999 complex class I binding enhancer. J Exp Med. 2002 Oct Jan;11(1):29-35 7;196(7):911-21 Wende H, Colonna M, Ziegler A, Volz A. Organization of Martin MP, Gao X, Lee JH, Nelson GW, Detels R, Goedert the leukocyte receptor cluster (LRC) on human JJ, Buchbinder S, Hoots K, Vlahov D, Trowsdale J, Wilson chromosome 19q13.4. Mamm Genome. 1999 M, O'Brien SJ, Carrington M. Epistatic interaction between Feb;10(2):154-60 KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet. 2002 Aug;31(4):429-34 Witt CS, Dewing C, Sayer DC, Uhrberg M, Parham P, Christiansen FT. Population frequencies and putative Maxwell LD, Wallace A, Middleton D, Curran MD. A haplotypes of the killer cell immunoglobulin-like receptor common KIR2DS4 deletion variant in the human that sequences and evidence for recombination. predicts a soluble KIR molecule analogous to the KIR1D Transplantation. 1999 Dec 15;68(11):1784-9 molecule observed in the rhesus monkey. Tissue Antigens. 2002 Sep;60(3):254-8 Boyington JC, Motyka SA, Schuck P, Brooks AG, Sun PD. Crystal structure of an NK cell immunoglobulin-like Moffett-King A. Natural killer cells and pregnancy. Nat Rev receptor in complex with its class I MHC ligand. Nature. Immunol. 2002 Sep;2(9):656-63 2000 Jun 1;405(6786):537-43 Norman PJ, Carrington CV, Byng M, Maxwell LD, Curran Crum KA, Logue SE, Curran MD, Middleton D. MD, Stephens HA, Chandanayingyong D, Verity DH, Development of a PCR-SSOP approach capable of Hameed K, Ramdath DD, Vaughan RW. Natural killer cell defining the natural killer cell inhibitory receptor (KIR) gene immunoglobulin-like receptor (KIR) locus profiles in African sequence repertoires. Tissue Antigens. 2000 and South Asian populations. Genes Immun. 2002 Oct;56(4):313-26 Apr;3(2):86-95 Khakoo SI, Rajalingam R, Shum BP, Weidenbach K, Shilling HG, Guethlein LA, Cheng NW, Gardiner CM, Flodin L, Muir DG, Canavez F, Cooper SL, Valiante NM, Rodriguez R, Tyan D, Parham P. Allelic polymorphism Lanier LL, Parham P. Rapid evolution of NK cell receptor synergizes with variable gene content to individualize systems demonstrated by comparison of chimpanzees and human KIR genotype. J Immunol. 2002 Mar humans. Immunity. 2000 Jun;12(6):687-98 1;168(5):2307-15 Martin AM, Freitas EM, Witt CS, Christiansen FT. The Shum BP, Flodin LR, Muir DG, Rajalingam R, Khakoo SI, genomic organization and evolution of the natural killer Cleland S, Guethlein LA, Uhrberg M, Parham P. immunoglobulin-like receptor (KIR) gene cluster. Conservation and variation in human and common Immunogenetics. 2000 Apr;51(4-5):268-80 chimpanzee CD94 and NKG2 genes. J Immunol. 2002 Jan 1;168(1):240-52 Wilson MJ, Torkar M, Haude A, Milne S, Jones T, Sheer D, Beck S, Trowsdale J. Plasticity in the organization and Uhrberg M, Parham P, Wernet P. Definition of gene sequences of human KIR/ILT gene families. Proc Natl content for nine common group B haplotypes of the Acad Sci U S A. 2000 Apr 25;97(9):4778-83 Caucasoid population: KIR haplotypes contain between seven and eleven KIR genes. Immunogenetics. 2002 Fan QR, Long EO, Wiley DC. Crystal structure of the Jul;54(4):221-9 human natural killer cell inhibitory receptor KIR2DL1-HLA- Cw4 complex. Nat Immunol. 2001 May;2(5):452-60 Vilches C, Parham P. KIR: diverse, rapidly evolving receptors of innate and adaptive immunity. Annu Rev Gardiner CM, Guethlein LA, Shilling HG, Pando M, Carr Immunol. 2002;20:217-51 WH, Rajalingam R, Vilches C, Parham P. Different NK cell surface phenotypes defined by the DX9 antibody are due Yawata M, Yawata N, Abi-Rached L, Parham P. Variation to KIR3DL1 gene polymorphism. J Immunol. 2001 Mar within the human killer cell immunoglobulin-like receptor 1;166(5):2992-3001 (KIR) gene family. Crit Rev Immunol. 2002;22(5-6):463-82 Norman PJ, Stephens HA, Verity DH, Chandanayingyong Yawata M, Yawata N, McQueen KL, Cheng NW, Guethlein D, Vaughan RW. Distribution of natural killer cell LA, Rajalingam R, Shilling HG, Parham P. Predominance of group A KIR haplotypes in Japanese associated with

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 802

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

diverse NK cell repertoires of KIR expression. Rajagopalan S, Bryceson YT, Kuppusamy SP, Geraghty Immunogenetics. 2002 Nov;54(8):543-50 DE, van der Meer A, Joosten I, Long EO. Activation of NK cells by an endocytosed receptor for soluble HLA-G. PLoS Martin MP, Bashirova A, Traherne J, Trowsdale J, Biol. 2006 Jan;4(1):e9 Carrington M. Cutting edge: expansion of the KIR locus by unequal crossing over. J Immunol. 2003 Sep Sambrook JG, Bashirova A, Andersen H, Piatak M, 1;171(5):2192-5 Vernikos GS, Coggill P, Lifson JD, Carrington M, Beck S. Identification of the ancestral killer immunoglobulin-like Pando MJ, Gardiner CM, Gleimer M, McQueen KL, receptor gene in primates. BMC Genomics. 2006 Aug Parham P. The protein made from a common allele of 15;7:209 KIR3DL1 (3DL1*004) is poorly expressed at cell surfaces due to substitution at positions 86 in Ig domain 0 and 182 VandenBussche CJ, Dakshanamurthy S, Posch PE, in Ig domain 1. J Immunol. 2003 Dec 15;171(12):6640-9 Hurley CK. A single polymorphism disrupts the killer Ig-like receptor 2DL2/2DL3 D1 domain. J Immunol. 2006 Oct Carrington M, Cullen M. Justified chauvinism: advances in 15;177(8):5347-57 defining meiotic recombination through sperm typing. Trends Genet. 2004 Apr;20(4):196-205 Yawata M, Yawata N, Draghi M, Little AM, Partheniou F, Parham P. Roles for HLA and KIR polymorphisms in Hansasuta P, Dong T, Thananchai H, Weekes M, Willberg natural killer cell repertoire selection and modulation of C, Aldemir H, Rowland-Jones S, Braud VM. Recognition of effector function. J Exp Med. 2006 Mar 20;203(3):633-45 HLA-A3 and HLA-A11 by KIR3DL2 is peptide-specific. Eur J Immunol. 2004 Jun;34(6):1673-9 Guethlein LA, Abi-Rached L, Hammond JA, Parham P. The expanded cattle KIR genes are orthologous to the Hiby SE, Walker JJ, O'shaughnessy KM, Redman CW, conserved single-copy KIR3DX1 gene of primates. Carrington M, Trowsdale J, Moffett A. Combinations of Immunogenetics. 2007 Jun;59(6):517-22 maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med. 2004 Kollnberger S, Chan A, Sun MY, Chen LY, Wright C, di Oct 18;200(8):957-65 Gleria K, McMichael A, Bowness P. Interaction of HLA-B27 homodimers with KIR3DL1 and KIR3DL2, unlike HLA-B27 Khakoo SI, Thio CL, Martin MP, Brooks CR, Gao X, heterotrimers, is independent of the sequence of bound Astemborski J, Cheng J, Goedert JJ, Vlahov D, Hilgartner peptide. Eur J Immunol. 2007 May;37(5):1313-22 M, Cox S, Little AM, Alexander GJ, Cramp ME, O'Brien SJ, Rosenberg WM, Thomas DL, Carrington M. HLA and NK Martin MP, Qi Y, Gao X, Yamada E, Martin JN, Pereyra F, cell inhibitory receptor genes in resolving hepatitis C virus Colombo S, Brown EE, Shupert WL, Phair J, Goedert JJ, infection. Science. 2004 Aug 6;305(5685):872-4 Buchbinder S, Kirk GD, Telenti A, Connors M, O'Brien SJ, Walker BD, Parham P, Deeks SG, McVicar DW, Jiang K, Zhu FM, Lv QF, Yan LX. Distribution of killer cell Carrington M. Innate partnership of HLA-B and KIR3DL1 immunoglobulin-like receptor genes in the Chinese Han subtypes against HIV-1. Nat Genet. 2007 Jun;39(6):733-40 population. Tissue Antigens. 2005 Jun;65(6):556-63 Single RM, Martin MP, Gao X, Meyer D, Yeager M, Kidd Kelley J, Walter L, Trowsdale J. Comparative genomics of JR, Kidd KK, Carrington M. Global diversity and evidence natural killer cell receptor gene clusters. PLoS Genet. 2005 for coevolution of KIR and HLA. Nat Genet. 2007 Aug;1(2):129-39 Sep;39(9):1114-9 Kikuchi-Maki A, Catina TL, Campbell KS. Cutting edge: Long EO. Negative signaling by inhibitory receptors: the KIR2DL4 transduces signals into human NK cells through NK cell paradigm. Immunol Rev. 2008 Aug;224:70-84 association with the Fc receptor gamma protein. J Immunol. 2005 Apr 1;174(7):3859-63 Martin MP, Single RM, Wilson MJ, Trowsdale J, Carrington M. KIR haplotypes defined by segregation analysis in 59 Kim S, Poursine-Laurent J, Truscott SM, Lybarger L, Song Centre d'Etude Polymorphisme Humain (CEPH) families. YJ, Yang L, French AR, Sunwoo JB, Lemieux S, Hansen Immunogenetics. 2008 Dec;60(12):767-74 TH, Yokoyama WM. Licensing of natural killer cells by host major histocompatibility complex class I molecules. Nature. Moesta AK, Norman PJ, Yawata M, Yawata N, Gleimer M, 2005 Aug 4;436(7051):709-13 Parham P. Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger Kirwan SE, Burshtyn DN. Killer cell Ig-like receptor- receptor for HLA-C than KIR2DL3. J Immunol. 2008 Mar dependent signaling by Ig-like transcript 2 15;180(6):3969-79 (ILT2/CD85j/LILRB1/LIR-1). J Immunol. 2005 Oct 15;175(8):5006-15 Romero V, Azocar J, Zúñiga J, Clavijo OP, Terreros D, Gu X, Husain Z, Chung RT, Amos C, Yunis EJ. Interaction of Stewart CA, Laugier-Anfossi F, Vély F, Saulquin X, NK inhibitory receptor genes with HLA-C and MHC class II Riedmuller J, Tisserant A, Gauthier L, Romagné F, alleles in Hepatitis C virus infection outcome. Mol Immunol. Ferracci G, Arosa FA, Moretta A, Sun PD, Ugolini S, Vivier 2008 May;45(9):2429-36 E. Recognition of peptide-MHC class I complexes by activating killer immunoglobulin-like receptors. Proc Natl Yawata M, Yawata N, Draghi M, Partheniou F, Little AM, Acad Sci U S A. 2005 Sep 13;102(37):13224-9 Parham P. MHC class I-specific inhibitory receptors and their ligands structure diverse human NK-cell repertoires Whang DH, Park H, Yoon JA, Park MH. Haplotype toward a balance of missing self-response. Blood. 2008 analysis of killer cell immunoglobulin-like receptor genes in Sep 15;112(6):2369-80 77 Korean families. Hum Immunol. 2005 Feb;66(2):146-54 Averdam A, Petersen B, Rosner C, Neff J, Roos C, Eberle Bashirova AA, Martin MP, McVicar DW, Carrington M. The M, Aujard F, Münch C, Schempp W, Carrington M, Shiina killer immunoglobulin-like receptor gene cluster: tuning the T, Inoko H, Knaust F, Coggill P, Sehra H, Beck S, Abi- genome for defense. Annu Rev Genomics Hum Genet. Rached L, Reinhardt R, Walter L. A novel system of 2006;7:277-300 polymorphic and diverse NK cell receptors in primates. MacFarlane AW 4th, Campbell KS. Signal transduction in PLoS Genet. 2009 Oct;5(10):e1000688 natural killer cells. Curr Top Microbiol Immunol. Cadavid LF, Lun CM. Lineage-specific diversification of 2006;298:23-57 killer cell Ig-like receptors in the owl monkey, a New World primate. Immunogenetics. 2009 Jan;61(1):27-41

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 803

NK cell receptors: evolution and diversity Borhis G, Khakoo SI

Gendzekhadze K, Norman PJ, Abi-Rached L, Graef T, Fadda L, Borhis G, Ahmed P, Cheent K, Pageon SV, Moesta AK, Layrisse Z, Parham P. Co-evolution of Cazaly A, Stathopoulos S, Middleton D, Mulder A, Claas KIR2DL3 with HLA-C in a human population retaining FH, Elliott T, Davis DM, Purbhoo MA, Khakoo SI. Peptide minimal essential diversity of KIR and HLA class I ligands. antagonism as a mechanism for NK cell activation. Proc Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18692-7 Natl Acad Sci U S A. 2010 Jun 1;107(22):10160-5 Graef T, Moesta AK, Norman PJ, Abi-Rached L, Vago L, Hiby SE, Apps R, Sharkey AM, Farrell LE, Gardner L, Older Aguilar AM, Gleimer M, Hammond JA, Guethlein LA, Mulder A, Claas FH, Walker JJ, Redman CW, Morgan L, Bushnell DA, Robinson PJ, Parham P. KIR2DS4 is a Tower C, Regan L, Moore GE, Carrington M, Moffett A. product of gene conversion with KIR3DL2 that introduced Maternal activating KIRs protect against human specificity for HLA-A*11 while diminishing avidity for HLA- reproductive failure mediated by fetal HLA-C2. J Clin C. J Exp Med. 2009 Oct 26;206(11):2557-72 Invest. 2010 Nov 1;120(11):4102-10 Hammond JA, Guethlein LA, Abi-Rached L, Moesta AK, Knapp S, Warshow U, Hegazy D, Brackenbury L, Guha IN, Parham P. Evolution and survival of marine carnivores did Fowell A, Little AM, Alexander GJ, Rosenberg WM, Cramp not require a diversity of killer cell Ig-like receptors or Ly49 ME, Khakoo SI. Consistent beneficial effects of killer cell NK cell receptors. J Immunol. 2009 Mar 15;182(6):3618-27 immunoglobulin-like receptor 2DL3 and group 1 human leukocyte antigen-C following exposure to hepatitis C Norman PJ, Abi-Rached L, Gendzekhadze K, Hammond virus. Hepatology. 2010 Apr;51(4):1168-75 JA, Moesta AK, Sharma D, Graef T, McQueen KL, Guethlein LA, Carrington CV, Chandanayingyong D, Orr MT, Murphy WJ, Lanier LL. 'Unlicensed' natural killer Chang YH, Crespí C, Saruhan-Direskeneli G, Hameed K, cells dominate the response to cytomegalovirus infection. Kamkamidze G, Koram KA, Layrisse Z, Matamoros N, Milà Nat Immunol. 2010 Apr;11(4):321-7 J, Park MH, Pitchappan RM, Ramdath DD, Shiau MY, Stephens HA, Struik S, Tyan D, Verity DH, Vaughan RW, Parham P, Abi-Rached L, Matevosyan L, Moesta AK, Davis RW, Fraser PA, Riley EM, Ronaghi M, Parham P. Norman PJ, Older Aguilar AM, Guethlein LA. Primate- Meiotic recombination generates rich diversity in NK cell specific regulation of natural killer cells. J Med Primatol. receptor genes, alleles, and haplotypes. Genome Res. 2010 Aug;39(4):194-212 2009 May;19(5):757-69 Pyo CW, Guethlein LA, Vu Q, Wang R, Abi-Rached L, Pende D, Marcenaro S, Falco M, Martini S, Bernardo ME, Norman PJ, Marsh SG, Miller JS, Parham P, Geraghty DE. Montagna D, Romeo E, Cognet C, Martinetti M, Maccario Different patterns of evolution in the centromeric and R, Mingari MC, Vivier E, Moretta L, Locatelli F, Moretta A. telomeric regions of group A and B haplotypes of the Anti-leukemia activity of alloreactive NK cells in KIR ligand- human killer cell Ig-like receptor locus. PLoS One. 2010 mismatched haploidentical HSCT for pediatric patients: Dec 29;5(12):e15115 evaluation of the functional role of activating KIR and Rajagopalan S, Long EO. Antagonizing inhibition gets NK redefinition of inhibitory KIR specificity. Blood. 2009 Mar cells going. Proc Natl Acad Sci U S A. 2010 Jun 26;113(13):3119-29 8;107(23):10333-4 Purdy AK, Campbell KS. Natural killer cells and cancer: Traherne JA, Martin M, Ward R, Ohashi M, Pellett F, regulation by the killer cell Ig-like receptors (KIR). Cancer Gladman D, Middleton D, Carrington M, Trowsdale J. Biol Ther. 2009 Dec;8(23):2211-20 Mechanisms of copy number variation and hybrid gene VandenBussche CJ, Mulrooney TJ, Frazier WR, formation in the KIR immune gene complex. Hum Mol Dakshanamurthy S, Hurley CK. Dramatically reduced Genet. 2010 Mar 1;19(5):737-51 surface expression of NK cell receptor KIR2DS3 is Vidal-Castiñeira JR, López-Vázquez A, Díaz-Peña R, attributed to multiple residues throughout the molecule. Alonso-Arias R, Martínez-Borra J, Pérez R, Fernández- Genes Immun. 2009 Mar;10(2):162-73 Suárez J, Melón S, Prieto J, Rodrigo L, López-Larrea C. Abi-Rached L, Kuhl H, Roos C, ten Hallers B, Zhu B, Effect of killer immunoglobulin-like receptors in the Carbone L, de Jong PJ, Mootnick AR, Knaust F, Reinhardt response to combined treatment in patients with chronic R, Parham P, Walter L. A small, variable, and irregular hepatitis C virus infection. J Virol. 2010 Jan;84(1):475-81 killer cell Ig-like receptor locus accompanies the absence Gonzalez-Galarza FF, Christmas S, Middleton D, Jones of MHC-C and MHC-G in gibbons. J Immunol. 2010 Feb AR. Allele frequency net: a database and online repository 1;184(3):1379-91 for immune gene frequencies in worldwide populations. Abi-Rached L, Moesta AK, Rajalingam R, Guethlein LA, Nucleic Acids Res. 2011 Jan;39(Database issue):D913-9 Parham P. Human-specific evolution and adaptation led to IPD KIR polymorphism database. major qualitative differences in the variable receptors of human and chimpanzee natural killer cells. PLoS Genet. http://www.ebi.ac.uk/ipd/kir/stats.html 2010 Nov 4;6(11):e1001192 This article should be referenced as such: Alter G, Jost S, Rihn S, Reyor LL, Nolan BE, Ghebremichael M, Bosch R, Altfeld M, Lauer GM. Borhis G, Khakoo SI. NK cell receptors: evolution and Reduced frequencies of NKp30+NKp46+, CD161+, and diversity. Atlas Genet Cytogenet Oncol Haematol. 2011; NKG2D+ NK cells in acute HCV infection may predict viral 15(9):795-804. clearance. J Hepatol. 2011 Aug;55(2):278-88 Davidson CL, Li NL, Burshtyn DN. LILRB1 polymorphism and surface phenotypes of natural killer cells. Hum Immunol. 2010 Oct;71(10):942-9

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 804

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Deep Insight Section

Somatostatin (SS), SS receptors and SS analog treatment in tumorigenesis Liliana Steffani, Luca Passafaro, Diego Ferone, Paolo Magni, Massimiliano Ruscica Department of Endocrinology, Pathophysiology and Applied Biology, Universita degli Studi di Milano, Milan, Italy (LS, LP, PM, MR), Department of Internal Medicine, Universita degli Studi di Genova, Genoa, Italy (DF)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Deep/SomatostatinID20094.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI SomatostatinID20094.txt

phospholipase A2, mitogen activated protein Abstract kinase, NO/cGMP, and serine-, threonine, and Somatostatin (SS) is an inhibitory tetradecapeptide phosphotyrosyl- protein phosphatase). hormone with exocrine, endocrine, paracrine, and Interestingly, SS and SS analogs can control tumor autocrine activities, which plays an important development and progression/metastatization by regulatory role in several cell functions, including direct actions, mediated by the ssts, and indirect inhibition of endocrine secretion and cell actions, independent of receptor involvement. The proliferation. Most of the effects of SS and of its direct antiproliferative effects include inhibition of currently available analogs are mediated via five autocrine/paracrine growth-promoting different G protein-coupled receptor (GPCRs), hormone/growth factor synthesis, arrest of cell codenamed sst1-5. SS receptors (ssts) are expressed division (by blockade of growth factor-mediated in a tissue- and subtype-selective manner in both mitogenic signals), suppression of cell invasion and normal and neoplastic cells, and the majority of SS induction of apoptosis (programmed cell death). target tissues express multiple ssts. Recent data Indirect antitumor effects of SS include suppression suggest that when ssts are coexpressed, they may of synthesis or/and release of growth factors and interact forming homo- and hetero-dimers also with growth-promoting hormones, such as insulin, other GPCRs, thus altering their original prolactin, insulin like-growth factor 1, epidermal pharmacological and functional profiles. The growth factor, transforming growth factor-, gastrin, formation of dimers can be not only constitutive, cholecystokinin and growth hormone. A specific but also ligand-promoted: hence, compounds with pattern of ssts activation thus seems to elicit high affinity for the different receptor subtypes can relevant antitumoral actions and deserves further be used to achieve effects elicited by specific exploitation with the aim of validating novel dimers. A feature common to most GPCRs is the therapeutic approaches to cancer. cyclic process of signaling, desensitization, internalization, resensitization, and recycling to the 1. Somatostatin plasma membrane. These events prevent cells from Somatostatin (SS) was first identified in the ovine undergoing excessive receptor stimulation or hypothalamus as a tetradecapeptide that inhibited periods of prolonged inactivity. SS receptors the release of growth hormone (Brazeau et al., differently internalize after agonist binding and, 1973). SS-producing cells are present at high specifically, sst2, sst3 and sst5 are internalized to a densities throughout the central and peripheral greater extent than sst1 or sst4. ssts are linked to nervous systems. In the periphery, SS is also several second messenger systems which are secreted by pancreas and gut and in a lesser extent involved in their downstream intracellular response by thyroid, adrenals and submandibular glands, (i.e., adenylyl cyclase, calcium and potassium ion kidneys, prostate, and placenta (Polak et al., 1975). channels, Na+/H+ antiporter, phospholipase C, SS mediates a variety of biological effects, the most

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 805

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis

Figure 1. Amino acid sequences of the human prosomatostatin. important occurring at the pituitary (inhibition of proteins of 391 aa, 369 aa, 418 aa, 388 aa, and 363 growth hormone (GH) and tireotropic stimulating aa, respectively (Yamada et al., 1992; Corness et hormone (TSH) secretion) and al., 1993; Rohrer et al., 1993; Panetta et al., 1994). gastroenteropancreatic (GEP) levels (inhibition of Structurally, those receptors belong to the so-called insulin, glucagon, and secretin secretion; inhibition "superfamily" of G protein-coupled receptors of hydrochloric acid production and intestinal fluid (GPCRs). All sst isoforms possess a highly absorption) (Konturek et al., 1976). In addition to conserved sequence motif, YANSCANPI/VLY, in the inhibition of hormone secretion, SS also shows seventh transmembrane region, which serves as a antiproliferative and anti-angiogenetic properties, signature sequence for this receptor family that have been largely investigated both in cell lines (Kreienkamp et al., 2002). On the other hand, genes (i.e., human prostate cancer cells, human non small for sst1, 3, 4, and sst5 lack classical introns. lung cell carcinomas and pituitary adenomas) and Interestingly, the estimated sequence identity GH-secreting tumors. The SS form originally between sst1 and sst2 receptors is 46%. The deduced identified in the hypothalamus was SS-14, while aa sequence of human sst3 receptors displays the SS-28, a congener of SS-14 extended at the N- following degrees of similarity with other members terminus, was discovered subsequently (Shen et al., of the sst family: 62% (sst1), 64% (sst2), and 58% 1982). The single human SS gene is located on (sst4). Moreover, four ssts have been identified in chromosome 3q28 and the correlate SS mRNA fish and variant forms of several ssts also exist: codes for a 116-amino acids (aa) prepro-SS protein sst3a, sst3b, sst5a, sst5b, and sst5c in goldfish (Canosa (MW 12,727 Da). Prepro-SS has a sequence of et al., 2004), and sst1a and sst1b in trout (Slagter and hydrophobic aa at the N-terminus which is cleaved Sheridan, 2004). As was the case with SS genes, at the gly-ala junction at position -78 (from the N- phylogenetic analysis suggests that sst genes appear terminus to the C-terminus). Pro-SS undergoes both to have arisen from a series of gene duplication monobasic (Arg-15) and dibasic (Arg-2Lys-1) events. cleavages to release the two biofunctional 2.1 Homo- and hetero-dimerization of hormones SS-28 and SS-14 (Funckes et al., 1983; somatostatin receptor subtypes Brakch et al., 2002) (Figure 1). When ssts in the cell membrane are coexpressed, they may interact forming homo- and hetero-dimers 2. Somatostatin receptors also with other GPCRs, thus altering their original In mammals, the biological actions of SS are pharmacological and functional profiles. A series of mediated by at least six G protein-coupled SS studies, carried out on transfected cell lines, have receptors (sst) encoded by five different genes, shown that dimers can consist of two identical sst named sst1-sst5. Sst2 exists in two splice variants, subtypes (homodimers) or two different subtypes sst2A (a long form) and sst2B (a short form), which (heterodimers), with a range of possible differ only in the length of the cytoplasmic tail. Sst2 combinations depending on the specific subtype displays a cryptic intron at the 3' end of the coding and, probably, on the specific sst-expressing region, which gives rise to the two spliced variants population (Baragli et al., 2007). (Baumeister and Meyerhof, 2000; Olias et al., The five SS receptor isoforms can be involved in 2004). In the human gene, the spliced exon encodes the formation of different dimers, namely, sst1 and for 25 aa residues compared to 38 residues in the sst5 bind efficiently together, while stable sst4-sst5 unspliced form. The encoded receptor proteins dimers have not been observed. These interactions range in size from 356 to 391 aa residues, showing are capable to provide greater signalling diversity, the greatest sequence similarity in the putative affecting the downstream intracellular effects transmembrane region, and diverge at their N- and mediated by receptor activation, such as ligand C-terminal segments (Patel, 1999). Human ssts binding affinity, agonist-induced regulation and genes are localized to chromosome 14q13 (sst1), trafficking. In fact, sst1 endocytosis is enhanced 17q24 (sst2), 22q13.1 (sst3), 20p11.2 (sst4), and when sst1 and sst5 are co-expressed in the same cell 16p13.3 (sst5) (Yamada et al., 1993) encoding for and sst5 is activated; conversely, the internalization

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 806

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis

of sst2 is delayed by sst5 and sst2 co-expression. stable complex, which is internalized into the same Moreover, ssts can form also heterodimers with endocytotic vesicles. Interestingly, the binding other GPCRs: sst2 interacts with the μ-opioid affinity of the agonist plays an important role in the receptor, and sst5 binds to the D2 dopamine receptor degree of receptor internalization. A high binding (D2R). Interestingly, the sst5-D2R dimer enhances affinity of the agonist is a prerequisite for triggering the effects of both receptors, leading to a more sst2 internalization. In fact, the bi-specific sst2/sst5 potent inhibition of adenylyl cyclase (AC) (Møller analog BIM23244, which has a greater sst2 affinity et al., 2003). compared to L-817/818 analog is able to induce a The dimer formation can be not only constitutive, greater internalization (Jacobs and Schulz, 2008). but also ligand-promoted: hence, compounds with Sst5 differs from sst2A in its cellular localization and high affinity for the different receptor subtypes can appears to be predominantly located in intracellular be used to achieve effects elicited by specific components even without agonist treatment, dimers. In the last years, a variety of mono-, bi- and whereas after stimulation, a large amount of pan-specific SS analogs has been synthesized, intracellular receptors is recruited to the cell allowing the characterization of the intracellular surface. The sst5 third intracellular (i3) loop and the effectors involved in the downstream signalling of C-terminal tail have been found to regulate receptor the different ssts (Saveanu et al., 2001). The new internalization, which occurs via clathrin-dependent receptor specific compounds showed to be useful mechanisms. In cultured pituitary cell lines, where under many aspects; among them, the sst5 underwent different kinds of point mutations understanding of the synergistic effect caused by within the i3 loop, there is a reduction of receptor the simultaneous activation of different receptors. internalization upon SS-28 treatment. Moreover, by In cultured pituitary cells, a sst2-D2R chimeric using different C-terminus truncated forms of the compound (BIM-23A387) showed a more potent receptor, an enhanced sst5 internalization has been action in inhibiting prolactin (PRL) and GH observed, thus showing that the sst5 C-terminal tail, secretion compared to the related mono-specific or at least a part of it, has an inhibitory role in analogs, either alone or in combination (Ferone et receptor internalization (Peverelli et al., 2008). al., 2007). A similar pattern of action has been Sst3, which shows high affinity for SS-14, observed for the anti-secretory and anti- internalizes efficiently after agonist stimulation proliferative activity in prostate and lung in vitro through a clathrin-dependent mediated pathway. models, where the treatment with the chimeric sst2- Without stimulation, sst3 is almost exclusively sst5 and sst2-sst5-D2R compounds were more located at the plasma membrane, whereas after effective than the respective mono-specific SS and agonist withdrawal only a small amount of sst3 is D2R analogs (Arvigo et al., 2010). This evidence recycled to the cell surface (Peverelli et al., 2008). suggests that the concurrent activation of different Hence, due to the differential expression of SS GPCRs triggers their dimerization, leading to an receptors in tumors, the comparison of their ability enhanced effect. to undergo agonist-induced desensitization and 2.2 Trafficking of somatostatin receptor internalization may provide important clues for the subtypes clinical use of SS analogs. In this context, an in A feature common to most GPCRs is the cyclic vitro study demonstrated that short-term process of signaling, desensitization, administration of the multiligand (sst1/sst2/sst3/sst5) internalization, resensitization, and recycling to the pasireotide (SOM230) modulates SS receptor plasma membrane. These events prevent cells from trafficking in a manner clearly distinct from undergoing excessive receptor stimulation or octreotide (sst2/sst5) (Tulipano and Schulz, 2007). periods of prolonged inactivity (van Koppen et al., SOM230 was less potent than octreotide in 2004). inducing signaling and internalization of the sst2 SS receptors differently internalize after agonist receptor. Whereas octreotide-activated sst2 binding and, specifically, sst2, sst3 and sst5 are receptors cointernalized with β-arrestin-2 into the internalized to a higher extent than sst1 or sst4. same endocytic vesicles, SOM230-mediated sst2 Among all subtypes, the agonist-mediated activation led to the formation of unstable trafficking of both sst2 splicing isoforms are the complexes that dissociated at or near the plasma mostly described (Jacobs and Schulz, 2008). membrane. Sst2 receptors recycled faster to the Investigations in neuroendocrine tumors showed plasma membrane in SOM230- than in octreotide- that both sst2A and sst2B isoforms are rapidly treated cells. The accelerated recycling of SOM230- desensitized and internalized after agonist-mediated activated receptors may counteract homologous phosphorylation. Receptor phosphorylation, which desensitization in sst2-expressing cells and, hence, involves sites located in the third intracellular loop result in longer lasting functional responses of and in the C-terminal tail, is followed by SOM230 (Lesche et al., 2009). recruitment of β-arrestin to the receptor forming a

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 807

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis

Figure 2. Schematic representation of a ligand-driven somatostatin receptor internalization. GRK: GPCR kinase; CCV: clathrin-coated vesicle (modified from van Koppen et al., 2004).

2.3 Somatostatin receptor signalling pathways exist about the inhibition of the MAPK signalling All five SS isoforms (ssts) bind/interact to G cascade by three of the five sst subtypes: sst2, sst3 proteins to activate their signalling pathways. They and sst5. In AtT-20 and in transfected CHO-K1 couple to all three Gi subunits (Gi1, Gi2, and Gi3) cells, sst5 constitutively restrains ERK1/2 leading to a potent inhibition of AC activation, and phosphorylation (Ben-Shlomo et al., 2007), and sst2 then of cyclic AMP (cAMP) synthesis. Specifically, and sst3 mediated the same inhibitory signal in sst1 is coupled to AC via Gi3; sst2A is able to SHSY-5Y neuroblastoma cells and in NIH3T3 associate with Gi1, Gi2, Gi3, and Gao2; sst3 interacts cells, respectively. Conversely, sst1 and sst4 with Gi1, Gi2, Gi4, and Gi6 (Reisine and Bell, 1995). stimulate the MAPK pathway (Patel, 1999). Several second messenger systems are involved in Glutamate receptor ion channels are also involved their downstream intracellular response: AC, in ssts signalling: sst2 inhibits AMPA/kainate 2+ + calcium (Ca ) and potassium (K ) ion channels, receptor-mediated glutamate currents, while sst1 sodium (Na+)/H+ antiporter, phospholipase C stimulates AMPA/kainate receptor activity in (PLC), phospholipase A2 (PLA2), mitogen cultured mouse hypothalamic neurons. activated protein kinase (MAPK), NO/cGMP, and Inositol 1,4,5-trisphosphate (IP3) represents another serine-, threonine-, and phosphotyrosyl-protein intracellular signalling pathway linked to sst2. In phosphatase (PTP) (Patel, 1999). CHO-DG44 cells, it takes place via sst2-mediated Sst2 and sst4 are the main receptors that activate activation of phosphatidylinositol 3-kinases (PI3K), voltage-gated K+ current (Yang and Chen, 2007). whereas in astrocytes and in intestinal smooth As a result of their activation, membrane muscle cells it is driven by PLC (Florio, 2008a). hyperpolarization occurs, hindering any subsequent Experimental data in rat pituitary F4C1 cells spontaneous membrane potential and leading to a indicate that the activation of sst2, but not sst1, 2+ reduction in intracellular Ca . Ssts can differently stimulates PLC activity and increases cytosolic modify Ca2+ currents; in AtT-20 murine cell line, Ca2+level, due to Ca2+ release from intracellular both sst2 and sst5 can couple negatively to an L-type stores (Rosskopf et al., 2003). Ca2+ channel reducing Ca2+ influx (Tallent et al., In hippocampal neurons, SS effect on PLA-2- 1996), whereas, conversely, in the GH3 rat pituitary dependent stimulation of arachidonate production 2+ tumor cell line, only sst2 blocks voltage-gated Ca has been associated with sst4, which is able to elicit current (Yang and Chen, 2007). arachidonate synthesis through phospholipase A2 The human ssts also stimulate PTP through a (PLA-2) activation (Patel, 1999). pertussis toxin-sensitive pathway involving Gi2, but In colon carcinoma, enteric endocrine and hepatic differencies among the various species have been cells, the Na+/H+ exchangers can be also activated found, since sst5 in rat does not regulate PTP. The by sst1, sst3 and sst4, but not by sst2 and sst5 (Florio, first evidence of ssts-mediated activation of PTP 2008a). was given by the counteraction driven by ssts on Interestingly, in human sst5 there are two regions, tyrosine kinase receptors-mediated proliferative the BBXXB domain and the DRY motif, located in effect (Florio, 2008a). One of the main downstream the third intracellular (i3) and second intracellular effects of ssts-mediated PTP activation is the (i2) loops, respectively, which are needed to inhibition of MAPK ERK1/2 activity. Several data activate the signalling pathways mediated by this

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 808

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis receptor subtype. Namely, the BBXXB domain, different receptors/signalling pathways and that the although being required in the subtype 5 antiproliferative effect of these synthetic downstream effectors generation, is not directly compounds may depend on tumor ssts profile, but involved in interactions with Gi protein, since a also on the specific target cell intracellular mutation in the first BBXXB residue does not affect signalling (Pyronnet et al., 2008). the receptor ability of inhibiting cAMP SS and SS analogs can control tumor development accumulation. Conversely, the DRY motif was and progression/metastatization by two separate found to be crucial in coupling with Gi protein, mechanisms: direct actions, mediated by the ssts, since mutations in the DRY sequence do not impair and indirect actions, independent of the receptors. sst5-driven inhibition of cAMP production. 3.1.1 Direct somatostatin antitumor actions However, both regions are necessary to mediate the Direct effects of SS and its analogs on tumor cell other sst5 intracellular responses, such as growth and spread derive from interaction with cytoplasmic Ca2+ reduction and inhibition of specific tumor cell membrane receptors. The direct ERK1/2 phosphorylation (Peverelli et al., 2009). antiproliferative actions include inhibition of autocrine/paracrine growth-promoting 3. Tumorigenesis hormone/growth factor synthesis, arrest of cell Tumorigenesis is a collection of complex genetic division (by blockade of growth factor-mediated diseases characterized by multiple defects in the mitogenic signals), suppression of cell invasion and homeostatic mechanisms that regulate cell growth, induction of apoptosis (programmed cell death) proliferation and differentiation. In humans, several (Pyronnet et al., 2008). The exact antitumoral lines of evidence indicate that tumorigenesis is a mechanism initiated by SS analogs depends on the multistep process which reflects genetic alterations tumor cell type and the ssts to which it binds. In this that drive the progressive transformation of normal way, each receptor subtype is able to mediate cells into highly malignant derivatives. different biological actions (Susini and Buscail, Tumorigenesis is thought to require four to six 2006). stochastic rate-limiting mutation events to occur in Cell cycle arrest is mediated by interaction of SS the lineage of one cell. Hanahan and Weinberg with its five receptors and the consequent initiation (Hanahan and Weinberg, 2000) suggest that six of several intracellular signalling pathways, which cellular alterations, or hallmarks, collectively drive are either activated or inhibited according to the sst a population of normal cells to become a cancer. subtype, the downstream recruited enzyme and cell The six hallmarks are (i) self-sufficiency in growth environment. These pathways include activation of signals (SG), (ii) insensitivity to antigrowth signals tyrosine kinases (JAK, c-src) and tyrosine (IA), (iii) evasion of apoptosis (EA), (iv) limitless phosphatases (SHP1, SHP2, PTP), replicative potential (LR), (v) sustained activation/inhibition of nitric oxide angiogenesis (SA), and (vi) tissue invasion and synthase/cGMP-dependent protein kinase, metastasis. Genetic instability (GI) is defined as an Ras/ERK pathway and inhibition of PI3 kinase/Akt "enabling characteristic" that facilitates the pathway, which in turn lead to induction of the acquisition of other mutations due to defects in cyclin-dependent kinase inhibitor p27kip1 or p21Cip1 DNA repair. These hallmarks form a candidate set and cell cycle arrest (Pyronnet et al., 2008). SS also of rules that underlie the transformation of a normal induces cell growth inhibition through restoration tissue to a cancerous one and are shared in common of functional gap junctions. These structures are by most and perhaps all types of human tumors composed of connexins and play a pivotal role in (Spencer et al., 2006). maintaining the differentiated state and cell-contact 3.1 Antitumor actions of somatostatin and inhibition. Actually, in most cancer cells, it has somatostatin analogs been observed an impaired expression of connexins SS has been shown to display several biological (Lahlou et al., 2005). It has been demonstrated that actions which include inhibition of exocrine and SS is also a potent anti-migratory and anti-invasive endocrine secretions, gut motility, cell proliferation, agent for various tumor cells. Inhibition of cell cell survival and angiogenesis. SS analogs show invasion occurs through molecular mechanisms antineoplastic and antiproliferative activity in many which are cell type specific and depend on sst experimental in vivo and in vitro models and this expression pattern, on sst effector coupling as well activity is principally attributed to activation of sst2 as on the signalling cascade involved in target cells and sst5. SS analogs treatment can be effective in (Pola et al., 2003). the control of tumor growth in humans and in 37- SS analogs are also thought to inhibit cell 82% of patients receiving SS analogs, as primary proliferation by inducing apoptosis. The receptor medical therapy, tumor shrinkage has been subtypes primarily involved in SS-induced observed. The antiproliferative and antitumoral apoptosis are sst3 and sst2. Apoptotic effect is effects of SS analogs occur independently of their achieved by regulation of the two main signalling antisecretory and antihormonal effects. From these pathways, the cell-extrinsic pathway (triggered by results we can infer that antisecretory and antitumor death receptors) and the cell-intrinsic pathway (also effects of SS and SS analogs are mediated by

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 809

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis called the mitochondrial pathway) (Pyronnet et al., inhibits cell viability in most NFPA primary 2008; Florio, 2008b). cultures, as well as BIM-23A760, a compound with SS and its chemically designed analogs are high affinity for DR2, sst2 and sst5 significantly potential therapeutic agents, in particular for the suppresses DNA synthesis in the 60% of the NFPA treatment of endocrine diseases that cause hormone cultures tested (Florio et al., 2008). hypersecretory syndromes. SS and its commercially It has been observed that SS and its analogs can available analogs exert antisecretory and decrease plasma calcitonin levels and improve antiproliferative effects by interacting with one or symptoms in patients with medullary thyroid more of the five ssts, which then trigger various carcinoma (MTC), but their antiproliferative effects intracellular signalling pathways according to the remain controversial. As a matter of fact, in TT tissue, thus possibly leading to different actions. cells, a human MTC cell line expressing all ssts, sst2 The tissue expression patterns of ssts, the binding activation leads to inhibition of DNA synthesis and profile of agonists and ssts effector coupling confer cell proliferation, whereas sst5 activation has an functional and therapeutic specificity to ligand opposite effect. Thus, we can infer that sst2 and sst5 activity (Zatelli and degli Uberti, 2009). agonists can antagonize the activity of one another The two SS analogs currently used in the clinics are in contrast to what happens in pituitary adenomas. Octreotide and Lanreotide. They have demonstrated Potent sst1-selective ligands (BIM-23296 and BIM- efficacy in reducing GH and IGF-1 levels in up to 23745) could have a therapeutic role in MTC 60% of patients with acromegaly and therefore have because they are effective in reducing DNA been widely used in the treatment of GH synthesis, the viability of TT cells, calcitonin hypersecretion (Shimon et al., 1997). The main secretion and gene expression (Zatelli et al., 2006). pharmacological target of these compounds is sst2, Ssts are also highly expressed in most the receptor subtype which is the most frequently neuroendocrine tumors with a variable expression expressed in human GH-secreting pituitary patterns. Treatment with Octreotide and Lanreotide adenomas, but they also bind, with a lesser affinity, is ineffective in inhibiting hormone secretion in to sst5. However, a significant proportion of patients some patients with neuroendocrine tumors because with acromegaly is resistant to the treatment with they develop tachyphylaxis. Conversely, pasireotide ocreotide. has shown a considerable reduction of symptoms in Pasireotide (SOM-230), a compound that interacts the majority of patients with metastatic with multiple ssts (sst1-2-3-5) is able to inhibit GH gastroenteropancreatic endocrine tumors (Desai et secretion in octreotide-resistant pituitary adenomas, al., 2009). representing a potential therapy for octreotide- Experimental data on prostate cancer showed that resistant acromegaly patients (Petersenn et al., four (sst1-2-3-5) out of five ssts receptors were found 2010). The efficacy of pasireotide in overcoming to be expressed in the LNCaP cell line, an in vitro octreotide resistance has been attributed to its model of human androgen-dependent PCa. Their ability of binding to all ssts and, in particular, to its activation by selective SS agonists resulted in a greater affinity for sst5, which is up-regulated in significant anti-proliferative effect with a peculiar such tumors. This ssts multiligand compound pattern according to receptor subtype, ligand showed in vitro a significant reduction of cell affinity and, possibly, receptor dimerization. viability in many non-functioning pituitary Moreover, such treatments were also able to adenomas (NFPAs) probably through the inhibition modulate the profile of the IGF system, known to of VEGF secretion. Several results suggested that be involved in PCa progression. Interestingly, these pasireotide could be a potential therapeutic agent data provide strong evidence for an inhibitory role for conditions characterized by an excess of ACTH. of sst1 activation on PCa cell proliferation, In patients with Cushing's disease, the suggesting that SS agonists with enhanced sst1 administration of pasireotide decreased urinary free affinity and selectivity may have great potentiality cortisol levels and significantly improved as pharmacological tools for at least androgen- symptoms associated with the disease. Moreover, in dependent PCa treatment. In addition, the ACTH-secreting pituitary tumor cells, pasireotide antiproliferative effect of sst1 and sst5 mono- reduced ACTH secretion and cell proliferation specific agonists may be due, at least in part, to the (Bode et al., 2010). NFPAs represent a possible inhibition of IGF-I secretion (Ruscica et al., 2010). therapeutic target also for selective sst1 agonists as 3.1.2 Indirect somatostatin antitumor mechanisms these tumors have been demonstrated to express Indirect antitumor effects of SS include suppression sst1. The sst1 agonist BIM-23926 has exhibited of synthesis or/and release of growth factors and antisecretory and antiproliferative effects in a group growth-promoting hormones such as insulin, of NFPAs in vitro. Moreover, several findings prolactin, IGF-1, epidermal growth factor (EGF), support the hypothesis that chimeric ssts/DR transforming growth factor- (TGF-), gastrin, agonists can be effective in suppressing in vitro cell cholecystokinin (CCK) and GH. proliferation in the majority of NFPAs. Indeed, Several experimental in vitro and in vivo results BIM-23A387, a chimeric sst2/DR2 selective agonist indicate that another indirect action of SS and SS

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 810

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis analogs on tumor growth may be the inhibition of Polak JM, Pearse AG, Grimelius L, Bloom SR. Growth- angiogenesis. Angiogenesis, the formation of new hormone release-inhibiting hormone in gastrointestinal and pancreatic D cells. Lancet. 1975 May 31;1(7918):1220-2 blood vessels from an existing capillary network, is necessary for tumor neovascularization, which is Konturek SJ, Tasler J, Obtulowicz W, Coy DH, Schally AV. Effect of growth hormone-release inhibiting hormone on essential for tumor growth, invasion and for hormones stimulating exocrine pancreatic secretion. J Clin dissemination of metastasis. By limiting the blood Invest. 1976 Jul;58(1):1-6 supply, tumor growth can be effectively controlled Shen LP, Pictet RL, Rutter WJ. Human somatostatin I: (Kvols and Woltering, 2006). SS and SS analogs sequence of the cDNA. Proc Natl Acad Sci U S A. 1982 exert antiangiogenic actions through different Aug;79(15):4575-9 mechanisms like suppression of endothelial cell Funckes CL, Minth CD, Deschenes R, Magazin M, proliferation and arrest of monocyte and endothelial Tavianini MA, Sheets M, Collier K, Weith HL, Aron DC, cell migration. Normal endothelial cells lack sst2 Roos BA, Dixon JE. Cloning and characterization of a receptors and the expression of this receptor mRNA-encoding rat preprosomatostatin. J Biol Chem. subtype on endothelial cells uniquely appears as 1983 Jul 25;258(14):8781-7 they proliferate to form new blood vessels (Kvols Yamada Y, Post SR, Wang K, Tager HS, Bell GI, Seino S. and Woltering, 2006). So, the inhibition of Cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in angiogenesis may result from the up-regulation of brain, gastrointestinal tract, and kidney. Proc Natl Acad Sci sst2 during the angiogenic switch from resting to U S A. 1992 Jan 1;89(1):251-5 proliferating endothelium. However, other sst such s Corness JD, Demchyshyn LL, Seeman P, Van Tol HH, as sst3 and sst5 may also play a role. At the Srikant CB, Kent G, Patel YC, Niznik HB. A human molecular level, this effect results from SS- somatostatin receptor (SSTR3), located on chromosome mediated inhibition of MAP kinase activity and 22, displays preferential affinity for somatostatin-14 like endothelial NO synthase (eNOS) activity. Another peptides. FEBS Lett. 1993 Apr 26;321(2-3):279-84 mechanism by which SS analogs suppress Rohrer L, Raulf F, Bruns C, Buettner R, Hofstaedter F, angiogenesis is through a broad inhibition of both Schüle R. Cloning and characterization of a fourth human somatostatin receptor. Proc Natl Acad Sci U S A. 1993 the release and the effect of growth factors, some of May 1;90(9):4196-200 which are angiogenic, including vascular endothelial growth factor (VEGF), platelet-derived Yamada Y, Stoffel M, Espinosa R 3rd, Xiang KS, Seino M, Seino S, Le Beau MM, Bell GI. Human somatostatin growth factor, IGF-1 and basic fibroblast growth receptor genes: localization to human chromosomes 14, factor. These growth factors, secreted by tumor 17, and 22 and identification of simple tandem repeat cells and infiltrating inflammatory cells, stimulate polymorphisms. Genomics. 1993 Feb;15(2):449-52 endothelial and smooth muscle cell proliferation Panetta R, Greenwood MT, Warszynska A, Demchyshyn and migration, important processes in angiogenesis LL, Day R, Niznik HB, Srikant CB, Patel YC. Molecular (Zatelli et al., 2007). cloning, functional characterization, and chromosomal localization of a human somatostatin receptor 4. Somatostatin receptor (somatostatin receptor type 5) with preferential affinity for activation and tumorigenesis: somatostatin-28. Mol Pharmacol. 1994 Mar;45(3):417-27 Reisine T, Bell GI. Molecular biology of somatostatin future directions receptors. Endocr Rev. 1995 Aug;16(4):427-42 According to the evidence reported in the present Tallent M, Liapakis G, O'Carroll AM, Lolait SJ, Dichter M, paper, a specific pattern of ssts activation seems to Reisine T. Somatostatin receptor subtypes SSTR2 and elicit important antitumoral actions with potential SSTR5 couple negatively to an L-type Ca2+ current in the relevance to some solid tumors expressing these pituitary cell line AtT-20. Neuroscience. 1996 Apr;71(4):1073-81 receptor isoforms. In addition to the well- established anti-secretory effects, which may affect Shimon I, Yan X, Taylor JE, Weiss MH, Culler MD, Melmed S. Somatostatin receptor (SSTR) subtype- the cancer-associated paraneoplastic syndrome as selective analogues differentially suppress in vitro growth well as the possible autotrophic actions of tumor- hormone and prolactin in human pituitary adenomas. produced secretory proteins, a consistent body of Novel potential therapy for functional pituitary tumors. J data indicates that stimulation of tumor-expressed Clin Invest. 1997 Nov 1;100(9):2386-92 ssts results in a multi-step restrain of tumorigenesis. Patel YC. Somatostatin and its receptor family. Front These mechanisms thus deserve further exploitation Neuroendocrinol. 1999 Jul;20(3):157-98 with the aim of validating novel therapeutic Baumeister H, Meyerhof W. Gene regulation of approaches to cancer. somatostatin receptors in rats. J Physiol Paris. 2000 May- Aug;94(3-4):167-77 References Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000 Jan 7;100(1):57-70 Brazeau P, Vale W, Burgus R, Ling N, Butcher M, Rivier J, Guillemin R. Hypothalamic polypeptide that inhibits the Saveanu A, Gunz G, Dufour H, Caron P, Fina F, Ouafik L, secretion of immunoreactive pituitary growth hormone. Culler MD, Moreau JP, Enjalbert A, Jaquet P. Bim-23244, Science. 1973 Jan 5;179(68):77-9 a somatostatin receptor subtype 2- and 5-selective analog with enhanced efficacy in suppressing growth hormone (GH) from octreotide-resistant human GH-secreting adenomas. J Clin Endocrinol Metab. 2001 Jan;86(1):140-5

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 811

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis

Brakch N, Lazar N, Panchal M, Allemandou F, Boileau G, Ferone D, Saveanu A, Culler MD, Arvigo M, Rebora A, Cohen P, Rholam M. The somatostatin-28(1-12)-NPAMAP Gatto F, Minuto F, Jaquet P. Novel chimeric somatostatin sequence: an essential helical-promoting motif governing analogs: facts and perspectives. Eur J Endocrinol. 2007 prosomatostatin processing at mono- and dibasic sites. Apr;156 Suppl 1:S23-8 Biochemistry. 2002 Feb 5;41(5):1630-9 Tulipano G, Schulz S. Novel insights in somatostatin Kreienkamp HJ, Larusson HJ, Witte I, Roeder T, Birgul N, receptor physiology. Eur J Endocrinol. 2007 Apr;156 Suppl Honck HH, Harder S, Ellinghausen G, Buck F, Richter D. 1:S3-11 Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster Yang SK, Chen C. Involvement of somatostatin receptor and their ligands by reverse pharmacology. J Biol Chem. subtypes in membrane ion channel modification by 2002 Oct 18;277(42):39937-43 somatostatin in pituitary somatotropes. Clin Exp Pharmacol Physiol. 2007 Dec;34(12):1221-7 Møller LN, Stidsen CE, Hartmann B, Holst JJ. Somatostatin receptors. Biochim Biophys Acta. 2003 Sep Zatelli MC, Piccin D, Vignali C, Tagliati F, Ambrosio MR, 22;1616(1):1-84 Bondanelli M, Cimino V, Bianchi A, Schmid HA, Scanarini M, Pontecorvi A, De Marinis L, Maira G, degli Uberti EC. Pola S, Cattaneo MG, Vicentini LM. Anti-migratory and Pasireotide, a multiple somatostatin receptor subtypes anti-invasive effect of somatostatin in human ligand, reduces cell viability in non-functioning pituitary neuroblastoma cells: involvement of Rac and MAP kinase adenomas by inhibiting vascular endothelial growth factor activity. J Biol Chem. 2003 Oct 17;278(42):40601-6 secretion. Endocr Relat Cancer. 2007 Mar;14(1):91-102 Rosskopf D, Schürks M, Manthey I, Joisten M, Busch S, Florio T. Molecular mechanisms of the antiproliferative Siffert W. Signal transduction of somatostatin in human B activity of somatostatin receptors (SSTRs) in lymphoblasts. Am J Physiol Cell Physiol. 2003 neuroendocrine tumors. Front Biosci. 2008 Jan 1;13:822- Jan;284(1):C179-90 40 Canosa LF, Cerdá-Reverter JM, Peter RE. Brain mapping Florio T. Somatostatin/somatostatin receptor signalling: of three somatostatin encoding genes in the goldfish. J phosphotyrosine phosphatases. Mol Cell Endocrinol. 2008 Comp Neurol. 2004 Jun 14;474(1):43-57 May 14;286(1-2):40-8 Olias G, Viollet C, Kusserow H, Epelbaum J, Meyerhof W. Florio T, Barbieri F, Spaziante R, Zona G, Hofland LJ, van Regulation and function of somatostatin receptors. J Koetsveld PM, Feelders RA, Stalla GK, Theodoropoulou Neurochem. 2004 Jun;89(5):1057-91 M, Culler MD, Dong J, Taylor JE, Moreau JP, Saveanu A, Gunz G, Dufour H, Jaquet P. Efficacy of a dopamine- Slagter BJ, Sheridan MA. Differential expression of two somatostatin chimeric molecule, BIM-23A760, in the somatostatin receptor subtype 1 mRNAs in rainbow trout control of cell growth from primary cultures of human non- (Oncorhynchus mykiss). J Mol Endocrinol. 2004 functioning pituitary adenomas: a multi-center study. Feb;32(1):165-77 Endocr Relat Cancer. 2008 Jun;15(2):583-96 van Koppen CJ, Jakobs KH. Arrestin-independent Jacobs S, Schulz S. Intracellular trafficking of somatostatin internalization of G protein-coupled receptors. Mol receptors. Mol Cell Endocrinol. 2008 May 14;286(1-2):58- Pharmacol. 2004 Sep;66(3):365-7 62 Lahlou H, Fanjul M, Pradayrol L, Susini C, Pyronnet S. Peverelli E, Mantovani G, Calebiro D, Doni A, Bondioni S, Restoration of functional gap junctions through internal Lania A, Beck-Peccoz P, Spada A. The third intracellular ribosome entry site-dependent synthesis of endogenous loop of the human somatostatin receptor 5 is crucial for connexins in density-inhibited cancer cells. Mol Cell Biol. arrestin binding and receptor internalization after 2005 May;25(10):4034-45 somatostatin stimulation. Mol Endocrinol. 2008 Kvols LK, Woltering EA. Role of somatostatin analogs in Mar;22(3):676-88 the clinical management of non-neuroendocrine solid Pyronnet S, Bousquet C, Najib S, Azar R, Laklai H, Susini tumors. Anticancer Drugs. 2006 Jul;17(6):601-8 C. Antitumor effects of somatostatin. Mol Cell Endocrinol. Spencer SL, Gerety RA, Pienta KJ, Forrest S. Modeling 2008 May 14;286(1-2):230-7 somatic evolution in tumorigenesis. PLoS Comput Biol. Desai KK, Khan MS, Toumpanakis C, Caplin ME. 2006 Aug 18;2(8):e108 Management of gastroentero-pancreatic neuroendocrine Susini C, Buscail L. Rationale for the use of somatostatin tumors (GEP-NETs). Minerva Gastroenterol Dietol. 2009 analogs as antitumor agents. Ann Oncol. 2006 Dec;55(4):425-43 Dec;17(12):1733-42 Lesche S, Lehmann D, Nagel F, Schmid HA, Schulz S. Zatelli MC, Piccin D, Tagliati F, Bottoni A, Luchin A, Vignali Differential effects of octreotide and pasireotide on C, Margutti A, Bondanelli M, Pansini GC, Pelizzo MR, somatostatin receptor internalization and trafficking in vitro. Culler MD, Degli Uberti EC. Selective activation of J Clin Endocrinol Metab. 2009 Feb;94(2):654-61 somatostatin receptor subtypes differentially modulates Peverelli E, Lania AG, Mantovani G, Beck-Peccoz P, secretion and viability in human medullary thyroid Spada A. Characterization of intracellular signaling carcinoma primary cultures: potential clinical perspectives. mediated by human somatostatin receptor 5: role of the J Clin Endocrinol Metab. 2006 Jun;91(6):2218-24 DRY motif and the third intracellular loop. Endocrinology. Baragli A, Alturaihi H, Watt HL, Abdallah A, Kumar U. 2009 Jul;150(7):3169-76 Heterooligomerization of human dopamine receptor 2 and Zatelli MC, degli Uberti E. The significance of new somatostatin receptor 2 Co-immunoprecipitation and somatostatin analogs as therapeutic agents. Curr Opin fluorescence resonance energy transfer analysis. Cell Investig Drugs. 2009 Oct;10(10):1025-31 Signal. 2007 Nov;19(11):2304-16 Arvigo M, Gatto F, Ruscica M, Ameri P, Dozio E, Albertelli Ben-Shlomo A, Pichurin O, Barshop NJ, Wawrowsky KA, M, Culler MD, Motta M, Minuto F, Magni P, Ferone D. Taylor J, Culler MD, Chesnokova V, Liu NA, Melmed S. Somatostatin and dopamine receptor interaction in Selective regulation of somatostatin receptor subtype prostate and lung cancer cell lines. J Endocrinol. 2010 signaling: evidence for constitutive receptor activation. Mol Dec;207(3):309-17 Endocrinol. 2007 Oct;21(10):2565-78

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 812

Somatostatin (SS), SS receptors and SS analog treatment in Steffani L, et al. tumorigenesis

Bode H, Seiz M, Lammert A, Brockmann MA, Back W, Ruscica M, Arvigo M, Gatto F, Dozio E, Feltrin D, Culler Hammes HP, Thomé C. SOM230 (pasireotide) and MD, Minuto F, Motta M, Ferone D, Magni P. Regulation of temozolomide achieve sustained control of tumour prostate cancer cell proliferation by somatostatin receptor progression and ACTH secretion in pituitary carcinoma activation. Mol Cell Endocrinol. 2010 Feb 5;315(1-2):254- with widespread metastases. Exp Clin Endocrinol 62 Diabetes. 2010 Nov;118(10):760-3 This article should be referenced as such: Petersenn S, Schopohl J, Barkan A, Mohideen P, Colao A, Abs R, Buchelt A, Ho YY, Hu K, Farrall AJ, Melmed S, Steffani L, Passafaro L, Ferone D, Magni P, Ruscica M. Biller BM. Pasireotide (SOM230) demonstrates efficacy Somatostatin (SS), SS receptors and SS analog treatment and safety in patients with acromegaly: a randomized, in tumorigenesis. Atlas Genet Cytogenet Oncol Haematol. multicenter, phase II trial. J Clin Endocrinol Metab. 2010 2011; 15(9):805-813. Jun;95(6):2781-9

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 813

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Case Report Section Paper co-edited with the European LeukemiaNet

A new case of translocation t(14;14)(q11;q32) in B lineage ALL Elvira D Rodrigues Pereira Velloso, Priscila Pereira dos Santos Teixeira, Karina Prandi Melillo, Luciana J Rodrigues da Silva, Cristina Alonso Ratis, Daniela Borri, Cristóvão LP Mangueira Clinical Laboratory, Hospital Israelita Albert Einstein, Sao Paulo, Brazil (EDRPV, PPdST, LJRdS, CAR, DB, CLPM); CASE Intermedica, Sao Paulo, Brazil (KPM)

Published in Atlas Database: February 2011 Online updated version : http://AtlasGeneticsOncology.org/Reports/t1414BALLVellosoID100048.html Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t1414BALLVellosoID100048.txt

Clinics Electron microscopy not done Age and sex Diagnosis 43 years old male patient. common B-ALL Previous history no preleukemia ; no previous malignancy ; no Survival inborn condition of note. Date of diagnosis: 02-2009 Organomegaly hepatomegaly , splenomegaly , no enlarged lymph Treatment: Cancer and Leukemia Group B nodes , central nervous system involvement. (CALGB) protocol Complete remission : not evaluated Blood Treatment related death : Neutropenia and lung infection 9 WBC : 227X 10 /l Relapse : no HB : 5,9g/dl Status: Death. Last follow up: 03-2009 Platelets : 51X 109/l Blasts : 12% Survival: 20 days Bone marrow : aspirate: 90% lymphoblast% Karyotype Cyto-Pathology Sample: Bone marrow Classification Culture time: 24 and 48 hours without stimulating agents Cytology Banding: G LLA-L2 Results: 46,XY,t(14;14)(q11;q32.1)[20] Immunophenotype HLA-DR+, TdT+, CD79a+, CD19+, cyCD22+, Karyotype at Relapse: not done CD20+, CD10+ Other molecular cytogenetics technics Rearranged Ig Tcr FISH using IGH Break Apart Rearrangement rearranged IGH (FISH) Probe, Vysis Pathology Other molecular cytogenetics results not done nuc ish(IGHx2)(5'IGH sep 3'IGHx1)[154/200]/ (5'IGHx2,3'IGHx1)(5'IGH con 3'IGHx1)[33/200]

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 814

A new case of translocation t(14;14)(q11;q32) in B lineage Rodrigues Pereira Velloso ED, et al. ALL

Han et al showed through FISH analysis the presence of trisomy 4 as a simultaneous involvement of IGH and CEPBE genes. The t(14;14)(q11;q32) CEBPE/IGH may be associated with good prognosis in B-ALL. In 4 cases with clinical follow-up, complete remission G- banded partial karyotypes showing the t(14;14). was achieved and those were alive at the time of report. In the case described herein, the t(14;14) was the sole anomaly, IGH rearrangement was detected but CEBPE involvement was not studied. This patient has well known bad prognostic features as high WBC count and CNS involvement and died few days after diagnosis. References Liu S, Bo L, Liu X, Li C, Qin S, Wang J. IGH gene involvement in two cases of acute lymphoblastic leukemia with t(14;14)(q11;q32) identified by sequential R-banding and fluorescence in situ hybridization. Cancer Genet Cytogenet. 2004 Jul 15;152(2):141-5 Akasaka T, Balasas T, Russell LJ, Sugimoto KJ, Majid A, Walewska R, Karran EL, Brown DG, Cain K, Harder L, Gesk S, Martin-Subero JI, Atherton MG, Brüggemann M, Calasanz MJ, Davies T, Haas OA, Hagemeijer A, Kempski H, Lessard M, Lillington DM, Moore S, Nguyen-Khac F, Radford-Weiss I, Schoch C, Struski S, Talley P, Welham MJ, Worley H, Strefford JC, Harrison CJ, Siebert R, Dyer MJ. Five members of the CEBP transcription factor family are targeted by recurrent IGH translocations in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Blood. 2007 Apr 15;109(8):3451-61 Interphase FISH shows IGH gene rearrangements (IGH Han Y, Xue Y, Zhang J, Wu Y, Pan J, Wang Y, Shen J, Dual Color, Break Apart Rearrangement probe). Dai H, Bai S. Translocation (14;14)(q11;q32) with simultaneous involvement of the IGH and CEBPE genes in B-lineage acute lymphoblastic leukemia. Cancer Genet Comments Cytogenet. 2008 Dec;187(2):125-9

Translocation t(14;14)(q11;q32) in B lineage acute This article should be referenced as such: lymphoblastic leukemia was described in few cases, some of them associated with other recurrent Rodrigues Pereira Velloso ED, Pereira dos Santos Teixeira P, Prandi Melillo K, Rodrigues da Silva LJ, Alonso rearrangements such as t(4;11) and t(8;14). Lui et Ratis C, Borri D, Mangueira CLP. A new case of al, in 2004 showed IGH rearrangement in two translocation t(14;14)(q11;q32) in B lineage ALL. Atlas cases, although the partner was unknown. Akasaka Genet Cytogenet Oncol Haematol. 2011; 15(9):814-815. et al in 2007, described CEBPE involvement in a patient with B-ALL and t(14;14)(q11;q32). In 2008,

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 815

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Instructions to Authors Manuscripts submitted to the Atlas must be submitted solely to the Atlas. Iconography is most welcome: there is no space restriction. The Atlas publishes "cards", "deep insights", "case reports", and "educational items". Cards are structured review articles. Detailed instructions for these structured reviews can be found at: http://AtlasGeneticsOncology.org/Forms/Gene_Form.html for reviews on genes, http://AtlasGeneticsOncology.org/Forms/Leukaemia_Form.html for reviews on leukaemias, http://AtlasGeneticsOncology.org/Forms/SolidTumour_Form.html for reviews on solid tumours, http://AtlasGeneticsOncology.org/Forms/CancerProne_Form.html for reviews on cancer-prone diseases. According to the length of the paper, cards are divided, into "reviews" (texts exceeding 2000 words), "mini reviews" (between), and "short communications" (texts below 400 words). The latter category may not be accepted for indexing by bibliographic databases. Deep Insights are written as traditional papers, made of paragraphs with headings, at the author's convenience. No length restriction. Case Reports in haematological malignancies are dedicated to recurrent -but rare- chromosomes abnormalities in leukaemias/lymphomas. Cases of interest shall be: 1- recurrent (i.e. the chromosome anomaly has already been described in at least 1 case), 2- rare (previously described in less than 20 cases), 3- with well documented clinics and laboratory findings, and 4- with iconography of chromosomes. It is mandatory to use the specific "Submission form for Case reports": see http://AtlasGeneticsOncology.org/Reports/Case_Report_Submission.html. Educational Items must be didactic, give full information and be accompanied with iconography. Translations into French, German, Italian, and Spanish are welcome.

Subscription: The Atlas is FREE!

Corporate patronage, sponsorship and advertising Enquiries should be addressed to [email protected].

Rules, Copyright Notice and Disclaimer Conflicts of Interest: Authors must state explicitly whether potential conflicts do or do not exist. Reviewers must disclose to editors any conflicts of interest that could bias their opinions of the manuscript. The editor and the editorial board members must disclose any potential conflict. Privacy and Confidentiality – Iconography: Patients have a right to privacy. Identifying details should be omitted. If complete anonymity is difficult to achieve, informed consent should be obtained. Property: As "cards" are to evolve with further improvements and updates from various contributors, the property of the cards belongs to the editor, and modifications will be made without authorization from the previous contributor (who may, nonetheless, be asked for refereeing); contributors are listed in an edit history manner. Authors keep the rights to use further the content of their papers published in the Atlas, provided that the source is cited. Copyright: The information in the Atlas of Genetics and Cytogenetics in Oncology and Haematology is issued for general distribution. All rights are reserved. The information presented is protected under international conventions and under national laws on copyright and neighbouring rights. Commercial use is totally forbidden. Information extracted from the Atlas may be reviewed, reproduced or translated for research or private study but not for sale or for use in conjunction with commercial purposes. Any use of information from the Atlas should be accompanied by an acknowledgment of the Atlas as the source, citing the uniform resource locator (URL) of the article and/or the article reference, according to the Vancouver convention. Reference to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favouring. The views and opinions of contributors and authors expressed herein do not necessarily state or reflect those of the Atlas editorial staff or of the web site holder, and shall not be used for advertising or product endorsement purposes. The Atlas does not make any warranty, express or implied, including the warranties of merchantability and fitness for a particular purpose, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, and shall not be liable whatsoever for any damages incurred as a result of its use. In particular, information presented in the Atlas is only for research purpose, and shall not be used for diagnosis or treatment purposes. No responsibility is assumed for any injury and/or damage to persons or property for any use or operation of any methods products, instructions or ideas contained in the material herein. See also: "Uniform Requirements for Manuscripts Submitted to Biomedical Journals: Writing and Editing for Biomedical Publication - Updated October 2004": http://www.icmje.org.

http://AtlasGeneticsOncology.org

Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS