Number 3 March 2015

Atlas of Genetics and Cytogenetics in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Volume 19 - Number 3 March 2015

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 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, and also more traditional review articles (“deep insights”) on the above subjects and on surrounding topics. It also present 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 [email protected] or [email protected]

Staff Mohammad Ahmad, Mélanie Arsaban, Marie-Christine Jacquemot-Perbal, Vanessa Le Berre, Anne Malo, Carol Moreau, Catherine Morel-Pair, Laurent Rassinoux, Alain Zasadzinski. Philippe Dessen is the Database Director (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

OPEN ACCESS JOURNAL 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 Antonio Cuneo (Ferrara, Italy) Leukaemia Section Paola Dal Cin (Boston, Massachussetts) Genes / Solid Tumours Section Brigitte Debuire (Villejuif, France) Deep Insights Section Marc De Braekeleer (Brest, France) Genes / Leukaemia Sections 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 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 Adriana Zamecnikova (Kuwait) Leukaemia Section

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) Atlas of Genetics and Cytogenetics in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Volume 19, Number 3, March 2015

Table of contents

Gene Section

CASP7 (caspase 7, apoptosis-related cysteine peptidase) 160 Cláudia Malheiros Coutinho-Camillo, Fernando Augusto Soares GADD45A (growth arrest and DNA-damage-inducible, alpha) 164 Sirma Damla User, Mesut Muyan ID2 (inhibitor of DNA binding 2, dominant negative helix-loop-helix protein) 168 Menno C van Zelm KLRK1 (killer cell lectin-like receptor subfamily K, member 1) 172 Lewis L Lanier ORAI3 (ORAI calcium release-activated calcium modulator 3) 176 Jessy Hasna, Nazim Benzerdjeb, Malika Faouzi, Anne-Sophie Ay, Philippe Kischel, Frédéric Hague, Henri Sevestre, Ahmed Ahidouch, Halima Ouadid-Ahidouch PCSK4 (proprotein convertase subtilisin/kexin type 4) 189 Majid Khatib, Beatrice Demoures PCSK5 (proprotein convertase subtilisin/kexin type 5) 191 Majid Khatib, Beatrice Demoures PDGFRA (platelet-derived growth factor receptor, alpha polypeptide) 194 Adriana Zamecnikova, Soad Al Bahar PF4V1 (platelet factor 4 variant 1) 198 Katrien Van Raemdonck, Paul Proost, Jo Van Damme, Sofie Struyf SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), member 3) 202 Cristian Turato, Patrizia Pontisso SNCG (synuclein, gamma (breast cancer-specific protein 1)) 210 Andrei Surguchov TWIST2 (twist family bHLH transcription factor 2) 217 Daniela Gasparotto, Erica Lorenzetto

Leukaemia Section t(9;17)(p13;p12) PAX5/NCOR1 225 Jean-Loup Huret t(11;14)(p15;q22) AP2A2/NID2 227 Nathalie Douet-Guilbert, Etienne De Braekeleer, Corinne Tous, Nadia Guéganic, Audrey Basinko, Marie-Josée Le Bris, Frédéric Morel, Marc De Braekeleer

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3)

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Solid Tumour Section

Lung: Translocations in Small Cell Carcinoma 230 Jean-Loup Huret

Deep Insight Section

FOXP3 expression in tumor cells and its role in cancer progression 234 Valentina Uva, Lucia Sfondrini, Tiziana Triulzi, Patrizia Casalini, Elda Tagliabue, Andrea Balsari

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3)

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

CASP7 (caspase 7, apoptosis-related cysteine peptidase) Cláudia Malheiros Coutinho-Camillo, Fernando Augusto Soares Department of Anatomic Pathology, AC Camargo Cancer Center, Sao Paulo, Brazil (CMCC, FAS)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/CASP7ID924ch10q25.html DOI: 10.4267/2042/56404 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

apoptosis (Cohen, 1997; Denault and Salvesen, Abstract 2002; Boatright and Salvesen, 2003). Apoptosis is a selective process for deleting cells in Caspase-7 is highly related to caspase-3, and these various biological systems and plays an essential two caspases are activated by both death receptor- role in the development and maintenance of tissue and mitochondria-induced apoptosis (Soung et al., homeostasis in multicellular organisms and 2003). inappropriate regulation of apoptosis is believed to Besides its activation during apoptosis, proteolytic be the cause of many human diseases, including maturation of caspase-7 has also been observed cancer (Thompson, 1995). under inflammatory conditions (Lamkanfi and Apoptosis relies on cysteine proteases called Kanneganti, 2010). caspases (CASPs). Caspases are synthesized as Keywords proforms and become activated by cleavage at Caspase-7, apoptosis, cancer, SNPs aspartate residues. Initiator caspases (1, 2, 4, 5, 8, 9, 10, 11, and 12) integrate molecular signals and Identity activate the downstream effector caspases (3, 6, 7, and 14). Because caspases cleave and activate each Other names: CASP-7, CMH-1, ICE-LAP3, other, the protease cascade amplifies, ensuring LICE2, MCH3 proper apoptotic cell death. In addition, caspases HGNC (Hugo): CASP7 cleave numerous substrates, such as nuclear lamins, inhibitors of DNase, and cytoskeletal proteins, Location: 10q25.3 resulting in the typical morphological alterations of Local order: Plus strand.

CASP7 transcript variants. Coding exons are marked by blue blocks, and non-coding exons, 5- and 3-UTRs are marked by white blocks.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 160 CASP7 (caspase 7, apoptosis-related cysteine peptidase) Coutinho-Camillo CM, Soares FA

Structure of procaspase-7. Numbers below the stick represents the approximate MWs of the resulting subunits and pro- regions. The proenzymes are cleaved at specific Asp residues (Dn, where n is the position in the protein).

DNA/RNA Mutations Description Soung et al. (2003) detected CASP7 mutations in 2 of 98 colon carcinomas (2%), 1 of 50 esophageal CASP7 gene contains 8 exons and spans 51.748 Kb carcinomas (2%), and 1 of 33 head/neck of genomic DNA. carcinomas (3%). Transcription Expression of the tumor-derived CASP7 mutants in CASP7 gene has 9 transcripts (splice variants): 293T cells showed that apoptosis was reduced - CASP7-201: 2694 bp (8 exons; 7 coding exons) compared to the wild-type caspase-7, suggesting - CASP7-005: 2659 bp (8 exons; 6 coding exons) that inactivating mutations of CASP7 might - CASP7-002: 2421 bp (8 exons; 6 coding exons) contribute to the pathogenesis of some human solid - CASP7-003: 2380 bp (8 exons; 7 coding exons) cancers. - CASP7-001: 2377 bp (7 exons; 6 coding exons) Genetic polymorphisms in the CASP7 gene may - CASP7-202: 1148 bp (6 exons; 6 coding exons) affect cancer risk through altering expression levels - CASP7-004: 834 bp (6 exons; 5 coding exons) and functions of this gene. Several polymorphisms - CASP7-007: 607 bp (3 exons; 0 coding exons) have been associated with susceptibility of cancer - CASP7-008: 799 bp (5 exons; 0 coding exons). development, as will be discussed later (Yan et al., 2013). Pseudogene A detailed list of genetic variations could be found Not identified. at: Ensembl. Protein Implicated in Description Lung cancer Caspase-7 is an effector caspase and plays an Note central role in the execution phase of the apoptosis. Lee et al. (2009) showed that the CASP7 rs2227310 Expression g.C>G polymorphism was associated with the risk of lung cancer. Caspase-7 is widely expressed in human tissues. Yoo et al. (2009) also demonstrated that the CASP7 Highly expressed in lung, skeletal muscle, liver, rs2227310 polymorphism may affect survival in kidney, spleen and heart, and moderately in testis. early-stage non-small cell lung cancer (NSCLC), Localisation suggesting that the analysis of this polymorphism Mainly in the cytoplasm, but also observed in the can help identify patients at high risk for a poor nucleus. disease outcome. Qian et al. (2012) also provided evidence that Function genetic variations of CASP7 may modulate overall Effector caspases are responsible for initiating the survival and progression-free survival of patients hallmarks of the degradation phase of apoptosis, with advanced NSCLC treated with platinum-based including DNA fragmentation, cell shrinkage and chemotherapy. membrane blebbing. Esophageal cancer Besides its activation during apoptosis, proteolytic maturation of caspase-7 has also been observed Note under inflammatory conditions. Liu et al. (2010) described that polymorphisms in CASP7 gene was associated with increased risk of Homology esophageal cancer. CASP7 (P. troglodytes, M. mulatta, C. lupus, B. taurus, G. gallus), Casp7 (M. musculus, R. Childhood leukemia norvegicus), casp7 (X. tropicalis, D. rerio), Ice (D. Note melanogaster), Dcp-1 (D. melanogaster), CASPS7 Park et al. (2012) suggested that three SNPs in (A. gambiae). CASP7 acts as a strong apoptosis signal that block

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 161 CASP7 (caspase 7, apoptosis-related cysteine peptidase) Coutinho-Camillo CM, Soares FA

or delay the apoptosis of childhood leukemia cancer Huntington's disease cells. Note Colorectal cancer Hermel et al. (2004) reported that caspase-7 Note immunoreactivity in post-mortem tissue from Palmerini et al. (2001) described a loss of caspase-7 Huntington's disease (HD) patients is dramatically in 84% of colon cancers, suggesting that caspase-7 enhanced in the medium spiny neurons of the deficiency might be used as a new caudate nucleus and neurons in the putamen when immunohistochemical marker of colonic neoplasia. compared to age-matched controls. Chae et al. (2011) reported that CASP7 rs2227310 Caspase-7 is able to bind full-length huntingtin polymorphism may be useful marker to predict the (Htt), accelerating the production of Htt fragments prognosis of patients with surgically resected and resulting in the eventual induction of apoptosis colorectal cancer. both in the neuronal processes and somata. Gastric cancer Rheumatoid arthritis Note Note Yoo et al. (2004) observed loss of capase-2, capase- CASP-7 gene is associated with the susceptibility to rheumatoid arthritis (RA). Genotyping of three 6 and capase-7 expression in gastric cancers single nucleotide polymorphisms (SNPs) of the irrespective of depth of invasion and histological CASP7 gene: rs11593766 (G/T), rs2227310 (C/G) subtypes suggesting a role in the development of gastric cancers. and rs2227309 (G/A) revealed that rs2227309 SNP was found to be associated with susceptibility to Endometrial cancer RA. Note Frequency of the G allele was significantly higher The AA genotype of rs11196445b, the CC among RA patients and a higher frequency of GG genotype of rs3124740, and the GG genotype of homozygous individuals was found in the RA rs10787498 in the CASP7 gene were associated patient group (Garcia-Lozano et al., 2007). with increased risk compared with homozygotes of Teixeira et al. (2008) found that CASP7 rs2227309 the major alleles, suggesting that genetic variants in SNP was not associated with RA in a European CASP7 may play a role in endometrial cancer Caucasian population. Nevertheless, CASP7 susceptibility in a Chinese population (Xu et al., isoforms alpha and beta could be involved in the 2009). apoptosis process in RA. Renal carcinoma Insulin-dependent diabetes mellitus Note Note Vilella-Arias et al. (2013) reported loss of CASP7 Babu et al. (2003) studied 18 SNPs in CASP7 and protein expression in renal cell carcinoma clear cell reported that 1 (SNP144692) differed significantly subtype (ccRCC) and this loss was associated with in frequency in the haplotypes found in affected the agressiveness of ccRCC, suggesting the individuals compared to control Bedouin Arab potential use of CASP7 as a prognostic marker. family haplotypes. This same SNP showed evidence of association with diabetes in a subset of Oral squamous cell carcinoma patients (DR3/DR4*0302) from Human Biological Note Data Interchange (HBDI) families, although these Coutinho-Camillo et al. (2011) reported that high results are in conflict with other studies. expression level of CASP7 protein was associated with poor prognosis in oral squamous cell References carcinoma (OSCC) patients. Thompson CB. Apoptosis in the pathogenesis and Alzheimer's disease treatment of disease. Science. 1995 Mar 10;267(5203):1456-62 Note Cohen GM. Caspases: the executioners of apoptosis. Elevated mRNA levels of caspases-7 and 8 Biochem J. 1997 Aug 15;326 ( Pt 1):1-16 measured by a quantitative PCR method were observed in the Alzheimer's disease (AD) temporal Palmerini F, Devilard E, Jarry A, Birg F, Xerri L. Caspase 7 downregulation as an immunohistochemical marker of neocortex as compared to the control brains, colonic carcinoma. Hum Pathol. 2001 May;32(5):461-7 suggesting that the transcriptional activation of key Denault JB, Salvesen GS. Caspases: keys in the ignition components of the apoptotic cascade correlates with of cell death. Chem Rev. 2002 Dec;102(12):4489-500 accumulation of Abeta42. Thus, a principal caspase pathway from caspase-8 Babu SR, Bao F, Roberts CM, Martin AK, Gowan K, Eisenbarth GS, Fain PR. Caspase 7 is a positional to caspase-3 and/or 7 may contribute to neuron loss candidate gene for IDDM 17 in a Bedouin Arab family. Ann in AD brain (Matsui et al., 2006). N Y Acad Sci. 2003 Nov;1005:340-3

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 162 CASP7 (caspase 7, apoptosis-related cysteine peptidase) Coutinho-Camillo CM, Soares FA

Boatright KM, Salvesen GS. Mechanisms of caspase Polymorphisms in the CASPASE genes and survival in activation. Curr Opin Cell Biol. 2003 Dec;15(6):725-31 patients with early-stage non-small-cell lung cancer. J Clin Oncol. 2009 Dec 1;27(34):5823-9 Soung YH, Lee JW, Kim HS, Park WS, Kim SY, Lee JH, Park JY, Cho YG, Kim CJ, Park YG, Nam SW, Jeong SW, Lamkanfi M, Kanneganti TD. Caspase-7: a protease Kim SH, Lee JY, Yoo NJ, Lee SH. Inactivating mutations of involved in apoptosis and inflammation. Int J Biochem Cell CASPASE-7 gene in human cancers. Oncogene. 2003 Biol. 2010 Jan;42(1):21-4 Sep 11;22(39):8048-52 Liu CY, Wu MC, Chen F, Ter-Minassian M, Asomaning K, Hermel E, Gafni J, Propp SS, Leavitt BR, Wellington CL, Zhai R, Wang Z, Su L, Heist RS, Kulke MH, Lin X, Liu G, Young JE, Hackam AS, Logvinova AV, Peel AL, Chen SF, Christiani DC. A Large-scale genetic association study of Hook V, Singaraja R, Krajewski S, Goldsmith PC, Ellerby esophageal adenocarcinoma risk. Carcinogenesis. 2010 HM, Hayden MR, Bredesen DE, Ellerby LM. Specific Jul;31(7):1259-63 caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease. Chae YS, Kim JG, Sohn SK, Lee SJ, Kang BW, Moon JH, Cell Death Differ. 2004 Apr;11(4):424-38 Park JY, Jeon SW, Bae HI, Choi GS, Jun SH. RIPK1 and CASP7 polymorphism as prognostic markers for survival in Yoo NJ, Lee JW, Kim YJ, Soung YH, Kim SY, Nam SW, patients with colorectal cancer after complete resection. J Park WS, Lee JY, Lee SH. Loss of caspase-2, -6 and -7 Cancer Res Clin Oncol. 2011 Apr;137(4):705-13 expression in gastric cancers. APMIS. 2004 Jun;112(6):330-5 Coutinho-Camillo CM, Lourenço SV, Nishimoto IN, Kowalski LP, Soares FA. Caspase expression in oral Matsui T, Ramasamy K, Ingelsson M, Fukumoto H, squamous cell carcinoma. Head Neck. 2011 Conrad C, Frosch MP, Irizarry MC, Yuan J, Hyman BT. Aug;33(8):1191-8 Coordinated expression of caspase 8, 3 and 7 mRNA in temporal cortex of Alzheimer disease: relationship to Park C, Han S, Lee KM, Choi JY, Song N, Jeon S, Park formic acid extractable abeta42 levels. J Neuropathol Exp SK, Ahn HS, Shin HY, Kang HJ, Koo HH, Seo JJ, Choi JE, Neurol. 2006 May;65(5):508-15 Kang D. Association between CASP7 and CASP14 genetic polymorphisms and the risk of childhood leukemia. García-Lozano JR, Torres B, Fernández O, Orozco G, Hum Immunol. 2012 Jul;73(7):736-9 Alvarez-Márquez A, García A, González-Gay MA, García A, Núñez-Roldán A, Martín J, González-Escribano MF. Qian J, Gu S, Wu Q, Zhao X, Wu W, Gao Z, Zhang W, Tan Caspase 7 influences susceptibility to rheumatoid arthritis. X, Wang H, Wang J, Fan W, Chen H, Han B, Lu D, Wei Q, Rheumatology (Oxford). 2007 Aug;46(8):1243-7 Jin L. Association of CASP7 polymorphisms and survival of patients with non-small cell lung cancer with platinum- Teixeira VH, Jacq L, Lasbleiz S, Hilliquin P, Oliveira CR, based chemotherapy treatment. Chest. 2012 Cornelis F, Petit-Teixeira E. Genetic and expression Sep;142(3):680-9 analysis of CASP7 gene in a European Caucasian population with rheumatoid arthritis. J Rheumatol. 2008 Vilella-Arias SA, Rocha RM, da Costa WH, Zequi Sde C, Oct;35(10):1912-8 Guimarães GC, Verjovski-Almeida S, Soares FA, Reis EM. Loss of caspase 7 expression is associated with poor Lee WK, Kim JS, Kang HG, Cha SI, Kim DS, Hyun DS, prognosis in renal cell carcinoma clear cell subtype. Kam S, Kim CH, Jung TH, Park JY. Polymorphisms in the Urology. 2013 Oct;82(4):974.e1-7 Caspase7 gene and the risk of lung cancer. Lung Cancer. 2009 Jul;65(1):19-24 Yan S, Li YZ, Zhu XW, Liu CL, Wang P, Liu YL. HuGE systematic review and meta-analysis demonstrate Xu HL, Xu WH, Cai Q, Feng M, Long J, Zheng W, Xiang association of CASP-3 and CASP-7 genetic YB, Shu XO. Polymorphisms and haplotypes in the polymorphisms with cancer risk. Genet Mol Res. 2013 May caspase-3, caspase-7, and caspase-8 genes and risk for 13;12(2):1561-73 endometrial cancer: a population-based, case-control study in a Chinese population. Cancer Epidemiol This article should be referenced as such: Biomarkers Prev. 2009 Jul;18(7):2114-22 Coutinho-Camillo CM, Soares FA. CASP7 (caspase 7, Yoo SS, Choi JE, Lee WK, Choi YY, Kam S, Kim MJ, Jeon apoptosis-related cysteine peptidase). Atlas Genet HS, Lee EB, Kim DS, Lee MH, Kim IS, Jheon S, Park JY. Cytogenet Oncol Haematol. 2015; 19(3):160-163.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 163

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

GADD45A (growth arrest and DNA-damage- inducible, alpha) Sirma Damla User, Mesut Muyan Department of Biological Sciences, Middle East Technical University, Ankara, Turkey (SDU, MM)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/GADD45AID339ch1p31.html DOI: 10.4267/2042/56405 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

from senescence and apoptosis. Abstract Novel approaches are therefore being developed to Growth Arrest and DNA-damage-inducible, alpha regulate GADD45 α levels to combat malignancies. (GADD45 α) is a member of the GADD family Keywords proteins that also include GADD45 β and GADD45 α, DDIT1, DNA damage, cell cycle GADD45 γ. The highly conserved GADD45 proteins are small Identity (18 kDa) and primarily localized in the nucleus. The GADD45 proteins acting as sensors of Other names: DDIT1, GADD45 environmental and physiological stress interact with HGNC (Hugo): GADD45A and/or modulate the activities of partner proteins Location: 1p31.3 involved in cell cycle, cell survival, apoptosis, maintenance of genomic stability and DNA repair. DNA/RNA GADD45 proteins also act as sensors of oncogenic stress in the initiation of tumors and in tumor Description responses to different therapeutics. The human GADD45 α is localized on chromosome The expression of GADD45 α in response to DNA 1 and comprises four exons (NCBI, 2014). damage is mediated by p53-dependent and p53- independent mechanisms, the latter which involves Transcription Wilms tumor 1 (WT1) protein. GADD45 α Depending on the splicing of the GADD45 α pre- subsequently inhibits G2/M transition of cell cycle mRNA, there are three mRNA variants. and induces apoptosis. GADD45 α also has a role in DNA-demethylation to promote genome stability. Pseudogene In many malignancies, GADD45 α levels are down- Homologous sequence on chromosome 12q may be regulated, likely allowing tumor cells to escape a retro-pseudogene (Papathanasiou et al., 1991).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 164 GADD45A (growth arrest and DNA-damage-inducible, alpha) User SD, Muyan M

Human GADD45 α located on chromosome 1 is on the forward strand. It consists of four exons represented as boxes and introns shown as lines. Darker grey boxes indicate the open reading frame (ORF) of 498 base pairs (bp). The first methionine (ATG) and the stop codon (TGA) of ORF are marked. The length of each exon and intron is shown in base pairs.

GADD45 α promoter (Zhan, 2005; Johnson et al., Protein 2013). Rapid and transient expression of GADD45 α Note can be induced by DNA damaging agents including Based on mRNA sequences, it is predicted that UV (p53-independent), ionizing radiation (p53- there are three isoforms of GADD45 α protein dependent), methylmethane sulfonate (MMS), (NCBI, 2014). While the isoform 1 utilizes 498 bp nitrogen mustard, melphalan, hydrogen peroxide ORF encoding 165 amino acids (aa)-long protein and hypoxia as well as by the withdrawal of growth (protein ID: NP_001915.1), the absence of the factors (Hollander and Fornace, 2002; Zhan, 2005; second in-frame exon in the isoform 2 results in a Rosemary Siafakas and Richardson, 2009). 396 bp ORF giving rise to a 131 aa-long protein GADD45 α expression is also activated by breast (protein ID: NP_001186670.1). In the isoform 3, cancer 1, early onset, (BRCA1) in various cell lines the second and third exons partially exist forming a (Harkin et al., 1999). 183 bp mRNA that encodes a 60 aa-long protein Localisation (protein ID: NP_001186671.1). GADD45 α, as other members of GADD family Description proteins, is predominantly localized in the nucleus GADD45 α protein is a member of the GADD (Zhan, 2005; Rosemary Siafakas and Richardson, family that includes GADD45 β and GADD45 γ. 2009). In glioblastoma and breast tumors, Forming a homodimer as well as heterodimers with GADD45 α is also observed to localize in the other family members, GADD45 α is involved in the cytoplasm (Reddy et al., 2008; Tront et al., 2013). maintenance of genomic integrity, growth arrest Function and apoptosis (Rosemary Siafakas and Richardson, 2009; Sytnikova et al., 2011) through interactions The maintenance of genome integrity is essential to with various proteins including Cdc2 protein prevent the development of cancer, which is kinase, Waf1/Cip1 ID: 139> protein, core histone associated with genomic instability. GADD45 α proteins, proliferating cell nuclear antigen (PCNA) plays an important role in maintaining genomic and MTK/MEKK4 (Zerbini and Libermann, 2005; integrity by promoting nucleotide-excision repair Zhan, 2005; Rosemary Siafakas and Richardson, (NER), cell cycle arrest and apoptosis (Hollander 2009; Johnson et al., 2013). GADD45 α also acts as and Fornace, 2002; Barreto et al., 2007; Sytnikova an RNA binding protein (Sytnikova et al., 2011). et al., 2011). Ubiquitination appears to be involved in the GADD45 α mediates NER by binding to repair turnover of GADD45 α (Leung et al., 2001). endonuclease xeroderma pigmentosum G (XPG) protein (Hollander and Fornace, 2002; Barreto et Expression al., 2007; Sytnikova et al., 2011). While the Protein levels of GADD45 α varies during the cell disruption of Cdc2/Cyclin B1 interactions by cycle, the highest being in G1 phase and the lowest GADD45 α is critical for the blockage of G2/M in S phase (Zhan, 2005; Rosemary Siafakas and transition (Zhan et al., 1999; Rosemary Siafakas Richardson, 2009). The expression of GADD45 α is and Richardson, 2009), the activation of c-Jun N- regulated by p53-dependent and p53-independent terminal kinase (JNK) as a result of the interaction mechanisms. The p53-dependent pathway involves of GADD45 α with mitogen-activated protein three the direct binding of p53 to a cognate response kinase (MTK1) induces apoptosis (Zerbini and element located on the third intron of GADD45 α. Libermann, 2005). GADD45 α is also reported to Whereas, in the p53-independent signaling route, repress cell migration and invasion by suppressing p53 modulates GADD45 α expression by interacting β-catenin signaling through stress-mediated p38 with WT1, a transcription factor and a tumor mitogen activated protein kinase (MAPK) pathway suppressor, bound on GC-rich motifs of the (Hildesheim et al., 2004).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 165 GADD45A (growth arrest and DNA-damage-inducible, alpha) User SD, Muyan M

Homology Glioblastoma The human GADD45 α shows 90% aa identity to Note GADD45 α of other species including rhesus In diffuse-infiltrating astrocytomas, the expression monkey, domestic cat, hamster, mouse and rat of GADD45 α assessed by qPCR shows variations (Rosemary Siafakas and Richardson, 2009). depending on tumor grading. It appears that In addition, there is a nearly 50% aa identity among GADD45 α is expressed at higher levels in GADD45 α and other GADD45 proteins (Rosemary glioblastoma (GBM; WHO grade IV) compared to Siafakas and Richardson, 2009). astrocytoma (DA; WHO grade II) or to anaplastic The RNA-binding domain of GADD45 α displays astrocytoma (AP; WHO grade III) (Reddy et al., high aa homology to many RNA binding proteins 2008). that includes ribosomal proteins L7a, S12 and L30e (Sytnikova et al., 2011; Tian and Locker, 2013). References Papathanasiou MA, Kerr NC, Robbins JH, McBride OW, Implicated in Alamo I Jr, Barrett SF, Hickson ID, Fornace AJ Jr. Induction by ionizing radiation of the gadd45 gene in Colorectal carcinoma cultured human cells: lack of mediation by protein kinase Note C. Mol Cell Biol. 1991 Feb;11(2):1009-16 In primary colorectal carcinoma tissue samples Zhan Q, Antinore MJ, Wang XW, Carrier F, Smith ML, grouped according to tumor staging (group 1: Harris CC, Fornace AJ Jr. Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53- restricted to gut; group 2: restricted to gut but signs regulated protein Gadd45. Oncogene. 1999 May of malignancy on lymph nodes are present; group 3: 6;18(18):2892-900 in addition to group 2 characteristics, metastasis to Harkin DP, Bean JM, Miklos D, Song YH, Truong VB, distant tissue is present), it was observed that Englert C, Christians FC, Ellisen LW, Maheswaran S, GADD45 α expression decreases as staging Oliner JD, Haber DA. Induction of GADD45 and increases. JNK/SAPK-dependent apoptosis following inducible Whereas, GADD45 α expression in close proximity expression of BRCA1. Cell. 1999 May 28;97(5):575-86 or distant tissues remains unchanged (Štorcelová et Leung CH, Lam W, Zhuang WJ, Wong NS, Yang MS, al., 2013). Fong WF. PKCdelta-dependent deubiquitination and stabilization of Gadd45 in A431 cells overexposed to EGF. Breast cancer Biochem Biophys Res Commun. 2001 Jul 13;285(2):283-8 Note Hollander MC, Fornace AJ Jr. Genomic instability, centrosome amplification, cell cycle checkpoints and Intracellular level of GADD45 α assessed by Gadd45a. Oncogene. 2002 Sep 9;21(40):6228-33 immunocytochemistry is reported to positively Hildesheim J, Belova GI, Tyner SD, Zhou X, Vardanian L, correlate with the presence of estrogen and Fornace AJ Jr. Gadd45a regulates matrix progesterone receptors in primary breast cancer metalloproteinases by suppressing DeltaNp63alpha and samples (Tront et al., 2013). beta-catenin via p38 MAP kinase and APC complex It appears that GADD45 α levels are higher in activation. Oncogene. 2004 Mar 11;23(10):1829-37 Luminal A (ER+, PR+, HER2-) and Luminal B Zerbini LF, Libermann TA. Life and death in cancer. (ER+, PR+, HER2+) subgroups of tumors than GADD45 alpha and gamma are critical regulators of NF- HER2+ (HER2+, ER-,PR-) and Triple Negative kappaB mediated escape from programmed cell death. Cell Cycle. 2005 Jan;4(1):18-20 (ER-, PR-, HER2-) subgroups (Tront et al., 2013). Zhan Q. Gadd45a, a p53- and BRCA1-regulated stress Gastric cardia adenocarcinoma protein, in cellular response to DNA damage. Mutat Res. 2005 Jan 6;569(1-2):133-43 Note Relative mRNA expression evaluated by qPCR of Barreto G, Schäfer A, Marhold J, Stach D, Swaminathan SK, Handa V, Döderlein G, Maltry N, Wu W, Lyko F, gastric cardia adenocarcinoma (GCA) samples Niehrs C. Gadd45a promotes epigenetic gene activation suggests that the expression of GADD45 α is by repair-mediated DNA demethylation. Nature. 2007 Feb repressed compared to that of neighboring normal 8;445(7128):671-5 tissue samples. Based on TNM staging, GADD45 α Reddy SP, Britto R, Vinnakota K, Aparna H, Sreepathi HK, mRNA levels were found to be higher in Stage I Thota B, Kumari A, Shilpa BM, Vrinda M, Umesh S, and Stage II patient samples than Stage III and Samuel C, Shetty M, Tandon A, Pandey P, Hegde S, Stage IV patient samples. Higher degree of Hegde AS, Balasubramaniam A, Chandramouli BA, Santosh V, Kondaiah P, Somasundaram K, Rao MR. methylation of the GADD45 α gene promoter Novel glioblastoma markers with diagnostic and prognostic appears to be one reason for the decreased value identified through transcriptome analysis. Clin expression of GADD45 α in GCA. There were no Cancer Res. 2008 May 15;14(10):2978-87 other correlation between GADD45 α mRNA levels Rosemary Siafakas A, Richardson DR. Growth arrest and and clinicopathological characteristics (Guo et al., DNA damage-45 alpha (GADD45alpha). Int J Biochem 2013). Cell Biol. 2009 May;41(5):986-9

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 166 GADD45A (growth arrest and DNA-damage-inducible, alpha) User SD, Muyan M

Sytnikova YA, Kubarenko AV, Schäfer A, Weber AN, clock gene per2 in human colorectal carcinoma tissue. Mol Niehrs C. Gadd45a is an RNA binding protein and is Biol Rep. 2013 Nov;40(11):6351-61 localized in nuclear speckles. PLoS One. 2011 Jan 7;6(1):e14500 Tian J, Locker J. Gadd45 in the liver: signal transduction and transcriptional mechanisms. Adv Exp Med Biol. Guo W, Dong Z, Guo Y, Chen Z, Kuang G, Yang Z. 2013;793:69-80 Methylation-mediated repression of GADD45A and GADD45G expression in gastric cardia adenocarcinoma. Tront JS, Willis A, Huang Y, Hoffman B, Liebermann DA. Int J Cancer. 2013 Nov;133(9):2043-53 Gadd45a levels in human breast cancer are hormone receptor dependent. J Transl Med. 2013 May 24;11:131 Johnson D, Hastwell PW, Walmsley RM. The involvement of WT1 in the regulation of GADD45a in response to This article should be referenced as such: genotoxic stress. Mutagenesis. 2013 Jul;28(4):393-9 User SD, Muyan M. GADD45A (growth arrest and DNA- Štorcelová M, Vicián M, Reis R, Zeman M, Herichová I. damage-inducible, alpha). Atlas Genet Cytogenet Oncol Expression of cell cycle regulatory factors hus1, gadd45a, Haematol. 2015; 19(3):164-167. rb1, cdkn2a and mre11a correlates with expression of

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 167

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Short Communication

ID2 (inhibitor of DNA binding 2, dominant negative helix-loop-helix protein) Menno C van Zelm Dept Immunology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands (MCv)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/ID2ID436ch2p25.html DOI: 10.4267/2042/56406 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract DNA/RNA Review on ID2, with data on DNA/RNA, on the Description protein encoded and where the gene is implicated. The gene spans 5608 bp containing 5 exons of which 2 (exons 3 and 4) are protein-encoding. Identity Transcription Other names: GIG8, ID2A, ID2H, bHLHb26 The ID2 gene has 4 transcripts, of which 3 can generate functional protein: ID2-001 (2041bp) HGNC (Hugo): ID2 contains all 5 exons, ID2-201 (1362 bp) contains 3 Location: 2p25.1 exons and ID2-002 contains 2 exons. ID2-003 (474 bp) contains a retained intron.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 168 ID2 (inhibitor of DNA binding 2, dominant negative helix-loop- van Zelm MC helix protein)

Pseudogene Furthermore, female mice lacking ID2 show lactation defects (Mori et al., 2000), and male mice A pseudogene of ID2 is located on chromosome 3. have impaired spermatogenesis (Sablitzky et al., Protein 1998). Homology Description ID2 is highly conserved in vertebrates, including ID2 belongs to the helix-loop-helix (HLH) protein mammals, reptiles, and fish. family. It is composed by 134 aa and belongs to a subgroup HLH family members (ID1, ID2, ID3, Mutations ID4) that lack a basic DNA-binding domain. ID proteins form heterodimers with class I basic HLH- Germinal group members such as MyoD (Langlands et al., No germinal mutations have been reported. 1997), NEDD9 (Law et al., 1999), and E2A gene products E12 and E47. ID2 has a main domain Somatic located on 38-79 aa responsible for the helix-loop- No somatic mutations have been reported. helix conformation. In addition, Id2 contains a 10 aa motif that is responsible for the nuclear export Implicated in signaling. Neuroblastoma Expression Note Expression of ID2 is found in the brain, ovary, ID2 functions as a key regulator in the phenotypic liver, lung, thyroid gland and prostate and several transition of neuroblastoma tumor cells subsets of leukocytes. ID2 expression is especially (Chakrabarti et al., 2013). Anchorage-dependent high in Natural Killer (NK-) cells, but is also found (AD) neuroblastoma cells express much higher in CD4+ T cells, CD8+ T cells, monocytes and levels of ID2 than anchorage-independent (AI) precursor B cells. cells. Moreover, knockdown of ID2 in AD cells Localisation induces an AI phenotype, whereas the opposite is Nucleus. seen upon forced expression of ID2 in AI cells. The function of ID2 in this process is at least in part via Function negative regulation of the TGF β/Smad pathway. Although it does not bind directly to DNA, by binding basic helix-loop-helix transcription factors Colon carcinoma through its HLH motif, ID2 may control tissue- Note specific genes related to cell growth, proliferation ID2 expression is upregulated by enhanced beta- and differentiation (Hara et al., 1994; Iavarone et catenin signaling and subsequent beta-catenin /TCF al., 1994). ID2 functions in cell fate decisions in mediated transcription. early leukocyte development. Specifically, ID2 is The induction of ID2 expression increases required for NK-cell, innate lymphoid and anchorage-independent survival of these cells lymphoid tissue inducer cells (Boos et al., 2007; (Rockman et al., 2001). Moro et al., 2010; Yokota et al., 1999). Melanoma Furthermore, ID2 functions in the development of several dendritic cell subsets: Langerhans cells, Note cutaneous dendritic cells and splenic CD8a+ The transition of melanoma to a more aggressive dendritic cells (Hacker et al., 2003). Although ID2 malignancy is associated with the resistance to seems redundant for T-cell development in thymus, growth inhibition by TGF-β (Javelaud et al., 2008). ID2 promotes NKT-cell development (Verykokakis In susceptible cells, TGF-β suppresses ID2 et al., 2013), and it is involved in effector expression and allows p15 lnk4b to induce a cell cycle differentiation (Masson et al., 2013), as well as γδ T arrest (Schlegel et al., 2009). Upon obtaining cell homeostasis (Zhang et al., 2014). Finally, ID2 resistance to TGF-β, the tumor cells overexpress inhibits progression of precursor-B-cell ID2 and remain in cycle. development (Hara et al., 1997; Jensen et al., 2013), Retinoblastoma as well as activation-induced deaminase expression during B-cell responses (Gonda et al., 2003), likely Note through inhibition of E47 (Sayegh et al., 2003). ID2 is overexpressed due to transcriptional Besides leukocytes, ID2 has been found to function activation by oncoproteins of the Myc family in in erythrocyte development (Ji et al., 2008), retinoblastoma, where it is thought to inhibit enterocyte precursor and lung epithelial cell Retinoblastoma protein family members (Lasorella differentiation in mice (Rawlins et al., 2009). et al., 2000; Lasorella et al., 2005).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 169 ID2 (inhibitor of DNA binding 2, dominant negative helix-loop- van Zelm MC helix protein)

Hodgkin's lymphoma activities is the key to AID gene expression. J Exp Med. 2003 Nov 3;198(9):1427-37 Note Hacker C, Kirsch RD, Ju XS, Hieronymus T, Gust TC, Kuhl The majority of Hodgkin's lymphomas are derived C, Jorgas T, Kurz SM, Rose-John S, Yokota Y, Zenke M. from germinal center B cells. Still, Hodgkin- Transcriptional profiling identifies Id2 function in dendritic Reed/Sternberg (HRS) cells of classical Hodgkin's cell development. Nat Immunol. 2003 Apr;4(4):380-6 lymphoma have a very atypical phenotype. Sayegh CE, Quong MW, Agata Y, Murre C. E-proteins This is the result of overexpression of ABF-1 and directly regulate expression of activation-induced Id2, which inhibit the function of the B cell- deaminase in mature B cells. Nat Immunol. 2003 determining transcription factor E2A (Mathas et al., Jun;4(6):586-93 2006; Renne et al., 2006). Lasorella A, Rothschild G, Yokota Y, Russell RG, Iavarone The mechanism resulting in ID2 overexpression, A. Id2 mediates tumor initiation, proliferation, and angiogenesis in Rb mutant mice. Mol Cell Biol. 2005 nor the impact of ID2 on cell cycle progression in May;25(9):3563-74 HRS cells have been demonstrated experimentally yet (Cotta and Medeiros, 2008). Mathas S, Janz M, Hummel F, Hummel M, Wollert-Wulf B, Lusatis S, Anagnostopoulos I, Lietz A, Sigvardsson M, Jundt F, Jöhrens K, Bommert K, Stein H, Dörken B. References Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of Hara E, Yamaguchi T, Nojima H, Ide T, Campisi J, neoplastic B cells in Hodgkin lymphoma. Nat Immunol. Okayama H, Oda K. Id-related genes encoding helix-loop- 2006 Feb;7(2):207-15 helix proteins are required for G1 progression and are repressed in senescent human fibroblasts. J Biol Chem. Renné C, Martin-Subero JI, Eickernjäger M, Hansmann 1994 Jan 21;269(3):2139-45 ML, Küppers R, Siebert R, Bräuninger A. Aberrant expression of ID2, a suppressor of B-cell-specific gene Iavarone A, Garg P, Lasorella A, Hsu J, Israel MA. The expression, in Hodgkin's lymphoma. Am J Pathol. 2006 helix-loop-helix protein Id-2 enhances cell proliferation and Aug;169(2):655-64 binds to the retinoblastoma protein. Genes Dev. 1994 Jun 1;8(11):1270-84 Boos MD, Yokota Y, Eberl G, Kee BL. Mature natural killer cell and lymphoid tissue-inducing cell development Hara E, Hall M, Peters G. Cdk2-dependent requires Id2-mediated suppression of E protein activity. J phosphorylation of Id2 modulates activity of E2A-related Exp Med. 2007 May 14;204(5):1119-30 transcription factors. EMBO J. 1997 Jan 15;16(2):332-42 Cotta CV, Medeiros LJ. Expression of helix-loop-helix Langlands K, Yin X, Anand G, Prochownik EV. Differential proteins in classical hodgkin lymphoma: a possible interactions of Id proteins with basic-helix-loop-helix explanation for a characteristic immunophenotype. Adv transcription factors. J Biol Chem. 1997 Aug Anat Pathol. 2008 Mar;15(2):97-104 8;272(32):19785-93 Javelaud D, Alexaki VI, Mauviel A. Transforming growth Sablitzky F, Moore A, Bromley M, Deed RW, Newton JS, factor-beta in cutaneous melanoma. Pigment Cell Norton JD. Stage- and subcellular-specific expression of Id Melanoma Res. 2008 Apr;21(2):123-32 proteins in male germ and Sertoli cells implicates distinctive regulatory roles for Id proteins during meiosis, Ji M, Li H, Suh HC, Klarmann KD, Yokota Y, Keller JR. Id2 spermatogenesis, and Sertoli cell function. Cell Growth intrinsically regulates lymphoid and erythroid development Differ. 1998 Dec;9(12):1015-24 via interaction with different target proteins. Blood. 2008 Aug 15;112(4):1068-77 Law SF, Zhang YZ, Fashena SJ, Toby G, Estojak J, Golemis EA. Dimerization of the docking/adaptor protein Rawlins EL, Clark CP, Xue Y, Hogan BL. The Id2+ distal HEF1 via a carboxy-terminal helix-loop-helix domain. Exp tip lung epithelium contains individual multipotent Cell Res. 1999 Oct 10;252(1):224-35 embryonic progenitor cells. Development. 2009 Nov;136(22):3741-5 Yokota Y, Mansouri A, Mori S, Sugawara S, Adachi S, Nishikawa S, Gruss P. Development of peripheral Schlegel NC, Eichhoff OM, Hemmi S, Werner S, Dummer lymphoid organs and natural killer cells depends on the R, Hoek KS. Id2 suppression of p15 counters TGF-beta- helix-loop-helix inhibitor Id2. Nature. 1999 Feb mediated growth inhibition of melanoma cells. Pigment 25;397(6721):702-6 Cell Melanoma Res. 2009 Aug;22(4):445-53 Lasorella A, Noseda M, Beyna M, Yokota Y, Iavarone A. Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, Id2 is a retinoblastoma protein target and mediates Kawamoto H, Furusawa J, Ohtani M, Fujii H, Koyasu S. signalling by Myc oncoproteins. Nature. 2000 Oct Innate production of T(H)2 cytokines by adipose tissue- 5;407(6804):592-8 associated c-Kit(+)Sca-1(+) lymphoid cells. Nature. 2010 Jan 28;463(7280):540-4 Mori S, Nishikawa SI, Yokota Y. Lactation defect in mice lacking the helix-loop-helix inhibitor Id2. EMBO J. 2000 Chakrabarti L, Wang BD, Lee NH, Sandler AD. A Nov 1;19(21):5772-81 mechanism linking Id2-TGF β crosstalk to reversible adaptive plasticity in neuroblastoma. PLoS One. Rockman SP, Currie SA, Ciavarella M, Vincan E, Dow C, 2013;8(12):e83521 Thomas RJ, Phillips WA. Id2 is a target of the beta- catenin/T cell factor pathway in colon carcinoma. J Biol Jensen K, Rother MB, Brusletto BS, Olstad OK, Dalsbotten Chem. 2001 Nov 30;276(48):45113-9 Aass HC, van Zelm MC, Kierulf P, Gautvik KM. Increased ID2 levels in adult precursor B cells as compared with Gonda H, Sugai M, Nambu Y, Katakai T, Agata Y, Mori KJ, children is associated with impaired Ig locus contraction Yokota Y, Shimizu A. The balance between Pax5 and Id2 and decreased bone marrow output. J Immunol. 2013 Aug

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 170 ID2 (inhibitor of DNA binding 2, dominant negative helix-loop- van Zelm MC helix protein)

1;191(3):1210-9 Zhang B, Lin YY, Dai M, Zhuang Y. Id3 and Id2 act as a dual safety mechanism in regulating the development and Masson F, Minnich M, Olshansky M, Bilic I, Mount AM, population size of innate-like γδ T cells. J Immunol. 2014 Kallies A, Speed TP, Busslinger M, Nutt SL, Belz GT. Id2- Feb 1;192(3):1055-63 mediated inhibition of E2A represses memory CD8+ T cell differentiation. J Immunol. 2013 May 1;190(9):4585-94 This article should be referenced as such: Verykokakis M, Krishnamoorthy V, Iavarone A, Lasorella van Zelm MC. ID2 (inhibitor of DNA binding 2, dominant A, Sigvardsson M, Kee BL. Essential functions for ID negative helix-loop-helix protein). Atlas Genet Cytogenet proteins at multiple checkpoints in invariant NKT cell Oncol Haematol. 2015; 19(3):168-171. development. J Immunol. 2013 Dec 15;191(12):5973-83

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 171

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

KLRK1 (killer cell lectin-like receptor subfamily K, member 1) Lewis L Lanier UCSF, Department of Microbiology and Immunology, San Francisco, CA 94143-0414, USA (LLL)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/KLRK1ID41094ch12p13.html DOI: 10.4267/2042/56407 This article is an update of : Lanier LL. KLRK1 (killer cell lectin-like receptor subfamily K, member 1). Atlas Genet Cytogenet Oncol Haematol 2008;12(1):47- 49.

Abstract DNA/RNA KLRK1 encodes a type II transmembrane-anchored Note glycoprotein that is expressed as a disulfide-linked KLRK1 is present on chromosome 12 within a homodimer on the surface of Natural Killer (NK) cluster of genes referred to as the "NK complex" cells, gamma/delta TcR+ T cells, CD8+ T cells, and (NKC) because several genes that are preferentially a minor subset of CD4+ T cells. It associates non- expressed by Natural Killer (NK) cells are located covalently with the DAP10 signaling protein and in this region, including on the centromeric side provides activating or costimulatory signals to NK KLRD1 (CD94) and on the telomeric side KLRC4 cells and T cells. NKG2D binds to a family of (NKG2F), KLRC3 (NKG2E), KLRC2 (NKG2C), glycoproteins, in humans the MICA, MICB, and and KLRC1 (NKG2A) (Houchins et al., 1991). ULBP1-6 membrane proteins, which are frequently Description expressed on cells that have become infected with pathogens or undergone transformation. The KLRK1 gene is 17702 bases located on the negative strand of chromosome 12 spanning bases Keywords 10372353 to 103900054 with a predicted 7 exons. KLRK1, C-type lectin-like receptor Transcription Identity There is evidence for alternative splicing of KLRK1, but only one isoform encoding a Other names: D12S2489E, CD314, KLR, NKG2- functional protein has been described in humans. D, NKG2D In one of the KLRK1 splice variants the fourth HGNC (Hugo): KLRK1 exon of KLRC4 is spliced to the 5-prime end of Location: 12p13.2 KLRK1.

Schematic representation of the KLRC2 (NKG2C), KLR3 (NKG2E), KLRC4 (NKG2F), and KLRK1 genes on human chromosome 12p13.2 (taken from Glienke et al., 1998, figure 4).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 172 KLRK1 (killer cell lectin-like receptor subfamily K, member 1) Lanier LL

Amino acid sequence of KLRK1 is shown, with the predicted transmembrane domain underlined. The R residue in the transmembrane is required for association of KLRK1 with the DAP10 signaling protein to form the mature receptor complex. Three potential sites for N-linked glycosylation are in bold.

KLRK1 is transcribed by NK cells, gamma/delta- TcR+ T cells, CD8+ T cells and some CD4+ T cells (Bauer et al., 1999). Transcription of KLRK1 is enhanced by stimulation of NK cells with IL-2 or IL-15 and decreased by culture with TGF-beta. Pseudogene No known pseudogenes. Protein Note KLRK1 is a type II transmembrane-anchored Schematic representation of the KLRK1 (NKG2D) - DAP10 receptor complex (taken from Garrity et al., 2005, figure 7). membrane glycoprotein expressed as a disulfide- bonded homodimer on the cell surface. Expression Expression of KLRK1 on the cell surface requires its KLRK1 protein is expressed on the cell surface of association with DAP10, which is a type I adapter NK cells, gamma/delta-TcR+ T cells, CD8+ T cells, protein expressed as a disulfide-bonded homodimer and some CD4+ T cells (Bauer et al., 1999). (Wu et al., 1999). On the cell surface, the receptor complex is a hexamer; two disulfide-bonded Localisation KLRK1 homodimers each paired with two DAP10 KLRK1 is expressed as a type II integral membrane disulfide-bonded homodimers (Garrity et al., 2005). glycoprotein on the cell surface of NK cells, A charged amino acid residue (aspartic acid) gamma/delta-TcR+ T cells, CD8+ T cells, and centrally located within the transmembrane region some CD4+ T cells (Bauer et al., 1999). of DAP10 forms a salt bridge with a charged amino In the absence of DAP10, KLRK1 protein is acid residue (arginine) in the transmembrane region retained in the cytoplasm and degraded (Wu et al., of KLRK1 to stabilize the receptor complex (Wu et 1999). al., 1999). Function Description KLRK1 binds to at least eight distinct ligands: KLRK1 is a type II membrane protein comprising MICA, MICB, ULBP-1, ULBP-2, ULBP-3, ULBP- 216 amino acids with a predicted molecular weight 4, ULBP-5, and ULBP-6 (Bauer et al., 1999; of 25,143 kDa. The protein has an N-terminal Cosman et al., 2001; Raulet et al., 2013). intracellular region, a transmembrane domain, a These ligands are type I glycoproteins with membrane-proximal stalk region, and an homology to MHC class I. The KLRK1 ligands extracellular region with a single C-type lectin-like frequently are over-expressed on tumor cells, virus- domain. KLRK1 is expressed on the cell surface as infected cells, and "stressed" cells (Raulet et al., a disulfide-bonded homodimer with a molecular 2013). The crystal structure of KLRK1 bound to weight of approximately 42 kDa when analyzed MICA has been described (Li et al., 2001). After under reducing conditions and approximately 80 binding to its ligand, KLRK1 transmits an kDa under non-reducing conditions. A cysteine activating signal via the DAP10 adapter subunit. residue just outside the transmembrane region DAP10 has a YINM motif in its cytoplasmic forms the disulfide bond joining the two subunits of domain, which upon tyrosine phosphorylation binds the homodimer. There are three potential sites for to Vav and the p85 subunit of PI3-kinase (Billadeau N-linked glycosylation in the extracellular region of et al., 2003; Wu et al., 1999), causing a downstream KLRK1. Treatment of the KLRK1 glycoprotein cascade of signaling in T cells and NK cells, with N-glycanase reduces the molecular weight to resulting in the killing of ligand-bearing cells and approximately the size of the core polypeptide. the secretion of cytokines by NK cells and T cells.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 173 KLRK1 (killer cell lectin-like receptor subfamily K, member 1) Lanier LL

Mutations Note None identified. Implicated in Cancer Note Many types of cancer (carcinomas, sarcomas, lymphomas, and leukemias) over-express the ligands for KLRK1 (Raulet et al., 2013). In some cases, this renders the tumor cells susceptible to killing by activated KLRK1-bearing NK cells. Some tumors shed or secrete soluble ligands that bind to KLRK1, which downregulates expression of KLRK1 on NK cells and T cells (Groh et al., 2002), although the physiological relevance of the shed ligands is controversial. Mice in which the Klrk1 gene has been disrupted show increased susceptibility to certain cancers caused by transgenic expression of oncogenes (Guerra et al., 2008). Viral infection Note Viral infection of cells can induce transcription and cell surface expression of ligands for KLRK1, rendering these infected cells susceptible to attack by NK cells and T cells (Champsaur and Lanier, 2010; Raulet et al., 2013). Some viruses, for example cytomegalovirus, encode proteins that intercept the ligand proteins intracellularly and prevent their expression on the surface of virus- infected cells. Rheumatoid arthritis Note An expansion of CD4+,CD28- T cells expressing Structure of the KLRK1 homodimer (a) and its ligand MICA KLRK1 was observed in the joints of patients with (b) (taken from Li et al., 2001, figure 1). rheumatoid arthritis and KLRK1 ligands were Homology detected on synovial cells in the inflamed tissue (Groh et al., 2003). Pan troglodytes: NP_001009059 Macaca mulatta: NP_001028061 Type I diabetes Macaca fascicularis: CAD19993 Note Callithrix jacchus: ABN45890 Peripheral blood NK cells and T cells in patients Papio anubis: ABO09749 with type I diabetes demonstrate a slightly Pongo pygmaeus: Q8MJH1 decreased amount of expression of KLRK1 on the Bos taurus: CAJ27114 cell surface, independent disease duration (Rodacki Sus scrofa: Q9GLF5 et al., 2007), similar to prior observations in the Mus musculus: NP_149069 NOD mouse (Ogasawara et al., 2003). Mus musculus: NP_001076791 Rattus norvegicus: NP_598196 References Callithrix jacchus: A4GHD0 Neovison vison: U6DVF4 Houchins JP, Yabe T, McSherry C, Bach FH. DNA Microcebus murinus: D1GEY1 sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on Varecia variegate: D1GF00 human natural killer cells. J Exp Med. 1991 Apr Lithobates catesbeiana: C1C4X9 1;173(4):1017-20

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 174 KLRK1 (killer cell lectin-like receptor subfamily K, member 1) Lanier LL

Glienke J, Sobanov Y, Brostjan C, Steffens C, Nguyen C, expression of NKG2D and its MIC ligands in rheumatoid Lehrach H, Hofer E, Francis F. The genomic organization arthritis. Proc Natl Acad Sci U S A. 2003 Aug of NKG2C, E, F, and D receptor genes in the human 5;100(16):9452-7 natural killer gene complex. Immunogenetics. 1998 Aug;48(3):163-73 Ogasawara K, Hamerman JA, Hsin H, Chikuma S, Bour- Jordan H, Chen T, Pertel T, Carnaud C, Bluestone JA, Bauer S, Groh V, Wu J, Steinle A, Phillips JH, Lanier LL, Lanier LL. Impairment of NK cell function by NKG2D Spies T. Activation of NK cells and T cells by NKG2D, a modulation in NOD mice. Immunity. 2003 Jan;18(1):41-51 receptor for stress-inducible MICA. Science. 1999 Jul 30;285(5428):727-9 Garrity D, Call ME, Feng J, Wucherpfennig KW. The activating NKG2D receptor assembles in the membrane Wu J, Song Y, Bakker AB, Bauer S, Spies T, Lanier LL, with two signaling dimers into a hexameric structure. Proc Phillips JH. An activating immunoreceptor complex formed Natl Acad Sci U S A. 2005 May 24;102(21):7641-6 by NKG2D and DAP10. Science. 1999 Jul 30;285(5428):730-2 Rodacki M, Svoren B, Butty V, Besse W, Laffel L, Benoist C, Mathis D. Altered natural killer cells in type 1 diabetic Cosman D, Müllberg J, Sutherland CL, Chin W, Armitage patients. Diabetes. 2007 Jan;56(1):177-85 R, Fanslow W, Kubin M, Chalupny NJ. ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 Guerra N, Tan YX, Joncker NT, Choy A, Gallardo F, Xiong and stimulate NK cytotoxicity through the NKG2D receptor. N, Knoblaugh S, Cado D, Greenberg NM, Raulet DH. Immunity. 2001 Feb;14(2):123-33 NKG2D-deficient mice are defective in tumor surveillance in models of spontaneous malignancy. Immunity. 2008 Li P, Morris DL, Willcox BE, Steinle A, Spies T, Strong RK. Apr;28(4):571-80 Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA. Nat Champsaur M, Lanier LL. Effect of NKG2D ligand Immunol. 2001 May;2(5):443-51 expression on host immune responses. Immunol Rev. 2010 May;235(1):267-85 Groh V, Wu J, Yee C, Spies T. Tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell Raulet DH, Gasser S, Gowen BG, Deng W, Jung H. activation. Nature. 2002 Oct 17;419(6908):734-8 Regulation of ligands for the NKG2D activating receptor. Annu Rev Immunol. 2013;31:413-41 Billadeau DD, Upshaw JL, Schoon RA, Dick CJ, Leibson PJ. NKG2D-DAP10 triggers human NK cell-mediated This article should be referenced as such: killing via a Syk-independent regulatory pathway. Nat Immunol. 2003 Jun;4(6):557-64 Lanier LL. KLRK1 (killer cell lectin-like receptor subfamily K, member 1). Atlas Genet Cytogenet Oncol Haematol. Groh V, Bruhl A, El-Gabalawy H, Nelson JL, Spies T. 2015; 19(3):172-175. Stimulation of T cell autoreactivity by anomalous

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 175

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

ORAI3 (ORAI calcium release-activated calcium modulator 3) Jessy Hasna, Nazim Benzerdjeb, Malika Faouzi, Anne-Sophie Ay, Philippe Kischel, Frédéric Hague, Henri Sevestre, Ahmed Ahidouch, Halima Ouadid-Ahidouch University of Picardie Jules Verne, UFR Sciences, EA 4667, Laboratory of Cell and Molecular Physiology, SFR CAP-SANTE (FED 4231), Amiens, France (JH, NB, MF, ASA, PK, FH, HS, AA, HOA), University of Picardie Jules Verne, Amiens University Hospital, Department of Pathology and Tumor Bank of Picardie, Amiens, France (NB, HS), Department of Biology, Faculty of Sciences, University Ibn Zohr, Agadir, Morocco (AA)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/ORAI3ID51589ch16p11.html DOI: 10.4267/2042/56408 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Transcription Size of ORAI3 transcript: 2.2 kb; NCBI ORAI3 Review on ORAI3, with data on DNA/RNA, on the mRNA model: NM_152288. protein encoded and where the gene is implicated. All three ORAI isoforms are widely expressed at the mRNA level and can be incorporated into the Identity plasma membrane when ectopically expressed. Broad expression of ORAI3 transcripts has been Other names: TMEM142C shown by Northern blot analysis: ORAI3 transcripts HGNC (Hugo): ORAI3 are expressed in heart, brain, kidney, thymus, lung, Location: 16p11.2 spleen, skeletal muscle, small intestine, as well as Note in primary aortic endothelial cells and bone marrow derived mast cells (Gwack et al., 2007). ORAI3 ORAI3 is a member of the ORAI family proteins appears to be the only family member that is discovered in 2006 as the essential pore-forming 2+ strongly expressed at the RNA level in brain. components of the low-conductance, highly Ca - (ORAI2 transcripts are prominent in kidney, lung, selective CRAC channels whose activation is dependent on depletion of the endoplasmic and spleen (Gwack et al., 2007)). reticulum Ca 2+ stores (Feske et al., 2006; Vig et al., Transcripts expression In immune cells, transcripts taken from isolated 2006; Zhang et al., 2006). primary CD3 +/CD4 + cells (Th-lymphocytes), In Greek mythology, the ORAI are the keepers of CD3 +/CD8 + cells (Tc-lymphocytes), CD19 + cells the gates of heaven: Eunomia (Order or Harmony), (B-lymphocytes) and BMMC showed that ORAI3 Dike (Justice) and Eirene (Peace). expression is readily detectable in Th -, Tc -, and B - lymphocytes and BMMC (Gross et al., 2007). DNA/RNA mRNA expression in normal tissues has been assessed by different techniques (microarrays, Description RNAseq, SAGE). Microarrays analyses show that ORAI3 is encoded by the gene TMEM142C ORAI3 is overexpressed in prostate, lung, (HUGO Gene Nomenclature Committee). monocytes and whole blood The ORAI3 gene is located on chromosome 16 in (http://biogps.org/#goto=genereportid=93129, with the p11.2. overexpression defined as 3 times the mean

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 176 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

expression observed in the 83 tissues or cells tested 2008). ORAI3 has a second extracellular loop in this study). ORAI3 mRNA expression is least linking transmembrane domains 3 and 4 which is important in pancreas, brain (especially the longer than that of ORAI1 and ORAI2 (~72 amino occipital lobe) and T cells (CD4 + as well as CD8 +). acids in ORAI3 compared to only 38 amino acids in ORAI1). ORAI3 has a cluster of 22 positively Protein charged amino acid residues immediately prior to the first transmembrane region which is fully Description conserved among all three ORAI channels (H44- Description of the protein sequence. R66 in ORAI3 and H69-R91 in ORAI1), and has Molecular weight: 31499 Da. three conserved glutamates located at the C- Sequence length: 295 amino acids. terminus to which is attributed the fast Ca 2+ - ORAI3 is a plasma membrane protein containing dependent inactivation of ORAI3 (Lee et al., 2009). four transmembrane domains with intracellular N- The ORAI3 N-terminus appears critical for and C-termini. ORAI3 contains a binding domain switching a store-operated channel to an for calmodulin in its N-terminus, and a coiled-coil exclusively arachidonate regulated channel domain for protein interaction in its C-terminus. (Thompson et al., 2010). Examination of the overall protein sequence of The residues E81 and E165 in the transmembrane ORAI3 reveals high percentage of homology with domains 1 and 3, and E85, D87 and E89 in the the family members: 63.2% with ORAI1 and 66.4% extracellular 1-2 loop are critical determinants of a with ORAI2 (60.3% between ORAI1 and ORAI2). high Ca 2+ selectivity. Other studies using a These homology percentages increase when the cysteine-scanning mutagenesis approach in ORAI3 comparison concerns the transmembrane domains: revealed that Ca 2+ selectivity was exclusively 93.8% with both ORAI1 and ORAI2, (92.5% determined by the E81 residue alone (McNally et between ORAI1 and ORAI2) (Feske et al., 2006; al., 2009). Hewavitharana et al., 2007). The pore-forming Replacing the N-terminal cytosolic domain of transmembrane domains of all three ORAI proteins ORAI3 with the corresponding domain of ORAI1 show a high degree (~82%) of conservation. doubles the magnitude of the measured store- The amino acid sequence of ORAI3 shows marked operated Ca 2+ currents, whilst the reverse exchange differences from its isoforms, particularly in the virtually eliminates all currents. N-terminal deletion regions outside of the essential pore-forming experiments narrow the critical region essential for domains, which might explain its unique properties the activation of ORAI3 to amino acids 42-62 (Lis and the differences with other isoforms in the et al., 2010). The appearance of significant store- modes of regulation and modulation from its operated currents dependes on a single specific isoforms (Shuttleworth, 2012). lysine residue K60 in ORAI3, the conservation of The sequence identities between ORAI3 and this residue in ORAI1 and ORAI3 cannot explain ORAI1 in the cytosolic N- and C-termini are 34% the differences in the magnitude of store-operated and 46%, respectively, and is 21% in the Ca 2+ currents between these two ORAI family extracellular loop between transmembrane domains members. N-terminal deletions of residues between 3 and 4 (Shuttleworth, 2012). W51 and Y55 significantly increase store-operated The N-terminus of ORAI3 comprises ~65 amino ORAI3-dependent currents (Bergsmann et al., acids and has no clusters of prolines and arginines 2011). The only sequence difference between seen in ORAI1 (N-terminus domain containing ~90 ORAI1 and ORAI3 in this region is the substitution amino acids and rich in clusters of prolines and of a lysine in ORAI1 for an arginine at position 53 arginines) (Takahashi et al., 2007; Frischauf et al., in ORAI3.

Schematic representation of ORAI protein structure and organization. Domains of human ORAI1, 2 and 3. P: proline-rich region, R: arginin-rich region, R/K: arginine-lysine-rich region, TM: transmembrane domain, CC: coiled-coil domain (Derler et al., 2012).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 177 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

ORAI3 protein sequence of amino acids. ORAI3 protein (1 .. 295) has four helical transmembrane domains: T1 (63 .. 82) (20 amino acids), T2 (95 .. 115) (21 amino acids), T3 (157 .. 177) (21 amino acids), T4 (244 .. 264) (21 amino acids).

ORAI3 lacks C195, a reactive cysteine present in terminus. They are conserved throughout evolution ORAI1 that serve as a detection system primarily in all mammalian ORAI1 proteins. Mutations at for changes in the extracellular oxidative these phosphorylation sites increase store-operated environment, and contains two additional cysteines Ca 2+ entry (SOCE) and CRAC current suggesting within the extracellular loop between TM3 and that ORAI1 phosphorylation at these residues by TM4. The absence of C195 in ORAI3 makes it protein kinase C (PKC) suppresses SOCE and resistant to H 2O2-inactivation, since pre-incubation CRAC channel activation. However, Ser-27 and with H 2O2of ORAI1/STIM1 expressing cells (HEK; Ser-30 are not present in ORAI2 and ORAI3. T cells) inhibits activation of ORAI1, but not of A phosphorylation of ORAI3 peptide has been ORAI3, and reinsertion of C195 within ORAI3 revealed by a phosphoproteome analysis of human renders ORAI3 channels redox sensitive (Bogeski liver cells (Sui et al., 2008). This phosphorylation et al., 2010). site is located in the C-terminus of ORAI3 on a Post-translational modifications of the protein tyrosine residue (Y278). Experimental ORAI3 Glycosylation: phosphorylation has also been demonstrated in Unlike ORAI1, ORAI3 does not have a HEK293 cells (Kawasaki et al., 2010). glycosylation site on the asparagine residue (N223) To examine in vivo PKC-mediated situated between the transmembrane domains TM3 phosphorylation, HEK293 cells expressing FLAG- et TM4 (Frischauf et al., 2008; Prakriya et al., tagged ORAI were incubated with 32 P monosodium 2006). phosphate, and then stimulated with thapsigargin in ORAI1 has a putative N-glycosylation motif (NVS) the presence of extracellular Ca 2+ . Thapsigargin in its extracellular loop between predicted mobilizes Ca 2+ from the ER and the extracellular transmembrane segments 3 and 4. This motif is space and activates Ca 2+ /DAG-dependent PKC absent in ORAI2 and 3 (Gwack et al., 2007). isoforms. ORAI1 phosphorylation is enhanced in ORAI3 migration properties do not change by response to thapsigargin. The levels of ORAI3 tunicamycin treatment. Indeed, HEK293 cells phosphorylation have been less than half of that stably transfected with FLAG-tagged ORAI and observed for ORAI1 (Kawasaki et al., 2010). treated with 2 µg/ml tunicamycin, showed that Other phosphorylation sites on ORAI3 were ORAI3 migrated at positions close to their predicted by NetPhos2.0: 13 serine sites (S20, S45, predicted molecular masse (32.5 kDa). S50, S57, S64, S65, S68, S86, S191, S203, S213, Phosphorylation: S214 and S20), 3 threonine sites (T26, T183 and Since ORAI3 is a tetraspanning plasma membrane T190) and 2 tyrosine sites (Y146 and Y278). protein, it contains three intracellular regions that Expression can potentially be phosphorylated by intracellular protein kinases: the N-terminus, an intracellular ORAI3 is only expressed in mammals (Cai, 2007). loop between transmembrane domains 2 and 3, and ORAI3 seems to be ubiquitously expressed in the C-terminus, each intracellular region potentially human contains one or more phosphorylation sites. Ser-27 (http://www.proteinatlas.org/ENSG00000175938/ti and Ser-30 have been identified as the main ssue), and mouse, showing a minor presence in phosphorylation sites in ORAI1 within its N- skeletal muscle, spleen and colon (Cordeiro and

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 178 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

Strauss, 2011; Gao et al., 2010; Gross et al., 2007). whereas ORAI3 can complement partially (partly More specifically, ORAI3 expression has been compensate in the absence of functional ORAI1) reported in brain, heart, kidney, testis, intestine, and ORAI2 has a lesser role (Gwack et al., 2007). placenta, lung (Gwack et al., 2007; Motiani et al., Combined overexpression of ORAI3 and STIM1 2013a), vascular smooth muscle cells (Trebak, results in substantial reconstitution of Ca 2+ entry in 2012), airway smooth muscle in human (Peel et al., SCID fibroblasts (Gwack et al., 2007). ORAI3 2008) and macrophages. ORAI3 mRNA is usually expression also rescues normal store-operated Ca 2+ much less expressed compared to ORAI1 in cells of entry in cells in which such entry was reduced by lymphoid origin. ORAI1, ORAI2, and ORAI3 are knockdown of ORAI1 (Mercer et al., 2006; expressed at similar levels in rat microglia (Hoth DeHaven et al., 2007). and Niemeyer, 2013). ORAI1, ORAI2, and ORAI3 channels are all similarly inhibited by extracellular Ca 2+ , indicating Localisation 2+ similar affinities for Ca within the selectivity ORAI3 localizes to the plasma membrane and filter. ORAI3 channels seem to differ from ORAI1 functions as a Ca 2+ -selective ion channel (Feske et and ORAI2 in being somewhat resistant to the al., 2006; Vig et al., 2006; Zhang et al., 2006; process of Ca 2+ depotentiation (DeHaven et al., Prakriya et al., 2006). This has been confirmed by 2007). Moreover, like ORAI1, ORAI3 can immunocytochemistry of tagged proteins expressed potentiate store-operated Ca 2+ entry in HEK293 in Jurkat T cells and in HEK293 cells. All three cells expressing TRPC6 or TRPC3 (Liao et al., ORAI isoforms are expressed and localized at or 2007). near the plasma membrane, with little or no overlap ORAI3 and ORAI1 channels participate in store- with the ER marker ERP72. This localization was operated Ca 2+ influx in human airway smooth not grossly altered after store depletion with muscle cells (Peel et al., 2008). Cells transfected thapsigargin (Gwack et al., 2007). During meiosis, with siRNA against ORAI3 display abnormal ORAI proteins get internalized into intracellular (cyclopiazonic acid) CPA-mediated Ca 2+ signals. vesicles and store-operated currents are suppressed Both Ca 2+ release from the stores and Ca 2+ influx (Yu et al., 2009). are reduced in the ORAI3 knockdown cells, Function suggesting that cells with reduced ORAI3 expression have a lower Ca 2+ store content and that In SOC channels: ORAI3 plays a role in regulating basal Ca 2+ levels ORAI3 presents a single putative channel pore and or in Ca 2+ release from the stores (Peel et al., 2008). 2+ has a role as a store-operated Ca (SOC) channel. In addition, ORAI genes expression and CRAC SOC channels are the major route for Ca 2+ entry in activation has also reported in the human retinal non-excitable cells, and they include ORAI pigment epithelium (Potier et al., 2009; Darbellay 2+ channels characterized by high selectivity for Ca et al., 2009; Bisaillon et al., 2010). over monovalent cations, low single-channel ORAI3 upregulation contributes to vascular smooth conductance (<1 pS), and an inwardly rectifying muscle remodeling and neointimal hyperplasia current-voltage (I-V) relationship. Functional caused by vascular injury. CRAC/SOC channels are formed by a tetrameric ORAI3 has been shown to be an important assembly of ORAI1/2/3 subunits (Ji et al., 2008; component of store-independent arachidonate- Mignen et al., 2008a; Penna et al., 2008; Maruyama regulated Ca 2+ (ARC) entry in HEK293 cells et al., 2009). (Mignen and Shuttleworth, 2000), and more ORAI3 is different from its family members, recently of a store-independent leukotriene C4- notably because of its exclusive presence in regulated Ca 2+ (LRC) entry pathway in vascular mammals (Cai, 2007) and its receptivity to smooth muscle cells (Zhang et al., 2013). pharmacological modulation (Schindl et al., 2008). In ARC channels: 2+ All three isoforms are selective to Ca , ORAI3 ORAI3 has been identified as an essential being more permeant to monovalent cations such as component of the store-independent, arachidonic + Na (DeHaven et al., 2007). Indeed, the ORAI3 acid activated, Ca 2+ -selective ARC channels currents display a significantly increased (Mignen and Shuttleworth, 2000; Mignen et al., permeability to Na + when measured in the absence 2008b). These channels are found in a variety of of external divalent cations (Lis et al., 2007). different cell types, frequently co-existing with ORAI3 expression is capable of inducing a store- store-operated CRAC channels (Mignen et al., induced conductance, but its magnitude is 2003; Mignen et al., 2005; Li et al., 2008; Yeung- considerably smaller than that seen with ORAI1. Yam-Wah et al., 2010), and sharing similar basic In HEK293 cells, human SCID T cells and biophysical properties. They are pentameric fibroblasts, in which store depletion has been aggregates consisting of three ORAI1 and two induced with thapsigargin, ORAI1 was shown to be ORAI3 subunits that form a functional ARC 2+ the major regulator of store-operated Ca influx, channel pore (Mignen et al., 2008b; Mignen et al.,

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 179 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

2007; Thompson et al., 2010). Two ORAI3 (ARC) and leukotriene C4 (LTC4)-regulated subunits are required within the pentamer to make Ca 2+ (LRC) channels (ORAI1/3 heteromultimers). the ARC channel sensitive to activation by low ORAI3 activation and interaction with STIM concentrations of arachidonic acid. ARC channels proteins are characterized by being activated by low ORAI channels are activated by STIM1 or STIM2, concentrations (2-8 µM) of arachidonic acid, single-pass transmembrane proteins localized insensitive to 2-APB, and with an absolutely predominantly in the membrane of the endoplasmic dependence on the pool of STIM1 residing in the reticulum. STIM proteins have a long C-terminal plasma membrane for their activation (Mignen et cytoplasmic region and contain an N-terminal EF- al., 2009). The acquisition of selective activation by hand located in the ER lumen that functions as a arachidonic acid depends on the cytosolic N- sensor of ER Ca 2+ levels (Roos et al., 2005; Liou et terminal domain of ORAI3 (Thompson et al., al., 2005; Williams et al., 2001; Stathopulos et al., 2010). 2006). The ARC currents are distinguished from the co- The activation of ORAI channels by STIM depends existing CRAC channel currents by their store- on Ca 2+ store depletion and is reversible once the independent activation, and the absence of any stores are refilled (Luik et al., 2008; Soboloff et al., detectible fast inactivation. Expression of a 2006). STIM1 activates store-operated Ca 2+ dominant-negative mutant of ORAI3 (E81Q) had channels only when it is not fixing Ca 2+ , e.g. when no effect on store-operated CRAC channel currents, the stores are depleted (Zhang et al., 2005). One but reduced currents through the store-independent minute after store depletion, STIM proteins are ARC channels to negligible levels (Mignen et al., redistributed in puncta in close proximity to the 2008b). plasma membrane (Liou et al., 2005; Luik et al., A recent study indicates a role of ARC channels in 2008; Várnai et al., 2007; Baba et al., 2006), where insulin secretion by pancreatic β cells (Yeung-Yam- they co-localize with and activate ORAI channels, Wah et al., 2010). It has been shown that the known allowing Ca 2+ influx (Liou et al., 2005; Wu et al., ability of glucose and various insulin stimulants 2006; Muik et al., 2008). This process implies including acetylcholine and cholecystokinin to tetramerisation of STIM1 proteins using the N- induce increases in cellular arachidonic acid results terminus (Luik et al., 2008). It is thought that within in activation of ARC channels in the β cells, these puncta, STIM1 communicates with and opens increasing cytosolic Ca 2+ levels and enhancing the CRAC channels located to the plasma membrane subsequent insulin secretion (Yeung-Yam-Wah et (Luik et al., 2006; Parvez et al., 2008). al., 2010). The initial interaction of STIM1 with the ORAI In LRC channels: channels involves their cytosolic C-terminal region ORAI3 channels are also implicated in store- (Li et al., 2007; Muik et al., 2008; Frischauf et al., independent, leukotriene C4 (LTC4)-regulated Ca 2+ 2009). In all three ORAI subtypes, this region (LRC) channels. Comparison of AA (arachidonic contains a predicted coiled-coil domain that is acid)- and LTC4-activated currents in vascular critical for interactions with STIM1 (Muik et al., smooth muscle cells and in HEK293 cells using 2008). whole-cell and perforated patch-clamp recording Truncation analysis identified a cytoplasmic region shows indistinguishable non-additive LTC4- and of STIM1, termed the CRAC activation domain AA-activated currents that both require ORAI1 and (CAD)/STIM1 ORAI1 activating region (SOAR) to ORAI3. This suggests that ARC and LRC be sufficient to activate ORAI1 (Kawasaki et al., conductances are mediated by the same channel. 2009; Muik et al., 2009; Park et al., 2009; Yuan et Experiments using a non-metabolizable form of AA al., 2009). The cytoplasmic N and C termini of or an inhibitor of 5-lipooxygenase suggest that ORAI1 mediate channel opening by interaction ARC and LRC currents in both cell types can be with STIM1. activated by either LTC4 or AA, with LTC4 being The activation of ORAI3-induced store-operated more potent. Although the plasma membrane (PM)- currents is significantly slower than that seen with STIM1 was required for current activation by LTC4 ORAI1 and ORAI2 (Lis et al., 2007). Contrary to and AA under whole-cell patch-clamp recordings in ORAI1, both ORAI2 and ORAI3 exhibit a 15-17 both cell types, ER-STIM1 was sufficient with fold higher coiled-coil probability (Frischauf et al., perforated patch recordings. These results 2009). A single point mutation in the ORAI1 demonstrate that ARC and LRC currents are coiled-coil domain (L273S) abrogates mediated by the same cellular populations of communication with STIM1 C-terminus (Frischauf STIM1, ORAI1, and ORAI3 (Zhang et al., 2013). et al., 2009; Muik et al., 2008). A single point In summary, ORAI3 proteins contribute to Ca 2+ mutation (L285S) within ORAI3 coiled-coil entry into cells through both store-dependent, Ca 2+ domain results in a partial inhibition of the release-activated Ca 2+ (CRAC) channels and store- interaction with STIM1 and subsequent activation independent, arachidonic acid (AA)-regulated Ca 2+ of ORAI3 currents. Full inhibition of the ORAI3-

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 180 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

induced currents requires incorporation of an ORAI3 inactivation additional mutation (L292S) in the coiled-coil Fast inactivation of ORAI channels is mediated by domain. cooperative interplay of several structures within According to Bergsmann, activation of ORAI ORAI proteins, by the CRAC modulatory domain channels requires coupling of the C terminus of (CMD) of STIM1, and via calmodulin binding to STIM to the N and C termini of ORAI (Bergsmann the ORAI N terminus (Parekh and Putney, 2005; et al., 2011), since increasing N-terminal Lee et al., 2009; Frischauf et al., 2011; Derler et al., truncations causes a progressive decrease of ORAI3 2009). fast inactivation concomitant with diminished ORAI3 currents exhibit a marked fast inactivation binding to calmodulin. within the first 100 ms, while that of ORAI2 or Therefore, a fully conserved N-terminal ORAI ORAI1 show less robust feedback regulation (Lis et region (aa 48-65 in ORAI3) is essential for STIM1- al., 2007; Schindl et al., 2009; Lee et al., 2009). dependent STIMulation (Derler et al., 2009; Li et This effect depends on the presence of three al., 2007; Yuan et al., 2009; Fahrner et al., 2009; conserved glutamates (E281, E283, E284) in the C- Park et al., 2009; Lis et al., 2010). terminal region of ORAI3 (Lee et al., 2009). Moreover, a single lysine within this conserved According to Yamashita et al. (2007), fast region (K60E in ORAI3) represents a critical inactivation is determined by the same acidic residue for store-operated activation (Lis et al., residues involved in determining Ca 2+ selectivity. A 2010). STIM1 C-terminus domain that include an acidic Interaction between ORAI family members cluster (amino acids 475-483) termed CRAC ORAI3 can multimerize with ORAI1 to form cation Modulatory Domain (CMD) is also indispensable channels that conduct Ca 2+ to some degree, since for fast ORAI channel inactivation (Derler et al., HEK293 cells stably expressing FLAG-tagged 2009; Mullins et al., 2009; Lee et al., 2009), since ORAI2 and ORAI3 revealed co- mutations in the CMD results in ORAI3 currents immunoprecipitation of ORAI2 and ORAI3 with with attenuated or even abolished Ca 2+ -dependent transiently overexpressed Myc-ORAI1. Thus, inactivation (Derler et al., 2009; Lee et al., 2009). ORAI members form homomultimers and can also On the other hand, Litjens et al. (2004) suggest that form heteromultimers (Gwack et al., 2007). fast inactivation may be calmodulin (CaM) Protein Interactions other than STIM dependent and involves a region in the cytosolic N- In addition to STIM1, p45 renamed as CRACR2A terminal domain of ORAI3 (S45-K62) that binds (CRAC regulator 2A) is also shown to co- CaM in a Ca 2+ -dependent manner (Mullins et al., immunoprecipitate with ORAI1, ORAI2 and 2009; Frischauf et al., 2011). Transient CaM ORAI3, suggesting a conserved binding mechanism binding is assumed to mediate fast inactivation. The with all the ORAI proteins, and that the ORAI process may be that CaM transiently competes with channels, STIM1 and CRACR2A may form a STIM1 for the N-terminal interaction site on ORAI ternary complex though direct interaction. essential for channel gating. Various other proteins and lipids have been Not only the C- but also the N-terminus and the identified to interact with either STIM1 or ORAI3 second intracellular loop between TM2 and TM3 or both. Among them is calmodulin (Mullins et al., contribute to ORAI inactivation/gating in a 2009; Parvez et al., 2008; Bergsmann et al., 2011). cooperative manner (Frischauf et al., 2011) and Calmodulin binds to ORAI3 and, together with modulate fast and slow inactivation as revealed by STIM, contributes to fast calcium-dependent chimeric and mutational approaches (Srikanth et al., inactivation; the structural studies show that 2010). ORAI fast inactivation also involves the CRACR2A/B is also able to interact with ORAI3 pore region since mutations of negatively charged (Srikanth et al., 2010) but to date there is no residues within the pore of ORAI results in evidence of functional regulation, because ORAI3 attenuation of Ca 2+ -dependent inactivation is able to form some complex with STIM-1 (Faouzi (Yamashita et al., 2007). et al., 2011). Pharmacology All proteins that interact with STIM1 are able to To date there is no specific inhibitor of ORAI3 but modulate ORAI3 function indirectly. Thus, SARAF ORAI3 channels can be blocked by generic (Palty et al., 2012), MS4A4B (Howie et al., 2009), blockers of calcium entry channels such as La 3+ Golli (Walsh et al., 2010), adenylyl cyclase type 8 (50-100 µM) and Gd 3+ (1-5 µM). Other non- (AC8) (Martin et al., 2009), the polycystin-1 specific blockers include SKF96365, the myosin cleavage product P100 (Woodward et al., 2010), light chain kinase inhibitor ML-9 (Smyth et al., caveolin (Yu et al., 2010), SPCA2 (Feng et al., 2008), and the bistrifluoromethyl-pyrazole 2010) and the L-type Ca 2+ channel (Cav1.2) (Wang derivative BTP2 (Zitt et al., 2004) can be used. et al., 2010) or the phospholipids PIP2 and PIP3 Another compound extensively studied is 2- (Korzeniowski et al., 2009; Walsh et al., 2009) are aminoethoxydiphenyl borate (2-APB), originally able to modulate indirectly ORAI3 activity. characterized as an inhibitor of InsP 3 receptors

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 181 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

(Maruyama et al., 1997; Bilmen and Michelangeli, ethylphenol (4-CEP) blocks ORAI1/3 store- 2002), later shown to have multiple diverse effects operated channels. 4-CEP induces a significant Ca 2+ including both the inhibition and activation of release in rat L6 myoblasts, but inhibits SOCE. The various different members of the TRP channel inhibitory effect is concentration-dependent and family (Voets et al., 2001; Trebak et al., 2002; more potent than the one of its analogues 4-CmC Chung et al., 2004; Hu et al., 2004; Li et al., 2006; and 4-chlorophenol (4-ClP). In the HEK293 T-REx Juvin et al., 2007), and the inhibition of SERCA cells overexpressing STIM1/ORAI1-3, 4-CEP pumps (Missiaen et al., 2001; Peppiatt et al., 2003), inhibited the ORAI1, ORAI2 and ORAI3 currents as well as to affect store-operated Ca 2+ entry via evoked by thapsigargin. The 2-APB-induced CRAC channels (Gregory et al., 2001; Iwasaki et ORAI3 current was also blocked by 4-CEP. This al., 2001; Prakriya and Lewis, 2001). inhibitory effect was reversible and independent of 2-APB displays a bi-functional effect that is the Ca 2+ release. The two analogues, 4-CmC and 4- dependent on the concentration used. High ClP, also inhibited the ORAI1-3 channels. Excised concentrations of 2-APB were shown to increase patch and intracellular application of 4-CEP store-operated currents in cells expressing STIM1 demonstrated that the action site was located and ORAI3 (Lis et al., 2007; DeHaven et al., 2008; extracellularly (Zeng et al., 2014). Peinelt et al., 2008; Schindl et al., 2008), GSK-7975A and GSK-5503A are selective CRAC accompanied by marked changes in ion selectivity channel blockers that inhibit both ORAI1 and by increasing ORAI3 channel pore size from ~3.8 ORAI3 currents by acting downstream of STIM1 Å to more than 5.34 Å, an effect that was oligomerization and STIM1/ORAI1 interaction, apparently dependent on the E165 residue of potentially via an allosteric effect on the selectivity ORAI3 that lies in the third transmembrane domain filter of ORAI (Derler et al., 2012). Both GSK (Schindl et al., 2008). The residues that assist in compounds fully inhibited ORAI3 currents. formation of the 2-APB-activated ORAI3 pore are Similarly, Synta-66 inhibited ORAI3 currents at a lined by TM1 residues, but also allows for TM3 similar rate as the GSK compounds. By contrast, 10 E165 to approach the central axis of the channel µM La 3+ blocked ORAI3 currents more rapidly. that forms the conducting pathway, or pore The GSK compounds appeared to inhibit ORAI3 (Amcheslavsky et al., 2014). Transmembrane currents slightly faster than those of ORAI1. domains 2 and 3, together with the linking Overall these GSK compounds were equally intracellular loop, are required for 2-APB to effective at blocking ORAI1 and ORAI3, and directly activate ORAI3 channels (Zhang et al., inhibition occurred at a substantially slower rate 2008). than La 3+ . Inhibition of ORAI currents by GSK ORAI3 can be directly activated by high compounds is not readily reversible: neither ORAI1 concentrations of 2-APB, in a STIM1- and store nor ORAI3 currents showed substantial recovery depletion-independent manner (DeHaven et al., from block by GSK-7975A or GSK-5503A over a 2008; Peinelt et al., 2008; Schindl et al., 2008; 4-5 min wash-out period. Zhang et al., 2008; Wang et al., 2009). These direct 2-APB stimulated ORAI3 currents are less 2-APB induced currents display large inward and susceptible to GSK-7975A. 10 µM GSK-7975A outward currents (i.e. they show double was totally ineffective in inhibiting these ORAI3 rectification) and a leftward shift in the reversal currents in contrast to those activated via STIM1. potential, features that indicate a marked reduction 50 µM GSK-7975A caused 50% inhibition and 100 in Ca 2+ selectivity, and an increased permeability to µM GSK-7975A caused full inhibition. The GSK monovalent cations (DeHaven et al., 2008; Peinelt CRAC channel blockers did not differentiate et al., 2008; Schindl et al., 2008; Zhang et al., between ORAI1 and ORAI3 channels consistent 2008). with the conserved pore geometry and selectivity When ORAI3 forms a store operated channel, store- filter among the ORAI isoforms. operated ORAI3 currents are potentiated by 2-APB Homology at low concentrations (<10 µM) without affecting ion selectivity (Yamashita et al., 2011). This effect ORAI3 (encoding gene: MGC13024 located on requires the presence of STIM1, and is strictly chromosome 16) has two human homologs: ORAI1 dependent on store depletion. (FLJ14466, chromosome 12) and ORAI2 (C7orf19, The most obvious unique property of the channels chromosome 7) (Feske et al., 2006). ORAI3 made involving ORAI3 is their ability to be activated an evolutionary appearance in mammals, evolving independently of store depletion, either from ORAI1 rather than ORAI2 (Cai, 2007) and pharmacologically by 2-APB or, physiologically, manifesting conductances that display unique by agonist-generated increased levels of features in their gating, selectivity, regulation and intracellular arachidonic acid. mode of activation (Shuttleworth, 2012). A recent study by (Zeng et al., 2014) shows that the ORAI3 is the 'newest' ORAI family member in the ryanodine receptor (RyR) agonist 4-chloro-3- evolutionary tree (Shuttleworth, 2012). Orthologous

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 182 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

ORAI3 genes are found in the following species: involvement in cell proliferation/survival and cell chimpanzee (98.98% homology), dog (92.20% cycle progression may be at least partially linked to homology), cow (90.51% homology), rat (89.83% the calcium influx through the channels since the 2+ homology), mice (88.48% homology). reduction of external calcium concentration [Ca ]o to 0.2 mM decreases significantly BC cell Mutations proliferation (Faouzi et al., 2011). A subsequent study highlighted a correlation Note between ORAI3 and the oncogene c-myc Understanding of the role of ORAI1, and indeed its expression in tumor tissues and in BC cell lines: initial identification, came from the study of ORAI3 and c-myc were over-expressed in 70% and patients carrying functionally critical mutations in 80% cases respectively. Expression of c-myc, as this gene. To date, no equivalent identification of assessed by RT-qPCR, is higher in the MCF-7 patients bearing similar mutations in ORAI3 have cancer cell line than in the non-cancerous MCF- been identified. (Diseases associated to absence of 10A cell line. A similar over-expression pattern was ORAI2, ORAI3 or STIM2 function have not been shown for ORAI3 in these cell lines (Faouzi et al., identified in human yet). 2013). ORAI3 down-regulation reduces both c-myc expression and activity levels exclusively in BC Implicated in cells, whereas ORAI1 (one of the two mammalian Note homologs to ORAI3) induced an upregulation of c- myc mRNA. The involvement of c-myc in the ORAI3 overexpression is associated with breast, ORAI3 signaling was demonstrated when silencing lung, leukemia and prostate cancers. c-myc resulted in closely-similar and non-additive Breast cancer effects to the ones induced by ORAI3 Note downregulation: decreased cell proliferation, cell ORAI3 channels are reported to be highly cycle arrest with a significant accumulation of the expressed in breast cancer (BC) tissues and breast cells in the G0/G1 phase, increased cell mortality cancer cell lines MCF-7 and T47D compared to (Faouzi et al., 2013). adjacent non cancerous tissues and non cancerous Authors showed that ORAI3 channels affect c-myc, cell lines, respectively (Faouzi et al., 2011). They most likely via the MAP Kinase pathway, as are also shown to be involved in proliferation, cell demonstrated by decreased phosphorylation levels cycle progression and survival of BC cells by of extracellular signal-regulated kinases 1 and 2 regulating the G1 phase and G1/S transition (ERK1/ERK2) after ORAI3 downregulation regulatory proteins. Thus, ORAI3 knockdown by (Faouzi et al., 2013). Parallel studies also reported that ORAI3 mediates specific siRNA inhibits cell proliferation, arrests + cell cycle progression in G1 phase, and increases SOCE in estrogen-receptor-positive (ER ) BC cell lines (Motiani et al., 2010), whereas in estrogen- apoptosis in these cells (Faouzi et al., 2011). This - phenotype is associated with a reduction in CDK4 receptor-negative (ER ) BC cell lines, SOCE is and CDK2 (cyclin-dependent kinases) and cyclin E mediated by ORAI1. This study was the first to and cyclin D1 expression, an accumulation of describe SOCE and endogenous calcium release- p21Waf1/Cip1 (a cyclin-dependent kinase activated currents (CRAC) that are mediated by inhibitor) and p53 (a tumor-suppressor protein) native ORAI3 channels and highlights a potential together with an increase of Bax/Bcl-2 ratio. connection between estrogen receptor alpha (ER α) Interestingly, these effects seem to be specific to and ORAI3 (Motiani et al., 2010). Authors then cancer cells, since down-regulation of ORAI3 reported that knockdown of ER α decreases ORAI3 channels does not affect either cell proliferation or expression level leading to a decrease in ORAI3- cell survival of normal breast cells. Annexin V and mediated SOCE and CRAC current, while 7-AAD double staining and analysis of the anti- activation of ER α increased ORAI3 expression and apoptotic protein Bcl-2 to the pro-apoptotic protein SOCE in MCF7 cells (Motiani et al., 2013b). Bax ratio revealed that the induced cell mortality by Consistently with the above cited studies, ORAI3 ORAI3 knockdown was mainly apoptotic as knockdown inhibits SOCE-dependent demonstrated by the increased percentage of phosphorylation of both ERK1/2 and focal adhesion Annexin V-positive cells and the increased kinase (FAK). Bax/Bcl-2. It also decreases the transcriptional activity of The same study showed that ORAI3 contributes to nuclear factor of activated T-cells (NFAT), which 2+ was associated with decreased cell growth and Ca influx in BC cells where both Store Operated + Calcium Entry (SOCE) amplitude and resting Matrigel invasion of ER MCF7 cells in contrast to 2+ ER - MDA-MB231 cells where no effects were [Ca ]i decreased significantly with ORAI3 knockdown. The authors concluded that the ORAI3 observed (Motiani et al., 2013b).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 183 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

2+ Lung cancer ORAI3-mediated SOC leading to [Ca ]i increase. Moreover, ORAI3 functional expression was higher Note in 2-APB-sensitive leukemia and myeloid cells as An overexpression of ORAI3 was observed in compared to 2-APB-insensitive myeloid cells 66.7% of human tumor samples as compared to the (Yanamadra et al., 2011). These results suggest that human non-tumoral samples (40/60) as revealed by Tipifarnib-resistant cells express less ORAI3 immunohistochemistry. The 60 lung ORAI3 conferring protection against apoptotic adenocarcinomas were classified according to effect of Tipifarnib (Yanamadra et al., 2011). grading system proposed by Yoshizawa et al. 2011 (low, intermediate and high grades). The ORAI3 Prostate cancer staining score is reported to be highly expressed in Note higher tumor grade (high grade; n= 16) as ORAI3 mRNA expression levels are significantly compared to low tumor grades (Ay et al., 2013). reduced in tumours when compared to non-tumour ORAI3 is also expressed in non small cell lung tissues from 13 prostate cancer patients. mRNA carcinoma cells (NSCLCC) such as NCI-H23, NCI- expression levels of ORAI3 are decreased in both H460, A549 and Calu-1. In NCI-H23 and NCI- androgen-sensitive human prostate adenocarcinoma H460 cells, ORAI3 is a major actor of Store cell line (LNCaP) and androgen-insensitive prostate Operated Calcium Entry (Ay et al., 2013). Ay et al. cancer cell line (DU145), when compared to human (2013) demonstrated that ORAI3 is involved in prostate epithelial cells from healthy tissue. The NSCLCC proliferation. Indeed, ORAI3 inhibition pharmacological effects of 2-APB on CRAC induces a strong decrease in NSCLCC proliferation, channels in prostate cancer cells differ from those accumulating cells in G0/G1 phase of the cell cycle. in human prostate epithelial cells, and siRNA based This accumulation in G0/G1 phase is associated knock-down experiments indicate changed ORAI3 with a decrease in Cyclin D1/cdk4 and Cyclin channel levels are underlying the altered E/cdk2 proteins level. No effect is observed on pharmacological profile (Holzmann et al., 2013). apoptosis. The same study demonstrated that SOCE induces Akt phosphorylation in NSCLCC and References ORAI3 inhibition decreases this activation demonstrating ORAI3 can promote proliferation Maruyama T, Kanaji T, Nakade S, Kanno T, Mikoshiba K. 2APB, 2-aminoethoxydiphenyl borate, a membrane- through SOCE by activating Akt pathway. They penetrable modulator of Ins(1,4,5)P3-induced Ca2+ also showed that neither ORAI1 nor ORAI2 are release. J Biochem. 1997 Sep;122(3):498-505 involved in SOCE in NSCLC cell lines, suggesting Mignen O, Shuttleworth TJ. I(ARC), a novel arachidonate- that ORAI3 is the main component of SOCE in regulated, noncapacitative Ca(2+) entry channel. J Biol those cells (Ay et al., 2013). Chem. 2000 Mar 31;275(13):9114-9 The same type of mechanism is observed with Gregory RB, Rychkov G, Barritt GJ. Evidence that 2- TRPC1 in NSCLCC. Indeed Tajeddine and Gailly aminoethyl diphenylborate is a novel inhibitor of store- (2012) have demonstrated that TRPC1 is involved operated Ca2+ channels in liver cells, and acts through a in G1/S transition in A549 NSCLC cell line through mechanism which does not involve inositol trisphosphate SOCE. They showed that cell cycle arrest after receptors. Biochem J. 2001 Mar 1;354(Pt 2):285-90 TRPC1 inhibition induces a decrease in EGFR Iwasaki H, Mori Y, Hara Y, Uchida K, Zhou H, Mikoshiba activation and subsequent signaling (PI3K/Akt, K. 2-Aminoethoxydiphenyl borate (2-APB) inhibits capacitative calcium entry independently of the function of MAPK). inositol 1,4,5-trisphosphate receptors. Receptors Those two studies suggest that SOCE is an Channels. 2001;7(6):429-39 important mechanism in proliferation of NSCLCC. Missiaen L, Callewaert G, De Smedt H, Parys JB. 2- Indeed, EGFR signaling is overactivated in Aminoethoxydiphenyl borate affects the inositol 1,4,5- NSCLCC either by constitutive activation of EGFR trisphosphate receptor, the intracellular Ca2+ pump and or K-Ras mutation. ORAI3, able to activate this the non-specific Ca2+ leak from the non-mitochondrial pathway, hence can be a potential target for anti- Ca2+ stores in permeabilized A7r5 cells. Cell Calcium. 2001 Feb;29(2):111-6 cancer drug. Prakriya M, Lewis RS. Potentiation and inhibition of Ca(2+) Myeloid leukemia release-activated Ca(2+) channels by 2- Note aminoethyldiphenyl borate (2-APB) occurs independently of IP(3) receptors. J Physiol. 2001 Oct 1;536(Pt 1):3-19 The mRNA levels of ORAI3 in both human leukemia and human myeloma tipifarnib-sensitive Voets T, Prenen J, Fleig A, Vennekens R, Watanabe H, Hoenderop JG, Bindels RJ, Droogmans G, Penner R, cell lines were significantly higher than in the Nilius B. CaT1 and the calcium release-activated calcium tipifarnib-insensitive human myeloma cells. channel manifest distinct pore properties. J Biol Chem. Tipifarnib is a new apoptotic agent that inhibits 2001 Dec 21;276(51):47767-70 farnesyltransferase responsible for the transfer of a Williams RT, Manji SS, Parker NJ, Hancock MS, Van farnesyl group to Ras protein. Tipifarnib activates Stekelenburg L, Eid JP, Senior PV, Kazenwadel JS,

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 184 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

Shandala T, Saint R, Smith PJ, Dziadek MA. Identification Baba Y, Hayashi K, Fujii Y, Mizushima A, Watarai H, and characterization of the STIM (stromal interaction Wakamori M, Numaga T, Mori Y, Iino M, Hikida M, molecule) gene family: coding for a novel class of Kurosaki T. Coupling of STIM1 to store-operated Ca2+ transmembrane proteins. Biochem J. 2001 Aug 1;357(Pt entry through its constitutive and inducible movement in 3):673-85 the endoplasmic reticulum. Proc Natl Acad Sci U S A. 2006 Nov 7;103(45):16704-9 Bilmen JG, Michelangeli F. Inhibition of the type 1 inositol 1,4,5-trisphosphate receptor by 2- Feske S, Gwack Y, Prakriya M, Srikanth S, Puppel SH, aminoethoxydiphenylborate. Cell Signal. 2002 Tanasa B, Hogan PG, Lewis RS, Daly M, Rao A. A Nov;14(11):955-60 mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006 May Trebak M, Bird GS, McKay RR, Putney JW Jr. Comparison 11;441(7090):179-85 of human TRPC3 channels in receptor-activated and store- operated modes. Differential sensitivity to channel blockers Li M, Jiang J, Yue L. Functional characterization of homo- suggests fundamental differences in channel composition. and heteromeric channel kinases TRPM6 and TRPM7. J J Biol Chem. 2002 Jun 14;277(24):21617-23 Gen Physiol. 2006 May;127(5):525-37 Mignen O, Thompson JL, Shuttleworth TJ. Ca2+ selectivity Luik RM, Wu MM, Buchanan J, Lewis RS. The elementary and fatty acid specificity of the noncapacitative, unit of store-operated Ca2+ entry: local activation of CRAC arachidonate-regulated Ca2+ (ARC) channels. J Biol channels by STIM1 at ER-plasma membrane junctions. J Chem. 2003 Mar 21;278(12):10174-81 Cell Biol. 2006 Sep 11;174(6):815-25 Peppiatt CM, Collins TJ, Mackenzie L, Conway SJ, Mercer JC, Dehaven WI, Smyth JT, Wedel B, Boyles RR, Holmes AB, Bootman MD, Berridge MJ, Seo JT, Roderick Bird GS, Putney JW Jr. Large store-operated calcium HL. 2-Aminoethoxydiphenyl borate (2-APB) antagonises selective currents due to co-expression of Orai1 or Orai2 inositol 1,4,5-trisphosphate-induced calcium release, with the intracellular calcium sensor, Stim1. J Biol Chem. inhibits calcium pumps and has a use-dependent and 2006 Aug 25;281(34):24979-90 slowly reversible action on store-operated calcium entry channels. Cell Calcium. 2003 Jul;34(1):97-108 Prakriya M, Feske S, Gwack Y, Srikanth S, Rao A, Hogan PG. Orai1 is an essential pore subunit of the CRAC Chung MK, Lee H, Mizuno A, Suzuki M, Caterina MJ. 2- channel. Nature. 2006 Sep 14;443(7108):230-3 aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J Neurosci. 2004 Jun Soboloff J, Spassova MA, Dziadek MA, Gill DL. Calcium 2;24(22):5177-82 signals mediated by STIM and Orai proteins--a new paradigm in inter-organelle communication. Biochim Hu HZ, Gu Q, Wang C, Colton CK, Tang J, Kinoshita- Biophys Acta. 2006 Nov;1763(11):1161-8 Kawada M, Lee LY, Wood JD, Zhu MX. 2- aminoethoxydiphenyl borate is a common activator of Stathopulos PB, Li GY, Plevin MJ, Ames JB, Ikura M. TRPV1, TRPV2, and TRPV3. J Biol Chem. 2004 Aug Stored Ca2+ depletion-induced oligomerization of stromal 20;279(34):35741-8 interaction molecule 1 (STIM1) via the EF-SAM region: An initiation mechanism for capacitive Ca2+ entry. J Biol Litjens T, Harland ML, Roberts ML, Barritt GJ, Rychkov Chem. 2006 Nov 24;281(47):35855-62 GY. Fast Ca(2+)-dependent inactivation of the store- operated Ca2+ current (ISOC) in liver cells: a role for Vig M, Peinelt C, Beck A, Koomoa DL, Rabah D, Koblan- calmodulin. J Physiol. 2004 Jul 1;558(Pt 1):85-97 Huberson M, Kraft S, Turner H, Fleig A, Penner R, Kinet JP. CRACM1 is a plasma membrane protein essential for Zitt C, Strauss B, Schwarz EC, Spaeth N, Rast G, store-operated Ca2+ entry. Science. 2006 May Hatzelmann A, Hoth M. Potent inhibition of Ca2+ release- 26;312(5777):1220-3 activated Ca2+ channels and T-lymphocyte activation by the pyrazole derivative BTP2. J Biol Chem. 2004 Mar Wu MM, Buchanan J, Luik RM, Lewis RS. Ca2+ store 26;279(13):12427-37 depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J Cell Biol. Liou J, Kim ML, Heo WD, Jones JT, Myers JW, Ferrell JE 2006 Sep 11;174(6):803-13 Jr, Meyer T. STIM is a Ca2+ sensor essential for Ca2+- store-depletion-triggered Ca2+ influx. Curr Biol. 2005 Jul Zhang SL, Yeromin AV, Zhang XH, Yu Y, Safrina O, 12;15(13):1235-41 Penna A, Roos J, Stauderman KA, Cahalan MD. Genome- wide RNAi screen of Ca(2+) influx identifies genes that Mignen O, Thompson JL, Yule DI, Shuttleworth TJ. regulate Ca(2+) release-activated Ca(2+) channel activity. Agonist activation of arachidonate-regulated Ca2+- Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):9357-62 selective (ARC) channels in murine parotid and pancreatic acinar cells. J Physiol. 2005 May 1;564(Pt 3):791-801 Cai X. Molecular evolution and structural analysis of the Ca(2+) release-activated Ca(2+) channel subunit, Orai. J Parekh AB, Putney JW Jr. Store-operated calcium Mol Biol. 2007 May 18;368(5):1284-91 channels. Physiol Rev. 2005 Apr;85(2):757-810 DeHaven WI, Smyth JT, Boyles RR, Putney JW Jr. Roos J, DiGregorio PJ, Yeromin AV, Ohlsen K, Lioudyno Calcium inhibition and calcium potentiation of Orai1, Orai2, M, Zhang S, Safrina O, Kozak JA, Wagner SL, Cahalan and Orai3 calcium release-activated calcium channels. J MD, Veliçelebi G, Stauderman KA. STIM1, an essential Biol Chem. 2007 Jun 15;282(24):17548-56 and conserved component of store-operated Ca2+ channel function. J Cell Biol. 2005 May 9;169(3):435-45 Gross SA, Wissenbach U, Philipp SE, Freichel M, Cavalié A, Flockerzi V. Murine ORAI2 splice variants form Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman functional Ca2+ release-activated Ca2+ (CRAC) channels. MH, Stauderman KA, Cahalan MD. STIM1 is a Ca2+ J Biol Chem. 2007 Jul 6;282(27):19375-84 sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature. 2005 Oct Gwack Y, Srikanth S, Feske S, Cruz-Guilloty F, Oh-hora 6;437(7060):902-5 M, Neems DS, Hogan PG, Rao A. Biochemical and functional characterization of Orai proteins. J Biol Chem.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 185 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

2007 Jun 1;282(22):16232-43 regulated Ca2+-selective (ARC) channels. J Physiol. 2008a Jan 1;586(1):185-95 Hewavitharana T, Deng X, Soboloff J, Gill DL. Role of STIM and Orai proteins in the store-operated calcium Mignen O, Thompson JL, Shuttleworth TJ. Orai1 subunit signaling pathway. Cell Calcium. 2007 Aug;42(2):173-82 stoichiometry of the mammalian CRAC channel pore. J Physiol. 2008b Jan 15;586(2):419-25 Juvin V, Penna A, Chemin J, Lin YL, Rassendren FA. Pharmacological characterization and molecular Muik M, Frischauf I, Derler I, Fahrner M, Bergsmann J, determinants of the activation of transient receptor Eder P, Schindl R, Hesch C, Polzinger B, Fritsch R, Kahr potential V2 channel orthologs by 2-aminoethoxydiphenyl H, Madl J, Gruber H, Groschner K, Romanin C. Dynamic borate. Mol Pharmacol. 2007 Nov;72(5):1258-68 coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J Biol Chem. Li Z, Lu J, Xu P, Xie X, Chen L, Xu T. Mapping the 2008 Mar 21;283(12):8014-22 interacting domains of STIM1 and Orai1 in Ca2+ release- activated Ca2+ channel activation. J Biol Chem. 2007 Oct Parvez S, Beck A, Peinelt C, Soboloff J, Lis A, Monteilh- 5;282(40):29448-56 Zoller M, Gill DL, Fleig A, Penner R. STIM2 protein mediates distinct store-dependent and store-independent Liao Y, Erxleben C, Yildirim E, Abramowitz J, Armstrong modes of CRAC channel activation. FASEB J. 2008 DL, Birnbaumer L. Orai proteins interact with TRPC Mar;22(3):752-61 channels and confer responsiveness to store depletion. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4682-7 Peel SE, Liu B, Hall IP. ORAI and store-operated calcium influx in human airway smooth muscle cells. Am J Respir Lis A, Peinelt C, Beck A, Parvez S, Monteilh-Zoller M, Cell Mol Biol. 2008 Jun;38(6):744-9 Fleig A, Penner R. CRACM1, CRACM2, and CRACM3 are store-operated Ca2+ channels with distinct functional Peinelt C, Lis A, Beck A, Fleig A, Penner R. 2- properties. Curr Biol. 2007 May 1;17(9):794-800 Aminoethoxydiphenyl borate directly facilitates and indirectly inhibits STIM1-dependent gating of CRAC Mignen O, Thompson JL, Shuttleworth TJ. STIM1 channels. J Physiol. 2008 Jul 1;586(13):3061-73 regulates Ca2+ entry via arachidonate-regulated Ca2+- selective (ARC) channels without store depletion or Penna A, Demuro A, Yeromin AV, Zhang SL, Safrina O, translocation to the plasma membrane. J Physiol. 2007 Parker I, Cahalan MD. The CRAC channel consists of a Mar 15;579(Pt 3):703-15 tetramer formed by Stim-induced dimerization of Orai dimers. Nature. 2008 Nov 6;456(7218):116-20 Takahashi Y, Murakami M, Watanabe H, Hasegawa H, Ohba T, Munehisa Y, Nobori K, Ono K, Iijima T, Ito H. Schindl R, Bergsmann J, Frischauf I, Derler I, Fahrner M, Essential role of the N-terminus of murine Orai1 in store- Muik M, Fritsch R, Groschner K, Romanin C. 2- operated Ca2+ entry. Biochem Biophys Res Commun. aminoethoxydiphenyl borate alters selectivity of Orai3 2007 Apr 27;356(1):45-52 channels by increasing their pore size. J Biol Chem. 2008 Jul 18;283(29):20261-7 Várnai P, Tóth B, Tóth DJ, Hunyady L, Balla T. Visualization and manipulation of plasma membrane- Smyth JT, Dehaven WI, Bird GS, Putney JW Jr. Ca2+- endoplasmic reticulum contact sites indicates the presence store-dependent and -independent reversal of Stim1 of additional molecular components within the STIM1- localization and function. J Cell Sci. 2008 Mar 15;121(Pt Orai1 Complex. J Biol Chem. 2007 Oct 5;282(40):29678- 6):762-72 90 Sui S, Wang J, Yang B, Song L, Zhang J, Chen M, Liu J, Yamashita M, Navarro-Borelly L, McNally BA, Prakriya M. Lu Z, Cai Y, Chen S, Bi W, Zhu Y, He F, Qian X. Orai1 mutations alter ion permeation and Ca2+-dependent Phosphoproteome analysis of the human Chang liver cells fast inactivation of CRAC channels: evidence for coupling using SCX and a complementary mass spectrometric of permeation and gating. J Gen Physiol. 2007 strategy. Proteomics. 2008 May;8(10):2024-34 Nov;130(5):525-40 Zhang SL, Kozak JA, Jiang W, Yeromin AV, Chen J, Yu Y, DeHaven WI, Smyth JT, Boyles RR, Bird GS, Putney JW Penna A, Shen W, Chi V, Cahalan MD. Store-dependent Jr. Complex actions of 2-aminoethyldiphenyl borate on and -independent modes regulating Ca2+ release- store-operated calcium entry. J Biol Chem. 2008 Jul activated Ca2+ channel activity of human Orai1 and Orai3. 11;283(28):19265-73 J Biol Chem. 2008 Jun 20;283(25):17662-71 Frischauf I, Schindl R, Derler I, Bergsmann J, Fahrner M, Darbellay B, Arnaudeau S, König S, Jousset H, Bader C, Romanin C. The STIM/Orai coupling machinery. Channels Demaurex N, Bernheim L. STIM1- and Orai1-dependent (Austin). 2008 Jul-Aug;2(4):261-8 store-operated calcium entry regulates human myoblast differentiation. J Biol Chem. 2009 Feb 20;284(8):5370-80 Ji W, Xu P, Li Z, Lu J, Liu L, Zhan Y, Chen Y, Hille B, Xu T, Chen L. Functional stoichiometry of the unitary calcium- Derler I, Fahrner M, Muik M, Lackner B, Schindl R, release-activated calcium channel. Proc Natl Acad Sci U S Groschner K, Romanin C. A Ca2(+ )release-activated A. 2008 Sep 9;105(36):13668-73 Ca2(+) (CRAC) modulatory domain (CMD) within STIM1 mediates fast Ca2(+)-dependent inactivation of ORAI1 Li L, Li X, Yan J. Alterations of concentrations of calcium channels. J Biol Chem. 2009 Sep 11;284(37):24933-8 and arachidonic acid and agglutinations of microfilaments in host cells during Toxoplasma gondii invasion. Vet Fahrner M, Muik M, Derler I, Schindl R, Fritsch R, Parasitol. 2008 Oct 20;157(1-2):21-33 Frischauf I, Romanin C. Mechanistic view on domains mediating STIM1-Orai coupling. Immunol Rev. 2009 Luik RM, Wang B, Prakriya M, Wu MM, Lewis RS. Sep;231(1):99-112 Oligomerization of STIM1 couples ER calcium depletion to CRAC channel activation. Nature. 2008 Jul Frischauf I, Muik M, Derler I, Bergsmann J, Fahrner M, 24;454(7203):538-42 Schindl R, Groschner K, Romanin C. Molecular determinants of the coupling between STIM1 and Orai Mignen O, Thompson JL, Shuttleworth TJ. Both Orai1 and channels: differential activation of Orai1-3 channels by a Orai3 are essential components of the arachidonate-

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 186 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

STIM1 coiled-coil mutant. J Biol Chem. 2009 Aug channels. Proc Natl Acad Sci U S A. 2009 May 7;284(32):21696-706 5;106(18):7391-6 Howie D, Nolan KF, Daley S, Butterfield E, Adams E, Yu F, Sun L, Machaca K. Orai1 internalization and STIM1 Garcia-Rueda H, Thompson C, Saunders NJ, Cobbold SP, clustering inhibition modulate SOCE inactivation during Tone Y, Tone M, Waldmann H. MS4A4B is a GITR- meiosis. Proc Natl Acad Sci U S A. 2009 Oct associated membrane adapter, expressed by regulatory T 13;106(41):17401-6 cells, which modulates T cell activation. J Immunol. 2009 Oct 1;183(7):4197-204 Yuan JP, Zeng W, Dorwart MR, Choi YJ, Worley PF, Muallem S. SOAR and the polybasic STIM1 domains gate Kawasaki T, Lange I, Feske S. A minimal regulatory and regulate Orai channels. Nat Cell Biol. 2009 domain in the C terminus of STIM1 binds to and activates Mar;11(3):337-43 ORAI1 CRAC channels. Biochem Biophys Res Commun. 2009 Jul 17;385(1):49-54 Walsh CM, Chvanov M, Haynes LP, Petersen OH, Tepikin AV, Burgoyne RD. Role of phosphoinositides in STIM1 Korzeniowski MK, Popovic MA, Szentpetery Z, Varnai P, dynamics and store-operated calcium entry. Biochem J. Stojilkovic SS, Balla T. Dependence of STIM1/Orai1- 2009 Dec 14;425(1):159-68 mediated calcium entry on plasma membrane phosphoinositides. J Biol Chem. 2009 Jul Bisaillon JM, Motiani RK, Gonzalez-Cobos JC, Potier M, 31;284(31):21027-35 Halligan KE, Alzawahra WF, Barroso M, Singer HA, Jourd'heuil D, Trebak M. Essential role for STIM1/Orai1- Lee KP, Yuan JP, Zeng W, So I, Worley PF, Muallem S. mediated calcium influx in PDGF-induced smooth muscle Molecular determinants of fast Ca2+-dependent migration. Am J Physiol Cell Physiol. 2010 inactivation and gating of the Orai channels. Proc Natl May;298(5):C993-1005 Acad Sci U S A. 2009 Aug 25;106(34):14687-92 Bogeski I, Kummerow C, Al-Ansary D, Schwarz EC, Martin AC, Willoughby D, Ciruela A, Ayling LJ, Pagano M, Koehler R, Kozai D, Takahashi N, Peinelt C, Griesemer D, Wachten S, Tengholm A, Cooper DM. Capacitative Ca2+ Bozem M, Mori Y, Hoth M, Niemeyer BA. Differential redox entry via Orai1 and stromal interacting molecule 1 (STIM1) regulation of ORAI ion channels: a mechanism to tune regulates adenylyl cyclase type 8. Mol Pharmacol. 2009 cellular calcium signaling. Sci Signal. 2010 Mar Apr;75(4):830-42 30;3(115):ra24 Maruyama Y, Ogura T, Mio K, Kato K, Kaneko T, Kiyonaka Feng M, Grice DM, Faddy HM, Nguyen N, Leitch S, Wang S, Mori Y, Sato C. Tetrameric Orai1 is a teardrop-shaped Y, Muend S, Kenny PA, Sukumar S, Roberts-Thomson SJ, molecule with a long, tapered cytoplasmic domain. J Biol Monteith GR, Rao R. Store-independent activation of Orai1 Chem. 2009 May 15;284(20):13676-85 by SPCA2 in mammary tumors. Cell. 2010 Oct 1;143(1):84-98 McNally BA, Yamashita M, Engh A, Prakriya M. Structural determinants of ion permeation in CRAC channels. Proc Gao YD, Hanley PJ, Rinné S, Zuzarte M, Daut J. Calcium- Natl Acad Sci U S A. 2009 Dec 29;106(52):22516-21 activated K(+) channel (K(Ca)3.1) activity during Ca(2+) store depletion and store-operated Ca(2+) entry in human Mignen O, Thompson JL, Shuttleworth TJ. The molecular macrophages. Cell Calcium. 2010 Jul;48(1):19-27 architecture of the arachidonate-regulated Ca2+-selective ARC channel is a pentameric assembly of Orai1 and Orai3 Kawasaki T, Ueyama T, Lange I, Feske S, Saito N. Protein subunits. J Physiol. 2009 Sep 1;587(Pt 17):4181-97 kinase C-induced phosphorylation of Orai1 regulates the intracellular Ca2+ level via the store-operated Ca2+ Muik M, Fahrner M, Derler I, Schindl R, Bergsmann J, channel. J Biol Chem. 2010 Aug 13;285(33):25720-30 Frischauf I, Groschner K, Romanin C. A Cytosolic Homomerization and a Modulatory Domain within STIM1 C Lis A, Zierler S, Peinelt C, Fleig A, Penner R. A single Terminus Determine Coupling to ORAI1 Channels. J Biol lysine in the N-terminal region of store-operated channels Chem. 2009 Mar 27;284(13):8421-6 is critical for STIM1-mediated gating. J Gen Physiol. 2010 Dec;136(6):673-86 Mullins FM, Park CY, Dolmetsch RE, Lewis RS. STIM1 and calmodulin interact with Orai1 to induce Ca2+- Motiani RK, Abdullaev IF, Trebak M. A novel native store- dependent inactivation of CRAC channels. Proc Natl Acad operated calcium channel encoded by Orai3: selective Sci U S A. 2009 Sep 8;106(36):15495-500 requirement of Orai3 versus Orai1 in estrogen receptor- positive versus estrogen receptor-negative breast cancer Park CY, Hoover PJ, Mullins FM, Bachhawat P, Covington cells. J Biol Chem. 2010 Jun 18;285(25):19173-83 ED, Raunser S, Walz T, Garcia KC, Dolmetsch RE, Lewis RS. STIM1 clusters and activates CRAC channels via Srikanth S, Jung HJ, Ribalet B, Gwack Y. The intracellular direct binding of a cytosolic domain to Orai1. Cell. 2009 loop of Orai1 plays a central role in fast inactivation of Mar 6;136(5):876-90 Ca2+ release-activated Ca2+ channels. J Biol Chem. 2010 Feb 12;285(7):5066-75 Potier M, Gonzalez JC, Motiani RK, Abdullaev IF, Bisaillon JM, Singer HA, Trebak M. Evidence for STIM1- and Orai1- Thompson J, Mignen O, Shuttleworth TJ. The N-terminal dependent store-operated calcium influx through ICRAC in domain of Orai3 determines selectivity for activation of the vascular smooth muscle cells: role in proliferation and store-independent ARC channel by arachidonic acid. migration. FASEB J. 2009 Aug;23(8):2425-37 Channels (Austin). 2010 Sep-Oct;4(5):398-410 Schindl R, Frischauf I, Bergsmann J, Muik M, Derler I, Walsh CM, Doherty MK, Tepikin AV, Burgoyne RD. Lackner B, Groschner K, Romanin C. Plasticity in Ca2+ Evidence for an interaction between Golli and STIM1 in selectivity of Orai1/Orai3 heteromeric channel. Proc Natl store-operated calcium entry. Biochem J. 2010 Sep Acad Sci U S A. 2009 Nov 17;106(46):19623-8 15;430(3):453-60 Wang Y, Deng X, Zhou Y, Hendron E, Mancarella S, Wang Y, Deng X, Mancarella S, Hendron E, Eguchi S, Ritchie MF, Tang XD, Baba Y, Kurosaki T, Mori Y, Soboloff Soboloff J, Tang XD, Gill DL. The calcium store sensor, J, Gill DL. STIM protein coupling in the activation of Orai STIM1, reciprocally controls Orai and CaV1.2 channels.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 187 ORAI3 (ORAI calcium release-activated calcium modulator 3) Hasna J, et al.

Science. 2010 Oct 1;330(6000):105-9 Biol Chem. 2012 May 11;287(20):16146-57 Woodward OM, Li Y, Yu S, Greenwell P, Wodarczyk C, Shuttleworth TJ. Orai3--the 'exceptional' Orai? J Physiol. Boletta A, Guggino WB, Qian F. Identification of a 2012 Jan 15;590(Pt 2):241-57 polycystin-1 cleavage product, P100, that regulates store operated Ca entry through interactions with STIM1. PLoS Trebak M. STIM/Orai signalling complexes in vascular One. 2010 Aug 23;5(8):e12305 smooth muscle. J Physiol. 2012 Sep 1;590(Pt 17):4201-8 Yeung-Yam-Wah V, Lee AK, Tse FW, Tse A. Arachidonic Ay AS, Benzerdjerb N, Sevestre H, Ahidouch A, Ouadid- acid stimulates extracellular Ca(2+) entry in rat pancreatic Ahidouch H. Orai3 constitutes a native store-operated beta cells via activation of the noncapacitative calcium entry that regulates non small cell lung arachidonate-regulated Ca(2+) (ARC) channels. Cell adenocarcinoma cell proliferation. PLoS One. Calcium. 2010 Jan;47(1):77-83 2013;8(9):e72889 Yu F, Sun L, Machaca K. Constitutive recycling of the Faouzi M, Kischel P, Hague F, Ahidouch A, Benzerdjeb N, store-operated Ca2+ channel Orai1 and its internalization Sevestre H, Penner R, Ouadid-Ahidouch H. ORAI3 during meiosis. J Cell Biol. 2010 Nov 1;191(3):523-35 silencing alters cell proliferation and cell cycle progression via c-myc pathway in breast cancer cells. Biochim Biophys Bergsmann J, Derler I, Muik M, Frischauf I, Fahrner M, Acta. 2013 Mar;1833(3):752-60 Pollheimer P, Schwarzinger C, Gruber HJ, Groschner K, Romanin C. Molecular determinants within N terminus of Holzmann C, Kilch T, Kappel S, Armbrüster A, Jung V, Orai3 protein that control channel activation and gating. J Stöckle M, Bogeski I, Schwarz EC, Peinelt C. ICRAC Biol Chem. 2011 Sep 9;286(36):31565-75 controls the rapid androgen response in human primary prostate epithelial cells and is altered in prostate cancer. Cordeiro S, Strauss O. Expression of Orai genes and Oncotarget. 2013 Nov;4(11):2096-107 I(CRAC) activation in the human retinal pigment epithelium. Graefes Arch Clin Exp Ophthalmol. 2011 Hoth M, Niemeyer BA. The neglected CRAC proteins: Jan;249(1):47-54 Orai2, Orai3, and STIM2. Curr Top Membr. 2013;71:237- 71 Faouzi M, Hague F, Potier M, Ahidouch A, Sevestre H, Ouadid-Ahidouch H. Down-regulation of Orai3 arrests cell- Motiani RK, Stolwijk JA, Newton RL, Zhang X, Trebak M. cycle progression and induces apoptosis in breast cancer Emerging roles of Orai3 in pathophysiology. Channels cells but not in normal breast epithelial cells. J Cell Physiol. (Austin). 2013a Sep-Oct;7(5):392-401 2011 Feb;226(2):542-51 Motiani RK, Zhang X, Harmon KE, Keller RS, Matrougui K, Frischauf I, Schindl R, Bergsmann J, Derler I, Fahrner M, Bennett JA, Trebak M. Orai3 is an estrogen receptor α- ⁺ Muik M, Fritsch R, Lackner B, Groschner K, Romanin C. regulated Ca² channel that promotes tumorigenesis. Cooperativeness of Orai cytosolic domains tunes subtype- FASEB J. 2013b Jan;27(1):63-75 specific gating. J Biol Chem. 2011 Mar 11;286(10):8577-84 Zhang X, González-Cobos JC, Schindl R, Muik M, Ruhle Yamashita M, Somasundaram A, Prakriya M. Competitive B, Motiani RK, Bisaillon JM, Zhang W, Fahrner M, Barroso modulation of Ca2+ release-activated Ca2+ channel gating M, Matrougui K, Romanin C, Trebak M. Mechanisms of by STIM1 and 2-aminoethyldiphenyl borate. J Biol Chem. STIM1 activation of store-independent leukotriene C4- 2011 Mar 18;286(11):9429-42 regulated Ca2+ channels. Mol Cell Biol. 2013 Sep;33(18):3715-23 Yanamandra N, Buzzeo RW, Gabriel M, Hazlehurst LA, Mari Y, Beaupre DM, Cuevas J. Tipifarnib-induced Amcheslavsky A, Safrina O, Cahalan MD. State- apoptosis in acute myeloid leukemia and multiple myeloma dependent block of Orai3 TM1 and TM3 cysteine mutants: cells depends on Ca2+ influx through plasma membrane insights into 2-APB activation. J Gen Physiol. 2014 Ca2+ channels. J Pharmacol Exp Ther. 2011 May;143(5):621-31 Jun;337(3):636-43 Zeng B, Chen GL, Daskoulidou N, Xu SZ. The ryanodine Derler I, Madl J, Schütz G, Romanin C. Structure, receptor agonist 4-chloro-3-ethylphenol blocks ORAI store- regulation and biophysics of I(CRAC), STIM/Orai1. Adv operated channels. Br J Pharmacol. 2014 Exp Med Biol. 2012;740:383-410 Mar;171(5):1250-9 Palty R, Raveh A, Kaminsky I, Meller R, Reuveny E. This article should be referenced as such: SARAF inactivates the store operated calcium entry machinery to prevent excess calcium refilling. Cell. 2012 Hasna J, Benzerdjeb N, Faouzi M, Ay AS, Kischel P, Apr 13;149(2):425-38 Hague F, Sevestre H, Ahidouch A, Ouadid-Ahidouch H. ORAI3 (ORAI calcium release-activated calcium modulator Tajeddine N, Gailly P. TRPC1 protein channel is major 3). Atlas Genet Cytogenet Oncol Haematol. 2015; regulator of epidermal growth factor receptor signaling. J 19(3):176-188.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 188

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Short Communication

PCSK4 (proprotein convertase subtilisin/kexin type 4) Majid Khatib, Beatrice Demoures University Bordeaux 1, INSERM U1029, Avenue des Facultes, Batiment B2, Talence 33405, France (MK, BD)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/PCSK4ID50716ch19p13.html DOI: 10.4267/2042/56409 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Protein Review on PCSK4, with data on DNA/RNA, on the protein encoded and where the gene is implicated. Description PCSK4 is a member of the family of subtilisin-like Identity proprotein convertase (PCs) that process protein at basic residues. Other names: PC4, SPC5 This protein is produced in the inactive zymogen HGNC (Hugo): PCSK4 form and is activated by proteolytic removal of its Location: 19p13.3 prodomain in the N-terminal site. DNA/RNA Expression PCSK4 is restricted to the reproductive tract and Description expressed primarily in testicular germ cells and This gene can be found on chromosome 19 at sperm. location: at 1432427 and ends at 1441410. Low levels of PCSK4 mRNA have also been Transcription detected in ovaries and the placenta. The DNA sequence contains 15 exons and the Localisation transcript length: 2661 bps translated to a 755 PCSK4 exact intracellular location has not yet been residues protein. determined.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 189 PCSK4 (proprotein convertase subtilisin/kexin type 4) Khatib M, Demoures B

Function fluorogenic peptides. Biochem J. 2004 Jun 1;380(Pt 2):505-14 PCSK4 cleaves synthetic peptide substrates after an Bassi DE, Fu J, Lopez de Cicco R, Klein-Szanto AJ. Arg in a basic sequence context; most often after Proprotein convertases: "master switches" in the regulation paired basic residues (K/R-X-K/R), to release of tumor growth and progression. Mol Carcinog. 2005 mature proteins from their proproteins. PCSK4 Nov;44(3):151-61 substrates include growth factors (DEAF-1, Qiu Q, Basak A, Mbikay M, Tsang BK, Gruslin A. Role of proIGF2, proenkephalin, proNGF, proPACAP, pro-IGF-II processing by proprotein convertase 4 in human HGFR), receptors (IGF-1R, HGFR), and members placental development. Proc Natl Acad Sci U S A. 2005 of the ADAM (a-disintegrin-and-metalloproteinase) Aug 2;102(31):11047-52 family (ADAM-1, ADAM-2, ADAM-3, ADAM-5). Scamuffa N, Calvo F, Chrétien M, Seidah NG, Khatib AM. The activation/inactivation of these substrates Proprotein convertases: lessons from knockouts. FASEB implicated directly the latter to the regulation of J. 2006 Oct;20(12):1954-63 gonadal functions, sperm motility, and species Gyamera-Acheampong C, Mbikay M. Proprotein specific reproduction. convertase subtilisin/kexin type 4 in mammalian fertility: a review. Hum Reprod Update. 2009 Mar-Apr;15(2):237-47 Homology Lahlil R, Calvo F, Khatib AM. The potential anti- The PCSK4 catalytic domain has a high percentage tumorigenic and anti-metastatic side of the proprotein of homology with those of the other PCs: 70% convertases inhibitors. Recent Pat Anticancer Drug Discov. 2009 Jan;4(1):83-91 between PCSK4 and Furin. Artenstein AW, Opal SM. Proprotein convertases in health Implicated in and disease. N Engl J Med. 2011 Dec 29;365(26):2507-18 Debnath S, Chatterjee S, Arif M, Kundu TK, Roy S. Pregnancy difficulties Peptide-protein interactions suggest that acetylation of lysines 381 and 382 of p53 is important for positive Note coactivator 4-p53 interaction. J Biol Chem. 2011 Jul An aberrant processing of IGF-II by PCSK4 plays a 15;286(28):25076-87 role in inadequate trophoblast migration and, thus, Seidah NG. What lies ahead for the proprotein fetal growth restriction. convertases? Ann N Y Acad Sci. 2011 Mar;1220:149-61 Infertility Tardif S, Guyonnet B, Cormier N, Cornwall GA. Alteration in the processing of the ACRBP/sp32 protein and sperm Note head/acrosome malformations in proprotein convertase 4 The fertilizing ability of PCSK4 null spermatozoa (PCSK4) null mice. Mol Hum Reprod. 2012 Jun;18(6):298- was also found to be significantly reduced. 307 Moreover, PCSK4 cleavages lead to sperm Seidah NG, Sadr MS, Chrétien M, Mbikay M. The acquisition of fertilization competence. multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions. J Biol References Chem. 2013 Jul 26;288(30):21473-81 Turpeinen H, Ortutay Z, Pesu M. Genetics of the first Basak A, Touré BB, Lazure C, Mbikay M, Chrétien M, seven proprotein convertase enzymes in health and Seidah NG. Enzymic characterization in vitro of disease. Curr Genomics. 2013 Nov;14(7):453-67 recombinant proprotein convertase PC4. Biochem J. 1999 Oct 1;343 Pt 1:29-37 This article should be referenced as such: Basak S, Chrétien M, Mbikay M, Basak A. In vitro Khatib M, Demoures B. PCSK4 (proprotein convertase elucidation of substrate specificity and bioassay of subtilisin/kexin type 4). Atlas Genet Cytogenet Oncol proprotein convertase 4 using intramolecularly quenched Haematol. 2015; 19(3):189-190.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 190

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Short Communication

PCSK5 (proprotein convertase subtilisin/kexin type 5) Majid Khatib, Beatrice Demoures University Bordeaux 1, INSERM U1029, Avenue des Facultes, Batiment B2, Talence 33405, France (MK, BD)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/PCSK5ID52105ch9q21.html DOI: 10.4267/2042/56410 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

proprotein convertase (PCs) that process proteins at Abstract basic residues. Review on PCSK5, with data on DNA/RNA, on the This protease undergoes an initial autocatalytic protein encoded and where the gene is implicated. processing event in the ER to generate a heterodimer which exits the ER. Identity It then sorts to the trans-Golgi network where a Other names: PC5, PC6, PC6A, SPC6 second autocatalytic event takes place and the catalytic activity is acquired. HGNC (Hugo): PCSK5 Location: 9q21.13 Expression PCSK5 is widely expressed and encoded by two DNA/RNA alternatively spliced mRNAs: PC5A (which Description encodes a soluble 913-amino acid protein) and PC5B (which encodes a type I membrane-bound This gene can be found on chromosome 9 at 1860-amino acid enzyme). location: 77695406-78164112. PC5A is mostly found in the adrenal gland, uterus, Transcription ovary, aorta, brain and lung. The DNA sequence contains 37 exons and the PC5B is more limited with high expression in the transcript length: 9538 bps translated to 1860 intestine (jejunum, duodenum, ileum, colon), the residues protein. kidney and the liver. Protein Localisation Isoform PC6A: Secreted. Description Isoform PC6B: Endomembrane system: Type I PCSK5 is a member of the family of subtilisin-like membrane protein localized.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 191 PCSK5 (proprotein convertase subtilisin/kexin type 5) Khatib M, Demoures B

Function cells: the role of PC5, a member of the subtilisin family. Biochemistry. 1996 Mar 26;35(12):3797-802 PCSK5 is capable of cleavage at the R-X-(K/R)-R Decroly E, Wouters S, Di Bello C, Lazure C, Ruysschaert consensus motif to release mature proteins from JM, Seidah NG. Identification of the paired basic their proproteins. convertases implicated in HIV gp160 processing based on PCSK5 substrates include growth factors (GDF11, in vitro assays and expression in CD4(+) cell lines. J Biol TGFb, BMP2, CALD1, NGF, PDGF-A, PDGF-B, Chem. 1996 Nov 29;271(48):30442-50 VEGF-C), receptors (IGF-1R), prohormones (pro- Mercure C, Jutras I, Day R, Seidah NG, Reudelhuber TL. renin), ECM proteins (N-cadherin, alpha-integrins), Prohormone convertase PC5 is a candidate processing enzymes (phospholipase, pro-MT1-MMP, ADAM enzyme for prorenin in the human adrenal cortex. Hypertension. 1996 Nov;28(5):840-6 family), and viral protein (HIV-1 glycoprotein gp160). The activation/inactivation of these Lissitzky JC, Luis J, Munzer JS, Benjannet S, Parat F, Chrétien M, Marvaldi J, Seidah NG. Endoproteolytic substrates implicated directly the latter to processing of integrin pro-alpha subunits involves the homeostatic balance, HDL metabolism, pregnancy redundant function of furin and proprotein convertase (PC) establishment and development, and to cell 5A, but not paired basic amino acid converting enzyme adhesion, proliferation and migration. (PACE) 4, PC5B or PC7. Biochem J. 2000 Feb 15;346 Pt 1:133-8 Homology Yana I, Weiss SJ. Regulation of membrane type-1 matrix The PCSK5 catalytic domain has a high percentage metalloproteinase activation by proprotein convertases. of homology with those of the other PCs: 65% Mol Biol Cell. 2000 Jul;11(7):2387-401 between PCSK5 and Furin. Szumska D, Pieles G, Essalmani R, Bilski M, Mesnard D, Kaur K, Franklyn A, El Omari K, Jefferis J, Bentham J, Taylor JM, Schneider JE, Arnold SJ, Johnson P, Implicated in Tymowska-Lalanne Z, Stammers D, Clarke K, Neubauer S, Morris A, Brown SD, Shaw-Smith C, Cama A, Capra V, Gastric cancer Ragoussis J, Constam D, Seidah NG, Prat A, Note Bhattacharya S. VACTERL/caudal regression/Currarino syndrome-like malformations in mice with mutation in the Studies have shown that mice developing proprotein convertase Pcsk5. Genes Dev. 2008 Jun adenocarcinomas along the small intestine 1;22(11):1465-77 exhibited more tumours when they lack PCSK5 in Iatan I, Dastani Z, Do R, Weissglas-Volkov D, Ruel I, Lee enterocytes. JC, Huertas-Vazquez A, Taskinen MR, Prat A, Seidah NG, Pajukanta P, Engert JC, Genest J. Genetic variation at the Currarino syndrome proprotein convertase subtilisin/kexin type 5 gene Note modulates high-density lipoprotein cholesterol levels. Circ Exon sequencing of healthy individuals and patients Cardiovasc Genet. 2009 Oct;2(5):467-75 with VACTERL malformations linked mutations in Lahlil R, Calvo F, Khatib AM. The potential anti- the human PCSK5 gene to this syndrome. tumorigenic and anti-metastatic side of the proprotein convertases inhibitors. Recent Pat Anticancer Drug Viral infection Discov. 2009 Jan;4(1):83-91 Note Artenstein AW, Opal SM. Proprotein convertases in health The human immunodeficiency virus HIV envelope and disease. N Engl J Med. 2011 Dec 29;365(26):2507-18 glycoprotein gp160 is synthesized as an inactive Seidah NG. What lies ahead for the proprotein precursor, which is processed into its fusiogenic convertases? Ann N Y Acad Sci. 2011 Mar;1220:149-61 form gp120/gp41 by host cell PCSK5 during its Maret D, Sadr MS, Sadr ES, Colman DR, Del Maestro RF, intracellular trafficking. Seidah NG. Opposite roles of furin and PC5A in N- cadherin processing. Neoplasia. 2012 Oct;14(10):880-92 References Paule S, Aljofan M, Simon C, Rombauts LJ, Nie G. Cleavage of endometrial α-integrins into their functional Campan M, Yoshizumi M, Seidah NG, Lee ME, Bianchi C, forms is mediated by proprotein convertase 5/6. Hum Haber E. Increased proteolytic processing of protein Reprod. 2012 Sep;27(9):2766-74 tyrosine phosphatase mu in confluent vascular endothelial

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 192 PCSK5 (proprotein convertase subtilisin/kexin type 5) Khatib M, Demoures B

Seidah NG, Prat A. The biology and therapeutic targeting Chem. 2013 Jul 26;288(30):21473-81 of the proprotein convertases. Nat Rev Drug Discov. 2012 May;11(5):367-83 This article should be referenced as such: Seidah NG, Sadr MS, Chrétien M, Mbikay M. The Khatib M, Demoures B. PCSK5 (proprotein convertase multifaceted proprotein convertases: their unique, subtilisin/kexin type 5). Atlas Genet Cytogenet Oncol redundant, complementary, and opposite functions. J Biol Haematol. 2015; 19(3):191-193.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 193

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

PDGFRA (platelet-derived growth factor receptor, alpha polypeptide) Adriana Zamecnikova, Soad Al Bahar Kuwait Cancer Control Center, Department of Hematology, Laboratory of Cancer Genetics, Kuwait (AZ, SAB)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/PDGFRAID143ch4q12.html DOI: 10.4267/2042/56411 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Protein Review on PDGFRA, with data on DNA/RNA, on Description the protein encoded and where the gene is Size: 1089 amino acids; molecular weight: 122670 implicated. Da. Keywords Subunit: Interacts with dimeric PDGFA, PDGFB tyrosine kinase, intracellular signaling, and/or PDGFC; heterodimers with PDGFRB. transmembrane receptor protein tyrosine kinase Present in an inactive conformation as a monomer activity. in the absence of bound ligand. Identity Expression PDGFR expression is characteristic of various Other names: CD140A, PDGFR-2, PDGFR2, mesodermal derivatives; specially expressed in the RHEPDGFRA urinary tracts and in male and female genitals. HGNC (Hugo): PDGFRA Localisation Location: 4q12 Subcellular location: cell membrane. Note Function Size: 69151 bases; Orientation: plus strand. Member of the type III class of tyrosine kinase receptors which also includes c-KIT, FLT3 and the DNA/RNA macrophage-colony-stimulating factor receptor; Description characterized by five immunoglobuline-like extracellular domains; a single-spanning The gene encoding the α-subunit of the PDGFRA transmembrane domain and an intracellular split maps to band q12 of chromosome 4. kinase domain, connected by a flexible polypeptide The gene contains 23 exons spanning about 65 kb. insert; structurally homologous to c-KIT. PDGFA The first noncoding exon is followed by a large has transmembrane receptor protein tyrosine kinase intron of approximately 23 kb (Gronwald et al., activity and acts as a cell-surface receptor for 1990; Kawagishi et al., 1995). members of the platelet-derived growth factor An important paralog of PDGFRA is FLT4. family: PDGFA, PDGFB and PDGFC, which are Transcription mitogens for fibroblasts and cells of mesenchymal 6.4-kb transcript; coexpressed with the 5.3-kb origin origin. It plays an essential role in the PDGF receptor mRNA. regulation of many biological processes including cell proliferation, survival, differentiation and cell

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 194 PDGFRA (platelet-derived growth factor receptor, alpha Zamecnikova A, Al Bahar S polypeptide)

migration. Plays an important role in embryonic PDGFRA mutations also have been described in development, in the adult control tissue somatic and familial gastrointestinal stromal homeostasis in various organs including kidney, tumors, synovial sarcomas, glioblastoma, malignant epidydimis, lung and pancreas; required for normal peripheral nerve sheath tumors, melanoma and a in development of intestinal villi in the gastrointestinal a variety of other cancers (Chompret et al., 2004; tract, plays a role in platelet activation, wound Heinrich et al., 2003). healing and angiogenesis (Demoulin et al., 2012; Heldin et al., 2013). Implicated in Regulation: Function as homo- and/or heterodimers depending on the cell type; activated by ligand- Gastrointestinal stromal tumor (GIST) induced dimerization and autophosphorylation on Note specific tyrosine residues upon binding. Activation Activating mutations of PDGFRA are found in 5- of the intracellular kinase activity of the receptor 8% of patients with gastrointestinal stromal tumors leads to creation of docking sites for signal (GISTs) but their frequency increases to 30% to transduction molecules. Subsequent 40% in gastric GISTs lacking KIT mutations phosphorylation of its substrates initiates a variety (Corless et al., 2005; Lasota et al., 2008). The of signal transduction cascades that promotes cell majority of these mutations are "substitution proliferation, survival and migration through the missense", that can arise by various mechanisms PI3K-AKT-mTOR and RAS-MAPK pathways as (Figure 1). well as promotes activation of STAT family These include mutation hot spots in exon 18 of the members (JAK/STAT) (Demoulin et al., 2012). PDGFRA gene such as the Asp-to-Val substitution at codon 842 (D842V) encoding the activation loop. Mutations Other activating mutations are less frequent such as Note mutations in exons 12 encoding the juxtamembrane Mutations in the PDGFRA gene contribute to the domain and in exon 14 encoding the tyrosine kinase pathophysiology of various diseases such as 1 domain of PDGFRA (Chompret et al., 2004; atherosclerosis, abnormalities of the tubal neural Heinrich et al., 2003). development and fibrotic diseases. In cancer, PDGFRA mutations except for D842V in exon 18 activating point mutations, gene amplifications and are sensitive to imatinib inhibition. chromosomal rearrangements including gene However, despite initial clinical responses to fusions and chromosomal deletions have been tyrosine kinase inhibitors (imatinib, nilotinib, found in certain malignancies. These include sorafenib and sunatinib), the majority of these hematological malignancies such as acute myeloid patient develops resistance to the drug limiting the leukemia, atypical chronic myelogenous leukemia, long-term benefit of tyrosine kinase inhibitors in chronic myelomonocytic leukemia, eosinophilic this group of patients (Gramza et al., 2009; Pierotti disorders and mastocytosis (Gotlib et al., 2008). et al., 2011).

Figure 1. Schematic representation of the most frequent activating mutations of the homologous platelet-derived growth factor receptor alpha (PDGFRA) kinase in patients with gastrointestinal stromal tumors. Most common mutations are in exon 18, such as the D842V substitution that shows resistance to imatinib. Mutations in the juxtamembrane domain (exon 12; V561D most common) and in exon 14 tyrosine kinase 1 (TK1) domain (e.g., N659K) are less common. Abbreviations: JM, juxtamembrane; TK, tyrosine kinase. Adopted and modified from Pierott et al., 2011).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 195 PDGFRA (platelet-derived growth factor receptor, alpha Zamecnikova A, Al Bahar S polypeptide)

Figure 2. The structure and mechanism of activation of PDGFRA fusions in hematological disorders. In the fusion oncogene, the partner gene always replaces the 5 part of exon 12 of PDGFRA creating an in frame fusion. As the 5 part of exon 12 of PDGFRA containing the inhibitory domain is truncated, its expression is controlled by the partner gene promoter resulting in constitutive activation of the PDGFRA kinase domain. NH2: N-terminal site; COOH: C-terminal site; TM: transmembrane domain; JM: juxtamembrane domain. Adopted and modified from Cools et al., 2003 and Gotlib et al., 2008).

The D842V mutation results in an amino acid breakpoints in PDGFRA invariably involve exon 12 substitution at position 842 in PDGFRA, from an encoding a portion of the juxtamembrane domain aspartic acid (D) to a valine (V). with autoinhibitory function (Baxter et al., 2002; This mutation occurs within the TK2 domain Gotlib et al., 2008); the disruption of which (Figure 1). activates the fusion protein (Figure 2). PDGFRA D842V mutation has been found in a The most investigated of these fusion genes is distinct subset of GIST, typically from the stomach. FIP1L1-PDGFRA that arise as a result of a cryptic The D842V mutation is known to be associated interstitial deletion on chromosome 4q12. FIP1L1- with tyrosine kinase inhibitor resistance. PDGFRA fusion protein is involved in the Hematologic disorders with primary pathogenesis of uncommon hematologic disorders with primary eosinophilia like chronic eosinophilic eosinophilia leukemia (CEL) hyperseosinophilic syndrome Note (HES) and systemic mastocytosis (SM). Similar to Several chromosomal rearrangements generating other fusion tyrosine kinases, FIP1L1-PDGFRA is fusion genes causing PDGFRA activation have a constitutively active tyrosine kinase that was been described in a variety of uncommon shown to be sensitive to kinase inhibitors (Cools et hematologic disorders that are often accompanied al., 2003; Jain et al., 2013). with a related condition called hypereosinophilic Lung adenocarcinoma syndrome. These rearrangements activate PDGFRA by fusion Cytogenetics to various partner genes: STRN (2p24) in the A t(4;12)(q12;q12) was found in a case of lung t(2;4)(p22;q12), FIP1L1 (interstitial 4q12 deletion), adenocarcinoma (Seo et al., 2012). CDK5RAP2 (9q33) in the ins(9;4)(q33;q12q25), Hybrid/Mutated gene KIF5B (10p11) in the t(4;10)(q12;p11), ETV6 SCAF11/PDGFRA (12p13) in the t(4;12)(q12;p13), and BCR (22q11) in the t(4;22)(q12;q11). Breakpoints In each of these rearrangements, the breakpoints in PDGFRA partner genes are variable, but the See figure below.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 196 PDGFRA (platelet-derived growth factor receptor, alpha Zamecnikova A, Al Bahar S polypeptide)

References Clin Oncol. 2005 Aug 10;23(23):5357-64 Gotlib J, Cools J. Five years since the discovery of Gronwald RG, Adler DA, Kelly JD, Disteche CM, Bowen- FIP1L1-PDGFRA: what we have learned about the fusion Pope DF. The human PDGF receptor alpha-subunit gene and other molecularly defined eosinophilias. Leukemia. maps to chromosome 4 in close proximity to c-kit. Hum 2008 Nov;22(11):1999-2010 Genet. 1990 Aug;85(3):383-5 Lasota J, Miettinen M. Clinical significance of oncogenic Kawagishi J, Kumabe T, Yoshimoto T, Yamamoto T. KIT and PDGFRA mutations in gastrointestinal stromal Structure, organization, and transcription units of the tumours. Histopathology. 2008 Sep;53(3):245-66 human alpha-platelet-derived growth factor receptor gene, PDGFRA. Genomics. 1995 Nov 20;30(2):224-32 Gramza AW, Corless CL, Heinrich MC. Resistance to Tyrosine Kinase Inhibitors in Gastrointestinal Stromal Baxter EJ, Hochhaus A, Bolufer P, Reiter A, Fernandez Tumors. Clin Cancer Res. 2009 Dec 15;15(24):7510-7518 JM, Senent L, Cervera J, Moscardo F, Sanz MA, Cross NC. The t(4;22)(q12;q11) in atypical chronic myeloid Pierotti MA, Tamborini E, Negri T, Pricl S, Pilotti S. leukaemia fuses BCR to PDGFRA. Hum Mol Genet. 2002 Targeted therapy in GIST: in silico modeling for prediction Jun 1;11(12):1391-7 of resistance. Nat Rev Clin Oncol. 2011 Mar;8(3):161-70 Cools J, DeAngelo DJ, Gotlib J, Stover EH, Legare RD, Demoulin JB, Montano-Almendras CP. Platelet-derived Cortes J, Kutok J, Clark J, Galinsky I, Griffin JD, Cross NC, growth factors and their receptors in normal and malignant Tefferi A, Malone J, Alam R, Schrier SL, Schmid J, Rose hematopoiesis. Am J Blood Res. 2012;2(1):44-56 M, Vandenberghe P, Verhoef G, Boogaerts M, Wlodarska Seo JS, Ju YS, Lee WC, Shin JY, Lee JK, Bleazard T, Lee I, Kantarjian H, Marynen P, Coutre SE, Stone R, Gilliland J, Jung YJ, Kim JO, Shin JY, Yu SB, Kim J, Lee ER, Kang DG. A tyrosine kinase created by fusion of the PDGFRA CH, Park IK, Rhee H, Lee SH, Kim JI, Kang JH, Kim YT. and FIP1L1 genes as a therapeutic target of imatinib in The transcriptional landscape and mutational profile of idiopathic hypereosinophilic syndrome. N Engl J Med. lung adenocarcinoma. Genome Res. 2012 2003 Mar 27;348(13):1201-14 Nov;22(11):2109-19 Heinrich MC, Corless CL, Duensing A, McGreevey L, Heldin CH, Lennartsson J. Structural and functional Chen CJ, Joseph N, Singer S, Griffith DJ, Haley A, Town properties of platelet-derived growth factor and stem cell A, Demetri GD, Fletcher CD, Fletcher JA. PDGFRA factor receptors. Cold Spring Harb Perspect Biol. 2013 activating mutations in gastrointestinal stromal tumors. Aug 1;5(8):a009100 Science. 2003 Jan 31;299(5607):708-10 Jain N, Khoury JD, Pemmaraju N, Kollipara P, Kantarjian Chompret A, Kannengiesser C, Barrois M, Terrier P, H, Verstovsek S. Imatinib therapy in a patient with Dahan P, Tursz T, Lenoir GM, Bressac-De Paillerets B. suspected chronic neutrophilic leukemia and FIP1L1- PDGFRA germline mutation in a family with multiple cases PDGFRA rearrangement. Blood. 2013 Nov of gastrointestinal stromal tumor. Gastroenterology. 2004 7;122(19):3387-8 Jan;126(1):318-21 Corless CL, Schroeder A, Griffith D, Town A, McGreevey This article should be referenced as such: L, Harrell P, Shiraga S, Bainbridge T, Morich J, Heinrich Zamecnikova A, Al Bahar S. PDGFRA (platelet-derived MC. PDGFRA mutations in gastrointestinal stromal tumors: growth factor receptor, alpha polypeptide). Atlas Genet frequency, spectrum and in vitro sensitivity to imatinib. J Cytogenet Oncol Haematol. 2015; 19(3):194-197.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 197

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

PF4V1 (platelet factor 4 variant 1) Katrien Van Raemdonck, Paul Proost, Jo Van Damme, Sofie Struyf Laboratory of Molecular Immunology, Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium (KVR, PP, JVD, SS)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/PF4V1ID44606ch4q13.html DOI: 10.4267/2042/56412 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

affinity of CXCL4L1 for glycosaminoglycans Abstract (GAG) is rather moderate (Dubrac et al., 2010; Review on PF4V1, with data on DNA/RNA, on the Struyf et al., 2011), strongly increasing its in vivo protein encoded and where the gene is implicated. half-life and diffusibility. Identity DNA/RNA Other names: CXCL4L1, CXCL4V1, PF4-ALT, Note PF4A, SCYB4V1 The gene and mRNA for CXCL4L1 are 1293 and HGNC (Hugo): PF4V1 741 bp in length, respectively. Location: 4q13.3 Description Note The CXCL4L1 mRNA is encoded by three exons. The CXC chemokine CXCL4L1 is a nonallelic Duplication of the CXCL4 gene, giving rise to the variant of the earlier identified platelet factor homologous CXCL4L1 gene, is conserved in CXCL4. These rather atypical chemokines display human and other primates including gorilla, a less prominent leukocyte chemoattractant activity, chimpanzee, orangutan, gibbon and macaque. yet influence a large range of other processes. Transcription CXCL4L1 was characterized as an especially potent angiostatic chemokine (Struyf et al., 2004). The existence of a CXCL4 variant was first Consequently, this platelet factor is an inhibitor of evidenced by Eisman et al. (1990) and Green et al. tumor growth and metastasis. The therapeutic (Eisman et al., 1990; Green et al., 1989). The potential of CXCL4L1 has been evidenced in CXCL4L1 mRNA is predominantly present in preclinical B16 melanoma, Lewis lung carcinoma platelets, but has also been detected in vascular and A549 adenocarcinoma animal models, as it smooth muscle cells and to a lesser extent in T inhibited both tumor growth and metastasis by cells, monocytes and endothelial cells (Lasagni et preventing tumor neovascularization (Struyf et al., al., 2007). CXCL4L1 mRNA detected in ovarian 2007). Furthermore, the carboxy-terminal peptide tissue has been attributed to macrophage CXCL4L1 CXCL4L1 47-70 retains its potential to suppress B16 expression (Furuya et al., 2012). CXCL4L1 melanoma growth in mice (Vandercappellen et al., expression was also observed in the HCT-8 colon 2010). Additionally, the recently highlighted impact adenocarcinoma cell line as evidenced by qPCR of CXCL4L1 on lymphatic endothelial cells in analysis (Verbeke et al., 2010). vitro, corroborates a potential inhibitory effect on Pseudogene tumor dissemination in vivo (Prats et al., 2013; Van Raemdonck et al., 2014). Compared to CXCL4, the None.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 198 PF4V1 (platelet factor 4 variant 1) Van Raemdonck K, et al.

Figure 1. Structure of the human CXCL4L1 gene. This figure schematically depicts the structure of the human CXCL4L1 gene as described in the NCBI database (NM_002620). Lines represent the introns, whereas rectangular exons are coloured blue, yellow and green to represent the non-coding domains, the signal peptide and the mature protein, respectively. Grey numbers indicate the basepairs (bp) spanning the exons. Red numbers apply to the amino acids (aa) encoded.

Protein Function CXCL4L1 has been described to be a strong Note inhibitor of angiogenesis. Together with its CXCL4L1 precursor: 104 amino acids (aa), 11553 potential to chemoattract T cells, natural killer cells Da; CXCL4L1 mature: 70 aa, 7805.8 Da. and immature dendritic cells, the vascular effects Description contribute to the antitumoral action of CXCL4L1 (Struyf et al., 2011). Struyf et al. (Struyf et al., CXCL4L1 is a member of the CXC chemokine 2007) indeed indicated the angiostatic platelet family of chemoattractant cytokines. factor to exert an antitumoral effect by inhibiting CXCL4L1 is a non-ELR CXC chemokine, meaning branching of the vascular network and metastasis. that it lacks the sequence glutamic acid-leucine- Considering neutrophils and monocytes, CXCL4L1 arginine just in front of the two NH -terminally 2 as opposed to CXCL4 would not attract these pro- located conserved cysteine residues. tumoral phagocytes (Vandercappellen et al., 2007). Expression Lasagni et al. identified a splice variant of CXCR3, Blood platelets release both CXCL4 and CXCL4L1 which was named CXCR3B, as a functional GPCR after activation. for CXCL4 (Lasagni et al., 2003). Currently, both The exact location of CXCL4L1 inside the platelet CXCL4 and CXCL4L1 are known to activate is not yet determined, whereas platelet CXCL4 is CXCR3A, as well as CXCR3B (Mueller et al., stored in the alpha-granules. 2008; Struyf et al., 2011; Van Raemdonck et al., In other cell types as well, CXCL4 is stored in 2014). In general, proliferative and positive secretory granules and released in response to migratory effects are supposed to be mediated by protein kinase C activation, whereas CXCL4L1 is CXCR3A, whereas inhibition of chemotaxis, anti- continuously synthesized and secreted through a proliferative and apoptotic effects are postulated to constitutive pathway (Lasagni et al., 2007). be provoked via CXCR3B (Lasagni et al., 2003). For instance, human aortic smooth muscle cells and Besides endothelial cells and T cells, CXCR3 human coronary smooth muscle cells constitutively expressing cell types can be extended to fibroblasts, release CXCL4L1. mesangial cells, airway epithelial and smooth Specific cancer cell lines have also been shown to muscle cells, pneumocytes and several sarcoma, produce CXCL4L1. carcinoma and myeloma cell types (Billottet et al., Secretion of CXCL4L1 in tumoral tissue was 2013). evidenced in vitro on stimulated osteosarcoma cells CXCL4 exerts its action through many different through the use of ELISA and further corroborated mechanisms, including binding to GAG and by immunohistochemical staining of different heteromultimerisation with other chemokines and human sarcoma tissue sections (osteosarcoma, growth factors, whereas in the case of CXCL4L1 its leiomyosarcoma and liposarcoma) distorted structure and unique protruding C- (Vandercappellen et al., 2007). terminal helix are assumed to conflict with this Furthermore, CXCL4L1 was strongly detected in mode of action. The open formation characteristic colorectal adenocarcinoma biopsy specimens of CXCL4L1 decreases GAG-binding, however (Verbeke et al., 2010). simultaneously enhancing anti-angiogenic and antitumoral effects (Dubrac et al., 2010; Kuo et al., Localisation 2013). Additionally, CXCL4L1 forms more stable Secreted. homodimers due to a loss in positive charge.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 199 PF4V1 (platelet factor 4 variant 1) Van Raemdonck K, et al.

This gained stability is likely to interfere with the endometriosis. CXCL4L1 is expressed in normal ability to form heteromers which requires initial ovaries and especially during endometriosis dissociation of the homomultimers (Kuo et al., (Furuya et al., 2012). However, CXCL4L1 mRNA 2013). levels were significantly lower in cancerous lesions. Homology Endometriosis-associated ovarian cancers (EAOC) were reported to be infiltrated by CD68+ tumor- CXCL4L1 is a non-allelic variant of CXCL4. associated macrophages. CXCL4 and CXCL4L1 Unlike CXCL4, its variant appears only in expression by those macrophages was studied at the primates. In men, mature proteins only differ in 3 protein level by Furuya and colleagues. However, amino acids. On the other hand, the signal peptide antibodies not distinguishing CXCL4 from its of human CXCL4L1 displays 38% amino acid variant were used. The tumor-associated divergence compared to human CXCL4, affecting macrophages displayed an impaired expression of its subcellular localization and regulated secretion either CXCL4, CXCL4L1 or possibly both. In mechanism, as was described by Lasagni et al. conclusion, macrophage expression of the platelet (Lasagni et al., 2007). factors appears to be associated with EAOC disease Implicated in state and may prove to be a useful disease marker. Coronary artery disease Osteosarcoma Prognosis Disease Recently, a possible prognostic significance for Secretion of CXCL4L1 in tumoral tissue was CXCL4L1 was evaluated in patients suffering from evidenced in vitro on stimulated osteosarcoma cells coronary artery disease (CAD) (De Sutter et al., through the use of ELISA and further corroborated 2012). Specifically in a selection of patients with by immunohistochemical staining of different stable CAD and preserved left ventricular function, human sarcoma tissue sections (osteosarcoma, CXCL4L1 levels significantly correlated to age, leiomyosarcoma and liposarcoma) creatinine and circulating platelet number, as well (Vandercappellen et al., 2007). On the other hand, as to N-terminal pro-B-type natriuretic peptide osteosarcoma cells also express the CXCR3 (NT-proBNP), a well validated prognostic marker receptor guiding initial tumor dissemination to in stable CAD. More importantly, CXCL4L1 metastatic sites were CXCR3 ligands such as showed an additional prognostic value on top of CXCL4L1 are expressed (Pradelli et al., 2009). NT-proBNP as lower levels of CXCL4L1 predicted a higher event rate and worse outcome. Colorectal cancer Surprisingly, in these patients with stable CAD the Disease prognostic value of CXCL4L1 is independent of Study of CXCL4L1 expression in human epithelial NT-proBNP. tumors revealed a distinct presence of CXCL4L1 in colorectal cancer cells, whereas its expression in References esophageal cancer was weak to undetectable Green CJ, Charles RS, Edwards BF, Johnson PH. (Verbeke et al., 2010). ELISA, qRT-PCR, Identification and characterization of PF4varl, a human immunocytochemistry as well as ex vivo gene variant of platelet factor 4. Mol Cell Biol. 1989 immunohistochemistry support the hypothesis that Apr;9(4):1445-51 CXCL4L1 is secreted by colorectal Eisman R, Surrey S, Ramachandran B, Schwartz E, Poncz adenocarcinoma cells and may affect the complex M. Structural and functional comparison of the genes for process of tumor development. However, no human platelet factor 4 and PF4alt. Blood. 1990 Jul correlation was found between the intensity or 15;76(2):336-44 extent of CXCL4L1 staining of patient biopsies and Lasagni L, Francalanci M, Annunziato F, Lazzeri E, the TNM stage. On the other hand, intratumorally Giannini S, Cosmi L, Sagrinati C, Mazzinghi B, Orlando C, administered CXCL4L1 has been shown to reduce Maggi E, Marra F, Romagnani S, Serio M, Romagnani P. An alternatively spliced variant of CXCR3 mediates the tumor vascularization and, consequently, tumor inhibition of endothelial cell growth induced by IP-10, Mig, growth and metastasis of A549 adenocarcinoma in and I-TAC, and acts as functional receptor for platelet mice, similar to its therapeutic benefit observed in factor 4. J Exp Med. 2003 Jun 2;197(11):1537-49 preclinical studies on B16 melanoma and Lewis Struyf S, Burdick MD, Proost P, Van Damme J, Strieter lung carcinoma (Struyf et al., 2007). RM. Platelets release CXCL4L1, a nonallelic variant of the chemokine platelet factor-4/CXCL4 and potent inhibitor of Endometriosis-associated ovarian angiogenesis. Circ Res. 2004 Oct 29;95(9):855-7 cancer (EAOC) Lasagni L, Grepin R, Mazzinghi B, Lazzeri E, Meini C, Oncogenesis Sagrinati C, Liotta F, Frosali F, Ronconi E, Alain-Courtois N, Ballerini L, Netti GS, Maggi E, Annunziato F, Serio M, Both clear cell and endometrioid types of ovarian Romagnani S, Bikfalvi A, Romagnani P. PF-4/CXCL4 and cancers occasionally develop on the bases of CXCL4L1 exhibit distinct subcellular localization and a

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 200 PF4V1 (platelet factor 4 variant 1) Van Raemdonck K, et al.

differentially regulated mechanism of secretion. Blood. Jul;41(7):990-1001 2007 May 15;109(10):4127-34 Struyf S, Salogni L, Burdick MD, Vandercappellen J, Struyf S, Burdick MD, Peeters E, Van den Broeck K, Dillen Gouwy M, Noppen S, Proost P, Opdenakker G, Parmentier C, Proost P, Van Damme J, Strieter RM. Platelet factor-4 M, Gerard C, Sozzani S, Strieter RM, Van Damme J. variant chemokine CXCL4L1 inhibits melanoma and lung Angiostatic and chemotactic activities of the CXC carcinoma growth and metastasis by preventing chemokine CXCL4L1 (platelet factor-4 variant) are angiogenesis. Cancer Res. 2007 Jun 15;67(12):5940-8 mediated by CXCR3. Blood. 2011 Jan 13;117(2):480-8 Vandercappellen J, Noppen S, Verbeke H, Put W, Conings De Sutter J, Van de Veire NR, Struyf S, Philippé J, De R, Gouwy M, Schutyser E, Proost P, Sciot R, Geboes K, Buyzere M, Van Damme J. PF-4var/CXCL4L1 predicts Opdenakker G, Van Damme J, Struyf S. Stimulation of outcome in stable coronary artery disease patients with angiostatic platelet factor-4 variant (CXCL4L1/PF-4var) preserved left ventricular function. PLoS One. versus inhibition of angiogenic granulocyte chemotactic 2012;7(2):e31343 protein-2 (CXCL6/GCP-2) in normal and tumoral mesenchymal cells. J Leukoc Biol. 2007 Dec;82(6):1519- Furuya M, Tanaka R, Miyagi E, Kami D, Nagahama K, 30 Miyagi Y, Nagashima Y, Hirahara F, Inayama Y, Aoki I. Impaired CXCL4 expression in tumor-associated Mueller A, Meiser A, McDonagh EM, Fox JM, Petit SJ, macrophages (TAMs) of ovarian cancers arising in Xanthou G, Williams TJ, Pease JE. CXCL4-induced endometriosis. Cancer Biol Ther. 2012 Jun;13(8):671-80 migration of activated T lymphocytes is mediated by the chemokine receptor CXCR3. J Leukoc Biol. 2008 Billottet C, Quemener C, Bikfalvi A. CXCR3, a double- Apr;83(4):875-82 edged sword in tumor progression and angiogenesis. Biochim Biophys Acta. 2013 Dec;1836(2):287-95 Pradelli E, Karimdjee-Soilihi B, Michiels JF, Ricci JE, Millet MA, Vandenbos F, Sullivan TJ, Collins TL, Johnson MG, Kuo JH, Chen YP, Liu JS, Dubrac A, Quemener C, Prats Medina JC, Kleinerman ES, Schmid-Alliana A, Schmid- H, Bikfalvi A, Wu WG, Sue SC. Alternative C-terminal helix Antomarchi H. Antagonism of chemokine receptor CXCR3 orientation alters chemokine function: structure of the anti- inhibits osteosarcoma metastasis to lungs. Int J Cancer. angiogenic chemokine, CXCL4L1. J Biol Chem. 2013 May 2009 Dec 1;125(11):2586-94 10;288(19):13522-33 Dubrac A, Quemener C, Lacazette E, Lopez F, Zanibellato Prats AC, Van den Berghe L, Rayssac A, Ainaoui N, C, Wu WG, Bikfalvi A, Prats H. Functional divergence Morfoisse F, Pujol F, Legonidec S, Bikfalvi A, Prats H, between 2 chemokines is conferred by single amino acid Pyronnet S, Garmy-Susini B. CXCL4L1-fibstatin change. Blood. 2010 Nov 25;116(22):4703-11 cooperation inhibits tumor angiogenesis, lymphangiogenesis and metastasis. Microvasc Res. 2013 Vandercappellen J, Liekens S, Bronckaers A, Noppen S, Sep;89:25-33 Ronsse I, Dillen C, Belleri M, Mitola S, Proost P, Presta M, Struyf S, Van Damme J. The COOH-terminal peptide of Van Raemdonck K, Gouwy M, Lepers SA, Van Damme J, platelet factor-4 variant (CXCL4L1/PF-4var47-70) strongly Struyf S. CXCL4L1 and CXCL4 signaling in human inhibits angiogenesis and suppresses B16 melanoma lymphatic and microvascular endothelial cells and growth in vivo. Mol Cancer Res. 2010 Mar;8(3):322-34 activated lymphocytes: involvement of mitogen-activated protein (MAP) kinases, Src and p70S6 kinase. Verbeke H, De Hertogh G, Li S, Vandercappellen J, Angiogenesis. 2014 Jul;17(3):631-40 Noppen S, Schutyser E, El-Asrar AA, Opdenakker G, Van Damme J, Geboes K, Struyf S. Expression of angiostatic This article should be referenced as such: platelet factor-4var/CXCL4L1 counterbalances angiogenic impulses of vascular endothelial growth factor, interleukin- Van Raemdonck K, Proost P, Van Damme J, Struyf S. 8/CXCL8, and stromal cell-derived factor 1/CXCL12 in PF4V1 (platelet factor 4 variant 1). Atlas Genet Cytogenet esophageal and colorectal cancer. Hum Pathol. 2010 Oncol Haematol. 2015; 19(3):198-201.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 201

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), member 3) Cristian Turato, Patrizia Pontisso Department of Medicine, University of Padua, Padua, Italy (CT, PP)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/SERPINB3ID42265ch18q21.html DOI: 10.4267/2042/56413 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Note SERPINB3, also known as SCCA1, encodes Review on SERPINB3, with data on DNA/RNA, members of the serpins family. The serpins are a on the protein encoded and where the gene is family of protease inhibitors originally grouped implicated. together as serine protease inhibitors, most of which are secreted (Silverman et al., 2004). The clade B Identity serpins comprise a number of proteins including SERPINB3. In the early 1990's (Takeshima et al., Other names: HsT1196, SCC, SCCA-1, SCCA- 1992) it was recognized as circulating "squamous PD, SCCA1, T4-A cell carcinoma antigen" (SCCA1) that was present HGNC (Hugo): SERPINB3 in a substantial fraction of sera from patients Location: 18q21.33 bearing squamous cell cancers of the cervix. Later on it was found to be associated with other types of Local order: According to Entrez-Gene, cancer of epithelial or endodermal origins, SERPINB3 maps to NC_000018.10 in the region including lung cancer, head and neck cancer, and between 63655197 and 63661963, complement and hepatocellular carcinoma (Schneider et al., 1995; span 6767 bases. Flanked by SERPINB4 and Pontisso et al., 2004). SERPINB11.

Figure 1. A. SERPINB3 maps in chromosome 18q21.3 (NC_000018.10) in the region between 63655197 and 63661963. Local order and flanked genes are reported. B. Map of a SERPINB3 transcript mRNA (NM_006919.2) showing its organization in 8 exon.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 202 SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), Turato C, Pontisso P member 3)

Figure 2. SERPINB3 protein mature chain. Site of Reactive Center Loop (RSL) (blue), and described variants (green) are indicated. Potential site of splicing variant missing amino acids 205-256 are also reported (yellow).

being the most variable region, located in an DNA/RNA exposed loop (Hunt and Dayhoff, 1980). Description The mechanism of protease inhibition by serpins involves a profound change in conformation, According to the NCBI map viewer, the gene is initiated by interaction of the protease with the located on chromosome 18q21.3 (NCBI Reference reactive site loop of the serpin (RSL) (amino acids Sequence: NC_000018.10) and encompasses 6767 340-368). RSL consists of a loop projecting from bp. the body of the protein, comprising a hinge region Transcription and a variable RSL (Huntington et al., 2000). Biochemical analysis of recombinant SERPINB3 The SERPINB3 gene comprises eight exons and shows that it is a potent cross-class inhibitor of seven introns which commonly encoded a 1,793 kb papain-like cysteine proteinases such as cathepsin mRNA. The ATG start is located in exon 2 with the L, cathepsin S and cathepsin K (Schick et al., stop codon in exon 8. 1998). Transcription control is regulated by STAT3. An isoform produced by alternative splicing has STAT3 occupies the promoter of SERPINB3/B4 been reported. The sequence of this isoform differs and siRNA removal of SERPINB3/B4 mRNA from the canonical sequence for 205-256 amino caused cell death in HN13 head and neck cancer acid missing (Fig.2). cells. Thus persistently activated STAT3 is a required part of the continuous activation of Expression SERPINB3/B4 genes, which protects tumor cells SERPINB3 is expressed in the spinous and granular from dying (Ahmed and Darnell, 2009). layers of normal squamous epithelium, in several Moreover recent mechanistic experiments and ChIP organs including: epithelium of the tongue, assays reveal that SERPINB3 increased expression esophagus, tonsil, cervix uterine, vagina, Hassal's in response to hypoxic conditions is specifically corpuscles of the thymus and some areas of the mediated by the binding of HIF-2α to the skin. SERPINB3 was also detected in saliva, SERPINB3 promoter (Cannito et al., 2013). respiratory secretions and amniotic fluid samples Pseudogene from healthy individuals (Kato, 1996; Cataltepe et al., 2000). Moreover, SERPINB3 was recently No known pseudogene. reported to be expressed on CD27+ B lymphocytes (Vidalino et al., 2012). Protein In particular, immunohistochemistry analysis revealed positive staining in sweat glands in the Description dermis of the skin, endothelial cells of the veins and SERPINB3 encodes a 390 amino acid 44,56 Kda arteries walls in the intestine. protein, which shows sequence homology to the Within the normal liver, SERPINB3 protein ovalbumin family of serine protease inhibitors (Ov- expression was seen in portal interlobular ducts, in serpin) (Remold-O'Donnell, 1993), a subfamily of the walls (myocytes of the media) of the large and the large serpin superfamily. Serpins have a highly medium sized hepatic arteries and sometimes in the ordered tertiary structure defined by the crystal endothelial cells of the portal veins. structure of the prototype molecule α1-antitrypsin, Normal hepatocytes, sinusoidal cells and Kupffer consisting of nine α-helices and three β-sheets cells do not exhibit any reactivity, except some arranged in a stressed configuration with the hepatocytes in the limiting plate that can show focal reactive center, which has the unusual feature of faint positivity (Turato et al., 2012).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 203 SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), Turato C, Pontisso P member 3)

HepCAM positive liver stem cells from both foetal MAP family of c-Jun terminal kinase (JNK) and adult livers also express SERPINB3 (Villano et (Katagiri et al., 2006) and the interaction between al., 2014). SERPINB3 and JNK1 was also supported by the Initially, SERPINB3 was discovered as a crystallographic study (Zheng et al., 2009); or with serological marker for advanced squamous cell a decreased phosphorylation of the proapoptotic tumors in the cervix (Uemura et al., 2000), and was p38 mitogen-activated protein kinase on p38 later found to be associated with other types of (Murakami et al., 2001). In epithelial ovarian cancer with epithelial or endodermal origins. cancer cells exposed to cisplatin, SERPINB3 Moreover, elevated expression of SERPINB3 is expression is associated with drug resistance and associated with high-grade breast carcinoma and poor progression-free survival (Ullman et al., 2011; correlates with estrogen receptor/progesterone Lim et al., 2012), whereas it inhibits the release of receptor double negative tumors as well as with a mitochondrial cytochrome c in squamous cell poor prognosis for breast cancer patients (Catanzaro carcinoma after treatment with TNF-α (Hashimoto et al., 2011). et al., 2005), or with DNA alkylating agents (Ullman et al., 2011). Localisation Moreover, SERPINB3 expression is associated with SERPINB3 may be found in cytoplasmic and poor survival in patients with breast cancer treated pericellular locations (Uemura et al., 2000). with anthracycline-based neoadjuvant Moreover an additional surface localization for this chemotherapy (Collie-Duguid et al., 2012) and in serpin has been reported (De Falco et al., 2001; patients with epithelial ovarian cancer a high Vidalino et al., 2012). SERPINB3 expression is a prognostic factor for Although it was initially reported that SERPINB3 is platinum resistance and shorter progression-free a cytosolic protein, its nuclear localization has been survival (Lim et al., 2012). also described recently, expanding the potential In addition, recent results, reported that SERPINB3 range of physiological functions of this molecule. protects from oxidative damage by Under certain conditions, such as following chemotherapeutics through inhibition of exposure to UV irradiation, SERPINB3 is mitochondrial respiratory complex I (Ciscato et al., translocated into the nucleus. Although SERPINB3 2014). does not possess a nuclear localization signal, it Experiments carried out with serum-derived HBV binds with c-Jun NH2-terminal kinase-1 (JNK1), particles have demonstrated that isolated and upon JNK1 activation SERPINB3 enters the SERPINB3 protein is able to bind preS1 encoded nucleus (Katagiri et al., 2006). sequence HBV surface protein, allowing virus entry In addition, other studies have shown that into human hepatocytes and also peripheral blood SERPINB3 may be secreted in serum and can mononuclear cells, underlying its potential predict HCC development in patients with cirrhosis biological role in HBV infection (De Falco et al., (Pontisso et al., 2006). 2001; Pontisso et al., 1991; Ruvoletto et al., 2004). Function This serpin induces also cell proliferation and deregulation of adhesion processes, leading to SERPINB3 is physiologically involved in the epithelial-mesenchymal transition (EMT) with regulation of differentiation in normal squamous increased invasiveness potential (Quarta et al., epithelium and is overexpressed in neoplastic tissue 2010). In addition, it has been reported that it of epithelial origin, where it might be involved in induces TGF-β expression (Calabrese et al., 2008; the apoptotic pathway as a protease inhibitor Turato et al., 2010) and promotes fibrogenesis in (Suminami et al., 1998). experimental models (Lunardi et al., 2011). Regarding their role in normal epithelia, it has been In addition, SERPINB3 may enhance its oncogenic suggested that SCCA isoforms may protect from potential through inhibition of several tumor bacterial, viral cystein proteases (Suminami et al., suppressive miRNAs (Turato et al., 2014a) and 1998), and mast cell chymase (Schick et al., 1997). could play a role in the development of cancer As a protease inhibitor, SERPINB3 is able to phenotype. inhibit cysteine proteases (cathepsins L, S, K and More recently, it has been reported that increased papain) (Schick et al., 1998), and in cancer cells it SERPINB3 expression leads to inhibition of protein confers resistance to drug-induced apoptosis by turnover, unfolded protein response, activation of inhibiting lysosomal cathepsin proteases (Suminami NF-kB and is essential for Ras-mediated cytokine et al., 2000). However, under a variety of stress production and tumour growth (Catanzaro et al., conditions SERPINB3 displays an anti-apoptotic 2014). function unrelated to its proteinase inhibition activity (Vidalino et al., 2009; Ciscato et al., 2014). Homology Indeed, SERPINB3 protects cells from exposure to SERPINB3 and SERPINB4 isoforms, also known radiation through an inhibitory effect either on the as squamous cell carcinoma antigen 1 and 2

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 204 SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), Turato C, Pontisso P member 3)

(SCCA1 and SCCA2) are highly homologous, 91% SERPINB3 has been proposed as a serological identical at the amino acid level (Suminami et al., biomarker that, alone or in combination with α- 1991), share conserved tertiary structure, and use a fetoprotein, may improve the sensitivity of HCC unique conformational rearrangement for their diagnosis (Beneduce et al., 2005; Giannelli et al., inhibitory activity (Stein et al., 1990). 2007a). However, SERPINB3 and SERPINB4 show distinct Circulating SERPINB3-IgM immuno-complexes properties and substrates: SERPINB3 inhibits have been described in cirrhotic patients at higher papain-like cysteine proteinases, cathepsins K, L, risk of HCC development (Pontisso et al., 2006) and S while SCCA2 inhibits chymotrypsin-like and in patients with HCC diagnosis (Beneduce et serine proteinases, cathepsin G and mast cell al., 2005). chymase (Takeda et al., 1995; Schick et al., 1997). Moreover, it has been reported that human In mouse, SCCA locus (SERPINB3 and hepatoma cells, stably transfected in order to over- SERPINB4) was expanded and contained four express SERPINB3, exhibited a significant increase genes, Serpinb3a, -b3b, -b3c, and -b3d, and three in proliferation rate and unequivocal evidence of pseudogenes, Serpinb3-ps1, -ps-2, and -ps-3. invasiveness and Epithelial to Mesenchimal Percentage protein sequences identity: 55-59%) Transition, then potentially acting as a putative (Askew et al., 2004). paracrine mediator able to favor cancer cell growth Moreover similarity-to-human data found for and metastatic invasiveness (Quarta et al., 2010). SERPINB3 was found in: worm (Caenorhabditis In addition a very recent study revealed that high elegans), fruit fly (Drosophila melanogaster), levels of SERPINB3 were detectable in 22% of mosquito (Anopheles gambiae), chicken (Gallus HCCs specimens from cirrhotic patients and were gallus), mouse (Mus musculus), rattus (Rattus found to be significantly associated with early norvegicus) and chimpanzee (Pan troglodytes). tumor recurrence, then representing a candidate prognostic marker able to identify the subset of Mutations most aggressive HCCs (Turato et al., 2014b). Note Lung cancer Maps of different Single-nucleotide polymorphisms Note (SNP) in human SERPINB3 are reported in Fig.2. Elevated levels of Squamous cell carcinoma antigen Only SNP rs3180227 has beeen well characterised (SCC-Ag) is secreted by non-small cell lung (Turato et al., 2009). tumours (NSCLC) and can be detected in serum This polymorphic variant (also know as SCCA-PD) (Mino et al., 1988). presents the 351G/A mutation in the variable It has been reported that preoperative SCC-Ag level reactive site loop (RSL) of the protein (GenBank in serum and its postoperative decrease have accession number: AY190327). prognostic significance in NSCLC The prevalence of SCCA-PD was 24% in the (Vassilakopoulos et al., 2001). normal population, while in patients with cirrhosis Moreover, in another study, tumor transcriptome it was 45%, supporting the hypothesis of a higher analysis has revealed the predictive and prognostic contribution of this isoform to liver disease impact of lysosomal protease inhibitors progression. (SERPINB3 and cystatin C) with clinical response In addition, the specific amino acid change detected in platinum-based chemotherapy - treated in in the reactive center of this SNP might confer a NSCLC patients (Petty et al., 2006). These different biological behavior to the serpin molecules potentially represent novel targets for improving the antiprotease activity of SERPINB3 NSCLC therapeutics. (Turato et al., 2011). Ovarian cancer Implicated in Note Hepatocellular carcinoma In a model of human epithelial ovarian cancer (EOC) using chickens, the most relevant animal Note model, SERPINB3 mRNA was induced in Several papers have documented the key role of cancerous, but not normal ovaries, and it was SERPINB3 in Hepatocellular Carcinoma (HCC). abundant only in the glandular epithelium of SERPINB3 is almost undetectable in normal liver cancerous ovaries of chickens. but it is expressed in HCC cells and in cells of In addition, strong expression of SERPINB3 highly dysplastic nodules and hepatocytes of peri- protein was reported as prognostic factor for tumoral cirrhotic tissue, suggesting that SERPINB3 platinum drug resistance and for poor progression- expression may represent a relatively early event in free survival in patients with EOC (Lim et al., hepatocarcinogenesis (Guido et al., 2008). 2012).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 205 SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), Turato C, Pontisso P member 3)

Breast cancer contribute to the apoptotic deregulation seen in SLE, thereby increasing the autoantigen burden Note (Vidalino et al., 2009). Elevated expression of SERPINB3 is associated Then, SERPINB3 expression and CD27 positivity with both high grade and advanced stage human were found to be directly related, suggesting that breast carcinomas. In addition, it has been reported this serpin might also be implicated in normal B that SCCA-positive breast tumors have a worse cell activation. clinical outcome, including decreased overall It has to be noted that the peripheral B cell survival and recurrence free survival (Catanzaro et repertoire and particularly CD27+ B cell number is al., 2011). heterogeneously altered in SLE (Korganow et al., Furthermore, SERPINB3 positivity predicted poor 2010). survival in patients treated with anthracycline-based In summary, these results may suggest a role for chemotherapy (Collie-Duguid et al., 2012). SERPINB3 in maintaining immune homeostasis, The implication of SERPINB3 in breast cancer may and that the impairment in serpin function may provide a novel diagnostic approach that will help contribute to the development of autoimmune to understand the initiation and advancement of disorders. breast cancer and provide new therapeutic options. Hepatoblastoma Lung fibrosis Note Note Hepatoblastoma (HB) is the most common liver Idiopathic pulmonary fibrosis (IPF), with its malignancy in early childhood and it is considered histopathological signature of usual interstitial an embryonal tumour of the liver. pneumonia is a chronic progressive disorder of According to a recent paper, SERPINB3 was interstitial lung disease of unknown etiology with a overexpressed in 79% of the cases of HBs. poor prognosis. Moreover, by immunohistochemistry SERPINB3 It has been reported overexpression of SERPINB3 expression was found mainly in the embryonic, in lung tissue of IPF patients compared with other blastemal, small cell undifferentiated (SCUD) forms of interstitial lung diseases and normal lungs. components of HB. High SERPINB3 reactivity was In IPF, SERPINB3 was abnormally secreted by also detected in neoplastic cell clusters of portal metaplastic epithelial cells other than bronchial vein tumour thrombosis. Furthermore a direct cells where it is normally expressed (Calabrese et correlation was observed between SERPINB3 gene al., 2008). expression and tumour extension, suggesting that Moreover, mice transgenic for human SERPINB3, this serpin might help in defining the risk profile of showed higher TGF-β expression and more children affected by this neoplasm (Turato et al., extended pulmonary fibrosis than controls (Lunardi 2012). et al., 2011). In addition, it has been reported that SERPINB3 Autoimmune disorders immunocomplexed is increased in scleroderma Note patients with lung fibrosis (Giannelli et al., 2007b). Alteration in serpin function was shown to Cholesteatoma associate with deregulation of cell survival as well as with some autoimmune traits, meaning that Note people carrying serpin dysfunction often display an Cholesteatoma is a destructive and expanding altered immune response. A recent study explored growth consisting of keratinizing squamous SERPINB3 expression in patients with impaired epithelium in the middle ear and/or mastoid immune response to assess the potential process. involvement of SERPINB3 in the deregulation of Recent data suggest that SERPINB3, STAT3, B-cell reactivity. cathepsin K, and cathepsin L are associated with the Although serpins mainly act at the intracellular proliferation and growth of cholesteatoma and that level, membrane-bound expression of SERPINB3 these proteins may be influential factors in was recently demonstrated also on peripheral blood cholesteatoma growth (Ho et al., 2012). mononuclear cells, especially on CD27+ B cells. Skin disease Interestingly, SERPINB3 was found to be absent on autoimmune diseases as SLE (systemic lupus Note erythematosus) CD27+ B lymphocytes, consistent Many intrinsic and extrinsic factors are associated with its expression being suppressed by high levels with the stratum corneum (SC) barrier disruption. of type I interferon, which is a typical finding in In the study of Katagiri C, it has been reported a SLE (Vidalino et al., 2012). high correlation between SERPINB3 and Since SERPINB3 displays an antiapoptotic transepidermal water loss (TEWL). This finding behavior, alterations in its expression might was confirmed by means of a barrier disruption

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 206 SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), Turato C, Pontisso P member 3)

study with a topical oleic acid treatment: subjects Mino N, Iio A, Hamamoto K. Availability of tumor-antigen 4 with high levels of SERPINB3 readily developed as a marker of squamous cell carcinoma of the lung and other organs. Cancer. 1988 Aug 15;62(4):730-4 impaired SC barrier function. Furthermore, SERPINB3 content showed a very high correlation Stein PE, Leslie AG, Finch JT, Turnell WG, McLaughlin PJ, Carrell RW. Crystal structure of ovalbumin as a model with the number of parakeratotic cells in the for the reactive centre of serpins. Nature. 1990 Sep cornified layer of the skin. These findings indicate 6;347(6288):99-102 that SERPINB3 can be considered a marker of Pontisso P, Morsica G, Ruvoletto MG, Zambello R, parakeratosis and may play an inhibitory role in the Colletta C, Chemello L, Alberti A. Hepatitis B virus binds to process of nuclear digestion (Katagiri et al., 2010). peripheral blood mononuclear cells via the pre S1 protein. Psoriatic Skin J Hepatol. 1991 Mar;12(2):203-6 It has been also reported that Cathepsin L (a target Suminami Y, Kishi F, Sekiguchi K, Kato H. Squamous cell of SERPINB3) is one of the lysosomal acid carcinoma antigen is a new member of the serine protease proteinases recently identified in psoriatic inhibitors. Biochem Biophys Res Commun. 1991 Nov epidermis (Kawada et al., 1997) together with 27;181(1):51-8 various proteinases including tryptase, chymase, Takeshima N, Suminami Y, Takeda O, Abe H, Okuno N, and cathepsin G (targets of SERPINB4: see Kato H. Expression of mRNA of SCC antigen in squamous homology in previous paragraph) released by cells. Tumour Biol. 1992;13(5-6):338-42 degranulation in psoriatic lesion (Harvima et al., Harvima IT, Naukkarinen A, Paukkonen K, Harvima RJ, 1993). Aalto ML, Schwartz LB, Horsmanheimo M. Mast cell tryptase and chymase in developing and mature psoriatic Takeda A, et al., have shown that SERPINB3/4 lesions. Arch Dermatol Res. 1993;285(4):184-92 isoforms (SERPINB3 and SERPINB4) are indeed Remold-O'Donnell E. The ovalbumin family of serpin predominantly present along the periphery of the proteins. FEBS Lett. 1993 Jan 4;315(2):105-8 intercellular space in the upper spinous cell layer of psoriatic epidermis from patients with a high serum Schneider SS, Schick C, Fish KE, Miller E, Pena JC, Treter SD, Hui SM, Silverman GA. A serine proteinase SERPINB3/4 level. In addition, SERPINB3/4 inhibitor locus at 18q21.3 contains a tandem duplication of immunoreactivity was detected around the the human squamous cell carcinoma antigen gene. Proc degranulated cells near the dermo-epidermal Natl Acad Sci U S A. 1995 Apr 11;92(8):3147-51 junction as well as in the granules of filtrated cells. Takeda A, Yamamoto T, Nakamura Y, Takahashi T, Hibino Furthermore, strong positive staining for T. Squamous cell carcinoma antigen is a potent inhibitor of SERPINB3/4 was also found in nuclei of granular cysteine proteinase cathepsin L. FEBS Lett. 1995 Feb layer cells and a considerable number of cells in the 6;359(1):78-80 elongated rete rete ridges of psoriatic epidermis. In Kato H. Expression and function of squamous cell particular, SERPINB3 mRNA was ubiquitously carcinoma antigen. Anticancer Res. 1996 Jul- expressed in all normal skin, and significantly Aug;16(4B):2149-53 overexpressed in psoriatic skin tissues. On the other Kawada A, Hara K, Kominami E, Hiruma M, Noguchi H, hand, SERPINB4 mRNA expression was specific Ishibashi A. Processing of cathepsins L, B and D in psoriatic epidermis. Arch Dermatol Res. 1997 for psoriatic skin tissues, while it was absent in Jan;289(2):87-93 normal epidermis (Takeda et al., 2002) Schick C, Kamachi Y, Bartuski AJ, Cataltepe S, Schechter Asthma related pathology NM, Pemberton PA, Silverman GA. Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the Note chymotrypsin-like proteinases cathepsin G and mast cell A protective role of SERPINB3 in asthma was chymase. J Biol Chem. 1997 Jan 17;272(3):1849-55 initially suggested by a microarray analysis of Schick C, Pemberton PA, Shi GP, Kamachi Y, Cataltepe human bronchial epithelial cell cultures after S, Bartuski AJ, Gornstein ER, Brömme D, Chapman HA, stimulation with IL-4 and IL-13 (Hansel and Diette, Silverman GA. Cross-class inhibition of the cysteine 2007). This serpin may exert its protective role by proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis. inhibiting endogenous proteases associated with the Biochemistry. 1998 Apr 14;37(15):5258-66 inflammatory response. Further studies indicated that SERPINB3 serum Suminami Y, Nawata S, Kato H. Biological role of SCC antigen. Tumour Biol. 1998;19(6):488-93 levels were increased in patients with bronchial asthma, asthma exacerbation and in patients with Cataltepe S, Gornstein ER, Schick C, Kamachi Y, Chatson K, Fries J, Silverman GA, Upton MP. Co-expression of the allergic rhinitis (Izuhara, 2003; Nishi et al., 2005; squamous cell carcinoma antigens 1 and 2 in normal adult Suzuki et al., 2010). human tissues and squamous cell carcinomas. J Histochem Cytochem. 2000 Jan;48(1):113-22 References Huntington JA, Read RJ, Carrell RW. Structure of a serpin- protease complex shows inhibition by deformation. Nature. Hunt LT, Dayhoff MO. A surprising new protein superfamily 2000 Oct 19;407(6806):923-6 containing ovalbumin, antithrombin-III, and alpha 1- proteinase inhibitor. Biochem Biophys Res Commun. 1980 Suminami Y, Nagashima S, Vujanovic NL, Hirabayashi K, Jul 31;95(2):864-71 Kato H, Whiteside TL. Inhibition of apoptosis in human

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 207 SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), Turato C, Pontisso P member 3)

tumour cells by the tumour-associated serpin, SCC carcinoma-related antigen in children with acute asthma. antigen-1. Br J Cancer. 2000 Feb;82(4):981-9 Ann Allergy Asthma Immunol. 2005 Mar;94(3):391-7 Uemura Y, Pak SC, Luke C, Cataltepe S, Tsu C, Schick C, Katagiri C, Nakanishi J, Kadoya K, Hibino T. Serpin Kamachi Y, Pomeroy SL, Perlmutter DH, Silverman GA. squamous cell carcinoma antigen inhibits UV-induced Circulating serpin tumor markers SCCA1 and SCCA2 are apoptosis via suppression of c-JUN NH2-terminal kinase. J not actively secreted but reside in the cytosol of squamous Cell Biol. 2006 Mar 27;172(7):983-90 carcinoma cells. Int J Cancer. 2000 Jul 20;89(4):368-77 Petty RD, Kerr KM, Murray GI, Nicolson MC, Rooney PH, De Falco S, Ruvoletto MG, Verdoliva A, Ruvo M, Raucci A, Bissett D, Collie-Duguid ES. Tumor transcriptome reveals Marino M, Senatore S, Cassani G, Alberti A, Pontisso P, the predictive and prognostic impact of lysosomal protease Fassina G. Cloning and expression of a novel hepatitis B inhibitors in non-small-cell lung cancer. J Clin Oncol. 2006 virus-binding protein from HepG2 cells. J Biol Chem. 2001 Apr 10;24(11):1729-44 Sep 28;276(39):36613-23 Pontisso P, Quarta S, Caberlotto C, Beneduce L, Marino Murakami A, Suminami Y, Hirakawa H, Nawata S, Numa M, Bernardinello E, Tono N, Fassina G, Cavalletto L, Gatta F, Kato H. Squamous cell carcinoma antigen suppresses A, Chemello L. Progressive increase of SCCA-IgM radiation-induced cell death. Br J Cancer. 2001 Mar immune complexes in cirrhotic patients is associated with 23;84(6):851-8 development of hepatocellular carcinoma. Int J Cancer. 2006 Aug 15;119(4):735-40 Vassilakopoulos T, Troupis T, Sotiropoulou C, Zacharatos P, Katsaounou P, Parthenis D, Noussia O, Troupis G, Giannelli G, Fransvea E, Trerotoli P, Beaugrand M, Papiris S, Kittas C, Roussos C, Zakynthinos S, Gorgoulis Marinosci F, Lupo L, Nkontchou G, Dentico P, Antonaci S. V. Diagnostic and prognostic significance of squamous cell Clinical validation of combined serological biomarkers for carcinoma antigen in non-small cell lung cancer. Lung improved hepatocellular carcinoma diagnosis in 961 Cancer. 2001 May;32(2):137-44 patients. Clin Chim Acta. 2007a Aug;383(1-2):147-52 Takeda A, Higuchi D, Takahashi T, Ogo M, Baciu P, Giannelli G, Iannone F, Fransvea E, Chialà A, Lapadula G, Goetinck PF, Hibino T. Overexpression of serpin Antonaci S. Squamous cellular carcinoma squamous cell carcinoma antigens in psoriatic skin. J immunocomplexed is increased in scleroderma patients Invest Dermatol. 2002 Jan;118(1):147-54 with lung fibrosis. Clin Exp Rheumatol. 2007b Sep- Oct;25(5):794-5 Izuhara K. The role of interleukin-4 and interleukin-13 in the non-immunologic aspects of asthma pathogenesis. Hansel NN, Diette GB. Gene expression profiling in human Clin Chem Lab Med. 2003 Jul;41(7):860-4 asthma. Proc Am Thorac Soc. 2007 Jan;4(1):32-6 Askew DJ, Askew YS, Kato Y, Turner RF, Dewar K, Calabrese F, Lunardi F, Giacometti C, Marulli G, Gnoato Lehoczky J, Silverman GA. Comparative genomic analysis M, Pontisso P, Saetta M, Valente M, Rea F, Perissinotto E, of the clade B serpin cluster at human chromosome Agostini C. Overexpression of squamous cell carcinoma 18q21: amplification within the mouse squamous cell antigen in idiopathic pulmonary fibrosis: clinicopathological carcinoma antigen gene locus. Genomics. 2004 correlations. Thorax. 2008 Sep;63(9):795-802 Jul;84(1):176-84 Guido M, Roskams T, Pontisso P, Fassan M, Thung SN, Pontisso P, Calabrese F, Benvegnù L, Lise M, Belluco C, Giacomelli L, Sergio A, Farinati F, Cillo U, Rugge M. Ruvoletto MG, Marino M, Valente M, Nitti D, Gatta A, Squamous cell carcinoma antigen in human liver Fassina G. Overexpression of squamous cell carcinoma carcinogenesis. J Clin Pathol. 2008 Apr;61(4):445-7 antigen variants in hepatocellular carcinoma. Br J Cancer. 2004 Feb 23;90(4):833-7 Ahmed ST, Darnell JE Jr. Serpin B3/B4, activated by STAT3, promote survival of squamous carcinoma cells. Ruvoletto MG, Tono N, Carollo D, Vilei T, Trentin L, Biochem Biophys Res Commun. 2009 Jan 23;378(4):821- Muraca M, Marino M, Gatta A, Fassina G, Pontisso P. 5 Surface expression of squamous cell carcinoma antigen (SCCA) can be increased by the preS1(21-47) sequence Turato C, Ruvoletto MG, Biasiolo A, Quarta S, Tono N, of hepatitis B virus. J Gen Virol. 2004 Mar;85(Pt 3):621-4 Bernardinello E, Beneduce L, Fassina G, Cavalletto L, Chemello L, Merkel C, Gatta A, Pontisso P. Squamous cell Silverman GA, Whisstock JC, Askew DJ, Pak SC, Luke carcinoma antigen-1 (SERPINB3) polymorphism in chronic CJ, Cataltepe S, Irving JA, Bird PI. Human clade B serpins liver disease. Dig Liver Dis. 2009 Mar;41(3):212-6 (ov-serpins) belong to a cohort of evolutionarily dispersed intracellular proteinase inhibitor clades that protect cells Vidalino L, Doria A, Quarta S, Zen M, Gatta A, Pontisso P. from promiscuous proteolysis. Cell Mol Life Sci. 2004 SERPINB3, apoptosis and autoimmunity. Autoimmun Rev. Feb;61(3):301-25 2009 Dec;9(2):108-12 Beneduce L, Castaldi F, Marino M, Quarta S, Ruvoletto M, Zheng B, Matoba Y, Kumagai T, Katagiri C, Hibino T, Benvegnù L, Calabrese F, Gatta A, Pontisso P, Fassina G. Sugiyama M. Crystal structure of SCCA1 and insight about Squamous cell carcinoma antigen-immunoglobulin M the interaction with JNK1. Biochem Biophys Res Commun. complexes as novel biomarkers for hepatocellular 2009 Feb 27;380(1):143-7 carcinoma. Cancer. 2005 Jun 15;103(12):2558-65 Katagiri C, Iida T, Nakanishi J, Ozawa M, Aiba S, Hibino T. Hashimoto K, Kiyoshima T, Matsuo K, Ozeki S, Sakai H. Up-regulation of serpin SCCA1 is associated with Effect of SCCA1 and SCCA2 on the suppression of TNF- epidermal barrier disruption. J Dermatol Sci. 2010 alpha-induced cell death by impeding the release of Feb;57(2):95-101 mitochondrial cytochrome c in an oral squamous cell Korganow AS, Knapp AM, Nehme-Schuster H, Soulas- carcinoma cell line. Tumour Biol. 2005 Jul-Aug;26(4):165- Sprauel P, Poindron V, Pasquali JL, Martin T. Peripheral B 72 cell abnormalities in patients with systemic lupus Nishi N, Miyazaki M, Tsuji K, Hitomi T, Muro E, Zaitsu M, erythematosus in quiescent phase: decreased memory B Yamamoto S, Inada S, Kobayashi I, Ichimaru T, Izuhara K, cells and membrane CD19 expression. J Autoimmun. 2010 Nagumo F, Yuyama N, Hamasaki Y. Squamous cell Jun;34(4):426-34

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 208 SERPINB3 (serpin peptidase inhibitor, clade B (ovalbumin), Turato C, Pontisso P member 3)

Quarta S, Vidalino L, Turato C, Ruvoletto M, Calabrese F, 10.1371/journal.pone.0049869. Epub 2012 Nov 21. Valente M, Cannito S, Fassina G, Parola M, Gatta A, Pontisso P. SERPINB3 induces epithelial-mesenchymal Turato C, Buendia MA, Fabre M, Redon MJ, Branchereau transition. J Pathol. 2010 Jul;221(3):343-56 S, Quarta S, Ruvoletto M, Perilongo G, Grotzer MA, Gatta A, Pontisso P.. Over-expression of SERPINB3 in Suzuki K, Inokuchi A, Miyazaki J, Kuratomi Y, Izuhara K. hepatoblastoma: a possible insight into the genesis of this Relationship between squamous cell carcinoma antigen tumour? Eur J Cancer. 2012 May;48(8):1219-26. doi: and the clinical severity of allergic rhinitis caused by 10.1016/j.ejca.2011.06.004. Epub 2011 Jul 5. Dermatophagoides farinae and Japanese cedar pollen. Ann Otol Rhinol Laryngol. 2010 Jan;119(1):22-6 Vidalino L, Doria A, Quarta SM, Crescenzi M, Ruvoletto M, Frezzato F, Trentin L, Turato C, Parolin MC, Ghirardello A, Turato C, Calabrese F, Biasiolo A, Quarta S, Ruvoletto M, Iaccarino L, Cavalletto L, Chemello L, Gatta A, Pontisso Tono N, Paccagnella D, Fassina G, Merkel C, Harrison TJ, P.. SERPINB3 expression on B-cell surface in autoimmune Gatta A, Pontisso P. SERPINB3 modulates TGF-beta diseases and hepatitis C virus-related chronic liver expression in chronic liver disease. Lab Invest. 2010 infection. Exp Biol Med (Maywood). 2012 Jul;237(7):793- Jul;90(7):1016-23 802. doi: 10.1258/ebm.2012.012024. Epub 2012 Jul 24. Catanzaro JM, Guerriero JL, Liu J, Ullman E, Sheshadri N, Cannito, S., Turato, C., Paternostro, C., Quarta, S., Novo, Chen JJ, Zong WX. Elevated expression of squamous cell E., Villano, G., Colombatto, S., David, E., Pontisso, P., and carcinoma antigen (SCCA) is associated with human Parola, M.. SERPIN-B3 induces HIF2 alpha nuclear breast carcinoma. PLoS One. 2011 Apr 19;6(4):e19096 translocation in hepatic cancer cells: a paracrine loop able to affect cancer cell behaviour. J. Hepatol. 58 (suppl 1), Lunardi F, Villano G, Perissinotto E, Agostini C, Rea F, S424-S425 Meeting Abstract: 1033 2013 Gnoato M, Bradaschia A, Valente M, Pontisso P, Calabrese F. Overexpression of SERPIN B3 promotes Catanzaro JM, Sheshadri N, Pan JA, Sun Y, Shi C, Li J, epithelial proliferation and lung fibrosis in mice. Lab Invest. Powers RS, Crawford HC, Zong WX.. Oncogenic Ras 2011 Jun;91(6):945-54 induces inflammatory cytokine production by upregulating the squamous cell carcinoma antigens SerpinB3/B4. Nat Turato C, Biasiolo A, Pengo P, Frecer V, Quarta S, Commun. 2014 Apr 23;5:3729. doi: 10.1038/ncomms4729. Fasolato S, Ruvoletto M, Beneduce L, Zuin J, Fassina G, Gatta A, Pontisso P. Increased antiprotease activity of the Ciscato F, Sciacovelli M, Villano G, Turato C, Bernardi P, SERPINB3 polymorphic variant SCCA-PD. Exp Biol Med Rasola A, Pontisso P.. SERPINB3 protects from oxidative (Maywood). 2011 Mar;236(3):281-90 damage by chemotherapeutics through inhibition of mitochondrial respiratory complex I. Oncotarget. 2014 May Ullman E, Pan JA, Zong WX.. Squamous cell carcinoma 15;5(9):2418-27. antigen 1 promotes caspase-8-mediated apoptosis in response to endoplasmic reticulum stress while inhibiting Turato C, Simonato D, Quarta S, Gatta A, Pontisso P.. necrosis induced by lysosomal injury. Mol Cell Biol. 2011 MicroRNAs and SerpinB3 in hepatocellular carcinoma. Life Jul;31(14):2902-19. doi: 10.1128/MCB.05452-11. Epub Sci. 2014a Mar 28;100(1):9-17. doi: 2011 May 16. 10.1016/j.lfs.2014.01.073. Epub 2014 Feb 2. (REVIEW) Collie-Duguid ES, Sweeney K, Stewart KN, Miller ID, Turato C, Vitale A, Fasolato S, Ruvoletto M, Terrin L, Smyth E, Heys SD.. SerpinB3, a new prognostic tool in Quarta S, Ramirez Morales R, Biasiolo A, Zanus G, Zali N, breast cancer patients treated with neoadjuvant Tan PS, Hoshida Y, Gatta A, Cillo U, Pontisso P.. chemotherapy. Breast Cancer Res Treat. 2012 SERPINB3 is associated with TGF-β1 and cytoplasmic β- Apr;132(3):807-18. doi: 10.1007/s10549-011-1625-9. Epub catenin expression in hepatocellular carcinomas with poor 2011 Jun 22. prognosis. Br J Cancer. 2014b May 27;110(11):2708-15. doi: 10.1038/bjc.2014.246. Epub 2014 May 8. Ho KY, Huang HH, Hung KF, Chen JC, Chai CY, Chen WT, Tsai SM, Chien CY, Wang HM, Wu YJ.. Villano G, Turato C, Quarta S, Ruvoletto M, Ciscato F, Cholesteatoma growth and proliferation: relevance with Terrin L, Semeraro R, Paternostro C, Parola M, Alvaro D, serpin B3. Laryngoscope. 2012 Dec;122(12):2818-23. doi: Bernardi P, Gatta A, Pontisso P.. Hepatic progenitor cells 10.1002/lary.23547. express SerpinB3. BMC Cell Biol. 2014 Feb 11;15:5. doi: 10.1186/1471-2121-15-5. Lim W, Kim HS, Jeong W, Ahn SE, Kim J, Kim YB, Kim MA, Kim MK, Chung HH, Song YS, Bazer FW, Han JY, This article should be referenced as such: Song G.. SERPINB3 in the chicken model of ovarian cancer: a prognostic factor for platinum resistance and Turato C, Pontisso P. SERPINB3 (serpin peptidase survival in patients with epithelial ovarian cancer. PLoS inhibitor, clade B (ovalbumin), member 3). Atlas Genet One. 2012;7(11):e49869. doi: Cytogenet Oncol Haematol. 2015; 19(3):202-209.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 209

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

SNCG (synuclein, gamma (breast cancer- specific protein 1)) Andrei Surguchov Department of Neurology, Kansas University Medical Center, Kansas City, KS and VA Medical Center, Kansas City, MO, USA (AS)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/SNCGID42343ch10q23.html DOI: 10.4267/2042/56414 This article is an update of : Czekierdowski A, Czekierdowska S. SNCG (synuclein, gamma (breast cancer-specific protein 1)). Atlas Genet Cytogenet Oncol Haematol 2008;12(1):12-16.

Abstract Keywords Invasion, metastasis, cancer, tumorigenesis, Expression of synuclein-gamma (SNCG) protein is transcriptional regulation, microRNA elevated in the advanced stages of many types of cancers, including breast, ovarian, lung, gastric, Identity liver, esophagus, colon, prostate and others. In breast carcinoma, SNCG is causatively linked to Other names: BCSG1, gamma-synuclein, stimulated proliferation, metastasis and drug PERSYN, PRSN, Synoretin resistance. HGNC (Hugo): SNCG A clinical follow-up study indicates that patients Location: 10q23.2 with an SNCG-positive breast cancer have a Note significantly shorter disease-free survival and Synuclein-gamma is a member of the synuclein overall survival than patients with SNCG-negative family of proteins which are believed to be tumors. involved in the pathogenesis of neurodegenerative Overexpression of SNCG compromises normal diseases. High levels of SNCG have been identified mitotic checkpoint controls, resulting in in several types of cancer suggesting the association multinucleation as well as faster cell growth. of its overexpression and cancer development. SNCG has also been shown to promote invasion and metastasis in in vitro assays as well as in animal models. SNCG overexpression also DNA/RNA interferes with drug-induced apoptotic responses. Expression of SNCG in cancer cells results in a Description more malignant phenotype with increased cell Human SNCG gene consists of five exons that span motility, enhanced transcriptional activity of steroid about 5 kbp. The intron 1 contains two closely receptors and accelerated rate of chromosomal located AP1 recognition sequences. Deletion of instability. these motifs greatly diminished the SNCG promoter Two closely located AP1 binding sites residing in activity, suggesting that AP1 is an important the first intron of the SNCG gene are important transactivator for SNCG transcription SNCG regulators of the promoter activity. Other factors transcription is primarily controlled by regulatory regulating SNCG expression are methylation- sequences located in intron 1 and exon 1 and in a demethylation of exon and post-transcriptional lesser extent by 5' flanking region. Synuclein regulation by microRNAs. expression is regulated predominantly at the level

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 210 SNCG (synuclein, gamma (breast cancer-specific protein 1)) Surguchov A

of SNCG gene transcription, SNCG mRNA Localisation stability and by micro-RNA (miRs). The three human synuclein genes are expressed in the thalamus, the substantia nigra, the caudate Protein nucleus, and the amygdala. Only gamma-synuclein Description appears strongly expressed in the subthalamic nucleus. Although gamma-synuclein is not present Encoded by human SNCG gene (synuclein family), in senile plaques, Lewy bodies or neurofibrillary the highly conserved 127-amino acid 13 kD tangles, a high level of gamma-synuclein cytoplasmic gamma-synuclein is similar to two immunoreactivity is detectable in dot-like structures other members of the family, alpha-synuclein and and other deposits which are characteristic lesions beta-synuclein. The gamma-synuclein protein is the in the brains of patients with neurodegenerative least conserved of the synuclein proteins. The diseases, as well as in the retina and optic nerve. human gamma-synuclein is 87.7% and 83.8% SNCG mRNA and gamma-synuclein protein were identical to the mouse and rat proteins, respectively, also detected in unstimulated and which are 4-amino acids shorter. For all three phytohaemagglutinin (PHA)-stimulated cultured members of the family the region of highest lymphocytes from peripheral blood of normal homology is the amino-terminal region. The donors. It has been shown previously by in situ synuclein proteins contain several repeated domains hybridization that SNCG/BCSG1 mRNA is not that display variations of a KTKEGV consensus expressed in normal adult breast tissue, but high sequence. This motif, repeated six to seven times in levels of this mRNA are present in advanced the amino-terminal portion of the protein, is infiltrating breast tumours. In paraformaldehyde reminiscent of the alpha-helical domains of the fixed cells, SNCG displayed punctuate cytoplasmic apolipoproteins and suggests lipid binding staining, a pattern that is usually associated with properties. The very high conservation between markers of the endoplasmic reticulum or vesicular species for a specific repeated domain of a structures. particular protein suggests that the repeated domains have arisen from the duplication of a Function single domain within an ancestral synuclein gene. The normal cellular function of gamma-synuclein is Later, this ancestral gene may have undergone as yet unclear, but interestingly exogenous successive duplications to give rise to the three expression of the protein increases the invasive and synuclein genes in which the repeated domains may metastatic potential of breast tumors. The highly still be able to diverge. The third domain, however, conserved N-terminal region is known to be remained absolutely identical, KTKEGV, in all important for the lipid interactions of the synucleins genes throughout all species. A similar type of and the highly acidic C-terminal region has been domain is present in proteins of the rho family. suggested to possess chaperone-like activity, to However, as of today, the role of these domains regulate the aggregation of synucleins and to remains unknown. mediate protein-protein interactions. It seems that Expression gamma-synuclein plays a role in neurofilament Mammalian gamma-synuclein was first identified network integrity and may modulate axonal as the so-called breast cancer-specific gene 1 architecture, also, it may increase the susceptibility (BCSG1) in a high-throughput direct differential- of neurofilament-H to calcium-dependent proteases cDNA-sequencing screen for markers of breast and may also modulate the keratin network in skin. cancer. Northern blot analysis showed that the gene Phosphorylation by GRK5 appears to occur on is principally expressed in the brain, particularly in residues distinct from other kinase target residues. the substantia nigra. The protein is expressed in the Synuclein gamma is likely involved in the peripheral nervous system, mainly in primary pathogenesis of neurodegenerative and ocular sensory neurons, sympathetic neurons, and motor diseases, for example, glaucoma and SNCG is neurons. A high level of synuclein gamma was also expressed at very high level in advanced infiltrating detected in several types of tumors, and in the breast cancer. The dual role of SNCG in olfactory epithelium. A sequence dubbed synoretin neurodegeneration and malignancy could involve was independently isolated from ocular tissues in a common mechanisms. Changes in organization of screen for novel proteins regulating the cell cytoskeleton are among the most prominent phototransduction and is now thought to represent characteristics of both processes. The involvement the bovine ortholog of gamma-synuclein. SNCG is of gamma-synuclein in regulating neurofilament expressed in brain, heart, skeletal muscle, ovary, network integrity raises the possibility that it may testis, colon, spleen, pancreas, kidney and lung. also affect the intermediate filament network in MicroRNAs (miRs) are implicated in the regulation malignant breast epithelial cells. In addition, of SNCG expression. gamma-synuclein regulates the level of expression

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 211 SNCG (synuclein, gamma (breast cancer-specific protein 1)) Surguchov A

of several genes, for example matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9). Mutations Gamma-synuclein is readily oxidized at Note methionine-38 and after oxidation it aggregates and No tumor-specific mutations of the SNCG gene induces the aggregation of alpha-synuclein. were found in breast tumors and tumor cell lines, The binding of gamma-synuclein to phospholipase but two linked polymorphisms in the coding region C β2 (PLC β2) results in inhibition of enzymatic were detected, both in mRNA and in exons III and activity and therefore may regulate the intracellular IV of the gene from G243C and T377A. level of second messenger phosphatidylinositol 4,5 These results reflect the absence of two G--A (and bisphosphate (PI(4,5)P2). consequently Glu--Lys) in tumor cell lines, tumors Gamma-synuclein is a novel regulator of lipid and control tissues. metabolism in adipocytes and the deficiency of this The above mentioned two linked polymorphic sites protein has a significant effect on whole body discriminate two alleles of the human persyn gene. energy expenditure. The frequencies of the two alleles were the same in Homology genomes of breast cancer and normal cells (20% G243/T377 and 80% C243/A377). There are currently almost 200 DNA and protein Both alleles are transcriptionally active and are sequences in the sequence databases with high expressed with similar efficiency in heterozygotes. homology to the synuclein gene or protein. All synuclein sequences available to date from Epigenetics Homo sapiens, Mus musculus, and Rattus Sequence analysis identified a CpG island in exon 1 norvegicus can be assigned to three distinct protein of SNCG that contains 15 CpG sites, covering the groups: alpha beta and gamma-synuclein. Synuclein region -169 to +81, relative to the translation start proteins have also been identified in other codon. CpG sites within the CpG island and its organisms: synelfin is the alpha-synuclein ortholog vicinity were partially and heterogeneously in Serinus, phospho-neuroprotein 14 (PNP14) is the methylated in SNCG-negative breast cancer cell beta-synuclein ortholog in Bos taurus, and the first lines but unmethylated in SNCG-positive cells. synuclein protein described in Torpedo californica SNCG expression correlates with complete corresponds to the human gamma-synuclein. demethylation of the exon 1 region. Specific Interestingly, synucleins are identified only in methylation at the CpG sites 2, 5, 7, and 10-15, was vertebrate and no homologous proteins have been sufficient to block the expression of SNCG gene in revealed in invertebrate or lower organisms. breast cell culture. Genomic sequencing and Each of the three family members is composed of methylation-specific PCR assays have shown that an N-terminal lipid-binding domain, containing a SNCG CpG island is fully methylated in normal series of 11-residue imperfect repeats, and an acidic tissues of liver, esophagus, prostate, cervix, C-terminal domain. stomach, colon, and lung, but only partially Among the human family members, gamma- methylated in breast tissue. Tumors from these synuclein 50% identical and 74% homologous to tissues contain completely demethylated SNCG. alpha-synuclein and 47% identical and 66% Universal loss of the epigenetic control of SNCG homologous beta-synuclein. gene expression in tumors and further The highly conserved N-terminal region is known demonstrating that the demethylation of SNCG to be important for the lipid interactions of the CpG island is primarily responsible for the aberrant synucleins and the highly acidic C-terminal region expression of SNCG protein in cancerous tissues has been suggested to possess chaperone-like have suggested an important role of gamma- activity, to regulate the aggregation of synuclein synuclein-related epigenectic events in various and to mediate protein-protein interactions. malignancies. Reactivation of SNCG gene The very high degree of conservation in the lipid- expression by DNA demethylation is a common binding N-terminal domains of all three synucleins critical contributing factor to malignant progression strongly suggests that both beta and gamma- of many solid tumors and its expression in primary synuclein like alpha-synuclein bind to lipid carcinomas is an effective molecular indicator of membranes and adopt a highly helical structure. distant metastasis. Nevertheless, differences in the sequences of the three proteins in both their central parts and C- Implicated in terminal domains must be responsible for those differences that do exist in their individual Note functions, as well as for their different roles in The possible involvement of gamma-synuclein in disease. tumorigenesis first came to light when a gene

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 212 SNCG (synuclein, gamma (breast cancer-specific protein 1)) Surguchov A

named BCSG1 (breast cancer-specific gene 1) was Breast cancer shown to be overexpressed in advanced infiltrating Note carcinoma of the breast. In fact, BCSG1 and Patients whose tumors expressed SNCG had a gamma-synuclein appear to be the same protein. significantly shorter disease-free survival and SNCG protein is highly expressed in diversified overall survival. They also had a high probability of cancer types, including the female hormone- death when compared with those whose tumors did sensitive cervical and breast cancers, male not express SNCG. Multivariate analysis hormone-sensitive prostate cancer, four cancer demonstrated that SNCG is an independent types of the digestive system, and lung cancer. predictive marker for recurrence and metastasis in These cancers are currently the leading cause of breast cancer progression. SNCG is expected to be mortality in both men and women. How SNCG a useful marker for breast cancer progression and a induces disease progression in different cancer potential target for breast cancer treatment. In one types remains elusive. Oncogenic activation of study it has been show that responses of 12 breast gamma-synuclein contributes to the development of cancer cell lines to paclitaxel-induced mitotic arrest breast and ovarian cancer by promoting tumor cell and cytotoxicity highly correlated with SNCG survival under adverse conditions and by providing expression status. SNCG-positive cells exhibited a resistance to certain chemotherapeutic drugs. significantly higher resistance to paclitaxel-induced Overexpression of gamma-synuclein leads to mitotic arrest than SNCG-negative cells. Down- constitutive activation of extracellular signal- regulation of SNCG expression directly increased regulated protein kinases (ERK1/ERK2) and down- the effectiveness of anti-microtubule drug-induced regulation of c-Jun N-terminal kinase 1(JNK1) in cytotoxicity in breast cancer cells without altering response to environmental stress signals. Gamma- cell responses to doxorubicin. These new findings synuclein is found in a wide variety of transformed suggest that SNCG expression in breast carcinomas cells and its overexpression leads to a significant is probably a causal factor contributing to the poor increase in proliferation, motility, invasiveness and patient response to paclitaxel treatment. metastasis. Cells expressing gamma-synuclein are significantly more resistant to the chemotherapeutic Ovarian cancer drugs paclitaxel and vinblastine as compared with Note the parental cells. Activation of JNK1 and its Several studies indicated that SNCG expression downstream caspase-3 by paclitaxel or vinblastine was not detectable in normal ovarian epithelium but is significantly down-regulated in gamma- was highly expressed in the vast majority of synuclein-expressing cells, indicating that the advanced staged ovarian carcinomas. Eighty-seven apoptosis pathway activated by vinblastine or percent of ovarian carcinomas were found to paclitaxel is blocked by gamma-synuclein. In breast express at least 1 type of synuclein, and 42% cancer cells, SNCG has been shown to act as a expressed all 3 synucleins (alpha, beta, and gamma) chaperon for estrogen receptor and stimulate simultaneously. Highly punctate gamma synuclein estrogen receptor-a signaling pathway that leads to expression was also observed in 20% of cell proliferation. On the other hand, the inhibitory preneoplastic lesions of the ovary, including effects of SNCG on mitotic checkpoint function are epithelial inclusion cysts, hyperplastic epithelium, mediated through the mitotic checkpoint kinase and papillary structures, suggesting that synuclein BubR1 and are independent of the expression status gamma up-regulation may occur early in the of estrogen receptor-alpha. The inhibitory effects of development of some ovarian tumors. SNCG on mitotic checkpoint can be overthrown by Demethylation is an important event in abnormal enforced overexpression of BubR1 in SNCG- synuclein-gamma expression. The methylation expressing cells. SNCG intracellularly associates pattern in ovarian cancer cells is different from that with BubR1 together. This observation suggests in breast cancer cells. In one of the studies that that SNCG expression compromises the mitotic examined SNCG-nonexpressing ovarian cancer checkpoint control by inhibition of the normal cells, all of the CpG sites were completely function of BubR1, thereby promoting genetic methylated instead of selective methylation at instability, a recognized and important contributing certain sites shown in breast cancer cells, thereby factor in tumorigenesis. Because all synucleins suggesting a tissue-specific methylation pattern. have chaperone-like activities, they may interact Recent studies indicated that the detection of SNCG with different proteins in different cellular mRNA in tumor-positive tumors was strongly background. Identifications of specific cellular associated with demethylation or hypometylation of targets of SNCG in different tumor types will SNCG gene. Methylation status was not correlated provide insight to delineate its oncogenic functions with FIGO stage or histological type of tumor. in human malignancies. Tumor grading was strongly associated with

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 213 SNCG (synuclein, gamma (breast cancer-specific protein 1)) Surguchov A

methylation status but due to relatively small group Esophageal cancer of studied samples (43 cases) this observation Note requires further confirmation. The examination of the serum γ-synuclein levels of Another interesting observation was that in 21% of patients with gastrointestinal and esophageal samples both products of amplification were squamous cell carcinomas, benign disease and present and all these cases were SNCG mRNA- healthy controls by a sandwich ELISA positive. demonstrated a positive correlation between serum This observation could suggested that partial γ-synuclein and the development of these types of methylation of SNCG probably does not influence cancer. From this study a conclusions was put on synuclein expression in ovarian cancer tissue. forward that serum γ-synuclein is a promising Comparison of the methylation status of SNCG and diagnostic biomarker for early detection of the expression of synuclein-gamma in breast and gastrointestinal and esophageal cancer. Serum γ- ovarian cancer cells lines in another study indicated synuclein SNCG may play an important role in a strong correlation between hypomethylation of invasion, infiltration and apoptosis of esophageal the CpG island and SNCG expression in cancer cell cancer and serve as target spots in the targeted lines. therapy of esophageal cancer. However, the The methylation pattern in ovarian cancer cells was analysis of expression pattern of SNCG in another different from that in breast cancer cells. study including 27 cases of esophageal cancer The analyzed CpG sites in ovarian cancer cells (ESC) demonstrated that it is downregulated in 16 were all methylated in contrary to a selective out of 27 cases of ESC. Overexpression of SNCG methylation at certain sites shown in breast cancer in ESC 9706 cell line has shown that the ectopic cells, thereby suggesting a tissue-specific expression of SNCG in ESC cell line inhibits cell methylation pattern. Moreover, when exon 1 was growth in dish and colony formation in soft agar. partially and heterogeneously methylated, then Therefore, unlike breast and ovarian cancers, a SNCG expression in breast cancer cells was not reversed correlation between SNCG expression and detected. ESC development was found, which led to a Lung cancer hypothesis that SNCG may play a role of a tumor suppressor in the development of human ESC. Note SNCG is not expressed in normal lung tissues, but Prostate cancer it is highly expressed in lung tumors. It has been Note demonstrated that cigarette smoke extract (CSE) A strong association between SNCG expression and has strong inducing effects on SNCG gene prostate cancer development is found. By expression in lung cancer cells through performing genomic sequencing and methylation- demethylation of SNCG CpG island. CSE treatment specific PCR assays, an inclusive demethylation of also augments the invasive capacity of cells in an CpG sites within the CpG island of SNCG gene in SNCG-dependent manner. These new findings prostate cancer samples was established. These demonstrate that tobacco exposure induces the results suggest a loss of the epigenetic control of abnormal expression of SNCG in lung cancer cells SNCG gene expression in tumors and demonstrate through downregulation of expression levels of that the demethylation of SNCG CpG island is DNA methyltransferases. primarily responsible for the aberrant expression of Gastric cancer SNCG in prostate cancerous tissues. A conclusion is drawn that that reactivation of SNCG gene Note expression by DNA demethylation is a common For the gastric cancer cell lines, SNCG mRNA critical contributing factor to malignant progression expression strongly correlated with demethylation of tumors and its expression in primary carcinomas of SNCG exon 1 CpG islands. Whereas SNCG was is an effective molecular indicator of distant not expressed in non-neoplastic gastric mucosal metastasis. Therefore, the methylation status of tissues obtained at autopsy, partial demethylation SNCG gene can be used as a sensitive molecular was present in these tissues. Demethylation occurs tool in early detections of tumorigenesis. Silencing before malignant transformation and that only SNCG by siRNA in LNCaP cells contributes to the partial demethylation does not result in up- inhibition of cellular proliferation, the induction of regulated SNCG mRNA expression. Thus, it cell-cycle arrest at the G1 phase, the suppression of appears that partial SNCG demethylation can occur cellular migration and invasion in vitro, as well as in normal gastric mucosa, which then extends in the decrease of tumor growth in vivo with the some cases to become to fully demethylated, notable exception of castrated mice. SNCG is a resulting in up-regulated SNCG mRNA expression. novel androgen receptor (AR) coactivator.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 214 SNCG (synuclein, gamma (breast cancer-specific protein 1)) Surguchov A

It interacts with AR and promotes prostate cancer gene, BCSG1, by direct differential cDNA sequencing. cellular growth and proliferation by activating AR Cancer Res. 1997 Feb 15;57(4):759-64 transcription in an androgen-dependent manner. Lavedan C, Buchholtz S, Auburger G, Albin RL, Athanassiadou A, Blancato J, Burguera JA, Ferrell RE, Endometrial cancer Kostic V, Leroy E, Leube B, Mota-Vieira L, Note Papapetropoulos T, Pericak-Vance MA, Pinkus J, Scott WK, Ulm G, Vasconcelos J, Vilchez JJ, Nussbaum RL, SNCG expression is associated with poor outcome Polymeropoulos MH. Absence of mutation in the beta- and in endometrial adenocarcinoma. There is a positive gamma-synuclein genes in familial autosomal dominant association between SNCG expression and tumor Parkinson's disease. DNA Res. 1998 Dec 31;5(6):401-2 grade, tumor stage, type II carcinomas, deep Ninkina NN, Alimova-Kost MV, Paterson JW, Delaney L, myometrial invasion and lymphovascular invasion. Cohen BB, Imreh S, Gnuchev NV, Davies AM, Buchman A correlation between SNCG and adverse VL. Organization, expression and polymorphism of the human persyn gene. Hum Mol Genet. 1998 Sep;7(9):1417- outcomes, such as shorter overall survival and 24 disease free survival is found. The expression level of SNCG in endometrioid Surguchov A, Surgucheva I, Solessio E, Baehr W. Synoretin--A new protein belonging to the synuclein family. endometrial carcinoma is closely associated with Mol Cell Neurosci. 1999 Feb;13(2):95-103 International Federation of Gynecology and Bruening W, Giasson BI, Klein-Szanto AJ, Lee VM, Obstetrics (IFGO) stages, the depth of myometrial Trojanowski JQ, Godwin AK. Synucleins are expressed in invasion and lymph nodes metastases (p the majority of breast and ovarian carcinomas and in SNCG is considered a useful marker for preneoplastic lesions of the ovary. Cancer. 2000 May endometrioid endometrial carcinoma invasion, 1;88(9):2154-63 metastasis and prognosis in endometrioid Lu A, Zhang F, Gupta A, Liu J. Blockade of AP1 endometrial carcinoma. transactivation abrogates the abnormal expression of breast cancer-specific gene 1 in breast cancer cells. J Biol Gallbladder cancer Chem. 2002 Aug 30;277(35):31364-72 Note Pan ZZ, Bruening W, Giasson BI, Lee VM, Godwin AK. SNCG is highly expressed in human gallbladder Gamma-synuclein promotes cancer cell survival and inhibits stress- and chemotherapy drug-induced apoptosis cancer, and its abnormal expression is associated by modulating MAPK pathways. J Biol Chem. 2002 Sep with tumor aggressiveness. SNCG gene silencing in 20;277(38):35050-60 NOZ cells inhibited cell growth, colony formation, Surgucheva I, McMahan B, Ahmed F, Tomarev S, Wax and invasion. In addition, it directly increased the MB, Surguchov A. Synucleins in glaucoma: implication of effectiveness of paclitaxel in inducing G2/M cell- gamma-synuclein in glaucomatous alterations in the optic cycle arrest and cell apoptosis. A decrease in tumor nerve. J Neurosci Res. 2002 Apr 1;68(1):97-106 growth and weight was found in mice injected with Gupta A, Godwin AK, Vanderveer L, Lu A, Liu J. SNCG-silenced NOZ cells. Together, these findings Hypomethylation of the synuclein gamma gene CpG island suggest that SNCG plays an important role in the promotes its aberrant expression in breast carcinoma and progression of human gallbladder cancer. ovarian carcinoma. Cancer Res. 2003 Feb 1;63(3):664-73 Gupta A, Inaba S, Wong OK, Fang G, Liu J. Breast Colon cancer cancer-specific gene 1 interacts with the mitotic checkpoint Note kinase BubR1. Oncogene. 2003 Oct 23;22(48):7593-9 Abnormal expression of SNCG protein has been Surgucheva IG, Sivak JM, Fini ME, Palazzo RE, demonstrated in colon cancer. SNCG predicts poor Surguchov AP. Effect of gamma-synuclein overexpression clinical outcome in colon cancer with normal levels on matrix metalloproteinases in retinoblastoma Y79 cells. Arch Biochem Biophys. 2003 Feb 1;410(1):167-76 of carcinoembryonic antigen (CEA). SNCG levels in colon adenocarcinoma were closely associated Wu K, Weng Z, Tao Q, Lin G, Wu X, Qian H, Zhang Y, Ding X, Jiang Y, Shi YE. Stage-specific expression of with intravascular embolus and tumor recurrence breast cancer-specific gene gamma-synuclein. Cancer but independent of preoperative serum CEA levels. Epidemiol Biomarkers Prev. 2003 Sep;12(9):920-5 SNCG expression was an independent prognostic Zhou CQ, Liu S, Xue LY, Wang YH, Zhu HX, Lu N, Xu NZ. factor of a shorter disease-free survival and overall Down-regulation of gamma-synuclein in human survival (P < 0.0001). SNCG is a new independent esophageal squamous cell carcinoma. World J predicator for poor prognosis in patients with colon Gastroenterol. 2003 Sep;9(9):1900-3 adenocarcinoma, including those with normal CEA Jiang Y, Liu YE, Goldberg ID, Shi YE. Gamma synuclein, a levels. Combination of CEA with SNCG improves novel heat-shock protein-associated chaperone, stimulates prognostic evaluation for patients with colon ligand-dependent estrogen receptor alpha signaling and adenocarcinoma. mammary tumorigenesis. Cancer Res. 2004 Jul 1;64(13):4539-46 References Yanagawa N, Tamura G, Honda T, Endoh M, Nishizuka S, Motoyama T. Demethylation of the synuclein gamma gene Ji H, Liu YE, Jia T, Wang M, Liu J, Xiao G, Joseph BK, CpG island in primary gastric cancers and gastric cancer Rosen C, Shi YE. Identification of a breast cancer-specific cell lines. Clin Cancer Res. 2004 Apr 1;10(7):2447-51

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 215 SNCG (synuclein, gamma (breast cancer-specific protein 1)) Surguchov A

Inaba S, Li C, Shi YE, Song DQ, Jiang JD, Liu J. Synuclein ganglion cells. Mol Vis. 2008 Aug 22;14:1540-8 gamma inhibits the mitotic checkpoint function and promotes chromosomal instability of breast cancer cells. Liu C, Dong B, Lu A, Qu L, Xing X, Meng L, Wu J, Eric Shi Breast Cancer Res Treat. 2005 Nov;94(1):25-35 Y, Shou C. Synuclein gamma predicts poor clinical outcome in colon cancer with normal levels of Liu H, Liu W, Wu Y, Zhou Y, Xue R, Luo C, Wang L, Zhao carcinoembryonic antigen. BMC Cancer. 2010 Jul W, Jiang JD, Liu J. Loss of epigenetic control of synuclein- 7;10:359 gamma gene as a molecular indicator of metastasis in a wide range of human cancers. Cancer Res. 2005 Sep Chen J, Jiao L, Xu C, Yu Y, Zhang Z, Chang Z, Deng Z, 1;65(17):7635-43 Sun Y. Neural protein gamma-synuclein interacting with androgen receptor promotes human prostate cancer Surgucheva I, Ninkina N, Buchman VL, Grasing K, progression. BMC Cancer. 2012 Dec 11;12:593 Surguchov A. Protein aggregation in retinal cells and approaches to cell protection. Cell Mol Neurobiol. 2005 Golebiewska U, Guo Y, Khalikaprasad N, Zurawsky C, Sep;25(6):1051-66 Yerramilli VS, Scarlata S. γ-Synuclein interacts with phospholipase C β2 to modulate G protein activation. PLoS Surgucheva I, Park BC, Yue BY, Tomarev S, Surguchov A. One. 2012;7(8):e41067 Interaction of myocilin with gamma-synuclein affects its secretion and aggregation. Cell Mol Neurobiol. 2005 Han S, She F, Wang D, Yao X, Jiang L, Chen Y. SNCG Sep;25(6):1009-33 gene silencing in gallbladder cancer cells inhibits key tumorigenic activities. Front Biosci (Landmark Ed). 2012 Czekierdowski A, Czekierdowska S, Wielgos M, Smolen A, Jan 1;17:1589-98 Kaminski P, Kotarski J. The role of CpG islands hypomethylation and abnormal expression of neuronal Liu C, Ma H, Qu L, Wu J, Meng L, Shou C. Elevated serum protein synuclein-gamma (SNCG) in ovarian cancer. synuclein-gamma in patients with gastrointestinal and Neuro Endocrinol Lett. 2006 Jun;27(3):381-6 esophageal carcinomas. Hepatogastroenterology. 2012 Oct;59(119):2222-7 Sung YH, Eliezer D. Secondary structure and dynamics of micelle bound beta- and gamma-synuclein. Protein Sci. Mhawech-Fauceglia P, Wang D, Syriac S, Godoy H, 2006 May;15(5):1162-74 Dupont N, Liu S, Odunsi K. Synuclein-γ (SNCG) protein expression is associated with poor outcome in endometrial Zhao W, Liu H, Liu W, Wu Y, Chen W, Jiang B, Zhou Y, adenocarcinoma. Gynecol Oncol. 2012 Jan;124(1):148-52 Xue R, Luo C, Wang L, Jiang JD, Liu J. Abnormal activation of the synuclein-gamma gene in hepatocellular Ninkina N, Peters OM, Connor-Robson N, Lytkina O, carcinomas by epigenetic alteration. Int J Oncol. 2006 Sharfeddin E, Buchman VL. Contrasting effects of α- May;28(5):1081-8 synuclein and γ-synuclein on the phenotype of cysteine string protein α (CSP α) null mutant mice suggest distinct Guo J, Shou C, Meng L, Jiang B, Dong B, Yao L, Xie Y, function of these proteins in neuronal synapses. J Biol Zhang J, Chen Y, Budman DR, Shi YE. Neuronal protein Chem. 2012 Dec 28;287(53):44471-7 synuclein gamma predicts poor clinical outcome in breast cancer. Int J Cancer. 2007 Sep 15;121(6):1296-305 Peters OM, Millership S, Shelkovnikova TA, Soto I, Keeling L, Hann A, Marsh-Armstrong N, Buchman VL, Ninkina N. Liu H, Zhou Y, Boggs SE, Belinsky SA, Liu J. Cigarette Selective pattern of motor system damage in gamma- smoke induces demethylation of prometastatic oncogene synuclein transgenic mice mirrors the respective pathology synuclein-gamma in lung cancer cells by downregulation of in amyotrophic lateral sclerosis. Neurobiol Dis. 2012 DNMT3B. Oncogene. 2007 Aug 30;26(40):5900-10 Oct;48(1):124-31 Wu K, Quan Z, Weng Z, Li F, Zhang Y, Yao X, Chen Y, Surgucheva I, Sharov VS, Surguchov A. γ-Synuclein: Budman D, Goldberg ID, Shi YE. Expression of neuronal seeding of α-synuclein aggregation and transmission protein synuclein gamma gene as a novel marker for between cells. Biochemistry. 2012 Jun 12;51(23):4743-54 breast cancer prognosis. Breast Cancer Res Treat. 2007 Mar;101(3):259-67 Millership S, Ninkina N, Rochford JJ, Buchman VL. γ- synuclein is a novel player in the control of body lipid Surgucheva I, Shestopalov VI, Surguchov A. Effect of metabolism. Adipocyte. 2013 Oct 1;2(4):276-80 gamma-synuclein silencing on apoptotic pathways in retinal ganglion cells. J Biol Chem. 2008 Dec Surgucheva I, Gunewardena S, Rao HS, Surguchov A. 26;283(52):36377-85 Cell-specific post-transcriptional regulation of γ-synuclein gene by micro-RNAs. PLoS One. 2013;8(9):e73786 Surgucheva I, Surguchov A. Gamma-synuclein: cell-type- specific promoter activity and binding to transcription This article should be referenced as such: factors. J Mol Neurosci. 2008 Jul;35(3):267-71 Surguchov A. SNCG (synuclein, gamma (breast cancer- Surgucheva I, Weisman AD, Goldberg JL, Shnyra A, specific protein 1)). Atlas Genet Cytogenet Oncol Surguchov A. Gamma-synuclein as a marker of retinal Haematol. 2015; 19(3):210-216.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 216

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Gene Section Review

TWIST2 (twist family bHLH transcription factor 2) Daniela Gasparotto, Erica Lorenzetto Experimental Oncology 1, (CRO) National Cancer Institute, Aviano 33081, Italy (DG, EL)

Published in Atlas Database: June 2014 Online updated version : http://AtlasGeneticsOncology.org/Genes/TWIST2ID43639ch2q37.html DOI: 10.4267/2042/56415 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract DNA/RNA Review on TWIST2, with data on DNA/RNA, on Description the protein encoded and where the gene is implicated. This gene is localized at chromosome 2q37.3 (Perrin-Schmitt et al., 1997) and has 2 exons (Šošic Identity et al., 2003). Other names: DERMO1, FFDD3, SETLSS, Transcription bHLHa39 TWIST2 DNA sequence contains 2 exons separated HGNC (Hugo): TWIST2 by a large intron, with the entire coding region located in exon 1 (Šošic et al., 2003). Two Location: 2q37.3 transcript variants encoding the same protein have Note been described: variant 1 of 1434 bp and variant 2 The gene maps on the long arm of Chromosome 2 of 1186 bp. Both variants encode a same protein but at position q37.3 (Perrin-Schmitt et al., 1997). have different 3' UTR (NCBI).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 217 TWIST2 (twist family bHLH transcription factor 2) Gasparotto D, Lorenzetto E

Structure of TWIST2 protein. The protein domains and their length (indicated by number of limiting residues) are reported. TWIST2 contains 2 nuclear localization signals, a basic helix loop helix (bHLH) domain and a TWIST box domain (Modified from Gong and Li, 2002).

Pseudogene Function No pseudogene of TWIST2 is known. TWIST2 is a transcription factor belonging to the bHLH (basic helix loop helix) family (Li et al., Protein 1995; Lee et al., 2000). bHLH proteins form homo- or heterodimers with other bHLH family members Description and, as dimers, bind DNA through the bHLH The TWIST2 gene encodes a 160 aminoacid protein domain at cis regulatory motifs, termed E-box with a predicted molecular weight of 19 kDa (Li et (CANNTG), leading to either activation or al., 1995; Lee et al., 2000; Gong and Li, 2002). inhibition of transcription (Murre et al., 1989; TWIST2 structure includes 2 nuclear localization Franco et al., 2011b). The specificity of the dimers signals at the N-terminus (aa 31-34, aa 49-53), a depends on several factors: partner choice, basic helix-loop-helix (bHLH) domain (aa 66-117) phosphorylation, other protein-protein interactions and a C-terminal TWIST box region (aa 141-160) and spatial-temporal expression (Murre et al., 1989; (Gong and Li, 2002). Franco et al., 2011b). The partner choice depends Post transcriptional phosphorylation at Ser55 is mainly on the availability and on the predicted for both human and rodent protein phosphorylation status of the partner (Firulli and (phosphosite). The bHLH domain includes highly Conway, 2008). conserved Thr and Ser residues whose TWIST2, similar to TWIST1, may also control posphorylation has been demonstrated to affect gene expression by epigenetic modulation of the dimer formation and DNA binding affinity in other promoter: TWIST1-2 interacts with and recruits TWIST family members (Firulli et al., 2005; Firulli histone acetyl transferases/histone deacetylases to and Conway, 2008). the promoter regulatory region, resulting in transcriptional repression through histone Expression modification (Hamamori et al., 1999; Gong and Li, TWIST2 is involved in mesodermal patterning and 2002; Lee et al., 2003). in differentiation of multiple cell lineages including Role in embryonic development muscle, cartilage, osteogenic, adipogenic and Murine Twist2 was identified through yeast-two- myeloid cells (Lee et al., 2000; Gong and Li, 2002; hybrid screen (Staudinger et al., 1993) and it was Lee et al., 2003; Sharabi et al., 2008). In human named Dermo1 for its expression pattern in the embryonic tissues TWIST2 protein is detectable in dermis of mouse embryo (Li et al., 1995). Dermo1 early chondroblast cells in cartilage plate and was later renamed Twist2 (Šošic et al., 2003) based surrounding mesenchymal cells, especially near the on its high homology and overlapping expression tip of the digits (Lee et al., 2000). In the skin, it is pattern with Twist1 (Li et al., 1995; Lee et al., expressed early in the undifferentiated 2000; ŠoŠic et al., 2003). During mouse mesenchymal layer beneath the epidermis that will embryogenesis Twist2 displays a spatial expression develop into dermis (Lee et al., 2000). TWIST2 is pattern similar to Twist1, but temporally delayed expressed in myeloid progenitors (Sharabi et al., (Li et al., 1995; Lee et al., 2000). Twist2 is 2008). In adult tissues, TWIST2 is expressed in the expressed at a lower level in the sclerotome and bone marrow (Ishikawa et al., 2013). A low level of dermatome of the somites, and in the limb buds at expression has been reported in glands and tubules Day 10.5, and accumulates predominantly in the (Lee et al., 2000) as well as liver, muscle, pancreas, dermatome, prevertebrae, and the derivatives of the and adipose tissue (Pettersson et al., 2010). It branchial arches by Day 13.5 (Li et al., 1995). As should be pointed out that many antibodies used to differentiation of prechondrial cells proceeds, detect the expression of TWIST proteins recognize Twist2 becomes restricted to the perichondrium (Li both members of the family, making it hard to et al., 1995). In the dermis, expression increases actually assess the specific contribution of each continuously through Day 17.5, is detectable in protein. newborns but is then downregulated in adult tissues Localisation (Li et al., 1995). An analogous role has been reported for Twist2 in avian limb outgrowth and Nuclear in embryonic tissues, predominantly patterning (Wade et al., 2012) as well as skin cytoplasmic in adult tissues (Lee et al., 2000). development (Scaal et al., 2001).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 218 TWIST2 (twist family bHLH transcription factor 2) Gasparotto D, Lorenzetto E

TWIST2 plays an important role in early bone Role in hematopoiesis and inflammation development, acting as a negative regulator of TWIST2 is constitutionally expressed in myeloid osteoblast differentiation. Indeed, TWIST2 progenitors where negatively controls myeloid represses osteoblast maturation maintaining cells in lineage differentiation into macrophages, a preosteoblast phenotype (Tamura and Noda, neutrophils and basophils (Sharabi et al., 2008). 1999; Lee et al., 2000) and it inhibits bone specific TWIST2 regulates the function of mature myeloid gene expression (Zhang et al., 2008). TWIST2 is cells by promoting the expression of the anti- involved in the modulation of the activity of Runx2, inflammatory cytokine interleukin-10 and inhibiting a master regulator of osteogenic program, both at the production of pro-inflammatory cytokines the transcriptional level and at the protein level interleukin-12 and interferon-γ (Sharabi et al., (Bialek et al., 2004; Zhang et al., 2008). By 2008). TWIST2 expression is also induced by NF- interacting with Runx2, TWIST2 inhibits the kB, as part of a negative feedback loop in which expression of Runx2 downstream targets, thus cytokines activate NF-kB and downstream blocking the osteogenesis program (Lee et al., activation of TWIST2 results in repression of NF- 2003). Runx2 can be downregulated by ERK, kB activation (Šošic et al., 2003). therefore ERK cooperates with TWIST2 in the In T lymphocytes and in immature thymocytes inhibition of osteoblast differentiation (Nakamura TWIST2 inhibits galectin-1-induced apoptosis, a et al., 2010). Insulin receptor signaling also controls process implicated in thymic negative selection osteoblast development by suppressing TWIST2 (Koh et al., 2008; Koh et al., 2009; Merindol et al., (Fulzele et al., 2010; Ulrich et al., 2013). In 2014). TWIST2 prevents NF-kB mediated osteoblast TWIST2 physically interacts with ATF4 expression of galectin receptor (CD7), therefore affecting its DNA binding function (Danciu et al., inhibiting galectin1/CD7-mediated apoptosis (Koh 2012). During cranial cell development TWIST2 is et al., 2008; Koh et al., 2009; Merindol et al., a mediator of Wnt signaling in specifying dermal 2014). cell fate and suppressing the cartilage cell fate Role in apoptosis and senescence (Tran et al., 2010). TWIST2 antagonizes oncogene-induced cell TWIST2 is furthermore involved in the inhibition failsafe programs, apoptosis and senescence. In a of muscle differentiation program acting as a genetic screen for antiapoptotic proteins, TWIST2 transcriptional repressor of MyoD, the activation of was demonstrated to protect cells from Myc and which typically requires the binding of MEF2 E1A-induced apoptosis by modulating the (Gong and Li, 2002). TWIST2 needs histone ARF/MDM2/p53 pathway (Maestro et al., 1999). deacetylases (HDAC) to exert Myo-MEF2 TWIST2 overcomes oncogene-induced premature inhibition (Gong and Li, 2002). TWIST2 is also a senescence by abrogating the transcription of critical regulator of adipose tissue homeostasis p16Ink4A and p21Cip1, key regulators of the p53- acting as a physical inhibitor of the transcription and RB-dependent pathways (Ansieau et al., 2008). factor ADD1/SREBP1c, involved in adipocyte Role in oxidative stress differentiation (Lee et al., 2003). TWIST2 participates in the control of reactive TWIST2 is required for normal corneal keratocyte oxygen species (ROS) and in cellular response to proliferation and eyelid morphogenesis. Loss of oxidative stress. TWIST2 lowers the level of TWIST2 leads to corneal thinning, due to early intracellular ROS and displays an antioxidant cessation of proliferation of corneal stromal activity in several cell types. TWIST-driven progenitors, resulting in fewer mature stromal inhibition of oxidative stress is involved in the keratocytes for the production of the corneal matrix protection of cells against c-Myc induced apoptosis (Weaving et al., 2010). (Floc'h et al., 2013). In myeloid lineage development, TWIST2 inhibits Role in stemness (cancer stem cells) the proliferation and the differentiation of TWIST2 mediates mesenchymal stem cell (MSC) granulocyte macrophage progenitors (Sharabi et al., self-renewal by maintaining the immature 2008). TWIST2-null 129/Sv mice undergo almost phenotype of human MSC and inhibiting normal embryonic development but die within the osteogenesis and chondrogenesis (Isenmann et al., first two weeks after birth due to cachexia and high 2009). In mouse, the inhibition of MSC levels of proinflammatory cytokines (Šošic et al., differentiation induced by fibroblast growth factor 2 2003). However, the phenotype is influenced by (Fgf2) is strongly correlated with the upregulation genetic background. In fact TWIST2-null mice in of TWIST2 and Spry4 and with the suppression of the 129/C57 mixed background survive with only a Erk1/2 activation (Lai et al., 2011). TWIST2- mild disease and facial signs reminiscent of facial expressing cells exhibit an increased expression of dermal dysplasia (Setleis syndrome), the human stem cell markers Bmi-1, Sox2, CD24, Nanog and disease associated with germline TWIST2 an increased capacity of self-renewal (Liu et al., inactivating mutations (Tukel et al., 2010). 2014).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 219 TWIST2 (twist family bHLH transcription factor 2) Gasparotto D, Lorenzetto E

Role in EMT syndrome), a developmental defect characterized by bitemporal or pre auricolar facial dermal dysplasia TWIST2 has been involved in epithelial- (Tukel et al., 2010; Cervantes-Barragán et al., 2011; mesenchymal transition (EMT), a process by which Franco et al., 2011a; Girisha et al., 2014). cells lose epithelial characteristics and acquire a migratory, mesenchymal phenotype. EMT is Somatic associated with downregulation of epithelial Missense mutations have been found in endometrial markers, such as E-cadherin, and induction of cancer (COSMIC). mesenchymal markers, such as N-cadherin. EMT has an essential role in embryonic development and Implicated in in the initial steps of tumor invasion and metastasis. Several bHLH transcription factors are involved in Breast cancer the regulation of EMT. TWIST2 has been Note implicated in EMT in several experimental models TWIST2 overexpression has been implicated in and cancer types (Tsuji et al., 2008; Katoh and breast cancer. TWIST2 protein was reported to be Katoh, 2009; Ponnusamy et al., 2010; Fang et al., expressed in 94% of breast cancers and in 100% of 2011; Akkari et al., 2012; Amatangelo et al., 2012; nodal metastases. In mammary cell lines TWIST2 Salnikov et al., 2012; Shimoda et al., 2012; Mao et ectopic overexpression induced EMT, enhanced al., 2012; Mao et al., 2013; Teng and Li, 2014; cell migration, colony formation, and tumor growth Díaz-Martín et al., 2014). A dynamic interaction in vivo. between EMT and stemness gene programs has TWIST2 induced the expression of stem cell been observed in prostate and bladder cancer cell markers thereby increasing the abundance of a experimental models (Celià-Terrassa et al., 2012). CD44high/CD24low subpopulation with stem-like self renewal capacities (Fang et al., 2011). Mao et al. (2012) observed TWIST2 expression, most frequently in ductal and in squamous cell breast carcinomas. Expression pattern was cytoplasmic in cells localized in tumor centre and in lymph node metastases, while TWIST2 displayed nuclear expression and associated with EMT features (fibroblast-like morphology, E-cadherin loss) in cells at the invasive front. TWIST2 cytoplasmic positive expression was associated with advanced TNM, advanced clinical stage and metastasis (Mao et al., 2012).

Cervical cancer Homology Note Li et al. (2012) investigated TWIST2 and E- TWIST genes are evolutionary conserved from cadherin protein expression in different stages and jellyfish to human. Duplications and deletions of grades of cervical dysplasia and squamous cell TWIST genes occurred during vertebrate evolution carcinoma (SCC). All SCCs were positive for (Germanguz et al., 2007; Gitelman, 2007). cytoplasmic TWIST2 expression, and in 87.1% The human TWIST2 protein shares 98.8% amino specimens staining were also nuclear. In contrast, acid identity to mouse and rat and 95% to chicken only 7.1% of healthy cervical samples showed Twist2 (Lee et al., 2000). weak/moderate cytoplasmic or nuclear positivity. Within the bHLH protein family, TWIST2 displays TWIST2 positivity was associated with aberrant E- the highest level of sequence similarity with Cadherin cytoplasmic localization. TWIST2 TWIST1. The two proteins are near identical in the cytoplasmic expression was associated with C-terminal Helix-Loop-Helix (HLH) and TWIST significantly increased risk of metastasis (Li et al., BOX domains, while they display some divergence 2012). in the N-terminal region (Lee et al., 2000; Gong and Li, 2002; Barnes and Firulli, 2009). Colorectal cancer Note Mutations TWIST2 expression was demonstrated by immunohistochemistry in 71% of colorectal Germinal cancers. The pattern of staining was mainly TWIST2 nonsense mutations have been associated cytoplasmic and was associated with reduced E- with Focal Facial Dermal Dyspasia type III (Setleis cadherin. Positive TWIST2 expression was

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 220 TWIST2 (twist family bHLH transcription factor 2) Gasparotto D, Lorenzetto E

associated with poor disease-free and overall transcription factor was related to the degree of survival (Yu et al., 2013). promoter methylation. In this study, TWIST2 Long et al. (2013) observed decreased expression of promoter methylation was more frequent in the mir-138 in colorectal cancers compared to healthy IgVH-mutated CLL subset (Raval et al., 2005). In tissues and provided evidence that mir-138 targeted acute lymphoblastic leukemia (ALL) the TWIST2 TWIST2. Indeed, overexpression of TWIST2 was promoter was hypermethylated in 50% of cases. In inversely correlated with mir-138 and associated ALL cell lines the restoration of TWIST2 with metastasis and poor prognosis (Long et al., expression reduced cell growth, induced apoptosis 2013). and increased sensitivity to chemotherapeutic Ovarian cancer agents (Thathia et al., 2012). Note Hepatocellular carcinomas (HCC) Mao et al. (2013) reported overexpression of Note TWIST2 in 16/22 ovarian tumors compared to TWIST2 was found to be overexpressed in human healthy tissue. The high level of TWIST2 was HCCs. Ectopic expression of TWIST2 in HCC cell positively correlated with HIF-1α. In the HO-8910 lines induced EMT, augmented cell migration, ovarian cell line, TWIST2 overexpression protected invasion and colony-forming abilities in vitro and cells from apoptosis under hypoxic condition promoted tumor growth in vivo. through activation of the PI3K/AKT survival Moreover, TWIST2 promoted cancer stem-like cell pathway. In a following study, the same authors self-renewal via upregulation of CD24 expression confirmed TWIST2 overexpression in 59/84 (70%) (Liu et al., 2014). ovarian cancers. The pattern of expression was both TWIST2 was also implicated in the process of nuclear and cytoplasmatic, and the fraction of oncogenic transformation of primary hepatocytes TWIST2-positive cases was increased along with by Hepatitis C virus (HCV). the FIGO stage. In the ovarian cell line SKOV-3 the The viral protein NS5A either alone or in the ectopic expression of TWIST2 induced EMT, β- context of other viral components in the course of catenin nuclear accumulation and activation of the infection, acted through TWIST2 activation to Wnt pathway (Mao et al., 2013). disrupt cell polarity, and, in cooperation with Ras, Head and Neck squamous cell induced cell transformation and metastastatic carcinomas (HNSCC) spread in vivo (Akkari et al., 2012). Note Sarcomas TWIST2 gene expression was increased in 87% Note esophageal squamous cell carcinomas (Ansieau et By investigating the role of TWIST in soft tissue al. 2008). In tongue squamous cell carcinomas sarcomas, Piccinin et al. (2012) demonstrated overexpression of TWIST2 was detected in 45% of overexpression of TWIST1 and TWIST2 proteins tumors and was associated with hypoxia. The in 60% and in 7% of tumors, respectively. concomitant expression of more than two of the TWIST1/2 contributed to transformation of primary markers TWIST2, HIF-1α, and SNIP1 was mesenchymal cells in vitro and to tumor growth in associated with poor survival (Liang et al., 2011). mice by antagonizing p53. In salivary adenoid cystic carcinoma the By directly interacting with p53, TWIST1/2 coexpression of TWIST2, HIF-2α and SIP1 was hindered p53 phosphorylation at Ser 392, thereby associated with perineural invasion, local facilitating MDM2-mediated p53 degradation recurrence, distant metastasis and shorter survival (Piccinin et al., 2012). (Zhou et al., 2012). In osteosarcoma TWIST2 demonstrated a tumor In HNSCCs TWIST2 overexpression was suppressive role (Ishikawa et al., 2013). In human correlated with high tumor grade and short survival osteosarcomas TWIST2 mRNA level was in oral cavity/pharynx cancer patients. In these downregulated compared with bone marrow stem tumors, TWIST2 expression was not associated cells. with EMT markers. Instead, within the N-positive In a murine model, TWIST2 correlated inversely group, identified subset of tumors with high risk of with tumorigenic potential. The tumor suppressor progression (Gasparotto et al., 2011). activity of TWIST2 was correlated with the Leukemias inhibition of the formation of a microenvironment favourable for tumor growth (Ishikawa et al., 2013). Note In leukemias TWIST2 has oncosuppressive role. Melanomas Raval et al. (2005) reported epigenetic silencing of Note TWIST2 in chronic lymphocytic leukemia (CLL), TWIST2 expression was significantly increased in and provided evidence that expression of this melanomas (60%) compared to their normal

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 221 TWIST2 (twist family bHLH transcription factor 2) Gasparotto D, Lorenzetto E

counterparts, as well as in melanoma cell lines Hamamori Y, Sartorelli V, Ogryzko V, Puri PL, Wu HY, (Ansieau et al., 2008). Wang JY, Nakatani Y, Kedes L. Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein TWIST1/2 inhibited premature senescence by twist and adenoviral oncoprotein E1A. Cell. 1999 Feb abrogating p16 INK4A and p21 cip activation and 5;96(3):405-13 promoted EMT in cooperation with mitogenic Maestro R, Dei Tos AP, Hamamori Y, Krasnokutsky S, signaling activation (Ansieau et al., 2008). Sartorelli V, Kedes L, Doglioni C, Beach DH, Hannon GJ. Twist is a potential oncogene that inhibits apoptosis. Focal facial dermal dysplasia type III, Genes Dev. 1999 Sep 1;13(17):2207-17 or Setleis syndrome Tamura M, Noda M. Identification of DERMO-1 as a Note member of helix-loop-helix type transcription factors Focal facial dermal dysplasia type III, or Setleis expressed in osteoblastic cells. J Cell Biochem. 1999 Feb 1;72(2):167-76 syndrome (OMIM entry 607556). The disease was first described in consanguineous patients in Puerto Lee MS, Lowe G, Flanagan S, Kuchler K, Glackin CA. Rican families (Setleis et al., 1963). Human Dermo-1 has attributes similar to twist in early bone development. Bone. 2000 Nov;27(5):591-602 Patients exhibit bilateral temporal preauricular marks and other facial abnormalities, including Scaal M, Füchtbauer EM, Brand-Saberi B. cDermo-1 expression indicates a role in avian skin development. absent eyelashes on both lids or multiple rows on Anat Embryol (Berl). 2001 Jan;203(1):1-7 the upper lids, slanted eyebrows chin clefting, and other nonfacial manifestations (Tukel et al., 2010, Gong XQ, Li L. Dermo-1, a multifunctional basic helix-loop- helix protein, represses MyoD transactivation via the HLH Cervantes-Barragán et al., 2011). domain, MEF2 interaction, and chromatin deacetylation. J The disease is panethnic (Tukel et al., 2010). The Biol Chem. 2002 Apr 5;277(14):12310-7 mode of inheritance of Setleis syndrome has been Lee YS, Lee HH, Park J, Yoo EJ, Glackin CA, Choi YI, reported as autosomal recessive or autosomal Jeon SH, Seong RH, Park SD, Kim JB. Twist2, a novel dominant with decreased manifestations in ADD1/SREBP1c interacting protein, represses the heterozygotes (Tukel et al., 2010, Cervantes- transcriptional activity of ADD1/SREBP1c. Nucleic Acids Barragán et al., 2011). Res. 2003 Dec 15;31(24):7165-74 Šoši ć D, Richardson JA, Yu K, Ornitz DM, Olson EN. Twist 2q37 deletion syndrome regulates cytokine gene expression through a negative Note feedback loop that represses NF-kappaB activity. Cell. 2003 Jan 24;112(2):169-80 TWIST2 has been proposed as candidate gene of the 2q37 deletion syndrome due to its localization Bialek P, Kern B, Yang X, Schrock M, Sosic D, Hong N, in the smallest deleted chromosome region (Leroy Wu H, Yu K, Ornitz DM, Olson EN, Justice MJ, Karsenty G. A twist code determines the onset of osteoblast et al., 2013). differentiation. Dev Cell. 2004 Mar;6(3):423-35 Disease Firulli BA, Krawchuk D, Centonze VE, Vargesson N, Patients exhibit facial dysmorphism and Virshup DM, Conway SJ, Cserjesi P, Laufer E, Firulli AB. brachydactyly, behavioural problems, autism of Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities. Nat varying severity and overweight or obesity (Leroy Genet. 2005 Apr;37(4):373-81 et al., 2013). Raval A, Lucas DM, Matkovic JJ, Bennett KL, Liyanarachchi S, Young DC, Rassenti L, Kipps TJ, Grever References MR, Byrd JC, Plass C. TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain SETLEIS H, KRAMER B, VALCARCEL M, EINHORN AH. mutated and unmutated chronic lymphocytic leukemia. J CONGENITAL ECTODERMAL DYSPLASIA OF THE Clin Oncol. 2005 Jun 10;23(17):3877-85 FACE. Pediatrics. 1963 Oct;32:540-8 Germanguz I, Lev D, Waisman T, Kim CH, Gitelman I. Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan Four twist genes in zebrafish, four expression patterns. YN, Cabrera CV, Buskin JN, Hauschka SD, Lassar AB. Dev Dyn. 2007 Sep;236(9):2615-26 Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common Gitelman I. Evolution of the vertebrate twist family and DNA sequence. Cell. 1989 Aug 11;58(3):537-44 synfunctionalization: a mechanism for differential gene loss through merging of expression domains. Mol Biol Evol. Staudinger J, Perry M, Elledge SJ, Olson EN. Interactions 2007 Sep;24(9):1912-25 among vertebrate helix-loop-helix proteins in yeast using the two-hybrid system. J Biol Chem. 1993 Mar Ansieau S, Bastid J, Doreau A, Morel AP, Bouchet BP, 5;268(7):4608-11 Thomas C, Fauvet F, Puisieux I, Doglioni C, Piccinin S, Maestro R, Voeltzel T, Selmi A, Valsesia-Wittmann S, Li L, Cserjesi P, Olson EN. Dermo-1: a novel twist-related Caron de Fromentel C, Puisieux A. Induction of EMT by bHLH protein expressed in the developing dermis. Dev twist proteins as a collateral effect of tumor-promoting Biol. 1995 Nov;172(1):280-92 inactivation of premature senescence. Cancer Cell. 2008 Perrin-Schmitt F, Bolcato-Bellemin AL, Bourgeois P, Jul 8;14(1):79-89 Stoetzel C, Danse JM. The locations of the H-twist and H- Firulli AB, Conway SJ. Phosphoregulation of Twist1 dermo-1 genes are distinct on the human genome. provides a mechanism of cell fate control. Curr Med Chem. Biochim Biophys Acta. 1997 Feb 27;1360(1):1-2 2008;15(25):2641-7

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 222 TWIST2 (twist family bHLH transcription factor 2) Gasparotto D, Lorenzetto E

Koh HS, Lee C, Lee KS, Ham CS, Seong RH, Kim SS, Weaving L, Mihelec M, Storen R, Sosic D, Grigg JR, Tam Jeon SH. CD7 expression and galectin-1-induced PP, Jamieson RV. Twist2: role in corneal stromal apoptosis of immature thymocytes are directly regulated by keratocyte proliferation and corneal thickness. Invest NF-kappaB upon T-cell activation. Biochem Biophys Res Ophthalmol Vis Sci. 2010 Nov;51(11):5561-70 Commun. 2008 May 23;370(1):149-53 Cervantes-Barragán DE, Villarroel CE, Medrano- Sharabi AB, Aldrich M, Sosic D, Olson EN, Friedman AD, Hernández A, Durán-McKinster C, Bosch-Canto V, Del- Lee SH, Chen SY. Twist-2 controls myeloid lineage Castillo V, Nazarenko I, Yang A, Desnick RJ. Setleis development and function. PLoS Biol. 2008 Dec syndrome in Mexican-Nahua sibs due to a homozygous 16;6(12):e316 TWIST2 frameshift mutation and partial expression in heterozygotes: review of the focal facial dermal dysplasias Tsuji T, Ibaragi S, Shima K, Hu MG, Katsurano M, Sasaki and subtype reclassification. J Med Genet. 2011 A, Hu GF. Epithelial-mesenchymal transition induced by Oct;48(10):716-20 growth suppressor p12CDK2-AP1 promotes tumor cell local invasion but suppresses distant colony growth. Fang X, Cai Y, Liu J, Wang Z, Wu Q, Zhang Z, Yang CJ, Cancer Res. 2008 Dec 15;68(24):10377-86 Yuan L, Ouyang G. Twist2 contributes to breast cancer progression by promoting an epithelial-mesenchymal Zhang Y, Hassan MQ, Li ZY, Stein JL, Lian JB, van Wijnen transition and cancer stem-like cell self-renewal. AJ, Stein GS. Intricate gene regulatory networks of helix- Oncogene. 2011 Nov 24;30(47):4707-20 loop-helix (HLH) proteins support regulation of bone-tissue related genes during osteoblast differentiation. J Cell Franco HL, Casasnovas JJ, Leon RG, Friesel R, Ge Y, Biochem. 2008 Oct 1;105(2):487-96 Desnick RJ, Cadilla CL. Nonsense mutations of the bHLH transcription factor TWIST2 found in Setleis Syndrome Barnes RM, Firulli AB. A twist of insight - the role of Twist- patients cause dysregulation of periostin. Int J Biochem family bHLH factors in development. Int J Dev Biol. Cell Biol. 2011a Oct;43(10):1523-31 2009;53(7):909-24 Franco HL, Casasnovas J, Rodríguez-Medina JR, Cadilla Isenmann S, Arthur A, Zannettino AC, Turner JL, Shi S, CL. Redundant or separate entities?--roles of Twist1 and Glackin CA, Gronthos S. TWIST family of basic helix-loop- Twist2 as molecular switches during gene transcription. helix transcription factors mediate human mesenchymal Nucleic Acids Res. 2011b Mar;39(4):1177-86 stem cell growth and commitment. Stem Cells. 2009 Oct;27(10):2457-68 Fu J, Qin L, He T, Qin J, Hong J, Wong J, Liao L, Xu J. The TWIST/Mi2/NuRD protein complex and its essential Katoh M, Katoh M. Integrative genomic analyses of ZEB2: role in cancer metastasis. Cell Res. 2011 Feb;21(2):275- Transcriptional regulation of ZEB2 based on SMADs, 89 ETS1, HIF1alpha, POU/OCT, and NF-kappaB. Int J Oncol. 2009 Jun;34(6):1737-42 Gasparotto D, Polesel J, Marzotto A, Colladel R, Piccinin S, Modena P, Grizzo A, Sulfaro S, Serraino D, Barzan L, Koh HS, Lee C, Lee KS, Park EJ, Seong RH, Hong S, Doglioni C, Maestro R. Overexpression of TWIST2 Jeon SH. Twist2 regulates CD7 expression and galectin-1- correlates with poor prognosis in head and neck squamous induced apoptosis in mature T-cells. Mol Cells. 2009 Dec cell carcinomas. Oncotarget. 2011 Dec;2(12):1165-75 31;28(6):553-8 Lai WT, Krishnappa V, Phinney DG. Fibroblast growth Fulzele K, Riddle RC, DiGirolamo DJ, Cao X, Wan C, factor 2 (Fgf2) inhibits differentiation of mesenchymal stem Chen D, Faugere MC, Aja S, Hussain MA, Brüning JC, cells by inducing Twist2 and Spry4, blocking extracellular Clemens TL. Insulin receptor signaling in osteoblasts regulated kinase activation, and altering Fgf receptor regulates postnatal bone acquisition and body expression levels. Stem Cells. 2011 Jul;29(7):1102-11 composition. Cell. 2010 Jul 23;142(2):309-19 Liang X, Zheng M, Jiang J, Zhu G, Yang J, Tang Y. Nakamura T, Toita H, Yoshimoto A, Nishimura D, Takagi Hypoxia-inducible factor-1 alpha, in association with T, Ogawa T, Takeya T, Ishida-Kitagawa N. Potential TWIST2 and SNIP1, is a critical prognostic factor in involvement of Twist2 and Erk in the regulation of patients with tongue squamous cell carcinoma. Oral Oncol. osteoblastogenesis by HB-EGF-EGFR signaling. Cell 2011 Feb;47(2):92-7 Struct Funct. 2010;35(1):53-61 Akkari L, Grégoire D, Floc'h N, Moreau M, Hernandez C, Pettersson AT, Laurencikiene J, Mejhert N, Näslund E, Simonin Y, Rosenberg AR, Lassus P, Hibner U. Hepatitis Bouloumié A, Dahlman I, Arner P, Rydén M. A possible C viral protein NS5A induces EMT and participates in inflammatory role of twist1 in human white adipocytes. oncogenic transformation of primary hepatocyte Diabetes. 2010 Mar;59(3):564-71 precursors. J Hepatol. 2012 Nov;57(5):1021-8 Ponnusamy MP, Lakshmanan I, Jain M, Das S, Amatangelo MD, Goodyear S, Varma D, Stearns ME. c- Chakraborty S, Dey P, Batra SK. MUC4 mucin-induced Myc expression and MEK1-induced Erk2 nuclear epithelial to mesenchymal transition: a novel mechanism localization are required for TGF-beta induced epithelial- for metastasis of human ovarian cancer cells. Oncogene. mesenchymal transition and invasion in prostate cancer. 2010 Oct 21;29(42):5741-54 Carcinogenesis. 2012 Oct;33(10):1965-75 Tran TH, Jarrell A, Zentner GE, Welsh A, Brownell I, Danciu TE, Li Y, Koh A, Xiao G, McCauley LK, Franceschi Scacheri PC, Atit R. Role of canonical Wnt signaling/ß- RT. The basic helix loop helix transcription factor Twist1 is catenin via Dermo1 in cranial dermal cell development. a novel regulator of ATF4 in osteoblasts. J Cell Biochem. Development. 2010 Dec;137(23):3973-84 2012 Jan;113(1):70-9 Tukel T, Šoši ć D, Al-Gazali LI, Erazo M, Casasnovas J, Franco HL, Richardson JA, Olson EN, Cadilla CL, Desnick Li Y, Wang W, Wang W, Yang R, Wang RJ. Homozygous nonsense mutations in TWIST2 cause T, Su T, Weng D, Tao T, Li W, Ma D, Setleis syndrome. Am J Hum Genet. 2010 Aug 13;87(2):289-96 Wang S. Correlation of TWIST2 upregulation and epithelial-mesenchymal

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 223 TWIST2 (twist family bHLH transcription factor 2) Gasparotto D, Lorenzetto E

transition during tumorigenesis and Leroy C, Landais E, Briault S, David A, Tassy O, Gruchy N, Delobel B, Grégoire MJ, Leheup B, Taine L, Lacombe progression of cervical carcinoma. D, Delrue MA, Toutain A, Paubel A, Mugneret F, Thauvin- Gynecol Oncol. 2012 Jan;124(1):112-8 Robinet C, Arpin S, Le Caignec C, Jonveaux P, Beri M, Leporrier N, Motte J, Fiquet C, Brichet O, Mozelle-Nivoix Mao Y, Zhang N, Xu J, Ding Z, Zong R, Liu Z. Significance M, Sabouraud P, Golovkine N, Bednarek N, Gaillard D, of heterogeneous Twist2 expression in human breast Doco-Fenzy M. The 2q37-deletion syndrome: an update of cancers. PLoS One. 2012;7(10):e48178 the clinical spectrum including overweight, brachydactyly Piccinin S, Tonin E, Sessa S, Demontis S, Rossi S, and behavioural features in 14 new patients. Eur J Hum Pecciarini L, Zanatta L, Pivetta F, Grizzo A, Sonego M, Genet. 2013 Jun;21(6):602-12 Rosano C, Dei Tos AP, Doglioni C, Maestro R. A "twist Long L, Huang G, Zhu H, Guo Y, Liu Y, Huo J. Down- box" code of p53 inactivation: twist box: p53 interaction regulation of miR-138 promotes colorectal cancer promotes p53 degradation. Cancer Cell. 2012 Sep metastasis via directly targeting TWIST2. J Transl Med. 11;22(3):404-15 2013 Oct 30;11:275 Salnikov AV, Liu L, Platen M, Gladkich J, Salnikova O, Mao Y, Xu J, Li Z, Zhang N, Yin H, Liu Z. The role of Ryschich E, Mattern J, Moldenhauer G, Werner J, nuclear β-catenin accumulation in the Twist2-induced Schemmer P, Büchler MW, Herr I. Hypoxia induces EMT ovarian cancer EMT. PLoS One. 2013;8(11):e78200 in low and highly aggressive pancreatic tumor cells but only cells with cancer stem cell characteristics acquire Ulrich C, Rolauffs B, Abele H, Bonin M, Nieselt K, Hart ML, pronounced migratory potential. PLoS One. Aicher WK. Low osteogenic differentiation potential of 2012;7(9):e46391 placenta-derived mesenchymal stromal cells correlates with low expression of the transcription factors Runx2 and Shimoda M, Sugiura T, Imajyo I, Ishii K, Chigita S, Seki K, Twist2. Stem Cells Dev. 2013 Nov 1;22(21):2859-72 Kobayashi Y, Shirasuna K. The T-box transcription factor Brachyury regulates epithelial-mesenchymal transition in Yu H, Jin GZ, Liu K, Dong H, Yu H, Duan JC, Li Z, Dong association with cancer stem-like cells in adenoid cystic W, Cong WM, Yang JH. Twist2 is a valuable prognostic carcinoma cells. BMC Cancer. 2012 Aug 29;12:377 biomarker for colorectal cancer. World J Gastroenterol. 2013 Apr 21;19(15):2404-11 Thathia SH, Ferguson S, Gautrey HE, van Otterdijk SD, Hili M, Rand V, Moorman AV, Meyer S, Brown R, Díaz-Martín J, Díaz-López A, Moreno-Bueno G, Castilla Strathdee G. Epigenetic inactivation of TWIST2 in acute MÁ, Rosa-Rosa JM, Cano A, Palacios J. A core microRNA lymphoblastic leukemia modulates proliferation, cell signature associated with inducers of the epithelial-to- survival and chemosensitivity. Haematologica. 2012 mesenchymal transition. J Pathol. 2014 Feb;232(3):319-29 Mar;97(3):371-8 Girisha KM, Bidchol AM, Sarpangala MK, Satyamoorthy K. Wade C, Brinas I, Welfare M, Wicking C, Farlie PG. Twist2 A novel Frameshift mutation in TWIST2 gene causing contributes to termination of limb bud outgrowth and Setleis syndrome. Indian J Pediatr. 2014 Mar;81(3):302-4 patterning through direct regulation of Grem1. Dev Biol. 2012 Oct 1;370(1):145-53 Liu AY, Cai Y, Mao Y, Lin Y, Zheng H, Wu T, Huang Y, Fang X, Lin S, Feng Q, Huang Z, Yang T, Luo Q, Ouyang Zhou C, Liu J, Tang Y, Zhu G, Zheng M, Jiang J, Yang J, G. Twist2 promotes self-renewal of liver cancer stem-like Liang X. Coexpression of hypoxia-inducible factor-2α, cells by regulating CD24. Carcinogenesis. 2014 TWIST2, and SIP1 may correlate with invasion and Mar;35(3):537-45 metastasis of salivary adenoid cystic carcinoma. J Oral Pathol Med. 2012 May;41(5):424-31 Merindol N, Riquet A, Szablewski V, Eliaou JF, Puisieux A, Bonnefoy N. The emerging role of Twist proteins in Floc'h N, Kolodziejski J, Akkari L, Simonin Y, Ansieau S, hematopoietic cells and hematological malignancies. Blood Puisieux A, Hibner U, Lassus P. Modulation of oxidative Cancer J. 2014 Apr 25;4:e206 stress by twist oncoproteins. PLoS One. 2013;8(8):e72490 Teng Y, Li X. The roles of HLH transcription factors in Ishikawa T, Shimizu T, Ueki A, Yamaguchi SI, Onishi N, epithelial mesenchymal transition and multiple molecular Sugihara E, Kuninaka S, Miyamoto T, Morioka H, mechanisms. Clin Exp Metastasis. 2014 Mar;31(3):367-77 Nakayama R, Kobayashi E, Toyama Y, Mabuchi Y, Matsuzaki Y, Yamaguchi R, Miyano S, Saya H. Twist2 This article should be referenced as such: functions as a tumor suppressor in murine osteosarcoma cells. Cancer Sci. 2013 Jul;104(7):880-8 Gasparotto D, Lorenzetto E. TWIST2 (twist family bHLH transcription factor 2). Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3):217-224.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 224

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Leukaemia Section Short Communication t(9;17)(p13;p12) PAX5/NCOR1 Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: July 2014 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0917p13p12ID1597.html DOI: 10.4267/2042/56416 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

activates B-cell specific genes and repress genes Abstract involved in other lineage commitments. Activates Review on t(9;17)(p13;p12) PAX5/NCOR1, with the surface cell receptor CD19 and repress FLT3. data on clinics, and the genes implicated. Pax5 physically interacts with the RAG1/RAG2 complex, and removes the inhibitory signal of the Clinics and pathology lysine-9-methylated histone H3, and induces V-to- DJ rearrangements. Genes repressed by PAX5 Disease expression in early B cells are restored in their B-cell acute lymphoblastic leukemia (B-ALL) function in mature B cells and plasma cells, and Epidemiology PAX5 repressed (Fuxa et al., 2004; Johnson et al., 2004; Zhang et al., 2006; Cobaleda et al., 2007; One case to date, a 2-year-old boy with a CD10+ Medvedovic et al., 2011). (B-II, common) ALL (Coyaud et al., 2010). NCOR1 Prognosis Location No data. 17p12 Genes involved and Protein 2440 amino acids; from N-term to C-term, NCOR1 proteins contains: a repression domains (responsible for the PAX5 repressive activity of the corepressor) (aa 1-312); a poly-Gln stretch (aa 58-64), two coiled coil Location domains (aa 174-216 and aa 299-328); a SANT 9p13.2 (Swi3, Ada2, NCoR1, TFIIB) domain (deacetylase Protein activation domain (DAD)) (aa 435-486); a coiled 391 amino acids; from N-term to C-term, PAX5 coil domain (aa 501-557); a poly-Ala stretch (aa contains: a paired domain (aa: 16-142); an 593-603); a Pro-rich stretch (aa 607-617); a SANT octapeptide (aa: 179-186); a partial homeodomain domain (histone interaction domain (HID)) (aa 623- (aa: 228-254); a transactivation domain (aa: 304- 674); a poly-Pro stretch (aa 1032-1035); a poly-Ala 359); and an inhibitory domain (aa: 359-391). stretch (aa 1707-1712); a CoRNR box motif (Leu- Lineage-specific transcription factor; recognizes the x-x-Leu-Leu motifs) (aa 1933-1937); a poly-Ser concensus recognition sequence stretch (aa 1952-1963); a nuclear receptor- GNCCANTGAAGCGTGAC, where N is any interacting domain ID1 (aa 2032-2115); a CoRNR nucleotide. Involved in B-cell differentiation. Entry box motif (aa 2055-2059); another nuclear receptor- of common lymphoid progenitors into the B cell interacting domain ID2 (aa 2212-2273); and a lineage depends on E2A, EBF1, and PAX5; CoRNR box motif (aa 2263-2267).

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 225 t(9;17)(p13;p12) PAX5/NCOR1 Huret JL

PAX5-NCOR1 fusion protein.

NCOR1 and NCOR2 (12q24, also called SMRT) octapeptide of PAX5 (201 aa), and most the ID2 are transcriptional corepressors that associate with region (containing the CoRNR box motif) of nuclear hormone receptors (thyroid hormone NCOR1 (214 aa). receptors and retinoic acid receptors) in the absence of ligand. References Thyroid hormone receptors interact specifically Li H, Leo C, Schroen DJ, Chen JD. Characterization of with the ID3 and ID2 domains of NCOR1 (ID receptor interaction and transcriptional repression by the domains contain an isoleucine-rich domain, named corepressor SMRT. Mol Endocrinol. 1997 the CoRNR box, this CoRNR box is required for Dec;11(13):2025-37 nuclear hormone receptors interaction). SANT-like Fuxa M, Skok J, Souabni A, Salvagiotto G, Roldan E, domains promote histone deacetylation. Repression Busslinger M. Pax5 induces V-to-DJ rearrangements and is mediated by formation of a large corepressor locus contraction of the immunoglobulin heavy-chain gene. complex that contains SIN3A/B (15q24 and Genes Dev. 2004 Feb 15;18(4):411-22 19p13), recruiting multiple histone deacetylase Johnson K, Pflugh DL, Yu D, Hesslein DG, Lin KI, Bothwell enzymes such as HDAC1 (1p35), HDAC2 (6q21), AL, Thomas-Tikhonenko A, Schatz DG, Calame K. B cell- specific loss of histone 3 lysine 9 methylation in the V(H) HDAC3 (5q31), HDAC4 (2q37), HDAC7 (12q13), locus depends on Pax5. Nat Immunol. 2004 Aug;5(8):853- and SIRT1 (10q21), resulting in the formation of 61 repressive chromatin structures. This complex Zhang Z, Espinoza CR, Yu Z, Stephan R, He T, Williams associates with the thyroid hormone receptor and GS, Burrows PD, Hagman J, Feeney AJ, Cooper MD. the retinoic acid receptor. Transcription factor Pax5 (BSAP) transactivates the RAG- NCOR1 and NCOR2 also interact with many other mediated V(H)-to-DJ(H) rearrangement of immunoglobulin transcription factors including: BCL6 (3q27), genes. Nat Immunol. 2006 Jun;7(6):616-24 RUNX1T1 (8q21, also called ETO), SPEN (1p36, Cobaleda C, Schebesta A, Delogu A, Busslinger M. Pax5: also called SHARP), ZBTB33 (Xq24), HEXIM1 the guardian of B cell identity and function. Nat Immunol. (17q21), TBL1XR1 (3q26), MEF2C (5q14), 2007 May;8(5):463-70 CNOT2 (12q15), and RBJP (4p15), Jun proteins Coyaud E, Struski S, Prade N, Familiades J et al.. Wide and the NFKB pathway (Li et al., 1997; Watson et diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogenetique Hematologique study. al., 2012; Mottis et al., 2013). Blood. 2010 Apr 15;115(15):3089-97 Medvedovic J, Ebert A, Tagoh H, Busslinger M. Pax5: a Result of the chromosomal master regulator of B cell development and anomaly leukemogenesis. Adv Immunol. 2011;111:179-206 Watson PJ, Fairall L, Schwabe JW. Nuclear hormone Hybrid gene receptor co-repressors: structure and function. Mol Cell Endocrinol. 2012 Jan 30;348(2):440-9 Description Fusion of PAX5 exon 5 to NCOR1 exon 43. Mottis A, Mouchiroud L, Auwerx J. Emerging roles of the corepressors NCoR1 and SMRT in homeostasis. Genes Fusion protein Dev. 2013 Apr 15;27(8):819-35 Description This article should be referenced as such: 415 amino acids. The predicted fusion protein Huret JL. t(9;17)(p13;p12) PAX5/NCOR1. Atlas Genet contains the DNA binding paired domain, and the Cytogenet Oncol Haematol. 2015; 19(3):225-226.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 226

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Leukaemia Section Short Communication t(11;14)(p15;q22) AP2A2/NID2 Nathalie Douet-Guilbert, Etienne De Braekeleer, Corinne Tous, Nadia Guéganic, Audrey Basinko, Marie-Josée Le Bris, Frédéric Morel, Marc De Braekeleer Cytogenetics Laboratory, Faculty of Medicine, University of Brest, France (NDG, EDB, CT, NG, AB, MJLB, FM, MDB)

Published in Atlas Database: August 2014 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t1114p15q22ID1677.html DOI: 10.4267/2042/56417 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Review on t(11;14)(p15;q22) AP2A2/NID2, with data on clinics, and the genes implicated. Clinics and pathology Disease Myelodysplastic syndrome Epidemiology A single case of Philadelphia chromosome-positive chronic myeloid leukemia with t(11;14)(p15;q22) following allogeneic stem cell transplantation in a 32 year-old male is reported in the literature. The translocation is associated with a complex karyotype. No molecular characterization was RHG banding showing chromosomes 11 and 14 and the performed (Karrman et al., 2007). derivatives der(11) and der(14). Clinics Cytogenetics morphological A 71-year-old woman seen because of macrocytic t(11;14)(p15;q22) is identified by banding anemia without etiology. cytogenetics. Cytology Cytogenetics molecular Bone marrow aspirate showing dysgranulopoiesis To determine the position of the breakpoints on and dyserythropoiesis. chromosomes 11 and 14, BACs located in the Presence of ring sideroblasts (24%) signing a bands of interest were used as probes in FISH refractory anemia with ring sideroblasts (RARS). experiments. Evolution Analysis with RP11-51L17 showed that one signal Patient alive two years later. hybridized to the normal chromosome 11, and the other hybridized to the der(14). Cytogenetics Analysis with RP11-963I11 showed that one signal hybridized to the normal chromosome 14, and the The t(11;14)(p15;q22) involves two genes, the other split and hybridized to both der(11) and AP2A2 and NID2 genes, that have never been der(14). Co-hybridization with both BAC clones shown to form a fusion gene. showed one fusion signal.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 227 t(11;14)(p15;q22) AP2A2/NID2 Douet-Guilbert N, et al.

FISH with BACs RP11-51L17 (spectrum orange, located in 11p15 and containing AP2A2) and RP11-963I11 (spectrum green, located in 14q22 and containing NID2) showing co-hybridization. During the FISH analyses with BAC clones on chromosome 11, we found that RP11-51L17 was translocated to der(14) and RP11-613G2 was deleted. RP11-51L17 is mapped between positions 774448 and 952590 and RP11-613G2 between positions 952562 and 1128625. The AP2A2 is mapped from positions 925809 to 1012245. The region of overlap between both BAC clones is included between exons 1 and 2 of AP2A2 (UCSC Genome Browser on Human Feb. 2009 (GRCh37/hg19) Assembly). Deletion of RP11-613G2 explains why only one fusion signal was observed.

RP11-51L17 contains the AP2A2 (adaptor-related DNA/RNA protein complex 2, alpha 2 subunit) gene and RP11- The AP2A2 gene contains 22 coding exons, 963I11 the NID2 (Nidogen 2) gene. spanning 86.4 kb. Three alternative transcripts are known (Nagase et al., 1998). Genes involved and Protein proteins The protein has 939 amino acids. It is a component of the adaptor protein complex 2 (AP-2) (Ohno, AP2A2 2006). AP-2 is involved in clathrin-dependent Location endocytosis in which proteins are incorporated into 11p15.5 vesicles surrounded by clathrin (clathrin-coated

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 228 t(11;14)(p15;q22) AP2A2/NID2 Douet-Guilbert N, et al.

vesicles) which are destined for fusion with the Nomura N, Ohara O. Prediction of the coding sequences early endosome. AP-2 is involved in a wide range of unidentified human genes. IX. The complete sequences of 100 new cDNA clones from brain which can code for of biological processes, notably signalling mediated large proteins in vitro. DNA Res. 1998 Feb 28;5(1):31-9 by Notch, Wnt, EGF and transforming growth Yao D, Ehrlich M, Henis YI, Leof EB. Transforming growth factor-b (TGF-b) superfamily members (Foerster et factor-beta receptors interact with AP2 by direct binding to al., 2013; Mizutani et al., 2010; Sorkin and von beta2 subunit. Mol Biol Cell. 2002 Nov;13(11):4001-12 Zastrow, 2009; Yao et al., 2002; Yu et al., 2007). Ohno H. Clathrin-associated adaptor protein complexes. J AP2 was also found to be essential for Cell Sci. 2006 Sep 15;119(Pt 18):3719-21 thrombopoietin (Tpo)-stimulated clathrin-mediated internalization of its receptor c-Mpl. As Tpo Karrman K, Sallerfors B, Lenhoff S, Fioretos T, Johansson B. Cytogenetic evolution patterns in CML post-SCT. Bone promotes proliferation and survival of Marrow Transplant. 2007 Feb;39(3):165-71 hematopoietic stem and progenitor cells and Ulazzi L, Sabbioni S, Miotto E, Veronese A, Angusti A, regulates megakaryocyte lineage differentiation and Gafà R, Manfredini S, Farinati F, Sasaki T, Lanza G, maturation, Tpo signal transduction has to be Negrini M. Nidogen 1 and 2 gene promoters are aberrantly controlled (Hitchcock et al., 2008). More recently, a methylated in human gastrointestinal cancer. Mol Cancer. role for AP2A2 has been suggested in asymmetric 2007 Feb 28;6:17 cell division and self-renewal of hematopoietic Yu A, Rual JF, Tamai K, Harada Y, Vidal M, He X, stem and progenitor cells (Ting et al., 2012). Kirchhausen T. Association of Dishevelled with the clathrin AP-2 adaptor is required for Frizzled endocytosis and NID2 planar cell polarity signaling. Dev Cell. 2007 Jan;12(1):129-41 Location 14q22.1 Hitchcock IS, Chen MM, King JR, Kaushansky K. YRRL motifs in the cytoplasmic domain of the thrombopoietin Note receptor regulate receptor internalization and degradation. NID2 expression was shown to be downregulated Blood. 2008 Sep 15;112(6):2222-31 in tumor tissues from patients with hepatocellular Sorkin A, von Zastrow M. Endocytosis and signalling: carcinoma (Cheng et al., 2012). Aberrant intertwining molecular networks. Nat Rev Mol Cell Biol. methylation of NID2 promoter induces loss of gene 2009 Sep;10(9):609-22 expression in gastrointestinal tumors (stomach and Mizutani A, Saitoh M, Imamura T, Miyazawa K, Miyazono colon) and in oral squamous cell carcinoma K. Arkadia complexes with clathrin adaptor AP2 and (Guerrero-Preston et al., 2011; Ulazzi et al., 2007). regulates EGF signalling. J Biochem. 2010 Dec;148(6):733-41 DNA/RNA Guerrero-Preston R, Soudry E, Acero J, Orera M, Moreno- The NID2 gene contains 21 coding exons, spanning López L, Macía-Colón G, Jaffe A, Berdasco M, Ili-Gangas 64.4 kb (Kohfeldt et al., 1998). C, Brebi-Mieville P, Fu Y, Engstrom C, Irizarry RA, Esteller M, Westra W, Koch W, Califano J, Sidransky D. NID2 and Protein HOXA9 promoter hypermethylation as biomarkers for The NID2 gene encodes a 1375 amino acids protein prevention and early detection in oral cavity squamous cell that is a member of the nidogen family of basement carcinoma tissues and saliva. Cancer Prev Res (Phila). membranes that control diverse cellular activities, 2011 Jul;4(7):1061-72 including adhesion, migration, differentiation, gene Cheng ZX, Huang XH, Wang Q, Chen JS, Zhang LJ, Chen expression and apoptosis. Its three dimensional XL. Clinical significance of decreased nidogen-2 structure consists of three globular domains expression in the tumor tissue and serum of patients with hepatocellular carcinoma. J Surg Oncol. 2012 connected by a flexible link and a rod (Kohfeldt et Jan;105(1):71-80 al., 1998). Disruption of the integrity of the Ting SB, Deneault E, Hope K, Cellot S, Chagraoui J, basement membrane creates an invasion-permissive Mayotte N, Dorn JF, Laverdure JP, Harvey M, Hawkins environment, often promoting cancer cell ED, Russell SM, Maddox PS, Iscove NN, Sauvageau G. proliferation and invasion (metastasis) (Lester and Asymmetric segregation and self-renewal of hematopoietic McCarthy, 1992). stem and progenitor cells with endocytic Ap2a2. Blood. 2012 Mar 15;119(11):2510-22 References Foerster S, Kacprowski T, Dhople VM, Hammer E, Herzog S, Saafan H, Bien-Möller S, Albrecht M, Völker U, Ritter Lester BR, McCarthy JB. Tumor cell adhesion to the CA. Characterization of the EGFR interactome reveals extracellular matrix and signal transduction mechanisms associated protein complex networks and intracellular implicated in tumor cell motility, invasion and metastasis. receptor dynamics. Proteomics. 2013 Nov;13(21):3131-44 Cancer Metastasis Rev. 1992 Mar;11(1):31-44 This article should be referenced as such: Kohfeldt E, Sasaki T, Göhring W, Timpl R. Nidogen-2: a new basement membrane protein with diverse binding Douet-Guilbert N, De Braekeleer E, Tous C, Guéganic N, properties. J Mol Biol. 1998 Sep 11;282(1):99-109 Basinko A, Le Bris MJ, Morel F, De Braekeleer M. t(11;14)(p15;q22) AP2A2/NID2. Atlas Genet Cytogenet Nagase T, Ishikawa K, Miyajima N, Tanaka A, Kotani H, Oncol Haematol. 2015; 19(3):227-229.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 229

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Solid Tumour Section Review

Lung: Translocations in Small Cell Carcinoma Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: August 2014 Online updated version : http://AtlasGeneticsOncology.org/Tumors/TranslocLungSmallCellCarcID6651.html DOI: 10.4267/2042/56418 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Cytology Small round or oval cells with a finely granular Review on translocations in small cell carcinoma, nucleus and frequent mitoses. with data on clinics, genetics and cytogenetics. Pathology Classification Immunohistochemistry is often positive for neuroendocrine markers, such as chromogranin, In the group of malignant epithelial tumours of the synaptophysin, and CD56; SCLCs are also positive lungs, small cell carcinomas (small cell lung cancer for NKX2-1 (14q13, also called TTF-1, a tissue- (SCLC)) are usually opposed to non-small cell specific transcription factor in lung epithelial cells) carcinomas (non-small cell lung cancer (NSCLC)). in most cases. Small cell carcinoma is a pulmonary neuroendocrine tumour. Prognosis Other neuroendocrine tumours of the lungs are Although SCLC is extremely sensitive to initial large cell neuroendocrine carcinomas, typical chemotherapy and radiotherapy, most patients carcinoids, and atypical carcinoids. eventually relapse. SCLC is an aggressive disease with poor prognosis, with a five years survival of Clinics and pathology 5%. High expression of SOX2 (3q26) and FGFR1 (8p11) are associated with the worst outcome Disease (Yang et al., 2013). Lung small cell carcinoma Epidemiology Genetics Small cell carcinomas comprise about 15-20% of Note lung cancers. TP53 (17p13) mutations are detected in 70 to 90% Small cell carcinoma is more often associated with of SCLCs. RB1 (13q14) and the retinoblastoma tobacco smoking than adenocarcinoma, and less pathway are inactivated in most SCLCs. PTEN than squamous cell carcinoma. (10q23) is mutated in 2 to 10%. MYC (8q24) amplifications and amplification of MYC family Clinics members are found in 30% of SCLCs in pre- Patients are typically men older than 60-70 years. invasive stages. Loss of heterozygocity (LOH) on Small cell carcinoma more often presents with chromosome arm 3p is found in more than 80% of symptoms of early metastases. SCLCs, including the loss of FHIT (3p14) protein Location of the tumour is usually central, but SCLC expression. FHIT controls the invasive phenotype may occur in a peripheral location. of lung cancer cells by regulating the expression of Bronchoscopic biopsy is often positive (van genes associated with epithelial-mesenchymal Meerbeeck et al., 2011). transition.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 230 Lung: Translocations in Small Cell Carcinoma Huret JL

FHIT loss confers cisplatin resistance in lung t(1;1)(p36;p34) RLF/FAM132A (Iwakawa et al., cancer via the AKT/NF-KB/SLUG pathway (Wu et 2013) al., 2014). In wide genomic analyses, an elevated t(1;1)(p36;p34) RLF/UBE2J2 (Iwakawa et al., rate of C:G>A:T transversions were found, 2013) compared to neutral mutations, consistent with t(1;1)(p35;p35) SERINC2/ZCCHC17 (Rudin et al., demonstrated effects of tobacco smoke carcinogens 2012) on DNA. SOX2, a transcription factor, one of the inv(1)(p34p34) BMP8B/RLF (Rudin et al., 2012) genes able to reprogram human somatic cells to inv(1)(p34p34) CAP1/BMP8B (Rudin et al., 2012) pluripotent stem cells (Lin et al., 2011) was inv(1)(p34p34) RLF/PPT1 (Rudin et al., 2012) amplified in 27% of the samples. A recurrent t(1;1)(p34;p34) CAP1/MACF1 (Iwakawa et al., RLF/MYCL translocation was found in 9% of 2013) SCLCs, and the RLF/MYCL fusion overexpressed t(1;1)(p34;p34) INPP5B/SF3A3 (Rudin et al., MYCL (1p34). FGFR1 was amplified in 6%. 2012) Chromatin-modifying enzymes such as EP300 t(1;1)(p34;p34) inv(1)(p34p34) RLF/MYCL (Rudin (22q13) were frequently mutated (Peifer et al., et al., 2012; Iwakawa et al., 2013) 2012; Rudin et al., 2012). To be noted that SOX2 t(1;1)(p34;p34) RLF/COL9A2 (Iwakawa et al., and FGFR1 are also known to be implicated in a 2013) subset of squamous cell carcinoma of the lung t(1;1)(p34;p34) RLF/SMAP2 (Iwakawa et al., (Pietanza and Ladanyi, 2012). In contrast with lung 2013) adenocarcinomas, there is no molecularly targeted t(1;1)(p34;p34) SF3A3/GNL2 (Iwakawa et al., agents yet for small cell carcinomas of the lung. 2013) t(1;1)(p34;p34) SMAP2/MYCL (Iwakawa et al., Cytogenetics 2013) t(1;1)(p34;p34) ZMPSTE24/MFSD2A (Iwakawa et Note al., 2013) Only a few genes have been found implicated in t(1;1)(q21;q21) TXNIP/NOTCH2NL (Rudin et al., many different translocations: 2012) - RLF (1p34), a zinc finger protein, which may be t(1;1)(q25;q25) XPR1/TRMT1L (Iwakawa et al., involved in transcriptional regulation. Depletion of 2013) Rlf leads to DNA hypermethylation in the mouse. del(1)(q44q44) ZNF695/TFB2M (Rudin et al., RLF is likely to be involved in epigenetic processes 2012) (Daxinger et al., 2013), RLF is translocated with 10 t(1;12)(p34;q24) TRIT1/EP400 (Iwakawa et al., different partners (herein below) in SCLCs; 2013) - BCL2L1 (20q11) an inhibitor of cell death, t(1;19)(p36;q13) UBE4B/TBCB (Iwakawa et al., involved in various cancers, translocated with 7 2013) different partners (herein below) in SCLCs; t(1;20)(p34;q11) BCL2L1/BMP8B (Iwakawa et al., - PVT1 (8q24), a non-protein coding and oncofetal 2013) gene, translocated with various partners in: breast t(1;20)(p34;q11) BCL2L1/DEM1 (Iwakawa et al., cancer, Ewing/PNET spectrum, and hematological 2013) malignancies, and with 7 different partners (herein t(1;20)(p34;q11) BCL2L1/RIMS3 (Iwakawa et al., below) in SCLCs; PVT1 is a hotspot for 2013) chromosomal breaks during MYC amplification t(1;20)(p34;q11) BCL2L1/RLF (Iwakawa et al., (L'Abbate et al., 2014). 2013) - Other genes recurrently found are HM13 (20q11), t(1;20)(p34;q11) BCL2L1/ZNF643 (Iwakawa et al., translocated with 3 different partners, and MYCL, 2013) BMP8B (1p34), CAP1 (1p34), CREBBP (16p13), t(1;20)(p34;q11) BCL2L1/ZNF684 (Iwakawa et al., and DNM2 (19p13), implicated twice each. 2013) Cytogenetics Morphological t(1;20)(p34;q11) PPT1/BCL2L1 (Iwakawa et al., 2013) Fusion transcripts were often found in amplified t(1;20)(p34;q11) RLF/HM13 (Iwakawa et al., 2013) regions in 1p34, 2p24, 8q24, and 9p24, suggesting t(2;5)(p22;q14) BIRC6/BHMT2 (Rudin et al., that amplification and fusion of genes occur 2012) simultaneously by chromothripsis (Iwakawa et al., t(2;6)(q31;p12) RCAN2/RAPGEF4 (Rudin et al., 2013). One hundred translocations have so far been 2012) reported in small cell carcinoma of the lung. They t(2;12)(p23;p13) CACNA2D4/WDR43 (Campbell are the following: et al., 2008) t(1;1)(p36;p36) RERE/SLC2A5 (Iwakawa et al., t(3;3)(p21;p21) NEK4/SFMBT1 (Rudin et al., 2013) 2012)

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 231 Lung: Translocations in Small Cell Carcinoma Huret JL

t(3;3)(p21;q23) RASA2/NICN1 (Iwakawa et al., 2013) t(8;14)(q24;q11) PVT1/SLC7A7 (Iwakawa et al., t(3;3)(q13;q22) STAG1/STXBP5L (Iwakawa et al., 2013) 2013) t(8;18)(q24;q12) PVT1/NOL4 (Iwakawa et al., del(3)(q23q23) SPSB4/ACPL2 (Rudin et al., 2012) 2013) t(3;3)(q23;q24) SLC25A36/PLSCR1 (Iwakawa et t(9;9)(p24;p24) KANK1/DOCK8 (Rudin et al., al., 2013) 2012) t(3;3)(q26;q26) GPR160/NCEH1 (Peifer et al., t(9;9)(p24;p24) RIC1/JAK2 (Iwakawa et al., 2013) 2012) t(10;10)(p12;p12) WAC/GPR158 (Iwakawa et al., inv(3)(q26q27) DCUN1D1/ATP11B (Rudin et al., 2013) 2012) t(10;10)(p11;q21) CCDC7/UBE2D1 (Rudin et al., del(3)(q28q28) LPP/TPRG1 (Rudin et al., 2012) 2012) t(3;11)(p24;p15) NGLY1/CCKBR (Iwakawa et al., t(10;10)(q21;q21) CCDC6/CTNNA3 (Rudin et al., 2013) 2012) t(3;11)(p22;q14) DLEC1/ODZ4 (Iwakawa et al., t(11;11)(p15;q23) ATP5L/TEAD1 (Iwakawa et al., 2013) 2013) t(3;11)(p21;p15) SFMBT1/AP2A2 (Iwakawa et al., t(11;11)(q14;q14) PICALM/CCDC81 (Iwakawa et 2013) al., 2013) t(3;12)(q13;p11) NAA50/MRPS35 (Rudin et al., t(11;11)(q14;q14) GAB2/NARS2 (Rudin et al., 2012) 2012) t(3;16)(q21;p13) TXNDC11/RUVBL1 (Rudin et t(12;12)(p13;p13) ENO2/ACRBP (Iwakawa et al., al., 2012) 2013) t(3;17)(q11;q21) NPEPPS/EPHA6 (Rudin et al., inv(12)(p13p13) ERC1/ANO2 (Rudin et al., 2012) 2012) inv(12)(p13p13) C12orf4/CD9 (Rudin et al., 2012) t(5;5)(q13;q13) NAIP/OCLN (Iwakawa et al., del(12)(p13p13) ANO2/FBXL14 (Rudin et al., 2013) 2012) t(5;5)(q31;q31) SKP1/CDKL3 (Rudin et al., 2012) del(12)(q12q13) RPAP3/SCAF11 (Rudin et al., t(5;5)(q31;q31) JADE2/UBE2B (Iwakawa et al., 2012) 2013) t(12;12)(q14;q21) PAWR/GNS (Iwakawa et al., del(6)(q21q22) CEP85L/SCML4 (Rudin et al., 2013) 2012) t(12;12)(q23;q23) CHPT1/UTP20 (Rudin et al., t(6;8)(p21;p21) HMBOX1/ZFAND3 (Iwakawa et 2012) al., 2013) t(12;12)(q24;q24) CIT/RFC5 (Iwakawa et al., t(6;20)(p21;p12) CRLS1/KCNK17 (Iwakawa et al., 2013) 2013) t(12;12)(q24;q24) NCOR2/SCARB1 (Iwakawa et del(7)(p21p21) ANKMY2/ISPD (Rudin et al., al., 2013) 2012) t(14;14)(q23;q23) MTHFD1/SYNE2 (Rudin et al., t(8;8)(q12;q24) PVT1/CHD7 (Campbell et al., 2012) 2008; Pleasance et al., 2010) t(14;16)(q32;q12) SMEK1/HEATR3 (Iwakawa et t(8;8)(q12;q24) PVT1/CLVS1 (Iwakawa et al., al., 2013) 2013) t(15;15)(q21;q21) SPG11/SORD (Iwakawa et al., inv(8)(q22q23) OXR1/COX6C (Rudin et al., 2012) 2013) inv(8)(q22q23) YWHAZ/OXR1 (Rudin et al., t(16;16)(p13;p13) CREBBP/RHBDF1 (Peifer et al., 2012) 2012; Iwakawa et al., 2013) t(8;8)(q23;q23) NUDCD1/SYBU (Iwakawa et al., t(16;16)(p13;p13) del(16)(p13p13) CREBBP/SLX4 2013) (Pleasance et al., 2010) t(8;8)(q23;q24) CSMD3/MYC (Iwakawa et al., t(17;17)(p11;p13) MPRIP/TP53 (Peifer et al., 2012) 2013) t(17;17)(q25;q25) LRRC45/GCGR (Iwakawa et al., t(8;8)(q23;q24) PTK2/PKHD1L1 (Iwakawa et al., 2013) 2013) t(17;17)(q25;q25) FOXK2/HEXDC (Iwakawa et t(8;8)(q24;q24) PVT1/LY6H (Iwakawa et al., 2013) al., 2013) t(8;14)(p23;q22) AGPAT5/TXNDC16 (Rudin et t(18;18)(p11;q12) TWSG1/PIK3C3 (Iwakawa et al., 2012) al., 2013) t(8;14)(q24;q11) PVT1/CCNB1IP1 (Iwakawa et al., t(19;19)(p13;p13) NFIX/GATAD2A (Iwakawa et 2013) al., 2013) t(8;14)(q24;q11) PVT1/MYH7 (Iwakawa et al., t(19;19)(p13;p13) DNM2/ILF3 (Rudin et al., 2012) 2013) del(19)(p13p13) DNM2/KCNN1 (Rudin et al., 2012)

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 232 Lung: Translocations in Small Cell Carcinoma Huret JL

inv(19)(p13p13) GIPC1/PKN1 (Rudin et al., 2012) Nurnberg P, Perner S, Heukamp LC, Brindle PK, Haas S, del(19)(q13q13) PPP1R37/KLC3 (Rudin et al., Thomas RK.. Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer. Nat 2012) Genet. 2012 Oct;44(10):1104-10. doi: 10.1038/ng.2396. t(20;20)(q11;q11) BCL2L1/HM13 (Iwakawa et al., Epub 2012 Sep 2. 2013) Pietanza MC, Ladanyi M.. Bringing the genomic landscape t(20;20)(q11;q11) TPX2/HM13 (Iwakawa et al., of small-cell lung cancer into focus. Nat Genet. 2012 2013) Oct;44(10):1074-5. doi: 10.1038/ng.2415. inv(20)(q11q13) CEP250/DPM1 (Rudin et al., Rudin CM, Durinck S, Stawiski EW, Poirier JT, Modrusan 2012) Z, Shames DS, Bergbower EA, Guan Y, Shin J, Guillory J, inv(22)(q13q13) TTLL1/TSPO (Rudin et al., 2012) Rivers CS, Foo CK, Bhatt D, Stinson J, Gnad F, Haverty PM, Gentleman R, Chaudhuri S, Janakiraman V, Jaiswal BS, Parikh C, Yuan W, Zhang Z, Koeppen H, Wu TD, References Stern HM, Yauch RL, Huffman KE, Paskulin DD, Illei PB, Varella-Garcia M, Gazdar AF, de Sauvage FJ, Bourgon R, Campbell PJ, Stephens PJ, Pleasance ED, O'Meara S, Li Minna JD, Brock MV, Seshagiri S.. Comprehensive H, Santarius T, Stebbings LA, Leroy C, Edkins S, Hardy C, genomic analysis identifies SOX2 as a frequently amplified Teague JW, Menzies A, Goodhead I, Turner DJ, Clee CM, gene in small-cell lung cancer. Nat Genet. 2012 Quail MA, Cox A, Brown C, Durbin R, Hurles ME, Edwards Oct;44(10):1111-6. doi: 10.1038/ng.2405. Epub 2012 Sep PA, Bignell GR, Stratton MR, Futreal PA. Identification of 2. somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing. Daxinger L, Harten SK, Oey H, Epp T, Isbel L, Huang E, Nat Genet. 2008 Jun;40(6):722-9 Whitelaw N, Apedaile A, Sorolla A, Yong J, Bharti V, Sutton J, Ashe A, Pang Z, Wallace N, Gerhardt DJ, Blewitt Pleasance ED, Stephens PJ, O'Meara S, McBride DJ, ME, Jeddeloh JA, Whitelaw E.. An ENU mutagenesis Meynert A, Jones D, Lin ML, Beare D, Lau KW, Greenman screen identifies novel and known genes involved in C, Varela I, Nik-Zainal S, Davies HR, Ordoñez GR, Mudie epigenetic processes in the mouse. Genome Biol. LJ, Latimer C, Edkins S, Stebbings L, Chen L, Jia M, Leroy 2013;14(9):R96. C, Marshall J, Menzies A, Butler A, Teague JW, Mangion J, Sun YA, McLaughlin SF, Peckham HE, Tsung EF, Costa Iwakawa R, Takenaka M, Kohno T, Shimada Y, Totoki Y, GL, Lee CC, Minna JD, Gazdar A, Birney E, Rhodes MD, Shibata T, Tsuta K, Nishikawa R, Noguchi M, Sato-Otsubo McKernan KJ, Stratton MR, Futreal PA, Campbell PJ. A A, Ogawa S, Yokota J.. Genome-wide identification of small-cell lung cancer genome with complex signatures of genes with amplification and/or fusion in small cell lung tobacco exposure. Nature. 2010 Jan 14;463(7278):184-90 cancer. Genes Chromosomes Cancer. 2013 Sep;52(9):802-16. doi: 10.1002/gcc.22076. Epub 2013 Lin B, Huang X, Han X, Foltz G.. SOX2 (SRY (sex May 28. determining region Y)-box 2). Atlas Genet Cytogenet Oncol Haematol. 2011;15(12):1054-1057. URL : Yang F, Gao Y, Geng J, Qu D, Han Q, Qi J, Chen G.. http://AtlasGeneticsOncology.org/Genes/SOX2ID44064ch Elevated expression of SOX2 and FGFR1 in correlation 3q26.html. with poor prognosis in patients with small cell lung cancer. Int J Clin Exp Pathol. 2013 Nov 15;6(12):2846-54. van Meerbeeck JP, Fennell DA, De Ruysscher DK.. Small- eCollection 2013. cell lung cancer. Lancet. 2011 Nov 12;378(9804):1741-55. doi: 10.1016/S0140-6736(11)60165-7. Epub 2011 May 10. L'Abbate A, Macchia G, D'Addabbo P, Lonoce A, Tolomeo (REVIEW) D, Trombetta D, Kok K, Bartenhagen C, Whelan CW, Palumbo O, Severgnini M, Cifola I, Dugas M, Carella M, Peifer M, Fernandez-Cuesta L, Sos ML, George J, Seidel De Bellis G, Rocchi M, Carbone L, Storlazzi CT.. Genomic D, Kasper LH, Plenker D, Leenders F, Sun R, Zander T, organization and evolution of double Menon R, Koker M, Dahmen I, Muller C, Di Cerbo V, minutes/homogeneously staining regions with MYC Schildhaus HU, Altmuller J, Baessmann I, Becker C, de amplification in human cancer. Nucleic Acids Res. Wilde B, Vandesompele J, Bohm D, Ansen S, Gabler F, 2014;42(14):9131-45. doi: 10.1093/nar/gku590. Epub 2014 Wilkening I, Heynck S, Heuckmann JM, Lu X, Carter SL, Jul 17. Cibulskis K, Banerji S, Getz G, Park KS, Rauh D, Grutter C, Fischer M, Pasqualucci L, Wright G, Wainer Z, Russell Wu DW, Lee MC, Hsu NY, Wu TC, Wu JY, Wang YC, P, Petersen I, Chen Y, Stoelben E, Ludwig C, Schnabel P, Cheng YW, Chen CY, Lee H.. FHIT loss confers cisplatin Hoffmann H, Muley T, Brockmann M, Engel-Riedel W, resistance in lung cancer via the AKT/NF-kB/Slug- Muscarella LA, Fazio VM, Groen H, Timens W, Sietsma H, mediated PUMA reduction. Oncogene. 2014 Jul 7. doi: Thunnissen E, Smit E, Heideman DA, Snijders PJ, 10.1038/onc.2014.184. [Epub ahead of print] Cappuzzo F, Ligorio C, Damiani S, Field J, Solberg S, Brustugun OT, Lund-Iversen M, Sanger J, Clement JH, This article should be referenced as such: Soltermann A, Moch H, Weder W, Solomon B, Soria JC, Validire P, Besse B, Brambilla E, Brambilla C, Lantuejoul Huret JL. Lung: Translocations in Small Cell Carcinoma. S, Lorimier P, Schneider PM, Hallek M, Pao W, Meyerson Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3):230- M, Sage J, Shendure J, Schneider R, Buttner R, Wolf J, 233.

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 233

Atlas of Genetics and Cytogenetics

in Oncology and Haematology

OPEN ACCESS JOURNAL INIST -CNRS

Deep Insight Section

FOXP3 expression in tumor cells and its role in cancer progression Valentina Uva, Lucia Sfondrini, Tiziana Triulzi, Patrizia Casalini, Elda Tagliabue, Andrea Balsari Molecular Targeting Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy (VU, TT, PC, ET), Dipartimento di Scienze Biomediche per la Salute, Universita degli Studi di Milano, Milan, Italy (LS, AB)

Published in Atlas Database: October 2014 Online updated version : http://AtlasGeneticsOncology.org/Deep/FOXP3andCancerID20138.html DOI: 10.4267/2042/56419 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2015 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Abstract Forkhead box P3 (FOXP3), a gene member of the forkhead/winged-helix family of transcription regulators, is implicated in regulating immune system development and function. This gene has been found to be of crucial importance for the generation of CD4 +CD25 + regulatory T cells (Tregs). In addition to its expression in the lymphocyte lineage, studies have recently described FOXP3 expression in normal and cancer cells non- hematopoietic-derived, suggesting that FOXP3 exerts a broader function than that on Tregs. A role for FOXP3 as an onco-suppressor gene in human cancers has been suggested based on in vitro studies showing that FOXP3 is implicated in repressing various oncogenes and enhancing expression of tumor- suppressor genes. However, numerous studies in samples from human cancer patients showed a positive correlation between FOXP3 expression and poor prognosis, especially with metastasis. Further investigations are required to clarify the significance of FOXP3 expression in tumor cells and to identify the mechanisms by which it affects prognosis.

Introduction molecular weight of 47 kDa. The isoform ∆2FOXP3, lacking exon 2 (aa 71-105), has been Forkhead box P3 (FOXP3) is a member of the proposed to act as a dominant negative isoform (Li forkhead/winged-helix family of transcription et al., 2007). Another splice variant of FOXP3, regulators. This gene is involved in immune system + called ∆7FOXP3, has been identified in both CD4 responses and in the development and function of + and CD8 regulatory T cell clones. ∆7FOXP3 lacks regulatory T cells (Tregs) (Fontenot et al., 2005; the 81 bp region that encodes exon 7. The absence Sakaguchi et al., 2008). The FOXP3 gene is located of this exon abrogates the suppressive function of on the X chromosome at Xp11.23 and it is Tregs (Kaur et al., 2010). Human Tregs can also submitted to X chromosome inactivation (Wang et express ∆2∆7FOXP3 isoform that lacks both exon al., 2009). This gene is highly conserved across 2 and exon 7 (aa 245-272) (Mailer et al., 2009). mammals (Ziegler, 2006) and contains 11 coding exons and 3 non-coding exons (Bennett and Ochs, FOXP3 expression in regulatory T 2001). FOXP3 protein contains four potential cells functional domains; repressor, zinc finger, leucine The nuclear expression of FOXP3 is considered as zipper and forkhead. Humans express both full- the most specific marker for Tregs and a key length protein and three splice variants (Allan et al., determinant of their immunosuppressive functions 2005; Kaur et al., 2010; Smith et al., 2006). The (Grimmig et al., 2013). The molecular mechanisms full-lenght form consists of 431-amino acids with a of Treg-mediated immunosuppression are still not

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 234 FOXP3 expression in tumor cells and its role in cancer Uva V, et al. progression

completely understood. Genome-wide analyses of submitted to neoadiuvant chemotherapy (Ladoire et FOXP3 + T cells revealed about 700 genes and al., 2008). many microRNAs differentially expressed in Contrary to the putative pro-tumorigenic effect, the FOXP3 + Tregs. In these cells FOXP3 has a dual presence of Tregs has been associated with a good role as both transcriptional activator and repressor prognosis in colorectal and head and neck cancers (Sadlon et al., 2010; Zheng et al., 2007). (Badoual et al., 2006; Ladoire et al., 2011). Regulatory T cells represent about 5% of Although Tregs can potentially promote cancer circulating CD4 + T lymphocytes in the human progression, they can also attenuate inflammation. peripheral blood. An increased number of Tregs has Because chronic inflammation is one of the critical been observed in the blood, in the tumor mass and processes promoting carcinogenesis and tumor in the draining lymph nodes of patients with growth, Tregs are able to down-regulate the pro- different solid tumors (Bergmann et al., 2008; tumorigenic inflammatory responses. It has been Mougiakakos et al., 2010; Petersen et al., 2006; hypothesized that colorectal cancer growth can be Strauss et al., 2007; Whiteside, 2012). The promoted by a Th17-cell mediated inflammatory increased frequency of tumor-infiltrating Tregs response. Human Tregs are able to limit Th17- have been associated with poor survival in breast related pro-tumorigenic effects through inhibition (Bates et al., 2006), gastric (Sasada et al., 2003), of their activation and function (Crome et al., ovarian (Sato et al., 2005), lung (Petersen et al., 2010). 2006), hepatocellular (Gao et al., 2007), renal (Li et FOXP3 expression in malignant al., 2009), and pancreatic (Hiraoka et al., 2006) cancers. cells After migrating to tumor site, Tregs suppress FOXP3 expression has been recently described in antitumor immune response interfering with the normal cells and in non-hematopoietic-derived activation and expansion of tumor-antigens-specific cancer cells, suggesting that FOXP3 biological effector T cells through different mechanisms not effects are not restricted to Tregs. FOXP3 fully understood yet (Whiteside, 2008). expression has been observed in different cancer In breast cancer, the percentage of Treg cells histotypes (breast, urinary bladder, tongue, gastric, increases in parallel with the disease stage, from esophageal, pancreas, colorectal, stomach, thyroid, normal to ductal carcinoma in situ (DCIS) and from glioma, non-small cell lung cancers and DCIS to invasive carcinoma. A high frequency of melanoma). Studies performed on samples from tumor-infiltrating FOXP3 + cells correlates with human cancer patients have produced data showing worse disease-free survival and decreased overall FOXP3 expression restricted only to cancer cells, survival in patients with invasive breast carcinoma, whereas weak or no FOXP3 expression was suggesting that the presence of Treg cells promotes detectable in their respective normal counterpart tumour progression by creating an (Ebert et al., 2008; Fu et al., 2013; Hinz et al., immunosuppressive environment (Bates et al., 2007; Wang et al., 2012; Wang et al., 2014; Won et 2006). al., 2013). Tan and colleagues proposed a further explanation In contrast, Zuo and colleagues reported a higher for the association of Treg cells with an aggressive expression of FOXP3 in normal epithelial breast phenotype in advanced breast cancers by cells than in tumor cells (Zuo et al., 2007b). Similar + demonstrating that tumor-infiltrating FOXP3 findings have been reported in samples of prostate Tregs are responsible for high RANKL expression and ovarian cancer, where FOXP3 protein was within the tumor microenvironment, and this clearly identifiable in normal epithelial samples, expression stimulates the metastatic progression of while the majority of malignant cells resulted RANK-expressing breast carcinoma cells (Tan et negative for this protein (Wang et al., 2009; Zhang al., 2011). and Sun, 2010). Tregs are also directly involved in promoting To note in most carcinomas, FOXP3 staining was angiogenesis in the tumor microenvironment localized predominantly in the cytoplasm of tumor (Facciabene et al., 2012), therefore Tregs might cells, whereas in the studies by Zuo and Wang only promote cancer growth both through tumor immune nuclear positivity for FOXP3 was scored as a escape and angiogenesis. positive result (Wang et al., 2009; Zuo et al., In addition to their potential role in favoring disease 2007b). progression and relapse, FOXP3 + Tregs have been suggested as possible biomarker to monitor the Tumor suppressive role of therapeutic response. For example, it has been FOXP3 + observed that the decrease of FOXP3 tumor- In vitro studies demonstrated an important role of infiltrating cells is associated with the pathological FOXP3 modulating the expression of various genes complete response in breast cancer patients

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 235 FOXP3 expression in tumor cells and its role in cancer Uva V, et al. progression

implicated in cancer development, including tumor them to escape from immune surveillance, thereby suppressors and oncogenes. resulting in cancer progression (Yoshii et al., 2012). For instance, FOXP3 represses the expression of A correlation between FOXP3 expression and HER2 and SKP2 in breast cancer cells (Zuo et al., lymph node metastases incidence was also reported 2007a; Zuo et al., 2007b) and an inverse correlation for esophageal squamous carcinoma (Xue et al., between FOXP3 and HER2 mRNA was observed 2010), where FOXP3 mRNA and protein in this type of tumor. Similarly, FOXP3 silencing in expression was not only higher in tumors than in normal mammary epithelial cells (where FOXP3 is normal mucosa, but also higher in advanced stages expressed at higher level compared to tumor tissue) than in early stages. FOXP3 negative patients determines an increase in HER2 expression (Zuo et experienced significantly better overall survival al., 2007b). FOXP3 can also down-regulate than the FOXP3-overexpressing group. Cox BRCA1, interfering with the BRCA1-mediated regression analysis showed that tumor stage and DNA repair processes (Li et al., 2013). FOXP3 protein expression were independent Moreover, FOXP3 is involved in the transcriptional prognostic risk factors (Wang et al., 2012). control of tumor-suppressor genes such as p21 and Two different studies on non-small-cell lung cancer LATS2 (Li et al., 2011). In prostate cancer, FOXP3 patients demonstrated that FOXP3 expression in is also able to repress the expression of c-Myc cancer cells positively correlated with both lymph whose overexpression contributes to a more node metastases and tumor staging aggressive cancer phenotype (Wang et al., 2009). (Dimitrakopoulos et al., 2011; Fu et al., 2013). FOXP3 has been demonstrated to play a tumor- FOXP3 expression has been correlated with poor suppressor role also in gastric cancer cells. In these prognosis even in colorectal (Kim M et al., 2013), cells the up-regulation of FOXP3 expression tongue (Liang et al., 2011), urinary bladder significantly inhibit cell growth and promote (Winerdal et al., 2011) cancer patients, and glioma apoptosis, through the induction of pro-apoptotic (Wang et al., 2014) patients. genes (PARP, caspase-3 and caspase-9), suggesting Finally, in vitro studies demonstrated that FOXP3 that endogenous FOXP3 might act as a positive expression in tumor cells correlates with the modulator in apoptotic pathway (Ma et al., 2013). inhibition of T-cell proliferation, indicating that Furthermore, in gastric cancer as in other FOXP3-positive cancer cells may acquire growth- malignancies, COX-2 has been shown to play an suppressive functions, similar to Tregs, and that important role in both carcinogenesis and cancer mimicking Tregs functions may represent a novel progression. By inhibiting NF-κB activity, which is mechanism of immune evasion (Grimmig et al., a major modulator of COX-2 expression, FOXP3 2013; Hinz et al., 2007). inhibits the expression of COX2 and hence cell All this data highlights the association between metastasis (Hao et al., 2014). FOXP3 expression in tumor cells and poor patient FOXP3 expression and prognosis prognosis. Notably, FOXP3 has not been associated with local recurrence but only with a possible role in human cancer in driving metastatic spread. Although FOXP3 has been described as an onco- FOXP3 localization in cancer suppressor gene, recent evidences point out the positive correlation between FOXP3 expression and cells and patient prognosis poor prognosis. FOXP3 protein is synthesized in the cytoplasm of In 2009, Merlo and colleagues demonstrated, for cells and then actively transported to the nucleus. the first time, that FOXP3 expression in breast As a transcription factor, the nuclear localization is carcinoma was inversely associated with patient required for its transcriptional repression function survival and that the risk increased with FOXP3 (Lopes et al., 2006). staining intensity. FOXP3 was also a strong FOXP3 is expressed constitutively within the prognostic factor for distant metastasis-free nucleus of Tregs and of those normal FOXP3- survival, but not for local recurrence incidence risk expressing epithelial cells (Sakaguchi, 2005). By (Merlo et al., 2009). These results were confirmed contrast, several studies demonstrated that FOXP3 by other 2 independent studies (Kim MH et al., cytoplasmic staining was more aboundant, 2013; Nair et al., 2013). compared to nuclear expression, in several types of The frequency of FOXP3 positive cancer cells in cancer (Hinz et al., 2007; Karanikas et al., 2008; primary gastric tumors correlated with the Ladoire et al., 2011; Merlo et al., 2009; Tao et al., incidence of lymph node metastases (Yoshii et al., 2012; Winerdal et al., 2011). 2012; Wang et al., 2010) and the 3-year survival The fact that many tumors display cytoplasmic rate, indicating a potential association of FOXP3 staining may be a result of defects in the nuclear expression with poor prognosis. In this regard, it localization signals of FOXP3, possibly due to has been demonstrated that gastric carcinoma cells acquired mutations. Frequent FOXP3 gene might have a Treg-like activity, which would allow mutations and deletions, together with post-

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 236 FOXP3 expression in tumor cells and its role in cancer Uva V, et al. progression

translational modifications and splicing variations Allan SE, Passerini L, Bacchetta R, Crellin N, Dai M, may result in cytoplasmic localization of FOXP3 Orban PC, Ziegler SF, Roncarolo MG, Levings MK. The role of 2 FOXP3 isoforms in the generation of human protein in cancer cells (Wang et al., 2009; Hancock CD4+ Tregs. J Clin Invest. 2005 Nov;115(11):3276-84 and Ozkaynak, 2009). Since the role of FOXP3 is Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A transcription regulation, a cytoplasmic FOXP3 function for interleukin 2 in Foxp3-expressing regulatory T localization could affect its biological role. The cells. Nat Immunol. 2005 Nov;6(11):1142-51 concept that FOXP3 cytoplasmic localization Sakaguchi S. Naturally arising Foxp3-expressing interferes with its onco-suppressive functions has CD25+CD4+ regulatory T cells in immunological tolerance been suggested by a recent study (Takenaka et al., to self and non-self. Nat Immunol. 2005 Apr;6(4):345-52 2013). Nuclear FOXP3 expression has been Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, Qian F, associated significantly with an improved overall Jungbluth AA, Frosina D, Gnjatic S, Ambrosone C, Kepner survival in breast cancer patients, whereas J, Odunsi T, Ritter G, Lele S, Chen YT, Ohtani H, Old LJ, cytoplasmic FOXP3 expression in tumor cells was Odunsi K. Intraepithelial CD8+ tumor-infiltrating significantly associated with poor clinical outcome lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. (Takenaka et al., 2013). A predominant cytoplasmic Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18538-43 FOXP3 localization has also been described in Badoual C, Hans S, Rodriguez J, Peyrard S, Klein C, melanoma and colorectal cancer, where FOXP3 Agueznay Nel H, Mosseri V, Laccourreye O, Bruneval P, expression correlated with poor prognosis (Kim et Fridman WH, Brasnu DF, Tartour E. Prognostic value of al., 2013; Quaglino et al., 2011). tumor-infiltrating CD4+ T-cell subpopulations in head and In contrast with these findings cytoplasmic FOXP3 neck cancers. Clin Cancer Res. 2006 Jan 15;12(2):465-72 expression was associated with better overall Bates GJ, Fox SB, Han C, Leek RD, Garcia JF, Harris AL, survival and disease-free survival in primary Banham AH. Quantification of regulatory T cells enables invasive HER2-overexpressing breast cancer the identification of high-risk breast cancer patients and those at risk of late relapse. J Clin Oncol. 2006 Dec patients (Ladoire et al., 2011). 1;24(34):5373-80 Conclusions Hiraoka N, Onozato K, Kosuge T, Hirohashi S. Prevalence of FOXP3+ regulatory T cells increases during the Since Treg cells are significant mediators of tumor progression of pancreatic ductal adenocarcinoma and its progression, targeting Tregs is under active premalignant lesions. Clin Cancer Res. 2006 Sep investigation. Many studies have already 15;12(18):5423-34 demonstrated that Tregs depletion is a promising Lopes JE, Torgerson TR, Schubert LA, Anover SD, way to promote antitumor immunity and tumor Ocheltree EL, Ochs HD, Ziegler SF. Analysis of FOXP3 regression (Jarry et al., 2014; Keenan et al., 2014; reveals multiple domains required for its function as a Reginato et al., 2013; Zhou et al., 2013). transcriptional repressor. J Immunol. 2006 Sep 1;177(5):3133-42 In contrast, despite the increasing knowledge about the biology of FOXP3, the prognostic value of its Petersen RP, Campa MJ, Sperlazza J, Conlon D, Joshi expression in human cancer cells remains still MB, Harpole DH Jr, Patz EF Jr. Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in controversial. The mechanism by which FOXP3 pathologic stage I NSCLC patients. Cancer. 2006 Dec expression in tumor cells affects prognosis has not 15;107(12):2866-72 been fully elucidated yet. However, immune Smith EL, Finney HM, Nesbitt AM, Ramsdell F, Robinson evasion, via FOXP3 expression in tumor cells, may MK. Splice variants of human FOXP3 are functional represent the main strategy for cancer progression. inhibitors of human CD4+ T-cell activation. Immunology. Further investigations are needed to clarify the 2006 Oct;119(2):203-11 significance of FOXP3 expression, its regulatory Ziegler SF. FOXP3: of mice and men. Annu Rev Immunol. mechanism and its association with prognosis of 2006;24:209-26 human cancer. Moreover, additional studies should Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, be carried out to clarify whether FOXP3 sub- Li YW, Tang ZY. Intratumoral balance of regulatory and cellular localization in tumor cells could be cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol. functionally relevant to the clinical prognosis. 2007 Jun 20;25(18):2586-93 Hinz S, Pagerols-Raluy L, Oberg HH, Ammerpohl O, References Grüssel S, Sipos B, Grützmann R, Pilarsky C, Ungefroren Bennett CL, Ochs HD. IPEX is a unique X-linked syndrome H, Saeger HD, Klöppel G, Kabelitz D, Kalthoff H. Foxp3 characterized by immune dysfunction, polyendocrinopathy, expression in pancreatic carcinoma cells as a novel enteropathy, and a variety of autoimmune phenomena. mechanism of immune evasion in cancer. Cancer Res. Curr Opin Pediatr. 2001 Dec;13(6):533-8 2007 Sep 1;67(17):8344-50 Sasada T, Kimura M, Yoshida Y, Kanai M, Takabayashi A. Li B, Samanta A, Song X, Iacono KT, Bembas K, Tao R, CD4+CD25+ regulatory T cells in patients with Basu S, Riley JL, Hancock WW, Shen Y, Saouaf SJ, gastrointestinal malignancies: possible involvement of Greene MI. FOXP3 interactions with histone regulatory T cells in disease progression. Cancer. 2003 acetyltransferase and class II histone deacetylases are Sep 1;98(5):1089-99 required for repression. Proc Natl Acad Sci U S A. 2007 Mar 13;104(11):4571-6

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 237 FOXP3 expression in tumor cells and its role in cancer Uva V, et al. progression

Strauss L, Bergmann C, Gooding W, Johnson JT, Somatic single hits inactivate the X-linked tumor Whiteside TL. The frequency and suppressor function of suppressor FOXP3 in the prostate. Cancer Cell. 2009 Oct CD4+CD25highFoxp3+ T cells in the circulation of patients 6;16(4):336-46 with squamous cell carcinoma of the head and neck. Clin Cancer Res. 2007 Nov 1;13(21):6301-11 Crome SQ, Clive B, Wang AY, Kang CY, Chow V, Yu J, Lai A, Ghahary A, Broady R, Levings MK. Inflammatory Zheng Y, Josefowicz SZ, Kas A, Chu TT, Gavin MA, effects of ex vivo human Th17 cells are suppressed by Rudensky AY. Genome-wide analysis of Foxp3 target regulatory T cells. J Immunol. 2010 Sep 15;185(6):3199- genes in developing and mature regulatory T cells. Nature. 208 2007 Feb 22;445(7130):936-40 Kaur G, Goodall JC, Jarvis LB, Hill Gaston JS. Zuo T, Liu R, Zhang H, Chang X, Liu Y, Wang L, Zheng P, Characterisation of Foxp3 splice variants in human CD4+ Liu Y. FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2. J Clin Invest. 2007a and CD8+ T cells--identification of Foxp3 ∆7 in human Dec;117(12):3765-73 regulatory T cells. Mol Immunol. 2010 Nov-Dec;48(1- 3):321-32 Zuo T, Wang L, Morrison C, Chang X, Zhang H, Li W, Liu Y, Wang Y, Liu X, Chan MW, Liu JQ, Love R, Liu CG, Mougiakakos D, Choudhury A, Lladser A, Kiessling R, Godfrey V, Shen R, Huang TH, Yang T, Park BK, Wang Johansson CC. Regulatory T cells in cancer. Adv Cancer CY, Zheng P, Liu Y. FOXP3 is an X-linked breast cancer Res. 2010;107:57-117 suppressor gene and an important repressor of the HER- Sadlon TJ, Wilkinson BG, Pederson S, Brown CY, Bresatz 2/ErbB2 oncogene. Cell. 2007b Jun 29;129(7):1275-86 S, Gargett T, Melville EL, Peng K, D'Andrea RJ, Glonek Bergmann C, Strauss L, Wang Y, Szczepanski MJ, Lang GG, Goodall GJ, Zola H, Shannon MF, Barry SC. S, Johnson JT, Whiteside TL. T regulatory type 1 cells in Genome-wide identification of human FOXP3 target genes squamous cell carcinoma of the head and neck: in natural regulatory T cells. J Immunol. 2010 Jul mechanisms of suppression and expansion in advanced 15;185(2):1071-81 disease. Clin Cancer Res. 2008 Jun 15;14(12):3706-15 Wang LH, Su L, Wang JT. Correlation between elevated Ebert LM, Tan BS, Browning J, Svobodova S, Russell SE, FOXP3 expression and increased lymph node metastasis Kirkpatrick N, Gedye C, Moss D, Ng SP, MacGregor D, of gastric cancer. Chin Med J (Engl). 2010 Davis ID, Cebon J, Chen W. The regulatory T cell- Dec;123(24):3545-9 associated transcription factor FoxP3 is expressed by Xue L, Lu HQ, He J, Zhao XW, Zhong L, Zhang ZZ, Xu ZF. tumor cells. Cancer Res. 2008 Apr 15;68(8):3001-9 Expression of FOXP3 in esophageal squamous cell Karanikas V, Speletas M, Zamanakou M, Kalala F, Loules carcinoma relating to the clinical data. Dis Esophagus. G, Kerenidi T, Barda AK, Gourgoulianis KI, Germenis AE. 2010 May;23(4):340-6 Foxp3 expression in human cancer cells. J Transl Med. Zhang HY, Sun H. Up-regulation of Foxp3 inhibits cell 2008 Apr 22;6:19 proliferation, migration and invasion in epithelial ovarian Ladoire S, Arnould L, Apetoh L, Coudert B, Martin F, cancer. Cancer Lett. 2010 Jan 1;287(1):91-7 Chauffert B, Fumoleau P, Ghiringhelli F. Pathologic Dimitrakopoulos FI, Papadaki H, Antonacopoulou AG, complete response to neoadjuvant chemotherapy of breast Kottorou A, Gotsis AD, Scopa C, Kalofonos HP, Mouzaki carcinoma is associated with the disappearance of tumor- A. Association of FOXP3 expression with non-small cell infiltrating foxp3+ regulatory T cells. Clin Cancer Res. 2008 lung cancer. Anticancer Res. 2011 May;31(5):1677-83 Apr 15;14(8):2413-20 Ladoire S, Martin F, Ghiringhelli F. Prognostic role of Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory FOXP3+ regulatory T cells infiltrating human carcinomas: T cells and immune tolerance. Cell. 2008 May the paradox of colorectal cancer. Cancer Immunol 30;133(5):775-87 Immunother. 2011 Jul;60(7):909-18 Whiteside TL. The tumor microenvironment and its role in Li W, Wang L, Katoh H, Liu R, Zheng P, Liu Y. promoting tumor growth. Oncogene. 2008 Oct Identification of a tumor suppressor relay between the 6;27(45):5904-12 FOXP3 and the Hippo pathways in breast and prostate Hancock WW, Ozkaynak E. Three distinct domains cancers. Cancer Res. 2011 Mar 15;71(6):2162-71 contribute to nuclear transport of murine Foxp3. PLoS Liang YJ, Liu HC, Su YX, Zhang TH, Chu M, Liang LZ, One. 2009 Nov 18;4(11):e7890 Liao GQ. Foxp3 expressed by tongue squamous cell Li JF, Chu YW, Wang GM, Zhu TY, Rong RM, Hou J, Xu carcinoma cells correlates with clinicopathologic features M. The prognostic value of peritumoral regulatory T cells and overall survival in tongue squamous cell carcinoma and its correlation with intratumoral cyclooxygenase-2 patients. Oral Oncol. 2011 Jul;47(7):566-70 expression in clear cell renal cell carcinoma. BJU Int. 2009 Quaglino P, Osella-Abate S, Marenco F, Nardò T, Gado C, Feb;103(3):399-405 Novelli M, Savoia P, Bernengo MG. FoxP3 expression on Mailer RK, Falk K, Rötzschke O. Absence of leucine zipper melanoma cells is related to early visceral spreading in in the natural FOXP3Delta2Delta7 isoform does not affect melanoma patients treated by electrochemotherapy. dimerization but abrogates suppressive capacity. PLoS Pigment Cell Melanoma Res. 2011 Aug;24(4):734-6 One. 2009 Jul 1;4(7):e6104 Tan W, Zhang W, Strasner A, Grivennikov S, Cheng JQ, Merlo A, Casalini P, Carcangiu ML, Malventano C, Triulzi Hoffman RM, Karin M. Tumour-infiltrating regulatory T cells T, Mènard S, Tagliabue E, Balsari A. FOXP3 expression stimulate mammary cancer metastasis through RANKL- and overall survival in breast cancer. J Clin Oncol. 2009 RANK signalling. Nature. 2011 Feb 24;470(7335):548-53 Apr 10;27(11):1746-52 Winerdal ME, Marits P, Winerdal M, Hasan M, Rosenblatt Wang L, Liu R, Li W, Chen C, Katoh H, Chen GY, McNally R, Tolf A, Selling K, Sherif A, Winqvist O. FOXP3 and B, Lin L, Zhou P, Zuo T, Cooney KA, Liu Y, Zheng P. survival in urinary bladder cancer. BJU Int. 2011 Nov;108(10):1672-8

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 238 FOXP3 expression in tumor cells and its role in cancer Uva V, et al. progression

Facciabene A, Motz GT, Coukos G. T-regulatory cells: key Patel P, Williams MM, Boczkowski D, Lyerly HK, Morse players in tumor immune escape and angiogenesis. MA, Devi GR. Immunologic targeting of FOXP3 in Cancer Res. 2012 May 1;72(9):2162-71 inflammatory breast cancer cells. PLoS One. 2013;8(1):e53150 Tao H, Mimura Y, Aoe K, Kobayashi S, Yamamoto H, Matsuda E, Okabe K, Matsumoto T, Sugi K, Ueoka H. Reginato E, Mroz P, Chung H, Kawakubo M, Wolf P, Prognostic potential of FOXP3 expression in non-small cell Hamblin MR. Photodynamic therapy plus regulatory T- celldepletion produces immunity against a mouse tumour lung cancer cells combined with tumor-infiltrating that expresses a self-antigen. Br J Cancer. 2013 Oct regulatory T cells. Lung Cancer. 2012 Jan;75(1):95-101 15;109(8):2167-74 Wang G, Liu G, Liu Y, Li X, Su Z. FOXP3 expression in Takenaka M, Seki N, Toh U, Hattori S, Kawahara A, esophageal cancer cells is associated with poor prognosis Yamaguchi T, Koura K, Takahashi R, Otsuka H, Takahashi in esophageal cancer. Hepatogastroenterology. 2012 H, Iwakuma N, Nakagawa S, Fujii T, Sasada T, Oct;59(119):2186-91 Yamaguchi R, Yano H, Shirouzu K, Kage M. FOXP3 Whiteside TL. What are regulatory T cells (Treg) regulating expression in tumor cells and tumor-infiltrating in cancer and why? Semin Cancer Biol. 2012 lymphocytes is associated with breast cancer prognosis. Aug;22(4):327-34 Mol Clin Oncol. 2013 Jul;1(4):625-632 Yoshii M, Tanaka H, Ohira M, Muguruma K, Iwauchi T, Won KY, Kim HS, Sung JY, Kim GY, Lee J, Park YK, Kim Lee T, Sakurai K, Kubo N, Yashiro M, Sawada T, YW, Suh JH, Lim SJ. Tumoral FOXP3 has potential Hirakawa K. Expression of Forkhead box P3 in tumour oncogenic function in conjunction with the p53 tumor cells causes immunoregulatory function of signet ring cell suppressor protein and infiltrated Tregs in human breast carcinoma of the stomach. Br J Cancer. 2012 May carcinomas. Pathol Res Pract. 2013 Dec;209(12):767-73 8;106(10):1668-74 Zhou S, Chen L, Qin J, Li R, Tao H, Zhen Z, Chen H, Chen Fu HY, Li C, Yang W, Gai XD, Jia T, Lei YM, Li Y. FOXP3 G, Yang Y, Liu B, She Z, Zhong C, Liang C. Depletion of and TLR4 protein expression are correlated in non-small CD4+ CD25+ regulatory T cells promotes CCL21- cell lung cancer: implications for tumor progression and mediated antitumor immunity. PLoS One. escape. Acta Histochem. 2013 Mar;115(2):151-7 2013;8(9):e73952 Grimmig T, Kim M, Germer CT, Gasser M, Waaga-Gasser Hao Q, Zhang C, Gao Y, Wang S, Li J, Li M, Xue X, Li W, AM. The role of FOXP3 in disease progression in Zhang W, Zhang Y. FOXP3 inhibits NF-κB activity and colorectal cancer patients. Oncoimmunology. 2013 Jun hence COX2 expression in gastric cancer cells. Cell 1;2(6):e24521 Signal. 2014 Mar;26(3):564-9 Kim M, Grimmig T, Grimm M, Lazariotou M, Meier E, Jarry U, Donnou S, Vincent M, Jeannin P, Pineau L, Rosenwald A, Tsaur I, Blaheta R, Heemann U, Germer Fremaux I, Delneste Y, Couez D. Treg depletion followed CT, Waaga-Gasser AM, Gasser M. Expression of Foxp3 in by intracerebral CpG-ODN injection induce brain tumor colorectal cancer but not in Treg cells correlates with rejection. J Neuroimmunol. 2014 Feb 15;267(1-2):35-42 disease progression in patients with colorectal cancer. Keenan BP, Saenger Y, Kafrouni MI, Leubner A, Lauer P, PLoS One. 2013;8(1):e53630 Maitra A, Rucki AA, Gunderson AJ, Coussens LM, Kim MH, Koo JS, Lee S. FOXP3 expression is related to Brockstedt DG, Dubensky TW Jr, Hassan R, Armstrong high Ki-67 index and poor prognosis in lymph node- TD, Jaffee EM. A Listeria vaccine and depletion of T- positive breast cancer patients. Oncology. 2013;85(2):128- regulatory cells activate immunity against early stage 36 pancreatic intraepithelial neoplasms and prolong survival of mice. Gastroenterology. 2014 Jun;146(7):1784-94.e6 Li W, Katoh H, Wang L, Yu X, Du Z, Yan X, Zheng P, Liu Y. FOXP3 regulates sensitivity of cancer cells to irradiation Wang L, Zhang B, Xu X, Zhang S, Yan X, Kong F, Feng X, by transcriptional repression of BRCA1. Cancer Res. 2013 Wang J. Clinical significance of FOXP3 expression in Apr 1;73(7):2170-80 human gliomas. Clin Transl Oncol. 2014 Jan;16(1):36-43 Ma GF, Chen SY, Sun ZR, Miao Q, Liu YM, Zeng XQ, Luo This article should be referenced as such: TC, Ma LL, Lian JJ, Song DL. FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating Uva V, Sfondrini L, Triulzi T, Casalini P, Tagliabue E, the apoptotic signaling pathway. Biochem Biophys Res Balsari A. FOXP3 expression in tumor cells and its role in Commun. 2013 Jan 11;430(2):804-9 cancer progression. Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3):234-239. Nair S, Aldrich AJ, McDonnell E, Cheng Q, Aggarwal A,

Atlas Genet Cytogenet Oncol Haematol. 2015; 19(3) 239 Atlas of Genetics and Cytogenetics in Oncology and Haematology

OPEN ACCESS JOURNAL 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