Volume 15 - Number 8 August 2011

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Scope

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

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

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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.

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Editor

Jean-Loup Huret (Poitiers, France) Editorial Board

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

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Volume 15, Number 8, August 2011

Table of contents

Gene Section

ING4 (inhibitor of growth family, member 4) 620 Angela Greco, Claudia Miranda LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) 625 Dongsheng Guo, Baofeng Wang MAPK14 (mitogen-activated protein kinase 14) 628 Almudena Porras, Carmen Guerrero RAD51L3 (RAD51-like 3 (S. cerevisiae)) 632 Mary K Taylor, Michael K Bendenbaugh, Susan M Brown, Brian D Yard, Douglas L Pittman SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) 637 Wendy S McDonough, Michael E Berens USP15 (ubiquitin specific peptidase 15) 645 Monica Faronato, Sylvie Urbé, Judy M Coulson CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) 652 Joanna Fares, Linda Wolff, Juraj Bies DLX4 (distal-less homeobox 4) 658 Patricia E Berg, Saurabh Kirolikar IL17A (interleukin 17A) 662 Norimitsu Inoue, Takashi Akazawa MYBBP1A (MYB binding protein (P160) 1a) 667 Claudia Perrera, Riccardo Colombo PLCD1 (phospholipase C, delta 1) 670 Xiaotong Hu PYY (peptide YY) 674 Maria Braoudaki, Fotini Tzortzatou-Stathopoulou SIAH2 (seven in absentia homolog 2 (Drosophila)) 677 Jianfei Qi, Ze'ev Ronai TP53BP2 (tumor protein p53 binding protein, 2) 681 Kathryn Van Hook, Zhiping Wang, Charles Lopez

Leukaemia Section

8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome) 686 Paula Aranaz, José Luis Vizmanos i(5)(p10) in acute myeloid leukemia 695 Nathalie Douet-Guilbert, Angèle Herry, Audrey Basinko, Marie-Josée Le Bris, Nadia Guéganic, Clément Bovo, Frédéric Morel, Marc De Braekeleer

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) Atlast(11;14)(q13;q32) of Genetics in multiple myeloma and Cytogenetics Huret JL, Laï JL in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

+20 or trisomy 20 (solely) 697 Jean-Loup Huret

Deep Insight Section

TMPRSS2:ETS gene fusions in prostate cancer 699 Julia L Williams, Maisa Yoshimoto, Alexander H Boag, Jeremy A Squire, Paul C Park

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) Atlas of Genetics and Cytogenetics

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Gene Section Review

ING4 (inhibitor of growth family, member 4) Angela Greco, Claudia Miranda Dept Experimental Oncology, Molecular Mechanisms Unit, Istituto Nazionale Tumori IRCCS Foundation - via Venezian 1 - 20133 Milan Italy (AG, CM)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/ING4ID40978ch12p13.html DOI : 10.4267/2042/45999 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

homology search for expressed tag clones with a PHD Identity finger motif (Shiseki et al., 2003). ING4 gene is located Other names: MGC12557; my036; p29ING4 on chromosome 12p13.31 and consists of eight exons HGNC (Hugo): ING4 encoding a 29-kDa protein expressed in multiple human tissues. Location: 12p13.31 Transcription DNA/RNA Multiple alternatively spliced transcript variants have been observed using different splice sites in the coding Description region; transcript variants span from 1461 bp to 1313 ING4 belongs to family of highly homologous five bp. members containing PHD domain and has been Wobble splicing events have been described at exon 4 identified through a computational sequence and 5 boundary.

Figure adapted from Atlas of Genetics and Cytogenetics in Oncology and Haematology.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 620 ING4 (inhibitor of growth family, member 4) Greco A, Miranda C

Different splicing variants have been identified (among regulation, chromatin remodeling, and regulation of them -v1, -v2, -v3 and -v4/ ∆4AA) involving 12 bp gene expression. Several ING4 partners have been (379-390) and resulting in in frame deletions of one to described. Similarly to the other ING members, ING4 four aminoacids in NLS (Tsai and Lin, 2006). was described to interact with p53 and to modulate p53 Several splicing variants have been described lacking transcriptional activity (Shiseki et al., 2003). The exon 2, 3 and 6 (entirely or in part), and named ING4- interaction of ING4 with p53 is mediated by the ∆Ex2, -∆Ex3, -∆Ex6A and -∆Ex6B, respectively (Raho bipartite ING4 nuclear localization signal (NLS) et al., 2007). (Zhang et al., 2005) and drives an increase of p53 Splicing variants have been detected in all tissues acetylation at lysine 382 (Shiseki et al., 2003). ING4 is analysed, indicating that are not tissue specific (Tsai a critical regulator of chromatin acetylation required for and Lin, 2006; Raho et al., 2007). gene expression. In particular, ING4 associates with the More recently five novel spliced variants of ING4-v1 HAT complex HBO1 and it is required for the majority and -v2 were identified, causing codon frame shift and of histone 4 acetylation and for normal progression eventually deletion of NLS or PHD domains. Increased through S phase (Doyon et al., 2006; Shi et al., 2006). expression of these variants was found in gastric Recently a critical role for specific recognition of adenocarcinomas compared to normal tissue (Li M et histone H3 trimethylated at lysine 4 (H3K4me3) by the al., 2009). ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions has been shown (Hung Protein et al., 2009). ING4 can also function as repressor of factors Note mediating angiogenesis. It was demonstrated that ING4 249 aminoacids, 29 kDa protein. plays an inhibitory role on NF-kappaB activity by Description interaction with p65NF-kappaB and that the lack of inhibition of the NF-kappaB pathway by ING4 results ING4 protein contains several conserved regions: i) a in increased angiogenesis in glioblastomas (Garkavtsev leucine zipper-like (LZL) domain, probably involved in et al., 2004). More recently, it has been described that protein interactions, located at the N-terminus; ii) a physiologic levels of ING4 govern innate immunity in functional bipartite nuclear localization signal (NLS1); mice by regulating the levels of IkappaB and NF- iii) a C-terminal plant homeo-domain (PHD), a Cys4- kappaB proteins and the activation of select cytokine His-Cys3 zinc finger motif spanning 50-80 residues, promoters (Coles et al., 2010). found in many nuclear proteins, such as transcription ING4 was also described to repress the ability of factors and proteins regulating chromatin structure; iv) hypoxia inducible factor (HIF)-1 to activate a non functional NLS located at the C-terminal end. transcription of its downstream target genes by Expression interacting with the HPH-2 prolyl hydroxylase. Under Ubiquitous. hypoxic conditions, ING4 may act as an adapter protein recruiting transcriptional repressors to mediate HIF Localisation activity (Ozer et al., 2005). p29ING4 is a nuclear protein. It possesses a bipartite Involvement of ING4 in regulation of apoptosis has nuclear localization signal. ING4 splicing variants have been demonstrated in several cellular systems. Its been described involving the NLS1 domain; most/all of overexpression can induce apoptosis through the them retains nuclear localization. Furthermore ING4-v1 downregulation of Bcl-2 and the upregulation of p21 is translocated to the nucleolus and such subcellular and Bax expression (Shiseki et al., 2003; Yu et al., localization is modulated by two wobble-splicing 2007; Li X et al., 2009b; Cai et al., 2009). events at the exon 4-5 boundary, causing displacement from the nucleolus to the nucleus. Homology ING4 protein shares homology with other ING family Function members with respect to the following regions: i) a ING4 was also isolated through a screening for genes leucine zipper-like (LZL) domain, probably involved in able to suppress loss of contact inhibition, thus interaction with proteins, located at the N-terminus of suggesting its tumor suppressor role. all the ING proteins except for ING1; ii) a nuclear ING4 is a nuclear protein participating to a variety of localization signal (NLS); iii) a C-terminal plant cellular functions, such as apoptosis, cell-cycle homeo-domain (PHD) involved in chromatin.

LZL: leucine zipper-like; NLS1: nuclear localization signal 1; PHD: plant homology domain; NLS2: nuclear localization signal 2.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 621 ING4 (inhibitor of growth family, member 4) Greco A, Miranda C

tumors. Decreased ING4 expression correlated Mutations significantly with tumor progression, with lower Note expression levels of ING4 observed in cases of high- The following ING4 point mutations have been found grade neoplasms. A statistical significant negative in lung adenocarcinoma and small cell lung carcinoma correlation between expression of ING4 and expression (H23 and H28, respectively) human cancer cell lines. of nuclear p65 was noticed (Klironomos et al., 2010). N214D: it alters ING4 capability of inhibition of Hepatocellular carcinoma (HCC) proliferation, anchorage independent cell migration Prognosis reducing protein stability by proteasome mediated degradation. Survival and metastasis analysis indicated that HCC Y121N: it does not alter ING4 functions (Moreno et al., patients with lower ING4 expression had poorer overall 2010). and disease-free survival than those with high expression. Multivariable Cox regression analysis Implicated in revealed that the ING4 expression level was an independent factor for prognosis (Fang et al., 2009). Breast cancer Oncogenesis Cytogenetics The ING4 mRNA and protein levels were significantly Analysis of CGH data revealed that 10-20% of primary lower in HCC than paracarcinomatous liver tissue. breast tumors present deletions in 12p13. The deletions ING4 expression level correlates with prognosis and appear to affect only one copy of the gene; no genomic metastatic potential, suggesting that ING4 as a mutations were found in the remaining allele of ING4 candidate prognostic marker of HCC (Fang et al., (Kim et al., 2004). 2009). Head and neck squamous cell Multiple myeloma (MM) carcinoma (HNSCC) Prognosis Cytogenetics MM patients with high IL-8 production and microvascular density (MVD) have significantly lower LOH of 12p13. ING4 levels compared with those with low IL-8 and Oncogenesis MVD. Loss of heterozygosity at 12p12-13 region was found in Oncogenesis 66% (33/50) of head and neck squamous cell ING4 suppression in MM cells up-regulated IL-8 and carcinomas by using six highly polymorphic OPN under hypoxic conditions, increasing the hypoxia microsatellite markers. No mutations of the ING4 gene inducible factor-1alpha (HIF-1alpha) activity and its were found. target gene NIP-3 expression. ING4 suppression in Quantitative real-time RT-PCR analysis demonstrated MM cells significantly increased vessel formation in decreased expression of ING4 mRNA in 76% of vitro, blunted by blocking IL-8 or OPN (Colla et al., primary tumors compared to matched normal samples 2007). (Gunduz et al., 2005). Glioma Lung cancer Oncogenesis Oncogenesis Reduced ING4 nuclear and cytoplasmic expression Expression of ING4 is significantly reduced in gliomas were both revealed in lung cancer and associated with as compared with normal human brain tissue, and the tumour grade. ING4 expression in the cytoplasm was extent of reduction correlates with the progression from found higher than in the nucleus in a high percentage of lower to higher grades of tumours. ING4 regulates tumors. Nuclear ING4 inhibition correlated with the brain tumour angiogenesis through transcriptional tumour stage and lymph node metastasis, thus repression of NF-kB-responsive genes (Garkavtsev et suggesting that ING4 is involved in the initiation and al., 2004). progression of lung cancers (Wang et al., 2010). Astrocytoma Gastric cancer Prognosis Oncogenesis A potential of role of ING4 as a biomarker for the ING4 RNA and protein were drastically reduced in prediction of the grade of astrocytic neoplasms has stomach adenocarcinoma cell lines and tissues, been suggested (Klironomos et al., 2010). significantly less in female than male patients. Novel Oncogenesis spliced forms of ING4-v1 and -v2 were identified in Significantly reduced levels of ING4 were observed in both normal and tumor tissue; increased expression of human astrocytomas compared to normal brain tissue, the novel spliced variants was observed in tumors; suggesting that down-regulation of this protein might however no correlation with clinical parameters was be involved in the pathogenesis of human astrocytic observed (Li M et al., 2009).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 622 ING4 (inhibitor of growth family, member 4) Greco A, Miranda C

Li X, Cai L, Liang M, Wang Y, Yang J, Zhao Y. ING4 induces References cell growth inhibition in human lung adenocarcinoma A549 cells by means of Wnt-1/beta-catenin signaling pathway. Anat Shiseki M, Nagashima M, Pedeux RM, Kitahama-Shiseki M, Rec (Hoboken). 2008 May;291(5):593-600 Miura K, Okamura S, Onogi H, Higashimoto Y, Appella E, Yokota J, Harris CC. p29ING4 and p28ING5 bind to p53 and Nozell S, Laver T, Moseley D, Nowoslawski L, De Vos M, p300, and enhance p53 activity. Cancer Res. 2003 May Atkinson GP, Harrison K, Nabors LB, Benveniste EN. The 15;63(10):2373-8 ING4 tumor suppressor attenuates NF-kappaB activity at the promoters of target genes. Mol Cell Biol. 2008 Garkavtsev I, Kozin SV, Chernova O, Xu L, Winkler F, Brown Nov;28(21):6632-45 E, Barnett GH, Jain RK. The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis. Palacios A, Muñoz IG, Pantoja-Uceda D, Marcaida MJ, Torres Nature. 2004 Mar 18;428(6980):328-32 D, Martín-García JM, Luque I, Montoya G, Blanco FJ. Molecular basis of histone H3K4me3 recognition by ING4. J Kim S, Chin K, Gray JW, Bishop JM. A screen for genes that Biol Chem. 2008 Jun 6;283(23):15956-64 suppress loss of contact inhibition: identification of ING4 as a candidate tumor suppressor gene in human cancer. Proc Natl Tsai KW, Tseng HC, Lin WC. Two wobble-splicing events Acad Sci U S A. 2004 Nov 16;101(46):16251-6 affect ING4 protein subnuclear localization and degradation. Exp Cell Res. 2008 Oct 15;314(17):3130-41 Gunduz M, Nagatsuka H, Demircan K, Gunduz E, Cengiz B, Ouchida M, Tsujigiwa H, Yamachika E, Fukushima K, Beder L, Cai L, Li X, Zheng S, Wang Y, Wang Y, Li H, Yang J, Sun J. Hirohata S, Ninomiya Y, Nishizaki K, Shimizu K, Nagai N. Inhibitor of growth 4 is involved in melanomagenesis and Frequent deletion and down-regulation of ING4, a candidate induces growth suppression and apoptosis in melanoma cell tumor suppressor gene at 12p13, in head and neck squamous line M14. Melanoma Res. 2009 Feb;19(1):1-7 cell carcinomas. Gene. 2005 Aug 15;356:109-17 Fang F, Luo LB, Tao YM, Wu F, Yang LY. Decreased Ozer A, Bruick RK. Regulation of HIF by prolyl hydroxylases: expression of inhibitor of growth 4 correlated with poor recruitment of the candidate tumor suppressor protein ING4. prognosis of hepatocellular carcinoma. Cancer Epidemiol Cell Cycle. 2005 Sep;4(9):1153-6 Biomarkers Prev. 2009 Feb;18(2):409-16 Ozer A, Wu LC, Bruick RK. The candidate tumor suppressor Hung T, Binda O, Champagne KS, Kuo AJ, Johnson K, Chang ING4 represses activation of the hypoxia inducible factor (HIF). HY, Simon MD, Kutateladze TG, Gozani O. ING4 mediates Proc Natl Acad Sci U S A. 2005 May 24;102(21):7481-6 crosstalk between histone H3 K4 trimethylation and H3 acetylation to attenuate cellular transformation. Mol Cell. 2009 Zhang X, Wang KS, Wang ZQ, Xu LS, Wang QW, Chen F, Wei Jan 30;33(2):248-56 DZ, Han ZG. Nuclear localization signal of ING4 plays a key role in its binding to p53. Biochem Biophys Res Commun. Li M, Jin Y, Sun WJ, Yu Y, Bai J, Tong DD, Qi JP, Du JR, 2005 Jun 17;331(4):1032-8 Geng JS, Huang Q, Huang XY, Huang Y, Han FF, Meng XN, Rosales JL, Lee KY, Fu SB. Reduced expression and novel Doyon Y, Cayrou C, Ullah M, Landry AJ, Côté V, Selleck W, splice variants of ING4 in human gastric adenocarcinoma. J Lane WS, Tan S, Yang XJ, Côté J. ING tumor suppressor Pathol. 2009 Sep;219(1):87-95 proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation. Mol Cell. 2006 Jan Li X, Cai L, Chen H, Zhang Q, Zhang S, Wang Y, Dong Y, 6;21(1):51-64 Cheng H, Qi J. Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG. Pathobiology. Shi X, Hong T, Walter KL, Ewalt M, Michishita E, Hung T, 2009a;76(4):181-92 Carney D, Peña P, Lan F, Kaadige MR, Lacoste N, Cayrou C, Davrazou F, Saha A, Cairns BR, Ayer DE, Kutateladze TG, Shi Li X, Zhang Q, Cai L, Wang Y, Wang Q, Huang X, Fu S, Bai J, Y, Côté J, Chua KF, Gozani O. ING2 PHD domain links Liu J, Zhang G, Qi J. Inhibitor of growth 4 induces apoptosis in histone H3 lysine 4 methylation to active gene repression. human lung adenocarcinoma cell line A549 via Bcl-2 family Nature. 2006 Jul 6;442(7098):96-9 proteins and mitochondria apoptosis pathway. J Cancer Res Clin Oncol. 2009b Jun;135(6):829-35 Tsai KW, Lin WC. Quantitative analysis of wobble splicing indicates that it is not tissue specific. Genomics. 2006 Tzouvelekis A, Aidinis V, Harokopos V, Karameris A, Zacharis Dec;88(6):855-64 G, Mikroulis D, Konstantinou F, Steiropoulos P, Sotiriou I, Froudarakis M, Pneumatikos I, Tringidou R, Bouros D. Down- Unoki M, Shen JC, Zheng ZM, Harris CC. Novel splice variants regulation of the inhibitor of growth family member 4 (ING4) in of ING4 and their possible roles in the regulation of cell growth different forms of pulmonary fibrosis. Respir Res. 2009 Feb and motility. J Biol Chem. 2006 Nov 10;281(45):34677-86 27;10:14 Colla S, Tagliaferri S, Morandi F, Lunghi P, Donofrio G, Coles AH, Gannon H, Cerny A, Kurt-Jones E, Jones SN. Martorana D, Mancini C, Lazzaretti M, Mazzera L, Ravanetti L, Inhibitor of growth-4 promotes IkappaB promoter activation to Bonomini S, Ferrari L, Miranda C, Ladetto M, Neri TM, Neri A, suppress NF-kappaB signaling and innate immunity. Proc Natl Greco A, Mangoni M, Bonati A, Rizzoli V, Giuliani N. The new Acad Sci U S A. 2010 Jun 22;107(25):11423-8 tumor-suppressor gene inhibitor of growth family member 4 (ING4) regulates the production of proangiogenic molecules by Kim S, Welm AL, Bishop JM. A dominant mutant allele of the myeloma cells and suppresses hypoxia-inducible factor-1 ING4 tumor suppressor found in human cancer cells alpha (HIF-1alpha) activity: involvement in myeloma-induced exacerbates MYC-initiated mouse mammary tumorigenesis. angiogenesis. Blood. 2007 Dec 15;110(13):4464-75 Cancer Res. 2010 Jun 15;70(12):5155-62 Raho G, Miranda C, Tamborini E, Pierotti MA, Greco A. Klironomos G, Bravou V, Papachristou DJ, Gatzounis G, Detection of novel mRNA splice variants of human ING4 tumor Varakis J, Parassi E, Repanti M, Papadaki H. Loss of inhibitor suppressor gene. Oncogene. 2007 Aug 9;26(36):5247-57 of growth (ING-4) is implicated in the pathogenesis and progression of human astrocytomas. Brain Pathol. 2010 Yu X, Zhang HF, Wang JZ, Xie YF, Yang JC, Miao JC. [Ad- Mar;20(2):490-7 ING4 inhibits K562 cell growth]. Zhonghua Xue Ye Xue Za Zhi. 2007 Jun;28(6):396-400

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 623 ING4 (inhibitor of growth family, member 4) Greco A, Miranda C

Moreno A, Palacios A, Orgaz JL, Jimenez B, Blanco FJ, Wang QS, Li M, Zhang LY, Jin Y, Tong DD, Yu Y, Bai J, Palmero I. Functional impact of cancer-associated mutations in Huang Q, Liu FL, Liu A, Lee KY, Fu SB. Down-regulation of the tumor suppressor protein ING4. Carcinogenesis. 2010 ING4 is associated with initiation and progression of lung Nov;31(11):1932-8 cancer. Histopathology. 2010 Aug;57(2):271-81

Palacios A, Moreno A, Oliveira BL, Rivera T, Prieto J, García This article should be referenced as such: P, Fernández-Fernández MR, Bernadó P, Palmero I, Blanco FJ. The dimeric structure and the bivalent recognition of Greco A, Miranda C. ING4 (inhibitor of growth family, member H3K4me3 by the tumor suppressor ING4 suggests a 4). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):620- mechanism for enhanced targeting of the HBO1 complex to 624. chromatin. J Mol Biol. 2010 Mar 5;396(4):1117-27

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 624 Atlas of Genetics and Cytogenetics

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Gene Section Mini Review

LRIG1 (leucine -rich repeats and immunoglobulin - like domains 1) Dongsheng Guo, Baofeng Wang Dept of Neurosurgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, 430030, People's Republic of China (DG, BW)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/LRIG1ID41198ch3p14.html DOI: 10.4267/2042/46009 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

by proteolytic processing, which also is a functional Identity molecule. Other names: DKFZp586O1624; LIG-1; LIG1 Expression HGNC (Hugo): LRIG1 LRIG1 is expressed ubiquitously in various epithelial Location: 3p14.1 cells, endothelial cells, heart, smooth and striated muscles, and in large neurons. DNA/RNA Localisation Description Differential subcellular distribution in a cell type- specific manner. Genomic DNA encoding LRIG1 spans a region of 122.89 kilobases on chromosome 3, at 3p14. LRIG1 Function gene is encoded on the reverse strand. LRIG1 acts as a suppressor of receptor tyrosine Transcription kinases, such as epidermal growth factor receptor (EGFR) family, MET (hepatocyte growth factor The pre-mRNA comprises 19 exons. receptor), and RET. The interaction of the extracellular Coding sequence: 4812 bp. LRR domain and immunoglobulin-like domains of LRIG1 with the extracellular parts of the human EGFR Protein results in recruitment of c-Cbl to the cytoplasmic Description domains, and induction of EGFR degradation. LRIG1 is involved in signal transduction, cell proliferation, LRIG1 is a transmembrane cell-surface protein cell apopotosis, cell cycle, cell migration, and cell consisting of 1093 amino acids. LRIG1 contains invasion. LRIG1 as a putative tumor suppressor gene extracellular part containing 15 leucine-rich repeats often be down-regulated in various human tumors. (LRR) and three C2-type immunoglobulin-like Soluble ectodomain of LRIG1 protein can modulate domains, a transmembrane region, and a cytoplasmic EGFR signaling and its growth-promoting activity in a tail. LRIG1 can be cut into soluble LRIG1 ectodomain paracrine fashion.

LRIG1 gene. Exons are represented by red boxes (in scale). Exons 1 to 19 are from the 5' to 3' direction.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 625 LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) Guo D, Wang B

LRIG1 protein. SP: signal peptide; NF: N-terminal cysteine-rich flanking domain; LRR: leucine-rich repeat; CF: C-terminal cysteine-rich flanking domain; Ig C2: C2-type immunoglobulin-like domains; TM: transmembrane domain; Cyto: cytoplasmic domain.

Implicated in surfaces in the spinous layers. Ependymoma References Note Nilsson J, Vallbo C, Guo D, Golovleva I, Hallberg B, Higher cytoplasmic immunoreactivity of LRIG1 Henriksson R, Hedman H. Cloning, characterization, and expression of human LIG1. Biochem Biophys Res Commun. correlates with older patient age and higher LRIG1 2001 Jun 29;284(5):1155-61 nuclear immunoreactivity with lower WHO grade. Nilsson J, Starefeldt A, Henriksson R, Hedman H. LRIG1 Prostate cancer protein in human cells and tissues. Cell Tissue Res. 2003 Apr;312(1):65-71 Note High LRIG1 expression is significantly associated with Thomasson M, Hedman H, Guo D, Ljungberg B, Henriksson R. LRIG1 and epidermal growth factor receptor in renal cell short overall and prostate cancer-specific survival for carcinoma: a quantitative RT--PCR and immunohistochemical 256 Swedish patients analysed. In contrast, in the U.S. analysis. Br J Cancer. 2003 Oct 6;89(7):1285-9 series, high LRIG1 expression is significantly Guo D, Holmlund C, Henriksson R, Hedman H. The LRIG gene associated with longer overall survival. family has three vertebrate paralogs widely expressed in human and mouse tissues and a homolog in Ascidiacea. Renal cell carcinoma (RCC) Genomics. 2004 Jul;84(1):157-65 Note Gur G, Rubin C, Katz M, Amit I, Citri A, Nilsson J, Amariglio N, LRIG1 expression is generally downregulated in Henriksson R, Rechavi G, Hedman H, Wides R, Yarden Y. conventional and papillary RCC but not in LRIG1 restricts growth factor signaling by enhancing receptor chromophobic RCC. ubiquitylation and degradation. EMBO J. 2004 Aug 18;23(16):3270-81 Cutaneous squamous cell carcinoma Laederich MB, Funes-Duran M, Yen L, Ingalla E, Wu X, (SCC) Carraway KL 3rd, Sweeney C. The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB family receptor tyrosine Note kinases. J Biol Chem. 2004 Nov 5;279(45):47050-6 LRIG-1 expression is highest in well-differentiated Ye F, Guo DS, Niu HQ, Tao SZ, Ou YB, Lu YP, Lei T. lesions of cutaneous SCC. LRIG-1 expression intensity [Molecular mechanism of LRIG1 cDNA-induced apoptosis in of tumor cells is significantly correlated with histologic human glioma cell line H4]. Ai Zheng. 2004 Oct;23(10):1149- differentiation of SCC. The SCC patients have 54 significant survival benefits in the high LRIG-1 Tanemura A, Nagasawa T, Inui S, Itami S. LRIG-1 provides a expression groups compared with low LRIG-1 novel prognostic predictor in squamous cell carcinoma of the expression groups. skin: immunohistochemical analysis for 38 cases. Dermatol Surg. 2005 Apr;31(4):423-30 Breast tumor Guo D, Nilsson J, Haapasalo H, Raheem O, Bergenheim T, Note Hedman H, Henriksson R. Perinuclear leucine-rich repeats and LRIG1 protein levels are significantly suppressed in the immunoglobulin-like domain proteins (LRIG1-3) as prognostic indicators in astrocytic tumors. Acta Neuropathol. 2006 majority of human breast tumors expressing ErbB2. Mar;111(3):238-46 Cervical squamous cell carcinoma Jensen KB, Watt FM. Single-cell expression profiling of human Note epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence. Proc Natl Acad Sci U S A. 2006 Aug LRIG1 appears to be a significant prognosis predictor 8;103(32):11958-63 in early-stage cervical cancer, independent of the other tumor markers. Yang WM, Yan ZJ, Ye ZQ, Guo DS. LRIG1, a candidate tumour-suppressor gene in human bladder cancer cell line Astrocytic tumor BIU87. BJU Int. 2006 Oct;98(4):898-902 Note Goldoni S, Iozzo RA, Kay P, Campbell S, McQuillan A, Agnew C, Zhu JX, Keene DR, Reed CC, Iozzo RV. A soluble Perinuclear staining of LRIG1 is associated with low ectodomain of LRIG1 inhibits cancer cell growth by attenuating WHO grade and better survival of the patients. basal and ligand-dependent EGFR activity. Oncogene. 2007 Psoriasis Jan 18;26(3):368-81 Guo D, Han L, Shu K, Chen J, Lei T. Down-regulation of Note leucine-rich repeats and immunoglobulin-like domain proteins In psoriasis, LRIG1 is mostly absent from the cell

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 626 LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) Guo D, Wang B

(LRIG1-3) in HP75 pituitary adenoma cell line. J Huazhong Suppression of the negative regulator LRIG1 contributes to Univ Sci Technolog Med Sci. 2007 Feb;27(1):91-4 ErbB2 overexpression in breast cancer. Cancer Res. 2008 Oct 15;68(20):8286-94 Hedman H, Henriksson R. LRIG inhibitors of growth factor signalling - double-edged swords in human cancer? Eur J Stutz MA, Shattuck DL, Laederich MB, Carraway KL 3rd, Cancer. 2007 Mar;43(4):676-82 Sweeney C. LRIG1 negatively regulates the oncogenic EGF receptor mutant EGFRvIII. Oncogene. 2008 Sep Ljuslinder I, Golovleva I, Palmqvist R, Oberg A, Stenling R, 25;27(43):5741-52 Jonsson Y, Hedman H, Henriksson R, Malmer B. LRIG1 expression in colorectal cancer. Acta Oncol. 2007;46(8):1118- Yi W, Haapasalo H, Holmlund C, Järvelä S, Raheem O, 22 Bergenheim AT, Hedman H, Henriksson R. Expression of leucine-rich repeats and immunoglobulin-like domains (LRIG) Shattuck DL, Miller JK, Laederich M, Funes M, Petersen H, proteins in human ependymoma relates to tumor location, Carraway KL 3rd, Sweeney C. LRIG1 is a novel negative WHO grade, and patient age. Clin Neuropathol. 2009 Jan- regulator of the Met receptor and opposes Met and Her2 Feb;28(1):21-7 synergy. Mol Cell Biol. 2007 Mar;27(5):1934-46 Thomasson M, Wang B, Hammarsten P, Dahlman A, Persson Karlsson T, Mark EB, Henriksson R, Hedman H. Redistribution JL, Josefsson A, Stattin P, Granfors T, Egevad L, Henriksson of LRIG proteins in psoriasis. J Invest Dermatol. 2008 R, Bergh A, Hedman H. LRIG1 and the liar paradox in prostate May;128(5):1192-5 cancer: a study of the expression and clinical significance of Ledda F, Bieraugel O, Fard SS, Vilar M, Paratcha G. Lrig1 is LRIG1 in prostate cancer. Int J Cancer. 2011 Jun an endogenous inhibitor of Ret receptor tyrosine kinase 15;128(12):2843-52 activation, downstream signaling, and biological responses to Yi W, Holmlund C, Nilsson J, Inui S, Lei T, Itami S, Henriksson GDNF. J Neurosci. 2008 Jan 2;28(1):39-49 R, Hedman H. Paracrine regulation of growth factor signaling Lindström AK, Ekman K, Stendahl U, Tot T, Henriksson R, by shed leucine-rich repeats and immunoglobulin-like domains Hedman H, Hellberg D. LRIG1 and squamous epithelial uterine 1. Exp Cell Res. 2011 Feb 15;317(4):504-12 cervical cancer: correlation to prognosis, other tumor markers, sex steroid hormones, and smoking. Int J Gynecol Cancer. This article should be referenced as such: 2008 Mar-Apr;18(2):312-7 Guo D, Wang B. LRIG1 (leucine-rich repeats and Miller JK, Shattuck DL, Ingalla EQ, Yen L, Borowsky AD, immunoglobulin-like domains 1). Atlas Genet Cytogenet Oncol Young LJ, Cardiff RD, Carraway KL 3rd, Sweeney C. Haematol. 2011; 15(8):625-627.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 627 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Mini Review

MAPK14 (mitogen -activated protein kinase 14) Almudena Porras, Carmen Guerrero Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, UCM, Ciudad Universitaria, 28040 Madrid, Spain (AP), Centro de Investigacion del Cancer, IBMCC, Universidad de Salamanca-CSIC, 37007 Salamanca, Spain (CG)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/MAPK14ID41292ch6p21.html DOI: 10.4267/2042/46010 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Larger transcripts contain 12 or 13 exons. Identity (GATExplorer). Other names: CSBP1; CSBP2; CSPB1; EXIP; Mxi2; Transcription PRKM14; PRKM15; RK; SAPK2A; p38; p38ALPHA 9 types of transcripts have been described, although HGNC (Hugo): MAPK14 only 5 are protein coding transcripts. The larger 4319- Location: 6p21.31 nucleotide transcript encodes a protein of 360 amino acid residues. The first and last exons are partially DNA/RNA untranslated. Description Pseudogene The gene spans a region of 83.53 kb and the coding None described so far. part is divided into 41 different exons.

Schematic representation of human chromosome 6 indicating the position of MAPK14 locus (p21.31) (red bar).

MAPK14 gene locus. Representation of the MAPK14 gene organization indicating the position of the exons (coding region) and untranslated regions.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 628 MAPK14 (mitogen-activated protein kinase 14) Porras A, Guerrero C

MAPK14 protein domains. Schematic representation of MAPK14 protein indicating the position of its functional domains. 30-54: protein kinase ATP signature, ATP-binding region; 59-162: MAPK signature; 24-308: protein kinase domain.

development and it regulates different cellular Protein functions such as proliferation, differentiation, cell Description death, adhesion, migration, as well as the response to stress and many metabolic pathways, among others. It MAPK14 is a Ser/Thr kinase composed of 90 to 360 does so through regulation of transcription, mRNA residues depending on the transcript variant. stability, chromatin remodelling, protein synthesis, etc. Concerning cell death, although p38alpha plays an important role as a pro-apoptotic signal, it can play a dual role, acting as either a mediator of cell survival or of cell death, depending on the cell type and the stimuli. Related with its function as a negative regulator of proliferation and a mediator of apoptosis, p38alpha acts as a tumor suppressor in the initial stages of a tumorigenic process, while at later stages it can promote metastasis.

Crystal structure of MAPK14 at 2.3 A resolution. From PDB (access number: 1WFC). Expression p38alpha MAPK is ubiquitously expressed, being the p38 most abundant isoform. Localisation p38alpha is mainly present in the cytosol, but it can translocate to the nucleus. In addition, it can be localized in the mitochondria or in other subcellular compartments. Function p38alpha is mainly activated by various environmental Signaling through p38alphaMAPK. Signaling through stresses and proinflammatory cytokines, but many MAPK14 cascade and its role in the regulation of cellular other extracellular signals, including growth factors, functions. MAPK14 is involved in signaling pathways triggered also lead to p38alpha activation. The canonical by a variety of stimuli such as growth factors, oxidative stress, activation requires its phosphorylation in threonine and UV, cytokines and DNA damage. Depending on the stimulus, different receptors and intermediates (adaptors, GTPases or tyrosine residues by dual-specificity MAP kinase kinases) are activated leading to the activation of the p38alpha kinases (MKKs), MKK3, MKK6 and MKK4. MAPK cascade. This cascade is initiated by activation of Substrates of this kinase include transcription factors, MAPKKKs, which phosphorylate and activate MAPKKs such as ATF1, ATF2, ATF6, p53, MEF2 or C/EBPbeta (MKK3/6/4), which in turn lead to activation of MAPK14 through dual phosphorylation in Tyr and Thr. Once phosphorylated, and protein kinases, such as MAPKAP-K2 and MAPK14 phosphorylates a number of cytosolic and nuclear MAPKAP-K3 (also known as MK-2 and MK-3), MSK- substrates, including transcription factors, which lead to the 1, MNK-1/MNK-2 and other proteins. control of many cellular responses. p38alpha MAPK is essential for embryonic

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 629 MAPK14 (mitogen-activated protein kinase 14) Porras A, Guerrero C

p38alpha MAPK plays a role in dopaminergic neural Mutations apoptosis through the phosphorylation of p53 and Somatic expression of the pro-apoptotic protein Bax. 4 somatic mutations according to Ensembl: Amyotrophic lateral sclerosis COSM21366; COSM20563; COSM35409; Disease COSM12875. ALS is a progressive, lethal, degenerative disorder of motor neurons leading to paralysis of voluntary Implicated in muscles. Numerous evidences point to a role of p38 MAPK in the development and progression of ALS Hematopoietic malignancies induced by mutations in SOD1 (superoxide dismutase Disease 1) gene. Mutant SOD1 provokes aberrant oxyradical p38 MAPK, mainly the p38alpha isoform, is a key reactions that increase the activation of p38 MAPK in player in the maintenance of hematopoiesis motor neurons and glial cells. This increase in active homeostasis, as it balances both proliferative and p38 MAPK may phosphorylate cytoskeletal proteins growth inhibitory signals triggered by the growth and activate cytokines and nitric oxide, thus factors and cytokines that regulate normal contributing to neurodegeneration through different hematopoiesis. Alterations in this p38 MAPK- mechanisms including apoptosis. controlled balance may result in either overproduction or depletion of myelosuppressive cytokines leading to To be noted the development of certain bone marrow failure syndromes. For example, p38alpha is responsible for Note the enhanced stem cell apoptosis characteristic of low See also the Deep Insight: "Role of p38alpha in grade myeolodysplastic syndromes (MDSs). On the apoptosis: implication in cancer development and other hand, imbalance toward the proliferative side may therapy". conduct to the development of myeloproliferative syndromes (MPSs), such as leukemia, lymphomas and References myelomas. In particular, p38alpha MAPK plays a pro- Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares apoptotic role in chronic myeloid leukemia (CML). In A, Zamanillo D, Hunt T, Nebreda AR. A novel kinase cascade fact, p38alpha MAP kinase pathway mediates the triggered by stress and heat shock that stimulates MAPKAP growth inhibitory effects of IFNalpha and STI-571, two kinase-2 and phosphorylation of the small heat shock proteins. Cell. 1994 Sep 23;78(6):1027-37 drugs used in the CML treatment, which underscores the importance of this pathway in the generation of Wilson KP, Fitzgibbon MJ, Caron PR, Griffith JP, Chen W, McCaffrey PG, Chambers SP, Su MS. Crystal structure of p38 antileukemic responses. mitogen-activated protein kinase. J Biol Chem. 1996 Nov Alzheimer's disease 1;271(44):27696-700 Disease Huang Y, Yuan ZM, Ishiko T, Nakada S, Utsugisawa T, Kato T, Kharbanda S, Kufe DW. Pro-apoptotic effect of the c-Abl Alzheimer is an incurable, neurodegenerative disease tyrosine kinase in the cellular response to 1-beta-D- characterized by a progressive deterioration of the arabinofuranosylcytosine. Oncogene. 1997 Oct cognitive, memory and learning ability due to the 16;15(16):1947-52 accumulation of plaques containing amyloidogenic Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson Abeta proteins and tangles containing CW, Appella E, Fornace AJ Jr. Phosphorylation of human p53 hyperphosphorylated tau protein. The ASK1-MKK6- by p38 kinase coordinates N-terminal phosphorylation and p38 signaling pathway participates in amyeloid apoptosis in response to UV radiation. EMBO J. 1999 Dec 1;18(23):6845-54 precursor protein (APP) and tau phosphorylation in response to oxidative stress and contributes to the Adams RH, Porras A, Alonso G, Jones M, Vintersten K, Panelli S, Valladares A, Perez L, Klein R, Nebreda AR. Essential role expression of the beta-secretase gene and the induction of p38alpha MAP kinase in placental but not embryonic of neuronal apoptosis triggered by ROS. cardiovascular development. Mol Cell. 2000 Jul;6(1):109-16 Parkinson disease D'Amico M, Hulit J, Amanatullah DF, Zafonte BT, Albanese C, Bouzahzah B, Fu M, Augenlicht LH, Donehower LA, Takemaru Disease K, Moon RT, Davis R, Lisanti MP, Shtutman M, Zhurinsky J, Parkinson is a degenerative disorder of the central Ben-Ze'ev A, Troussard AA, Dedhar S, Pestell RG. The nervous system characterized by muscle rigidity, integrin-linked kinase regulates the cyclin D1 gene through tremor and loss of physical movement caused by a glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways. J Biol Chem. progressive loss of dopaminergic neurons. Mutations in 2000 Oct 20;275(42):32649-57 alpha-synuclein are one of the main causes of Sanchez-Prieto R, Rojas JM, Taya Y, Gutkind JS. A role for Parkinson. alpha-synuclein activates p38alpha MAPK the p38 mitogen-acitvated protein kinase pathway in the in human microglia promoting a potent inflammatory transcriptional activation of p53 on genotoxic stress by stimulation of microglial cells. Additionally, the chemotherapeutic agents. Cancer Res. 2000 May 1;60(9):2464-72

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 630 MAPK14 (mitogen-activated protein kinase 14) Porras A, Guerrero C

Park JI, Choi HS, Jeong JS, Han JY, Kim IH. Involvement of p38 mitogen-activated protein kinase. Neurodegener Dis. p38 kinase in hydroxyurea-induced differentiation of K562 2005;2(3-4):128-34 cells. Cell Growth Differ. 2001 Sep;12(9):481-6 Silva RM, Kuan CY, Rakic P, Burke RE. Mixed lineage kinase- Platanias LC. The p38 mitogen-activated protein kinase c-jun N-terminal kinase signaling pathway: a new therapeutic pathway and its role in interferon signaling. Pharmacol Ther. target in Parkinson's disease. Mov Disord. 2005 Jun;20(6):653- 2003 May;98(2):129-42 64 Tamagno E, Robino G, Obbili A, Bardini P, Aragno M, Parola Cuenda A, Rousseau S. p38 MAP-kinases pathway regulation, M, Danni O. H2O2 and 4-hydroxynonenal mediate amyloid function and role in human diseases. Biochim Biophys Acta. beta-induced neuronal apoptosis by activating JNKs and 2007 Aug;1773(8):1358-75 p38MAPK. Exp Neurol. 2003 Apr;180(2):144-55 Zhou L, Opalinska J, Verma A. p38 MAP kinase regulates Tortarolo M, Veglianese P, Calvaresi N, Botturi A, Rossi C, stem cell apoptosis in human hematopoietic failure. Cell Cycle. Giorgini A, Migheli A, Bendotti C. Persistent activation of p38 2007 Mar 1;6(5):534-7 mitogen-activated protein kinase in a mouse model of familial amyotrophic lateral sclerosis correlates with disease Zuluaga S, Alvarez-Barrientos A, Gutiérrez-Uzquiza A, Benito progression. Mol Cell Neurosci. 2003 Jun;23(2):180-92 M, Nebreda AR, Porras A. Negative regulation of Akt activity by p38alpha MAP kinase in cardiomyocytes involves Mathiasen JR, McKenna BA, Saporito MS, Ghadge GD, Roos membrane localization of PP2A through interaction with RP, Holskin BP, Wu ZL, Trusko SP, Connors TC, Maroney AC, caveolin-1. Cell Signal. 2007 Jan;19(1):62-74 Thomas BA, Thomas JC, Bozyczko-Coyne D. Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity Karunakaran S, Saeed U, Mishra M, Valli RK, Joshi SD, Meka in SH-SY5Y cells. Brain Res. 2004 Apr 2;1003(1-2):86-97 DP, Seth P, Ravindranath V. Selective activation of p38 mitogen-activated protein kinase in dopaminergic neurons of Porras A, Zuluaga S, Black E, Valladares A, Alvarez AM, substantia nigra leads to nuclear translocation of p53 in 1- Ambrosino C, Benito M, Nebreda AR. P38 alpha mitogen- methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. J activated protein kinase sensitizes cells to apoptosis induced Neurosci. 2008 Nov 19;28(47):12500-9 by different stimuli. Mol Biol Cell. 2004 Feb;15(2):922-33 Wagner EF, Nebreda AR. Signal integration by JNK and p38 Puig B, Gómez-Isla T, Ribé E, Cuadrado M, Torrejón- MAPK pathways in cancer development. Nat Rev Cancer. Escribano B, Dalfó E, Ferrer I. Expression of stress-activated 2009 Aug;9(8):537-49 kinases c-Jun N-terminal kinase (SAPK/JNK-P) and p38 kinase (p38-P), and tau hyperphosphorylation in neurites Cuadrado A, Nebreda AR. Mechanisms and functions of p38 surrounding betaA plaques in APP Tg2576 mice. Neuropathol MAPK signalling. Biochem J. 2010 Aug 1;429(3):403-17 Appl Neurobiol. 2004 Oct;30(5):491-502 This article should be referenced as such: Bendotti C, Bao Cutrona M, Cheroni C, Grignaschi G, Lo Coco D, Peviani M, Tortarolo M, Veglianese P, Zennaro E. Inter- and Porras A, Guerrero C. MAPK14 (mitogen-activated protein intracellular signaling in amyotrophic lateral sclerosis: role of kinase 14). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):628-631.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 631 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

RAD51L3 (RAD51 -like 3 (S. cerevisiae)) Mary K Taylor, Michael K Bendenbaugh, Susan M Brown, Brian D Yard, Douglas L Pittman South Carolina College of Pharmacy, University of South Carolina, Coker Life Sciences Building, 715 Sumter Street, Columbia, SC 29208, USA (MKT, MKB, SMB, BDY, DLP)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/RAD51L3ID347ch17q12.html DOI: 10.4267/2042/46011 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

result during DNA replication or from DNA-damaging Identity agents, e.g., cisplatin (Masson et al., 2001). Other names: HsTRAD; R51H3; RAD51D; Trad The RAD51L3 protein directly interacts with HGNC (Hugo): RAD51D RAD51L2 (RAD51C) and XRCC2. It does not directly interact with RAD51L1 (RAD51B) (Schild et al., Location: 17q12 2000). Note Of the five RAD51 paralog proteins, four come DNA/RNA together to form the BCDX2 complex, which includes RAD51L1 (RAD51B; chromosome 14), RAD51L2 Note (RAD51C; chromosome 17), RAD51L3 (RAD51D; The human gene is composed of 10 exons. The study chromosome 17), and XRCC2 (chromosome 14). The by Kawabata and Saeki (1999) describes alternative protein complex is involved in homologous splicing of the human gene using a numbering scheme recombination repair of double-stranded breaks that of 12 alternatively spliced exons. The exon alignment is illustrated below.

Human RAD51D alternative splicing. A. Exons 4 and 8 of the Kawabata and Saeki numbering scheme are considered "alternative exons" and not included in the reference sequence. B. Summary of splice variants and predicted translation products (for further details see the annexed document below).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 632 RAD51L3 (RAD51-like 3 (S. cerevisiae)) Taylor MK, et al.

Further descriptions of mouse alternatively spliced residues 54-77 in the amino terminus is required for variants are described in Gruver et al., 2009 and proper interactions with XRCC2. Together, these Kawabata et al., 2004. interactions aid in the repair of DNA damage (Miller et Transcription al., 2004; Gruver et al., 2009). The HsTRAD transcript is the predominant variant. It Expression is the full-length transcript and is made up of 2418 base According to the study by Kawabata and Saeki (1999), pairs. This transcript will be used as the reference for RAD51L3 transcripts are expressed to varying degrees the information that follows. There are multiple splice in the colon, prostate, spleen, testis, ovaries, thymus, variants for the RAD51L3 gene that translate into one small intestine and leukocytes. of seven putative protein isoforms. Localisation Protein Located in the nucleus. Specifically, RAD51L3 localizes to the telomeres during both mitosis and Note meiosis (Tarsounas et al., 2004). There is evidence that The Saccharomyces cerevisiae Rad51 protein is RAD51L3 is found in the cytoplasm as well (Gruver et homologous to the RecA protein of Escherichia coli. al., 2005). The RecA protein is known to promote repair via ATP- Function dependent mechanisms and is responsible for pairing and strand transfer between homologous DNA RAD51D is one of five members of the RAD51 gene sequences. This is similar to the actions of the RAD51 family that is known to participate in repair of double protein in repair pathways. There are 5 members of the stranded DNA breaks via homologous recombination. RAD51 family that share similar roles in recombination Without repair, the DNA damage can result in cell and DNA repair. RAD51D is one of these RecA-like death or chromosomal aberrations that can ultimately genes (Pittman et al., 1998; Cartwright et al., 1998). lead to cancer (Thacker, 2005). Knockout studies with The RAD51D gene is predicted to encode seven mice have shown a dramatic increase in levels of different protein isoforms through alternative splicing. chromosomal aberrations, most notably, chromatid and Isoform 1 is the predominant protein and is translated chromosome breaks that occur through unrepaired from the HsTRAD transcript mentioned previously replication forks (Smiraldo et al., 2005; Hinz et al., (Kawabata and Saeki, 1999). The diagram below is 2006). Proteomic studies have identified an interaction based on this predominant form. between RAD51D with the SFPQ protein (Rajesh et al., 2011). Exposure of mouse RAD51D-deficient cells to a Description strong alkylating agent results in G2/M cell cycle arrest The RAD51D protein contains regions necessary for and ultimately apoptosis (Rajesh et al., 2010). interactions with other RAD51 paralogs as well as RAD51D has recently been shown to play a diverse those that are required for proper function of the role in cellular processes through its interaction with protein. RAD51D contains an ATP binding domain proteins involved in cell division, embryo development, with highly conserved Walker A and B motifs (Pittman protein and carbohydrate metabolism, cellular et al., 1998; Cartwright et al., 1998). Mutations trafficking, protein synthesis, modification or folding, targeting the conserved residues of glycine and lysine and cellular structure (Rajesh et al., 2009). within the Walker A motif region resulted in a RAD51L3 is directly associated with telomeres reduction in RAD51C binding ability and were shown prevents their dysfunction (Tarsounas et al., 2004). In to be required for DNA repair (Gruver et al., 2005). mouse studies, RAD51L3 foci were present at The Walker B motif contains a "GGQRE" sequence telomeres in both meiosis and mitosis. Knockout between residues 219-223 that is also required for DNA studies showed that "RAD51D-deficient" mice repair (Wiese et al., 2006). Furthermore, RAD51D- exhibited an increase in end-to-end fusion and telomere XRCC2 complex formation is significantly reduced attrition (Smiraldo et al., 2005). In addition, human with mutations targeting a highly conserved aspartate studies using RAD51D-deficient cells have shown residue within the Walker B motif (Wiese et al., 2006). repeated shortening of the telomeric DNA, leading to A carboxyl terminal domain spanning amino acids 77- chromosomal instability. This suggests a role for 329 has been identified to be required for RAD51D to "RAD51D" in telomere capping. Failure to provide this interact with RAD51C. function can lead to chromosomal aberrations In addition, the "linker region" located between (Tarsounas and West, 2005).

RAD51L3 protein structure. Isoform 1 (from full-length transcript).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 633 RAD51L3 (RAD51-like 3 (S. cerevisiae)) Taylor MK, et al.

Homology

Official gene name: Canis lupus familiaris RAD51-like 3 (S. cerevisiae) Reference material: [Dog] Genomic location: no primary references found chromosome 9 Official gene name: Pan troglodytes RAD51-like 3 (S. cerevisiae) Reference material: [Chimpanzee] Genomic location: no primary references found chromosome 17 Official gene name: Bos taurus RAD51-like 3 (S. cerevisiae) Reference material: [Cow] Genomic location: Zimin et al., 2009 chromosome 19 Official gene name: Gallus gallus RAD51-like 3 (S. cerevisiae) Reference material: [Chicken] Genomic location: no primary references found chromosome 19 Official gene name: Rattus norvegicus RAD51-like 3 (S. cerevisiae) Reference material: [Rat] Genomic location: Strausberg et al., 2002 chromosome 10

Official gene name: Reference material: Mus musculus RAD51-like 3 (S. cerevisiae) Pittman et al., 1998 [Mouse] Genomic location: Cartwright et al., 1998 chromosome 11 Official gene name: RAD51D (ARABIDOPSIS HOMOLOG OF Arabidopsis thaliana Reference material: RAD51D) [Thale cress] Durrant et al., 2007 Genomic location: chromosome 1 Official gene name: Os09g0104200 Oryza sativa Reference material: Genomic location: [Rice] no primary references found chromosome 9 **Hypothetical protein** Official gene name: Danio rerio zgc:77165 Reference material: [Zebrafish] Genomic location: no primary references found chromosome 5

** Protein alignments and protein sequences are available at the HomoloGene database.

(SNP ID: rs28363284) results in an allele change to Mutations GGG (from the wild type GAG). This point mutation rd Note affects the 233 amino acid as a glycine residue is Single nucleotide polymorphisms have been identified observed in this particular mutation rather than the in RAD51L3. However, only a small number of the natural glutamic acid. This particular variation in amino major mutations occur in coding regions. The majority acid sequence has been implicated as a precursor to of the other mutations are present in various locations breast cancer (see "Implicated In" section below). within the introns. Of the mutations affecting the gene, Another mutation observed in the coding region is at only one has an observed clinical association. It is mRNA position 188 (SNP ID: rs1871892), resulting in observed that a mutation of the mRNA position 954 a change in the sequence to TCA (from the wild type

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 634 RAD51L3 (RAD51-like 3 (S. cerevisiae)) Taylor MK, et al.

CCA). This particular substitution results in the RAD51D-E233G variant have been shown to have insertion of proline at the 36 th protein position rather higher proliferative indices and a less favorable clinical than a serine. immunohistochemical pattern (Rodríguez-López et al., A third mutation observed is noted to occur at mRNA 2004). However, another study found no statistically positions 810 (SNP ID: rs4796033). A mutation at this significant evidence that this variant is associated with location results in a sequence of CAG (from the natural breast cancer risk. Yet, this study did find that it was CGG). The effect of this substitution is the insertion of plausible that the variable could lead to a small increase a glutamine residue at the 185 th amino acid position in the risk of breast cancer and that a small, yet rather than the arginine observed in the wild type gene. insignificant, effect was made by the variant on the risk It is noted, that this particular mutation also occurs in 2 of breast cancer (approximately 30%) (Dowty et al., additional transcripts of the gene at the mRNA 2008). th rd positions 750 and 414 affecting the 165 and 53 Prognosis amino acid residues respectively. Other mutations in It has been noted that the RAD51D-E223G variant the coding region include E237K (SNP ID: confers increased resistance to DNA damaging agents rs115031549), R252Q (SNP ID: rs28363283), A245T such as: mitomycin C, cisplatin, ultraviolet light, and (SNP ID: rs28363282), A210T (SNP ID: rs80116829), methyl methane sulfonate, and taxol. This presents E177D (SNP ID: rs55942401), and R24S (SNP ID: clinical implications as these are commonly utilized rs28363257). therapies. Furthermore, the variant has increased cellular proliferation and telomere maintenance Implicated in compared to the wild-type and exhibits reduced Cancer interaction with the binding partner RAD51C but does not affect binding to XRCC2 (Nadkarni et al., 2009b). Disease Cancer arises in part due to the accumulation of genetic Bloom's syndrome damage. Furthermore, such damage has a greater Disease tendency to be found in significant levels when genetic Bloom's syndrome is an autosomal recessive disorder repair pathways such as DNA mismatch repair and of rare occurrence. Characteristics include short stature, homologous recombination (HR) are defective. immunodeficiency, fertility defects, and increased risk Involved in the pathway of HR are numerous proteins for the development of various types of cancer. Cells that are known as the RAD51 paralogs (RAD51L1, associated with this disorder are noted for their RAD51L2, RAD51L3, XRCC2 and XRCC3). It is genomic instability. They exhibit an increase in sister believed that the lack of genetic stability created from chromatid and homologous chromosome exchanges. In the loss of this pathway, HR, is significant in initiation normal, healthy cells, BLM, a helicase of the RecQ and potentially the progression of cancer. In particular, family, interacts with the RAD51L3 portion of the defects in the HR pathway have been noted to be RAD51L3-XRCC2 heteromeric complex. Upon joining associated with breast and ovarian cancer (Thacker, with the complex, BLM disrupts synthetic 4-way 2005); however, it is plausible that such a defect could junctions that resemble Holliday junctions suggesting potentially lead to multiple forms of cancer due to the an important role for the protein-protein interaction in accumulation of genetic mutations (although it takes DNA repair. The mutated form of the gene encoding significant damage accumulation to lead to tumor for this protein, which occurs in Bloom's syndrome, formation). A RAD51L3 variant does have an results in the inability for BLM to bind to RAD51L3. association with increased familial breast cancer risk Absence of normal BLM function leads to the (Rodríguez-López et al., 2004). characteristic elevation in recombination events seen in Breast cancer Bloom's syndrome (Braybrooke et al., 2003). Note References Although conflicting data exist, the RAD51D-E233G variant allele has been identified as a potential Cartwright R, Dunn AM, Simpson PJ, Tambini CE, Thacker J. Isolation of novel human and mouse genes of the recA/RAD51 precursor to breast cancers in women with high familial recombination-repair gene family. Nucleic Acids Res. 1998 Apr risk but do not possess a BRCA1/BRCA2 mutation 1;26(7):1653-9 (Rodríguez-López et al., 2004; Dowty et al., 2008). Pittman DL, Weinberg LR, Schimenti JC. Identification, Disease characterization, and genetic mapping of Rad51d, a new In an initial study that screened for possible breast mouse and human RAD51/RecA-related gene. Genomics. 1998 Apr 1;49(1):103-11 cancer alleles, it was determined that the exon 8 mutation led to an increased frequency of breast cancer Kawabata M, Saeki K. Multiple alternative transcripts of the human homologue of the mouse TRAD/R51H3/RAD51D gene, in a specific group of cases (familial cancer cases) a member of the rec A/RAD51 gene family. Biochem Biophys versus the control group (Rodríguez-López et al., Res Commun. 1999 Apr 2;257(1):156-62 2004). Additionally, individuals expressing the

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 635 RAD51L3 (RAD51-like 3 (S. cerevisiae)) Taylor MK, et al.

Schild D, Lio YC, Collins DW, Tsomondo T, Chen DJ. Smiraldo PG, Gruver AM, Osborn JC, Pittman DL. Extensive Evidence for simultaneous protein interactions between human chromosomal instability in Rad51d-deficient mouse cells. Rad51 paralogs. J Biol Chem. 2000 Jun 2;275(22):16443-9 Cancer Res. 2005 Mar 15;65(6):2089-96 Masson JY, Tarsounas MC, Stasiak AZ, Stasiak A, Shah R, Tarsounas M, West SC. Recombination at mammalian McIlwraith MJ, Benson FE, West SC. Identification and telomeres: an alternative mechanism for telomere protection purification of two distinct complexes containing the five and elongation. Cell Cycle. 2005 May;4(5):672-4 RAD51 paralogs. Genes Dev. 2001 Dec 15;15(24):3296-307 Thacker J. The RAD51 gene family, genetic instability and Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner cancer. Cancer Lett. 2005 Mar 10;219(2):125-35 RD, Collins FS, Wagner L, Shenmen CM, Schuler GD, Altschul SF, Zeeberg B, Buetow KH, Schaefer CF, Bhat NK, Hopkins Hinz JM, Tebbs RS, Wilson PF, Nham PB, Salazar EP, RF, Jordan H, Moore T, Max SI, Wang J, Hsieh F, Diatchenko Nagasawa H, Urbin SS, Bedford JS, Thompson LH. L, Marusina K, Farmer AA, Rubin GM, Hong L, Stapleton M, Repression of mutagenesis by Rad51D-mediated homologous Soares MB, Bonaldo MF, Casavant TL, Scheetz TE, recombination. Nucleic Acids Res. 2006;34(5):1358-68 Brownstein MJ, Usdin TB, Toshiyuki S, Carninci P, Prange C, Wiese C, Hinz JM, Tebbs RS, Nham PB, Urbin SS, Collins Raha SS, Loquellano NA, Peters GJ, Abramson RD, Mullahy DW, Thompson LH, Schild D. Disparate requirements for the SJ, Bosak SA, McEwan PJ, McKernan KJ, Malek JA, Walker A and B ATPase motifs of human RAD51D in Gunaratne PH, Richards S, Worley KC, Hale S, Garcia AM, homologous recombination. Nucleic Acids Res. Gay LJ, Hulyk SW, Villalon DK, Muzny DM, Sodergren EJ, Lu 2006;34(9):2833-43 X, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madan A, Young AC, Durrant WE, Wang S, Dong X. Arabidopsis SNI1 and RAD51D Shevchenko Y, Bouffard GG, Blakesley RW, Touchman JW, regulate both gene transcription and DNA recombination Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz during the defense response. Proc Natl Acad Sci U S A. 2007 J, Myers RM, Butterfield YS, Krzywinski MI, Skalska U, Mar 6;104(10):4223-7 Smailus DE, Schnerch A, Schein JE, Jones SJ, Marra MA. Generation and initial analysis of more than 15,000 full-length Dowty JG, Lose F, Jenkins MA, Chang JH, Chen X, Beesley J, human and mouse cDNA sequences. Proc Natl Acad Sci U S Dite GS, Southey MC, Byrnes GB, Tesoriero A, Giles GG, A. 2002 Dec 24;99(26):16899-903 Hopper JL, Spurdle AB. The RAD51D E233G variant and breast cancer risk: population-based and clinic-based family Braybrooke JP, Li JL, Wu L, Caple F, Benson FE, Hickson ID. studies of Australian women. Breast Cancer Res Treat. 2008 Functional interaction between the Bloom's syndrome helicase Nov;112(1):35-9 and the RAD51 paralog, RAD51L3 (RAD51D). J Biol Chem. 2003 Nov 28;278(48):48357-66 Gruver AM, Yard BD, McInnes C, Rajesh C, Pittman DL. Functional characterization and identification of mouse Rad51d Kawabata M, Akiyama K, Kawabata T. Genomic structure and splice variants. BMC Mol Biol. 2009 Mar 27;10:27 multiple alternative transcripts of the mouse TRAD/RAD51L3/RAD51D gene, a member of the recA/RAD51 Nadkarni A, Furda A, Rajesh C, McInnes C, Ruch RJ, Pittman gene family. Biochim Biophys Acta. 2004 Aug 12;1679(2):107- DL. Functional characterization of the RAD51D E233G genetic 16 variant. Pharmacogenet Genomics. 2009a Feb;19(2):153-60 Miller KA, Sawicka D, Barsky D, Albala JS. Domain mapping of Nadkarni A, Rajesh P, Ruch RJ, Pittman DL. Cisplatin the Rad51 paralog protein complexes. Nucleic Acids Res. resistance conferred by the RAD51D (E233G) genetic variant 2004;32(1):169-78 is dependent upon p53 status in human breast carcinoma cell lines. Mol Carcinog. 2009b Jul;48(7):586-91 Rodríguez-López R, Osorio A, Ribas G, Pollán M, Sánchez- Pulido L, de la Hoya M, Ruibal A, Zamora P, Arias JI, Salazar Rajesh C, Gruver AM, Basrur V, Pittman DL. The interaction R, Vega A, Martínez JI, Esteban-Cardeñosa E, Alonso C, profile of homologous recombination repair proteins RAD51C, Letón R, Urioste Azcorra M, Miner C, Armengod ME, RAD51D and XRCC2 as determined by proteomic analysis. Carracedo A, González-Sarmiento R, Caldés T, Díez O, Proteomics. 2009 Aug;9(16):4071-86 Benítez J. The variant E233G of the RAD51D gene could be a Zimin AV, Delcher AL, Florea L, Kelley DR, Schatz MC, Puiu low-penetrance allele in high-risk breast cancer families D, Hanrahan F, Pertea G, Van Tassell CP, Sonstegard TS, without BRCA1/2 mutations. Int J Cancer. 2004 Jul Marçais G, Roberts M, Subramanian P, Yorke JA, Salzberg 20;110(6):845-9 SL. A whole-genome assembly of the domestic cow, Bos Sasaki MS, Takata M, Sonoda E, Tachibana A, Takeda S. taurus. Genome Biol. 2009;10(4):R42 Recombination repair pathway in the maintenance of Rajesh P, Rajesh C, Wyatt MD, Pittman DL. RAD51D protects chromosomal integrity against DNA interstrand crosslinks. against MLH1-dependent cytotoxic responses to O(6)- Cytogenet Genome Res. 2004;104(1-4):28-34 methylguanine. DNA Repair (Amst). 2010 Apr 4;9(4):458-67 Tarsounas M, Muñoz P, Claas A, Smiraldo PG, Pittman DL, Rajesh C, Baker DK, Pierce AJ, Pittman DL. The splicing- Blasco MA, West SC. Telomere maintenance requires the factor related protein SFPQ/PSF interacts with RAD51D and is RAD51D recombination/repair protein. Cell. 2004 Apr necessary for homology-directed repair and sister chromatid 30;117(3):337-47 cohesion. Nucleic Acids Res. 2011 Jan;39(1):132-45 Gruver AM, Miller KA, Rajesh C, Smiraldo PG, Kaliyaperumal S, Balder R, Stiles KM, Albala JS, Pittman DL. The ATPase This article should be referenced as such: motif in RAD51D is required for resistance to DNA interstrand Taylor MK, Bendenbaugh MK, Brown SM, Yard BD, Pittman crosslinking agents and interaction with RAD51C. DL. RAD51L3 (RAD51-like 3 (S. cerevisiae)). Atlas Genet Mutagenesis. 2005 Nov;20(6):433-40 Cytogenet Oncol Haematol. 2011; 15(8):632-636.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 636 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) Wendy S McDonough, Michael E Berens The Translational Genomics Research Institute, 445 N Fifth Street, Phoenix, Arizona 85004, USA (WSM, MEB)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/SLC9A3R1ID46023ch17q25.html DOI: 10.4267/2042/46012 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Transcription The SLC9A3R1 gene encodes a 1978 bp mRNA Other names: EBP50; NHERF; NHERF1; NPHLOP2 transcript. HGNC (Hugo): SLC9A3R1 Reported regulatory transcription factor binding sites Location: 17q25.1 upstream of the SLC9A3R1 promoter region include: NF-kappaB1, HNF-4alpha2, COUP-TF1, NF-kappaB, DNA/RNA NRSF form 2, NRSF form 1, FOXD1, PPAR-gamma2, PPAR-gamma1, GATA-1. Description The SLC9A3R1 gene is comprised of 6 exons and spans approximately 20.7 kb of genomic DNA.

SLC9A3R1 Physical Map.

DNA size 20.71 Kb; mRNA size 1978 bp; 6 exons.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 637 SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) McDonough WS, Berens ME

NHERF (human)-358 aminos acids.

Induction: SLC9A3R1 can be induced by estrogen. Protein SLC9A3R1 has been show to have binary interaction Description with the following proteins: CFTR, CLCN3, MSN, NF2, RDX. The SLC9A3R1 protein is composed of 353 amino acids (389 kDa). Mutations Post-transcriptional regulation of SLC9A3R1 occurs via Serines S77-p, S162-p, S339-p, S340-p. Note SLC9A3R1 is also phosphroylated on T95-p. SLC9A3R1 has been shown to have three natural SLC9A3R1 has two PDZ (DHR) domains. variants. SLC9A3R1 can exist as a homodimer or heterodimer Natural variant 110L --> V in NPHLOP2; the mutant with SLC9A3R2. expressed in cultured renal cells increases the Expression generation of cyclic AMP (cAMP) by parathyroid hormone (PTH) and inhibits phosphate transport. SLC9A3R1 is expressed in liver, salivary glands, Natural variant 153R --> Q in NPHLOP2; the mutant kidney, pancreas, trachea, small intestine, stomach, expressed in cultured renal cells increases the prostate and brain. generation of cAMP by PTH and inhibits phosphate Localisation transport. SLC9A3R1 is a cytoplasmic protein. SLC9A3R1 Natural variant 225E --> K in NPHLOP2; the mutant translocates from the cytoplasm to the apical cell expressed in cultured renal cells increases the membrane in a PODXL-dependent manner. generation of cAMP by PTH and inhibits phosphate SLC9A3R1 colocalizes with actin in microvilli-rich transport. apical regions of the syncytiotrophoblast. SLC9A3R1 has been found in microvilli, ruffling membrane and Implicated in filopodia of HeLa cells. SLC9A3R1 is also been Cancer progression discovered in lipid rafts of T-cells. Subcellular localization is present in cells with apical Note specialized structure such as micorvili and cilia. There is growing evidence SLC9A3R1 plays an SLC9A3R1 also has a membranous expression in cells important role in cancer progression. SLC9A3R1 of non-epithelial origin (astrocytes) and hematopoietic functions as an adaptor protein to control cell stem cells and has been found in membrane rafts in transformation. In addition, recent evidence suggests lymphocytes and at the rear edge of neutrophils. that SLC9A3R1 has a dual role either acting as a tumor suppressor when it is localized as the cell membrane or Function as an oncogenic protein when it is localized in the SLC9A3R1 is a scaffold protein that connects plasma cytoplasm (Georgescu et al., 2008). membrane proteins with members of the ezrin/moesin/radixin family and thereby helps to link Glioblastoma them to the actin cytoskeleton to regulate their surface Note expression. SLC9A3R1 has been shown to be The invasive nature of glioblastoma multiforme necessary for recycling of internalized ADRB2. presents a clinical problem rendering tumors incurable SLC9A3R1 regulates SLC9A3 as well as its subcellular by conventional treatment modalities such as surgery, location. SLC9A3R1 is required for cAMP-mediated ionizing radiation, and temozolomide. phosphorylation and inhibition of SLC9A3R1. SLC9A3R1 has been implicated to play a role in SLC9A3R1 interacts with MCC. SLC9A3R1 may sustaining glioma cell migration and invasion. participate in HTR4 targeting to microvilli. SLC9A3R1 SLC9A3R1 has been shown to be over-expressed in has been shown to play a role in the WNT signaling invading glioma cells as compared to the tumor core pathway. (Kislin et al., 2009).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 638 SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) McDonough WS, Berens ME

Breast cancer SJ. The NHERF1 PDZ2 domain regulates PKA-RhoA-p38- mediated NHE1 activation and invasion in breast tumor cells. Note Mol Biol Cell. 2007 May;18(5):1768-80 Increased cytoplasmic expression of SLC9A3R1 in Weinman EJ, Steplock D, Wang Y, Shenolikar S. breast tumors suggests a key role of its localization and Characterization of a protein cofactor that mediates protein compartmentalization in defining cancerogenesis, kinase A regulation of the renal brush border membrane Na(+)- progression, and invasion. SLC9A3R1 overexpression H+ exchanger. J Clin Invest. 1995 May;95(5):2143-9 has been associated with increasing tumor Reczek D, Berryman M, Bretscher A. Identification of EBP50: cytohistological grade, aggressive clinical behavior, A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J Cell Biol. 1997 unfavorable prognosis, and increased tumor hypoxia. Oct 6;139(1):169-79 Moreover, SLC9A3R1 co-localizes with the oncogenic receptor HER2/neu in HER2/neu-overexpressing Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ, Donowitz M. cAMP-mediated inhibition of the carcinoma and in distant metastases (Mangia et al., epithelial brush border Na+/H+ exchanger, NHE3, requires an 2009). associated regulatory protein. Proc Natl Acad Sci U S A. 1997 The switch from apical membranous to cytoplasmic Apr 1;94(7):3010-5 expression is compatible with a dual role for NHERF1 Hall RA, Ostedgaard LS, Premont RT, Blitzer JT, Rahman N, as a tumour suppressor or tumour promoter dependent Welsh MJ, Lefkowitz RJ. A C-terminal motif found in the beta2- on its subcellular localization (Georgescu et al., 2008). adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to Cystic fibrosis the Na+/H+ exchanger regulatory factor family of PDZ proteins. Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8496-501 Note The inherited disease cystic fibrosis is one of the most Hall RA, Premont RT, Chow CW, Blitzer JT, Pitcher JA, Claing A, Stoffel RH, Barak LS, Shenolikar S, Weinman EJ, Grinstein common chronic lung diseases in children and young S, Lefkowitz RJ. The beta2-adrenergic receptor interacts with adults and may lead to an early death. the Na+/H+-exchanger regulatory factor to control Na+/H+ Cystic fibrosis transmembrane regulator (CFTR) exchange. Nature. 1998 Apr 9;392(6676):626-30 functions as a cAMP-regulated chloride channel, and Murthy A, Gonzalez-Agosti C, Cordero E, Pinney D, Candia C, mutations in CFTR are contributory in cystic fibrosis. Solomon F, Gusella J, Ramesh V. NHE-RF, a regulatory CFTR contains a C-terminal SLC9A3R1 consensus cofactor for Na(+)-H+ exchange, is a common interactor for sequence affording the two proteins to bind with high merlin and ERM (MERM) proteins. J Biol Chem. 1998 Jan 16;273(3):1273-6 affinity. Recent experiments have postulated two roles for SLC9A3R1 in CFTR function. Guggino, Stanton, Short DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, Milgram SL. An apical PDZ protein and coworkers have proposed that NHERF functions as anchors the cystic fibrosis transmembrane conductance a membrane retention signal for CFTR (Moyer et al., regulator to the cytoskeleton. J Biol Chem. 1998 Jul 1999). 31;273(31):19797-801 Raghuram et al. suggest that SLC9A3R1 facilitates the Wang S, Raab RW, Schatz PJ, Guggino WB, Li M. Peptide dimerization of CFTR leading to its full expression of binding consensus of the NHE-RF-PDZ1 domain matches the chloride channel activity (Raghuram et al., 2001). C-terminal sequence of cystic fibrosis transmembrane Lastly, ss2-adrenoceptors have been shown to conductance regulator (CFTR). FEBS Lett. 1998 May 1;427(1):103-8 physically interact with CFTR Na+/H+ Exchanger Regulatory Factor 1 SLC9A3R1 protein. This function Cao TT, Deacon HW, Reczek D, Bretscher A, von Zastrow M. of SLC9A3R1 could be a new therapeutic target in CF A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor. Nature. 1999 Sep patients to facilitate the trafficking of mutated CFTR to 16;401(6750):286-90 plasma membrane (Bossard et al., 2011). Hall RA, Spurney RF, Premont RT, Rahman N, Blitzer JT, Hypophosphatemia and nephrolithiasis Pitcher JA, Lefkowitz RJ. G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via Note a PDZ domain-mediated interaction. J Biol Chem. 1999 Aug SLC9A3R1 plays an important role in tumor 20;274(34):24328-34 phosphorous transport. Inactivating missense mutations Mohler PJ, Kreda SM, Boucher RC, Sudol M, Stutts MJ, in SLC9AR1 have been identified in patients with Milgram SL. Yes-associated protein 65 localizes p62(c-Yes) to hypercalciuria and neprolithiasis (Karim et al., 2008). the apical compartment of airway epithelia by association with EBP50. J Cell Biol. 1999 Nov 15;147(4):879-90 To be noted Moyer BD, Denton J, Karlson KH, Reynolds D, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, Stanton BA. A Note PDZ-interacting domain in CFTR is an apical membrane We acknowledge the support of Michael Northrop for polarization signal. J Clin Invest. 1999 Nov;104(10):1353-61 scientific illustrations. Breton S, Wiederhold T, Marshansky V, Nsumu NN, Ramesh V, Brown D. The B1 subunit of the H+ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B- References intercalated cells. J Biol Chem. 2000 Jun 16;275(24):18219-24 Cardone RA, Bellizzi A, Busco G, Weinman EJ, Dell'Aquila ME, Casavola V, Azzariti A, Mangia A, Paradiso A, Reshkin

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 639 SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) McDonough WS, Berens ME

Maudsley S, Zamah AM, Rahman N, Blitzer JT, Luttrell LM, Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun CC, Kalbacher Lefkowitz RJ, Hall RA. Platelet-derived growth factor receptor H, Gregor M, Seidler U. The down regulated in adenoma (dra) association with Na(+)/H(+) exchanger regulatory factor gene product binds to the second PDZ domain of the NHE3 potentiates receptor activity. Mol Cell Biol. 2000 kinase A regulatory protein (E3KARP), potentially linking Nov;20(22):8352-63 intestinal Cl-/HCO3- exchange to Na+/H+ exchange. Biochemistry. 2002 Oct 15;41(41):12336-42 Moyer BD, Duhaime M, Shaw C, Denton J, Reynolds D, Karlson KH, Pfeiffer J, Wang S, Mickle JE, Milewski M, Cutting Li JG, Chen C, Liu-Chen LY. Ezrin-radixin-moesin-binding GR, Guggino WB, Li M, Stanton BA. The PDZ-interacting phosphoprotein-50/Na+/H+ exchanger regulatory factor domain of cystic fibrosis transmembrane conductance (EBP50/NHERF) blocks U50,488H-induced down-regulation of regulator is required for functional expression in the apical the human kappa opioid receptor by enhancing its recycling plasma membrane. J Biol Chem. 2000 Sep 1;275(35):27069- rate. J Biol Chem. 2002 Jul 26;277(30):27545-52 74 Liedtke CM, Yun CH, Kyle N, Wang D. Protein kinase C Tang Y, Tang J, Chen Z, Trost C, Flockerzi V, Li M, Ramesh V, epsilon-dependent regulation of cystic fibrosis transmembrane Zhu MX. Association of mammalian trp4 and phospholipase C regulator involves binding to a receptor for activated C kinase isozymes with a PDZ domain-containing protein, NHERF. J (RACK1) and RACK1 binding to Na+/H+ exchange regulatory Biol Chem. 2000 Dec 1;275(48):37559-64 factor. J Biol Chem. 2002 Jun 21;277(25):22925-33 Brdicková N, Brdicka T, Andera L, Spicka J, Angelisová P, Mahon MJ, Donowitz M, Yun CC, Segre GV. Na(+)/H(+ ) Milgram SL, Horejsí V. Interaction between two adapter exchanger regulatory factor 2 directs parathyroid hormone 1 proteins, PAG and EBP50: a possible link between membrane receptor signalling. Nature. 2002 Jun 20;417(6891):858-61 rafts and actin cytoskeleton. FEBS Lett. 2001 Oct 26;507(2):133-6 Ogura T, Furukawa T, Toyozaki T, Yamada K, Zheng YJ, Katayama Y, Nakaya H, Inagaki N. ClC-3B, a novel ClC-3 Gisler SM, Stagljar I, Traebert M, Bacic D, Biber J, Murer H. splicing variant that interacts with EBP50 and facilitates Interaction of the type IIa Na/Pi cotransporter with PDZ expression of CFTR-regulated ORCC. FASEB J. 2002 proteins. J Biol Chem. 2001 Mar 23;276(12):9206-13 Jun;16(8):863-5 He J, Lau AG, Yaffe MB, Hall RA. Phosphorylation and cell Park M, Ko SB, Choi JY, Muallem G, Thomas PJ, Pushkin A, cycle-dependent regulation of Na+/H+ exchanger regulatory Lee MS, Kim JY, Lee MG, Muallem S, Kurtz I. The cystic factor-1 by Cdc2 kinase. J Biol Chem. 2001 Nov fibrosis transmembrane conductance regulator interacts with 9;276(45):41559-65 and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3. J Biol Chem. 2002 Karthikeyan S, Leung T, Birrane G, Webster G, Ladias JA. Dec 27;277(52):50503-9 Crystal structure of the PDZ1 domain of human Na(+)/H(+) exchanger regulatory factor provides insights into the Rochdi MD, Watier V, La Madeleine C, Nakata H, Kozasa T, mechanism of carboxyl-terminal leucine recognition by class I Parent JL. Regulation of GTP-binding protein alpha q (Galpha PDZ domains. J Mol Biol. 2001 May 18;308(5):963-73 q) signaling by the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50). J Biol Chem. 2002 Oct Karthikeyan S, Leung T, Ladias JA. 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Identification of EPI64, a TBC/rabGAP SJ, Bosak SA, McEwan PJ, McKernan KJ, Malek JA, domain-containing microvillar protein that binds to the first PDZ Gunaratne PH, Richards S, Worley KC, Hale S, Garcia AM, domain of EBP50 and E3KARP. J Cell Biol. 2001 Apr Gay LJ, Hulyk SW, Villalon DK, Muzny DM, Sodergren EJ, Lu 2;153(1):191-206 X, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madan A, Young AC, Ediger TR, Park SE, Katzenellenbogen BS. Estrogen receptor Shevchenko Y, Bouffard GG, Blakesley RW, Touchman JW, inducibility of the human Na+/H+ exchanger regulatory Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz factor/ezrin-radixin-moesin binding protein 50 (NHE- J, Myers RM, Butterfield YS, Krzywinski MI, Skalska U, RF/EBP50) gene involving multiple half-estrogen response Smailus DE, Schnerch A, Schein JE, Jones SJ, Marra MA. elements. 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Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 643 SLC9A3R1 (solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1) McDonough WS, Berens ME

Bossard F, Silantieff E, Lavazais-Blancou E, Robay A, Sagan Frizzled regulates β-catenin signaling. Oncogene. 2011 Jan C, Rozec B, Gauthier C. β1, β2, and β3 adrenoceptors and 6;30(1):32-42 Na+/H+ exchanger regulatory factor 1 expression in human bronchi and their modifications in cystic fibrosis. Am J Respir This article should be referenced as such: Cell Mol Biol. 2011 Jan;44(1):91-8 McDonough WS, Berens ME. SLC9A3R1 (solute carrier family Wheeler DS, Barrick SR, Grubisha MJ, Brufsky AM, Friedman 9 (sodium/hydrogen exchanger), member 3 regulator 1). Atlas PA, Romero G. Direct interaction between NHERF1 and Genet Cytogenet Oncol Haematol. 2011; 15(8):637-644.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 644 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

USP15 (ubiquitin specific peptidase 15) Monica Faronato, Sylvie Urbé, Judy M Coulson Physiology Department, School of Biomedical Sciences, Faculty of Health and Life Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK (MF, SU, JMC)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Genes/USP15ID44585ch12q14.html DOI: 10.4267/2042/46013 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Description The USP15 gene spans 145 kb of genomic DNA. Other names: KIAA0529; UNPH4; Unph-2 Transcription HGNC (Hugo): USP15 Four transcripts of the human USP15 gene are Location: 12q14.1 described by Ensembl and are summarized in the accompanying diagram and table. According to DNA/RNA Entrezgene, USP15 encodes a single reference Note sequence mRNA of 4611bp (NM_006313.1) composed USP15 is a member of the ubiquitin-specific protease of 21 exons, which corresponds to USP15-203. (USP) family; these cysteine proteases comprise the However, three other USP15 splice variants utilise largest sub-group of deubiquitinase enzymes (DUBs). several alternative-splicing sites between exon 5 and USP15 cleaves the isopeptide bonds of polyubiquitin exon 7 of this reference sequence. USP15-201, a 4698 chains, and can cleave linear ubiquitin fusion proteins bp mRNA comprised of 22 exons, is expressed at (Baker et al., 1999). similar levels to the reference sequence (Angelats et al., 2003). Expression of the remaining variants, USP15- 204 and the truncated USP15-202, is less well studied.

Schematic illustrating four human USP15 transcripts. The USP15 reference sequence mRNA (USP15-203) and three alternative splice variants are illustrated. The approximate position and size of exons within the USP15 gene, according to Ensembl, is shown for each splice variant.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 645 USP15 (ubiquitin specific peptidase 15) Faronato M, et al.

Summary table of USP15 transcripts.

Name Ensembl Entrez Aceview Size (bp) Exons

USP15-203 ENST00000353364 NM_006313.1 Variant b 4611 21

USP15-201 ENST00000280377 Variant a 4698 22

USP15-204 ENST00000393654 4626 23

USP15-202 ENST00000312635 Variant e 717 7

Schematic illustrating four human USP15 isoforms. The domain structure is shown for the reference sequence protein (USP15-203) and three alternative isoforms according to Ensembl. DUSP, domain present in ubiquitin-specific proteases; UBL, ubiquitin-like fold; UCH, ubiquitin carboxyl-terminal hydrolase. The cysteine motifs that form the zinc-binding site are shown in purple and the amino acids comprising the catalytic triad are shown in red. The approximate location of nuclear export sequences (triangles) and a putative nuclear localisation signal (inverted triangle) are shown above isoform USP15-203. Differences in amino acid sequence between isoforms are shown in light blue. The UCH is absent in isoform USP15-202, but USP15-201, USP15-203 and USP15-204 are predicted to be catalytically active.

Summary table of USP15 protein isoforms.

Name Ensembl Entrez Size (aa) MW (kDa)

USP15-203 ENSP00000258123 NP_006304.1 952 109

USP15-201 ENSP00000280377 981 112

USP15-204 ENSP00000377264 957 109

USP15-202 ENSP00000309240 235 40

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 646 USP15 (ubiquitin specific peptidase 15) Faronato M, et al.

dependent on a C-terminal NLS (Park et al., 2000) that Protein is conserved across species. Using an Usp15/USP15- Description specific polyclonal antibody, Soboleva et al. demonstrated that in HeLa (human cervical cancer All USP15 isoforms encompass a single N-terminal cells) endogenous USP15 localised to the cytoplasm DUSP (domain in USPs) characterised by a novel and nucleolus, but was largely excluded from the tripod-like fold with a conserved hydrophobic surface nucleoplasm; whilst in NIH3T3 (mouse fibroblast patch that is predicted to participate in protein-protein cells), Usp15 localised in the cytoplasm and was interaction (de Jong et al., 2006). The ubiquitin enriched proximal to the plasma membrane (Soboleva carboxyl-terminal hydrolase (UCH) catalytic core of et al., 2005). Interestingly, GFP-tagged USP15 the USPs is typically around 350 amino acids, but (isoform USP15-203) adopts a largely cytoplasmic consists of six conserved boxes, interspersed by distribution in human cancer cell lines (Urbé, insertion sites for additional sequence that can confer unpublished observation). diversity (Ye et al., 2009). The major insertion in USP15 is between boxes 3/4, and embeds an ubiquitin- Function like fold (UBL) domain within the catalytic domain. Human USP15 was cloned and characterized in 1999 UBLs are commonly found in the USPs and in certain (Baker et al., 1999) and belongs to the largest ubiquitin other DUB families (Zhu et al., 2007; Komander et al., specific protease (USP) group of deubiquitinating 2009a). They exhibit low sequence conservation, but enzymes (DUBs). Protein ubiquitination occurs at have high structural similarity with ubiquitin and have lysine residues through the concerted action of E1 been proposed to play important roles in regulating activating, E2 conjugating and E3 ligase enzymes. DUB catalytic function or interactions (Zhu et al., Ubiquitin contains seven lysine residues (K6, K11, 2007; Ye et al., 2009). The intercalation of a UBL K27, K29, K33, K48 and K63), which allow poly- between boxes 3 and 4 of the catalytic domain ubiquitin chains to assemble through alternative increases the spacing between two sets of zinc- isopeptide bond linkages. In addition, linear ubiquitin coordinating cysteine motifs, which form a functional chains may be assembled through the amino-terminus zinc finger that is required for activity (Hetfeld et al., and substrate proteins may also be mono-ubiquitinated. 2005). In the case of USP15, a second UBL is located Consequently, in addition to the classical K48-poly- directly adjacent to the DUSP (Zhu et al., 2007; Ye et ubiquitin tag that targets substrates for proteasome- al., 2009). mediated degradation, ubiquitination has multiple The four USP15 splice variants encode four distinct cellular functions including regulation of protein protein isoforms, which are illustrated in the diagram localisation and activity (Pickart and Eddins, 2004). and summarised in the accompanying table. As a The general role of the DUBs, in addition to processing consequence of alternative splicing, isoform USP15- inactive ubiquitin precursors and keeping the 26S 201 has a 29 amino acid insert within the unstructured proteasome free of inhibitory ubiquitin chains, is to region between the first UBL domain and the start of reverse the ubiquitination of substrate proteins (Amerik the UCH domain, whereas USP15-204 has a and Hochstrasser, 2004). There are approximately 80 substitution of 3 amino acids for 8 residues within the active human DUBs that are divided into five families first UBL. Otherwise the three isoforms that retain the (Komander et al., 2009a). These DUBs are steadily catalytic domain are identical. They also retain being assigned to specific substrates (Ventii and predicted nuclear export signals (NES) (Soboleva et al., Wilkinson, 2008), which is increasingly revealing 2005) and, by homology with rat, a functional nuclear associations with signalling pathways in cancer (Sacco localisation signal (NLS) (Park et al., 2000). et al., 2010). Expression USP15 has activity against both mono-ubiquitinated and poly-ubiquitinated substrates; the zinc-binding USP15 messenger RNA (mRNA) expression is domain is necessary for USP15 to process poly- prevalent throughout the tissues of the body, although ubiquitin chains, but is not required for USP15 to its levels vary. Human USP15 is least abundant in remove ubiquitin from linear ubiquitin-GFP fusion brain, lung and kidney, consistent with observations for proteins (Hetfeld et al., 2005). Although USP15 is mouse Usp15 and the rat ortholog UBP109 (Park et al., relatively promiscuous in showing little specificity 2000; Angelats et al., 2003). In each species, USP15 is between K48- and K63-linked poly-ubiquitin chains, or most abundant in testes, and is variously enriched in between K63 and K11 di-ubiquitin linkages, it has spleen, heart, skeletal muscle or peripheral blood limited activity against K11-linked poly-ubiquitin leukocytes. chains or linear ubiquitin (Komander et al., 2009b; Localisation Bremm et al., 2010). As USP15 harbours both putative NES and NLS, its A recent endeavour to map protein partners of the sub-cellular distribution may in part depend on the DUBs by mass spectroscopy reported that, in common cellular context. Rat UBP109 localises to both the with USP4 and USP39, USP15 interacts with several cytoplasm and the nuclear compartment, with the latter proteins involved in mRNA processing and so may

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 647 USP15 (ubiquitin specific peptidase 15) Faronato M, et al.

play a role in ubiquitin-dependent control of splicing or activate transcription (Karin and Ben-Neriah, 2000). In mRNA decay (Sowa et al., 2009). In addition, various response to TNFalpha, IkBa has been reported to cancer-signalling pathways have been associated with interact with the CSN leading to its deubiquitination USP15. For example, USP15 was one of twelve DUBs and stabilisation by CSN-associated USP15 identified from an siRNA screen that impact on the (Schweitzer et al., 2007). hepatocyte growth factor (HGF)-dependent cell The adenomatous polyposis coli (APC) tumour scattering response in non-small cell lung cancer and suppressor and the beta-catenin oncogene are pancreatic cancer cells (Buus et al., 2009). A number of frequently mutated in cancers, particularly of the specific USP15 substrates have also been described, intestine, leading to constitutive wingless and Int-1 including the human papilloma virus (HPV) E6 (Wnt) signalling (Clevers, 2006). The CSN is proposed oncoprotein (Vos et al., 2009), the RING-box protein to control the balance of beta-catenin and APC through Rbx1 (Hetfeld et al., 2005), the adenomatous polyposis formation of a regulatory super-complex. coli (APC) tumour suppressor (Huang et al., 2009), and Deneddylation by the CSN promotes assembly of the the NF-kB inhibitor IkBa (Schweitzer et al., 2007). beta-catenin destruction complex, whilst CSN- The latter three examples are all connected with the associated USP15 stabilises APC (Huang et al., 2009). COP9-signalosome (CSN), a conserved multi-protein The APC also plays a role in mitotic fidelity through complex that regulates the cullin-RING ligase (CRL) interaction with the plus end-binding protein EB1 that superfamily of ubiquitin E3 ligases (Wei et al., 2008). controls microtubule growth and dynamics. In contrast CRLs have a core complex comprised of a cullin to APC, EB1 is destabilised by USP15, suggesting that scaffold and the RING-box protein Rbx1 that recruit this is not a direct substrate, but rather that USP15 alternative adapter and substrate recognition proteins to stabilises a CRL that accelerates ubiquitination and form diverse E3 complexes with different substrate degradation of EB1 (Peth et al., 2007). It is interesting specificities. The primary function of the CSN is to to speculate that such links with microtubule regulation remove the ubiquitin-like modifier Nedd8 from the may underpin recent reports that USP15 levels can cullin component. This both terminates E3 activity and influence the taxol sensitivity of cancer cells (Xu et al., is required for the reassembly of new CRLs (Wei et al., 2009; Xie et al., 2010). 2008). The CSN plays a role in many cancer-associated VCP/p97 is a large AAA+-type ATPase that acts as a pathways including the cell cycle and DNA damage chaperone in many cellular processes. Its basic function repair, and both CSN and CRL components may be is to segregate ubiquitinated proteins from dysregulated in tumours (Richardson and Zundel, macromolecular complexes, and VCP plays an 2005). Ubp12p, an S. pombe ortholog of human important role in recognizing and handling misfolded USP15, was shown through a systematic mass proteins, which are then either handed over for spectrometry screen to bind the CSN (Zhou et al., degradation or recycled. The CSN directly interacts 2003). This targets Ubp12p to nuclear cullins, where it with VCP and USP15 can process VCP-bound poly- is proposed to protect against auto-ubiquitination and ubiquitinated substrates, which accumulate following degradation of CRL components, in particular the USP15 depletion (Cayli et al., 2009). VCP is substrate-specific adaptors. (Zhou et al., 2003; Wee et implicated in human neurodegenerative disorders al., 2005). where it co-localises with poly-glutamine aggregates Human USP15 also co-purifies with the CSN complex and is proposed to act as both an aggregate-formase and was reported to stabilise the CRL core component and an unfoldase (Kakizuka, 2008). Another Rbx1 (Hetfeld et al., 2005), thereby acting as a positive established VCP-associated cofactor, the DUB Ataxin- regulator of these E3 ligase complexes. In contrast, 3, is subject to polyglutamine repeat expansion, which other studies suggest USP15 may directly oppose CRL causes Machado-Joseph disease (Madsen et al., 2009). E3 ligase activity by deubiquitinating specific Although the mechanism is as yet unclear, USP15 was substrates. For example, the CSN is involved in recently associated with this same disorder (Menzies et ubiquitin-dependent turnover of the IkBa inhibitor that al., 2010). retains NF-kB in the cytosol (Schweitzer et al., 2007). Homology Phosphorylation of IkBa triggers CRL-mediated poly- ubiquitination of IkBa and subsequent proteasomal USP15 belongs to the peptidase C19 family. The degradation, allowing NF-kB to enter the nucleus and closest paralogs based on sequence homology are USP4 and USP11.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 648 USP15 (ubiquitin specific peptidase 15) Faronato M, et al.

USP15 and the paralogs USP4 and USP11. The highly similar domain structure is illustrated for USP15 (NP_006304.1), USP4 (NP_003354.2) and USP11 (NP_004642.2). The degree of identity (Id) or similarity (Sim) was derived using ClustalW (EMBL-EBI); note that the indicated UCH homology includes the internal UBL domain. Overall, USP15 shares 56.9% identity (71.2% similarity) with USP4, and 42.4% identity (60.2% similarity) with USP11.

Mutations Ovarian cancer Note Somatic USP15 was identified from a genome-wide siRNA No mutations have yet been reported for USP15 screen for Paclitaxel-resistance in the cervical cancer according to the COSMIC database, which includes cell line HeLa and, in ovarian cancer samples, data from four studies of 595 renal carcinoma, Paclitaxel-resistant cases (n=3) showed lower glioblastoma, breast and colon cancers. expression of USP15 mRNA than drug-sensitive cases (n=6) (Xu et al., 2009). Moreover, USP15 appeared to Implicated in stabilise caspase-3, suggesting that reduced levels of USP15 may promote cell survival rather than apoptosis Cervical cancer in response to drug treatment. Note Gastro-intestinal cancers USP15 plays an oncogenic role in cervical cancer. Note Specific HPV strains are associated with cervical carcinoma and two HPV oncoproteins, E6 and E7, are USP15 was also amongst four genes, identified by expressed in these cancers. E6 hijacks a cellular E3 expression profiling of Docetaxel-sensitive versus ubiquitin ligase and forms a complex with p53, whilst resistant cells, which correlated with drug-sensitivity in E7 binds the retinoblastoma (Rb) protein; in each case a panel of gastric cell lines. However, no statistical the viral oncoproteins facilitate degradation of the correlation was established between elevated USP15 cellular tumour suppressor. It was recently found that transcript levels and Docetaxel-sensitivity in 25 gastric USP15 interacts with the oncogenic HPV16 E6 protein cancer tissues (Xie et al., 2010). (Vos et al., 2009). siRNA mediated depletion of USP15 Germline mutations in APC lead to inherited colon led to a decrease in E6 protein, whilst overexpression cancer and sporadic tumours are associated with beta- of wild-type but not catalytically inactive USP15 catenin stabilisation. Huang et al. show a role for promoted the stabilisation of E6. Interesting, another USP15 in stabilizing APC levels through the action of group has shown that E7 is regulated in a similar the CSN (Huang et al., 2009). fashion by USP11 (Lin et al., 2008). Intriguingly, USP4 Machado-Joseph disease also has functional Rb binding motifs (Blanchette et al., Note 2001; DeSalle et al., 2001) that are conserved in USP11 USP15 was identified from microarray analysis of a and USP15 (Baker et al., 1999). mouse model of spinocerebellar ataxia type 3. In this

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 649 USP15 (ubiquitin specific peptidase 15) Faronato M, et al.

study, USP15 transcript and protein levels were de Jong RN, Ab E, Diercks T, Truffault V, Daniëls M, Kaptein decreased in both the ataxin-3 model, and in a second R, Folkers GE. Solution structure of the human ubiquitin- specific protease 15 DUSP domain. J Biol Chem. 2006 Feb huntingtin transgenic model of a polyglutamine 24;281(8):5026-31 disorder; although overexpression of USP15 promoted Peth A, Boettcher JP, Dubiel W. Ubiquitin-dependent the accumulation of protein aggregates, this was proteolysis of the microtubule end-binding protein 1, EB1, is independent of its activity on poly-ubiquitin chains controlled by the COP9 signalosome: possible consequences (Menzies et al., 2010). for microtubule filament stability. J Mol Biol. 2007 Apr 27;368(2):550-63 References Schweitzer K, Bozko PM, Dubiel W, Naumann M. CSN controls NF-kappaB by deubiquitinylation of IkappaBalpha. Baker RT, Wang XW, Woollatt E, White JA, Sutherland GR. EMBO J. 2007 Mar 21;26(6):1532-41 Identification, functional characterization, and chromosomal localization of USP15, a novel human ubiquitin-specific Zhu X, Ménard R, Sulea T. High incidence of ubiquitin-like protease related to the UNP oncoprotein, and a systematic domains in human ubiquitin-specific proteases. Proteins. 2007 nomenclature for human ubiquitin-specific proteases. Oct 1;69(1):1-7 Genomics. 1999 Aug 1;59(3):264-74 Kakizuka A. Roles of VCP in human neurodegenerative Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: disorders. Biochem Soc Trans. 2008 Feb;36(Pt 1):105-8 the control of NF-[kappa]B activity. Annu Rev Immunol. Lin CH, Chang HS, Yu WC. USP11 stabilizes HPV-16E7 and 2000;18:621-63 further modulates the E7 biological activity. J Biol Chem. 2008 Park KC, Choi EJ, Min SW, Chung SS, Kim H, Suzuki T, Jun 6;283(23):15681-8 Tanaka K, Chung CH. Tissue-specificity, functional Ventii KH, Wilkinson KD. Protein partners of deubiquitinating characterization and subcellular localization of a rat ubiquitin- enzymes. Biochem J. 2008 Sep 1;414(2):161-75 specific processing protease, UBP109, whose mRNA expression is developmentally regulated. Biochem J. 2000 Jul Wei N, Serino G, Deng XW. The COP9 signalosome: more 15;349(Pt 2):443-53 than a protease. Trends Biochem Sci. 2008 Dec;33(12):592- 600 Blanchette P, Gilchrist CA, Baker RT, Gray DA. Association of UNP, a ubiquitin-specific protease, with the pocket proteins Buus R, Faronato M, Hammond DE, Urbé S, Clague MJ. pRb, p107 and p130. Oncogene. 2001 Sep 6;20(39):5533-7 Deubiquitinase activities required for hepatocyte growth factor- induced scattering of epithelial cells. Curr Biol. 2009 Sep DeSalle LM, Latres E, Lin D, Graner E, Montagnoli A, Baker 15;19(17):1463-6 RT, Pagano M, Loda M. The de-ubiquitinating enzyme Unp interacts with the retinoblastoma protein. Oncogene. 2001 Sep Cayli S, Klug J, Chapiro J, Fröhlich S, Krasteva G, Orel L, 6;20(39):5538-42 Meinhardt A. COP9 signalosome interacts ATP-dependently with p97/valosin-containing protein (VCP) and controls the Angelats C, Wang XW, Jermiin LS, Copeland NG, Jenkins NA, ubiquitination status of proteins bound to p97/VCP. J Biol Baker RT. Isolation and characterization of the mouse Chem. 2009 Dec 11;284(50):34944-53 ubiquitin-specific protease Usp15. Mamm Genome. 2003 Jan;14(1):31-46 Huang X, Langelotz C, Hetfeld-Pechoc BK, Schwenk W, Dubiel W. The COP9 signalosome mediates beta-catenin Zhou C, Wee S, Rhee E, Naumann M, Dubiel W, Wolf DA. degradation by deneddylation and blocks adenomatous Fission yeast COP9/signalosome suppresses cullin activity polyposis coli destruction via USP15. J Mol Biol. 2009 Aug through recruitment of the deubiquitylating enzyme Ubp12p. 28;391(4):691-702 Mol Cell. 2003 Apr;11(4):927-38 Komander D, Clague MJ, Urbé S. Breaking the chains: Amerik AY, Hochstrasser M. Mechanism and function of structure and function of the deubiquitinases. Nat Rev Mol Cell deubiquitinating enzymes. Biochim Biophys Acta. 2004 Nov Biol. 2009 Aug;10(8):550-63 29;1695(1-3):189-207 Komander D, Reyes-Turcu F, Licchesi JD, Odenwaelder P, Pickart CM, Eddins MJ. Ubiquitin: structures, functions, Wilkinson KD, Barford D. Molecular discrimination of mechanisms. Biochim Biophys Acta. 2004 Nov 29;1695(1- structurally equivalent Lys 63-linked and linear polyubiquitin 3):55-72 chains. EMBO Rep. 2009 May;10(5):466-73 Hetfeld BK, Helfrich A, Kapelari B, Scheel H, Hofmann K, Madsen L, Seeger M, Semple CA, Hartmann-Petersen R. New Guterman A, Glickman M, Schade R, Kloetzel PM, Dubiel W. ATPase regulators--p97 goes to the PUB. Int J Biochem Cell The zinc finger of the CSN-associated deubiquitinating enzyme Biol. 2009 Dec;41(12):2380-8 USP15 is essential to rescue the E3 ligase Rbx1. Curr Biol. 2005 Jul 12;15(13):1217-21 Sowa ME, Bennett EJ, Gygi SP, Harper JW. Defining the human deubiquitinating enzyme interaction landscape. Cell. Richardson KS, Zundel W. The emerging role of the COP9 2009 Jul 23;138(2):389-403 signalosome in cancer. Mol Cancer Res. 2005 Dec;3(12):645- 53 Vos RM, Altreuter J, White EA, Howley PM. The ubiquitin- specific peptidase USP15 regulates human papillomavirus type Soboleva TA, Jans DA, Johnson-Saliba M, Baker RT. Nuclear- 16 E6 protein stability. J Virol. 2009 Sep;83(17):8885-92 cytoplasmic shuttling of the oncogenic mouse UNP/USP4 deubiquitylating enzyme. J Biol Chem. 2005 Jan 7;280(1):745- Xu M, Takanashi M, Oikawa K, Tanaka M, Nishi H, Isaka K, 52 Kudo M, Kuroda M. USP15 plays an essential role for caspase-3 activation during Paclitaxel-induced apoptosis. Wee S, Geyer RK, Toda T, Wolf DA. 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Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 650 USP15 (ubiquitin specific peptidase 15) Faronato M, et al.

Bremm A, Freund SM, Komander D. Lys11-linked ubiquitin Sacco JJ, Coulson JM, Clague MJ, Urbé S. Emerging roles of chains adopt compact conformations and are preferentially deubiquitinases in cancer-associated pathways. IUBMB Life. hydrolyzed by the deubiquitinase Cezanne. Nat Struct Mol Biol. 2010 Feb;62(2):140-57 2010 Aug;17(8):939-47 Xie L, Wei J, Qian X, Chen G, Yu L, Ding Y, Liu B. CXCR4, a Menzies FM, Huebener J, Renna M, Bonin M, Riess O, potential predictive marker for docetaxel sensitivity in gastric Rubinsztein DC. Autophagy induction reduces mutant ataxin-3 cancer. Anticancer Res. 2010 Jun;30(6):2209-16 levels and toxicity in a mouse model of spinocerebellar ataxia type 3. Brain. 2010 Jan;133(Pt 1):93-104 This article should be referenced as such: Faronato M, Urbé S, Coulson JM. USP15 (ubiquitin specific peptidase 15). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):645-651.

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CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) Joanna Fares, Linda Wolff, Juraj Bies Lab Cell Oncology, National Cancer Institute NIH, 37 Convent Dr, Bethesda MD 20892, USA (JF, LW, JB); Biochemistry and Molecular Biology Department, Georgetown University, Washington DC 20037, USA (JF)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/CDKN2BID187ch9p21.html DOI: 10.4267/2042/46014 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

member of the polycomb repressive complex 2 (PRC2) Identity as well as BMI1 and M33 members of the polycomb Other names: CDK4I; INK4B; MTS2; P15; TP15; repressive complex 1 (PRC1) are strongly expressed p15INK4b and are found to localize to the INK4/ARF RD. BMI1 HGNC (Hugo): CDKN2B has been shown to interact specifically with CDC6. These polycomb group (PcG) complexes repress the Location: 9p21.3 locus activity through the establishment of repressive chromatin modifications such as H3K27 trimethylation. DNA/RNA During senescence, binding of these complexes to RD Description is lost and correlates with increased expression of the INK4/ARF genes (Figure 1). The p15INK4B gene encompasses 6.41 Kb of DNA The p15INK4B gene is also silenced by a long non and has 2 coding exons. It is tandemly linked to coding RNA, called antisense non-coding RNA in the p16INK4A and p14ARF within 42 Kb of genomic INK4 locus (ANRIL), whose expression was found to locus located on chromosome 9p21. The locus is be inversed to the expression of p15INK4B in leukemia commonly referred to as INK4/ARF locus. cell lines. It was shown that ANRIL induces the Transcription silencing of p15INK4B in cis and trans by triggering heterochromatin formation in a Dicer-independent CDKN2B gene encodes 2 distinct transcript variants: manner. PcG complexes are recruited to the INK4/ARF p15 and p10. p10 arises from an alternative 5' splice locus by ANRIL and modulate its repression (Figure donor site within intron 1 of: 1). Additionally, a naturally occurring antisense - p15. circular ANRIL RNAs (cANRIL) has also been - p15: 3.82 Kb of mRNA. described. Different forms of cANRIL are produced in - p10: 0.86 Kb of mRNA. most INK4/ARF expressing cells, suggesting that Regulation: A conserved DNA element with the ability alternative splicing events leading to different ANRIL to regulate the entire INK4/ARF locus has been structures can contribute to changes in PcG-mediated identified in close proximity of the locus and named INK4/ARF repression. regulatory domain (RD). It appears to promote Specific transcription regulators of p15INK4B have transcriptional repression of all three genes encoded by also been reported (see Figure 2). These include TGF- the locus, in a manner dependent on CDC6. In b, MIZ-1, SMAD3/SMAD4 complex, SP1, c-MYC, proliferating embryonic fibroblasts (MEFs), EZH2 a IRF8, PU.1, SNAIL and EGR1 factors among others.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 652 CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) Fares J, et al.

Figure 1. p15INK4B expression is dramatically induced by TGF- SNAIL, SP1 and EGR-1 was also described for its b, suggesting that it is a potent downstream effector of ability to trigger the p15INK4b promoter activation TGF-b mediated growth arrest. upon TPA treatment. MIZ-1 is a transcription factor that has been shown to In murine myeloid cells specifically, the interferon bind the initiator element (Inr) in the promoter region consensus sequence-binding protein/interferon and induce the transcription of p15INK4B in epithelial regulatory factor 8 (ICSBP/IRF-8) in combination with cells. However it can also recruit transcriptional co- PU.1 were shown to bind p15Ink4b promoter and repressors such as c-MYC and GFI-1 to the promoter activate the transcription of the gene in response to region by binding to them and forming inhibitory IFN-b treatment. complexes. TGF-b has been reported to re-activate the In AML patients with inv(16), p15INK4B silencing core promoter through downregulation of C-MYC and was found to be caused by inv(16)-encoded core GFI-1, thereby releasing endogenous MIZ-1 from binding factor beta-smooth muscle myosin heavy chain inhibition. (CBFb-SMMHC). CBFb-SMMHC was shown to The SMAD3/4 complex readily forms following TGF-b displace RUNX1 from a newly determined CBF site in treatment and physical interaction between this the promoter of p15INK4B. complex, MIZ-1 and promoter-bound SP1 protein has been described. These interactions have been proposed Protein to constitute a platform for the recruitment of co- activators, and do not seem to be affected by the Description suppressor activity of c-MYC. The inhibitory function p15INK4B transcript encodes two protein isoforms p15 of c-MYC seems to be cell-type dependent, as it was and p15.5 that are functionally indistinguishable. p15.5 confirmed in epithelial cells but not in the is an N-terminally extended variant of p15 initiated hematopoietic lineage. In myeloid cells, the from an upstream alternative in frame initiation codon. transcription factor c-MYB was shown to prevent the p15 protein is 138 aa long and its mass is 14.72 KDa. transcription and the upregulation of p15INK4B which p10 transcript encodes the shorter variant. The protein is normally associated with the differentiation process. consists of 78 aa only and its mass is 10 KDa. It shares The mechanism by which C-MYB does this is unclear a similar NH2 terminus to p15 but contains a different but it is not through upregulation of c-MYC, a known basic COOH terminus that is translated from the target of c-MYB. p15Ink4b intronic region (Figure 2). A tri-component transcriptional complex consisting of

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 653 CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) Fares J, et al.

Figure 2.

Expression early hematopoietic progenitors has also been described. In knockout mice, loss of p15INK4B was p15INK4B is expressed at very low levels under shown to favor the differentiation of common myeloid normal physiological conditions. Its expression seems progenitors (CMP) into granulocyte macrophage to be lineage restricted. In bone marrow cells the progenitors (GMP) resulting in an imbalance between highest level of p15INK4B is mainly detected in the myeloid and the erythroid compartments. maturing monocytes/macrophages and lymphocytes. III- Function during cellular senescence. The gene expression has also been reported to be Cellular senescence is accompanied by hallmark normally up-regulated during megakaryocytic features that include the up-regulation of cell cycle differentiation. Increased expression of p15INK4B is inhibitors like p15INK4B, p16INK4A and p21CIP. also detected during stress and senescence of cells. When overexpressed, p15INK4B engages the RB Localisation pathway to promote a stable senescent state which has Nucleus and cytoplasm. been shown to occur in part through a process that involves alterations in heterochromatin and the stable Function silencing of E2F target genes. I- Function in the cell cycle. Another mechanism that has been described is the p15INK4B belongs to the INK4 family of protein inactivation of c-MYC which results in the induction of kinase inhibitors named for their high and exclusive p15INK4B expression and correlates with the global specificity towards the catalytic activity of cyclin changes in heterochromatin structure known to be dependent kinases 4 (CDK4) or 6 (CDK6). Structural associated with cellular senescence. studies have demonstrated that the protein performs its inhibitory activity by allosteric competition with the D- Homology type cyclins to bind CDK4/6 kinases and prevents the p15INK4B is highly conserved. Its sequence in homo formation of active CDK4/6-cyclin-Ds complexes. This sapiens is > 85% similar to bos taurus, mus musculus keeps the retinoblastoma protein (RB), which is and rattus norvegicus; and > 70% similar to gallus downstream of this pathway, in its hypophosphorylated gallus. state. Hypophosporylated RB binds and inactivates the E2F transcription factors required for the Mutations transcriptional activation of genes necessary for entry Note into the S phase of the cell cycle and DNA synthesis. Intragenic p15INK4B mutations are highly infrequent. Three other members of the INK4 family of CDK inhibitors: p16INK4A, p18INK4C and p19INK4D are encoded by unique genes and share roughly 40% Implicated in homology. They have similar protein structure Various hematological disorders and characterized by the presence of four ankyrin-like motif tandem repeats that are predicted to be engaged in malignancies protein-protein interactions. Note II- Function during hematopoietic cell differentiation. p15INK4B is frequently epigenetically silenced in Another role for p15INK4B during differentiation of leukemias, myelodysplastic syndromes and

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 654 CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) Fares J, et al.

myeloproliferative diseases by mechanisms involving marrow. In this way the mice develop symptoms that aberrant DNA methylation and/or histone closely resemble CMML in human patients. modifications. These diseases are subcategorized by the Acute myeloid leukemia (AML) French-American-British (FAB) co-operative group, based on the percentage of blast cells in bone marrow Disease and peripheral blood, degree of cytopenia, and in AML is the most common type of leukemia among accordance to the direction of differentiation along the adults with 14000 new cases diagnosed each year, and myeloid or lymphoid lineages as well as the degree of with 9000 deaths per year in the United States. AML maturation of the hematopoietic cells. classification into ten different subtypes was originally defined by the FAB cooperative group according to the Myelodysplastic syndromes (MDS) direction of differentiation along the different myeloid Disease lineages as well as the degree of maturation of the cells. Myelodysplastic syndromes are heterogeneous clonal However, AML exemplifies a genetically hematologic disorders characterized by dysplasia of the heterogeneous cancer with more than a hundred genetic myeloid bone marrow cells accompanied with aberrations implicated in the disease. peripheral blood cytopenia and increased risk of Prognosis transformation to acute myeloid leukemia (AML). Despite the great genetic and phenotypic heterogeneity MDS transforms into AML once the percentage of of AML, hypermethylation of the p15INK4B promoter blasts in the bone marrow has exceeded 30% (FAB). region (CpG island) is found to occur in up to 80% of MDS can arise in patients de novo (primary MDS), or AML cases across all FAB subtypes. It correlates with following chemotherapy or exposure to toxins a loss of p15INK4B expression, poor prognosis and (secondary MDS). According to the Leukemia and shorter survival time in patients. The p15INK4B Lymphoma Society reports, MDS most commonly methylation status in AML patients in clinical affects males aged 70 and above, and is considered to remission is now monitored and used as a reliable be a disease of the elderly. About 11000 new cases are prognostic marker for relapse. These findings were diagnosed each year, resulting in an incidence rate of 4 further experimentally confirmed in a conditional cases per 100000 population for both genders. knockout mouse model where myeloid-specific gene Prognosis inactivation resulted in an increased susceptibility to p15INK4B is silenced by promoter hypermethylation in retrovirus-induced myeloid leukemia. > 50% of MDS cases. Levels of p15INK4B Acute lymphoblastic leukemia (ALL) methylation increase as the disease progresses and provide a marker that can predict occurrence of AML. Disease There are about 4000 new cases of ALL in the United Chronic myelomonocytic leukemia States each year. It appears most often in children (CMML) younger than age 10. ALL is the most common Disease leukemia in children. However, it can appear in people The defining features of CMML are an absolute of any age. About one-third of cases are adults. monocytosis in peripheral blood (> 1x10 9/L), increased Prognosis numbers of monocytes in bone marrow, a variable In B and T acute lymphoblastic leukemia the degree of dysplasia and less than 5% and 20% of blasts p15INK4B promoter methylation as well as deletion of in peripheral blood and bone marrow, respectively. the entire locus has been reported. There are two types of CMML: proliferative and dysplastic. Roughly half of CMML diagnosed patients Chronic leukemia have an elevated white blood cell count commonly Disease associated with hepatomegaly and splenomegaly Chronic leukemia can be subdivided into two subtypes, (myeloproliferative form of the disease). Patients chronic myelogenous leukemia (CML) and chronic lacking these features are generally considered to have lymphocytic leukemia (CLL). CLL is primarily an the myelodysplastic form of the disease. adult disease; it is very rare in children and young Prognosis adults. The median age of diagnosis is 72 years, and Hypermethylation is found in up to 60% of CMML about 60% of patients are male. In the United States, cases and correlates with a more aggressive form of about 15000 people are diagnosed with CLL each year. disease. Experimentally, a LysMCre mouse model was This disease is also commonly referred to as B-cell developed in which p15INK4B gene is deleted chronic lymphocytic leukemia (B-CLL). specifically in cells of the myeloid lineage, to better Prognosis mimic the loss of the gene expression the way it is Promoter hypermethylation has been reported in a observed in humans. The mice develop non-reactive small subset of B-CLL (11%) at all stages of the monocytosis of the peripheral blood as well as disease. In CML, silencing of p15INK4B either by increased myeloid blast progenitors in the bone

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 655 CDKN2B (cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)) Fares J, et al.

deletion or hypermethylation of its promoter was not Aoki E, Uchida T, Ohashi H, Nagai H, Murase T, Ichikawa A, found to be a very frequent event. Yamao K, Hotta T, Kinoshita T, Saito H, Murate T. Methylation status of the p15INK4B gene in hematopoietic progenitors and Glioblastoma multiforme (GBM) peripheral blood cells in myelodysplastic syndromes. Leukemia. 2000 Apr;14(4):586-93 Disease Fuxe J, Raschperger E, Pettersson RF. Translation of GBM is the most common and very aggressive brain p15.5INK4B, an N-terminally extended and fully active form of tumor in adults. It involves glial cells and accounts for p15INK4B, is initiated from an upstream GUG codon. more than 50% of parenchymal brain tumors Oncogene. 2000 Mar 23;19(13):1724-8 approximately 20% of all intracranial tumors. Jeffrey PD, Tong L, Pavletich NP. Structural basis of inhibition Glioblastoma growth is characterized by a high motility of CDK-cyclin complexes by INK4 inhibitors. Genes Dev. 2000 of tumor cells that display broad chemoresistance Dec 15;14(24):3115-25 leading to frequent post-surgical tumor recurrence. It is Latres E, Malumbres M, Sotillo R, Martín J, Ortega S, Martín- one of the most dreaded cancer diagnoses due to its Caballero J, Flores JM, Cordón-Cardo C, Barbacid M. Limited poor prognosis and the limited treatment options, with overlapping roles of P15(INK4b) and P18(INK4c) cell cycle the median survival duration after diagnosis varying inhibitors in proliferation and tumorigenesis. EMBO J. 2000 Jul 3;19(13):3496-506 from 6 months to 2 years. Malumbres M, Ortega S, Barbacid M. Genetic analysis of Prognosis mammalian cyclin-dependent kinases and their inhibitors. Biol Homozygous deletion of the Chem. 2000 Sep-Oct;381(9-10):827-38 p15INK4B/p14ARF/p16INK4A locus on chromosome Teofili L, Morosetti R, Martini M, Urbano R, Putzulu R, Rutella 9p21.3 is a signature genetic event that drives the S, Pierelli L, Leone G, Larocca LM. Expression of cyclin- pathogenesis of GBM. The deletion of this locus is the dependent kinase inhibitor p15(INK4B) during normal and most common homozygous deletion present in GBM (> leukemic myeloid differentiation. Exp Hematol. 2000 May;28(5):519-26 75% of samples). Specific p15INK4B promoter methylation was also detected in 37% of patients Amati B. Integrating Myc and TGF-beta signalling in cell-cycle diagnosed with glioblastoma and it correlated with control. Nat Cell Biol. 2001 May;3(5):E112-3 shorter survival. Schmidt M, Koller R, Haviernik P, Bies J, Maciag K, Wolff L. Deregulated c-Myb expression in murine myeloid leukemias Hepatocellular carcinoma (HCC) prevents the up-regulation of p15(INK4b) normally associated Disease with differentiation. Oncogene. 2001 Sep 27;20(43):6205-14 HCC is a primary malignancy of the liver that mostly Seoane J, Pouponnot C, Staller P, Schader M, Eilers M, arises secondary to hepatitis B or C viral infections. Massagué J. TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b. Nat Cell Biol. 2001 Outcome of the disease is poor, because only 10 - 20% Apr;3(4):400-8 of hepatocellular carcinomas can be removed Staller P, Peukert K, Kiermaier A, Seoane J, Lukas J, completely using surgery, and the cancer is usually Karsunky H, Möröy T, Bartek J, Massagué J, Hänel F, Eilers deadly within 3 to 6 months. M. Repression of p15INK4b expression by Myc through Prognosis association with Miz-1. Nat Cell Biol. 2001 Apr;3(4):392-9 The suppression of the C-MYC oncogene induces Teofili L, Martini M, Di Mario A, Rutella S, Urbano R, Luongo cellular senescence in diverse tumor types including M, Leone G, Larocca LM. Expression of p15(ink4b) gene during megakaryocytic differentiation of normal and hepatocellular carcinoma and correlates with increased myelodysplastic hematopoietic progenitors. Blood. 2001 Jul p15INK4b expression. In primary HCC, p15INK4B 15;98(2):495-7 promoter is hypermethylated in about 50% of the cases, Wolff L, Schmidt M, Koller R, Haviernik P, Watson R, Bies J, and homozygous deletions of both p16INK4A and Maciag K. Three genes with different functions in p15INK4B have been reported in 30% HCC patients transformation are regulated by c-Myb in myeloid cells. Blood and cell lines. This suggests that p15INK4B might be Cells Mol Dis. 2001 Mar-Apr;27(2):483-8 contributing to human hepatocarcinogenesis through a Narita M, N ũnez S, Heard E, Narita M, Lin AW, Hearn SA, pathway associated with cellular senescence. Spector DL, Hannon GJ, Lowe SW. Rb-mediated heterochromatin formation and silencing of E2F target genes References during cellular senescence. Cell. 2003 Jun 13;113(6):703-16 Tessema M, Länger F, Dingemann J, Ganser A, Kreipe H, Jen J, Harper JW, Bigner SH, Bigner DD, Papadopoulos N, Lehmann U. Aberrant methylation and impaired expression of Markowitz S, Willson JK, Kinzler KW, Vogelstein B. Deletion of the p15(INK4b) cell cycle regulatory gene in chronic p16 and p15 genes in brain tumors. Cancer Res. 1994 Dec myelomonocytic leukemia (CMML). Leukemia. 2003 15;54(24):6353-8 May;17(5):910-8 Tsubari M, Tiihonen E, Laiho M. Cloning and characterization Qin Y, Liu JY, Li B, Sun ZL, Sun ZF. Association of low of p10, an alternatively spliced form of p15 cyclin-dependent p16INK4a and p15INK4b mRNAs expression with their CpG kinase inhibitor. Cancer Res. 1997 Jul 15;57(14):2966-73 islands methylation with human hepatocellular carcinogenesis. Drexler HG. Review of alterations of the cyclin-dependent World J Gastroenterol. 2004 May 1;10(9):1276-80 kinase inhibitor INK4 family genes p15, p16, p18 and p19 in Schmidt M, Bies J, Tamura T, Ozato K, Wolff L. The interferon human leukemia-lymphoma cells. 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regulates expression of tumor suppressor p15(Ink4b) in murine Yu W, Gius D, Onyango P, Muldoon-Jacobs K, Karp J, myeloid cells. Blood. 2004 Jun 1;103(11):4142-9 Feinberg AP, Cui H. Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature. 2008 Jan Aggerholm A, Holm MS, Guldberg P, Olesen LH, Hokland P. 10;451(7175):202-6 Promoter hypermethylation of p15INK4B, HIC1, CDH1, and ER is frequent in myelodysplastic syndrome and predicts poor Basu S, Liu Q, Qiu Y, Dong F. Gfi-1 represses CDKN2B prognosis in early-stage patients. Eur J Haematol. 2006 encoding p15INK4B through interaction with Miz-1. Proc Natl Jan;76(1):23-32 Acad Sci U S A. 2009 Feb 3;106(5):1433-8 Markus J, Garin MT, Bies J, Galili N, Raza A, Thirman MJ, Le Bies J, Sramko M, Fares J, Rosu-Myles M, Zhang S, Koller R, Beau MM, Rowley JD, Liu PP, Wolff L. Methylation- Wolff L. Myeloid-specific inactivation of p15Ink4b results in independent silencing of the tumor suppressor INK4b (p15) by monocytosis and predisposition to myeloid leukemia. Blood. CBFbeta-SMMHC in acute myelogenous leukemia with 2010 Aug 12;116(6):979-87 inv(16). Cancer Res. 2007 Feb 1;67(3):992-1000 Burd CE, Jeck WR, Liu Y, Sanoff HK, Wang Z, Sharpless NE. Papageorgiou SG, Lambropoulos S, Pappa V, Economopoulou Expression of linear and novel circular forms of an INK4/ARF- C, Kontsioti F, Papageorgiou E, Tsirigotis P, Dervenoulas J, associated non-coding RNA correlates with atherosclerosis Economopoulos T. Hypermethylation of the p15INK4B gene risk. PLoS Genet. 2010 Dec 2;6(12):e1001233 promoter in B-chronic lymphocytic leukemia. Am J Hematol. 2007 Sep;82(9):824-5 Hu CT, Chang TY, Cheng CC, Liu CS, Wu JR, Li MC, Wu WS. Snail associates with EGR-1 and SP-1 to upregulate Wu CH, van Riggelen J, Yetil A, Fan AC, Bachireddy P, transcriptional activation of p15INK4b. FEBS J. 2010 Felsher DW. Cellular senescence is an important mechanism Mar;277(5):1202-18 of tumor regression upon c-Myc inactivation. Proc Natl Acad Sci U S A. 2007 Aug 7;104(32):13028-33 Kotake Y, Nakagawa T, Kitagawa K, Suzuki S, Liu N, Kitagawa M, Xiong Y. Long non-coding RNA ANRIL is required for the Peters G. An INKlination for epigenetic control of senescence. PRC2 recruitment to and silencing of p15(INK4B) tumor Nat Struct Mol Biol. 2008 Nov;15(11):1133-4 suppressor gene. Oncogene. 2011 Apr 21;30(16):1956-62

Solomon DA, Kim JS, Jean W, Waldman T. Conspirators in a This article should be referenced as such: capital crime: co-deletion of p18INK4c and p16INK4a/p14ARF/p15INK4b in glioblastoma multiforme. Fares J, Wolff L, Bies J. CDKN2B (cyclin-dependent kinase Cancer Res. 2008 Nov 1;68(21):8657-60 inhibitor 2B (p15, inhibits CDK4)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):652-657.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 657 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

DLX4 (distal -less homeobox 4) Patricia E Berg, Saurabh Kirolikar The George Washington University Medical Center, Washington DC 20037, USA (PEB), The George Washington University, Department of Biochemistry and Molecular Biology, Washington DC 20037, USA (SK)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/DLX4ID49827ch17q21.html DOI: 10.4267/2042/46015 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

17q21-22 (Nakamura et al., 1996). We then mapped Identity BP1, which also mapped to the same chromosomal Other names: BP1; DLX7; DLX8; DLX9 region (Fu et al., 2001). In fact, our BAC included HGNC (Hugo): DLX4 sequences from both BP1 and DLX7. When we published the paper showing that BP1 is a repressor of Location: 17q21.33 the beta-globin gene and identifying BP1, DLX4 and DLX7 as isoforms, to prevent confusion in the DNA/RNA literature we gave the gene a single name, DLX4, based Note on the fact that the DLX4 DNA sequence was BP1, DLX4 and DLX7 are not interchangeable names published first (Chase et al., 2002). Thus, the DLX4 for the same gene, as sometimes claimed. We cloned a gene encodes at least three different proteins with cDNA encoding BP1 from a library made from K562 presumably different functions, DLX4, BP1 and DLX7. erythroleukemia cells, often used as a model for The NCBI Database is somewhat confusing in this hemoglobin switching. After it was sequenced it was regard - the gene is called DLX4, but BP1 is named apparent that part of the BP1 sequence was identical to DLX4 variant 1 and DLX7 is called DLX4 variant 2. that of two other published "genes", DLX4 and DLX7; Description upstream of nucleotide 565 of BP1, all three had The DLX4 gene is located at 17q21.33 and is about entirely different sequences, while downstream of that 5761 bp in length (chr17:48,046,562-48,052,322). site the sequences were identical (Fu et al., 2001; Chase et al., 2002). This suggested that the three might be Transcription isoforms of one gene, differing only in the first exon; Three different mRNAs are expressed by DLX4, BP1 this can occur by alternative splicing or by use of and DLX7. BP1 mRNA is about 2012 bp. alternative promoters. DLX7 had been mapped to

The red lines indicate the predicted ORFs. Number 565 indicates the nucleotide of BP1 where divergence occurs among BP1, DLX4, and DLX7. HB is the homeobox region. The regions between the two vertical lines indicate the regions of DNA identity. The complete ORF is not available for DLX4.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 658 DLX4 (distal-less homeobox 4) Berg PE, Kirolikar S

Amino acid sequence alignment between BP1 (Q92988-1), DLX7 (Q92988-2) and an unidentified isoform of DLX4 (Q92988-3). The sequences were obtained from the UniProt database which has incorrectly identified the BP1 sequence as DLX4.

high BP1 expression can lead to decreased cell death Protein and, as shown below, increased proliferation. Description BP1 appears to be a repressor of BRCA1. Three breast cancer cell lines engineered to overexpress BP1 show The BP1 protein is an alternatively spliced isoform decreased BRCA1 RNA and protein, while cells in derived from the DLX4 gene. The protein is about 240 which BP1 is knocked down by siRNA treatment show aa in length with the calculated molecular weight of 26 increased BRCA1 expression, suggesting that BP1 kDa. The observed weight of the protein is about 36 activity may contribute to reduced BRCA1 in some kDa on Western blots using lysates from breast cancer breast cancers (Kluk et al., 2010). and prostate cancer cell lines. This difference may be due to post-translational modifications of BP1 protein. Implicated in BP1 protein has a 116 aa N-terminal region, 60 aa homeobox domain and a 64 aa C-terminal domain, Breast cancer while DLX7 is 168 aa. Note Expression BP1 is activated in about 80% of invasive ductal In normal tissue: BP1 protein is expressed in adult breast (IDC) tumors. Aberrant expression of BP1 was kidney and placenta and in low levels in normal breast shown by semi-quantitative RT-PCR, where 80% of and fetal liver (Chase et al., 2002). tumors were BP1 positive, and by immunostaining, where 81% of tumors were BP1 positive, a remarkable Localisation agreement between mRNA and protein expression (Fu Both nuclear and cytoplasmic immunostaining are seen et al., 2003; Man et al., 2005). Surprisingly, 89% of the in BP1 positive breast tumors and prostate tumors (Man tumors of African American women (AAW) were BP1 et al., 2005; Schwartz et al., 2009). positive, compared with 57% of the tumors of Function Caucasian women (p=0.04). In addition, 100% of ER negative tumors were BP1 positive, compared with Functionally, we demonstrated that BP1 is a repressor 73% of ER positive (p=0.03). Both tumors of AAW of the beta-globin gene, while DLX7 binds to the same and ER negative tumors are associated with DNA sequence upstream of the beta-globin gene but aggressiveness. A group in China quantitated BP1 lacks the ability to repress it (Fu et al., 2001; Chase et mRNA in the tumors of 142 Chinese women, al., 2002). Thus, the functions of BP1 and DLX7 are discovering that 65% of their tumors were BP1 clearly different in this context. BP1 acts to repress positive, and confirming an association between high embryonic and fetal globin genes during early BP1 mRNA expression and ER negative tumors (Yu et development but is itself repressed during normal adult al., 2008b). Inflammatory breast cancer (IBC) is an erythropoiesis. extremely aggressive breast cancer, with approximately BP1 overexpression induces increased Bcl-2 expression half the survival seen in IDC; 100% of the forty-six and decreased apoptosis. pBP1 binds to the regulatory cases of IBC we examined were highly BP1 region of the bcl-2 gene, an anti-apoptotic gene, immunoreactive, suggesting an association between resulting in elevated expression of Bcl-2 protein and aggressiveness, frequency of BP1 positivity, and BP1 resistance to TNF-alpha in MCF-7 breast cancer cells protein (pBP1) staining intensity (Man et al., 2009). (Stevenson et al., 2007). Increased BP1 is associated pBP1 expression correlates with breast cancer with decreased cleavage of caspase-7, caspase-8 and progression. The frequency of pBP1 positivity, caspase-9, and increased expression of PARP. Thus,

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distribution and intensity of BP1 expression all Next we compared the growth-inhibitory and cyto- increased with the progression of tumor development differentiating activities of all-trans retinoic acid from 0% (normal) to 21% in hyperplasia, 46% in ductal (ATRA) in two acute promyelocytic leukemia (APL) carcinoma in situ, and 81% in IDC (p <0.0001) (Man et cells lines, NB4 (ATRA-responsive) and R4 (ATRA- al., 2005). This suggests BP1 expression may be an resistant) cells relative to BP1 levels (Awwad et al., important upstream factor in an oncogenic pathway and 2008). NB4 cells and R4 cells both expressed BP1; may contribute to tumor progression. BP1 was repressed after ATRA treatment of NB4 cells Expression of BP1 is associated with larger tumor but not R4 cells. In NB4 cells engineered to size. We have recently shown a correlation between overexpress BP1, proliferation was no longer inhibited BP1 mRNA or protein expression and tumor size in and differentiation was reduced two- to three-fold. In women with invasive ductal breast cancer. This patients, BP1 levels were increased in all pre-treatment correlation is also true in a mouse model (submitted). APL patients tested, while BP1 expression was BP1 positivity correlates with increased decreased in 91% of patients after combined ATRA proliferation. BP1 positive cells were significantly and chemotherapy treatment. Two patients underwent more frequently positive for Ki67, a proliferation disease relapse during follow up; one patient exhibited marker, when 5000 BP1 positive tumor cells were a 42-fold increase in BP1 expression, while the other compared with 5000 BP1 negative tumor cells (Man et showed no change. This suggests BP1 may be part of a al., 2005). pathway involved in resistance to therapy. BP1 appears to be associated with metastasis. Prostate cancer Among the IBC cases, nine had metastasized. The lymph nodes corresponding to these cases were all BP1 Note positive, providing evidence that BP1 is expressed in Prostate cancer, another hormone dependent solid metastasis (Man et al., 2009). Moreover, BP1 positive tumor, was examined for activation of BP1 (Schwartz cells were observed in lymphatic ducts of patients with et al., 2008). Significant BP1 immunoreactivity was metastatic IBC. A correlation between high BP1 identified in 70% of prostatic tumors, whether the mRNA levels and metastasis in invasive ductal breast analysis was performed on tissue sections (50 cases) or cancer was observed by Yu et al. (2008b). tissue microarray platforms (123 cases). We also BP1 mRNA levels are associated with survival. observed low BP1 immunostaining in 42% of Kaplan-Meier curves revealed that patients with grade hyperplastic cells, similar to the 46% BP1 positivity in III tumors expressing high BP1 mRNA levels showed hyperplastic breast cells. Compared to normal and decreased survival compared with patients whose grade hyperplastic tissues, the malignant tissues consistently III tumors contained lower BP1 mRNA levels (Yu et showed the highest number of BP1 positive cells and al., 2008b). the highest intensity of BP1 immunostaining, similar to BP1 is activated by DNA amplification. It is our observations in breast. In tissue sections, twelve important to determine the factors that activate BP1. cases with paired carcinoma and prostatic Approximately 33% of the tumors we examined from intraepithelial neoplasia (PIN) showed agreement, both women with metastatic breast cancer exhibited DNA components exhibiting strong immunoreactivity. amplification of BP1. Amplification was associated Tumor proliferation, assayed with Ki67 with BP1 positivity by immunostaining in all cases immunostaining, was higher in cancer cells that were (Cavalli et al., 2008). BP1 positive relative to those that were BP1 negative, Overall, the data strongly suggest that BP1 may be a in agreement with the data in breast cancer cells. These useful new biomarker in early detection of breast findings suggest that BP1 is an important upstream cancer and a potential therapeutic target. factor in the carcinogenic pathway of prostate cancer and that the expression of BP1 may reflect or directly Leukemia contribute to tumor progression and/or invasion. Note Non-small cell lung cancer (NSCLC) We examined BP1 in the bone marrow of leukemia patients by semi-quantitative RT-PCR, finding that Note BP1 was activated in 63% of acute myeloid leukemias An interesting study by Yu et al. (2008a) demonstrated (AML), including 81% of pediatric and 47% of adult that high BP1 mRNA levels occur in NSCLC tumors, patients with AML, in 32% of T-cell acute lymphocytic compared with adjacent normal cells or normal lung leukemias (ALL) but not in the pre-B ALL cases (Haga samples. High mRNA levels are associated with stage et al., 2000). Expression of BP1 occurred in primitive III tumors, lower disease free survival (DFS) and lower leukemia cells and in CD34 positive progenitors. In the overall survival. In fact, high BP1 mRNA is an same study we examined expression of DLX4 and independent predictor of DFS. DLX7 by designing primers specific for each isoform. Interestingly, the three isoforms were frequently co- References expressed in the same cases. Nakamura S, Stock DW, Wydner KL, Bollekens JA, Takeshita K, Nagai BM, Chiba S, Kitamura T, Freeland TM, Zhao Z,

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Minowada J, Lawrence JB, Weiss KM, Ruddle FH. Genomic homeobox gene, is associated with resistance to all-trans analysis of a new mammalian distal-less gene: Dlx7. retinoic acid in acute promyelocytic leukemia cells. Ann Genomics. 1996 Dec 15;38(3):314-24 Hematol. 2008 Mar;87(3):195-203 Haga SB, Fu S, Karp JE, Ross DD, Williams DM, Hankins WD, Cavalli LR, Man YG, Schwartz AM, Rone JD, Zhang Y, Urban Behm F, Ruscetti FW, Chang M, Smith BD, Becton D, CA, Lima RS, Haddad BR, Berg PE. Amplification of the BP1 Raimondi SC, Berg PE. BP1, a new homeobox gene, is homeobox gene in breast cancer. Cancer Genet Cytogenet. frequently expressed in acute leukemias. Leukemia. 2000 2008 Nov;187(1):19-24 Nov;14(11):1867-75 Yu M, Wan Y, Zou Q. Prognostic significance of BP1 mRNA Fu S, Stevenson H, Strovel JW, Haga SB, Stamberg J, Do K, expression level in patients with non-small cell lung cancer. Berg PE. Distinct functions of two isoforms of a homeobox Clin Biochem. 2008a Jul;41(10-11):824-30 gene, BP1 and DLX7, in the regulation of the beta-globin gene. Gene. 2001 Oct 31;278(1-2):131-9 Yu M, Yang Y, Shi Y, Wang D, Wei X, Zhang N, Niu R. Expression level of beta protein 1 mRNA in Chinese breast Chase MB, Fu S, Haga SB, Davenport G, Stevenson H, Do K, cancer patients: a potential molecular marker for poor Morgan D, Mah AL, Berg PE. BP1, a homeodomain-containing prognosis. Cancer Sci. 2008b Jan;99(1):173-8 isoform of DLX4, represses the beta-globin gene. Mol Cell Biol. 2002 Apr;22(8):2505-14 Man YG, Schwartz A, Levine PH, Teal C, Berg PE. BP1, a putative signature marker for inflammatory breast cancer and Fu SW, Schwartz A, Stevenson H, Pinzone JJ, Davenport GJ, tumor aggressiveness. Cancer Biomark. 2009;5(1):9-17 Orenstein JM, Gutierrez P, Simmens SJ, Abraham J, Poola I, Stephan DA, Berg PE. Correlation of expression of BP1, a Schwartz AM, Man YG, Rezaei MK, Simmens SJ, Berg PE. homeobox gene, with estrogen receptor status in breast BP1, a homeoprotein, is significantly expressed in prostate cancer. Breast Cancer Res. 2003;5(4):R82-7 adenocarcinoma and is concordant with prostatic intraepithelial neoplasia. Mod Pathol. 2009 Jan;22(1):1-6 Man YG, Fu SW, Schwartz A, Pinzone JJ, Simmens SJ, Berg PE. Expression of BP1, a novel homeobox gene, correlates Kluk BJ, Fu Y, Formolo TA, Zhang L, Hindle AK, Man YG, with breast cancer progression and invasion. Breast Cancer Siegel RS, Berg PE, Deng C, McCaffrey TA, Fu SW. BP1, an Res Treat. 2005 Apr;90(3):241-7 isoform of DLX4 homeoprotein, negatively regulates BRCA1 in sporadic breast cancer. Int J Biol Sci. 2010 Sep 10;6(5):513-24 Stevenson HS, Fu SW, Pinzone JJ, Rheey J, Simmens SJ, Berg PE. BP1 transcriptionally activates bcl-2 and inhibits This article should be referenced as such: TNFalpha-induced cell death in MCF7 breast cancer cells. Breast Cancer Res. 2007;9(5):R60 Berg PE, Kirolikar S. DLX4 (distal-less homeobox 4). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):658-661. Awwad RT, Do K, Stevenson H, Fu SW, Lo-Coco F, Costello M, Campbell CL, Berg PE. Overexpression of BP1, a

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 661 Atlas of Genetics and Cytogenetics

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Gene Section Review

IL17A (interleukin 17A) Norimitsu Inoue, Takashi Akazawa Department of Molecular Genetics, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Osaka 537-8511, Japan (NI, TA)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/IL17AID40945ch6p12.html DOI: 10.4267/2042/46016 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Pseudogene No pseudogene. Other names: CTLA8; IL-17; IL-17A; IL17 HGNC (Hugo): IL17A Protein Location: 6p12.2 Note Local order: Centromere-PKHD1 (polycystic kidney The IL17A protein is a disulfide-linked homodimeric and hepatic diseases 1)-MIR206 (microRNA 206)- glycoprotein. Members of the IL17 protein family MIR133B (microRNA 133b)-IL17A-IL17F (IL17A to F) have four highly conserved cysteine (interleukin 17F)-SLC25A20P1 (solute carrier family residues on each of the monomeric peptides (Moseley 25, member 20 pseudogene 1)-MCM3 et al., 2003; Kolls et al., 2004; Korn et al., 2009). (minichromosome maintenance complex component Structural analysis of the IL17F protein indicates that 3)-Telomere. these four cysteines participate in the characteristic cysteine-knot formation found in certain other growth DNA/RNA factors such as nerve growth factor (NGF), bone Description morphogenetic proteins (BMPs), and transforming growth factor-beta1 (TGFbeta1) (Hymowitz et al., 3 exons. 2001). Two additional cysteine residues participate in Transcription homodimer formation via inter-chain disulfide-bonds. The IL17F peptide can also form a functional The transcript is 1859 bp and has a 45 bp 5' UTR, a 468 heterodimer with IL17A. bp coding sequence, and a 1346 bp 3' UTR.

IL17A gene. The IL17A gene spans a region of 4252 bp composed of three exons (untranslated region (UTR), light blue; coding region, blue) and two introns (brown). Exons 1, 2, and 3 are 72 bp (45 bp 5' UTR plus 27 bp coding region), 203 bp (all coding region), and 1584 bp (238 bp coding region plus 1346 bp 3' UTR) in length, respectively. The two introns are 1144 bp and 1249 bp in length, respectively.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 662 IL17A (interleukin 17A) Inoue N, Akazawa T

IL-17A protein. IL17A protein (155 amino acids) is composed of a signal peptide (light green, 23 amino acids) and a mature peptide (green, 132 amino acids). The four conserved cysteines (Cys) form the intra-chain disulfide bonds indicated by black lines (Cys94/Cys144 and Cys99/Cys146) (Hymowitz et al., 2001). The two cysteines indicated by asterisks (Cys33 and Cys129) participate in homodimer formation via inter-chain disulfide bonds. Asparagine 68 (Asn68, black circle) is predicted to be glycosylated.

Description hydrocarbon receptor (AHR, a nuclear receptor for a number of low-molecular weight chemicals such as the Each IL17A monomer is a 19.9-kD peptide that tryptophan photoproduct 6-formylindolo[3,2- consists of 155 amino acids. The IL17A peptide b]carbazole (FICZ)) all positively regulate Th17 cell comprises a 23-amino acid signal peptide and a 132- differentiation (Korn et al., 2009; Hirahara et al., 2010). amino acid mature peptide (IL17A homodimer, 35 kD). Moreover, prostaglandin E2, ATP, and C-type lectin Expression ligands act on antigen-presenting cells to facilitate IL17A is secreted by CD4-positive T cells (Th17 cells), Th17 cell differentiation. In contrast, IL4, Interferon- which also produce IL17F, IL21, and IL22 (Korn et al., gamma (IFNgamma), IL27, suppressor of cytokine 2009; Eyerich et al., 2010). CD8-positive T cells, signaling 3 (SOCS3), and STAT5 suppress Th17 cell gamma delta T cells, natural killer (NK) cells, NKT differentiation. Finally, high levels of lactic acid cells, and lymphoid tissue inducer (LTi) cells also secreted from tumors via the Warburg effect act on secret IL17A. These leukocytes all express the retinoic macrophages to mediate increased IL17A production acid receptor-related orphan nuclear receptor C but not Th17 cell differentiation (Shime et al., 2008; (RORC, the human analogue of mouse RORgammat Yabu et al., 2011). that is a splice variant of the Rorc gene). RORC is Th17 cells in both the mouse and the human have essential for IL17A production. recently been shown to differentiate from naïve CD4 T Th17 cells are the third subset of helper T cells, with cells independently of TGFbeta1 signaling. These effector functions distinct from Th1 and Th2 cells. TGFbeta1-independent Th17 cells instead differentiate Th17 cells are differentiated from naïve T cells in the in the presence of IL6, IL23 and IL1beta (Hirahara et presence of IL6 plus TGFbeta1 (Bettelli et al., 2007; al., 2010; Ghoreschi et al., 2010). TGFbeta1- McGeachy et al., 2008; Awasthi et al., 2009). In the independent Th17 cells co-express RORgammat and T- presence of TGFbeta1 alone, naïve T cells express the bet (TBX21, T-box protein 21) and exhibit more transcriptional factor forkhead box P3 (FOXP3) and pathogenic potential than TGFbeta1-dependent Th17 differentiate into induced regulatory T cells (iTreg cells in the development of experimental allergic cells). In the presence of IL6 alone, the cells express encephalomyelitis (EAE). the transcriptional factor BCL6 and differentiate into T Function follicular helper cells (Tfh cells) (Nurieva et al., 2009). Interleukin 17A is a pro-inflammatory cytokine and act Interleukin 21 is secreted from Th17 cells and amplifies on a variety of cells (e.g., fibroblasts, epithelial cells, Th17 cell generation by an autocrine mechanism. and monocytes) to induce the production of cytokines Interleukin 21 also induces the expression of the IL23 (IL6, tumor necrosis factor-alpha TNFalpha, receptor in the Th17 cells (Bettelli et al., 2007; granulocyte-macrophage colony-stimulating-factor McGeachy and Cua, 2008; Awasthi and Kuchroo, (GMCSF), granulocyte colony-stimulating-factor 2009). Interleukin 23 is secreted from dendritic cells (GCSF)), chemokines (chemokine (C-X-C motif) and macrophages following stimulation by Toll-like ligand 1 (CXCL1), CXCL2, CXCL5, CXCL8) and receptor ligands. IL23 in turn mediates the stabilization matrix metalloproteinases (MMP2, MMP13) to mediate and maintenance of the Th17 cell phenotype, inducing the recruitment, activation and migration of neutrophils IL17A production by Th17 cells (Stritesky et al., 2008; and myeloid cells (Kolls and Linden, 2004; Eyerich et McGeachy et al., 2009). Interleukin 1beta is also al., 2010). involved in the induction of IL17A secretion and the IL17A, IL17F, and the IL17A-IL17F heterodimer bind promotion of Th17 differentiation (Chung et al., 2009). to a heteromeric receptor complex composed of IL17 In addition to RORC and the aforementioned cytokines, receptor A (IL17RA) and IL17 receptor C (IL17RC). signal transducer and activator of transcription 3 IL17RA is expressed at high levels in hematopoietic (STAT3), interferon regulatory factor 4 (IRF4), runt- cells and at low levels in epithelial cells, fibroblasts and related transcriptional factor 1 (RUNX1), and aryl

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 663 IL17A (interleukin 17A) Inoue N, Akazawa T

endothelial cells (Gaffen, 2009). On the other hand, addition, Immunotherapy is more effective in patients IL17RC is expressed at low levels in hematopoietic with prostate cancer that have a higher number of Th17 cells and at high levels in the adrenal gland, prostate, cells. liver, and thyroid. Although cytokines secreted by most In the mouse system, the overexpression of IL17A in activated helper T cells generally stimulate the Janus tumor cells suppresses tumor growth in a cytotoxic T kinase (JAK)/STAT pathway, the IL17 family lymphocyte-dependent manner (Benchetrit et al., cytokines stimulate signal pathways that are common 2002). The transfer of tumor antigen-specific T cells in the innate immune system, such as the Toll-like polarized to the IL17-producing phenotype also induces receptor signaling pathway. the eradication of tumor cells by inducing strong CD8- IL17 receptors have a conserved domain termed the positive T cell activation (Martin-Orozco et al., 2009). "similar expression to fibroblast growth factor/IL-17R Furthermore, the deficiency of IL17A in mice promotes (SEFIR)" domain in the cytoplasmic region. This the growth and metastasis of tumors (Martin-Orozco et domain is similar to the Toll-/IL-1R (TIR) domain al., 2009; Kryczek et al., 2009b). Interleukin 17A- (Gaffen, 2009). When the IL17 receptor is activated, producing T cells are predicted to induce the the adaptor molecule actin related gene 1 (Act1, a U- recruitment of other effector cells (e.g., cytotoxic CD8- box E3 ubiquitin ligase) is recruited to the SEFIR positive T cells and NK cells) to the tumors by domain and mediates the lysine63-linked ubiquitination inducing the expression of CXCL9 and CXCL10 by of tumor necrosis factor receptor-associated factor 6 tumors (Kryczek et al., 2009a). Moreover, Th17 cells (TRAF6). Ubiquitinated TRAF6 then activates the induce the expression of chemokine (C-C motif) ligand transcriptional factor nuclear factor-kappaB 20 (CCL20, a ligand for chemokine (C-C motif) (NFkappaB), various mitogen-activated protein (MAP) receptor 6 (CCR6)) in tumor tissues. Chemokine (C-C kinases including Erk and p38, and CCAAT/enhancer- motif) ligand 20 recruits dendritic cells to mediate anti- binding proteins (C/EBP beta and C/EBP gamma). tumor effects in a CCL20/CCR6-depedent manner Homology (Martin-Orozco et al., 2009). Pro-tumor effects. IL17A is a prototypical member of the IL17 family. The proportion of Th17 cells in the peripheral blood is This family includes six proteins, termed IL17A, increased in patients with advanced stage gastric cancer IL17B, IL17C, IL17D, IL17E (also called IL25), and compared with patients with early stage diseases IL17F. Interleukin 17A to F are not homologous to any (Zhang et al., 2008). In patients with hepatocellular other known proteins. IL17A shows the highest carcinoma, increased intratumoral accumulation of homology with IL17F (55%). It is less similar to the IL17A-producing cells is significantly associated with a other IL17 family members (IL17B, 29%; IL17C, 23%; poor prognosis (Zhang et al., 2009). IL17D, 25%; and IL17E, 17%) (Kolls and Linden, In the mouse system, the overexpression of IL17A in 2004). tumors facilitates tumor growth via the induction of angiogenesis in the tumor microenvironment Implicated in (Numasaki et al., 2003; Numasaki et al., 2005). Furthermore, IL17A-deficient or IL17RA-deficient Various cancers mouse models were used to show that IL17A was Note involved in the promotion of tumor growth via Infiltration of IL17A-producing T cells in tumors. induction of myeloid derived suppressor cells (MDSC) IL17A-producing T cells and/or IL17A expression are (He et al., 2010), activation of IL6-STAT3 pathway detected in many human tumor tissues, including (Wang et al., 2009), and production of IL17A by ovarian, pancreatic, renal cell, prostate, gastric, and tumor-infiltrating gamma delta T cells (Wakita et al., hepatocellular cancers (Zou et al., 2010; Maniati et al., 2010). 2010). Although IL17A-producing cells are not the The discrepancies between anti-tumor and pro-tumor dominant T cell subset in the tumor microenvironment, effects may be due to distinct roles of IL17A and they are increased to greater extent in the tumor site IL17A-producing cells in different tumors. than in the peripheral blood of the patients (Kryczek et Gastric cancer al., 2009a). Anti-tumor effects. Note In some human tumors, such as ovarian and prostate The single nucleotide polymorphism (SNP) in the cancer, IL17A and IL17A-producing cells are IL17A gene promoter region, which is located at a associated with antitumorigenic actions. Increased position -197 from the start codon (rs2275913, G/A IL17A levels in ascites are well-correlated with better SNPs, a position at 52051033 bp from pter), has been patient survival and lower grading stages of ovarian examined in Japanese gastric cancer patients (Shibata cancer (Kryczek et al., 2009a). An increased population et al., 2009). The frequency of the A-allele (odds ratio, of Th17 cells is also associated with lower grading 1.42) and the A/A homozygote (odds ratio, 3.02) is stages of prostate cancer (Sfanos et al., 2008). In significantly increased in gastric cancer patients compared with healthy controls.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 664 IL17A (interleukin 17A) Inoue N, Akazawa T

Autoimmune and inflammatory Roark CL, Simonian PL, Fontenot AP, Born WK, O'Brien RL. gammadelta T cells: an important source of IL-17. Curr Opin diseases Immunol. 2008 Jun;20(3):353-7 Note Sfanos KS, Bruno TC, Maris CH, Xu L, Thoburn CJ, DeMarzo Interleukin 17A and IL17-producing cells are AM, Meeker AK, Isaacs WB, Drake CG. Phenotypic analysis of associated with the pathogenesis of many autoimmune prostate-infiltrating lymphocytes reveals TH17 and Treg skewing. Clin Cancer Res. 2008 Jun 1;14(11):3254-61 and inflammatory diseases such as EAE/multiple sclerosis, inflammatory skin diseases/psoriasis, Shime H, Yabu M, Akazawa T, Kodama K, Matsumoto M, Seya T, Inoue N. Tumor-secreted lactic acid promotes IL-23/IL- inflammatory bowel diseases, and experimental 17 proinflammatory pathway. J Immunol. 2008 Jun arthritis/rheumatoid arthritis in humans as well as mice 1;180(11):7175-83 (Korn et al., 2009; Awasthi and Kuchroo, 2009). Stritesky GL, Yeh N, Kaplan MH. IL-23 promotes maintenance Infections but not commitment to the Th17 lineage. J Immunol. 2008 Nov 1;181(9):5948-55 Note Zhang B, Rong G, Wei H, Zhang M, Bi J, Ma L, Xue X, Wei G, Both IL17A and IL17F are preferentially produced Liu X, Fang G. The prevalence of Th17 cells in patients with during infections with the Gram-negative bacteria gastric cancer. Biochem Biophys Res Commun. 2008 Sep Klebsiella pneumonia, Borrelia burgdorferi, and 26;374(3):533-7 Salmonella enterica enteritidis; the Gram-positive Awasthi A, Kuchroo VK. Th17 cells: from precursors to players bacterium Listeria monocytogenes; the acid-fast in inflammation and infection. Int Immunol. 2009 bacterium Mycobacterium tuberculosis; and the yeast- May;21(5):489-98 like fungi Pneumocystis jirovecii and Candida albicans Chung Y, Chang SH, Martinez GJ, Yang XO, Nurieva R, Kang (Korn et al., 2009; O'Connor et al., 2010). In an early HS, Ma L, Watowich SS, Jetten AM, Tian Q, Dong C. Critical response to the infection, IL17A is predominantly regulation of early Th17 cell differentiation by interleukin-1 secreted by gamma delta T cells (Roark et al., 2008; signaling. Immunity. 2009 Apr 17;30(4):576-87 Cua et al., 2010). This results in the rapid recruitment Gaffen SL. Structure and signalling in the IL-17 receptor family. of neutrophils to sites of infection for efficient Nat Rev Immunol. 2009 Aug;9(8):556-67 pathogen clearance. Later, antigen-specific Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 alphabetaTh17 cells contribute to the response. Cells. Annu Rev Immunol. 2009;27:485-517 Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, References Huang E, Finlayson E, Simeone D, Welling TH, Chang A, Coukos G, Liu R, Zou W. Phenotype, distribution, generation, Hymowitz SG, Filvaroff EH, Yin JP, Lee J, Cai L, Risser P, and functional and clinical relevance of Th17 cells in the Maruoka M, Mao W, Foster J, Kelley RF, Pan G, Gurney AL, human tumor environments. Blood. 2009a Aug 6;114(6):1141- de Vos AM, Starovasnik MA. IL-17s adopt a cystine knot fold: 9 structure and activity of a novel cytokine, IL-17F, and implications for receptor binding. EMBO J. 2001 Oct Kryczek I, Wei S, Szeliga W, Vatan L, Zou W. Endogenous IL- 1;20(19):5332-41 17 contributes to reduced tumor growth and metastasis. Blood. 2009b Jul 9;114(2):357-9 Benchetrit F, Ciree A, Vives V, Warnier G, Gey A, Sautès- Fridman C, Fossiez F, Haicheur N, Fridman WH, Tartour E. Martin-Orozco N, Muranski P, Chung Y, Yang XO, Yamazaki Interleukin-17 inhibits tumor cell growth by means of a T-cell- T, Lu S, Hwu P, Restifo NP, Overwijk WW, Dong C. T helper dependent mechanism. Blood. 2002 Mar 15;99(6):2114-21 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity. 2009 Nov 20;31(5):787-98 Moseley TA, Haudenschild DR, Rose L, Reddi AH. Interleukin- 17 family and IL-17 receptors. Cytokine Growth Factor Rev. McGeachy MJ, Chen Y, Tato CM, Laurence A, Joyce-Shaikh 2003 Apr;14(2):155-74 B, Blumenschein WM, McClanahan TK, O'Shea JJ, Cua DJ. The interleukin 23 receptor is essential for the terminal Numasaki M, Fukushi J, Ono M, Narula SK, Zavodny PJ, Kudo differentiation of interleukin 17-producing effector T helper cells T, Robbins PD, Tahara H, Lotze MT. Interleukin-17 promotes in vivo. Nat Immunol. 2009 Mar;10(3):314-24 angiogenesis and tumor growth. Blood. 2003 Apr 1;101(7):2620-7 Nurieva RI, Chung Y, Martinez GJ, Yang XO, Tanaka S, Matskevitch TD, Wang YH, Dong C. Bcl6 mediates the Kolls JK, Lindén A. Interleukin-17 family members and development of T follicular helper cells. Science. 2009 Aug inflammation. Immunity. 2004 Oct;21(4):467-76 21;325(5943):1001-5 Numasaki M, Watanabe M, Suzuki T, Takahashi H, Nakamura Shibata T, Tahara T, Hirata I, Arisawa T. Genetic A, McAllister F, Hishinuma T, Goto J, Lotze MT, Kolls JK, polymorphism of interleukin-17A and -17F genes in gastric Sasaki H. IL-17 enhances the net angiogenic activity and in carcinogenesis. Hum Immunol. 2009 Jul;70(7):547-51 vivo growth of human non-small cell lung cancer in SCID mice through promoting CXCR-2-dependent angiogenesis. J Wang L, Yi T, Kortylewski M, Pardoll DM, Zeng D, Yu H. IL-17 Immunol. 2005 Nov 1;175(9):6177-89 can promote tumor growth through an IL-6-Stat3 signaling pathway. J Exp Med. 2009 Jul 6;206(7):1457-64 Bettelli E, Korn T, Kuchroo VK. Th17: the third member of the effector T cell trilogy. Curr Opin Immunol. 2007 Dec;19(6):652- Zhang JP, Yan J, Xu J, Pang XH, Chen MS, Li L, Wu C, Li SP, 7 Zheng L. Increased intratumoral IL-17-producing cells correlate with poor survival in hepatocellular carcinoma patients. J McGeachy MJ, Cua DJ. Th17 cell differentiation: the long and Hepatol. 2009 May;50(5):980-9 winding road. Immunity. 2008 Apr;28(4):445-53

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Cua DJ, Tato CM. Innate IL-17-producing cells: the sentinels of Maniati E, Soper R, Hagemann T. Up for Mischief? IL-17/Th17 the immune system. Nat Rev Immunol. 2010 Jul;10(7):479-89 in the tumour microenvironment. Oncogene. 2010 Oct 21;29(42):5653-62 Eyerich S, Eyerich K, Cavani A, Schmidt-Weber C. IL-17 and IL-22: siblings, not twins. Trends Immunol. 2010 O'Connor W Jr, Zenewicz LA, Flavell RA. The dual nature of Sep;31(9):354-61 T(H)17 cells: shifting the focus to function. Nat Immunol. 2010 Jun;11(6):471-6 Ghoreschi K, Laurence A, Yang XP, Tato CM, McGeachy MJ, Konkel JE, Ramos HL, Wei L, Davidson TS, Bouladoux N, Wakita D, Sumida K, Iwakura Y, Nishikawa H, Ohkuri T, Grainger JR, Chen Q, Kanno Y, Watford WT, Sun HW, Eberl Chamoto K, Kitamura H, Nishimura T. Tumor-infiltrating IL-17- G, Shevach EM, Belkaid Y, Cua DJ, Chen W, O'Shea JJ. producing gammadelta T cells support the progression of Generation of pathogenic T(H)17 cells in the absence of TGF-β tumor by promoting angiogenesis. Eur J Immunol. 2010 signalling. Nature. 2010 Oct 21;467(7318):967-71 Jul;40(7):1927-37 He D, Li H, Yusuf N, Elmets CA, Li J, Mountz JD, Xu H. IL-17 Zou W, Restifo NP. T(H)17 cells in tumour immunity and promotes tumor development through the induction of tumor immunotherapy. Nat Rev Immunol. 2010 Apr;10(4):248-56 promoting microenvironments at tumor sites and myeloid- derived suppressor cells. J Immunol. 2010 Mar 1;184(5):2281- Yabu M, Shime H, Hara H, Saito T, Matsumoto M, Seya T, 8 Akazawa T, Inoue N. IL-23-dependent and -independent enhancement pathways of IL-17A production by lactic acid. Int Hirahara K, Ghoreschi K, Laurence A, Yang XP, Kanno Y, Immunol. 2011 Jan;23(1):29-41 O'Shea JJ. Signal transduction pathways and transcriptional regulation in Th17 cell differentiation. Cytokine Growth Factor This article should be referenced as such: Rev. 2010 Dec;21(6):425-34 Inoue N, Akazawa T. IL17A (interleukin 17A). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):662-666.

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Gene Section Mini Review

MYBBP1A (MYB binding protein (P160) 1a) Claudia Perrera, Riccardo Colombo Department of Cell Biology-Oncology, Nerviano Medical Sciences, Viale Pasteur 10, Nerviano 20014, Italy (CP, RC)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/MYBBP1AID41467ch17p13.html DOI: 10.4267/2042/46000 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Protein Other names: FLJ37886; P160; PAP2 Description HGNC (Hugo): MYBBP1A Human MYBBP1A is a 1328 aa long protein. Murine Location: 17p13.2 MYBBP1A was originally identified as a protein interacting with the leucine zipper of c-Myb (Favier et DNA/RNA al., 1994). Subsequently, in 1998, the human gene homologue of MYBBP1A was cloned and its Note chromosomal location mapped to 17p13.3 (Keough et Total gene length 16491 bp, mRNA length 4122 bp, al., 1999). telomeric to SPNS2 and centromeric to GGT6. Two variants described. MYBBP1A gene is composed by 26 Expression exones and 28 introns. MYBBP1A is ubiquitously expressed (Tavner et al., Description 1998). The human MYBBP1A gene is located on chromosome Localisation 17p13.3 (Keough et al., 1999). MYBBP1A is a nuclear protein, predominantly Transcription localized in the nucleolus (Keough et al., 2003). MYBBP1A has been confirmed as a resident protein of The complete MYBBP1A cDNA is 4518 bp, including the nucleolus by three large-scale proteomic studies 25 bp of 5' UTR and 506 bp of 3' UTR up to the polyA that have established a protein inventory of this sub- tail. nuclear compartment

Schematic representation of Mybbp1A protein. aa 1-582 is the domain reported to interact in vitro with Myb. NLS: Nuclear and nucleolar localisation signal. The indicated S, T and Y are phosphorylated residues identified in several phospho-proteomic studies.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 667 MYBBP1A (MYB binding protein (P160) 1a) Perrera C, Colombo R

(Andersen et al., 2002; Scherl et al., 2002; Andersen et component of the proteome as well as the phospho- al., 2005). The nuclear/nucleolar localization signals proteome of the human mitotic spindle. MYBBP1A are present in the C-terminal tail of MYBBP1A. contains several consensus motifs for several kinases, Function but until now, only Ser1303 has been proven in vitro and in HeLa cells to be indeed phosphorylated by MYBBP1A functions have not yet been completely Aurora B kinase (Perrera et al., 2010). In this work, it clarified. It was originally identified as a protein able to has been shown that MYBBP1A depletion by RNAi interact with the negative regulatory domain (NRD) of causes a delay in progression through mitosis and c-Myb; however, it was later shown to lack any defects in mitotic spindle assembly and stability, significant effect in a Myb-dependent transcription indicating that, like other nucleolar proteins, reporter assay (Favier et al., 1994). MYBBP1A has MYBBP1A may have a role in insuring correct mitotic been found to interact with and regulate several progression (Perrera et al., 2010). transcription factors: it binds and represses both Prep1- MYBBP1A has been reported to be also sumoylated Pbx1, involved in development and organogenesis, and upon MG132 treatment (Matafora et al., 2009). also PGC-1a, a key regulator of metabolic processes such as mitochondrial biogenesis and respiration and Homology gluconeogenesis in liver (Fan et al., 2004; Diaz et al., Orthologous genes for MYBBP1A sharing a high 2007). MYBBP1A acts as a co-repressor for RelA/p65, degree of similarity are present in rat and mice. Protein a member of the NFkB family, by competing with the homologues have also been recognized in dog, bovine, co-activator p300 histone acetyltrasferase for and chicken and a MYBBP1A-like protein spanning interaction with the transcription activation domain 1269 residues and showing a 60% similarity to the (TAD) of RelA/p65. It is also a co-repressor on the human protein has been identified in zebrafish, Period2 promoter, repressing the expression of Per2, an suggesting that MYBBP1A is significantly conserved essential gene in the regulation of the circadian clock across vertebrate species (Amsterdam et al., 2004). (Owen et al., 2007; Hara et al., 2009). Conversely, MYBBP1A shares some homology to a yeast protein MYBBP1A is a positive regulator of the aromatic called POL5, reported to be an essential DNA hydrocarbon receptor (AhR) which mediates polymerase in Saccharomyces cerevisiae (Yang et al., transcriptional responses to certain hydrophobic 2003). ligands, such as dioxin, by enhancing the ability of AhR to activate transcription (Jones et al., 2002). Implicated in MYBBP1A has also been reported to be a component of macromolecular complexes such as the B-WICH Various cancers complex, a 3 MDa assembly made of proteins and Disease RNAs, formed during active transcription (Cavellan et MYBBP1A maps at 17p13.3, a region frequently lost in al., 2006) or part of large interactomes such as the many solid and haematological tumors, such as breast SMN interactome (Fuller et al., 2010). and ovarian cancer, medulloblastoma, astrocytoma, MYBBP1A can be post-translationally processed in leukemias, etc. This indicates that this chromosomal some type of cells to generate an amino-terminal band contains one or more tumor suppressor genes. fragments of 67 kDa (p67). Ribosomal stress induced However, MYBBP1A is unlikely a candidate for being by Actinomycin D (an inhibitor of ribosome a tumor suppressor gene, as it lies centromeric to the biogenesis) treatment causes MYBBP1A processing regions of LOH described (Keough et al., 1999). and translocation from the nucleolus to the nucloplasm (Diaz et al., 2007; Yamauchi et al., 2008), indicating a References possible MYBBP1A role in ribosome biogenesis. Several post-translational modifications have been Favier D, Gonda TJ. Detection of proteins that bind to the leucine zipper motif of c-Myb. Oncogene. 1994 Jan;9(1):305- described for MYBBP1A, even if their biological 11 significance is not yet clarified. MYBBP1A is reported to be a heavily phosphorylated protein in cells, Tavner FJ, Simpson R, Tashiro S, Favier D, Jenkins NA, Gilbert DJ, Copeland NG, Macmillan EM, Lutwyche J, Keough according to several large-scale mass spectrometry- RA, Ishii S, Gonda TJ. Molecular cloning reveals that the p160 based phosphoproteomic studies (Beausoleil et al., Myb-binding protein is a novel, predominantly nucleolar protein 2004; Beausoleil et al., 2006; Nousiainen et al., 2006; which may play a role in transactivation by Myb. Mol Cell Biol. Olsen et al., 2006; Cantin et al., 2008; Daub et al., 1998 Feb;18(2):989-1002 2008; Dephoure et al., 2008; Imami et al., 2008). The Keough R, Woollatt E, Crawford J, Sutherland GR, Plummer S, majority of the phosphosites mapped in MYBBP1A in Casey G, Gonda TJ. Molecular cloning and chromosomal mapping of the human homologue of MYB binding protein these studies (18 out of a total of 21) reside within the (P160) 1A (MYBBP1A) to 17p13.3. Genomics. 1999 Dec ~200 amino-acid long C-terminal portion of the 15;62(3):483-9 protein, which has been shown to be relevant for its Andersen JS, Lyon CE, Fox AH, Leung AK, Lam YW, Steen H, nuclear and nucleolar localization (Keough et al., Mann M, Lamond AI. Directed proteomic analysis of the human 2003). Notably, MYBBP1A was also found to be also a nucleolus. Curr Biol. 2002 Jan 8;12(1):1-11

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Jones LC, Okino ST, Gonda TJ, Whitlock JP Jr. Myb-binding Díaz VM, Mori S, Longobardi E, Menendez G, Ferrai C, protein 1a augments AhR-dependent gene expression. J Biol Keough RA, Bachi A, Blasi F. p160 Myb-binding protein Chem. 2002 Jun 21;277(25):22515-9 interacts with Prep1 and inhibits its transcriptional activity. Mol Cell Biol. 2007 Nov;27(22):7981-90 Scherl A, Couté Y, Déon C, Callé A, Kindbeiter K, Sanchez JC, Greco A, Hochstrasser D, Diaz JJ. Functional proteomic Owen HR, Elser M, Cheung E, Gersbach M, Kraus WL, analysis of human nucleolus. Mol Biol Cell. 2002 Hottiger MO. MYBBP1a is a novel repressor of NF-kappaB. J Nov;13(11):4100-9 Mol Biol. 2007 Feb 23;366(3):725-36 Keough RA, Macmillan EM, Lutwyche JK, Gardner JM, Tavner Cantin GT, Yi W, Lu B, Park SK, Xu T, Lee JD, Yates JR 3rd. FJ, Jans DA, Henderson BR, Gonda TJ. Myb-binding protein Combining protein-based IMAC, peptide-based IMAC, and 1a is a nucleocytoplasmic shuttling protein that utilizes CRM1- MudPIT for efficient phosphoproteomic analysis. J Proteome dependent and independent nuclear export pathways. Exp Cell Res. 2008 Mar;7(3):1346-51 Res. 2003 Sep 10;289(1):108-23 Daub H, Olsen JV, Bairlein M, Gnad F, Oppermann FS, Körner Yang W, Rogozin IB, Koonin EV. Yeast POL5 is an R, Greff Z, Kéri G, Stemmann O, Mann M. Kinase-selective evolutionarily conserved regulator of rDNA transcription enrichment enables quantitative phosphoproteomics of the unrelated to any known DNA polymerases. Cell Cycle. 2003 kinome across the cell cycle. Mol Cell. 2008 Aug 8;31(3):438- Mar-Apr;2(2):120-2 48 Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Dephoure N, Zhou C, Villén J, Beausoleil SA, Bakalarski CE, Hopkins N. Identification of 315 genes essential for early Elledge SJ, Gygi SP. A quantitative atlas of mitotic zebrafish development. Proc Natl Acad Sci U S A. 2004 Aug phosphorylation. Proc Natl Acad Sci U S A. 2008 Aug 31;101(35):12792-7 5;105(31):10762-7 Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Imami K, Sugiyama N, Kyono Y, Tomita M, Ishihama Y. Li J, Cohn MA, Cantley LC, Gygi SP. Large-scale Automated phosphoproteome analysis for cultured cancer cells characterization of HeLa cell nuclear phosphoproteins. Proc by two-dimensional nanoLC-MS using a calcined titania/C18 Natl Acad Sci U S A. 2004 Aug 17;101(33):12130-5 biphasic column. Anal Sci. 2008 Jan;24(1):161-6 Fan M, Rhee J, St-Pierre J, Handschin C, Puigserver P, Lin J, Yamauchi T, Keough RA, Gonda TJ, Ishii S. Ribosomal stress Jäeger S, Erdjument-Bromage H, Tempst P, Spiegelman BM. induces processing of Mybbp1a and its translocation from the Suppression of mitochondrial respiration through recruitment of nucleolus to the nucleoplasm. Genes Cells. 2008 Jan;13(1):27- p160 myb binding protein to PGC-1alpha: modulation by p38 39 MAPK. Genes Dev. 2004 Feb 1;18(3):278-89 Hara Y, Onishi Y, Oishi K, Miyazaki K, Fukamizu A, Ishida N. Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond Molecular characterization of Mybbp1a as a co-repressor on AI, Mann M. Nucleolar proteome dynamics. Nature. 2005 Jan the Period2 promoter. Nucleic Acids Res. 2009 6;433(7021):77-83 Mar;37(4):1115-26 Beausoleil SA, Villén J, Gerber SA, Rush J, Gygi SP. A Matafora V, D'Amato A, Mori S, Blasi F, Bachi A. Proteomics probability-based approach for high-throughput protein analysis of nucleolar SUMO-1 target proteins upon proteasome phosphorylation analysis and site localization. Nat Biotechnol. inhibition. Mol Cell Proteomics. 2009 Oct;8(10):2243-55 2006 Oct;24(10):1285-92 Fuller HR, Man NT, Lam le T, Thanh le T, Keough RA, Cavellán E, Asp P, Percipalle P, Farrants AK. The WSTF- Asperger A, Gonda TJ, Morris GE. The SMN interactome SNF2h chromatin remodeling complex interacts with several includes Myb-binding protein 1a. J Proteome Res. 2010 nuclear proteins in transcription. J Biol Chem. 2006 Jun Jan;9(1):556-63 16;281(24):16264-71 Perrera C, Colombo R, Valsasina B, Carpinelli P, Troiani S, Nousiainen M, Silljé HH, Sauer G, Nigg EA, Körner R. Modugno M, Gianellini L, Cappella P, Isacchi A, Moll J, Phosphoproteome analysis of the human mitotic spindle. Proc Rusconi L. Identification of Myb-binding protein 1A (MYBBP1A) Natl Acad Sci U S A. 2006 Apr 4;103(14):5391-6 as a novel substrate for aurora B kinase. J Biol Chem. 2010 Apr 16;285(16):11775-85 Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M. Global, in vivo, and site-specific phosphorylation This article should be referenced as such: dynamics in signaling networks. Cell. 2006 Nov 3;127(3):635- 48 Perrera C, Colombo R. MYBBP1A (MYB binding protein (P160) 1a). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):667-669.

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Gene Section Mini Review

PLCD1 (phospholipase C, delta 1) Xiaotong Hu Biomedical Research Center, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China (XH)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/PLCD1ID43927ch3p22.html DOI: 10.4267/2042/46001 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

in a shorter isoform 2 (NP_006216) with a different N- Identity terminus compared to isoform 1. HGNC (Hugo): PLCD1 Location: 3p22.2 Protein Local order: The PLCD1 gene is located between the Description VILL gene and the DLEC1 gene. The deduced 777-amino acid isoform 1 (NM_001130964) and 756-amino acid isoform 2 DNA/RNA (NP_006216) shares 95% sequence homology with the Description rat protein. They contain a N-terminal PH domain, 2 EF-hand1 domains, PI-PLC X-box, PI-PLC Y-box and The PLCD1 gene is a functioning gene and contains 15 C2 region. exons and spans 22.17 kb. Expression Transcription Expressed high or medium in CNS (brain), The variant 1 (NM_001130964) encodes the longer hematopoietic (blood), liver and pancreas, digestive isoform 1 (NP_001124436). The variant 2 (GI-tract), respiratory (lung), male and female tissues, (NM_006225.3) contains an alternate 5' terminal exon placenta, urinary tract (kidney) skin and soft tissues but compared to transcript variant 1, and initiates no expression in cardio vascular and endocrine tissues. translation from an in-frame upstream AUG, resulting

PLCD1 starts at 38048987 bp and ends at 38071253 bp from pter on Chr3p22-p21.3 and located between VILL and DLEC1 gene.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 670 PLCD1 (phospholipase C, delta 1) Hu X

Closed and opened boxes represent coding and non-coding sequences of PLCD1 gene, respectively.

Protein domain organization of the mammalian PLCD1.

Localisation cytoplasm, and nuclear import is mediated by its Ca2+ - dependent interaction with importin beta 1. Playing an Intracellular: cytoplasm and nucleus. important suppressive role in the development and Function progression of cancers such as esophageal squamous Catalyzing the hydrolysis of phosphatidylinositol 4,5 cell carcinoma (ESCC) and gastric cancer (GC). biphosphate to generate diacylglycerol and inositol Homology 1,4,5 triphosphate. Mediating a wide variety of cellular The PLCD1 gene is conserved in dog, cow, mouse, rat, stimuli. Shuttling between the nucleus and the chicken, zebrafish, and A. thaliana. Mutations

The mutation location is highlighted in red and this mutation occured in 17% (1/6) skin samples. AA Mutation: p.E226K (Substitution - Missense). CDS Mutation: c.676G>A (Substitution).

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Implicated in Gastric cancer Prognosis Esophageal squamous cell carcinoma Located at the important tumor suppressor locus, 3p22, Prognosis PLCD1 encodes an enzyme that mediates regulatory Firstly, four commonly deleted regions (CDRs) at signaling of energy metabolism, calcium homeostasis 3p26.3, 3p22, 3p21.3 and 3p14.2 were identified. and intracellular movements. We identified PLCD1 as Absent and down-regulated expression of several a downregulated gene in aerodigestive carcinomas candidate TSGs, including CHL1, PCAF, RBMS3, through expression profiling and epigenetic PLCD1 and CACNA2D3, were detected in primary characterization. We found that PLCD1 was expressed ESCC tumors and ESCC cell lines. These results in all normal adult tissues but low or silenced in 84% provided evidence that minimal deleted regions at (16/19) gastric cancer cell lines, well correlated with its 3p26.3, 3p22, 3p21.3 and 3p14.2 containing potential CpG island (CGI) methylation status. Methylation was TSGs may contribute to the pathogenesis of esophageal further detected in 62% (61/98) gastric primary tumors, cancer. but none of normal gastric mucosa tissues. PLCD1 Secondly, absent expression of PLC delta 1 was methylation was significantly correlated with tumor detected in 26 of 50 (52%) primary ESCCs and 4 of 9 high stage. Detailed methylation analysis of 37 CpG (44.4%) ESCC cell lines, which was significantly sites at the PLCD1 CGI by bisulfite genomic associated with DNA copy number loss and promoter sequencing confirmed its methylation. PLCD1 hypermethylation (P < 0.05). Functional studies silencing could be reversed by pharmacological showed that PLC delta 1 was able to suppress both in demethylation with 5-aza-2'-deoxycytidine, indicating a vitro and in vivo tumorigenic ability of ESCC cells, direct epigenetic silencing. Ectopic expression of including foci formation, colony formation in soft agar, PLCD1 in silenced gastric tumor cells dramatically and tumor formation in nude mice. The tumor- inhibited their clonogenicity and migration, possibly suppressive mechanism of PLC delta 1 was associated through downregulating MMP7 expression and with its role in the cell cycle arrest at the G(1)-S hampering the reorganization of cytoskeleton through checkpoint by up-regulation of p21 and down- cofilin inactivation by phosphorylation. Thus, regulation of phosphorylated Akt (Ser(473)). In epigenetic inactivation of PLCD1 is common and addition, down-regulation of PLC delta 1 protein was tumor-specific in gastric cancer, and PLCD1 acts as a significantly correlated with ESCC metastasis (P = functional tumor suppressor involved in gastric 0.014), which was associated with its function in carcinogenesis. increasing cell adhesion and inhibiting cell mobility. Colon carcinomas These results suggest that PLC delta 1 plays an important suppressive role in the development and Prognosis progression of ESCC. Decreased levels of the PLC delta 1 protein were seen in most colon carcinomas (12 of 13 paired samples) Breast carcinoma and PLC delta 1 protein was not detected in any of the Prognosis carcinoma cell lines. PLCD1 are more highly expressed in the transformed Rat colon neoplasms cell lines compared to MCF-10A. To test whether PLCd1 or PLCd3 played any role in tumor cell Prognosis proliferation or cell migration. RNAi mediated The expression of PLC-delta expression in rat colon knockdown of PLCD1 reduced proliferation of the neoplasms induced by methylazoxymethanol (MAM) MDA-MB-231 cells. Morphological changes including acetate was examined. Large-bowel neoplasms were cell rounding, and surface blebbing and nuclear observed in five of 10 rats given MAM acetate 40 fragmentation were observed. These changes were weeks after treatment. PLC-delta expression in the accompanied by reductions in cell migration activities. neoplasms was not detected by northern blot analysis, On the other hand, PLCD1 knockdown failed to cause and a low level of expression was detected by comparable morphological changes in the normal immunoblot analysis, although PLC-delta expression MCF-10A line, but did reduce cell proliferation and was apparent in the non-neoplastic colon mucosae of migration. Taken together, these data are consistent MAM acetate-treated rats as well as in the colon with the idea that PLCD1 support the growth and mucosae of control rats. migration of normal and neoplastic mammary epithelial Insulinoma cells in vitro. Note However there is contrasted results published in Insulinoma MIN6 cells. another paper. Their results suggested that PLCD1 is a functional tumor suppressor inducing G(2)/M arrest Prognosis and frequently methylated in breast cancer. To study the effects of enhanced phosphoinositide

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 672 PLCD1 (phospholipase C, delta 1) Hu X

hydrolysis on insulin secretion, phosphoinositide- and delta 1 in primary human colon carcinomas and colon specific phospholipase Cbeta1 (PLCbeta1) or carcinoma cell lines. Mol Carcinog. 1995 Mar;12(3):146-52 PLCdelta1 was overexpressed in insulinoma MIN6 Ishikawa S, Takahashi T, Ogawa M, Nakamura Y. Genomic cells via adenoviral vectors. Inositol phosphate structure of the human PLCD1 (phospholipase C delta 1) locus production stimulated by KCl or glucose in both on 3p22-->p21.3. Cytogenet Cell Genet. 1997;78(1):58-60 PLCbeta1- and PLCdelta1-overexpressing cells were Ishihara H, Wada T, Kizuki N, Asano T, Yazaki Y, Kikuchi M, greater than that in control cells, reduced Oka Y. Enhanced phosphoinositide hydrolysis via overexpression of phospholipase C beta1 or delta1 inhibits phosphatidylinositol-4,5-bisphosphate levels were stimulus-induced insulin release in insulinoma MIN6 cells. observed in these cells stimulated by NaF or KCl. Biochem Biophys Res Commun. 1999 Jan 8;254(1):77-82 These data suggest that excessive phosphoinositide Fu L, Qin YR, Xie D, Hu L, Kwong DL, Srivastava G, Tsao SW, hydrolysis inhibits secretagogue-induced insulin release Guan XY. Characterization of a novel tumor-suppressor gene in MIN6 cells. PLC delta 1 at 3p22 in esophageal squamous cell carcinoma. Pheochromocytoma Cancer Res. 2007 Nov 15;67(22):10720-6 Qin YR, Fu L, Sham PC, Kwong DL, Zhu CL, Chu KK, Li Y, Note Guan XY. Single-nucleotide polymorphism-mass array reveals Pheochromocytoma PC12 cells. commonly deleted regions at 3p22 and 3p14.2 associate with poor clinical outcome in esophageal squamous cell carcinoma. Prognosis Int J Cancer. 2008 Aug 15;123(4):826-30 PLCD1 is recruited from the cytoplasm to lipid rafts 2+ Yamaga M, Kawai K, Kiyota M, Homma Y, Yagisawa H. after CCH-induced Ca mobilization following the Recruitment and activation of phospholipase C (PLC)-delta1 in activation of PLCbeta by GPCR and PLCD1 is lipid rafts by muscarinic stimulation of PC12 cells: contribution activated only in lipid rafts by localized capacitative of p122RhoGAP/DLC1, a tumor-suppressing PLCdelta1 entry of extracellular Ca 2+ . PLCD1, p122RhoGAP and binding protein. Adv Enzyme Regul. 2008;48:41-54 RhoA in combination could constitute a unique Hu XT, Zhang FB, Fan YC, Shu XS, Wong AH, Zhou W, Shi functional unit for the regulation of both QL, Tang HM, Fu L, Guan XY, Rha SY, Tao Q, He C. phosphoinositide/Ca 2+ signaling and the actin Phospholipase C delta 1 is a novel 3p22.3 tumor suppressor involved in cytoskeleton organization, with its epigenetic cytoskeleton in the periphery of specified membrane silencing correlated with high-stage gastric cancer. Oncogene. domains. This would provide further insights into the 2009 Jul 2;28(26):2466-75 molecular mechanisms of cancer development. Rebecchi MJ, Raghubir A, Scarlata S, Hartenstine MJ, Brown T, Stallings JD. Expression and function of phospholipase C in References breast carcinoma. Adv Enzyme Regul. 2009;49(1):59-73 Yoshimi N, Wang A, Makita H, Suzui M, Mori H, Okano Y, Xiang T, Li L, Fan Y, Jiang Y, Ying Y, Putti TC, Tao Q, Ren G. Banno Y, Nozawa Y. Reduced expression of phospholipase C- PLCD1 is a functional tumor suppressor inducing G(2)/M arrest delta, a signal-transducing enzyme, in rat colon neoplasms and frequently methylated in breast cancer. Cancer Biol Ther. induced by methylazoxymethanol acetate. Mol Carcinog. 1994 2010 Sep;10(5):520-7 Dec;11(4):192-6 This article should be referenced as such: Nomoto K, Tomita N, Miyake M, Xhu DB, LoGerfo PR, Weinstein IB. Expression of phospholipases gamma 1, beta 1, Hu X. PLCD1 (phospholipase C, delta 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):670-673.

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Gene Section Mini Review

PYY (peptide YY) Maria Braoudaki, Fotini Tzortzatou-Stathopoulou University Research Institute for the Study and Treatment of Childhood Genetic and Malignant Diseases, University of Athens, "Aghia Sophia" Children's Hospital, Athens, Greece (MB), Hematology/Oncology Unit, First Department of Pediatrics, University of Athens, "Aghia Sophia" Children's Hospital, Athens, Greece (FTS) Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/PYYID46182ch17q21.html DOI: 10.4267/2042/46002 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Expression PYY is expressed predominantly in endocrine L-cells Other names: PYY1 that line the distal small bowel and colon. HGNC (Hugo): PYY Localisation Location: 17q21.31 Extracellular, subcellular location: secreted granules. DNA/RNA Co-localized with proglucagon products, glicentin and glucagon-like peptide-1 (GLP-1) and GLP-2. PYY is a Description gastrointestinal track-derived hormone synthesized by endocrine cells of terminal ileum and colon, involved PYY gene is composed of 4 exons and 3 introns that in the regulation of food intake. span approximately 51732 bases (start 42030106 bp to end 42081837 bp from pter) oriented at the minus Function strand. Enteroendocrine L-cells release two circulating forms Transcription of PYY in the distal gut: PYY1-36 and PYY3-36. The latter form is considered the predominant form in both Two transcript variants (1048 bp and 1048 bp in fasted and fed states and is produced by the cleavage of length). the N-terminal Tyr-Pro residues from PYY1-36 by dipeptidyl-peptidase IV (DPPIV). Protein Description Size: 97 amino acids; 11046 Da.

Human peptide YY (PYY). Adapted from Shih et al., 2009.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 674 PYY (peptide YY) Braoudaki M, Tzortzatou-Stathopoulou F

PYY exerts its inhibitory actions via various Y remains unknown. Previous studies have proposed that receptors, including Y1 receptor-mediated epithelial PYY reduces intracellular levels of cAMP in breast responses and Y2 receptor-mediated neuronal effects. It carcinoma cells. Moreover, it has been reported that inhibits food intake via NPY-2 receptors expressed by combination of PYY with vitamin E results in a neurons of the arcuate nucleus of the hypothalamus. significant additive inhibition of breast carcinoma cells. Generally, it is considered to act in the hypothalamus as Cancer cachexia a signal of satiety. Other inhibitory actions include slowing gastric emptying; increasing nutrient Note absorption, inducing intestinal anion and electrolytic Cancer cachexia is generally characterized by secretion as well as slowing small intestine motility. In decreased protein synthesis and loss in the small bowel. addition, it has been shown to decrease exocrine PYY has been shown to increase small bowel weight pancreatic secretion and act as an appetite suppressant and protein content. However, the exact role of PYY on in the fasting state at physiological concentrations. cancer cachexia has not yet been clarified. Homology Body weight PPY or PNP or PP and NPY. Note PYY-36 plays a role in long-term body weight Mutations regulation, due to the negative correlation between PYY concentrations and adiposity markers in humans, Note such that PYY levels increase with weight loss and Common polymorphisms: Arg72Thr, which has been when leptin levels are low. associated with type-2 diabetes and in some cases with enhanced body mass. Other variants: Gln62Pro and Obesity and type II diabetes Leu73Pro associated with body mass and obesity, Note respectively as well as A-23G,C888T and 3' UTR Low endogenous PYY levels in obese individuals, have variant C+1134A. The latter has been related to previously suggested that PYY deficiency may enhanced body mass. contribute to hyperinsulinemia and insulin resistance and predispose obesity and type II diabetes. Implicated in References Colon cancer Conn MP, Melmed S.. Endocrinology: Basic and clinical Note principles. Humana Press. 1997. Loss of PYY expression has been implicated in the Grise KR, Rongione AJ, Laird EC, McFadden DW.. Peptide YY development and progression to colon adenocarcinoma. inhibits growth of human breast cancer in vitro and in vivo. J PYY expression has been associated with elevated Surg Res. 1999 Apr;82(2):151-5. differentiation, whilst PYY treatment of colon cancers Heisler T, Towfigh S, Simon N, Liu C, McFadden DW.. Peptide resulted in selected overexpression of enzymes YY augments gross inhibition by vitamin E succinate of human frequently identified in the normal colonocytic pancreatic cancer cell growth. J Surg Res. 2000a phenotype. The colon cancer growth regulatory effects Jan;88(1):23-5. of PYY might be dose dependent. Heisler T, Towfigh S, Simon N, McFadden DW.. Peptide YY and vitamin E inhibit hormone-sensitive and -insensitive breast Pancreatic cancer and pancreatitis cancer cells. J Surg Res. 2000b Jun 1;91(1):9-14. Note Tseng WW, Liu CD.. Peptide YY and cancer: current findings PYY suppresses growth and levels of intracellular and potential clinical applications. Peptides. 2002 cyclic adenosine monophosphate (cAMP) in pancreatic Feb;23(2):389-95. (REVIEW) adenocarcinoma. It is considered to have a therapeutic Vona-Davis L, Yu A, Magabo K, Evans T, Jackson B, Riggs D, value for pancreatic cancer and pancreatitis, since it McFadden D.. Peptide YY attenuates transcription factor exerts its immune function by altering transcription activity in tumor necrosis factor-alpha-induced pancreatitis. J Am Coll Surg. 2004 Jul;199(1):87-95. factors vital for cell signaling pathways. In addition, administration of PYY has been shown to improve Torekov SS, Larsen LH, Glumer C, Borch-Johnsen K, amylase and cytokine release in pancreatitis cases. It Jorgensen T, Holst JJ, Madsen OD, Hansen T, Pedersen O.. Evidence of an association between the Arg72 allele of the has also been suggested that PYY in combination with peptide YY and increased risk of type 2 diabetes. Diabetes. vitamin E exhibit a significantly increased inhibitory 2005 Jul;54(7):2261-5. effect on pancreatic cancer in vitro. Ahituv N, Kavaslar N, Schackwitz W, Ustaszewska A, Collier Breast cancer JM, Hebert S, Doelle H, Dent R, Pennacchio LA, McPherson R.. A PYY Q62P variant linked to human obesity. Hum Mol Note Genet. 2006 Feb 1;15(3):387-91. Epub 2005 Dec 20. PYY inhibits in vitro growth of breast cancer cells, Boey D, Heilbronn L, Sainsbury A, Laybutt R, Kriketos A, however the exact mechanism of antitumor activity Herzog H, Campbell LV.. Low serum PYY is linked to insulin

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 675 PYY (peptide YY) Braoudaki M, Tzortzatou-Stathopoulou F

resistance in first-degree relatives of subjects with type 2 and fat on ghrelin and peptide YY secretion in prepubertal diabetes. Neuropeptides. 2006 Oct;40(5):317-24. Epub 2006 children. J Clin Endocrinol Metab. 2009 Nov;94(11):4463-71. Oct 12. Epub 2009 Oct 9. Pfluger PT, Kampe J, Castaneda TR, Vahl T, D'Alessio DA, Shih PA, Wang L, Chiron S, Wen G, Nievergelt C, Mahata M, Kruthaupt T, Benoit SC, Cuntz U, Rochlitz HJ, Moehlig M, Khandrika S, Rao F, Fung MM, Mahata SK, Hamilton BA, Pfeiffer AF, Koebnick C, Weickert MO, Otto B, Spranger J, O'Connor DT.. Peptide YY (PYY) gene polymorphisms in the Tschop MH.. Effect of human body weight changes on 3'-untranslated and proximal promoter regions regulate cellular circulating levels of peptide YY and peptide YY3-36. J Clin gene expression and PYY secretion and metabolic syndrome Endocrinol Metab. 2007 Feb;92(2):583-8. Epub 2006 Nov 21. traits in vivo. J Clin Endocrinol Metab. 2009 Nov;94(11):4557- 66. Epub 2009 Oct 9. Vona-Davis L, McFadden DW.. PYY and the pancreas: inhibition of tumor growth and inflammation. Peptides. 2007 Cox HM, Tough IR, Woolston AM, Zhang L, Nguyen AD, Feb;28(2):334-8. Epub 2006 Dec 27. (REVIEW) Sainsbury A, Herzog H.. Peptide YY is critical for acylethanolamine receptor Gpr119-induced activation of Cox HM.. Endogenous PYY and NPY mediate tonic Y1- and gastrointestinal mucosal responses. Cell Metab. 2010 Jun Y2-mediated absorption in human and mouse colon. Nutrition. 9;11(6):532-42. 2008 Sep;24(9):900-6. Epub 2008 Jul 26. Kirchner H, Tong J, Tschop MH, Pfluger PT.. Ghrelin and PYY Moschovi M, Trimis G, Vounatsou M, Katsibardi K, Margeli A, in the regulation of energy balance and metabolism: lessons Dimitriadi F, Papassotiriou I, Chrousos G, Tzortzatou- from mouse mutants. Am J Physiol Endocrinol Metab. 2010 Stathopoulou F.. Serial plasma concentrations of PYY and May;298(5):E909-19. Epub 2010 Feb 23. (REVIEW) ghrelin during chemotherapy in children with acute lymphoblastic leukemia. J Pediatr Hematol Oncol. 2008 This article should be referenced as such: Oct;30(10):733-7. Braoudaki M, Tzortzatou-Stathopoulou F. PYY (peptide YY). Lomenick JP, Melguizo MS, Mitchell SL, Summar ML, Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):674-676. Anderson JW.. Effects of meals high in carbohydrate, protein,

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Gene Section Review

SIAH2 (seven in absentia homolog 2 (Drosophila)) Jianfei Qi, Ze'ev Ronai Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, CA, 92037, USA (JQ, ZR)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/SIAH2ID46199ch3q25.html DOI: 10.4267/2042/46003 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

an N-terminal ring domain, followed by two zinc finger Identity motifs, and a C-terminal substrate binding domain Other names: hSiah2 (SBD). The ring domain is the catalytic domain that HGNC (Hugo): SIAH2 recruits E2 ubiquitin-conjugating enzymes, while the SBD mediates the binding of adaptor proteins or some Location: 3q25.1 Siah substrate proteins. The structure of murine Siah1a SBD has been solved. The structure reveals that Siah is DNA/RNA a dimeric protein, and the SBD adopts an eight- Description stranded beta-sandwich fold (Polekhina et al., 2001). The substrate binding groove is formed by the beta- The human Siah2 gene is composed of 2 exons sandwich fold and the beta-strand that connects to the spanning a genomic region of about 22.4 kb. second zinc finger domain (House et al., 2005). Transcription Expression The transcript length of human Siah2 is 2632 bp. The Siah2 mRNA is widely expressed in the embryonic and open reading frame of the coding region is 975 bp. adult mouse tissues. It is expressed at a higher level in Pseudogene the olfactory epithelium, retina, forebrain and proliferating cartilage of developing bone (Della et al., No pseudogene of Siah2 has been reported. 1993). Siah2 mRNA is also expressed in most human Protein tissues (Hu et al., 1997). Localisation Description Siah protein can be localized in both cytoplasm and Human Siah2 protein consists of 324 amino acids, with nucleus. a molecular weight of 36 kDa. Siah protein consists of

Genomic organization of human Siah2. The line indicates intron and boxes indicate coding regions (exon 1-2) of the gene. Exon and intron lengths, the ATG transcription start site and the TGA stop codon are indicated.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 677 SIAH2 (seven in absentia homolog 2 (Drosophila)) Qi J, Ronai Z

Domains of human Siah2 protein.

Function Homology Siah2 is the mammalian homolog of Drosophila SINA Homologs: Human has two Siah genes (Siah1 and (seven in absentia), which interacts with transcriptional Siah2) (Hu et al., 1997), while mouse has three Siah repressor Tramtrack via adaptor protein PHYL genes (Siah2, Siah1a, Siah1b) (Della et al., 1993). (Phyllopod) and induces the proteasomal degradation Human Siah2 shares 77% identity with human Siah1 of Tramtrack, thereby determining R7 cell fate (Li et (Hu et al., 1997). al., 1997; Tang et al., 1997). As a ring-finger E3 Orthologs: Highly conserved Siah2 orthologs have ubiquitin ligase, Siah targets the degradation of diverse been identified in all multicellular organisms examined substrates via ubiquitin-proteasome pathway, and (Nakayama et al., 2009). affects multiple signaling pathways such as HIF (Nakayama et al., 2004), Ras (Nadeau et al., 2007; Mutations Schmidt et al., 2007), NF-kB (Polekhina et al., 2002; Habelhah et al., 2002), and beta-catenin (Liu et al., Note 2001; Matsuzawa and Reed, 2001). Siah2 transcription No SIAH2 mutations have been reported. is upregulated by hypoxia (Nakayama et al., 2004); p38-mediated phosphorylation of mouse Siah2 on Implicated in Thr24 and Ser29 alters its subcellular localization (Khurana et al., 2006); HIPK2-mediated Lung cancer phosphorylation of human Siah2 on Thr 26, Ser 28 and Note Ser 68 decreases the stability of Siah2 and impairs its Ahmed et al. showed that Siah2 knockdown in human interaction with HIPK2 (Calzado et al., 2009). lung cancer cell lines (BZR, A549, H727, and UMC11) Over 20 Siah substrates have been reported (Nakayama inhibited MAPK-ERK signaling, reduced cell et al., 2009) and some of them can be degradated by proliferation and increased apoptosis; Siah2 Siah2, Siah1 or both of them. In contrast to Siah1a knockdown also reduced anchorage-independent knockout mice which exibit growth retardation and growth of A549 cells in soft agar, and blocked the spermatogenesis defect, Siah2 knockout mice display growth of A549 xenograft tumors in nude mice no apparent phenotype, whereas Siah2 and Siah1a (Ahmed et al., 2007). double knockout mice are embryonic or neonatal lethal, Melanoma suggesting that the two Siah homologs have both overlapping and distinct functions in vivo (Frew et al., Note 2003). Despite the diverse substrates of Siah identified Qi et al. showed that inhibition of Siah2 activity using in vitro, loss of Siah2 (or both Siah2 and Siah1a) in different inhibitory proteins blocked tumor formation vivo largely has no effect on the levels of many Siah or metastasis of SW1 melanoma cells in a syngeneic substrates and the physiological processes associated mouse model due to the inhibition of Ras and HIF with these substrates (Frew et al., 2002; Frew et al., pathways, respectively (Qi et al., 2008). Similary, Shah 2003). et al. showed that a putative chemical inhibitor of Siah2 is implicated in the regulation of hypoxia Siah2, menadione, decreased the levels of HIF-1alpha response through its effect on HIF prolyl hydroxylases and phospho-ERK in human melanoma cell line or HIPK2 (Nakayama et al., 2004; Calzado et al., UACC903 and abolished the growth of xenograft 2009). Siah2 knockout mice subject to hypoxia showed tumor in nude mice (Shah et al., 2009). impaired respiratory response and defect to adjust Breast cancer levels of red blood cells (Nakayama et al., 2004). Siah2 Note has been shown to be required for development and Möller et al. showed that inhibition of Siah in a mouse progression of several types of cancers via its breast cancer cell line reduced the xenograft tumor regulation of HIF or Ras pathways (House et al., 2009). growth and prolonged the survival of mice due to Siah2-dependent degradation of Pard3A is found to inhibition of HIF pathway (Möller et al., 2009). control germinal zone exit of neuronal progenitors or Behling et al. examined the SIAH staining in 65 immature neurons in mice (Famulski et al., 2010).

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 678 SIAH2 (seven in absentia homolog 2 (Drosophila)) Qi J, Ronai Z

patients of ductal carcinoma in situ (DCIS). Higher deficient for Siah genes. Mol Cell Biol. 2002 Dec;22(23):8155- level of Siah staining was observed in tumors compared 64 with the normal adjacent tissues, and in tumors with Habelhah H, Frew IJ, Laine A, Janes PW, Relaix F, Sassoon more aggressive features. D, Bowtell DD, Ronai Z. Stress-induced decrease in TRAF2 stability is mediated by Siah2. EMBO J. 2002 Nov There was also higher Siah staining in specimens from 1;21(21):5756-65 patients with recurrence as compared to patients without recurrence. This study stuggests that Siah may Polekhina G, House CM, Traficante N, Mackay JP, Relaix F, Sassoon DA, Parker MW, Bowtell DD. Siah ubiquitin ligase is serve as a prognostic biomarker that predicts DCIS structurally related to TRAF and modulates TNF-alpha progression to invasive breast cancer (Behling et al., signaling. Nat Struct Biol. 2002 Jan;9(1):68-75 2010). Frew IJ, Hammond VE, Dickins RA, Quinn JM, Walkley CR, Pancreatic cancer Sims NA, Schnall R, Della NG, Holloway AJ, Digby MR, Janes PW, Tarlinton DM, Purton LE, Gillespie MT, Bowtell DD. Note Generation and analysis of Siah2 mutant mice. Mol Cell Biol. Schmidt et al. showed that inhibition of Siah activity 2003 Dec;23(24):9150-61 attenuated MAPK-ERK signaling, blocked RAS- Nakayama K, Frew IJ, Hagensen M, Skals M, Habelhah H, induced focus formation in fibroblasts, abolished Bhoumik A, Kadoya T, Erdjument-Bromage H, Tempst P, anchorage-independent growth of human pancreatic Frappell PB, Bowtell DD, Ronai Z. Siah2 regulates stability of prolyl-hydroxylases, controls HIF1alpha abundance, and cancer cells in soft agar and xenograft tumor growth in modulates physiological responses to hypoxia. Cell. 2004 Jun nude mice (Schmidt et al., 2008). 25;117(7):941-52 Prostate cancer House CM, Hancock NC, Möller A, Cromer BA, Fedorov V, Bowtell DD, Parker MW, Polekhina G. Elucidation of the Note substrate binding site of Siah ubiquitin ligase. Structure. 2006 Qi et al. showed that knockout of Siah2 in the TRAMP Apr;14(4):695-701 model abolished the formation of prostate Khurana A, Nakayama K, Williams S, Davis RJ, Mustelin T, neuroendocrine tumor, inhibition of Siah2 activity Ronai Z. Regulation of the ring finger E3 ligase Siah2 by p38 blocked hypoxia-induced neuroendocrine MAPK. J Biol Chem. 2006 Nov 17;281(46):35316-26 differentiation (NED) in prostate cancer cells or in the Nadeau RJ, Toher JL, Yang X, Kovalenko D, Friesel R. xenogaft tumors, and Siah2 protein levels were higher Regulation of Sprouty2 stability by mammalian Seven-in- in high-grade PCa that expresss NE markers. This Absentia homolog 2. J Cell Biochem. 2007 Jan 1;100(1):151- study suggests that Siah2 plays a key role in 60 development of prostate NE tumor and NED of human Schmidt RL, Park CH, Ahmed AU, Gundelach JH, Reed NR, PCa by controling a cooperation between HIF and NE- Cheng S, Knudsen BE, Tang AH. Inhibition of RAS-mediated specific transcription factor FoxA2 (Qi et al., 2010). transformation and tumorigenesis by targeting the downstream E3 ubiquitin ligase seven in absentia homologue. Cancer Res. Breakpoints 2007 Dec 15;67(24):11798-810 Ahmed AU, Schmidt RL, Park CH, Reed NR, Hesse SE, Note Thomas CF, Molina JR, Deschamps C, Yang P, Aubry MC, Tang AH. Effect of disrupting seven-in-absentia homolog 2 There is no breakpoint reported. function on lung cancer cell growth. J Natl Cancer Inst. 2008 Nov 19;100(22):1606-29 References Qi J, Nakayama K, Gaitonde S, Goydos JS, Krajewski S, Eroshkin A, Bar-Sagi D, Bowtell D, Ronai Z. The ubiquitin Hu G, Chung YL, Glover T, Valentine V, Look AT, Fearon ER. ligase Siah2 regulates tumorigenesis and metastasis by HIF- Characterization of human homologs of the Drosophila seven dependent and -independent pathways. Proc Natl Acad Sci U in absentia (sina) gene. Genomics. 1997 Nov 15;46(1):103-11 S A. 2008 Oct 28;105(43):16713-8 Li S, Li Y, Carthew RW, Lai ZC. Photoreceptor cell House CM, Möller A, Bowtell DD. Siah proteins: novel drug differentiation requires regulated proteolysis of the targets in the Ras and hypoxia pathways. Cancer Res. 2009 transcriptional repressor Tramtrack. Cell. 1997 Aug Dec 1;69(23):8835-8 8;90(3):469-78 Calzado MA, de la Vega L, Möller A, Bowtell DD, Schmitz ML. Tang AH, Neufeld TP, Kwan E, Rubin GM. PHYL acts to down- An inducible autoregulatory loop between HIPK2 and Siah2 at regulate TTK88, a transcriptional repressor of neuronal cell the apex of the hypoxic response. Nat Cell Biol. 2009 fates, by a SINA-dependent mechanism. Cell. 1997 Aug Jan;11(1):85-91 8;90(3):459-67 Möller A, House CM, Wong CS, Scanlon DB, Liu MC, Ronai Z, Liu J, Stevens J, Rote CA, Yost HJ, Hu Y, Neufeld KL, White Bowtell DD. Inhibition of Siah ubiquitin ligase function. RL, Matsunami N. Siah-1 mediates a novel beta-catenin Oncogene. 2009 Jan 15;28(2):289-96 degradation pathway linking p53 to the adenomatous polyposis coli protein. Mol Cell. 2001 May;7(5):927-36 Nakayama K, Qi J, Ronai Z. The ubiquitin ligase Siah2 and the hypoxia response. Mol Cancer Res. 2009 Apr;7(4):443-51 Matsuzawa SI, Reed JC. Siah-1, SIP, and Ebi collaborate in a novel pathway for beta-catenin degradation linked to p53 Shah M, Stebbins JL, Dewing A, Qi J, Pellecchia M, Ronai ZA. responses. Mol Cell. 2001 May;7(5):915-26 Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma Frew IJ, Dickins RA, Cuddihy AR, Del Rosario M, Reinhard C, tumorigenesis. Pigment Cell Melanoma Res. 2009 O'Connell MJ, Bowtell DD. Normal p53 function in primary cells Dec;22(6):799-808

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 679 SIAH2 (seven in absentia homolog 2 (Drosophila)) Qi J, Ronai Z

Behling KC, Tang A, Freydin B, Chervoneva I, Kadakia S, regulates formation of neuroendocrine phenotype and Schwartz GF, Rui H, Witkiewicz AK. Increased SIAH neuroendocrine prostate tumors. Cancer Cell. 2010 Jul expression predicts ductal carcinoma in situ (DCIS) 13;18(1):23-38 progression to invasive carcinoma. Breast Cancer Res Treat. 2010 Nov 19; Della NG, Senior PV, Bowtell DD. Isolation and characterisation of murine homologues of the Drosophila Famulski JK, Trivedi N, Howell D, Yang Y, Tong Y, Gilbertson seven in absentia gene (sina). Development. 1993 R, Solecki DJ. Siah regulation of Pard3A controls neuronal cell Apr;117(4):1333-43 adhesion during germinal zone exit. Science. 2010 Dec 24;330(6012):1834-8 This article should be referenced as such: Qi J, Nakayama K, Cardiff RD, Borowsky AD, Kaul K, Williams Qi J, Ronai Z. SIAH2 (seven in absentia homolog 2 R, Krajewski S, Mercola D, Carpenter PM, Bowtell D, Ronai (Drosophila)). Atlas Genet Cytogenet Oncol Haematol. 2011; ZA. Siah2-dependent concerted activity of HIF and FoxA2 15(8):677-680.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 680 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

TP53BP2 (tumor protein p53 binding protein, 2) Kathryn Van Hook, Zhiping Wang, Charles Lopez Department of Medicine, Division of Hematology and Medical Oncology, Oregon Health and Science University, Portland, OR, USA (KV, ZW, CL)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/TP53BP2ID42667ch1q42.html DOI: 10.4267/2042/46004 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Pseudogene Not known. Other names: 53BP2; ASPP2; BBP; P53BP2; PPP1R13A Protein HGNC (Hugo): TP53BP2 Location: 1q41 Description ASPP2 is a pro-apoptotic protein with a predicted size DNA/RNA of approximately 135 kDa. It is the founding member of a family of ASPP proteins that all share the common Description motifs of four Ankyrin-repeats, a Src-homology 3 The TP53BP2 gene spans about 66 kb on chromosome (SH3) domain, and a Polyproline domain in their C- 1q42.1 on the minus strand (Yang et al., 1997). There terminus (Iwabuchi et al., 1994). The N-terminus of are two transcripts as a result of alternative splicing ASPP2 is thought to be important for regulating its (Takahashi et al., 2004). The transcript variant 1, which apoptotic function and contains a putative Ras- is shorter (4670 bp), does not contain exon 3 and gives association domain as well as a ubiquitin-like fold rise to a longer form of the protein named TP53BPL (Tidow et al., 2007). ASPP2 has been most widely (long) or ASPP2. The transcript variant 2, which is studied for its ability to interact with and stimulate the longer (4802 bp), contains exon 3 which harbors a stop apoptotic function of the tumor suppressor p53 (and codon. As a result, the transcription initiates at exon 6 p63/p73) but several studies have also demonstrated giving rise to a shorter form of the protein named p53-independent as well as apoptosis-independent TP53BPS (short) or BBP. functions for ASPP2 as well (Kampa et al., 2009a). ASPP2 was originally pulled out of a yeast two-hybrid Transcription screen using the p53-binding domain as bait as a partial ASPP2 is a serum inducible protein and subject to C-terminal clone named 53BP2 (Iwabuchi et al., 1994). transcriptional regulation by E2F and its family members (Chen et al., 2005; Fogal et al., 2005).

TSS=transcription start site.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 681 TP53BP2 (tumor protein p53 binding protein, 2) Van Hook K, et al.

ASPP2 protein domains. RA=Ras-association domain; PP=polyproline domain; AR=ankyrin repeats.

In 1996, Naumovski and Cleary determined that 53BP2 evidence to indicate ASPP2 as a player in was a partial clone of a longer transcript they named mitochondrial-mediated apoptosis (Kobayashi et al., Bcl-2 binding protein (Bbp or Bbp/53BP2) for its 2005). ability to bind the anti-apoptotic protein Bcl-2. It was Tumor suppressor. Several clinical studies later determined that Bbp is a splice isoform of the full demonstrate low ASPP2 expression in a variety of length gene product from this locus, ASPP2 (Samuels- human tumors (breast, lung, lymphoma) and this low Lev et al., 2001). expression often correlates with poor clinical outcome, Expression suggesting that ASPP2 may function as a tumor suppressor (Mori et al., 2000; Samuels-Lev et al., 2001; Northern blot analysis, using a C-terminal probe, shows Lossos et al., 2002; Cobleigh et al., 2005). In support of elevated levels of ASPP2 mRNA in several human this concept, Iwabuchi et al. demonstrated in 1998 that tissues including heart, testis, and peripheral blood transfection of 53BP2 inhibits Ras/E1A-mediated leukocytes (Yang et al., 1999). ASPP2 protein levels transformation in rat embryonic fibroblasts. Since then are controlled by proteasomal degradation (Zhu et al., two separate mouse models targeting the ASPP2 locus 2005). via homologous recombination have demonstrated that Localisation loss of only one copy of ASPP2 increases spontaneous and irradiation-induced tumor formation in vivo (Vives ASPP2 contains a nuclear localization signal within its et al., 2006; Kampa et al., 2009b). Taken together these ankyrin repeat domain (amino acid residues 795-894) data strongly suggest that ASPP2 is a haplo-insufficient that when expressed alone or as a fusion with other tumor suppressor. proteins localizes in the nucleus of cells (Sachdev et al., Cell cycle. Bbp, a splice isoform of ASPP2, can induce 1998; Yang et al., 1999). Despite this signal however, accumulation of cells in G /M and thus impede cell full length ASPP2 is predominantly located in the 2 cycle progression (Naumovski and Cleary, 1996). cytoplasm and often seen near the cell periphery Additionally, ASPP2 appears to play a role in the G /G (Naumovski and Cleary, 1996; Iwabuchi et al., 1998; 0 1 cell cycle checkpoint in response to gamma-irradiation Yang et al., 1999). as murine thymocytes that lack one copy of the ASPP2 Function locus did not arrest at G 0/G 1 as efficiently as wild type Apoptosis. Before ASPP2 was known to be the full thymocytes (Kampa et al., 2009b). length gene product from the TP53BP2 locus, Yang Cell polarity. ASPP2 is often seen near the cell and colleagues showed that overexpression of periphery and has been shown to co-localize with and Bbp/53BP2 in cells induces apoptosis (Yang et al., bind to the tight junction protein PAR-3. Furthermore, 1999). In 2000, Lopez et al. demonstrated that loss of ASPP2 expression correlates with a loss of tight Bbp/53BP2 was UV-damage inducible and that loss of junction integrity and an impaired ability to maintain this endogenous protein promotes cell survival in apical domains in polarized cells in culture (Cong et al., response to damage, thus implicating a function in the 2010). Interestingly these findings hold true in vivo as damage response pathway. In 2001, Samuels-Lev et al. well. ASPP2 co-localizes with the PAR-3 complex and provided evidence that not only does full length ASPP2 apical junctions in the brain and is necessary for tight promote apoptosis but that it does so, at least in part, junction integrity. Targeted deletion of ASPP2 in the through a p53-mediated mechanism that may involve mouse leads to defects associated with a loss of preferential binding of p53 to its apoptotic target genes. structural organization in the brain and retina ASPP2 has also been shown to modulate the apoptotic (Sottocornola et al., 2010). activity of the p53 family members, p63/p73 Senescence. Senescence, a type of irreversible cell (Bergamaschi et al., 2004), and is known to bind other cycle arrest, is considered an intrinsic protective proteins involved in apoptosis such as Bcl-2 and NF- response against malignant transformation. Wang et al. kappaB (Naumovski and Cleary, 1996; Yang et al., recently identified ASPP2 as a mediator of Ras-induced 1999). However, the functional ramifications of these senescence by demonstrating that mouse embryonic interactions remain unclear. Additionally, there is fibroblasts with a targeted deletion of

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 682 TP53BP2 (tumor protein p53 binding protein, 2) Van Hook K, et al.

Potential functions and putative interacting partners of ASPP2. Modified from Kampa et al., 2009a. exon 3 of the ASPP2 gene (TP53BP2) are less prone to TP53BP2 have been found associated with gastric senescence in the presence of activated Ras as cancer susceptibility (Ju et al., 2005) and epigenetic compared to wild type fibroblasts (as measured by silencing of the promoter by methylation is frequently beta-galactosidase staining). Data also suggests that observed (Sarraf and Stancheva, 2004; Liu et al., 2005; Ras-induced senescence may be mediated by ASPP2 Zhao et al., 2010). through its ability to inhibit Ras from inducing accumulation of cyclin D1 in the nucleus (Wang et al., Implicated in 2011). Breast cancer Homology Note ASPP2 is a member of the ASPP family of proteins that ASPP2 mRNA expression is frequently downregulated share a significant amount of homology in their C- in human breast cancer samples as compared to terminal domains. ASPP1, ASPP2, and the splice adjacent normal tissue (Sgroi et al., 1999; Samuels-Lev isoform of ASPP2, BBP, share homology in both their et al., 2001; Cobleigh et al., 2005). Reduced levels of N-terminal and C-terminal domains while the family ASPP2 expression are seen in both invasive and member iASPP only retains C-terminal homology metastatic breast tumor tissue (Sgroi et al., 1999) and (Samuels-Lev et al., 2001; Bergamaschi et al., 2003). ASPP2 downregulation may be favored in tumor cells expressing wild type but not mutant p53 (Samuels-Lev Mutations et al., 2001). Note Prognosis No mutations at the ASPP2 locus, TP53BP2, have been Elevated levels of ASPP2 mRNA were correlated with reported. However, single nucleotide polymorphisms in a lower risk of distant recurrence of disease among a

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 683 TP53BP2 (tumor protein p53 binding protein, 2) Van Hook K, et al.

panel of 78 patients with extensive lymph node p53-binding proteins, 53BP1 and 53BP2. J Biol Chem. 1998 involvement (Cobleigh et al., 2005). Oct 2;273(40):26061-8 Sachdev S, Hoffmann A, Hannink M. Nuclear localization of Non-Hodgkin's lymphoma specifically IkappaB alpha is mediated by the second ankyrin repeat: the diffuse large B-cell lymphoma, follicular IkappaB alpha ankyrin repeats define a novel class of cis- acting nuclear import sequences. Mol Cell Biol. 1998 center lymphoma, and Burkitt's May;18(5):2524-34 lymphoma Sgroi DC, Teng S, Robinson G, LeVangie R, Hudson JR Jr, Note Elkahloun AG. In vivo gene expression profile analysis of Overall, ASPP2 expression (as measured by Real-time human breast cancer progression. Cancer Res. 1999 Nov 15;59(22):5656-61 RT-PCR) was found to be significantly higher in diffuse large B-cell lymphoma as compared to Yang JP, Hori M, Takahashi N, Kawabe T, Kato H, Okamoto T. follicular center lymphoma. However, the variability of NF-kappaB subunit p65 binds to 53BP2 and inhibits cell death induced by 53BP2. Oncogene. 1999 Sep 16;18(37):5177-86 ASPP2 expression in diffuse large B-cell lymphoma was much greater than that seen in follicular center Lopez CD, Ao Y, Rohde LH, Perez TD, O'Connor DJ, Lu X, Ford JM, Naumovski L. Proapoptotic p53-interacting protein lymphoma. ASPP2 expression appeared inversely 53BP2 is induced by UV irradiation but suppressed by p53. proportional to serum lactate dehydrogenase levels. Mol Cell Biol. 2000 Nov;20(21):8018-25 Additionally, levels of ASPP2 expression are extremely Mori T, Okamoto H, Takahashi N, Ueda R, Okamoto T. low or undetectable in cell lines derived from Burkitt's Aberrant overexpression of 53BP2 mRNA in lung cancer cell lymphoma (Lossos et al., 2002). lines. FEBS Lett. 2000 Jan 14;465(2-3):124-8 Prognosis Nakagawa H, Koyama K, Murata Y, Morito M, Akiyama T, In general, patients with high ASPP2 expression tended Nakamura Y. APCL, a central nervous system-specific to have a longer median survival than those with low homologue of adenomatous polyposis coli tumor suppressor, binds to p53-binding protein 2 and translocates it to the ASPP2 expression (Lossos et al., 2002). perinucleus. Cancer Res. 2000 Jan 1;60(1):101-5 Gastric cancer Espanel X, Sudol M. Yes-associated protein and p53-binding Note protein-2 interact through their WW and SH3 domains. J Biol Chem. 2001 Apr 27;276(17):14514-23 Four single nucleotide polymorphisms within the ASPP2 gene locus, TP53BP2, show significant Samuels-Lev Y, O'Connor DJ, Bergamaschi D, Trigiante G, Hsieh JK, Zhong S, Campargue I, Naumovski L, Crook T, Lu correlation with gastric cancer susceptibility (Ju et al., X. ASPP proteins specifically stimulate the apoptotic function 2005). of p53. Mol Cell. 2001 Oct;8(4):781-94 Hepatitis B virus-positive hepatocellular Lossos IS, Natkunam Y, Levy R, Lopez CD. Apoptosis stimulating protein of p53 (ASPP2) expression differs in diffuse carcinoma large B-cell and follicular center lymphoma: correlation with Note clinical outcome. Leuk Lymphoma. 2002 Dec;43(12):2309-17 Downregulation of ASPP2 (and ASPP1) as a result of Bergamaschi D, Samuels Y, O'Neil NJ, Trigiante G, Crook T, promoter hypermethylation (as measured by Hsieh JK, O'Connor DJ, Zhong S, Campargue I, Tomlinson methylation-specific PCR) is frequently observed in ML, Kuwabara PE, Lu X. iASPP oncoprotein is a key inhibitor of p53 conserved from worm to human. Nat Genet. 2003 human patient samples of HBV-positive hepatocellular Feb;33(2):162-7 carcinoma as compared to surrounding non-tumor tissue (Zhao et al., 2010). Chen Y, Liu W, Naumovski L, Neve RL. ASPP2 inhibits APP- BP1-mediated NEDD8 conjugation to cullin-1 and decreases APP-BP1-induced cell proliferation and neuronal apoptosis. J References Neurochem. 2003 May;85(3):801-9 Iwabuchi K, Bartel PL, Li B, Marraccino R, Fields S. Two Bergamaschi D, Samuels Y, Jin B, Duraisingham S, Crook T, cellular proteins that bind to wild-type but not mutant p53. Proc Lu X. ASPP1 and ASPP2: common activators of p53 family Natl Acad Sci U S A. 1994 Jun 21;91(13):6098-102 members. Mol Cell Biol. 2004 Feb;24(3):1341-50 Helps NR, Barker HM, Elledge SJ, Cohen PT. Protein Cao Y, Hamada T, Matsui T, Date T, Iwabuchi K. Hepatitis C phosphatase 1 interacts with p53BP2, a protein which binds to virus core protein interacts with p53-binding protein, the tumour suppressor p53. FEBS Lett. 1995 Dec 53BP2/Bbp/ASPP2, and inhibits p53-mediated apoptosis. 27;377(3):295-300 Biochem Biophys Res Commun. 2004 Mar 19;315(4):788-95 Naumovski L, Cleary ML. The p53-binding protein 53BP2 also Meek SE, Lane WS, Piwnica-Worms H. Comprehensive interacts with Bc12 and impedes cell cycle progression at proteomic analysis of interphase and mitotic 14-3-3-binding G2/M. Mol Cell Biol. 1996 Jul;16(7):3884-92 proteins. J Biol Chem. 2004 Jul 30;279(31):32046-54 Yang JP, Ono T, Sonta S, Kawabe T, Okamoto T. Assignment Sarraf SA, Stancheva I. Methyl-CpG binding protein MBD1 of p53 binding protein (TP53BP2) to human chromosome band couples histone H3 methylation at lysine 9 by SETDB1 to DNA 1q42.1 by in situ hybridization. Cytogenet Cell Genet. replication and chromatin assembly. Mol Cell. 2004 Aug 1997;78(1):61-2 27;15(4):595-605 Iwabuchi K, Li B, Massa HF, Trask BJ, Date T, Fields S. Takahashi N, Kobayashi S, Jiang X, Kitagori K, Imai K, Hibi Y, Stimulation of p53-mediated transcriptional activation by the Okamoto T. Expression of 53BP2 and ASPP2 proteins from

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 684 TP53BP2 (tumor protein p53 binding protein, 2) Van Hook K, et al.

TP53BP2 gene by alternative splicing. Biochem Biophys Res Langton PF, Colombani J, Aerne BL, Tapon N. Drosophila Commun. 2004 Mar 5;315(2):434-8 ASPP regulates C-terminal Src kinase activity. Dev Cell. 2007 Dec;13(6):773-82 Chen D, Padiernos E, Ding F, Lossos IS, Lopez CD. Apoptosis-stimulating protein of p53-2 (ASPP2/53BP2L) is an Tidow H, Andreeva A, Rutherford TJ, Fersht AR. Solution E2F target gene. Cell Death Differ. 2005 Apr;12(4):358-68 structure of ASPP2 N-terminal domain (N-ASPP2) reveals a ubiquitin-like fold. J Mol Biol. 2007 Aug 24;371(4):948-58 Cobleigh MA, Tabesh B, Bitterman P, Baker J, Cronin M, Liu ML, Borchik R, Mosquera JM, Walker MG, Shak S. Tumor Sun WT, Hsieh PC, Chiang ML, Wang MC, Wang FF. p53 gene expression and prognosis in breast cancer patients with target DDA3 binds ASPP2 and inhibits its stimulation on p53- 10 or more positive lymph nodes. Clin Cancer Res. 2005 Dec mediated BAX activation. Biochem Biophys Res Commun. 15;11(24 Pt 1):8623-31 2008 Nov 14;376(2):395-8 Fogal V, Kartasheva NN, Trigiante G, Llanos S, Yap D, Kampa KM, Bonin M, Lopez CD. New insights into the Vousden KH, Lu X. ASPP1 and ASPP2 are new transcriptional expanding complexity of the tumor suppressor ASPP2. Cell targets of E2F. Cell Death Differ. 2005 Apr;12(4):369-76 Cycle. 2009a Sep 15;8(18):2871-6 Hershko T, Chaussepied M, Oren M, Ginsberg D. Novel link Kampa KM, Acoba JD, Chen D, Gay J, Lee H, Beemer K, between E2F and p53: proapoptotic cofactors of p53 are Padiernos E, Boonmark N, Zhu Z, Fan AC, Bailey AS, Fleming transcriptionally upregulated by E2F. Cell Death Differ. 2005 WH, Corless C, Felsher DW, Naumovski L, Lopez CD. Apr;12(4):377-83 Apoptosis-stimulating protein of p53 (ASPP2) heterozygous mice are tumor-prone and have attenuated cellular damage- Ju H, Lee KA, Yang M, Kim HJ, Kang CP, Sohn TS, Rhee JC, response thresholds. Proc Natl Acad Sci U S A. 2009b Mar Kang C, Kim JW. TP53BP2 locus is associated with gastric 17;106(11):4390-5 cancer susceptibility. Int J Cancer. 2005 Dec 20;117(6):957-60 Uhlmann-Schiffler H, Kiermayer S, Stahl H. The DEAD box Kobayashi S, Kajino S, Takahashi N, Kanazawa S, Imai K, Hibi protein Ddx42p modulates the function of ASPP2, a stimulator Y, Ohara H, Itoh M, Okamoto T. 53BP2 induces apoptosis of apoptosis. Oncogene. 2009 May 21;28(20):2065-73 through the mitochondrial death pathway. Genes Cells. 2005 Mar;10(3):253-60 Cong W, Hirose T, Harita Y, Yamashita A, Mizuno K, Hirano H, Ohno S. ASPP2 regulates epithelial cell polarity through the Liu ZJ, Lu X, Zhang Y, Zhong S, Gu SZ, Zhang XB, Yang X, PAR complex. Curr Biol. 2010 Aug 10;20(15):1408-14 Xin HM. Downregulated mRNA expression of ASPP and the hypermethylation of the 5'-untranslated region in cancer cell Sottocornola R, Royer C, Vives V, Tordella L, Zhong S, Wang lines retaining wild-type p53. FEBS Lett. 2005 Mar Y, Ratnayaka I, Shipman M, Cheung A, Gaston-Massuet C, 14;579(7):1587-90 Ferretti P, Molnár Z, Lu X. ASPP2 binds Par-3 and controls the polarity and proliferation of neural progenitors during CNS Takahashi N, Kobayashi S, Kajino S, Imai K, Tomoda K, development. Dev Cell. 2010 Jul 20;19(1):126-37 Shimizu S, Okamoto T. Inhibition of the 53BP2S-mediated apoptosis by nuclear factor kappaB and Bcl-2 family proteins. Zhao J, Wu G, Bu F, Lu B, Liang A, Cao L, Tong X, Lu X, Wu Genes Cells. 2005 Aug;10(8):803-11 M, Guo Y. Epigenetic silence of ankyrin-repeat-containing, SH3-domain-containing, and proline-rich-region- containing Zhu Z, Ramos J, Kampa K, Adimoolam S, Sirisawad M, Yu Z, protein 1 (ASPP1) and ASPP2 genes promotes tumor growth Chen D, Naumovski L, Lopez CD. Control of ASPP2/(53BP2L) in hepatitis B virus-positive hepatocellular carcinoma. protein levels by proteasomal degradation modulates p53 Hepatology. 2010 Jan;51(1):142-53 apoptotic function. J Biol Chem. 2005 Oct 14;280(41):34473- 80 Liu CY, Lv X, Li T, Xu Y, Zhou X, Zhao S, Xiong Y, Lei QY, Guan KL. PP1 cooperates with ASPP2 to dephosphorylate and Vives V, Su J, Zhong S, Ratnayaka I, Slee E, Goldin R, Lu X. activate TAZ. J Biol Chem. 2011 Feb 18;286(7):5558-66 ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth. Genes Dev. 2006 May Wang XD, Lapi E, Sullivan A, Ratnayaka I, Goldin R, Hay R, 15;20(10):1262-7 Lu X. SUMO-modified nuclear cyclin D1 bypasses Ras- induced senescence. Cell Death Differ. 2011 Feb;18(2):304-14 Hakuno F, Kurihara S, Watson RT, Pessin JE, Takahashi S. 53BP2S, interacting with insulin receptor substrates, This article should be referenced as such: modulates insulin signaling. J Biol Chem. 2007 Dec 28;282(52):37747-58 Van Hook K, Wang Z, Lopez C. TP53BP2 (tumor protein p53 binding protein, 2). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):681-685.

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 685 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Leukaemia Section Review

8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome) Paula Aranaz, José Luis Vizmanos Department of Genetics, School of Sciences, University of Navarra, E-31008 Pamplona, Spain (PA, JLV)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/8p11inMPDID1091.html DOI: 10.4267/2042/46005 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

responsible are not well known (Yang et al., 2004; Identity Garcia et al., 2005; Gelsi-Boyer et al., 2005; Pole et al., Alias 2006; Yang et al., 2006; Yang et al., 2010). Stem cell leukemia-lymphoma syndrome (SCLL); 8p11 Disease stem cell syndrome; 8p11 stem cell Clinical entity defined by the disruption of the FGFR1 leukemia/lymphoma syndrome; Myeloid and lymphoid gene located at 8p11 with generation of a fusion gene neoplasms FGFR1 abnormalities (WHO 2008 between the 3' part of FGFR1 and the 5' part of the proposal) partner gene that also provides its promoter. The Note partner gene is always expressed in the haematopietic Although "8p11 myeloproliferative syndrome" (EMS) system and codes for a protein with oligomerization (Macdonald et al., 1995) is the most frequent name for domains. As a result of oligomerization, chimeric this disease in the literature, it must be designated as proteins show constitutive and ligand-independent "myeloid and lymphoid neoplasm with FGFR1 activation of FGFR1 kinase activity. abnormalities" under the current 2008 World Health The 8p11 myeloproliferative syndrome (EMS) is a Organization classification (Tefferi and Vardiman, myeloproliferative disease with multilineage 2008; Tefferi et al., 2009). This disease has been involvement characterized by chronic myelomonocytic referred to as "stem cell leukemia/lymphoma" (SCLL) leukemia (CMML)-like myeloid hyperplasia, marked (Inhorn et al., 1995) which remark the coexistence of peripheral blood eosinophilia and associated with a lymphoma, myeloid malignancy and lymphoblastic high incidence of non-Hodgkin's lymphoma, usually of leukemia. the T-cell lymphoblastic subtype. Occasional cases also show a B-cell lymphoproliferative disorder (Macdonald Clinics and pathology et al., 2002). EMS cases were already described in the 1970s and Note 1980s, but cytogenetic and molecular analyses were not This disease is related to fusion genes between FGFR1, available (Manthorpe et al., 1977; Kjeldsberg et al., located in 8p11, and several partner genes. However 1979; Catovsky et al., 1980; Posner et al., 1982). In there are some other aberrations affecting this 1992, 3 cases of T-cell lymphoblastic lymphoma chromosomal band and 8p12 in other neoplasms. Some associated with eosinophilia that subsequently acute myeloid leukemia (AML) cases have been developed acute myeloid leukemia or described related to translocations affecting MYST3 myelodysplastic/myeloproliferative neoplasms were (also known as MOZ) (Borrow et al., 1996; Carapeti et reported (Abruzzo et al., 1992). One of them showed a al., 1998; Chaffanet et al., 2000; Murati et al., 2007; t(8;13) by conventional cytogenetics. Later it was Esteyries et al., 2008; Gervais et al., 2008) and shown that one of the breakpoints involved one 8p11 WHSC1L1 (also known as NSD3) (Rosati et al., 2002; locus (Xiao et al., 1998). In the same year, Rao et al. Romana et al., 2006; Taketani et al., 2009), both of reported a patient with t(8;13)(p11;q12) who presented them in 8p11. In addition, aberrations in 8p11-p12 are with leukocytosis, monocytosis, myeloid hyperplasia of also frequent events in breast cancer, but the loci

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bone marrow, and generalized lymphadenopathy due to leukopenia but some cases have been reported with T-cell lymphoblastic lymphoma (Rao et al., 1998). The normal leukocyte counts. Eosinophilia is frequent, but term 8p11 myeloproliferative syndrome was suggested monocytosis appears only in one third of patients. in 1995 by Macdonald et al. (Aguiar et al., 1995; Basophils are increased only in cases with the Macdonald et al., 1995) and confirmed as clinical entity t(8;22)(p11;q11). Blasts have been detected in half of (SCLL) by Inhorn et al. (Inhorn et al., 1995). the patients and some cases show blast counts typical Phenotype/cell stem origin of an acute leukemia. These blasts are mainly of a myeloid or myeloid and lymphoid (bilineal) lineage The presence of the cytogenetic aberration in 8p11 both although some of them are also of an immature in myeloid and lymphoid cells, suggest a bilineage lymphoid lineage. differentiation from a pluripotent and common stem Most of the patients show a hypercellular bone marrow cell (Macdonald et al., 2002). that leads to a diagnosis of myeloid hyperplasia or a Etiology myeloproliferative neoplasm. But in some cases, the dysplastic features lead to a diagnosis of The FGFR1 fusion proteins that result from myelodysplastic syndrome or a myelodisplastic chromosomal translocations affecting 8p11 have syndrome/myeloproliferative neoplasm. constitutive and ligand-independent FGFR1 enzymatic Most of the cases with lymph node biopsies reported activity. FGFR1 is a receptor tyrosine kinase that had T-lymphoblastic lymphoma and the rest had dimerizes upon ligand binding, activating multiple myeloid sarcoma. In some cases evidence of bilineal T- signalling pathways like Ras/MAPK, PI3K, cell/myeloid or B-and T- cell lymphoblastic lymphoma PLCgamma and STAT. These pathways could be has been reported. For a review see Jackson et al., abnormally activated as consequence of FGFR1 2010. aberration (Macdonald et al., 2002) resulting in cell transformation. In fact, expression of ZMYM2-FGFR1 Treatment and BCR-FGFR1 fusions in immunodeficient mice are This is a very aggressive disease with a high rate of capable of initiating an EMS-like disease (Agerstam et progression to an AML resistant to conventional al., 2010). Different fusion proteins could activate these chemotherapy with a median survival time of less than pathways in a different way and could explain the 12 months (Macdonald et al., 2002; Cross and Reiter, phenotypic variability of the disease (Roumiantsev et 2008; Jackson, 2010). al., 2004; Cross and Reiter, 2008; Jackson et al., 2010). There are very few cases (Martinez-Climent et al., Epidemiology 1998; Zhou et al., 2010) responding to interferon alpha, this treatment could be useful at early stages. However, This is a very rare disease with less than 100 patients to date, only stem cell transplant remains effective to reported around the world and it can be found at any eradicate or suppress the malignant clone (Macdonald age. It has been reported at ages ranging from 3 to 84 et al., 2002; Jackson et al., 2010). Median survival time years (median: 44 years). There is a slightly male-to- for patients who received transplant after female predominance (Macdonald et al., 2002; Jackson transformation to AML is 24 months (range 6-46 et al., 2010). months) but median survival time is 12 months for the Clinics patients who did not received transplant (range 0-60 Around 20-25% of patients show systemic and months) (Jackson et al., 2010). Currently there are no unspecific symptoms like fatigue, night sweats, weight specific inhibitors for clinical use effective in this loss and fever and around 20% are asymptomatic (and disease. Patients with FGFR1 fusions do not respond to the disease is detected in routine analyses). Near two drugs developed for other tyrosine kinases like thirds of patients show lymphadenopathy, generalized imatinib, although several FGFR1 inhibitors have been or localized. Hepato- and/or splenomegaly are also tested, some of them with promising effects (Zhang et frequent events in these patients. One of the distinctive al., 2010; Zhou et al., 2010; Bhide et al., 2010; Risuleo features of this disease is the high frequency of et al., 2009; Ma et al., 2008; Cai et al., 2008; Chase et lymphoblastic lymphoma, uncommon in other al., 2007; Kammasud et al., 2007; Klenke et al., 2007; myeloproliferative neoplasms (Macdonald et al., 2002; Chen et al., 2004; Aviezier et al., 2000). Jackson et al., 2010). Evolution Cytology This disease has a chronic phase characterized by It seems that neoplastic cells present in lymph nodes myeloid hyperplasia and overproduction of myeloid are predominantly small or medium lymphoblasts with cells that can differentiate, but without treatment the a small cytoplasm (Jackson et al., 2010). disease progresses rapidly (1 to 2 years after diagnosis) to an acute myeloid leukemia (AML) or sometimes to a Pathology B-lineage ALL (Macdonald et al., 1995; Inhorn et al., The blood counts reported are variable. More than 90% 1995; Macdonald et al., 2002; Jackson et al., 2010). of patients have leukocytosis and less than 10% have

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Gain of an additional copy of chromosome 21 is a non- (Macdonald et al., 2002; Cross and Reiter, 2008; random cytogenetic event apparently associated with Jackson et al., 2010). progression of this disease (Agerstam et al., 2007; t(6;8)(q27;p11) FGFR1OP-FGFR1 Goradia et al., 2008) but its role remains unclear This translocation was first described by Popovici et al. (Jackson et al., 2010). This abnormality is reported in (1999) and fuses FGFR1OP (previously known as FOP only 5 of 47 (10.6%) karyotypes at the time of -FGFR1 oncogene partner-) with FGFR1. diagnosis but in 10 of 13 (76.9%) karyotypes reported As other FGFR1 fusion variants, the chimeric in follow-up. These karyotypes were mostly derived FGFR1OP-FGFR1 protein retains the N-terminus during clinical deterioration. In addition, some findings leucine-rich region of FGFR1OP (an oligomerization at the time of transformation from EMS to acute domain) fused to the catalytic domain of FGFR1 leukemia include the addition of various marker driving the abnormal oligomerization of the chimeric chromosomes, as well as trisomy of chromosomes 8, 9, protein and a constitutive and ligand-independent 12 or 19 and deletions of chromosome 7 or either the activation. 7p or 7q arms, and derivative chromosome 9 (Jackson This translocation has been reported in 8 cases until et al., 2010). date (Vizmanos et al., 2004). Eosinophilia is frequent Prognosis in these patients. Four of these patients had features at presentation and/or a clinical course typical of EMS, As mentioned before, this is a devastating disease, but three showed polycythemia vera (PV) and another which transforms to acute leukemia in a few months if one B-ALL. left untreated, and in which the malignant clone cannot t(8;9)(p12;q33) CEP110-FGFR1 be eradicated by conventional chemotherapy. So at this This translocation was described in 1983 but moment, without specific FGFR1 inhibitors for clinical molecularly characterized by Guasch et al. in 2000 use, the stem cell transplant remains as the only (Guasch et al., 2000). possibility to a long-term survival (Macdonald et al., This translocation has been reported in more than ten 1995; Inhorn et al., 1995; Macdonald et al., 2002; cases until date (Mozziconazzi et al., 2008; Jackson et Jackson et al., 2010). al., 2010) and the MPD caused by this aberration transforms rapidly and always in myelomonocytic Genetics leukemia, with a possible B- or T-lymphoid Note involvement. In addition, tonsillar involvement and monocytosis also correlates with this variant This disease is defined by the fusion of FGFR1 (8p11) (Mozziconazzi et al., 2008; Jackson et al., 2010). with other partner genes, as consequence of a Recently a complete haematological and molecular cytogenetic aberration, mainly chromosomal remission has been reported after two years in a patient translocations. FGFR1 codes for a receptor tyrosine with this translocation treated early with interferon kinase. The gene fusion maintains the 3' terminal part alpha (Zhou et al., 2010). of the FGFR1 gene (from exon 9) joined to the 5' terminal part of the partner gene. Partner genes are t(8;22)(p11;q11) BCR-FGFR1 This translocation was reported simultaneously by two widely expressed and fusion genes have also this groups in 2001 (Fioretos et al., 2001; Demiroglu et al., expression pattern. The chimeric gene codes for a 2001) and fuses BCR with FGFR1. protein which retains the TK domain from FGFR1 and As in the case of the other FGFR1 fusions, BCR is also oligomerization domains provided by the partner gene. widely expressed and BCR-FGFR1 retains This protein has a constitutive and ligand-independent oligomerization domains from BCR and the catalytic activity and activates multiple signal transduction domain from FGFR1, leading to constitutive and pathways. ligand-independent activity of the chimeric protein. However it seems that BCR not only drives this Cytogenetics oligomerization but could also play some role in Variants triggering the downstream signalling pathways. Patients with BCR-FGFR1 fusions have a slightly t(8;13)(p11;q12) ZMYM2-FGFR1 different clinical phenotype from other FGFR1 fusion This is the first translocation described (Xiao et al., variants. In fact, these patients have a clinical and 1998; Reiter et al., 1998; Smedley et al., 1998; morphological picture similar to typical BCR-ABL Popovici et al., 1998) and the most common one. In positive chronic myeloid leukemia (Roumiantsev et al., fact, it has been described in 33 of the 65 cases 2004; Cross and Reiter, 2008; Baldazzi et al., 2010; described until now (Jackson et al., 2010). Jackson et al., 2010). This translocation generates a fusion gene ZMYM2- t(8;11)(p11;p15) NUP98-FGFR1 FGFR1. This translocation was described by Sohal et al. in Patients with this translocation develop 2001, in a patient with AML with additional lymphadenopathy and T-cell lymphoblastic lymphoma cytogenetic aberrations (patient UPN6,

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47,XY,t(8;11)(p11;p15),+8,-17,+i(17q)) (Sohal et al., The consequence of the t(7,8)(q34;p11) is the fusion 2001). Only interphase cells were available to perform gene TRIM24-FGFR1. FISH analysis, and the results obtained indicated a t(8;17)(p11;q23) MYO18A-FGFR1 breakpoint within or in the vicinity of NUP98 but This translocation was described by Walz et al. (2005) definite molecular characterization could not be done. in a 74-year-old female with a 2 years history of an Other patient with AML and the same translocation had unusual myelodysplastic/myeloproliferative disease been reported previously (Larson et al., 1983), (MDS/MPD) with thrombocytopenia, markedly indicating that this was a recurrent abnormality. reduced size and numbers of megakaryocytes and However no new cases have been described since these elevated numbers of monocytes, eosinophils and reports so NUP98-FGFR1 fusion has not been basophils. Her karyotype showed an additional trisomy confirmed definitely. NUP98 has a small coiled-coil 20 and she died after a treatment-resistant disease region that could drive the constitutive activation of the progression of two years. chimeric NUP98-FGFR1 protein but it is possible that This translocation fuses MYO18A, located in 17q11.2 disruption of NUP98 was also involved in the with FGFR1. However, the breakpoint in chromosome oncogenic process. 17 was cytogenetically located to 17q23. FISH and t(8;19)(p11;q13) HERVK-FGFR1 molecular analysis showed that this fusion gene was This aberration was firstly described in 2000, consequence of a complex cytogenetic aberration with associated with loss of the Y chromosome in a man an additional inversion in 17q region between 17q11 with an AML M0, probably secondary to a and 17q23. myeloproliferative disorder, who died 15 months after t(8;12)(p11;q15) CPSF6-FGFR1 diagnosis (Mugneret et al., 2000). Later, the same This translocation targeting FGFR1 was first described group identified the chromosome 19 partner showing by Sohal et al. (2001). Later the same group identified that a long terminal repeat of human endogenous the partner gene as CPSF6 (located at 12q15) (Hidalgo- retrovirus gene (HERV-K) was fused in frame with Curtis et al., 2008) and again, only this case has been FGFR1 (Guasch et al., 2003). This fusion has been described. The patient was a 75-year-old female with described only in this case. lymphadenopathy, splenomegaly, neutrophilia and ins(12;8)(p11;p11p22) FGFR1OP2-FGFR1 eosinophilia in peripheral blood and also an increase of This FGFR1 fusion is not caused by a chromosomal eosinophils and eosinophil precursors in the bone translocation but an inversion. Ins(12;8)(p11;p11p22) marrow. After a rapid clinical deterioration the patient targeting FGFR1 was first described by Sohal et al. died in 10 weeks. (2001) in a 75-years old patient diagnosed with a T-cell t(2;8)(q37;p11) LRRFIP1-FGFR1 lymphoblastic lymphoma and marked lymph node In 2009, Soler et al. identified and characterized the infiltration with atypical eosinophils. Whole blood t(2;8)(q37;p11) in an 82-year-old man with 10% count was normal except for very mild eosinophilia and eosinophils, 2-4% myelocytes and metamyelocytes, the bone marrow also showed some atypical and 8% circulating blasts and an hypocellular bone eosinophils. After complete remission, this patient marrow with moderate dysgranulopoiesis and 15% relapsed and transformed to an AML with the same blasts (Soler et al., 2009). Some years before, this chromosomal aberration and died. Later this aberration patient had displayed pancitopenia and a bone marrow was molecularly characterized by the same group showing a refractory anemia with an excess (Grand et al., 2004) as a fusion between FGFR1OP2 of blasts (15%). The disease transformed to AML in (from FGFR1 oncogene partner 2) located at 12p11.23 one year and the patient died. FISH analysis on and FGFR1. Fusion structure was identical to other retrospective samples showed that the t(2;8)(q37;p11) FGFR1 variants. was not present in early stages (pancitopenia) of the This fusion has been reported only in one case (Sohal et disease. al., 2001; Grand et al., 2004) but it has been also found in the cell line KG-1 (Gu et al., 2006; DSMZ ACC 14) Genes involved and proteins that can be used to assay in vitro specific FGFR1 inhibitors (Gu et al., 2006; Chase et al., 2007). This cell ZMYM2 line was derived from the bone marrow of a 59-year- Location old man with erythroleukemia transformed to AML at 13q12 relapse in 1977 (Koeffler and Golde, 1978). Protein t(7;8)(q34;p11) TRIM24-FGFR1 ZMYM2 (also known as ZNF198, RAMP -rearranged This translocation was described and molecularly in atypical myeloproliferative disorder-, or FIM - fused characterized by Belloni et al. (2005) in a 49-year-old in myeloproliferative disorder) codes for a zinc finger woman with a putative chronic MPD with eosinophilia protein that may act as a transcription factor involved which transformed to an AML-M4 and died in a few in ribosomal RNA transcription and also could be part days. As other FGFR1 fusions, this is the only case of a BHC histone deacetylase complex. The chimeric reported to date.

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protein retains the proline-rich domain of ZMYM2 (an Protein oligomerization domain) and the tyrosine kinase HERV-K is also ubiquitously expressed. The HERV- domain of FGFR1. The abnormal oligomerization of Ks are human specific endogenous retrovirus that have the chimeric protein leads to constitutive and ligand- been proposed as etiological cofactors in some chronic independent activation. In addition, this abnormal diseases like cancer because they are mobile elements protein is located in the cytoplasm and not in the that could disrupt tumor suppressor and/or DNA repair membrane as native FGFR1. genes. In this case, it seems that the part of the HERV- FGFR1OP K sequence fused showed similarities with a retroviral envelope protein whose dimerization would induce the Location constitutive activation of the chimeric protein HERVK- 6q27 FGFR1 (Guasch et al., 2003). Protein FGFR1OP2 FGFR1OP, widely expressed, codes for a hydrophilic centrosomal protein that could be a member of a Location leucine-rich protein family, and it is involved in the 12p11.23 anchoring of microtubules (MTS) to subcellular Protein structures. FGFR1OP could play a role in lung cancer As other FGFR1 partners, FGFR1OP2 is also widely growth and progression and has been proposed as a expressed but its function is unknown. It could code for prognostic biomarker for this disease (Mano et al., a cytoskeleton molecule (Lin et al., 2010). However, 2007). the putative protein coded by this gene has four CEP110 potential coiled-coil domains and the first two are retained in the chimeric protein, so they could mediate Location its oligomerization and constitutive activation (Grand et 9q33.2 al., 2004). Protein TRIM24 CEP110 encodes also a centrosomal protein with several leucine zipper motifs required for the Location centrosome to function as a microtubule organizing 7q34 center. CEP110 is also widely expressed and CEP110- Protein FGFR1 retains the leucine zipper motifs of CEP110 at TRIM24 (previously known as TIF1) codes for a its N-terminus which could mediate the consititutive protein of the tripartite motif (TRIM) family that activation of the FGFR1 catalytic domain at its C- mediates transcriptional control by interaction with terminus. In addition the CEP110-FGFR1 fusion several nuclear receptors and localizes to nuclear protein has been found in the cytoplasm, whereas both bodies. The tripartite motif includes three zinc-binding CEP110 and FGFR1 wild-type proteins are centrosome domains - a RING, a B-box type 1 and a B-box type 2 - and plasma membrane-bound proteins respectively and a coiled-coil region that is retained in the chimeric (Guasch et al., 2000). protein so it could promote, as other FGFR1 fusion BCR proteins, its constitutive and ligand-independent activation Location 22q11.2 MYO18A Protein Location BCR is, like ETV6, a common fusion partner of several 17q11.2 tyrosine kinase genes rearranged in myeloid disorders Protein (BCR-ABL, BCR-JAK2, BCR-PDGFRA and BCR- MYO18A is a widely expressed gene that codes for a FGFR1 have been described to date). However function protein of unknown function of the myosin of the protein encoded by this gene is not clear and its superfamily. It has been recently described that this name comes from breakpoint cluster region. protein is a novel PAK2 (p21-activated kinase 2) NUP98 binding partner (Hsu et al., 2010). PAK2 has many biological functions, including the regulation of actin Location reorganization and cell motility. MYO18A contains 11p15 several functional motifs that are retained in MYO18A- HERVK FGFR1, including an N-terminal PDZ (PSD- 95/Dlg/ZO-1) protein-protein interaction domain, a Location myosin head domain and a region that is predicted to 7p22.1

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 690 8p11 myeloproliferative syndrome (EMS, eight p11 myeloproliferative syndrome) Aranaz P, Vizmanos JL

form multiple coiled-coils. Some of these coiled-coils peripheral T and B lymphocytosis. Scand J Haematol. 1977 could drive oligomerization of MYO18A-FGFR1, with May;18(5):449-54 consequent constitutive activation of the FGFR1 kinase Koeffler HP, Golde DW. Acute myelogenous leukemia: a activity. human cell line responsive to colony-stimulating activity. Recently, MYO18A has also been found fused to Science. 1978 Jun 9;200(4346):1153-4 PDGFRB as consequence of a t(5;17)(q33-q34;q11) but Kjeldsberg CR, Nathwani BN, Rappaport H. Acute myeloblastic with a different breakpoint in which all the predicted leukemia developing in patients with mediastinal lymphoblastic lymphoma. Cancer. 1979 Dec;44(6):2316-23 coiled-coil domains of normal MYO18A are retained in the chimeric protein (Walz et al., 2009). Catovsky D, Bernasconi C, Verdonck PJ, Postma A, Hows J, van der Does-van den Berg A, Rees JK, Castelli G, Morra E, CPSF6 Galton DA. 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oncogene partner 2/wound inducible transcript-3.0 Gray NS. A structure-guided approach to creating covalent (FGFR1OP2/wit3.0), facilitates fibroblast-driven wound closure. FGFR inhibitors. Chem Biol. 2010 Mar 26;17(3):285-95 Am J Pathol. 2010 Jan;176(1):108-21 Zhou L, Fu W, Yuan Z, Hou J. Complete molecular remission Yang ZQ, Liu G, Bollig-Fischer A, Giroux CN, Ethier SP. after interferon alpha treatment in a case of 8p11 Transforming properties of 8p11-12 amplified genes in human myeloproliferative syndrome. Leuk Res. 2010 breast cancer. Cancer Res. 2010 Nov 1;70(21):8487-97 Nov;34(11):e306-7

Zhang J, Zhou J, Ren X, Diao Y, Li H, Jiang H, Ding K, Pei D. This article should be referenced as such: A new diaryl urea compound, D181, induces cell cycle arrest in the G1 and M phases by targeting receptor tyrosine kinases Aranaz P, Vizmanos JL. 8p11 myeloproliferative syndrome and the microtubule skeleton. Invest New Drugs. 2010 Nov 16; (EMS, eight p11 myeloproliferative syndrome). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8):686-694. Zhou W, Hur W, McDermott U, Dutt A, Xian W, Ficarro SB, Zhang J, Sharma SV, Brugge J, Meyerson M, Settleman J,

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 694 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Leukaemia Section Mini Review i(5)(p10) in acute myeloid leukemia Nathalie Douet-Guilbert, Angèle Herry, Audrey Basinko, Marie-Josée Le Bris, Nadia Guéganic, Clément Bovo, Frédéric Morel, Marc De Braekeleer Laboratory of Histology, Embryology, and Cytogenetics, Faculty of Medicine and Health Sciences, Université de Bretagne Occidentale, 22, avenue Camille Desmoulins, CS 93837, F-29238 Brest cedex 3, France (NDG, AH, AB, MJL, NG, CB, FM, MD)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/i5pID1376.html DOI: 10.4267/2042/46006 This article is an update of : Schoch C. i(5)(p10) in acute myeloid leukemia. Atlas Genet Cytogenet Oncol Haematol 2005;9(2):150-151

Identity

i(5)(p10) G-banding - Claudia Schoch (left), and R-banding - Nathalie Douet-Guilbert (right).

Note Type 2: classified as acute myeloid leukemia (5 cases), In literature, two types of i(5)(p10) are observed: predominantly AML M5a. Type 1: i(5)(p10) inducing a loss of the long arm of Etiology chromosome 5 (5q) and a trisomy of the short arm of Unclear the chromosome 5 (5p); Type 2: +i(5)(p10) (or supernumerary i(5)(p10) or gain Epidemiology of i(5)(p10)) inducing a tetrasomy of the short arm of Type 1: it is found in young adults in MDS (average chromosome 5 (5p). The i(5)(p10) occurred in addition age: 35 years; range: 19-67) and in older patients in to two normal chromosomes 5. AML (average age: 66 years; range: 50-85). The isochromosome of the short arm of chromosome 5 Type 2: the +i(5)(p10) is found in patients with an - i(5)(p10) - has only been described in a few cases of average age of 48.5 years (range : 24-78). myeloid leukemia. Prognosis Clinics and pathology Prognosis of patients with i(5)(p10) seems to be poor compared to patients with del(5q), but it is unclear due Phenotype/cell stem origin to the very small number of cases and the usually Type 1: classified as myelodysplastic syndrome (4 associated complex chromosomal abnormalities. cases), acute myeloid leukemia (4 cases) predominantly AML M2;

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 695 i(5)(p10) in acute myeloid leukemia Douet-Guilbert N, et al.

A - FISH with partial chromosome painting 5p (pcp 5p) (Green signal) and 5q (pcp 5q) (Red signal). B - FISH with LSI 5p15.2 (Green signal) / 5q31 (Red signal). Nathalie Douet-Guilbert.

Tamura S, Takemoto Y, Hashimoto-Tamaoki T, Mimura K, Cytogenetics Sugahara Y, Senoh J, Furuyama JI, Kakishita E. Cytogenetic analysis of de novo acute myeloid leukemia with trilineage Cytogenetics morphological myelodysplasia in comparison with myelodysplastic syndrome evolving to acute myeloid leukemia. Int J Oncol. 1998 The formation of i(5p) results from the loss of the long Jun;12(6):1259-62 arm of chromosome 5 and duplication of its short arm inducing trisomy 5p and monosomy 5q in type 1 and Markovic VD, Bouman D, Bayani J, Al-Maghrabi J, Kamel-Reid S, Squire JA. Lack of BCR/ABL reciprocal fusion in variant tetrasomy 5p in type 2. Philadelphia chromosome translocations: a use of double A metacentric del(5q) could be an isochromosome of fusion signal FISH and spectral karyotyping. Leukemia. 2000 the short arm of chromosome 5. FISH technique with Jun;14(6):1157-60 specific probes of chromosome 5p/5q used as a Schoch C, Bursch S, Kern W, Schnittger S, Hiddemann W, complement of conventional karyotype is necessary to Haferlach T. Gain of an isochromosome 5p: a new recurrent identify i(5)(p10). The i(5p) is a variant of del(5q). The chromosome abnormality in acute monoblastic leukemia. Cancer Genet Cytogenet. 2001 May;127(1):85-8 i(5p) is monocentric or dicentric. Christodoulou J, Schoch C, Schnittger S, Haferlach T. Additional anomalies Myelodysplastic syndrome (RARS) with +i(12p) abnormality in In one case, i(5)(p10) was the sole anomaly but rapidly a patient 10 months after diagnosis and successful treatment of a mediastinal germ cell tumor (MGCT). Ann Hematol. 2004 evolved into a complex karyotype. Complex Jun;83(6):386-9 karyotypes were present in the other cases: -12/del12p (3 cases), -17/del17p (2 cases), del9q (2 cases). Schmidt HH, Strehl S, Thaler D, Strunk D, Sill H, Linkesch W, Jäger U, Sperr W, Greinix HT, König M, Emberger W, Haas Supernumerary +i(5)(p10) was accompanied by several OA. RT-PCR and FISH analysis of acute myeloid leukemia additional anomalies, especially trisomy 8 with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications. Leukemia. Genes involved and proteins 2004 Jun;18(6):1115-21 Panani AD. Gain of an isochromosome 5p: a rare recurrent Note abnormality in acute myeloid leukemia. In Vivo. 2006 May- Type 1: to explain the specific phenotype of i(5)(p10), Jun;20(3):359-60 loss of tumor suppressor genes in the deleted region Herry A, Douet-Guilbert N, Morel F, Le Bris MJ, Guéganic N, (5q) associated with gene dosage effect of genes Berthou C, De Braekeleer M. Isochromosome 5p and related located on 5p is suggested. anomalies: a novel recurrent chromosome abnormality in myeloid disorders. Cancer Genet Cytogenet. 2010 Jul Type 2: gene dosage effect of genes located on the 15;200(2):134-9 short arm of chromosome 5. This article should be referenced as such: References Douet-Guilbert N, Herry A, Basinko A, Le Bris MJ, Guéganic N, Bovo C, Morel F, De Braekeleer M. i(5)(p10) in acute myeloid El-Rifai W, Elonen E, Larramendy M, Ruutu T, Knuutila S. leukemia. Atlas Genet Cytogenet Oncol Haematol. 2011; Chromosomal breakpoints and changes in DNA copy number 15(8):695-696. in refractory acute myeloid leukemia. Leukemia. 1997 Jul;11(7):958-63

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Leukaemia Section Mini Review

+20 or trisomy 20 (solely) Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: January 2011 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/tri20ID1572.html DOI: 10.4267/2042/46007 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

The 8 lympoid cases were: three acute lymphoblastic Clinics and pathology leukaemias (ALL) (two of which involved the T-cell Disease lineage), one chronic lymphocytic leukaemia (CLL), three non Hodgkin lymphomas (NHL) : one follicular Myeloid and lymphoid malignancies (Shabtai et al., (FL), one diffuse large B-cell (DLBL), and one T-cell 1978; Kristoffersson et al., 1985; Michalová et al., lymphoma); and one Waldenstrom macroglobulinemia. 1987; Palka et al., 1987; Speaks et al., 1987; Attas et al., 1989; Cuneo et al., 1989; Nayak et al., 1990; Cuneo Epidemiology et al., 1992; Cuneo et al., 1995; Hashimoto et al., 1995; In the myeloid group, there were 7 male and 5 female Rigolin et al., 1997; Jaing et al., 1999; Mauritzson et patients, median age was 68-72 years (range: 8-79 al., 2001; Tamura et al., 2001; Mikhail et al., 2002; years, 8 of the 10 documented cases were above 60 Farag et al., 2006; Paulsson et al., 2008). years). In the lymploid group, there was an unbalanced Note sex ratio: 6 male and 2 female patients; median age was Trisomy 20 solely has also been reported in various 33-53 years (range: 7-76 years). benign and malignant solid tumours, in particular in Prognosis desmoid fibromatosis, colonic adenomatous polyps, Data is very scarce, and not conclusive, inasmuch as colorectal adenocarcinomas, fibroadenomas of the the genes involved in these cases are unknown, and as breast, breast adenocarcinoma, transitional cell the trisomy 20 group is probably heterogeneous from carcinoma of the urinary tract, squamous cell that view point. carcinoma of the oro-pharynx and naso-pharynx; it has also been found more rarely in many other solid tumours (see records in the Mitelman Database). Genes involved and proteins Phenotype/cell stem origin Note Genes involved are unknown. Trisomy 20 solely has been described in 20 cases of hematological malignancies: This was a myeloid malignancy in 12 cases: six acute References myeloid leukaemias (AML), four myelodysplastic Shabtai F, Weiss S, van der Lijn E, Lewinski U, Djaldetti M, syndromes (MDS), and two myeloproliferative Halbrecht I. A new cytogenetic aspect of polycythemia vera. disorders (MPD). They were: two M4-AML, two M5- Hum Genet. 1978 Apr 24;41(3):281-7 AML, one M0-AML, one AML not otherwise specified Kristoffersson U, Heim S, Heldrup J, Akerman M, Garwicz S, (NOS), one refractory anaemia (RA), one RA with Mitelman F. Cytogenetic studies of childhood non-Hodgkin excess of blasts (RAEB), one chronic myelomonocytic lymphomas. Hereditas. 1985;103(1):77-84 leukaemia (CMML), one MDS-NOS, and two Michalová K, Kobylka P, Lukásová M, Neuwirt J. Cytogenetic polycytemia vera (PV). One AML, a M5-AML, study of circulating blasts in leukemias. Cancer Genet Cytogenet. 1987 Apr;25(2):329-39 appeared to be treatment-related, in a 8-year-old girl with neuroblastoma, diagnosed 32 months before onset Palka G, Spadano A, Geraci L, Fioritoni G, Dragani A, Calabrese G, Guanciali Franchi P, Stuppia L. Chromosome of the leukaemia. A cryptic rearrangement of MLL was changes in 19 patients with Waldenström's macroglobulinemia. found. Survival was short (Jaing et al., 1999). Cancer Genet Cytogenet. 1987 Dec;29(2):261-9

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 697 +20 or trisomy 20 (solely) Huret JL

Speaks SL, Sanger WG, Linder J, Johnson DR, Armitage JO, Mauritzson N, Johansson B, Rylander L, Albin M, Strömberg Weisenburger D, Purtilo D. Chromosomal abnormalities in U, Billström R, Ahlgren T, Mikoczy Z, Mitelman F, Hagmar L, indolent lymphoma. Cancer Genet Cytogenet. 1987 Nilsson PG. The prognostic impact of karyotypic subgroups in Aug;27(2):335-44 myelodysplastic syndromes is strongly modified by sex. Br J Haematol. 2001 May;113(2):347-56 Attas L, Lichtman SM, Budman DR, Verma RS. Trisomy 20 in acute myelogenous leukemia. Cancer Genet Cytogenet. 1989 Tamura A, Miura I, Iida S, Yokota S, Horiike S, Nishida K, Fujii May;39(1):25-8 H, Nakamura S, Seto M, Ueda R, Taniwaki M. Interphase detection of immunoglobulin heavy chain gene translocations Cuneo A, Tomasi P, Ferrari L, Balboni M, Piva N, Fagioli F, with specific oncogene loci in 173 patients with B-cell Castoldi G. Cytogenetic analysis of different cellular lymphoma. Cancer Genet Cytogenet. 2001 Aug;129(1):1-9 populations in chronic myelomonocytic leukemia. Cancer Genet Cytogenet. 1989 Jan;37(1):29-37 Mikhail FM, Serry KA, Hatem N, Mourad ZI, Farawela HM, El Kaffash DM, Coignet L, Nucifora G. AML1 gene over- Nayak BN, Sokal J, Ray M. Clonal chromosomal changes in expression in childhood acute lymphoblastic leukemia. chronic lymphocytic leukemia. Cancer Lett. 1990 Feb;49(2):99- Leukemia. 2002 Apr;16(4):658-68 105 Farag SS, Archer KJ, Mrózek K, Ruppert AS, Carroll AJ, Cuneo A, Fagioli F, Pazzi I, Tallarico A, Previati R, Piva N, Vardiman JW, Pettenati MJ, Baer MR, Qumsiyeh MB, Koduru Carli MG, Balboni M, Castoldi G. Morphologic, immunologic PR, Ning Y, Mayer RJ, Stone RM, Larson RA, Bloomfield CD. and cytogenetic studies in acute myeloid leukemia following Pretreatment cytogenetics add to other prognostic factors occupational exposure to pesticides and organic solvents. predicting complete remission and long-term outcome in Leuk Res. 1992 Aug;16(8):789-96 patients 60 years of age or older with acute myeloid leukemia: Cuneo A, Ferrant A, Michaux JL, Boogaerts M, Demuynck H, results from Cancer and Leukemia Group B 8461. Blood. 2006 Van Orshoven A, Criel A, Stul M, Dal Cin P, Hernandez J. Jul 1;108(1):63-73 Cytogenetic profile of minimally differentiated (FAB M0) acute Paulsson K, Cazier JB, Macdougall F, Stevens J, Stasevich I, myeloid leukemia: correlation with clinicobiologic findings. Vrcelj N, Chaplin T, Lillington DM, Lister TA, Young BD. Blood. 1995 Jun 15;85(12):3688-94 Microdeletions are a general feature of adult and adolescent Hashimoto K, Miura I, Chyubachi A, Saito M, Miura AB. acute lymphoblastic leukemia: Unexpected similarities with Correlations of chromosome abnormalities with histologic and pediatric disease. Proc Natl Acad Sci U S A. 2008 May immunologic characteristics in 49 patients from Akita, Japan 6;105(18):6708-13 with non-Hodgkin lymphoma. Cancer Genet Cytogenet. 1995 Mitelman F, Johansson B and Mertens F (Eds.).. Mitelman May;81(1):56-65 Database of Chromosome Aberrations and Gene Fusions in Rigolin GM, Cuneo A, Roberti MG, Bardi A, Castoldi G. Cancer (2011). http://cgap.nci.nih.gov/Chromosomes/Mitelman Myelodysplastic syndromes with monocytic component: hematologic and cytogenetic characterization. Haematologica. This article should be referenced as such: 1997 Jan-Feb;82(1):25-30 Huret JL. +20 or trisomy 20 (solely). Atlas Genet Cytogenet Jaing TH, Yang CP, Hung IJ. Acute monoblastic leukemia in a Oncol Haematol. 2011; 15(8):697-698. child following chemotherapy for neuroblastoma. J Formos Med Assoc. 1999 Oct;98(10):688-91

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Deep Insight Section Review

TMPRSS2:ETS gene fusions in prostate cancer Julia L Williams, Maisa Yoshimoto, Alexander H Boag, Jeremy A Squire, Paul C Park Department of Pathology and Molecular Medicine, Queen's University, Kingston, Ontario, Canada (JLW, MY, AHB, JAS), Kingston General Hospital, 76 Stuart Street, Douglas 4, Room 8-431, Kingston, Ontario, K7L 2V7 Canada (PCP)

Published in Atlas Database: December 2010 Online updated version : http://AtlasGeneticsOncology.org/Deep/TMPRSS2ERGinCancerID20091.html DOI: 10.4267/2042/46008 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Table of Contents I. Background I.i. Prostate cancer oncogenomics II. Discovery of TMPRSS2:ETS fusion genes in prostate cancer III. Frequency of TMPRSS2:ETS gene fusions in prostate cancer IV. TMPRSS2:ETS gene fusions: genes and protein structure IV.i. TMPRSS2 and the androgen receptor IV.ii. ETS transcription factors V. Fusion variants VI. Detection and classification VI.i. FISH VI.ii. Other methods of detection VII. Fusion gene formation and chromosomal instability VIII. Heterogeneity of multifocal disease IX. Prognostic significance X. Clinical utility XI. Role of ETS in prostate tumourigenesis: Driver? XII. Concluding remarks

I. Background Clinicopathological criteria, including Gleason grading, are not sufficient to differentiate men whose tumours Prostate cancer (CaP) is the most commonly diagnosed require immediate and aggressive therapy from those male malignancy and a leading cause of cancer deaths that would suffice with vigilant clinical observation, in developed countries. With one in six men diagnosed, thereby causing the latter group enormous amounts of CaP remains a serious global public health issue (Jemal unnecessary treatment (Yao and Lu-Yao, 2002). In this et al., 2008). CaP is a clinically heterogeneous disease, regard, the emerging data on the genetics of CaP hold with manifestations ranging from a rapid and often fatal great promise not only in stratifying this heterogeneous progression, to the typical, indolent disease which group of patients, but also in providing the groundwork remains relatively insignificant to a patient's health. for future development of targeted therapy.

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I. i. Prostate cancer oncogenomics qPCR analysis. Subsequent studies showed that the Advances in cytogenetics and genomics facilitated the frequency of the TMPRSS2:ERG fusion gene (or ERG characterization of common genomic alterations in rearrangement) is exceptionally variable and CaP, which are predominantly characterized by inconsistent in the literature, ranging from 27% to 79% deletions (10q, PTEN; 13q, RB1; 8p, NKX3.1 (a in radical prostatectomy (RP) and biopsy samples, prostate-specific tumour suppressor); 5q; 2q; 17p; and generally from prostate-specific antigen (PSA) less commonly 6q; 7p; 16q; 18q) with only a small screened cohorts (Tomlins et al., 2005; Yoshimoto et number of recurrent gains (8q, MYC; and chromosome al., 2008; Mehra et al., 2007b; Watson et al., 2009; 7). More complex patterns as well as an accumulation Barwick et al., 2010; Magi-Galluzzi et al., 2010). in the number of genomic gains and amplifications Some of the discrepancies in frequency relate to (Xq11.2-q12, androgen receptor (AR)) emerge as the differences in the patient cohort (race as well as global disease advances. Genomic rearrangements leading to geographical location or PSA-screened versus the formation of TMPRSS2:ETS gene fusions and population based) or the type of specimen examined, as deletion of the PTEN tumour suppressor are the two well as the technique used to detect the fusion gene. most frequent alterations observed in CaP (Tomlins et This concept is clearly illustrated when comparing al., 2005; Yoshimoto et al., 2007; Yoshimoto et al., population based cohorts, which have a lower reported 2008). The TMPRSS2:ERG gene fusion is the principle frequency of 15-35%, as compared to RP cohorts genomic alteration and a characteristic signature in (Attard et al., 2008a; Demichelis et al., 2007; Fitzgerald approximately half of prostatic malignancies. et al., 2008). The TMPRSS2:ERG gene fusion is found in approximately half of Caucasian patients, with a II. Discovery of TMPRSS2:ETS lower reported frequency in African-American men and fusion genes in prostate cancer is less common in Asian cohorts (Mosquera et al., The discovery of the TMPRSS2:ERG gene fusion 2009; Magi-Galluzzi et al., 2010; Lee K et al., 2010; exemplifies the current shift in the strategy in cancer Miyagi et al., 2010). An excellent example of genomics from experimental to bioinformatics discrepancies based on patient populations was approaches. By surveying 132 gene-expression CaP demonstrated in a study that compared patients from datasets from the Oncomine database (Compendia the United Kingdom (UK) and China, wherein 41.3% Bioscience) using the data transformation algorithm of the UK patients but only 7.5% of Chinese cohort cancer outlier profile analysis (COPA), Chinnaiyan and were found to harbour ERG rearrangements detected colleagues identified genes with aberrant expression by FISH (Mao et al., 2010). The recent, contradicting profiles in a subset of samples (Tomlins et al., 2005). report that 90% of the Chinese RP specimens COPA allowed the systematic investigation of cancer- harboured ETS rearrangements further emphasize the related genes known to participate in chromosomal multifactorial nature of the variability in the frequency rearrangements in haematological malignancies and of this gene fusion (Sun et al., 2010). sarcomas. Two mutually exclusive Erythroblastosis TMPRSS2:ERG gene fusions are reported in 10-21% virus E26 transformation-specific (ETS) transcription of high-grade prostatic intraepithelial neoplastic factors, ETS variant 1 (ETV1, 7p21.3) and ETS-related (HGPIN) lesions, but are identified almost exclusively gene (ERG, 21q22.2), were identified as high-ranking adjacent to fusion-positive cancer (Cerveira et al., outliers in several independent gene-expression 2006; Perner et al., 2007; Carver et al., 2009b; Han et profiling datasets. Exon-walking quantitative PCR al., 2009; Zhang et al., 2010; Mosquera et al., 2008). (qPCR) of patient samples found the 3' regions of ERG Benign prostatic hyperplasia (BPH) and normal and ETV1 to be overexpressed but the corresponding 5' epithelium are negative for ERG rearrangements and regions were absent. 5' RNA ligase-mediated rapid fusion transcripts, with the exception of the report by amplification of cDNA ends identified the 5' end of Clark and colleagues (Rajput et al., 2007; Wang et al., these transcripts as the promoter sequences belonging 2006; Cerveira et al., 2006; Clark et al., 2007; Perner et to the prostate-specific, androgen regulated al., 2007; Dai et al., 2008; Han et al., 2009; Mosquera transmembrane protease, serine II (TMPRSS2, et al., 2009; Zhang et al., 2010; Sun et al., 2010; Lu et 21q22.3). al., 2009). This study found that eight of 17 (47%) normal epithelial samples adjacent to fusion-positive III. Frequency of TMPRSS2:ETS CaP were also positive for TMPRSS2:ERG gene fusions in prostate cancer rearrangements and found a 6% fusion-positive rate in This breakthrough study reported that 79% of radical BPH samples (Clark et al., 2007). Hormone refractory prostatectomy (RP) samples harboured a fusion of the and/or metastatic CaP exhibits less variability in the 5' untranslated region (UTR) of TMPRSS2 with the occurrence of TMPRSS2:ERG rearrangements with coding sequences of either ERG or ETV1 (Tomlins et reported frequencies ranging from 29-59% (Perner et al., 2005). Interestingly, just months prior, Petrovics et al., 2007; Mehra et al., 2008; Gopalan et al., 2009; Han al. (2005) reported that ERG was the most commonly et al., 2009; Boormans et al., 2010; Saramaki et al., overexpressed oncogene in CaP by microarray and 2008; Attard et al., 2009; Stott et al., 2010). Additionally, minute prostatic adenocarcinoma, a form

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(8) 700 TMPRSS2:ETS gene fusions in prostate cancer Williams JL, et al.

of CaP that is considered less clinically significant, to the growth and development of the prostate gland harbours ETS fusions in approximately half of reported but also play an important role in the initiation and cases, underscoring the necessity of examining all progression of CaP (Balk et al., 2008). It is well prostatic malignancies for aggressive features such as established that despite chemical castration the AR the fusion gene (Albadine et al., 2009). functions to drive CaP, likely through a variety of Investigators have also evaluated this rearrangement in mechanisms such as hypersensitivity to low levels of the peripheral and transitional zones of the prostate. androgens, activation in absence of, or via Nearly half of the peripheral tumours (43.3%) derived unconventional ligands, or amplification of the AR from RP samples were ERG rearrangement positive, gene locus, resulting in elevated levels of AR protein. whereas all 30 corresponding transitional tumours All of these mechanisms could potentially allow AR displayed a normal ERG locus by FISH (Guo et al., activation in the presence of low androgen 2009). In contrast, other studies found TMPRSS2:ERG concentration (Ai et al., 2009; Kawata et al., 2010; Vis (13.3%) and ERG rearrangements (11.9%) are present and Schroder, 2009). Consequently, TMPRSS2 in turn in transitional zone tumours, despite being identified at plays an important role in CaP progression in spite of a lower rate compared to peripheral zone lesions hormonal ablation regimens as its promoter region (Bismar and Trpkov, 2010; Falzarano et al., 2010). drives the expression of the fused ETS gene. These data illustrate the enormous variability in the IV. ii. ETS transcription factors frequency of TMPRSS2:ERG fusion positivity with Twenty-seven human ETS transcription factor family respect to the cohort, disease stage, origin of sample as members have been identified, all of which share a well as the method of detection. conserved DNA binding domain that recognizes unique Interestingly, a comprehensive study of patient samples sequences containing GGA(A/T) (Nye et al., 1992). from 54 tumour types, including sarcomas and ERG (21q22.2) is the ETS transcription factor most haematological malignancies, for ETS gene fusions and commonly known to participate in CaP gene fusions. ERG rearrangements by FISH found these alterations The ERG gene contains 11 exons, with the to be exclusive to CaP samples (Scheble et al., 2010). transcriptional start site in exon 3. The ERG protein can The same result was obtained when RT-PCR was interact with ETS members as well as other performed to detect TMPRSS2:ERG and transcription factors, such as Jun and Fos, through its TMPRSS2:ETV1 fusion transcripts in gastric and protein-protein interacting domain, SAM-PNT (Carrere colorectal carcinomas (Yoo et al., 2007). These et al., 1998; Verger et al., 2001; Basuyaux et al., 1997). findings provide strong evidence that TMPRSS2:ETS The conserved ETS DNA binding domain permits gene fusions are specific to CaP. binding to purine rich DNA sequences (Reddy et al., IV. TMPRSS2:ETS gene fusions: 1991; Reddy et al., 1987), and in this manner, exert its genes and protein structure effects in numerous cellular processes including membrane remodelling, angiogenesis, differentiation, IV. i. TMPRSS2 and the androgen receptor proliferation, and tumourigenesis (Carver et al., 2009a; TMPRSS2, a 70 KDa serine protease family member is Kruse et al., 2009; Oikawa et al., 2003; Randi et al., associated with physiological and pathological 2009; Ellett et al., 2009; Birdsey et al., 2008; processes such as digestion, tissue remodelling, blood Mclaughlin et al., 2001; Sato et al., 2001). Emerging coagulation, fertility, inflammatory responses, tumour evidence suggests that formation of the fusion gene cell invasion and apoptosis. The normal function of this may promote prostatic tumourigenesis, progression, protein is not yet known but is composed of a type II and invasive disease and is associated with CaP-related transmembrane domain, receptor class A low density mortality (Demichelis et al., 2007; Klezovitch et al., lipoprotein domain, scavenger receptor cysteine-rich 2008; Wang et al., 2008; Hawksworth et al., 2010). domain, protease domain, and cytoplasmic domain Functionally, ERG overexpression in CaP is highly (Paoloni-Giacobino et al., 1997). The 32 KDa serine implicated in promoting motility and invasiveness protease domain undergoes autocleaveage, secretion (Perner et al., 2007; Singh et al., 2002; Trojanowska et into the prostate epithelia and interacts with cell surface al., 2000; Tomlins et al., 2008a; Sreekumar et al., 2009; proteins, the extracellular matrix and proteins of Schulz et al., 2010). In CaP, ERG expression has been neighbouring cells (Vaarala et al., 2001; Wilson et al., associated with elevated levels of histone deactylase 1 2005; Afar et al., 2001). The TMPRSS2 (21q22.3) gene (HDAC1) and subsequent down regulation of HDAC1 is composed of 14 exons, with protein coding target genes, upregulation of WNT pathway proteins, sequences only in the latter half. Importantly, and inhibition of apoptotic signalling (Iljin et al., 2006). TMPRSS2 harbours androgen responsive elements HDAC1 upregulation is common in CaP, but was (ARE) in its 5' UTR. Prostate epithelial cells express found to be uniformly increased in ERG rearranged TMPRSS2 at higher levels relative to other tissues, tumours (Iljin et al., 2006; Bjorkman et al., 2008). while TMPRSS2 gene expression is further elevated in Activation of the WNT pathway leads to transcription CaP relative to BPH and normal prostatic epithelium of numerous genes involved in tumourigenesis, (Afar et al., 2001). Androgens and the AR are essential including AR, MYC, JUN, cyclinD1, BMP4 and

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MMP7 (Terry et al., 2006; Schweizer et al., 2008). intensive study examining ETV1 rearrangements found Highlighting the importance of WNT pathway that only 9 of 23 samples had previously identified 5' activation in fusion-positive CaP is the resultant partners, demonstrating the dramatic variability in increase in AR transcription and expression levels. fusion partners pairing with ETS transcription factors Consequently transcription of the fusion gene is other than ERG (Attard et al., 2008b). The combined increased, further amplifying ERG expression. frequency of the remaining fusion variants accounts for Moreover, beta-catenin and the AR interact in an approximately 10% of cases. androgen-dependent manner to regulate AR target Five prime partners are divided into classes based on genes, whereas in androgen-insensitive tumours, both their tissue specificity and sensitivity to androgens beta-catenin and AR target genes are expressed (Tomlins et al., 2007). Class I is reserved for (Schweizer et al., 2008). TMPRSS2; Class II represents other prostate-specific ERG overexpression has recently been shown to androgen inducible 5' UTR or endogenous retroviral activate C-MYC and result in its overexpression. These elements; Class III represents the prostate-specific but experiments revealed that C-MYC is activated by ERG androgen repressed partners; Class IV represents the and together their co-overexpression results in the non-tissue-specific promoters that are ubiquitously suppression of prostate-epithelial differentiation genes expressed, (i.e. house-keeping genes-this class often and shorter time to biochemical recurrence forms a chimeric or fusion protein, unlike the previous (Hawksworth et al., 2010; Sun et al., 2008). ERG can divisions of gene fusions); and finally Class V consists also induce ICAM2 expression, resulting in AKT of ETV1-specific rearrangements, including the activation via PDK1 and subsequent inhibition of BAD localization of the entire ETV1 locus to prostate leading to suppression of apoptotic signals (Mclaughlin specific locus, 14q13.3-14q21.1 (Tomlins et al., 2007; et al., 2001). In addition, ERG can bind BRCA1, a co- Attard et al., 2008b). To date only a single study has activator of the AR, and together were shown to identified fusion genes in CaP devoid of ETS regulate IGFR expression (Chai et al., 2001; Schayek et transcription factor participation (Palanisamy et al., al., 2009). IGFR expression eventually leads to 2010). activation of AKT upon IGFR ligand-binding. ERG regulates MMPs thus influencing extracellular matrix VI. Detection and classification (ECM) remodelling and the invasive potential of the VI. i. FISH cell (Ellett et al., 2009; Hawksworth et al., 2010; Singh Two strategies for FISH experiments are frequently et al., 2002; Schulz et al., 2010). Recently, Carver and used to detect the TMPRSS2:ERG fusion gene in CaP. colleagues have identified ETS binding sites in the The three-colour break-apart strategy, developed by promoter regions of CXCR4 and ADAMTS1, two Yoshimoto et al. (2006), uses differentially labelled genes involved in cellular motility and invasion (Carver bacterial artificial chromosome (BAC) clones as probes et al., 2009a). Furthermore, it was shown that ERG for the 3' (RP11-476D17) and 5' (RP11-95I21) directly upregulates the expression level of CXCR4 and segments of ERG with an additional BAC probe ADAMTS1 (Carver et al., 2009b). Together, these specific for the 5' region of TMPRSS2 (RP11-535H11) studies provide a compelling evidence for a central, or for the transcriptional regulatory sequences functional role of the fusion gene in the biology of (telomeric) of TMPRSS2 (RP11-35C4, RP11-891L10; prostatic carcinoma. RP11-260O11; Figure 1) (Yoshimoto et al., 2006). Using this probe configuration enables not only the V. Fusion variants detection of ERG rearrangement, but also allows The TMPRSS2:ERG fusion gene constitutes the confirmation that ERG's coding sequences are majority (>85%) of ETS rearrangements in CaP, likely juxtaposed to the transcriptional regulatory region of owing to their close proximity (2.7 Mb) and identical TMPRSS2. Moreover, the mechanism of orientation on chromosome 21. Although, the rearrangement can also be deduced by this approach remainder of this review will predominately focus on (Yoshimoto et al., 2006; Yoshimoto et al., 2007). the TMPRSS2:ERG rearrangement, numerous Characterization of rearrangement method is essential alternative ETS members also can fuse to TMPRSS2, as differential clinical impacts are observed with the albeit at a much lower frequency. Similarly, variability various mechanisms resulting in ETS gene fusions. in 5' partners have also been identified (Table 1). An 1Table 1 5' partner Class 3' partner Initial reference TMPRSS2 I ERG Tomlins et al.(2005) TMPRSS2 I ETV1 Tomlins et al. (2005) TMPRSS2 I ETV4 Tomlins et al. (2006) HERV-K_22q11.23 II ETV1 Tomlins et al. (2007)

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SLC45A3 II ETV1 Tomlins et al (2007) C15orf21 III ETV1 Tomlins et al. (2007) HNRPA2B1 IV ETV1 Tomlins et al. (2007) KLK2 II ETV4 Hermans et al. (2008a) CANT1 II ETV4 Hermans et al. (2008a) ACSL3 II ETV1 Attard et al. (2008b) SLC45A3 II ERG Han et al. (2008) FLJ35294 II ETV1 Han et al. (2008) DDX5 IV ETV1 Han et al. (2008) TMPRSS2 I ETV5 Helgeson et al. (2008) SLC45A3 II ETV5 Helgeson et al. (2008) EST14 II ETV1 Hermans et al. (2008b) HERVK17 II ETV1 Hermans et al. (2008b)

FOXP1 II ETV1 Hermans et al. (2008b)

SLC45A3 II ELK4 Rickman et al. (2009)

NDRG1 II ERG Pflueger et al. (2009) SLC45A3 ETS neg BRAF* Palanisamy et al. (2010)

ESRP1 ETS neg RAF1* Palanisamy et al. (2010)

*Only 3' partners identified to date that are not a member of the ETS transcription factor family.

Figure 1: In house BAC probe configuration for three-colour break-apart TMPRSS2:ERG gene fusion FISH This schematic ideogram depicts the positions of differentially labelled bacterial artificial chromosome (BAC) clones specific for 3' and 5' regions of the ERG gene (RP11-476D17 in spectrum orange and RP11-95I21 in spectrum green, respectively), within the 21q22.2-3 region. Telomeric to this, the TMPRSS2 gene locus is represented by RP11-535H11 (spectrum red) which spans the gene, or by three BAC clones downstream (telomeric) of the TMPRSS2 gene (RP11-35C4, RP11-891L10 and RP11-260O11 in spectrum aqua). This probe design permits the accurate identification of TMPRSS2:ERG gene fusions as well as ERG rearrangements independent of fusion with TMPRSS2 fusion.

Work by Attard and colleagues classified the 1-3 and a partial deletion of exon 4, the known TMPRSS2:ERG rearrangement mechanisms according intervening genes (based on RefSeq Genes: to the pattern of interphase FISH signals (Attard et al., NCRNA00114, ETS2, PSMG1, BRWD1, 2008a). Class N describes the normal ERG locus, NCRNA00257, HMGN1, WRB, LCA5L, SH3BGR, therefore co-localization of the two ERG probe signals C21orf88, B3GALT5, IGSF5, PCP4, DSCAM, in close proximity to the TMPRSS2 signal (less than C21orf130, MIR3197, BACE2, PLAC4, FAM3B, one signal diameter) (Figure 2A). The majority of MX2, and MX1) and the coding exons of TMPRSS2. fusion-positive cases present with a heterozygous The three-colour FISH of an Edel rearrangement deletion at cytoband 21q22.2-3, termed Class Edel displays co-localization of the 3' ERG and TMPRSS2 (Figure 2B). The deletion typically spans ERG, exons signals, and absence of the 5' ERG signal. Less

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frequently a genomic rearrangement leading to (Figure 2C). In both types of TMPRSS2:ERG insertion of those sequences elsewhere in the genome rearrangements the unaffected chromosome 21 to an unknown chromosome location can occur generally display a Class N signal configuration. resulting in the separation of the 5' ERG signals from Finally, additional copies of TMPRSS2:ERG gene the co-localization of the 3' ERG and TMPRSS2 fusions is identified as Class 2+Edel (Attard et al., signals, thus described as ERG split or Class Esplit 2008a).

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Figure 2: Classification of TMPRSS2:ERG gene fusion by interphase FISH. Prostate cancer patient samples were hybridized with the three-colour probe set, described in Figure 1, and counterstained with DAPI. The nuclei of interest (in dashed boxes) are magnified in the insets. A) Class N, where no ERG rearrangement has occurred. Co- localization of 3' and 5' ERG probes (often visualized as a yellow signal), with the 5' TMPRSS2 BAC probe signals (red) indicates a normal Chr21q22.2-3 locus. Frequently, the signals for the TMPRSS2 probe, situated 2.7 Mb away from the ERG locus, may be separated from the ERG signals by up to one probe signal width. B) Class Edel is characterized by the co-localization of 3' ERG probe to the TMPRSS2 probe signals, and the absence of the 5' ERG signal. This represents rearrangement with the loss of the intervening sequence. The unaffected Chr21 displays Class N configuration. C) Class Esplit is characterized by the co-localization of the 3' ERG and TMPRSS2 signals, with the retention of the 5' ERG signal elsewhere in the nucleus. The unaffected Chr21 displays Class N configuration.

The second strategy employs a two-colour break-apart protein product by 39-99 amino acids, while the few FISH design to identify rearrangements in specific ETS that initiate translation from the native start codon in genes (Mehra et al., 2007; Zhang et al., 2010). By this exon 3 produce full length ERG (Tomlins et al., 2005; method, confirmation of fusion between two genes and Soller et al., 2006; Wang et al., 2006; Clark et al., identification of the specific 5' partner is not possible 2007; Clark et al., 2008a). One of the transcripts because the probes are specific for a single gene, and produces a genuine TMPRSS2:ERG fusion protein and therefore, this strategy is an indirect method for the eight contain premature stop codons and are unlikely to detection of fusion genes in CaP. result in ERG overexpression (Tomlins et al., 2005; VI.ii. Other methods of detection Soller et al., 2006; Wang et al., 2006; Clark et al., RT-PCR is another technique frequently employed to 2007; Clark et al., 2008a). These studies further determine the fusion status of prostatic tissue samples. revealed that elaborate heterogeneity exists in hybrid However, this approach is limited to the detection of transcripts present, between foci and within individual the hybrid transcripts, and is unable to obtain important tumour foci of the same patient. Variability in the cytogenetic information such as the genomic translation start site consequentially affects the size of mechanism that generated the gene fusion (Edel vs the mRNA transcript (373-885 bp) and therefore the Esplit). To date, as many as 17 fusion transcripts and protein product potentially modifying the functional splice variations have been characterized, the most capacity of the gene fusion product (Wang et al., 2006). common fusion transcript is composed of exon 1 of Distinct transcript variants display differential TMPRSS2 fused to exon 4 of ERG (T1:E4 (Wang et prognostic influence based on the resultant biological al., 2006; Jhavar et al., 2008)). The majority of the activities (Hermans et al., 2009; Wang et al., 2008). A remaining transcripts result in truncation of the ERG significant challenge remains in relating the clinical

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prognosis to the fusion gene and will be addressed in by sequencing, revealed the presence of consensus the subsequent section. sequences homologous to the human Alu-Sq and Alu- Indirect methods suggestive of gene fusions in CaP are Sp subfamily (Liu et al., 2006). The presence of these being employed to broadly determine ETS consensus sequences within intronic regions correlated rearrangement status with perhaps the anticipation of with the presence of the fusion gene and may be a implementing a high-throughput method of detection. factor contributing to the deletion at 21q22.2-3, Analysis of array comparative genomic hybridization resulting in the fusion gene. More recently, genotyping (aCGH) data, specifically the 21q22.2-3 region may of familial CaP revealed that the fusion gene associates permit identification of Class Edel TMPRSS2:ERG with polymorphisms in DNA repair genes, specifically gene fusions (Watson et al., 2009; Ishkanian et al., POLI and ESCO1 (Luedeke et al., 2009). 2009). Class Esplit retains the intervening sequences Using FISH, the AR was shown to induce within the nuclei, in a copy neutral manner and chromosomal proximity of TMPRSS2 and ERG by consequently aCGH cannot identify this rearrangement. binding to the promoter region of TMPRSS2 (Mani et Overexpression of ETS transcription factors by gene al., 2009). Subsequently, LnCaP cells were irradiated to expression microarrays may also imply an ETS induce double-strand breaks inducing the formation of rearrangement has occurred. Due to inconclusive the TMPRSS2:ERG gene fusion upon results, FISH or RT-PCR assays are necessary to dihydrotestosterone stimulation in the previously validate microarray findings pertaining to the ETS gene fusion-negative cell line (Mani et al., 2009). The DNA- fusions. A novel multiplexing technology recently bound AR is also implicated in chromatin architecture developed uses nanostructured microelectrodes modifications that can cause double-strand breaks, integrated onto a chip and has the capability to detect commonly repaired by non homologous end joining disease-specific biomarkers, including differentiation machinery, and may result in the formation of gene of various fusion transcripts (Fang et al., 2009). More fusions (Lin et al., 2009). Also recently, androgen recently, immunohistochemistry (IHC) is also being signalling was shown to recruit topoisomerase II beta explored as a means to detect the fusion by ERG with the AR to the breakpoints resulting protein overexpression, in the absence of confirming TMPRSS2:ERG fusion genes (Haffner et al., 2009). fusion at the genomic or transcript level (Furusato et Undoubtedly, specific nucleotide sequences within the al., 2010; Park et al., 2010). Of the numerous direct 21q22.2-3 region are of great importance and further (three-colour FISH, RT-PCR) and indirect (two-colour elucidation as to their role in the formation of gene FISH, microarrays, IHC) methods employed to fusions is essential. The TMPRSS2:ERG gene fusion is determine fusion status to date, the three-colour FISH an unique model to query sequence level yields the most cytogenetic and genomic information. polymorphisms that may lead to the formation of intra- In addition, to identifying the fusion status and class, chromosomal rearrangements largely due to the close the inherent cell-by-cell analysis addresses the proximity and orientation of the involved genes as well heterogeneous nature of the disease, and provides a as the high rate of recurrence. biological context for such information. Chromosomal instability, defined as the formation of novel chromosome alterations and rearrangements at an VII. Fusion gene formation and elevated rate, compared to normal cells may also be a chromosomal instability factor contributing to the formation of ETS gene There is increasing research interest and effort focusing fusions in CaP. It is well established that deletion of the on the genomic events and attributes that lead to the tumour suppressor PTEN (10q23.31), a common formation of ETS gene fusions. A complex genomic aberration in CaP, results in an elevated level TMPRSS2:ERG rearrangement found in a single of chromosomal instability through activation of AKT. patient was meticulously examined and described by One of sequelae of this change is the phosphorylation Yoshimoto et al. (2007). This patient had a and inhibition of the cell cycle check point kinase 1 microdeletion of the sequences from 5' ERG to and (Chk1), an important kinase preventing cell cycle including the TMPRSS2 coding sequences with a progression in response to DNA damage (Puc et al., concurrent translocation of the region immediately 2005; Sanchez et al., 1997). Furthermore, nuclear telomeric of 5' untranslated TMPRSS2 sequences. A PTEN interacts with kinetochore proteins and induces detailed multicolour FISH assay mapped the region the expression of RAD51, a protein required to reduce between ERG and TMPRSS2, revealing the complexity the incidence of spontaneous double-strand breaks of chromosomal rearrangements that can lead to the (Shen et al., 2007). PTEN deficiency ultimately alters formation of fusion genes in CaP. This case multiple cell cycle checkpoints, which could potentially demonstrates the valuable information available delay DNA damage repair and/or chromosome through the use of complex multicolour FISH assays. segregation (Gupta et al., 2009). Overall, PTEN has a The molecular mechanisms that underlie this recurrent variety of roles in maintaining chromosomal stability translocation are just beginning to be understood. For and integrity, and the recurrent PTEN loss in CaP may example, fine mapping of the deletion breakpoints represent an important trigger in the events leading to located within the ERG and TMPRSS2 loci, followed the formation of TMPRSS2:ETS gene fusions.

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VIII. Heterogeneity of multifocal suggests a trend towards unfavourable factors outcome in CaP progression. Notably, an interesting comparison disease of two studies looking at conservatively managed men CaP, one of the most heterogeneous epithelial had opposing results from an association with CaP- carcinomas, is also notoriously multifocal in nature. As specific death (Demichelis et al., 2007) to no reduction a multifocal disease, CaP permits the investigation and in CaP-specific survival for patients harbouring this comparison of recurrent genomic events within distinct rearrangement (Fitzgerald et al., 2008). foci of individual patients. In 32 RP specimens with The role of deletion (Edel) or retention (Esplit) of spatially distinct foci, ERG rearrangement status was intervening sequences in tumour biology has also been examined using two-colour FISH (Barry et al., 2007). scrutinized with relatively uniform results suggesting Nineteen samples displayed interfocal homogeneity, that Edel is not only the more prevalent mechanism but with 80% of the samples negative for ERG is also associated with more aggressive disease rearrangement. The remaining 13 samples exhibited (Mwamukonda et al., 2010; Attard et al., 2008a; Mehra interfocal heterogeneity but intrafocal homogeneity, et al., 2008). Edel was associated with worse prostate- with two samples harbouring three separate foci each specific survival and shorter time to biochemical with a different rearrangement status: Class N, Edel recurrence than Class Esplit (Yoshimoto et al., 2006; and Esplit. Similarly, whole mount examination of RP Attard et al., 2008a). While, Class 2+Edel was specimens for fusion transcripts yielded multiple fusion associated with lethal disease and significantly worse transcripts in individual CaP foci with an identical clinical outcome, particularly when combined with hybrid transcript profile in the adjacent HGPIN prostate-specific clinicopathological criteria (Furusato et al., 2008). Two other studies examining (Yoshimoto et al., 2006; Attard et al., 2008a). Another fusion transcript variants found different transcript study also demonstrated that Edel rearrangements have hybrids in discrete regions of a single prostate (Wang et a more aggressive tendency since all fusion-positive al., 2006; Clark et al., 2007). Multiple TMPRSS2:ETS androgen-independent metastases had lost 5' ERG gene fusion-positive foci may arise independently and sequences (Mehra et al., 2008) and Edel was associated exhibit interfocal heterogeneity. Investigation of ERG with clinically aggressive features of progression rearrangement in multifocal CaP and corresponding (Perner et al., 2006). These findings are consistent with metastases provides a glimpse of the potential elevated ERG expression and led investigators to biological impact of this aberration in the context of speculate that the loss of 5' ERG is associated with disease progression (Perner et al., 2009). This study aggressive CaP. reported that the metastatic lesion was always positive It must be considered that the commonly deleted 2.7 for ERG rearrangement through the same mechanism Mb between ERG and TMPRSS2 could contain (Edel vs Esplit) as that present in at least one of the important tumour suppressor genes (Yoshimoto et al., prostatic tumour foci (not necessarily the index focus). 2006) and its loss in the Edel rearrangement leads to The authors suggest that this rearrangement may be a haploinsufficiency of a tumour suppressor gene that factor contributing to the development of metastases, underlies the aggressive clinical course. One gene regardless of clinicopathological criteria of the candidate for this effect within this region, HMGN1, a individual foci. nucleosome binding protein has previously been IX. Prognostic significance associated with CaP progression (Birger et al., 2005). In agreement with this view, the Esplit rearrangement There exists appreciable controversy with respect to the has not yet been shown to associate with any particular prognostic significance of the TMPRSS2:ERG gene clinical outcome to date. However, given the low fusion in CaP, with studies suggesting that the fusion frequency of this event (~10% of TMPRSS2:ERG gene has a favourable (Winnes et al., 2007; Petrovics et rearrangements), Esplit rearrangements cannot be al., 2005; Winnes et al., 2007; Saramaki et al., 2008), excluded as a measure of prognosis until further studies unfavourable (Yoshimoto et al., 2008; Mehra et al., are completed (Attard et al., 2008a). 2007a; Perner et al., 2006; Wang et al., 2006; Nam et On the other hand, one investigation demonstrated no al., 2007a; Nam et al., 2007b; Attard et al., 2008a; statistically significant association with Mehra et al., 2008; Reid et al., 2010; Demichelis et al., clinicopathological criteria and Edel or Esplit, but 2007; Rostad et al., 2009; Lapointe et al., 2007a) or no evidence did suggest 2+Edel is a factor contributing to association (Mehra et al., 2008; Yoshimoto et al., 2006; CaP-specific mortality (Fitzgerald et al., 2008). Neill et al., 2007; Fletcher et al., 2008; Dai et al., 2008; Conversely, TMPRSS2:ERG rearrangement alone were Furusato et al., 2008; Darnel et al., 2009; Gopalan et associated with lower histological grade, but no other al., 2009; Rubio-Briones et al., 2010; Fitzgerald et al., clinical features or CaP-specific death (Gopalan et al., 2008; Lee et al., 2010; Sun et al., 2010; Rouzier et al., 2009). However, the same group also found that copy 2008) with clinical outcomes. When no association number increases (CNI) of the ERG locus with or between clinicopathological criteria and the presence of without TMPRSS2:ERG gene fusions was associated the fusion gene was found, it was often attributable to with high grade and advanced stage, while cancers with small sample sizes. The most compelling evidence CNI and 2+Edel rearrangements tended to be more

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clinically aggressive (Fine et al., 2010). While the comprehensive evaluation of biopsy or RP specimens above results are conflicting to the previous studies that providing well-informed judgments on diagnosis, showed no association with extra copies of the prognosis and treatment decisions. unrearranged ERG locus (Attard et al., 2008a), the In an attempt to optimize the clinical utility of this difference may be due to the deletion of the intervening biomarker, investigators are turning to less invasive genes potentially generating a more aggressive survey modes, such as urine specimens and circulating tumourigenic phenotype. tumour cells (CTCs). Post-digital rectal exam (DRE) SPINK1 (5q32) is a gene identified in a more recent urine analyzed by qRT-PCR found 17.2% positive rate COPA meta-analysis as being overexpressed in 10% of for ERG overexpression. Subsequent examination of CaP, all of which are exclusively ETS fusion negative the corresponding biopsy samples from the same cohort (Tomlins et al., 2008b). For this reason, SPINK1 revealed a 40% positive rate (Rice et al., 2010). The expression and the TMPRSS2:ERG fusion gene were clinical yield of this assay can be improved, as evaluated to determine their relative significance as indicated by another study which found a 69% positive prognostic biomarkers in biopsy samples from rate following prostatic message versus only 24% when hormonally treated men (Leinonen et al., 2010). In this no prostatic message was performed before collecting cohort, the fusion gene was associated with Ki-67 the urine samples (Rostad et al., 2009). Two additional staining, age at diagnosis and tumour area, but not with studies report similar results (42% and 59%) of fusion- any prostate-specific clinicopathological criteria positive transcripts in post-DRE urine (Hessels et al., (Leinonen et al., 2010). On the other hand, cases that 2007; Laxman et al., 2006). Expressed prostatic overexpressed SPINK1 had a significantly shorter time secretion has also been a successful specimen for the to biochemical recurrence, but no association with any non-invasive detection of fusion transcripts (Clark et other criteria. al., 2008b). These studies demonstrate the potential for X. Clinical utility ETS gene fusion detection using non-invasive approaches following physical evaluation of the Promptly following the emergence of adverse clinical prostate by DRE, adding valuable information to correlations of fusion-positive CaP Mosquera and disease stratification prior to radical treatments. A colleagues evaluated a series of CaP cases to determine handful of studies have also used FISH to detect ERG if there was a morphological phenotype associated with rearrangement in CTCs in effort to monitor disease this genomic alteration (Mosquera et al., 2007). recurrence or treatment efficacy (Mao et al., 2008; Blinded to ERG rearrangement status, the group Attard et al., 2009; Stott et al., 2010). identified five histologic criteria that were significantly related to ERG rearrangement positive samples: blue- XI. Role of ETS in prostate tinged mucin, cribriform growth, macronucleoli, tumourigenesis: Driver? intraductal tumour spread, and signet-ring cell features. Discrepancies regarding the fusion gene are not limited Only 24% of ERG rearranged samples did not identify to its diagnostic potential and prognostic significance. with any of the above mentioned morphological Controversy also exists in the sequence of genomic and features and 93% of samples with three features were molecular alterations in CaP initiation and progression. positive for ERG rearrangement. The authors speculate Several groups speculate that the formation of the that the morphological characteristics shared by ERG TMPRSS2:ERG fusion gene is required for CaP rearranged tumours could result from ETS dysregulated initiation in ETS positive CaP tumours, with other pathways and could be utilized in the routine aberrations occuring later in the course of disease assessment performed by pathologists. More recently, advancement and metastasis (Perner et al., 2007; the same group published a similar set of Tomlins et al., 2007; Attard et al., 2009). Recently, morphological features with the addition of collagenous Attard and colleagues demonstrated that CTCs from micronodules (Mosquera et al., 2009). A significant individual castration-resistant CaP patients were either association between perineural invasion, blue-tinged clonally positive or negative for ERG rearrangement, mucin, and intraductal tumor spread with a positive however, loss of PTEN and increase in AR gene copy gene fusion status has been documented (Nigwekar et number was heterogeneous in the CTCs derived from a al., 2008), while another study confirmed the single patient (Attard et al., 2009). These results association that ETS fusion-positive CaP samples were suggest that the formation of the fusion gene occurred more likely mucin-positive than mucin-negative (Tu et before PTEN loss and gain of the AR locus. However, al., 2007). These results should be taken, however, in this study was performed on a very small cohort of the context of somewhat contradictory reports, such castration-resistant CaP and may not necessarily reflect that the TMPRSS2:ERG gene fusion correlates with the sequence of accumulating genomic alterations in low Gleason grade and is inversely related to high- CaP. grade morphological features. These studies hold TMPRSS2:ETS fusion status in HGPIN occurs at a low promise that morphological markers routinely frequency, and almost exclusively only when examined by pathologists could be coupled with the juxtaposed to fusion-positive CaP. These HGPIN current clinicopathological criteria for a more lesions, however, do not exhibit the chromosomal copy

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number changes seen in 42% of the paired CaP as factors downstream of AKT as a result of PTEN assessed by CGH (Cerveira et al., 2006). These results deficiency, but on their own are not sufficient to propose that formation of the fusion gene may precede provoke the transition from benign to neoplastic. The chromosomal-level alterations and is consistent with implication is not that PTEN loss is the driver in CaP, the literature indicating that few gross chromosome- or but that ETS gene fusions are enhancer alterations arm-level chromosomal copy alterations are present in significantly affecting cellular processes further localized CaP. Another study investigating a range of progressing prostatic tumourigenesis when the prostate tissues comprising benign, precursor, background is primed first by a driver event capable of malignant and metastatic samples found that the initiating sufficient dysregulation leading to the majority of patient samples examined showed development of a preneoplastic lesion. However, homogeneity with respect to fusion status and because PTEN loss and the presence of the fusion gene mechanism (Edel vs Esplit). The presence of fusion- are significantly associated events in CaP (Yoshimoto positive HGPIN was interpreted as additional evidence et al., 2008; Carver et al., 2009a; Han et al., 2009; King that this entity is a true-precursor to CaP, a notion that et al., 2009; Bismar et al., 2010) it is likely that PTEN has eluded indisputable proof (Perner et al., 2007). In inactivation may be an important driver lesion for contrast, a recent study that examined both PTEN fusion-positive CaP. Together the driver event and ETS genomic loss and fusion status by FISH observed overexpression lead to a significantly aggressive and PTEN loss in benign and HGPIN lesions, while ERG invasive lesion. Therefore, elucidation of the cellular rearrangement was identified in HGPIN, albeit at a pathways affected by ETS overexpression is lower frequency than PTEN loss, and absent in BPH fundamental to the comprehension of the aggressive lesions (Bismar et al., 2010). Maintaining this model of nature observed in the majority of fusion-positive CaP, progression and accumulation of genomic changes in and to developing novel therapeutic strategies to CaP it is possible that heterozygous loss of PTEN may specifically target this subset of CaP. be a 'driver lesion', in a subset of CaP, and PTEN XII. Concluding remarks haploinsufficiency may facilitate the selective Difficulty in assigning prognostic significance, formation of the fusion gene (Figure 3). diagnostic and therapeutic utility to ETS gene fusions is TMPRSS2:ERG gene fusion may occur as a secondary a result of a myriad of factors including, the alteration and may function as an 'enhancer' permitting heterogeneity of the disease, the mechanism of the cell to achieve a higher level of aggressiveness and rearrangement (Edel vs Esplit), the technique used to invasiveness. When this sequence of events was assess the presence of the fusion as well as the cohort emulated in transgenic mice, ERG overexpression examined. Nevertheless, continued controversy resulted in marked acceleration of preneoplastic lesions between positive and negative clinical associations of to invasive CaP on a Pten deleted background, but ERG the gene fusion dictates further studies are required overexpression alone simply displayed slight atypical using larger cohorts to determine the absolute potential histology compared to control mice (Carver et al., of this genomic aberration as a biomarker for CaP 2009a; Carver et al., 2009b). In vitro experiments using diagnostic utility, prognostic significance, and cell lines demonstrated ERG overexpression provided stratification of patients to aid in treatment decisions. enhanced motility without affecting proliferation, in Furthermore, comprehension of the pathways affected agreement with Tomlins et al. (2007); Carver et al. by ETS overexpression will aid in the potential of (2009b). These findings corroborate the view that implementing ETS specific therapies to target this effectors of ERG overexpression affect cellular aggressive subtype of CaP and benefit many patients. processes which are complimentary to unregulated

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Figure 3: Model of prostate cancer progression showing ETS gene fusions as an enhancer lesion. Cooperation of unregulated pathways downstream of PTEN with effectors of ERG overexpression is likely a crucial event in the progression of an invasive and aggressive prostatic adenocarcinoma. Heterozygous genomic deletion of PTEN in benign prostatic precursors may represent an early event, and act as a driver lesion leading to proliferation, survival and genomic instability-all initial requisites of cancer. As a consequence of such heightened genomic instability, PTEN haploinsufficiency may facilitate the selective formation of the fusion gene with consequent acquisition of additional invasive properties. The presence of both rearrangements within a lesion is associated with accelerated disease progression and poor prognosis, indicating that synergistic molecular interactions exist between their complementary pathways. Continuing instability generates genotypic heterogeneity and diversity, such that subclones bearing PTEN homozygous deletions and amplified AR loci have further selective advantage for aggressive tumour progression, androgen escape and metastases.

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