Number 3 March 2012

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Volume 16 - Number 3 March 2012

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

Editorial correspondance

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

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

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

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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

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Volume 16, Number 3, March 2012

Table of contents

Editorial

Why breast cancer and prostate cancer are so frequent? A new genetic mechanism, involving hormones and viruses 187 Jean-Loup Huret

Gene Section

CUX1 (cut-like homeobox 1) 191 Benjamin Kühnemuth, Patrick Michl DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) 196 June L Traicoff, Stephen M Hewitt, Joon-Yong Chung MYEOV (myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas)) 205 Jérôme Moreaux PCNA (proliferating cell nuclear antigen) 208 Ivaylo Stoimenov, Thomas Helleday RASSF5 (Ras association (RalGDS/AF-6) domain family member 5) 212 Lee Schmidt, Geoffrey J Clark RGS17 (regulator of G-protein signaling 17) 216 Chenguang Li, Lei Wang, Yihua Sun, Haiquan Chen SLC39A1 (solute carrier family 39 (zinc transporter), member 1) 218 Renty B Franklin, Leslie C Costello CBX7 (chromobox homolog 7) 220 Ana O'Loghlen, Jesus Gil RPRM (reprimo, TP53 dependent G2 arrest mediator candidate) 223 Alejandro H Corvalan, Veronica A Torres VMP1 (vacuole membrane protein 1) 225 Alejandro Ropolo, Andrea Lo Ré, María Inés Vaccaro XPO1 (exportin 1 (CRM1 homolog, yeast)) 228 Alessandra Ruggiero, Maria Giubettini, Patrizia Lavia

Leukaemia Section t(11;18)(p15;q12) 233 Jean-Loup Huret t(11;21)(q21;q22) 234 Jean-Loup Huret t(8;17)(q24;q22) ???BCL3/MYC 236 Jean-Loup Huret

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 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

Deep Insight Section

Plasticity and Tumorigenicity 238 Elena Campos-Sanchez, Isidro Sanchez-Garcia, Cesar Cobaleda Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function 252 Xiaodong Lu, Wenxin Qin

Case Report Section

A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) 260 Jill D Kremer, Anwar N Mohamed Insertion as an alternative mechanism of CBFB-MYH11 gene fusion in a new case of acute myeloid leukemia with an abnormal chromosome 16 263 Yaser Hussein, Vandana Kulkarni, Anwar N Mohamed

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) Atlas of Genetics and Cytogenetics in Oncology and Haematology

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Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Editorial

Why breast cancer and prostate cancer are so frequent? A new genetic mechanism, involving hormones and viruses Jean-Loup Huret Atlas of Genetics and Cytogenetics in Oncology and Haematology Unit, University of Poitiers, Department of Medical Information, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

This work was presented at the 8th European Cytogenetics Conference, Porto, 2-5 July 2011

Abbreviated title: Hormones and viruses in breast and prostate cancers

Abstract Prostate and breast cancers, which are hormone-dependant cancers, are highly frequent (up to 1/3 of cancers in male, 1/3 of cancers in female patients) and often multifocal. Multifocality, in particular, rings the bell of a specific carcinogenetic agent (such as heritability is in retinoblastoma). Here we point a highly uneven distribution of genetic events (translocation breakpoints) in prostate and breast cancers, which favours the hypothesis of cooperation between viruses and hormone receptors to cut DNA at high rates, delete parts of it, facilitating oncogene translocations and oncogenesis. If our hypothesis turns out to be right, vaccination against breast cancer and prostate cancer might notably diminish the frequency of these cancers.

Looking at chromosomal rearrangements in prostate Such a non random close proximity of the two partner adenocarcinoma, using the Atlas of Genetics and breakpoints is even more striking at the base level (e.g. Cytogenetics in Oncology and Haematology (Huret et 6 kb in the 5q31-5q31 rearrangement; see Table 1). al., 2003), the Mitelman Database (Mitelman et al., The situation is very similar with breast 2012), and Goldenpath (Fujita et al., 2011) adenocarcinoma: Of 39 rearrangements, 20 have (http://atlasgeneticsoncology.org/, http://cgap.nci. breakpoints in close proximity (Table 2). The nih.gov/Chromosomes/Mitelman, and probability of such an event is p=6 x 10 -39 . We herein http://genome.ucsc.edu/ respectively), we noted that, uncover a highly significant non random distribution of out of 42 relevant rearrangements available in early breakpoints in breast and prostate cancer DNA 2011, 10 exhibited the two partner breakpoints in the rearragements and microdeletions (by comparison, only same chromosome band (e.g. 5q31 fused to 5q31). 1 of 10 rearrangements in lung adenocarcinoma exhibit Given that 312 chromosome bands are at risk of both breakpoints in the same band (R3HDM2/NFE2 in rearrangement, the probability of the observed the del(12)(q13q13)). A bias of publications may exist, distribution is p=1.5 x 10 -16 (binomial distribution). but cannot account for such a highly unexpected finding.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 187 Why breast cancer and prostate cancer are so frequent? A new genetic mechanism, involving hormones and viruses Huret JL

Table 1. Distance in base pairs in rearrangements with the two breakpoints on the same chromosomal band in prostate adenocarcinoma fusion gene rearrangement coordinates 1st gene coordinates 2nd gene distance WDR55/DND1 t(5;5)(q31;q31) 140044384 140050382 6 kb MBTPS2/YY2 t(X;X)(p22;p22) 21857656 21874603 17 kb ZNF649/ZNF577 t(19;19)(q13;q13) 52392489 52374553 18 kb C19ORF25/APC2 t(19;19)(p13;p13) 1473201 1450148 23 kb SLC45A3/ELK4 t(1;1)(q32;q32) 205626981 205588398 39 kb USP10/ZDHHC7 del(16)(q24q24) 84733555 85008067 275 kb RERE/PIK3CD del(1)(p36p36) 8412466 9711790 1,3 Mb HJURP/EIF4E2 t(2;2)(q37;q37) 234745487 233415357 1,3 Mb TMPRSS2/ERG t(21;21)(q22;q22) 42836479 39751952 3 Mb PIK3C2A/TEAD1 del(11)(p15p15) 17108126 12695969 4,4 Mb Note. These fusion genes were first described in Tomlins et al., 2005; Maher et al., 2009a; et al., 2009b; Rickman et al., 2009. Table 2. Distance in base pairs in rearrangements with the two breakpoints on the same chromosomal band in breast adenocarcinoma fusion gene rearrangement coordinates 1st gene coordinates 2nd gene distance EFTUD2/KIF18B del(17)(q21q21) 42927655 43003449 76 kb PLXND1/TMCC1 del(3)(q22q22) 129274056 129366637 93 kb PAPOLA/AK7 del(14)(q32q32) 96968720 96858448 110 kb SEPT8/AFF4 del(5)(q31q31) 132086509 132211072 125 kb SLC26A6/PRKAR2A del(3)(p21p21) 48663158 48788093 125 kb AC141586/CCNF del(16)(p13p13) 2653351 2479395 174 kb ERO1L/FERMT2 del(14)(q22q22) 53108607 53323990 215 kb HN1/USH1G del(17)(q25q25) 73131344 72912176 219 kb HMGXB3/PPARGC1B del(5)(q32q32) 149380169 149109864 270 kb INTS4/GAB2 del(11)(q14q14) 77589768 77926343 337 kb PLA2R1/RBMS1 del(2)(q24q24) 160798012 161128663 330 kb RASA2/ACPL2 del(3)(q23q23) 141205926 140950682 255 kb BC017255/TMEM49 t(17;17)(q22;q23) 57183959 57784863 600 kb LDHC/SERGEF del(11)(p15p15) 18433853 17809599 624 kb KCNQ5/RIMS1 del(6)(q13q13) 73331571 72596650 735 kb MYO9B/FCHO1 del(19)(p13p13) 17186591 17858527 672 kb STRADB/NOP58 del(2)(q33q33) 202316392 203130515 814 kb SMYD3/ZNF695 del(1)(q44q44) 245912645 247148625 1,2 Mb CYTH1/PRPSAP1 del(17)(q25q25) 76670131 74306868 2,4 Mb RAF1/DAZL t(3;3)(p24;p25) 12625102 16628303 4 Mb Note. These fusion genes were first described in Maher et al., 2009b; Stephens, P.J. et al., 2009.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 188 Why breast cancer and prostate cancer are so frequent? A new genetic mechanism, involving hormones and viruses Huret JL

Prostate and breast share two specificities: high cooperation of viruses and hormone receptors (together frequency of cancer, cancers that are frequently with other factors: genotoxic stress, inflammation...) to multifocal. Prostate cancer represents up to 1/3 of cut and saw DNA would greatly enhance the risk for an cancers in men, breast cancer 1/3 of cancers in female oncogenic event to occur. patients in western countries. Prostate cancer is a There is no reason a priori for an organ to fall "too" multicentric tumour in 75-80% of cases. At autopsy, up frequently into a cancerous process in the absence of an to 30 to 70 % of men aged 70-80 years have cancerous additional carcinogenetic agent. In skin cancer, foci in the prostate; after 80, 90% have hyperplasia, and ultraviolet radiation is the known major helper, and more than 70% have a neoplastic disease. Breast lung cancer is a rare cancer without a history of adenocarcinoma is a multifocal tumour in at least 10- smoking. The "abnormally" high frequency of prostate 15% of cases. Multifocality, in particular, rings the bell and breast cancers may well be due to this association of a specific "helper" carcinogenetic agent (such as in crime of viruses and hormone receptors. heritability in retinoblastoma). If our hypothesis turns to be right, inhibitors for Androgen and estrogen receptors are crucial for the androgens/estrogens receptors and vaccination against normal development as well as for cancer progression breast cancer prostate cancer (as it is now available for of the target organs, respectively the androgen receptor cervix cancer / papilloma virus) might abrogate an (AR) for the prostate and the estrogen receptor (ER) for important proportion of DSBs, and notably diminish the breast. Hormone receptors bind DNA at specific the frequency of these cancers and/or facilitate their motifs called hormone responsive elements: AR binds cure. to a specific TGT/AGGGA/T motif and ER binds to the consensus core element AGGGTCA. References Some retroviruses, such as the mouse mammary Cato AC, Henderson D, Pont H. The hormone response tumour virus (MMTV) and its human homolog HMTV element of the mouse mammary tumour virus DNA mediates contain hormone responsive elements (Cato et al., the progestin and androgen induction of transcription in the 1987; Pogo et al., 2010). A retrovirus containing proviral long terminal repeat region. EMBO J 1987; 6:363-638. androgen response elements remains to be found, since Fujita PA, Rhead B, Zweig AS, Hinrichs AS, Karolchik D, Cline the candidate, XMRV, appears now to be of MS, Goldman M, Barber GP, Clawson H, Coelho A, Diekhans experimental recombinant origin (Paprotka et al., M, Dreszer TR, Giardine BM, Harte RA, Hillman-Jackson J, 2011). Hsu F, Kirkup V, Kuhn RM, Learned K, Li CH, Meyer LR, Pohl A, Raney BJ, Rosenbloom KR, Smith KE, Haussler D, Kent Both androgen and estrogen receptors induce DNA WJ. The UCSC Genome Browser database: update 2011. double strands breaks (DSBs) (Lin et al., 2009, Nucleic Acids Res 2011; 39:D876-882 Williamson and Lees-Miller, 2011); such DSBs can Haffner MC, Aryee MJ, Toubaji A, Esopi DM, Albadine R, seed the formation of genomic/chromosome Gurel B, Isaacs WB, Bova GS, Liu W, Xu J, Meeker AK, Netto rearrangements, and, at random, the possible junction G, De Marzo AM, Nelson WG, Yegnasubramanian S. of oncogene loci normally separated (Lin et al., 2009, Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Nat Genet 2010; Mani et al., 2009). It has been found that androgen 42:668-675. receptor signaling and topoisomerase II mediate DSBs and TMPRSS2-ERG rearrangements in prostate cancer Huret JL, Dessen P, Bernheim A. An Internet database on genetics in oncology. Oncogene 2003; 22:1907. (Haffner et al., 2010). In case of a TMPRSS2-ERG rearrangement, a deletion of 3 mb occurs (Table 1). Lawson JS, Glenn WK, Salmons B, Ye Y, Heng B, Moody P, Johal H, Rawlinson WD, Delprado W, Lutze-Mann L, Whitaker HMTV is found in 40% of breast cancers in American NJ. Mouse mammary tumor virus-like sequences in human women, in 60% of milk from patients with a history of breast cancer. Cancer Res 2010; 70:3576-3585. breast cancer and in 5% of milk from normal subjects Lin C, Yang L, Tanasa B, Hutt K, Ju BG, Ohgi K, Zhang J, (cited in Pogo et al., 2010). HMTV/MMTV sequences Rose DW, Fu XD, Glass CK, Rosenfeld MG. Nuclear receptor- and enhanced Wnt-1 expression were found in breast induced chromosomal proximity and DNA breaks underlie ductal carcinoma (Lawson et al., 2010). However, other specific translocations in cancer. Cell 2009; 139:1069-1083. viruses may be implicated, and it must be kept in mind Maher CA, Kumar-Sinha C, Cao X, Kalyana-Sundaram S, Han that HBV, HPV, EBV, CMV have also been found to B, Jing X, Sam L, Barrette T, Palanisamy N, Chinnaiyan AM. be associated with breast cancer. Transcriptome sequencing to detect gene fusions in cancer. We Hypothesize that HMTV and other viruses can Nature 2009a; 458:97-101. integrate in the cell genome of breast or prostatic cells Maher CA, Palanisamy N, Brenner JC, Cao X, Kalyana- as proviruses in multiple sites, at random. Hormone Sundaram S, Luo S, Khrebtukova I, Barrette TR, Grasso C, Yu J, Lonigro RJ, Schroth G, Kumar-Sinha C, Chinnaiyan AM. receptors (ER or AR) would bind DNA at hormone Chimeric transcript discovery by paired-end transcriptome responsive elements sites, including those added in sequencing. Proc Natl Acad Sci USA 2009b; 106:12353- numerous copies by the proviruses. Hormone receptors 12358. would induce DNA breaks, as usually, DNA deletions Mani RS, Tomlins SA, Callahan K, Ghosh A, Nyati MK, would occur in a certain percentage of cases, Varambally S, Palanisamy N, Chinnaiyan AM. Induced facilitating oncogene translocations. Not all breaks, not chromosomal proximity and gene fusions in prostate cancer. all fusion genes are pathogenetically significant, but the Science 2009; 326:1230-1232.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 189 Why breast cancer and prostate cancer are so frequent? A new genetic mechanism, involving hormones and viruses Huret JL

Mitelman F, Johansson B. and Mertens F. (Eds.). 2011. AM, Martens JW, Silver DP, Langerød A, Russnes HE, Mitelman Database of Chromosome Aberrations and Gene Foekens JA, Reis-Filho JS, van 't Veer L, Richardson AL, Fusions in Cancer. Børresen-Dale AL, Campbell PJ, Futreal PA, Stratton MR. Complex landscapes of somatic rearrangement in human Paprotka T, Delviks-Frankenberry KA, Cingöz O, Martinez A, breast cancer genomes. Nature 2009; 462:1005-1010. Kung HJ, Tepper CG, Hu WS, Fivash MJ Jr, Coffin JM, Pathak VK. Recombinant Origin of the Retrovirus XMRV. Science Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra 2011; 333:97-101. R, Sun XW, Varambally S, Cao X, Tchinda J, Kuefer R, Lee C, Montie JE, Shah RB, Pienta KJ, Rubin MA, Chinnaiyan AM. Pogo BG, Holland JF, Levine PH. Human mammary tumor Recurrent fusion of TMPRSS2 and ETS transcription factor virus in inflammatory breast cancer. Cancer 2010; 116:2741- genes in prostate cancer. Science 2005; 310:644-648. 2744. Williamson LM, Lees-Miller SP. Estrogen receptor α-mediated Rickman DS, Pflueger D, Moss B, VanDoren VE, Chen CX, de transcription induces cell cycle-dependent DNA double-strand la Taille A, Kuefer R, Tewari AK, Setlur SR, Demichelis F, breaks. Carcinogenesis 2011; 32:279-285. Rubin MA. SLC45A3-ELK4 is a novel and frequent erythroblast transformation-specific fusion transcript in prostate cancer. This article should be referenced as such: Cancer Res 2009; 69: 2734-2738. Huret JL. Why breast cancer and prostate cancer are so Stephens PJ, McBride DJ, Lin ML,Varela I, Pleasance ED, frequent? A new genetic mechanism, involving hormones and Simpson JT, Stebbings LA, Leroy C, Edkins S, Mudie LJ, viruses. Atlas Genet Cytogenet Oncol Haematol. 2012; Greenman CD, Jia M, Latimer C, Teague JW, Lau KW, Burton 16(3):187-190. J, Quail MA, Swerdlow H, Churcher C, Natrajan R, Sieuwerts

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Gene Section Review

CUX1 (cut-like homeobox 1) Benjamin Kühnemuth, Patrick Michl Department of Gastroenterology and Endocrinology, University of Marburg, Marburg, Germany (BK, PM)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/CUX1ID403ch7q22.html DOI: 10.4267/2042/47275 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Protein Other names: CASP, CDP, CDP/Cut, CDP1, COY1, Description CUTL1, CUX, Clox, Cux/CDP, FLJ31745, GOLIM6, The human full length CUX1 protein (p200) consists of Nbla10317, p100, p110, p200, p75 1505 amino acids and contains four DNA binding HGNC (Hugo): CUX1 domains: three CUT-repeats and one CUT- Location: 7q22.1 homeodomain (Harada et al., 1994). Several shortened CUX1 isoforms have been described DNA/RNA that are named according to their molecular weight. CUX1 p75 is the product of a shortened mRNA that is Description generated by the use of an alternative transcription start The human CUX1 gene is located on chromosome site in exon 20 (Rong Zeng et al., 2000; Goulet et al., 7q22 (Scherer et al., 1993). It comprises 33 exons and 2002). CUX1 p150, p110, p90 and p80 are generated spans 468 kb. by proteolytic processing of the full length protein by a Five alternative splice variants have been identified. nuclear isoform of Cathepsin L and other not yet Most of the splicing sites are located in the regions identified proteases such as caspases (Goulet et al., downstream of exon 14 and 15 (Rong Zeng et al., 2004; Goulet et al., 2006; Maitra et al., 2006; Truscott 2000). Two alternative sites for transcript termination et al., 2007). have been identified. Termination at UGA in exon 24 The presence of DNA binding domains in the CUX1 leads to production of CUX1 mRNA comprising exon isoforms determines their interaction with DNA and 1-24. Elongation up to exon 33 results in alternative their transcriptional activity. The full length protein splicing and the production of CASP mRNA p200 shows unstable DNA binding, carries the comprising exon 1-15 and 25-33 (Lievens et al., 1997; CCAAT-displacement activity and functions Rong Zeng et al., 2000). predominantly as a transcriptional repressor. In The first transcriptional start site is located in exon 1 contrast, the p110, p90, p80 and p75 isoforms show but transcription can be initiated at several sites in a stable DNA binding and function both as 200 bp region upstream of exon 1 (Rong Zeng et al., transcriptional repressors or activators (Truscott et al., 2000). Initiation within intron 20 leads to production of 2004; Goulet et al., 2002; Goulet et al., 2006; Moon et an mRNA coding for the shortened p75 isoform al., 2001). According to Maitra et al., the p150 isoform (Goulet et al., 2002). is incapable of DNA binding (Maitra et al., 2006). Several putative translation initiation codons can be Several posttranslational modifications are known to found in exon 1 but ATG at position 550 has been modulate the DNA binding activities of the CUX1 described as the predominant initiation site (Rong Zeng proteins. et al., 2000).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 191 CUX1 (cut-like homeobox 1) Kühnemuth B, Michl P

Cux1 isoforms. The p75 isoform is the product of a shortened mRNA that is generated by the use of an alternative transcriptional start site. In contrast, the p150, p110, p90 and p80 isoforms are produced by proteolytic processing of the full length protein (p200). CR = cut repeat; HD = homeodomain.

Protein kinase C and Casein kinase II are able to Localisation phosphorylate serine or threonine residues within the cut repeats (Coqueret et al., 1998b; Li et al., 2007). Studies indicate that phosphorylated CUX1 is Protein kinase A and cyclin A/Cdk1 phosphorylate preferentially localized in the cytoplasm whereas specific serine residues in a region between the Cut dephosphorylation leads to translocation into the repeat 3 and the homeodomain (Michl et al., 2006; nucleus (Sansregret et al., 2010). Santaguida et al., 2001). PCAF acetyl-transferase is Function able to acetylate CUX1 on a lysine residue in the The vast majority of studies describes CUX1 as a homeodomain (Li et al., 2000). Both, phosphorylation transcriptional repressor (Lievens et al., 1995; Ai et al., and acetylation have been shown to inhibit CUX1 DNA 1999; Catt et al., 1999a; Catt et al., 1999b; Ueda et al., binding (Sansregret et al., 2010; Li et al., 2000). 2007). The repressor activity can be mediated by Consistent with this, dephosphorylation by Cdc 25A competition for DNA binding sites with transcriptional phosphatase is able to increase DNA binding of CUX1 activators (Kim et al., 1997; Stünkel et al., 2000), by (Coqueret et al, 1998a). recruitment of histone deacetylases (Li et al., 1999) or Expression by recruitment of histone lysine methyltransferases Early studies suggested that in mammalian cells, CUX1 (Nishio and Walsh, 2004). CUX1 may also negatively represses genes that are upregulated in differentiated regulate gene expression by binding to matrix tissues. Furthermore, the expression of CUX1 might be attachment regions and by modulating their association restricted to proliferating and undifferentiated cells and with the nuclear scaffold (Banan et al., 1997; Stünkel et is inversely related to the degree of differentiation al., 2000; Goebel et al., 2002; Kaul-Ghanekar et al., (vanden Heuvel et al., 1996; Pattison et al., 1997; van 2004). In contrast, the mechanisms underlying its Gurp et al., 1999). More recently however, studies in effects on transcriptional activation are less well mice revealed that CUX1 is also expressed in understood. terminally differentiated cells of many tissues (Khanna- CUX1 is involved in at least three cellular processes Gupta et al., 2001; Ellis et al., 2001). important for cancer progression: cell proliferation, cell Increased CUX1 expression was found in various motility/invasiveness and apoptosis. tumour types including multiple myelomas, acute Proliferation lymphoblastic leukaemia, breast carcinoma and Studies indicate that the pro-proliferative effects of pancreatic cancer (De Vos et al., 2002; Tsutsumi et al., CUX1 are mainly mediated by the p110 isoform. This 2003; Michl et al., 2005; Ripka et al., 2007). isoform is produced by proteolytic cleavage of the full It has been shown that the cellular expression of CUX1 length protein occuring during G1/S-transition in the mRNA and protein is elevated following TGF-beta cell cycle (Goulet et al., 2004; Moon et al., 2001). Cells stimulation in many cell types including fibroblasts, stably transfected with p110 CUX1 showed increased pancreatic cancer cells, breast cancer cells and proliferation due to a shortened G1-phase whereas malignant plasma cells (Fragiadaki et al., 2011; Michl embryonic fibroblasts obtained from CUX1 knockout et al., 2005; De Vos et al., 2002). This regulation of mice showed elongated G1-phase and less proliferation CUX1 expression by TGF-beta is probably mediated compared to cells isolated from wild-type mice by p38MAPK and Smad4 signalling (Michl et al., (Sansregret et al., 2006). 2005).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 192 CUX1 (cut-like homeobox 1) Kühnemuth B, Michl P

A genome-wide location array for p110 CUX1 binding et al., 1992; Valarché et al., 1993). In humans, a sites in transformed and non-transformed cell lines homologue gene, called CUX2, was described identified numerous CUX1 target genes that are related (Jacobsen et al., 2001). to proliferation and cell cycle progression (Harada et al., 2008). Most of these genes are activated by p110 Mutations CUX1 including DNA polymerase-alpha, cyclin A2 and cyclin E2. In contrast, other genes are repressed Note such as the CDK-inhibitor p21 (Truscott et al., 2003; A missense mutation affecting the homeodomain has Nishio and Walsh, 2004; Harada et al., 2008). been described in one patient suffering from acute Cell motility myeloid leukaemia, the significance of which remains First evidence that CUX1 plays a role in cell motility to be elucidated (Thoennissen et al., 2011). originates from knockdown studies in fibroblasts and a panel of human cancer cell lines that revealed that Implicated in depletion of CUX1 leads to decreased cell migration and invasion (Michl et al., 2005). In agreement with Pancreatic cancer this, cells stably expressing p110 and p75 CUX1 show Note increased cell migration and invasion (Kedinger et al., In pancreatic cancer CUX1 expression is elevated 2009; Cadieux et al., 2009). Additionally, tail vein compared to normal pancreas tissue (Ripka et al., injection of cells stably expressing shRNA against 2010a). Furthermore, an increased expression in high- CUX1 resulted in reduced formation of lung grade tumours compared to low grade tumours was metastases, whereas injection of cells stably described (Michl et al., 2005). overexpressing CUX1 led to increased lung metastases The expression of CUX1 is accompanied by the (Michl et al., 2005; Cadieux et al., 2009). overexpression of its downstream targets WNT5a and The molecular basis for these effects on cell motility GRIA3 that, at least in part, mediate the proinvasive was in part elucidated in a genome-wide location and proproliferative effects of CUX1 (Ripka et al., analysis in several cell lines (Kedinger et al., 2009). In 2006; Ripka et al., 2010b). this study, CUX1 was found to inhibit the expression of Antiapoptotic effects of CUX1 in pancreatic cancer, genes that repress cell migration (e.g. E-cadherin, that have been shown in in vitro studies and in occludin) and to turn on the expression of genes that xenograft models, are associated with a positive promote cell migration (e.g. FAK, N-cadherin, regulation of BCL2 and downregulation of tumour vimentin) (Kedinger et al., 2009). The regulation of necrosis factor alpha and are, at least in part, mediated these genes seems to be mediated both directly by by the glutamate receptor GRIA3 (Ripka et al., 2010a; binding of CUX1 to the gene promoters but also Ripka et al., 2010b). indirectly by modulation of transcription factors and Breast cancer signaling proteins involved in EMT (e.g. SNAI1, SNAI2, Src, Wnt5a) (Kedinger et al., 2009; Aleksic et Note al., 2007; Ripka et al., 2007). Additionally, several of In mammary carcinoma the CUX1 expression is the CUX1 target genes are known GTPases important increased in high-grade tumours compared to low grade for actin-cytoskeleton polymerization (Kedinger et al., tumours and a reverse correlation between CUX1 2009). mRNA levels and the relapse free- and overall-survival Apoptosis was shown (Michl et al., 2005). Furthermore, is has Studies in pancreatic cancer cell lines showed that been shown that the expression levels of the intron 20- depletion of CUX1 by siRNA increases TNFalpha- and initiated mRNA, that leads to the synthesis of the p75 TRAIL-induced apoptosis whereas overexpression of CUX1 isoform, is specifically expressed in breast CUX1 rescues from apoptosis. Additionally, treatment cancer and positively correlated with a diffuse of xenograft tumours with siRNA for CUX1 lead to infiltrative growth pattern (Goulet et al., 2002). retarded tumour growth and increased apoptosis. These Transgenic mice expressing p75 and p110 CUX1 under effects are at least in part explained by a positive the control of the mouse mammary tumour virus-long regulation of the antiapoptotic protein BCL2 by CUX1 terminal repeat developed breast cancer after a long (Ripka et al., 2010a). Subsequently, the glutamate latency period. This tumour development was receptor GRIA3 was identified as another downstream accompanied by an increased activity of WNT-β- target of CUX1 able to mediate its antiapoptotic effects catenin signalling (Cadieux et al., 2009). (Ripka et al., 2010b). References Homology Neufeld EJ, Skalnik DG, Lievens PM, Orkin SH. Human Cut homeodomain proteins are highly conserved in CCAAT displacement protein is homologous to the Drosophila evolution of metazoans. Homologues of the Drosophila homeoprotein, cut. Nat Genet. 1992 Apr;1(1):50-5 melanogaster Cut protein have been described at least Andres V, Nadal-Ginard B, Mahdavi V. Clox, a mammalian in human, dog and mouse (Neufeld et al., 1992; Andres homeobox gene related to Drosophila cut, encodes DNA-

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binding regulatory proteins differentially expressed during by CCAAT displacement protein/cut homolog, is associated development. Development. 1992 Oct;116(2):321-34 with histone deacetylation. J Biol Chem. 1999 Mar 19;274(12):7803-15 Scherer SW, Neufeld EJ, Lievens PM, Orkin SH, Kim J, Tsui LC. Regional localization of the CCAAT displacement protein van Gurp MF, Pratap J, Luong M, Javed A, Hoffmann H, gene (CUTL1) to 7q22 by analysis of somatic cell hybrids. Giordano A, Stein JL, Neufeld EJ, Lian JB, Stein GS, van Genomics. 1993 Mar;15(3):695-6 Wijnen AJ. The CCAAT displacement protein/cut homeodomain protein represses osteocalcin gene transcription Valarché I, Tissier-Seta JP, Hirsch MR, Martinez S, Goridis C, Brunet JF. The mouse homeodomain protein Phox2 regulates and forms complexes with the retinoblastoma protein-related Ncam promoter activity in concert with Cux/CDP and is a protein p107 and cyclin A. Cancer Res. 1999 Dec putative determinant of neurotransmitter phenotype. 1;59(23):5980-8 Development. 1993 Nov;119(3):881-96 Li S, Aufiero B, Schiltz RL, Walsh MJ. Regulation of the Harada R, Dufort D, Denis-Larose C, Nepveu A. Conserved homeodomain CCAAT displacement/cut protein function by cut repeats in the human cut homeodomain protein function as histone acetyltransferases p300/CREB-binding protein (CBP)- DNA binding domains. J Biol Chem. 1994 Jan 21;269(3):2062- associated factor and CBP. Proc Natl Acad Sci U S A. 2000 7 Jun 20;97(13):7166-71 Lievens PM, Donady JJ, Tufarelli C, Neufeld EJ. Repressor O'Connor MJ, Stünkel W, Koh CH, Zimmermann H, Bernard activity of CCAAT displacement protein in HL-60 myeloid HU. The differentiation-specific factor CDP/Cut represses leukemia cells. J Biol Chem. 1995 May 26;270(21):12745-50 transcription and replication of human papillomaviruses through a conserved silencing element. J Virol. 2000 Vanden Heuvel GB, Bodmer R, McConnell KR, Nagami GT, Jan;74(1):401-10 Igarashi P. Expression of a cut-related homeobox gene in developing and polycystic mouse kidney. Kidney Int. 1996 Rong Zeng W, Soucie E, Sung Moon N, Martin-Soudant N, Aug;50(2):453-61 Bérubé G, Leduy L, Nepveu A. Exon/intron structure and alternative transcripts of the CUTL1 gene. Gene. 2000 Jan Banan M, Rojas IC, Lee WH, King HL, Harriss JV, Kobayashi 4;241(1):75-85 R, Webb CF, Gottlieb PD. Interaction of the nuclear matrix- associated region (MAR)-binding proteins, SATB1 and Stünkel W, Huang Z, Tan SH, O'Connor MJ, Bernard HU. CDP/Cux, with a MAR element (L2a) in an upstream regulatory Nuclear matrix attachment regions of human papillomavirus region of the mouse CD8a gene. J Biol Chem. 1997 Jul type 16 repress or activate the E6 promoter, depending on the 18;272(29):18440-52 physical state of the viral DNA. J Virol. 2000 Mar;74(6):2489- 501 Kim EC, Lau JS, Rawlings S, Lee AS. Positive and negative regulation of the human thymidine kinase promoter mediated Ellis T, Gambardella L, Horcher M, Tschanz S, Capol J, by CCAAT binding transcription factors NF-Y/CBF, dbpA, and Bertram P, Jochum W, Barrandon Y, Busslinger M. The CDP/cut. Cell Growth Differ. 1997 Dec;8(12):1329-38 transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle. Genes Dev. Lievens PM, Tufarelli C, Donady JJ, Stagg A, Neufeld EJ. 2001 Sep 1;15(17):2307-19 CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA- Jacobsen NJ, Elvidge G, Franks EK, O'Donovan MC, binding domains, and interacts with full-length CDP in vitro. Craddock N, Owen MJ. CUX2, a potential regulator of NCAM Gene. 1997 Sep 15;197(1-2):73-81 expression: genomic characterization and analysis as a positional candidate susceptibility gene for bipolar disorder. Am Pattison S, Skalnik DG, Roman A. CCAAT displacement J Med Genet. 2001 Apr 8;105(3):295-300 protein, a regulator of differentiation-specific gene expression, binds a negative regulatory element within the 5' end of the Khanna-Gupta A, Zibello T, Sun H, Lekstrom-Himes J, Berliner human papillomavirus type 6 long control region. J Virol. 1997 N. C/EBP epsilon mediates myeloid differentiation and is Mar;71(3):2013-22 regulated by the CCAAT displacement protein (CDP/cut). Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):8000-5 Coqueret O, Bérubé G, Nepveu A. The mammalian Cut homeodomain protein functions as a cell-cycle-dependent Moon NS, Premdas P, Truscott M, Leduy L, Bérubé G, Nepveu transcriptional repressor which downmodulates A. S phase-specific proteolytic cleavage is required to activate p21WAF1/CIP1/SDI1 in S phase. EMBO J. 1998a Aug stable DNA binding by the CDP/Cut homeodomain protein. Mol 17;17(16):4680-94 Cell Biol. 2001 Sep;21(18):6332-45 Coqueret O, Martin N, Bérubé G, Rabbat M, Litchfield DW, Santaguida M, Ding Q, Bérubé G, Truscott M, Whyte P, Nepveu A. DNA binding by cut homeodomain proteins is down- Nepveu A. Phosphorylation of the CCAAT displacement modulated by casein kinase II. J Biol Chem. 1998b Jan protein (CDP)/Cux transcription factor by cyclin A-Cdk1 30;273(5):2561-6 modulates its DNA binding activity in G(2). J Biol Chem. 2001 Dec 7;276(49):45780-90 Ai W, Toussaint E, Roman A. CCAAT displacement protein binds to and negatively regulates human papillomavirus type 6 De Vos J, Thykjaer T, Tarte K, Ensslen M, Raynaud P, E6, E7, and E1 promoters. J Virol. 1999 May;73(5):4220-9 Requirand G, Pellet F, Pantesco V, Rème T, Jourdan M, Rossi JF, Ørntoft T, Klein B. Comparison of gene expression profiling Catt D, Hawkins S, Roman A, Luo W, Skalnik DG. between malignant and normal plasma cells with Overexpression of CCAAT displacement protein represses the oligonucleotide arrays. Oncogene. 2002 Oct 3;21(44):6848-57 promiscuously active proximal gp91(phox) promoter. Blood. 1999a Nov 1;94(9):3151-60 Goebel P, Montalbano A, Ayers N, Kompfner E, Dickinson L, Webb CF, Feeney AJ. High frequency of matrix attachment Catt D, Luo W, Skalnik DG. DNA-binding properties of CCAAT regions and cut-like protein x/CCAAT-displacement protein and displacement protein cut repeats. Cell Mol Biol (Noisy-le- B cell regulator of IgH transcription binding sites flanking Ig V grand). 1999b Dec;45(8):1149-60 region genes. J Immunol. 2002 Sep 1;169(5):2477-87 Li S, Moy L, Pittman N, Shue G, Aufiero B, Neufeld EJ, Goulet B, Watson P, Poirier M, Leduy L, Bérubé G, Meterissian LeLeiko NS, Walsh MJ. Transcriptional repression of the cystic S, Jolicoeur P, Nepveu A. 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CDP/Cux isoform, p75, activated in breast tumor cells. Cancer FIH-1 expression by the homeodomain protein CDP/Cut/Cux. Res. 2002 Nov 15;62(22):6625-33 Mol Cell Biol. 2007 Oct;27(20):7345-53 Truscott M, Raynal L, Premdas P, Goulet B, Leduy L, Bérubé Ripka S, König A, Buchholz M, Wagner M, Sipos B, Klöppel G, G, Nepveu A. CDP/Cux stimulates transcription from the DNA Downward J, Gress T, Michl P. WNT5A--target of CUTL1 and polymerase alpha gene promoter. Mol Cell Biol. 2003 potent modulator of tumor cell migration and invasion in Apr;23(8):3013-28 pancreatic cancer. Carcinogenesis. 2007 Jun;28(6):1178-87 Tsutsumi S, Taketani T, Nishimura K, Ge X, Taki T, Sugita K, Truscott M, Denault JB, Goulet B, Leduy L, Salvesen GS, Ishii E, Hanada R, Ohki M, Aburatani H, Hayashi Y. Two Nepveu A. Carboxyl-terminal proteolytic processing of CUX1 distinct gene expression signatures in pediatric acute by a caspase enables transcriptional activation in proliferating lymphoblastic leukemia with MLL rearrangements. Cancer cells. J Biol Chem. 2007 Oct 12;282(41):30216-26 Res. 2003 Aug 15;63(16):4882-7 Ueda Y, Su Y, Richmond A. CCAAT displacement protein Goulet B, Baruch A, Moon NS, Poirier M, Sansregret LL, regulates nuclear factor-kappa beta-mediated chemokine Erickson A, Bogyo M, Nepveu A. A cathepsin L isoform that is transcription in melanoma cells. Melanoma Res. 2007 devoid of a signal peptide localizes to the nucleus in S phase Apr;17(2):91-103 and processes the CDP/Cux transcription factor. Mol Cell. 2004 Apr 23;14(2):207-19 Harada R, Vadnais C, Sansregret L, Leduy L, Bérubé G, Robert F, Nepveu A. Genome-wide location analysis and Kaul-Ghanekar R, Jalota A, Pavithra L, Tucker P, expression studies reveal a role for p110 CUX1 in the Chattopadhyay S. SMAR1 and Cux/CDP modulate chromatin activation of DNA replication genes. Nucleic Acids Res. 2008 and act as negative regulators of the TCRbeta enhancer Jan;36(1):189-202 (Ebeta). Nucleic Acids Res. 2004;32(16):4862-75 Cadieux C, Kedinger V, Yao L, Vadnais C, Drossos M, Paquet Nishio H, Walsh MJ. CCAAT displacement protein/cut homolog M, Nepveu A. Mouse mammary tumor virus p75 and p110 recruits G9a histone lysine methyltransferase to repress CUX1 transgenic mice develop mammary tumors of various transcription. Proc Natl Acad Sci U S A. 2004 Aug histologic types. Cancer Res. 2009 Sep 15;69(18):7188-97 3;101(31):11257-62 Kedinger V, Sansregret L, Harada R, Vadnais C, Cadieux C, Truscott M, Raynal L, Wang Y, Bérubé G, Leduy L, Nepveu A. Fathers K, Park M, Nepveu A. p110 CUX1 homeodomain The N-terminal region of the CCAAT displacement protein protein stimulates cell migration and invasion in part through a (CDP)/Cux transcription factor functions as an autoinhibitory regulatory cascade culminating in the repression of E-cadherin domain that modulates DNA binding. J Biol Chem. 2004 Nov and occludin. J Biol Chem. 2009 Oct 2;284(40):27701-11 26;279(48):49787-94 Ripka S, Neesse A, Riedel J, Bug E, Aigner A, Poulsom R, Michl P, Ramjaun AR, Pardo OE, Warne PH, Wagner M, Fulda S, Neoptolemos J, Greenhalf W, Barth P, Gress TM, Poulsom R, D'Arrigo C, Ryder K, Menke A, Gress T, Michl P. CUX1: target of Akt signalling and mediator of Downward J. CUTL1 is a target of TGF(beta) signaling that resistance to apoptosis in pancreatic cancer. Gut. 2010a enhances cancer cell motility and invasiveness. Cancer Cell. Aug;59(8):1101-10 2005 Jun;7(6):521-32 Ripka S, Riedel J, Neesse A, Griesmann H, Buchholz M, Goulet B, Truscott M, Nepveu A. A novel proteolytically Ellenrieder V, Moeller F, Barth P, Gress TM, Michl P. processed CDP/Cux isoform of 90 kDa is generated by Glutamate receptor GRIA3--target of CUX1 and mediator of cathepsin L. Biol Chem. 2006 Sep;387(9):1285-93 tumor progression in pancreatic cancer. Neoplasia. 2010b Aug;12(8):659-67 Maitra U, Seo J, Lozano MM, Dudley JP. Differentiation- induced cleavage of Cutl1/CDP generates a novel dominant- Sansregret L, Gallo D, Santaguida M, Leduy L, Harada R, negative isoform that regulates mammary gene expression. Nepveu A. Hyperphosphorylation by cyclin B/CDK1 in mitosis Mol Cell Biol. 2006 Oct;26(20):7466-78 resets CUX1 DNA binding clock at each cell cycle. J Biol Chem. 2010 Oct 22;285(43):32834-43 Michl P, Downward J. CUTL1: a key mediator of TGFbeta- induced tumor invasion. Cell Cycle. 2006 Jan;5(2):132-4 Fragiadaki M, Ikeda T, Witherden A, Mason RM, Abraham D, Bou-Gharios G. High doses of TGF-β potently suppress type I Sansregret L, Goulet B, Harada R, Wilson B, Leduy L, collagen via the transcription factor CUX1. Mol Biol Cell. 2011 Bertoglio J, Nepveu A. The p110 isoform of the CDP/Cux Jun 1;22(11):1836-44 transcription factor accelerates entry into S phase. Mol Cell Biol. 2006 Mar;26(6):2441-55 Thoennissen NH, Lasho T, Thoennissen GB, Ogawa S, Tefferi A, Koeffler HP. Novel CUX1 missense mutation in association Aleksic T, Bechtel M, Krndija D, von Wichert G, Knobel B, with 7q- at leukemic transformation of MPN. Am J Hematol. Giehl K, Gress TM, Michl P. CUTL1 promotes tumor cell 2011 Aug;86(8):703-5 migration by decreasing proteasome-mediated Src degradation. Oncogene. 2007 Aug 30;26(40):5939-49 This article should be referenced as such: Li J, Wang E, Dutta S, Lau JS, Jiang SW, Datta K, Kühnemuth B, Michl P. CUX1 (cut-like homeobox 1). Atlas Mukhopadhyay D. Protein kinase C-mediated modulation of Genet Cytogenet Oncol Haematol. 2012; 16(3):191-195.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 195 Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Gene Section Review

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) June L Traicoff, Stephen M Hewitt, Joon-Yong Chung Center for Peer Review and Science Management, SRA International, Inc Maryland, USA (JLT), Applied Molecular Pathology Laboratory & Tissue Array Research Program, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA (SMH), Applied Molecular Pathology Laboratory, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA (JYC)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/DNAJA3ID40342ch16p13.html DOI: 10.4267/2042/47276 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity DNA/RNA Other names: FLJ45758, TID1, hTid-1 Note HGNC (Hugo): DNAJA3 DNAJA3 belongs to the evolutionarily conserved DNAJ/HSP40 family of proteins. There are 41 known Location: 16p13.3 DnaJ/Hsp40 proteins in the human genome (Qiu et al., Local order: According to NCBI Map Viewer, genes 2006). flanking DNAJA3 are COR07-PAM16, NMRAL1, and According to NCBI Gene, the DNAJA3 gene is HMOX2. conserved in human chimpanzee, cow, mouse, rat, Note chicken, zebrafish, fruit fly, mosquito, C. elegans, S. DNAJA3 was first identified by its ability to form pombe, S. cerevisiae, K. lactis, E. gossypii, M. grisea, complexes with the human papillomavirus E7 N. crassa, and rice. oncoprotein (Schilling et al., 1998) in a yeast-two Description hybrid screen. Sequence analysis revealed that DNAJA3 was the human homolog of the Drosophila The DNAJA3 gene is located on chromosome 16p13.3 tumor suppressor protein Tid56. Furthermore, between markers D16S521 and D16S418. This DNAJA3 contained a J-domain which is characteristic chromosomal region carries several loci implicated in of the family of DnaJ proteins which interact with and human proliferation disorders, including the tuberous stimulate the ATPase activity of heat shock cognate 70 sclerosis 2 gene (TSC2), polycystic kidney disease 1 (hsc70) family members (Schilling et al., 1998). gene (PKD1), and the CREB binding protein (CBP) locus (Yin and Rozakis-Adcock, 2001).

Chromosome 16 - NC_000016.9. Modified from NCBI Map Viewer.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 196 DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

DNAJA3 is approximately 34 kb and is composed of DNAJA3 expression also increased in pathological 12 exons separated by 11 introns. Exon sizes vary from cardiac hypertrophic states (Hayashi et al., 2006). 64 to 232 nucleotides, with the exception of exon 12 Pseudogene corresponding to the 3' untranslated region of DNAJA3, which extends over 1.1 kb. Intron sizes vary Paralogs. According to GeneCards, DNAJC16 is a from 618 to 8291 nucleotides (Yin and Rozakis- paralog for DNAJA3. DNAJC16 is located on Adcock, 2001). chromosome 1p36.1. Sequence encoding the DNAJ domain is present in exons 2, 3 and 4, sequence encoding the Cys-rich Protein domain is found in exons 5 an 6, and the COOH- Note terminal region is found in exons 7 through 11 (Yin and The DNAJA3 gene encodes three cytosolic (Tid50, Rozakis-Adcock, 2001). Tid48, Tid46) proteins and three mitochondrial (Tid43, Transcription Tid40, Tid38) proteins. Proteins encoded by the longer Promoter elements. DNAJA3 contains a putative splice variant DNAJA3L have often been designated in transcriptional start site 21 nucleotides upstream of the the literature as Tid1L. Proteins encoded by the shorter initiating methionine. The presumptive promoter is splice variant DNAJA3S have often been named Tid1S. characterized by the lack of TATA and CAAT motifs, In this review, Tid1L will be designated DNAJA3L, and a high G+C content. The 5' flanking region and Tid1S will be designated DNAJA3S. Specific contains several consensus binding sites for isoforms will be designated by size, e.g., Tid 50 will be transcription factors that regulate gene expression designated as DNAJA3 (50 kD). during tissue and organ development, such as myeloid Description zinc finger (MZF1), Ikaros 2 and homeodomain DNAJA3 protein is present in two isoforms, proteins, as well as factors implicated in cell growth corresponding to splice variants encoding them. The and survival responses, including AP-1, PEA3, E2F longer DNAJA3L isoform is a 480 amino acid protein and NF-kB. with a predicted size of 52 kD. The shorter DNAJA3S Splice variants. Alternative splicing of a single isoform is a 453 amino acid protein with a predicted heteronuclear RNA (hnRNA) species generates the size of 49 kD (Lu et al., 2006; UniProt). three DNAJA3 isoforms. The long form DNAJA3L (hTID1L) fully incorporates all exons. The Expression intermediate form DNAJA3I (hTID1I) is generated by DNAJA3 protein has been detected in human breast, splicing of exon 10 to exon 12. This results in the loss colon, ovarian, lung, and head and neck squamous cell of the 34 C-terminal-most amino acids as well as the carcinoma (HNSCC) tissues (Traicoff et al., 2007; stop codon; these are replaced with six amino acids Kurzik-Dumke et al., 2008; Chen et al., 2009). KRSTGN from exon 12. The short form DNAJA3S (hTID1S) results from an in-frame deletion of 50 amino Localisation acids that correspond precisely to exon 5 (Yin and DNAJA3 localizes to human mitochondrial nucleoids, Rozakis-Adcock, 2001). which are large protein complexes bound to RNA expression. DNAJA3 mRNA was detected in 50 mitochondrial DNA. Unlike other DnaJs, DNAJA3L different human fetal and adult tissues. However the and DNAJA3S form heterocomplexes; both relative abundance correlated with metabolic activity of unassembled and complexed DNAJA3 are observed in the tissues, with the highest levels observed in liver and human cells. DNAJA3L showed a longer residency skeletal muscle (Kurzik-Dumke and Czaja, 2007). time in the cytosol prior to mitochondrial import as Human tissues and cell lines showed differential compared with DNAJA3S; DNAJA3L was also expression of the three DNAJA3 splice variant significantly more stable in the cytosol than DNAJA3S, mRNAs. Fetal brain tissue predominantly expressed which is rapidly degraded (Lu et al., 2006). DNAJA3I, while breast tissues and T-cells Function predominantly expressed DNAJA3L. Cell lines derived from prostate epithelia, skin and lung fibroblasts, I. Binding partners normal astrocytes, and an osteosarcoma predominantly Human DNAJA3 protein has been shown to interact expressed DNAJA3I with low levels of DNAJA3L also with diverse partners, including viral proteins, heat present. DNAJA3S transcript was undetectable in all shock proteins, and key regulators of cell signaling and samples (Yin and Rozakis-Adcock, 2001). growth. DNAJA3 transcripts showed differential expression Viral proteins during development. Expression of DNAJA3 Hepatitis B virus core protein: DNAJA3 associated transcripts in mouse neonatal cardiomyocytes increased with the hepatitis B virus core protein, specifically with as development of the heart proceeded and reached a the carboxyl-terminal region (amino acids 94-185). The maximal level at 4 weeks of age, when cardiac N-terminal end of DNAJA3 (amino acids 1-447) was myocytes have matured (Hayashi et al., 2006). required for this interaction. Furthermore, the

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 197 DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

DNAJA3S precursor co-sedimented with viral capsid- terminus of DNAJA3L was required for this interaction like particles composed of the full-length core protein (Lu et al., 2006). Both DNAJA3S and DNAJA3L could (Sohn et al., 2006). Interaction between DNAJA3 and interact with Hsp70 (Kim et al., 2004). Endogenous the HBV core protein was confirmed in co- DNAJA3L and DNAJA3S coimmunoprecipitated with immunoprecipitation experiments using transfected mitochondrial Hsp70, but not Hsc70, in U2OS hepatoma cells (Sohn et al., 2006). osteosarcoma cells (Syken et al., 1999). Epstein-Barr virus-encoded BARF1 protein: Tumor suppressor proteins DNAJA3 (amino acids 149-320) associated with the Adenomatous polyposis coli (APC): endogenous Epstein-Barr virus-encoded BARF1 protein (amino cytosolic DNAJA3 proteins interacted with APC in acids 21-221). Interaction between DNAJA3 and normal colon epithelium and colorectal cancer cell BARF1 was confirmed in co-immunoprecipitation lines (HT-29, Caco-2, and HRT-18). The N-terminal experiments using transfected HeLa cells (Wang et al., Armadillo domain of APC was sufficient for binding to 2006). DNAJA3. The DNAJA3 and APC interaction Herpes simplex virus type 1 UL9 protein: DNAJA3 comprised part of a larger multi-component complex associated with the herpes simplex virus type 1 (HSV- that also contained Hsp70, Hsc70, Actin, Dvl, and 1) UL9 protein. UL9 protein is an origin-binding Axin. This complex functions independently of the protein. Interaction between DNAJA3 and UL9 was known roles of APC in beta-catenin degradation and confirmed by in vitro co-immunoprecipitation (Eom proliferation mediated by Wg/Wnt signaling (Kurzik- and Lehman, 2002). Dumke and Czaja, 2007). Human T cell leukemia virus type 1 (HTLV-1) Tax Endogenous DNAJA3 proteins were shown to interact protein: DNAJA3 associated with HTLV-1 Tax. The with the caspase-cleaved N-terminus of APC in interaction occurred through a central cysteine-rich zinc HCT116 cells (Qian et al., 2010). The caspase-cleaved finger-like region of DNAJA3 (amino acids 236 to APC protein has an important physiological role in 300). Interaction between DNAJA3 and Tax was mediating apoptosis (Qian et al., 2010). confirmed by co-immunoprecipitation experiments Patched: endogenous human Patched interacted with using transfected human embryonic kidney cells (HEK) the cytosolic forms of the DNAJA3 proteins in human (Cheng et al., 2001). Furthermore, the DNAJA3 and colon epithelium and colon tumor cells (Kurzik-Dumke Tax interaction occurred through a complex comprised and Czaja, 2007). The tumor-associated polymorphism of DNAJA3, Tax, and heat shock protein 70 (Hsp70), in Patched (Ptch FVB allele) was associated with poorer in which the cysteine-rich region of DNAJA3 binding to DNAJA3 (Wakabayashi et al., 2007). interacted with Tax, while the J domain of DNAJA3 INT6: endogenous human DNAJA3 interacted with interacted with Hsp70 (Cheng et al., 2001). INT6 (the p48 subunit of the eIF3 translation initiation Human papilloma virus-16 (HPV-16) E7 factor) in log phase, but not confluent, Jurkat T-cells oncoprotein: DNAJA3 was initially characterized (Traicoff et al., 2007). through its interaction with the HPV-16 E7 Von Hippel-Lindau protein (VHL): endogenous oncoprotein. DNAJA3 amino acids 1 to 235 and 297 to pVHL co-immunoprecipitated with DNAJA3L protein 342 independently interacted with HPV-16 E7. in HEK293 cells (Bae et al., 2005). Interaction between DNAJA3 and HPV-16 E7 was p53: DNAJA3 directly interacts with p53 through the confirmed by in vitro binding assays and co- DNAJA3 DNAJ domain. Either the N- or C- terminal immunoprecipitation experiments using transfected domains of p53 was sufficient for the interaction (Trinh human osteosarcoma (U2OS) cells (Schilling et al., et al., 2010). 1998). Receptors Heat shock proteins Interferon-gamma receptor (IFN-gammaR) subunit Hsp70 and Hsc70: endogenous DNAJA3 (specifically IFN-gammaR2: DNAJA3 interacted with IFN-gamma the cytosolic form) immunoprecipitated with the heat R2 in transfected COS cells. Furthermore, DNAJA3 shock proteins Hsp70 and Hsc70 in normal colon bound more efficiently to a IFN-gammaR2 chimera epithelium and colon cancer cell lines (Kurzik-Dumke with an active kinase domain than to a similar construct and Czaja, 2007). Endogenous DNAJA3 also interacted with an inactive kinase domain (Sarkar et al., 2001). with Hsp70/Hsc70 in HEp2 cells, and this interaction ErbB-2 (HER2/neu): endogenous ErbB-2 and was reduced in cells treated with interferon-gamma DNAJA3 co-immunoprecipitated in SK-BR-3 breast (Sarkar et al., 2001). The J domain of DNAJA3 was cancer cells (Kim et al., 2004). The cytoplasmic shown to be required for interaction with Hsp70 in domains of ErbB-2 and DNAJA3 were sufficient for HEK cells (Cheng et al., 2001). this interaction (Kim et al., 2004). Proteins encoded by the long and short splice forms of ErbB-2 co-immunoprecipiated with DNAJA3 and the DNAJA3, DNAJA3L and DNAJA3S, respectively, carboxyl terminus of heat shock cognate 70 interacting showed differential interactions with heat shock protein (CHIP). This complex was demonstrated in proteins. Unassembled DNAJA3L (the long splice tissue extracts from breast tumor specimens as well as variant) was shown to interact with Hsc70 specifically in transfected cell lines (Jan et al., 2011). in the cytosol (Lu et al., 2006). The unique carboxyl

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Trk receptor tyrosine kinases: the carboxyl-terminal linking to the subsynaptic cytoskeleton, as end of DNAJA3 (residues 224-429) bound to Trk at its demonstrated by knockdown and overexpression activation loop in a phosphotyrosine-dependent manner experiments. (Liu et al., 2005). Knockdown of DNAJA3 in skeletal muscle fibers Muscle-specific kinase (MuSK) component of the resulted in dispersed synaptic AchR clusters and agrin receptor: DNAJA3S, but not DNAJA3L, impaired neuromuscular transmission. Knockdown of associated with the cytoplasmic portion of MuSK in DNAJA3 in myotubes resulted in inhibition of AchR mouse skeletal muscle cells (Linnoila et al., 2008). clustering, inhibition of agrin-induced activation of the Signaling proteins Rac and Rho small GTPases and tyrosine NF-kappaB: DNAJA3 strongly associated with the phosphorylation of AchR, and decreased Dok-7- cytoplasmic protein complex of NF-kappaB-IkappaB induced clustering of AChRs. In contrast, through direct interaction with overexpression of the N-terminal half of DNAJA3 IkappaBalpha/IkappaBbeta and the IKKalpha/beta induced agrin-and MuSK-independent phosphorylation subunits of the IkappaB kinase complex. The and clustering of AChRs (Linnoila et al., 2008; Song endogenous interaction was observed in Jurkat, SAOS- and Balice-Gordon, 2008). 2, and HEK293 cells (Cheng et al., 2005). Neurite outgrowth: DNAJA3 regulated nerve growth JAK/STAT: Jak2 interacted with DNAJA3S as well as factor (NGF)-induced neurite outgrowth in PC12- DNAJA3L as shown by immunoprecipitation from derived nnr5 cells. DNAJA3 bound to Trk at the transfected COS-1 cells expressing these proteins. activation loop and DNAJA3 was tyrosine Endogenous DNAJA3 and Jak2 were shown to interact phosphorylated by Trk in yeast cells, transfected cells, in HEp2 cells (Sarkar et al., 2001). and in neurotophin-stimulated primary rat hippocampal The carboxyl terminus of endogenous DNAJA3L, but neurons. Overexpression of DNAJA3 led to NGF- not DNAJA3S, co-immunoprecipitated with STAT1 induced neurite outgrowth in TrkA-expressing nnr5 and with STAT3 in U2OS cells (Lu et al., 2006). cells. In contrast, knockdown of DNAJA3 resulted in DNAJA3L remained associated with activated reduced NGF-induced neurite growth in nnr5-TrkA phosphorylated STAT1 upon treatment with interferon- cells (Liu et al., 2005). gamma (Lu et al., 2006). Viral pathways The DNAJ domain of DNAJA3 interacted with the Hepatitis B virus replication: expression of DNAJA3 transactivation domain of Stat5b in hematopoietic cell suppressed replication of HBV in human hepatoma lines (Dhennin-Duthille et al., 2011). cells, while knockdown of DNAJA3 led to increased p120 GTPase-activating protein (GAP): both the HBV replication. The mechanism for inhibited cytoplasmic precursor and mitochondrial mature forms replication was through accelerated degradation and of murine DNAJA3 associated with GAP in vivo in destabilization of the viral core and HBx proteins (Sohn rodent cells. GAP selectively bound to the et al., 2006). unphosphorylated form of murine DNAJA3 (Trentin et Herpes simplex virus type 1 replication: the HSV-1 al., 2001). UL9 protein is an origin-binding protein that is DNA replication proteins essential for viral DNA replication. DNAJA3 DNA polymerase gamma (Polga) alpha subunit: modulates DNA replication by enhancing the binding endogenous DNAJA3 interacted with the alpha subunit of UL9 protein to an HSV-1 origin and facilitating of Polga in HEK293 cells. Polga is the only formation of the multimer from the dimeric UL9 mitochondrial DNA polymerase responsible for all protein, perhaps through a chaperone function. mitochondrial DNA synthetic reactions (Hayashi et al., However, DNAJA3 had no effect on the DNA- 2006). dependent ATPase or helicase activities associated with II. Signaling pathways and cellular effects the UL9 protein (Eom and Lehman, 2002). DNAJA3 modulates diverse signaling pathways and Epstein-Barr virus secretion: the EBV BARF1 gene cellular effects that are vital for cell growth and encodes a secretory protein with transforming and differentiation. mitogenic activities. Coexpression experiments with BARF1 and DNAJA3 showed that DNAJA3 could Neural pathways promote secretion of BARF1, perhaps through Neuromuscular synaptogenesis: DNAJA3 is an chaperone functions (Wang et al., 2006). essential component of the agrin signaling pathway that Motility and metastasis is crucial for synaptic development. Motoneuron- DNAJA3 was shown to negatively regulate the motility derived agrin clusters nicotinic acetylcholine receptors and metastasis of breast cancer cells through (AChRs) in mammalian cells. DNAJA3 binds to the attenuation of nuclear factor kappaB activity on the cytoplasmic domain of muscle-specific kinase (MuSK), promoter of the IL8 gene (Kim et al., 2005). a component of the agrin receptor and colocalizes with Reductions of DNAJA3 levels in MDA-MB231 breast AchRs at developing, adult, and denvervated motor cancer cells increased their migration as a result of endplates. DNAJA3 transduces signals from MuSK increased interleukin-8 (IL-8) secretion without activation to AchR clustering, culmintating in cross- affecting survival or growth rate. Furthermore,

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DNAJA3 was shown to negatively modulate de novo regardless of the p53 expression status. In contrast, synthesis of IL-8 through regulating NFkappaB cells transduced with a DNAJA3 mutant that has an N- activity. Additionally, DNAJA3 knockdown enhanced terminal J domain deletion and that lost suppressive the metastasis of breast cancer cells in animals (Kim et activity on IKK, continued to proliferate (Cheng et al., al., 2005). 2005). Other studies also indicate a potential role for DNAJA3 DNAJA3 mediates apoptosis through the Bcl-2 in inhibiting transformation and metastasis. Stable pathway. DNAJA3 induced apoptosis in SF767 glioma DNAJA3 knockdown cells exhibited an enhanced cells that contained a tumor-associated mutation at the ability for anchorage-independent growth, as measured DNAJA3 locus. Apoptosis resulted from caspase by an increase in soft-agar colony formation (Edwards activation and cytochrome c release from mitochondria. and Münger, 2004). In contrast, ectopic expression of However, Bcl-XL protected cells from hTid-1S- DNAJA3 in HNSCC cells was shown to significantly induced cell death and cytochrome c release. However, inhibit cell proliferation, migration, invasion, hTid1S caused S and G2/M arrest in cells with wild anchorage-independent growth, and type Tid1. Interestingly, hTid1L had no effect on xenotransplantation tumorigenicity (Chen et al., 2009). growth of glioma cells (Trentin et al., 2004). Expression of DNAJA3 inhibited the transformation Immature dnaja3(-/-) DN4 thymocytes exhibited phenotype of two human lung adenocarcinoma cell significantly reduced expression of the antiapoptotic lines (Cheng et al., 2001). bcl-2 gene (Lo et al., 2005). Apoptosis Expression of constitutively active AKT (pAKT) DNAJA3 encodes two mitochondrial matrix localized counteracted and inhibited DNAJA3-induced apoptosis splice variants: DNAJA3 (43 kD) and DNAJA3 (40 in HNSCC cells (Chen et al., 2009). kD). DNAJA3 (43 kD) and DNAJA3 (40 kD) do not DNAJA3 mediates apoptosis through APC. themselves induce apoptosis; instead they have DNAJA3 (40 kD) isoform inhibited apoptosis through opposing effects on apoptosis induced by exogenous antagonizing the apoptotic function of the N-terminal stimuli. region of the APC protein (Qian et al., 2010). Expression of DNAJA3 (43 kD) increases apoptosis DNAJA3 mediates apoptosis through p53. induced by both the DNA-damaging agent mitomycin c Overexpression of DNAJA3 enhanced p53-dependent and tumor necrosis factor-alpha. This activity is J apoptosis, and restored pro-apoptotic activity of mutant domain-dependent, since a J domain mutant of p53 in colon, breast, and glioma cell lines (Ahn et al., DNAJA3 (43 kD) suppressed apoptosis. Conversely, 2010). The mechanism is through direct interaction of expression of DNAJA3 (40 kD) suppressed apoptosis, the DNAJ domain of DNAJA3 and p53 (Trinh et al., while expression of a J domain mutant of DNAJA3 (40 2010). In contrast, depletion of DNAJA3 resulted in the kD) increased apoptosis (Syken et al., 1999). inhibition of hypoxia or genotoxic stress-induced p53 Cells lacking expression of DNAJA3 proteins were mitochondrial localization and apoptosis (Trinh et al., protected from cell death in response to multiple 2010). stimuli, including cisplatin, tumor necrosis factor Mitochondrial functions alpha/cycloheximide and mitomycin C (Edwards and Although DNAJA3 has many cellular functions, Münger, 2004). DNAJA3 often localizes to the mitochondria and also DNAJA3 regulates activation-induced cell death has important functions in the mitochondria. (AICD) in the Th2 subset of helper T cells. AICD is an Epidermal growth factor (EGF) response: GAP and apoptotic process induced by stimulation through the DNAJA3 were shown to colocalize at perinuclear T-cell receptor and Th2 cells are significantly less mitochondrial membranes in response to EGF prone to AICD than Th1 cells are. The antiapoptotic stimulation (Trentin et al., 2001). variant, Tid-1S was shown to be selectively induced in p53 localization and apoptotic function: depletion of murine Th2 cells following activation. Expression of a DNAJA3 prevented p53 accumulation at the dominant-negative mutant of hTid-1S in a Th2 cell line mitochondria and resulted in resistance to apoptosis strikingly enhanced activation of caspase 3 in response under hypoxic or genotoxic stresses (Trinh et al., to CD3 stimulation, and caused the cells to become 2010). DNAJA3 formed a complex with p53 under sensitive to AICD. Therefore, the accumulation of Tid- hypoxic conditions that directed p53 translocation to 1S in Th2 cells following activation may contribute to the mitochondria and the subsequent initiation of the induction of apoptosis resistance during the apoptosis (Ahn et al., 2010). Loss of DNAJA3 activation of Th2 cells (Syken et al., 2003). expression abrogated p53 translocation to the DNAJA3 mediates apoptosis through the nuclear mitochondria and inhibited apoptosis (Ahn et al., factor kappaB (NF-kappaB) pathway. DNAJA3 2010). Conversely, overexpression of DNAJA3 repressed the activity of NF-kappaB through physical promoted p53 mitochondrial localization and apoptosis and functional interactions with the IKK complex and (Ahn et al., 2010). IkappaB. Overexpression of DNAJA3 led to inhibition Viral protein localization: in the absence of Tax, of cell proliferation and induction of apoptosis of expression of the DNAJA3/Hsp70 molecular complex human osteosarcoma cells and human melanoma cells was targeted to perinuclear mitochondrial clusters. In

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the presence of Tax, DNAJA3 and its associated Hsp70 fibroblasts, as well as in premature senescence of are sequestered within a cytoplasmic "hot spot" REF52 cells triggered by activated ras. Conversely, structure, a subcellular distribution that is characteristic spontaneous immortalization of rat embryo fibroblasts of Tax in HEK cells (Cheng et al., 2001). was suppressed upon ectopic expression of DNAJA3. APC interaction: the amino terminus of APC Suppression of endogenous DNAJA3 activity interacted with DNAJA3 at the mitochondria in vivo in alleviated the suppression of tumor necrosis factor colorectal cancer cell lines (Qian et al., 2010). alpha-induced NF-kappaB activity by DNAJA3. These Chaperone function: DNAJA3 isoforms were also results suggest that DNAJA3 contributes to senescence shown to exhibit a conserved mitochondrial DnaJ-like by repressing NF-kappa B signaling (Tarunina et al., function substituting for the yeast mitochondrial DnaJ- 2004). like protein Mdj1p (Lu et al., 2006). DNAJA3 repressed NF-kappaB activity induced by Mitochondrial biogenesis: DNAJA3 was shown to be Tax, tumor necrosis factor alpha (TNFalpha), and crucial for mitochondrial biogenesis partly through Bcl10. DNAJA3 specifically suppressed serine chaperone activity on DNA polymerase gamma phosphorylation of IkappaBalpha by activated IkappaB (Hayashi et al., 2006). Mice deficient in Dnaja3 kinase beta (IKKbeta). developed dilated cardiomyopathy (DCM) and died The suppressive activity of DNAJA3 on IKKbeta before 10 weeks of age (Hayashi et al., 2006). required a functional J domain that mediates Progressive respiratory chain deficiency and decreased association with heat shock proteins and resulted in copy number of mitochondrial DNA were observed in prolonging the half-life of the NF-kappaB inhibitors cardiomyocytes lacking Dnaja3 (Hayashi et al., 2006). IkappaBalpha and IkappaBbeta (Cheng et al., 2002). Tumor suppressor pathways AKT: overexpression of DNAJA3 in HNSCC cells APC: DNAJA3 directly bound to the APC tumor inhibited cell proliferation, migration, invasion, suppressor protein and promoted a physiological anchorage-independent growth, and function for APC that was independent of APC's xenotransplantation tumorigenicity. Overexpression of involvement in beta-catenin degradation or regulation DNAJA3 attenuated EGFR activity and blocked the of the actin cytoskeleton (Kurzik-Dumke and Czaja, activation of AKT in HNSCC cells, which are known 2007). to be involved in the regulation of survival in HNSCC pVHL: TID1L directly interacted with von Hippel- cells. Conversely, ectopic expression of constitutively Lindau protein and enhanced the interaction between active AKT greatly reduced apoptosis induced by HIF-1 alpha and pVHL. This resulted in destabilization DNAJA3 overexpression (Chen et al., 2009). of HIF-1 alpha protein, decreased vascular endothelial JAK2: DNAJA3L and DNAJA3S interacted with Jak2 growth factor expression, and inhibition of in vivo in COS-1 cells. Interaction was primarily in the angiogenesis (Bae et al., 2005). cytoplasm and predominantly with the active kinase Interferon-gamma: DNAJA3L and DNAJA3S domain of Jak2 (Sarkar et al., 2001). interacted with the interferon-gamma receptor chain c-MET receptor tyrosine kinase (MetR): MetR IFN-gammaR2 and modulated IFN-gamma-mediated interacted with DNAJA3L and DNAJA3S, but showed transcriptional activity. Furthermore, IFN-gamma preferential binding to DNAJA3S. Interaction occurred treatment reduced the interaction between through the J domain. In RCC cells, overexpression of Hsp70/Hsc70 and DNAJA3 (Sarkar et al., 2001). DNAJA3S enhanced HGF-mediated MetR Oncogenic pathways autophosphorylation, while DNAJA3L showed modest Erb-B2/HER2: DNAJA3 physically interacted with inhibition of MetR activity. Modulation of MetR the signaling domain of ErbB-2 and ErbB-2 were phosphorylation levels was independent of pVHL. shown to colocalize in mammary carcinoma cells (SK- DNAJA3S enhanced HGF-mediated cell migration and BR-3). Overexpression of DNAJA3 induced growth modulated HGF-mediated MAPK phosphorylation. arrest and apoptosis in ErbB-2-overexpressing breast DNAJA3 knockdown inhibited MetR activation and cancer cells; the DNAJ and C-terminal domains of migration in response to HGF (Copeland et al., 2011). DNAJA3 were critical for mediating apoptosis. Signal transducers and activators of transcription Downregulation of ERK1/ERK2 and BMK1 MAPK (STAT) 5b: DNAJA3 specifically interacted with pathways also contributed to apoptosis. DNAJA3S STAT5b but not STAT5a in hematopoietic cell lines. negatively regulated ErbB-2 signaling pathways by Interaction involved the DNAJ domain. DNAJA3 enhancing the degradation of ErbB-2. Finally, negatively regulated the expression and transcriptional increased cellular DNAJA3 inhibited the growth of activity of STAT5b and suppressed the growth of ErbB-2-dependent tumors in mice (Kim et al., 2004). hematopoietic cells transformed by an oncogenic form Mammary tumor tissue from breast cancer patients and of STAT5b (Dhennin-Duthille et al., 2011). transgenic mice carrying the rat HER-2/neu oncogene Cell Fate suggest that DNAJA3 suppresses ErbB-2 in breast DNAJA3 was shown to be required for the T-cell cancers (Kurzik-Dumke et al., 2010). transition from double-negative 3 to double-positive NF-kappaB: expression of DNAJA3 was upregulated stages. Mice with dnaja3 specifically deleted in T cells upon cellular senescence in rat and mouse embryo developed thymic atrophy, with dramatic reduction of

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 201 DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

double-positive and single-positive thymocytes in the correlation with negative or weakly positive expression dnaja3(-/-) thymus. DNAJA3 deficiency inhibited cell of ErbB2 in human breast cancer tissue samples. High proliferation and enhanced cell death of DN4 cells. The DNAJA3 levels were strongly correlated with high expression profile of genes involved in cytokine levels of CHIP (carboxyl terminus of heat shock receptor signaling was altered in DN4 T-cells. cognate 70 interacting protein). Lower expression of Expression of human bcl-2 transgene restored T DNAJA3 had a higher risk of unfavorable tumor grade, lymphocyte proliferation and differentiation in the later pathological stage, larger tumor size, and dnaja3 knockout mice. These results suggest that microscopic features of a more malignant histology dnaja3 is critical in early thymocyte development, (Jan et al., 2011). Higher expression of DNAJA3 especially during transition from the DN3 to double- correlated with increased 10-year overall and disease- positive stages, possibly through its regulation of bcl-2 free survival rate (Jan et al., 2011). expression, which provides survival signals. The expression of the three DNAJA3 isoform Homology transcripts was examined in human breast cancer carcinomas by RT-PCR. Aberrant expression of all Mouse (laboratory): Dnaja3 three forms correlated with malignant transformation. Rat: Dnaja3 Furthermore, elevated DNAJA3L expression was Cattle: DNAJA3 associated with less aggressive tumors (Kurzik-Dumke Chimpanzee: DNAJA3 et al., 2010). Immunohistochemical analysis Dog (domestic): DNAJA3 demonstrated high levels of DNAJA3 protein in tumors of the luminal A subtype, but significantly lower levels Mutations of DNAJA3 protein in the luminal B subtype, triple Note negative tumors, and the HER-2 subtype which overexpresses HER-2 (Kurzik-Dumke et al., 2010). The SF767 glioma cell line exhibits an aberrant 52 kD Multiplex tissue immunoblotting of human breast molecular weight protein. Sequence analysis of cDNA tumor tissue microarrays was used to test correlations generated from this line showed two mutations: an between DNAJA3 protein levels and a set of tumor additional thymine at nucleotide position 1438 and an suppressor proteins. DNAJA3 protein levels showed additional cytosine at nucleotide position 1449. These strongly positive correlations with p53, Patched, and mutations alter the reading frame of the DNAJA3 INT6 proteins (Traicoff et al., 2007). Additionally, sequence, introducing an additional 71 amino acids DNAJA3 protein levels showed moderate positive following the penultimate threonine residue at position correlations with c-Jun and phospho-c-Jun proteins 479. The mutations appear to increase the steady-state (Traicoff et al., 2007). abundance of the mutant protein, resulting in aberrantly high levels of the DNAJA3 mutant variant (Trentin et Head and neck squamous cell al., 2004). carcinoma (HNSCC) Implicated in Disease The clinical association between DNAJA3 expression Colon cancer and progression of HNSCC was explored using immunohistochemical analysis of primary HNSCC Disease patient tumor tissue. DNAJA3 expression was DNAJA3 and INT6 protein levels, as well as DNAJA3 negatively associated with tumor T stage, overall stage, and Patched protein levels, were positively correlated survival, and recurrence. Patients with higher in human colon tumor tissues (Traicoff et al., 2007). expression of DNAJA3 were predicted to have better However, there were no correlations between DNAJA3 overall survival than those with low or undetectable and p53, c-Jun, or phospho-c-Jun protein levels expression of DNAJA3 protein (Chen et al., 2009). (Traicoff et al., 2007). These results were demonstrated Highly malignant HNSCC cell lines also demonstrated by multiplex tissue immunoblotting of tissue low or undetectable levels of DNAJA3, in contrast to microarrays (Traicoff et al., 2007). less aggressive lines where DNAJA3 protein was easily Progression of colorectal cancers correlated with detected (Chen et al., 2009). overexpression and loss of polarization of expression of DNAJA3. These changes were associated with Ovarian cancer upregulation of Hsp70 and loss of Disease compartmentalization of APC (Kurzik-Dumke et al., Multiplex tissue immunoblotting of ovarian tumor 2008). tissues demonstrated that DNAJA3 protein levels Breast cancer showed moderate positive correlations with INT6, c- Jun, phospho-c-Jun, and p53. No correlations were Disease observed between DNAJA3 and Patched (Traicoff et DNAJA3 protein expression showed a strong al., 2007).

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Lung cancer Edwards KM, Münger K. Depletion of physiological levels of the human TID1 protein renders cancer cell lines resistant to Disease apoptosis mediated by multiple exogenous stimuli. Oncogene. Multiplex tissue immunoblotting of lung tumor tissues 2004 Nov 4;23(52):8419-31 demonstrated that DNAJA3 protein levels were Kim SW, Chao TH, Xiang R, Lo JF, Campbell MJ, Fearns C, strongly correlated with INT6. DNAJA3 protein levels Lee JD. Tid1, the human homologue of a Drosophila tumor were moderately correlated with Patched, c-Jun, and suppressor, reduces the malignant activity of ErbB-2 in carcinoma cells. Cancer Res. 2004 Nov 1;64(21):7732-9 p53. However, DNAJA3 proteins showed negative correlation with phospho-c-Jun in these samples Tarunina M, Alger L, Chu G, Munger K, Gudkov A, Jat PS. Functional genetic screen for genes involved in senescence: (Traicoff et al., 2007). role of Tid1, a homologue of the Drosophila tumor suppressor Cardiomyopathy l(2)tid, in senescence and cell survival. Mol Cell Biol. 2004 Dec;24(24):10792-801 Note Trentin GA, He Y, Wu DC, Tang D, Rozakis-Adcock M. Mice deficient in Dnaja3 developed dilated Identification of a hTid-1 mutation which sensitizes gliomas to cardiomyopathy (DCM) and died before 10 weeks of apoptosis. FEBS Lett. 2004 Dec 17;578(3):323-30 age (Hayashi et al., 2006). Progressive respiratory Bae MK, Jeong JW, Kim SH, Kim SY, Kang HJ, Kim DM, Bae chain deficiency and decreased copy number of SK, Yun I, Trentin GA, Rozakis-Adcock M, Kim KW. Tid-1 mitochondrial DNA were observed in cardiomyocytes interacts with the von Hippel-Lindau protein and modulates lacking Dnaja3 (Hayashi et al., 2006). angiogenesis by destabilization of HIF-1alpha. 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Biochemical and functional characterization of Epstein-Barr virus-encoded BARF1 protein: Syken J, Macian F, Agarwal S, Rao A, Münger K. TID1, a interaction with human hTid1 protein facilitates its maturation mammalian homologue of the drosophila tumor suppressor and secretion. Oncogene. 2006 Jul 20;25(31):4320-31 lethal(2) tumorous imaginal discs, regulates activation-induced cell death in Th2 cells. Oncogene. 2003 Jul 24;22(30):4636-41 Kurzik-Dumke U, Czaja J. Htid-1, the human homolog of the Drosophila melanogaster l(2)tid tumor suppressor, defines a novel physiological role of APC. Cell Signal. 2007 Sep;19(9):1973-85

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Traicoff JL, Chung JY, Braunschweig T, Mazo I, Shu Y, breast cancers over expressing the receptor. J Transl Med. Ramesh A, D'Amico MW, Galperin MM, Knezevic V, Hewitt 2010 Jun 17;8:58 SM. Expression of EIF3-p48/INT6, TID1 and Patched in cancer, a profiling of multiple tumor types and correlation of Maselli RA, Arredondo J, Cagney O, Ng JJ, Anderson JA, expression. J Biomed Sci. 2007 May;14(3):395-405 Williams C, Gerke BJ, Soliven B, Wollmann RL. Mutations in MUSK causing congenital myasthenic syndrome impair MuSK- Wakabayashi Y, Mao JH, Brown K, Girardi M, Balmain A. Dok-7 interaction. Hum Mol Genet. 2010 Jun 15;19(12):2370-9 Promotion of Hras-induced squamous carcinomas by a polymorphic variant of the Patched gene in FVB mice. Nature. Qian J, Perchiniak EM, Sun K, Groden J. The mitochondrial 2007 Feb 15;445(7129):761-5 protein hTID-1 partners with the caspase-cleaved adenomatous polyposis cell tumor suppressor to facilitate Kurzik-Dumke U, Hörner M, Czaja J, Nicotra MR, Simiantonaki apoptosis. Gastroenterology. 2010 Apr;138(4):1418-28 N, Koslowski M, Natali PG. Progression of colorectal cancers correlates with overexpression and loss of polarization of Trinh DL, Elwi AN, Kim SW. Direct interaction between p53 expression of the htid-1 tumor suppressor. Int J Mol Med. 2008 and Tid1 proteins affects p53 mitochondrial localization and Jan;21(1):19-31 apoptosis. Oncotarget. 2010 Oct;1(6):396-404 Linnoila J, Wang Y, Yao Y, Wang ZZ. A mammalian homolog Copeland E, Balgobin S, Lee CM, Rozakis-Adcock M. hTID-1 of Drosophila tumorous imaginal discs, Tid1, mediates agrin defines a novel regulator of c-Met Receptor signaling in renal signaling at the neuromuscular junction. Neuron. 2008 Nov cell carcinomas. Oncogene. 2011 May 12;30(19):2252-63 26;60(4):625-41 Dhennin-Duthille I, Nyga R, Yahiaoui S, Gouilleux-Gruart V, Song Y, Balice-Gordon R. New dogs in the dogma: Lrp4 and Régnier A, Lassoued K, Gouilleux F. The tumor suppressor Tid1 in neuromuscular synapse formation. Neuron. 2008 Nov hTid1 inhibits STAT5b activity via functional interaction. J Biol 26;60(4):526-8 Chem. 2011 Feb 18;286(7):5034-42 Chen CY, Chiou SH, Huang CY, Jan CI, Lin SC, Hu WY, Chou Jan CI, Yu CC, Hung MC, Harn HJ, Nieh S, Lee HS, Lou MA, SH, Liu CJ, Lo JF. Tid1 functions as a tumour suppressor in Wu YC, Chen CY, Huang CY, Chen FN, Lo JF. Tid1, CHIP and head and neck squamous cell carcinoma. J Pathol. 2009 ErbB2 interactions and their prognostic implications for breast Nov;219(3):347-55 cancer patients. J Pathol. 2011 Nov;225(3):424-37 Ahn BY, Trinh DL, Zajchowski LD, Lee B, Elwi AN, Kim SW. This article should be referenced as such: Tid1 is a new regulator of p53 mitochondrial translocation and apoptosis in cancer. Oncogene. 2010 Feb 25;29(8):1155-66 Traicoff JL, Hewitt SM, Chung JY. DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3). Atlas Genet Cytogenet Kurzik-Dumke U, Hörner M, Nicotra MR, Koslowski M, Natali Oncol Haematol. 2012; 16(3):196-204. PG. In vivo evidence of htid suppressive activity on ErbB-2 in

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Gene Section Short Communication

MYEOV (myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas)) Jérôme Moreaux INSERM U1040, institut de recherche en biotherapie, CHRU Saint Eloi, 80 Av Augustain Fliche, 34295 Montpellier CEDEX 5, France (JM)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/MYEOVID395.html DOI: 10.4267/2042/47277 This article is an update of : Janssen JWG. MYEOV myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas). Atlas Genet Cytogenet Oncol Haematol 2003;7(1):23-24.

The presence of functional domains such as RNP-1 Identity (motif typical of RNA binding protein) and the studies Other names: OCIM of the short hydrophobic regions and of the C-terminal HGNC (Hugo): MYEOV leucine/isoleucine tail showed that MYEOV might be directed to the membrane. MYEOV small interfering Location: 11q13.3 RNA (siRNA) decreased proliferation of gastric cancer Local order: 350 kb centromeric of cyclin D1. cells, colon cancer cell lines and multiple myeloma Note cells in vitro. Detected by application of the NIH/3T3 tumorigenicity Description assay. However MYEOV cDNA was not positive in 2 exons; 3,5 kb transcript represents unspliced mRNA. NIH/3T3 assay. Transcription DNA/RNA Main transcript 2,8 kb (broad band because of alternative splice products); minor transcript 3,5 kb; Note coding sequence 313 or 255 amino acids. In normal The MYEOV gene was originally isolated by the tissues hardly any expression detectable. High application of the NIH/3T3 tumorigenicity assay with expression in a subset of multiple myeloma cell lines DNA from a gastric carcinoma. The chromosomal with a t(11;14)(q13;q32) and in breast tumors and region 11q13 is frequently associated with genetic esophageal squamous cell carcinomas with or without rearrangements in a large number of human 11q13 amplification. malignancies, including B-cell malignancies and overexpression of MYEOV is frequently observed in Pseudogene breast tumors and oral, esophageal squamous cell No pseudogenes have been reported for MYEOV. carcinomas and multiple myeloma.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 205 MYEOV (myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas)) Moreaux J

patients with newly diagnosed MM express MYEOV Protein gene. A treatment with 5-aza-2'-deoxycytidine of 2 absent Description MYEOV myeloma cell lines induced MYEOV expression without affecting that in the MYEOV present 313 or 255 amino acids; contains one RNP-1 motif and myeloma cells. MYEOV siRNA did not significantly 6 regions that might function as a transmembrane induce apoptosis in myeloma cell lines, but it blocked domain. Leucine-rich stretch at C-terminal. the cell cycle entry into the S-phase. Expression Colon cancer 5' UTR inhibits efficient translation of the protein. Oncogenesis Localisation Knockout of MYEOV RNA (siRNA) has been shown In endoplasmic reticulum and mitochondria. to decrease proliferation of colon cancer cell lines in vitro. Furthermore, in colon cancer, MYEOV Homology stimulates colorectal cancer cell migration in vitro. No known homology. MYEOV expression is enhanced by PGE2 treatment in colorectal cancer cells. Implicated in Gastric cancer t(11;14)(q13;q32) Oncogenesis Disease Knockout of MYEOV RNA (siRNA) has been shown Subset of multiple myeloma cell lines with to decrease proliferation and invasion of gastric cancer t(11;14)(q13;q32). cells in vitro. Cytogenetics Neuroblastoma MYEOV overexpression due to juxtaposition to the 5' Oncogenesis enhancer or the most telomeric 3' enhancer of the MYEOV is a candidate gene target in neuroblastoma immunoglobulin heavy chain (IgH). that was identified by chromosomal gain 11q13 11q13 amplification and/or through SNP analysis. MYEOV expression was overexpression analyzed in 55 neuroblastoma samples including 25 cell lines. MYEOV was shown to be upregulated in 11 Disease out of 25 neuroblastoma cell lines and 7 out of 20 fresh Breast cancer; esophageal squamous cell carcinomas. tumors. Knockout of MYEOV RNA (siRNA) has been Prognosis shown to decrease proliferation of neuroblastoma cell MYEOV DNA amplification correlated with estrogen line in vitro. and progesterone receptor-positive cancer, invasive Oral squamous cell carcinoma lobular carcinoma type and axillary nodal involvement. Oncogenesis In contrast to Cyclin D1 amplification, no association with disease outcome could be found. Gain of 11q13 was significantly associated with cervical lymph node metastasis in oral squamous cell Multiple myeloma carcinoma (54 patients included in the study). Copy Prognosis number amplification of MYEOV is associated with In a cohort of 171 myeloma patients, patients with cervical lymph node metastasis in oral squamous cell MYEOV absent MMC have an increased event-free carcinoma. Lymph node metastasis is associated with a survival compared to patients with MYEOVpresent significant decrease of 5-years survival in oral MMC, after high-dose therapy and stem cell squamous cell carcinoma. transplantation and a trend for increased overall survival. In a Cox proportional hazard model, MYEOV References expression in MMC is predictive for event-free survival Janssen JW, Vaandrager JW, Heuser T, Jauch A, Kluin PM, for patients independently of International Staging Geelen E, Bergsagel PL, Kuehl WM, Drexler HG, Otsuki T, System stage, t(4;14) translocation, albumin, or B2M Bartram CR, Schuuring E. Concurrent activation of a novel serum levels. In a second independent cohort of 208 putative transforming gene, myeov, and cyclin D1 in a subset of multiple myeloma cell lines with t(11;14)(q13;q32). Blood. patients (LR-TT3, from the University of Arkansas for 2000 Apr 15;95(8):2691-8 Medical Sciences (Little Rock, AR, USA)), MYEOV had a "present" call in MMCs of 73% of patients. Janssen JW, Cuny M, Orsetti B, Rodriguez C, Vallés H, absent Bartram CR, Schuuring E, Theillet C. MYEOV: a candidate Patients with MYEOV MMCs had a significant gene for DNA amplification events occurring centromeric to better overall survival in the LR-TT3 cohort. CCND1 in breast cancer. Int J Cancer. 2002 Dec 20;102(6):608-14 Oncogenesis In a cohort of 171 patients, MMC of 79% of the Janssen JW, Imoto I, Inoue J, Shimada Y, Ueda M, Imamura M, Bartram CR, Inazawa J. MYEOV, a gene at 11q13, is

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 206 MYEOV (myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas)) Moreaux J

coamplified with CCND1, but epigenetically inactivated in a multiple myeloma. Exp Hematol. 2010 Dec;38(12):1189- subset of esophageal squamous cell carcinomas. J Hum 1198.e3 Genet. 2002;47(9):460-4 Sugahara K, Michikawa Y, Ishikawa K, Shoji Y, Iwakawa M, Leyden J, Murray D, Moss A, Arumuguma M, Doyle E, Shibahara T, Imai T. Combination effects of distinct cores in McEntee G, O'Keane C, Doran P, MacMathuna P. Net1 and 11q13 amplification region on cervical lymph node metastasis Myeov: computationally identified mediators of gastric cancer. of oral squamous cell carcinoma. Int J Oncol. 2011 Br J Cancer. 2006 Apr 24;94(8):1204-12 Oct;39(4):761-9 Moss AC, Lawlor G, Murray D, Tighe D, Madden SF, Mulligan Takita J, Chen Y, Okubo J, Sanada M, Adachi M, Ohki K, AM, Keane CO, Brady HR, Doran PP, MacMathuna P. ETV4 Nishimura R, Hanada R, Igarashi T, Hayashi Y, Ogawa S. and Myeov knockdown impairs colon cancer cell line Aberrations of NEGR1 on 1p31 and MYEOV on 11q13 in proliferation and invasion. Biochem Biophys Res Commun. neuroblastoma. Cancer Sci. 2011 Sep;102(9):1645-50 2006 Jun 23;345(1):216-21 This article should be referenced as such: Lawlor G, Doran PP, MacMathuna P, Murray DW. MYEOV (myeloma overexpressed gene) drives colon cancer cell Moreaux J. MYEOV (myeloma overexpressed (in a subset of migration and is regulated by PGE2. J Exp Clin Cancer Res. t(11;14) positive multiple myelomas)). Atlas Genet Cytogenet 2010 Jun 22;29:81 Oncol Haematol. 2012; 16(3):205-207. Moreaux J, Hose D, Bonnefond A, Reme T, Robert N, Goldschmidt H, Klein B. MYEOV is a prognostic factor in

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Gene Section Review

PCNA (proliferating cell nuclear antigen) Ivaylo Stoimenov, Thomas Helleday Department of Genetics Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden (IS), Department of Genetics Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden; Gray Institute for Radiation Oncology & Biology, University of Oxford, Oxford, OX3 7DQ, UK (TH)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/PCNAID41670ch20p12.html DOI: 10.4267/2042/47278 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Transcription There are two reported gene transcripts, which encode Other names: MGC8367 the same protein. HGNC (Hugo): PCNA PCNA transcript variant 1 is 1355 bp long after the Location: 20p12.3 completion of mRNA splicing. It has NCBI Reference Sequence code NM_002592.2 (NCBI). The PCNA DNA/RNA transcript variant 1 has seven exons, six of which are contributing to the protein sequence. The first intron is Description relatively large in comparison with the other PCNA The PCNA gene is situated on human chromosome 20 transcript variant. Following the splicing the length of and it spans about 12 kb. It is a single-copy gene, the transcript is shortened to about 12% of that of the initial transcript. The translation starts from the middle however, several pseudogenes have been noted. The nd th precise localization of the PCNA gene is at the border of the 2 exon and ends in the beginning of 7 exon. of two histological G-bands (p12.3 and p13) (Webb et The product is a full length protein, designated as al., 1990), thus it is reported in both locations NP_002583.1 (NCBI), with 261 amino acids. depending on the probe used. The human PCNA gene PCNA transcript variant 2 is 1319 bp long after the was first cloned and characterized in 1989 by Travali completion of mRNA splicing. and co-workers (Travali et al., 1989).

The localisation of the PCNA gene (in red) at the interface between 20p12.3 and 20p13 histological bands on chromosome 20.

NCBI Reference Length Length Exons Protein AA Sequence (unspliced) (spliced) PCNA transcript variant 1 NM_002592.2 11670 bp 1355 bp 7 NP_002583.1 261 PCNA transcript variant 2 NM_182649.1 5049 bp 1319 bp 6 NP_872590.1 261

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 208 PCNA (proliferating cell nuclear antigen) Stoimenov I, Helleday T

It has NCBI Reference Sequence code NM_182649.1 Function (NCBI). The PCNA transcript variant 2 has six exons, PCNA was originally discovered as an antigen, reacting which are contributing to the protein sequence. After with antibodies derived from sera of patients with the splicing the length of the transcript is shortened to systemic lupus erythematosus (Miyachi et al., 1978). about 26% of that of the initial transcript. Translation The first assigned function of the PCNA protein is as starts from the end of the 1 st exon and ends in the an auxiliary factor of polymerase delta (Tan et al., beginning of 7 th exon. The product is a full length 1986; Prelich et al., 1987). Later it was suggested that protein, designated as NP_872590.1 (NCBI), with 261 PCNA functions as a cofactor to many other eukaryotic amino acids. polymerases such as polymerase epsilon, polymerase Pseudogene beta and several specialised polymerases known as PCNAP - one pseudogene on human chromosome X - translesion synthesis polymerases (eta, kappa, lambda, p11 (Ku et al., 1989; Webb et al., 1990). theta, etc.), with which PCNA is known to interact PCNAP1 and PCNAP2 - two pseudogenes in tandem (Naryzhny, 2008). The role of PCNA in DNA on human chromosome 4 - q24 (Taniguchi et al., 1996). replication is thoroughly investigated and PCNA is There are several other possible pseudogenes: proposed to serve as a switch between the priming LOC390102 on chromosome 11 - p15.1 (Webb et al., polymerase alpha and replicative polymerases (delta 1990), LOC392454 on chromosome X - p11.3 (Ku et and epsilon) and functioning as a cofator of the latter al., 1989; Webb et al., 1990). polymerases. Complementary to enhancing the processivity of DNA replication, PCNA is known to Protein coordinate the maturation of Okazaki fragments through interaction with FEN1 and stimulation of the Description flap endonuclease activity. PCNA interacts with large The human PCNA protein is a polypeptide of 261 number of proteins, suggesting many functions in vivo amino acids and theoretical molecular weight of about (Naryzhny, 2008; Stoimenov and Helleday, 2009). 29 kDa. The functional protein is a homotrimer, build There is evidence, derived from experiments in yeast, from three identical units interacting head-to-tail and that PCNA may be involved in the establishment of forming a doughnut shaped molecule. There is an sister chromatid cohesion in S phase of the cell cycle evidence for the existence of a double homotrimer in (Moldovan et al., 2006). PCNA is an indispensable vivo (Naryzhny et al., 2005). factor for different DNA repair pathways including mismatch repair, nucleotide excision repair and sub- Expression pathways of base excision repair. There is a growing Expressed in nearly all proliferating tissues with high body of evidence for the function of PCNA in the levels detected in thymus, bone marrow, foetal liver chromatin remodelling and organisation. The and certain cells of the small intestine and colon. interaction of PCNA and CAF1 is in the heart of the Localisation nucleosome assembly, while the chromatin modification is also known to be regulated by PCNA PCNA is exclusively localized in the nucleus. It can be through the known interaction with DNMT1 and detected by immunofluorescence in all proliferating HDAC1. nuclei as discrete nuclear foci, representing sites of ongoing DNA replication and/or DNA repair.

PCNA and mapped interactions with several proteins (D-type of cyclins, CDKN1A, FEN1, RFC complex, polymerase epsilon and polymerase delta). Two residues are highlighted, lysine at position 164 (site of ubiquitylation) and tyrosine at position 211 (site of phosphorylation).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 209 PCNA (proliferating cell nuclear antigen) Stoimenov I, Helleday T

One of the most stable interactions of PCNA is that stated to be an independent predictor in primary breast with the cyclin-kinase inhibitor CDKN1A, which cancer patients (Horiguchi et al., 1998) with a suggests a role of PCNA in the cell cycle progression. prognostic value (Chu et al., 1998). Another evidence for the involvement of PCNA in the Chronic lymphoid leukemia (CLL) cell cylcle control is the interaction with cyclin-D. Several amino-acid residues are post-translationally Note modified, suggesting even more complex functions There are attempts to correlate the levels of the PCNA (Stoimenov and Helleday, 2009). PCNA could be protein in cells derived from patients with chronic subjected to post-translational phosphorylation, lymphoid leukemia and the prognosis of survival (del acetylation, methylation, ubiquitylation and Giglio et al., 1992; Faderl et al., 2002). The high level SUMOylation. of PCNA in the cells of CLL patients suggests a higher proliferative activity and potentially shorter survival Implicated in (del Giglio et al., 1992). Intracellular levels of PCNA protein can be used as marker to predict clinical Note behaviour and overall survival in patients with CLL The absence of the proliferating nuclear cell antigen (Faderl et al., 2002). (PCNA) protein is embryonic lethal in mice (Roa et al., Non-Hodgkin's lymphoma 2008; Peled et al., 2008). The embryonic lethality in mice also suggests a critical importance of the PCNA Note protein for humans at least in proliferating tissues In studies conducting immunohistochemical staining of (Moldovan et al., 2007). The knockout mice for PCNA materials from patients with non-Hodgkin's lymphoma, (Pcna -/-) are dying in embryonic state, consistent with PCNA labeling index together with AgNOR score can the role of PCNA in orchestrating DNA replication be used to predict overall survival (Korkolopoulou et (Moldovan et al., 2007). In addition to this fact, there al., 1998). PCNA is the only independent predictor of are no known mutations of the PCNA protein in the post-relapse survival and the histologic grade, humans, which therefore leads to a speculation that which is the most important indicator of disease-free PCNA is so vital that any alternation of its sequence survival (Korkolopoulou et al., 1998). would have deleterious consequences. One suggestion Malignant and nonmalignant skin for such essential function is the fact that both diseases sequences of the PCNA protein and of the respective gene are highly conserved during evolution (Stoimenov Note and Helleday, 2009). Indeed, a human population study In one study of comparison between malignant skin of PCNA polymorphisms shows only 7 intronic single diseases (squamous cell carcinoma, adult T nucleotide polymorphisms (SNP) and 2 synonymous lymphotrophic leukemia, mycosis fungoides, malignant exonic SNPs (Ma et al., 2000). melanoma and malignant lymphoma) and According to OMIM and Human Locus Specific nonmalignant skin diseases (resistant atopic dermatitis, Mutation Databases there is no known disease, which is psoriasis vulgaris, verruca vulgaris) the anti-PCNA caused by mutation or loss of function of the PCNA staining was used as a prognostic marker (Kawahira, protein. 1999). The percentage of PCNA-positive cells reported The only implication of PCNA in human disease is as a in the study was higher for malignant skin diseases in prognostic or diagnostic marker, sometimes used comparison with the non-malignant skin deseases together with other markers. The utilisation of PCNA (Kawahira, 1999). The localization of PCNA-positive as a marker is very much restricted to an illustration of cells was found to be in the dermis and the basal layer proliferation potential and therefore cannot be specific in case of the malignant skin diseases, whereas in the for any disease. However, PCNA is indeed used as a nonmalignant skin diseases PCNA-positive cells were prognostic and diagnostic marker in several human detected only in the basal layer (Kawahira, 1999). The diseases in clinical practice, as shown below. The list is PCNA labeling index and the distribution of PCNA- far from complete since any human disease associated positive cells in the skin were suggested to be helpful in with proliferation could utilise PCNA as a marker. the early diagnosis of skin malignancies. Primary breast cancer Systemic lupus erythematosus (SLE) Note Note A group of patients with high PCNA labeling index The anti-PCNA antibodies were originally found in was associated with poor overall survival compared patients with systemic lupus erythematosus (Miyachi et with the low PCNA labeling index group in several al., 1978), most of whom had diffuse proliferative immunohistochemical studies (Horiguchi et al., 1998; glomerulonephritis in a small clinical study (Fritzler et Chu et al., 1998). PCNA labeling index is al., 1983).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 210 PCNA (proliferating cell nuclear antigen) Stoimenov I, Helleday T

Korkolopoulou P, Angelopoulou MK, Kontopidou F, Tsengas A, References Patsouris E, Kittas C, Pangalis GA. Prognostic implications of proliferating cell nuclear antigen (PCNA), AgNORs and P53 in Miyachi K, Fritzler MJ, Tan EM. Autoantibody to a nuclear non-Hodgkin's lymphomas. Leuk Lymphoma. 1998 Aug;30(5- antigen in proliferating cells. J Immunol. 1978 6):625-36 Dec;121(6):2228-34 Kawahira K. Immunohistochemical staining of proliferating cell Fritzler MJ, McCarty GA, Ryan JP, Kinsella TD. Clinical nuclear antigen (PCNA) in malignant and nonmalignant skin features of patients with antibodies directed against diseases. Arch Dermatol Res. 1999 Jul-Aug;291(7-8):413-8 proliferating cell nuclear antigen. Arthritis Rheum. 1983 Feb;26(2):140-5 Ma X, Jin Q, Försti A, Hemminki K, Kumar R. Single nucleotide polymorphism analyses of the human proliferating cell nuclear Tan CK, Castillo C, So AG, Downey KM. An auxiliary protein antigen (pCNA) and flap endonuclease (FEN1) genes. Int J for DNA polymerase-delta from fetal calf thymus. J Biol Chem. Cancer. 2000 Dec 15;88(6):938-42 1986 Sep 15;261(26):12310-6 Faderl S, Keating MJ, Do KA, Liang SY, Kantarjian HM, Prelich G, Tan CK, Kostura M, Mathews MB, So AG, Downey O'Brien S, Garcia-Manero G, Manshouri T, Albitar M. KM, Stillman B. Functional identity of proliferating cell nuclear Expression profile of 11 proteins and their prognostic antigen and a DNA polymerase-delta auxiliary protein. Nature. significance in patients with chronic lymphocytic leukemia 1987 Apr 2-8;326(6112):517-20 (CLL). Leukemia. 2002 Jun;16(6):1045-52 Ku DH, Travali S, Calabretta B, Huebner K, Baserga R. Human Naryzhny SN, Zhao H, Lee H. Proliferating cell nuclear antigen gene for proliferating cell nuclear antigen has pseudogenes (PCNA) may function as a double homotrimer complex in the and localizes to chromosome 20. Somat Cell Mol Genet. 1989 mammalian cell. J Biol Chem. 2005 Apr 8;280(14):13888-94 Jul;15(4):297-307 Moldovan GL, Pfander B, Jentsch S. PCNA controls Travali S, Ku DH, Rizzo MG, Ottavio L, Baserga R, Calabretta establishment of sister chromatid cohesion during S phase. B. Structure of the human gene for the proliferating cell nuclear Mol Cell. 2006 Sep 1;23(5):723-32 antigen. J Biol Chem. 1989 May 5;264(13):7466-72 Moldovan GL, Pfander B, Jentsch S. PCNA, the maestro of the Webb G, Parsons P, Chenevix-Trench G. Localization of the replication fork. Cell. 2007 May 18;129(4):665-79 gene for human proliferating nuclear antigen/cyclin by in situ hybridization. Hum Genet. 1990 Nov;86(1):84-6 Naryzhny SN. Proliferating cell nuclear antigen: a proteomics view. Cell Mol Life Sci. 2008 Nov;65(23):3789-808 del Giglio A, O'Brien S, Ford R, Saya H, Manning J, Keating M, Johnston D, Khetan R, el-Naggar A, Deisseroth A. Prognostic Peled JU, Kuang FL, Iglesias-Ussel MD, Roa S, Kalis SL, value of proliferating cell nuclear antigen expression in chronic Goodman MF, Scharff MD. The biochemistry of somatic lymphoid leukemia. Blood. 1992 May 15;79(10):2717-20 hypermutation. Annu Rev Immunol. 2008;26:481-511 Taniguchi Y, Katsumata Y, Koido S, Suemizu H, Yoshimura S, Roa S, Avdievich E, Peled JU, Maccarthy T, Werling U, Kuang Moriuchi T, Okumura K, Kagotani K, Taguchi H, Imanishi T, FL, Kan R, Zhao C, Bergman A, Cohen PE, Edelmann W, Gojobori T, Inoko H. Cloning, sequencing, and chromosomal Scharff MD. Ubiquitylated PCNA plays a role in somatic localization of two tandemly arranged human pseudogenes for hypermutation and class-switch recombination and is required the proliferating cell nuclear antigen (PCNA). Mamm Genome. for meiotic progression. Proc Natl Acad Sci U S A. 2008 Oct 1996 Dec;7(12):906-8 21;105(42):16248-53 Chu JS, Huang CS, Chang KJ. Proliferating cell nuclear Stoimenov I, Helleday T. PCNA on the crossroad of cancer. antigen (PCNA) immunolabeling as a prognostic factor in Biochem Soc Trans. 2009 Jun;37(Pt 3):605-13 invasive ductal carcinoma of the breast in Taiwan. Cancer Lett. 1998 Sep 25;131(2):145-52 This article should be referenced as such: Horiguchi J, Iino Y, Takei H, Maemura M, Takeyoshi I, Yokoe Stoimenov I, Helleday T. PCNA (proliferating cell nuclear T, Ohwada S, Oyama T, Nakajima T, Morishita Y. Long-term antigen). Atlas Genet Cytogenet Oncol Haematol. 2012; prognostic value of PCNA labeling index in primary operable 16(3):208-211. breast cancer. Oncol Rep. 1998 May-Jun;5(3):641-4

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Gene Section Short Communication

RASSF5 (Ras association (RalGDS/AF -6) domain family member 5) Lee Schmidt, Geoffrey J Clark University of Louisville, Room 119C, Baxter II Research Building, 580 S Preston Street, Louisville, KY 40202, USA (LS, GJC) Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/RASSF5ID42059ch1q32.html DOI: 10.4267/2042/47279 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity DNA/RNA Other names: MGC10823, MGC17344, Maxp1, Description NORE1, NORE1A, NORE1B, RAPL, RASSF3 The human gene for RASSF5 is 81 kb in length and is HGNC (Hugo): RASSF5 located on chromosome 1(q32.1). The gene can Location: 1q32.1 produce 4 protein isoforms, two via differential exon usage, a third via differential promoter usage and the genesis of the 4 th (which can be found as an EST clone) is not yet clear. The largest isoform, A, is 418 amino acids long and has a molecular weight of about 47 kD. The protein structure of RASSF5A contains a proline- rich region at the N-terminus which contains potential SH3 binding sites and a nuclear localization signal. This is followed by a cystein rich domain, sometimes Note referred to as a zinc finger. Next is the Ras association Murine RASSF5 originally named Nore1a. Nore1B domain and this is followed by sequence containing the independently identified and designated RAPL. Rat SARAH motif required for binding to the pro-apoptotic RASSF5 also cloned independently and designated kinases MST1 and MST2. A second nuclear Maxp1. localization sequence has been reported between amino acids 200-260 and a nuclear export sequence between amino acids 260-300.

Figure 1. Isoform A is shown as the longest isoform with 6 exons. Isoform B, without an alternate exon, shows that the frameshift gives a shortened and unique C-terminus. Isoform C is shown with a special 5' UTR and lacks an in-frame coding region leading to a unique N- terminus. The total coding sequence for Isoform A is about 1260 bases with the other isoforms being smaller.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 212 RASSF5 (Ras association (RalGDS/AF-6) domain family member 5) Schmidt L, Clark GJ

Figure 2. A figure showing the processed mRNA as well as the amino acid sequence for isoforms A-D followed by motif explanation of isoform A (Nore1a).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 213 RASSF5 (Ras association (RalGDS/AF-6) domain family member 5) Schmidt L, Clark GJ

Protein References Description Vavvas D, Li X, Avruch J, Zhang XF. Identification of Nore1 as a potential Ras effector. J Biol Chem. 1998 Mar The full length cDNA (for isoform A) encodes for a 47- 6;273(10):5439-42 kDa protein which contains a proline-rich region at the Khokhlatchev A, Rabizadeh S, Xavier R, Nedwidek M, Chen T, N-terminus followed by a putative Zhang XF, Seed B, Avruch J. Identification of a novel Ras- diacylglycerol/phorbol ester binding domain. This is regulated proapoptotic pathway. Curr Biol. 2002 Feb followed by the Ras association (RA) domain and then 19;12(4):253-65 the domain containing a SARAH motif. This later is Chen J, Lui WO, Vos MD, Clark GJ, Takahashi M, Schoumans responsible for binding to the pro-apoptotic kinases J, Khoo SK, Petillo D, Lavery T, Sugimura J, Astuti D, Zhang MST1 and MST2. C, Kagawa S, Maher ER, Larsson C, Alberts AS, Kanayama HO, Teh BT. The t(1;3) breakpoint-spanning genes LSAMP Expression and NORE1 are involved in clear cell renal cell carcinomas. Cancer Cell. 2003 Nov;4(5):405-13 Nore1a mRNA is expressed in the lung, kidney, liver, brain, spleen, thymus and heart. Hesson L, Dallol A, Minna JD, Maher ER, Latif F. NORE1A, a homologue of RASSF1A tumour suppressor gene is Localisation inactivated in human cancers. Oncogene. 2003 Feb 13;22(6):947-54 It can be detected on microtubules, in the centrosome, but appears most obvious in the nucleus. Vos MD, Martinez A, Ellis CA, Vallecorsa T, Clark GJ. The pro- apoptotic Ras effector Nore1 may serve as a Ras-regulated Function tumor suppressor in the lung. J Biol Chem. 2003 Jun 13;278(24):21938-43 RASSF5A is a pro-apoptotic Ras effector that can bind and relocalize the pro-apoptotic MST kinases in the Aoyama Y, Avruch J, Zhang XF. Nore1 inhibits tumor cell growth independent of Ras or the MST1/2 kinases. Oncogene. presence of activated Ras. It can also promote cell 2004 Apr 22;23(19):3426-33 cycle arrest and modulate the activity of p53 by Praskova M, Khoklatchev A, Ortiz-Vega S, Avruch J. regulating its' nuclear localization. Knockdown of Regulation of the MST1 kinase by autophosphorylation, by the RASSF5A promotes cellular proliferation and soft agar growth inhibitory proteins, RASSF1 and NORE1, and by Ras. growth. Thus, RASSF5A appears to function as a Ras Biochem J. 2004 Jul 15;381(Pt 2):453-62 regulated tumor suppressor. Analysis of human tumors Avruch J, Praskova M, Ortiz-Vega S, Liu M, Zhang XF. Nore1 has found little evidence of somatic mutation but the and RASSF1 regulation of cell proliferation and of the MST1/2 gene is frequently inactivated by promoter methylation kinases. Methods Enzymol. 2006;407:290-310 in a broad range of human tumors. RASSF5C (also Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee JS, known as Nore1b or RAPL) has been reported to Factor VM, Thorgeirsson SS. Ubiquitous activation of Ras and modulate cellular adhesion and to be regulated by the Jak/Stat pathways in human HCC. Gastroenterology. 2006 Ras related protein Rap1a. RASSF5C has also been Apr;130(4):1117-28 implicated as serving as an adaptor protein to facilitate Donninger H, Vos MD, Clark GJ. The RASSF1A tumor the interaction of Ras and CARMA1. suppressor. J Cell Sci. 2007 Sep 15;120(Pt 18):3163-72 Kumari G, Singhal PK, Rao MR, Mahalingam S. Nuclear Mutations transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for arginine-rich Note nuclear targeting signals. J Mol Biol. 2007 Apr 13;367(5):1294- 311 No tumor mutations yet reported. Calvisi DF, Donninger H, Vos MD, Birrer MJ, Gordon L, Leaner V, Clark GJ. NORE1A tumor suppressor candidate modulates Implicated in p21CIP1 via p53. Cancer Res. 2009 Jun 1;69(11):4629-37 Clear cell renal carcinoma Richter AM, Pfeifer GP, Dammann RH. The RASSF proteins in cancer; from epigenetic silencing to functional characterization. Note Biochim Biophys Acta. 2009 Dec;1796(2):114-28 RASSF5 is frequently down-regulated by promoter Bee C, Moshnikova A, Mellor CD, Molloy JE, Koryakina Y, methylation in a variety of tumors including clear cell Stieglitz B, Khokhlatchev A, Herrmann C. Growth and tumor renal carcinomas. Moreover, a rare hereditary form of suppressor NORE1A is a regulatory node between Ras kidney cancer has been reported that maps with a signaling and microtubule nucleation. J Biol Chem. 2010 May translocation inactivating the RASSF5 gene. 21;285(21):16258-66 Kumari G, Singhal PK, Suryaraja R, Mahalingam S. Functional Various cancers interaction of the Ras effector RASSF5 with the tyrosine kinase Note Lck: critical role in nucleocytoplasmic transport and cell cycle Nore1a is frequently inactivated by promoter regulation. J Mol Biol. 2010 Mar 19;397(1):89-109 methylation in renal carcinoma, breast cancer, lung Park J, Kang SI, Lee SY, Zhang XF, Kim MS, Beers LF, Lim cancer, liver cancer and neurological tumors. DS, Avruch J, Kim HS, Lee SB. Tumor suppressor ras

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 214 RASSF5 (Ras association (RalGDS/AF-6) domain family member 5) Schmidt L, Clark GJ

association domain family 5 (RASSF5/NORE1) mediates death This article should be referenced as such: receptor ligand-induced apoptosis. J Biol Chem. 2010 Nov 5;285(45):35029-38 Schmidt L, Clark GJ. RASSF5 (Ras association (RalGDS/AF- 6) domain family member 5). Atlas Genet Cytogenet Oncol Overmeyer JH, Maltese WA. Death pathways triggered by Haematol. 2012; 16(3):212-215. activated Ras in cancer cells. Front Biosci. 2011 Jan 1;16:1693-713

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Gene Section Short Communication

RGS17 (regulator of G -protein signaling 17) Chenguang Li, Lei Wang, Yihua Sun, Haiquan Chen Department of Thoracic Oncology, Fudan University Shanghai Cancer Center and Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China (CL, LW, YS, HC)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/RGS17ID47522ch6q25.html DOI: 10.4267/2042/47280 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Description The RGS17 protein consists of 210 amino acid Other names: RGS-17, RGSZ2, hRGS17 residues. This gene encodes a member of the regulator HGNC (Hugo): RGS17 of G-protein signaling family. This protein contains a Location: 6q25.2 conserved, 120 amino acid motif called the RGS domain and a cysteine-rich region. DNA/RNA Expression Description Widely expressed in human organs. The RGS17 gene spans over a region of 120 kbp DNA Localisation including 4 coding exons and 1 non-coding exon (exon Its cellular localization has not been formally 1). monitored to date. Transcription Function The RGS17 gene mRNA consists of about 1472 The protein attenuates the signaling activity of G- nucleotides with an open reading frame (ORF) of 633 proteins by binding to activated, GTP-bound G alpha bases. subunits and acting as a GTPase activating protein Pseudogene (GAP), increasing the rate of conversion of the GTP to GDP. RGS proteins are GTPase-activating proteins for RGS17P1 regulator of G-protein signaling 17 Gi and Gq class G-alpha proteins. They accelerate pseudogene 1. transit through the cycle of GTP binding and hydrolysis and thereby accelerate signaling kinetics and Protein termination. This hydrolysis allows the G alpha Note subunits to bind G beta/gamma subunit heterodimers, 210 amino acids; 24 kDa. forming inactive G-protein heterotrimers, thereby terminating the signal.

Diagram of the RGS17 protein in scale. The numbers represent specific residues. The regions are RGS_RZ-like (Regulator of G protein signaling (RGS) domain found in the RZ protein), putative G-alpha interaction site. C: Carboxyl-terminal; N: Amino-terminal.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 216 RGS17 (regulator of G-protein signaling 17) Li C, et al.

Homology Ovarian cancer The RGS17 gene is conserved in chimpanzee, dog, Disease cow, mouse, rat, chicken, and zebrafish. RGS2, RGS5, RGS10 and RGS17 transcripts are expressed at significantly lower levels in cells resistant Mutations to chemotherapy compared with parental, chemo- sensitive cells in ovarian cancer cells (Hooks et al., Germinal 2010). No germline mutations in this gene have been reported. Prognosis Somatic RGS17 loss of expression contributes to the A synonymous-coding somatic mutations of this gene development of chemoresistance in ovarian cancer is reported in pancreas cancer at codon 166, P166P cells. (COSMIC). References Implicated in James MA, Lu Y, Liu Y, Vikis HG, You M. RGS17, an overexpressed gene in human lung and prostate cancer, Various cancer induces tumor cell proliferation through the cyclic AMP-PKA- CREB pathway. Cancer Res. 2009 Mar 1;69(5):2108-16 Note Lung cancer, prostate cancer. You M, Wang D, Liu P, Vikis H, James M, Lu Y, Wang Y, Wang M, Chen Q, Jia D, Liu Y, Wen W, Yang P, et al. Fine Disease mapping of chromosome 6q23-25 region in familial lung cancer RSG17 is overexpressed in lung and prostate cancer families reveals RGS17 as a likely candidate gene. Clin Cancer Res. 2009 Apr 15;15(8):2666-74 (James et al., 2009). Expression of RGS17 is upregulated in 80% of lung tumors, and also up-regulated Hooks SB, Callihan P, Altman MK, Hurst JH, Ali MW, Murph in prostate tumors. Overexpression of RGS17 induce MM. Regulators of G-Protein signaling RGS10 and RGS17 regulate chemoresistance in ovarian cancer cells. Mol Cancer. and maintain cell proliferation. 2010 Nov 2;9:289 Lung cancer Sun Y, Fang R, Li C, Li L, Li F, Ye X, Chen H. Hsa-mir-182 suppresses lung tumorigenesis through down regulation of Disease RGS17 expression in vitro. Biochem Biophys Res Commun. hsa-mir-182 is involved in the down regulation of 2010 May 28;396(2):501-7 RGS17 expression through two conserved sites located in its 3' UTR region (Sun et al., 2010). This article should be referenced as such: Two SNPs in the first intron of RGS17 (rs4083914 and Li C, Wang L, Sun Y, Chen H. RGS17 (regulator of G-protein rs9479510) were found associated with familial lung signaling 17). Atlas Genet Cytogenet Oncol Haematol. 2012; cancer susceptibility (You et al., 2009). 16(3):216-217.

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Gene Section Short Communication

SLC39A1 (solute carrier family 39 (zinc transporter), member 1) Renty B Franklin, Leslie C Costello Department of Oncology and Diagnostic Sciences, Dental School and The Greenebaum Cancer Center, University of Maryland, Baltimore, MD, 21201, USA (RBF, LCC)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/SLC39A1ID46571ch1q21.html DOI: 10.4267/2042/47281 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Expression ZIP1 is ubiquitously expressed in mammalian cells Other names: ZIP1, ZIRTL (Gaither and Eide, 2001). Expression is down regulated HGNC (Hugo): SLC39A1 in prostate malignancy (Franklin et al., 2005). Its Location: 1q21.3 constitutive expression is reported to be regulated by SP and CREB1 (Makhov et al., 2009). Down regulation DNA/RNA of expression in prostate malignancy is reported due to transcription repression by ras response element Description binding protein-1 (RREB-1) (Milon et al., 2010). The SLC39A1 gene contains 5 exons, three of which Localisation are coding. The length of the gene is 8600 base pairs ZIP1 is located at the cell membrane. according to the Entrez Gene database. Several transcripts have been reported containing either 3, 4, or Function 5 exons. However, the coding sequence for SLC39A1 ZIP1 is a facilitated zinc uptake transporter (Franklin et is the same for all reported transcripts. al., 2003). Activity of the transporter results in the Transcription intracellular accumulation of zinc. ZIP1 is involved in apoptosis induction in prostate cancer cells. Only a single isoform has been reported; mRNA 2445 bases in length; coding region of 975 bases. Homology Pseudogene Mus musculus Slc39a1; Rattus norvegicus Slc39a1; Bos taurus Slc39a1; Danio rerio slc39a1. None reported. Protein Mutations Note Description No diseases related to mutation are reported. SLC39A1 encodes Zrt/Irt-like protein family member 1 (ZIP1). Implicated in ZIP1 is a 35 kDa molecular weight protein consisting of 324 amino acids. The protein contains 8 Prostate cancer transmembrane spanning domains and shows the Note characteristics of a zinc transporter (Gaither and Eide, SLC39A1 is down regulated in prostate cancer 2001).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 218 SLC39A1 (solute carrier family 39 (zinc transporter), member 1) Franklin RB, Costello LC

(Franklin et al., 2005). Knockdown of the SLC39A1 Golovine K, Makhov P, Uzzo RG, Shaw T, Kunkle D, Kolenko expression decreases cellular zinc and increases growth VM. Overexpression of the zinc uptake transporter hZIP1 inhibits nuclear factor-kappaB and reduces the malignant of PC-3 cells (Franklin et al., 2003). Over expression of potential of prostate cancer cells in vitro and in vivo. Clin SLC39A1 inhibits prostate tumor growth in a xenograft Cancer Res. 2008 Sep 1;14(17):5376-84 model (Golovine et al., 2008). Makhov P, Golovine K, Uzzo RG, Wuestefeld T, Scoll BJ, Kolenko VM. Transcriptional regulation of the major zinc References uptake protein hZip1 in prostate cancer cells. Gene. 2009 Feb 15;431(1-2):39-46 Gaither LA, Eide DJ. The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells. J Biol Chem. Milon BC, Agyapong A, Bautista R, Costello LC, Franklin RB. 2001 Jun 22;276(25):22258-64 Ras responsive element binding protein-1 (RREB-1) down- regulates hZIP1 expression in prostate cancer cells. Prostate. Franklin RB, Ma J, Zou J, Guan Z, Kukoyi BI, Feng P, Costello 2010 Feb 15;70(3):288-96 LC. Human ZIP1 is a major zinc uptake transporter for the accumulation of zinc in prostate cells. J Inorg Biochem. 2003 This article should be referenced as such: Aug 1;96(2-3):435-42 Franklin RB, Costello LC. SLC39A1 (solute carrier family 39 Franklin RB, Feng P, Milon B, Desouki MM, Singh KK, (zinc transporter), member 1). Atlas Genet Cytogenet Oncol Kajdacsy-Balla A, Bagasra O, Costello LC. hZIP1 zinc uptake Haematol. 2012; 16(3):218-219. transporter down regulation and zinc depletion in prostate cancer. Mol Cancer. 2005 Sep 9;4:32

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Gene Section Mini Review

CBX7 (chromobox homolog 7) Ana O'Loghlen, Jesus Gil Cell Proliferation Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Campus, London W12 0NN, UK (AO, JG)

Published in Atlas Database: November 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/CBX7ID43845ch22q13.html DOI: 10.4267/2042/47282 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

251 amino acids. Isoelectric point: 10,0228. Molecular Identity weight of the protein: 28209 Da. HGNC (Hugo): CBX7 Description Location: 22q13.1 CBX7 has a chromodomain region which is commonly Note found in proteins associated with the remodelling and Orientation: minus strand. Size: 32508 bases. manipulation of chromatin. In mammals, chromodomain-containing proteins are responsible for DNA/RNA aspects of gene regulation related to chromatin remodelling and formation of heterochromatin regions. Description Chromodomain-containing proteins also bind DNA size is 4081 bp with 6 exons. CBX7 is a highly methylated histones and appear in the RNA-induced conserved gene in chimpanzee, dog, cow, rat and transcriptional silencing complex. Specifically, CBX7 mouse. is involved in maintaining the transcriptionally repressive state of its target genes. The better Transcription characterized target of CBX7 is the INK4a/ARF locus, mRNA size: 3964 bp. which is repressed by CBX7 in order to overcome the senescent phenotype in several mouse and human cell Protein lines. Repression of other targets like E-cadherin has been also suggested. Note

Figure 1. Location of Cbx7 within Chromosome 22.

Figure 2. Diagram of Cbx7 transcript. Cbx7 has 6 exons. The black boxes indicate the consensus coding sequences (CCDS).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 220 CBX7 (chromobox homolog 7) O'Loghlen A, Gil J

Figure 3. Structure of Cbx7 protein. Cbx7 has a chromodomain motif and a Polycomb (Pc) box which are indicated in grey.

Expression CBX7 is expressed ubiquitously, but at higher levels in the nervous system, thyroid gland, prostate, fallopian tubes and bladder in normal tissue. CBX7 expression is also high in ES cells. Localisation In the nucleus. Function CBX7 is a member of the Polycomb group (PcG) genes, which are transcriptional repressors that play an essential role in development, cancer progression and stem cell maintenance. Mainly two different PcG complexes have been described: Polycomb Repressive Complex 1 (PRC1) and PRC2. PRC2 is the complex implicated in initiating the silencing of its target genes by methylating histone H3 on lysines 9 and 27. PRC1 is implicated in stabilizing this repressive state by recognizing the methylation marks through the Polycomb proteins and by ubiquitinating the histone H2A on Lys119. CBX7 belongs to the PRC1 complex and has been described to be a regulator of cellular lifespan by repressing the INK4a/ARF locus in several mouse and human cell lines. On the other hand, depletion of CBX7 from the cell induces a senescent phenotype by increasing the expression of the cell cycle regulators p16/ARF. X chromosome inactivation CBX7 has high affinity for binding H3K9me3 and H3K27me3. It associates with heterochromatin, binds RNA and it's enriched in the X chromosome, giving CBX7 a role in maintaining the repression of genes in Figure 4. 4a: Summary of Cbx7's mechanism in embryonic the X chromosome. stem cells (ESC). Cbx7 is essential for ESC self-renewal. Loss of Cbx7, either by differentiating ESC or by an Epigenetic regulation exogenous/endogenous induction of the microRNA (miR) CBX7, as part of the PRC1 complex, has a role in families miR-125 and miR-181, induces ESC differentiation. maintaining the repressive state of its target genes. This is accompanied by an increase in other Cbxs as they are CBX7 binds to the long non-coding RNA ANRIL in targets of Cbx7. On the other hand, overexpression of Cbx7 in ESC reinforces pluripotency and keeps the cells in an ESC-like order to represses the INK4a/ARF locus and this state when forced to differentiate. 4b and 4c: Summary of interaction is essential for CBX7's function. Both Cbx7's mechanism in human primary fibroblasts (IMR-90). CBX7 and ANRIL have been found to have high levels Ectopic expression of the miR families miR-125 and miR-181 in prostate cancer tissues. induces a degradation of Cbx7 mRNA in IMR-90. Depletion of Cbx7 induces the cells to senesce. Thus, overexpression of Stem cells self-renewal miR-125 and miR-181 induces senescence through CBX7 has been recently implicated to be essential for downregulation of Cbx7. maintaining the pluripotency state of stem cells (ES cells). Overexpression of CBX7 in ESC impairs cell Mutations differentation. On the other hand, depletion of CBX7 from ESC induces spontaneous differentiation. Two Note different miR families (miR-125 and miR-181) were Expression of CBX7 without the Pc box or with point identified in a screening for CBX7 regulators and have mutations in the chromodomain region (F11A, K31A, been described to have a role in ESC differentiation by W32A, W35A) does not extend the life span of human targeting the 3'UTR of CBX7.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 221 CBX7 (chromobox homolog 7) O'Loghlen A, Gil J

or mouse cells. The mutant R17Q, which affects the Gil J, Bernard D, Peters G. Role of polycomb group proteins in binding of CBX7 to RNA, stem cell self-renewal and cancer. DNA Cell Biol. 2005 Feb;24(2):117-25 extended the lifespan of cells, but to a lesser extent than CBX7 wt. Point mutations in the Pc box as F234D or Bernstein E, Duncan EM, Masui O, Gil J, Heard E, Allis CD. Mouse polycomb proteins bind differentially to methylated F244D result in loss or reduced interaction of CBX7 histone H3 and RNA and are enriched in facultative with RNF2. heterochromatin. Mol Cell Biol. 2006 Apr;26(7):2560-9 Scott CL, Gil J, Hernando E, Teruya-Feldstein J, Narita M, Implicated in Martínez D, Visakorpi T, Mu D, Cordon-Cardo C, Peters G, Beach D, Lowe SW. Role of the chromobox protein CBX7 in Various cancers lymphomagenesis. Proc Natl Acad Sci U S A. 2007 Mar Disease 27;104(13):5389-94 CBX7 has been implicated in several tumors such as Pallante P, Federico A, Berlingieri MT, Bianco M, Ferraro A, gastric cancer, follicular lymphoma, breast cancer, Forzati F, Iaccarino A, Russo M, Pierantoni GM, Leone V, Sacchetti S, Troncone G, Santoro M, Fusco A. Loss of the colon carcinoma, pancreatic cancer, tyroid cancer, CBX7 gene expression correlates with a highly malignant glioma. phenotype in thyroid cancer. Cancer Res. 2008 Aug Prognosis 15;68(16):6770-8 There is a controversy in the role of CBX7 in cancer, as Yap KL, Li S, Muñoz-Cabello AM, Raguz S, Zeng L, Mujtaba some papers associate CBX7 overexpression with poor S, Gil J, Walsh MJ, Zhou MM. Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by prognosis and advanced estate of the tumor and polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell. aggressiveness, while others state that depletion of 2010 Jun 11;38(5):662-74 CBX7 from certain cancers indicates the state of Morey L, Pascual G, Cozzuto L, Roma G, Wutz A, Benitah SA, malignancy of the tumor. The ability of CBX7 to Di Croce L. Nonoverlapping functions of the polycomb group regulate multiple targets and the relevance of those cbx family of proteins in embryonic stem cells. Cell Stem Cell. targets in different tumor types and stages probably 2012 Jan 6;10(1):47-62 explain those paradoxical findings. O'Loghlen A, Muñoz-Cabello AM, Gaspar-Maia A, Wu HA, Banito A, Kunowska N, Racek T, Pemberton HN, Beolchi P, Lavial F, Masui O, Vermeulen M, Carroll T, Graumann J, References Heard E, Dillon N, Azuara V, Snijders AP, Peters G, Bernstein Gil J, Bernard D, Martínez D, Beach D. Polycomb CBX7 has a E, Gil J. MicroRNA Regulation of Cbx7 Mediates a Switch of unifying role in cellular lifespan. Nat Cell Biol. 2004 Polycomb Orthologs during ESC Differentiation. Cell Stem Jan;6(1):67-72 Cell. 2012 Jan 6;10(1):33-46 Bernard D, Martinez-Leal JF, Rizzo S, Martinez D, Hudson D, This article should be referenced as such: Visakorpi T, Peters G, Carnero A, Beach D, Gil J. CBX7 controls the growth of normal and tumor-derived prostate cells O'Loghlen A, Gil J. CBX7 (chromobox homolog 7). Atlas Genet by repressing the Ink4a/Arf locus. Oncogene. 2005 Aug Cytogenet Oncol Haematol. 2012; 16(3):220-222. 25;24(36):5543-51

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Gene Section Short Communication

RPRM (reprimo, TP53 dependent G2 arrest mediator candidate) Alejandro H Corvalan, Veronica A Torres Laboratory of Molecular Pathology and Epidemiology, Department of Hemathology - Oncology, School of Medicine - P Universidad Catolica de Chile, 391 Marcoleta St - Santiago 8330074 Chile (AHC, TorresVAT)

Published in Atlas Database: November 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/RPRMID42082ch2q23.html DOI: 10.4267/2042/47283 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity in the cytoplasm. Function Other names: FLJ90327, REPRIMO Reprimo is a candidate tumor suppresor gene involved HGNC (Hugo): RPRM in the G2/M phase cell cycle arrest mediated by tumor Location: 2q23.3 protein p53. Reprimo induces cell cycle arrest by inhibiting the nuclear translocation of the Cdc2-Cyclin DNA/RNA B1 complex. Description Implicated in Reprimo gene consists of 1 exon. The gene spans 1,47 kb of genomic DNA on the chromosome 2 in the minus Various cancers strand. Note Transcription The aberrant methylation of the promoter region of Reprimo is a common event that may contribute to the The mRNA is 1496 bp in length. pathogenesis of some types of human cancer. Promoter methylation of Reprimo was found in pancreatic cancer Protein (91%), gastric cancer (90%), gallbladder cancer (62%), Description lymphomas (57%), colorectal cancer (56%) and esophageal adenocarcinomas (40%). In breast cancer, The open reading frame encodes a 109 amino acid leukemias and lung cancer, promoter methylation of protein with an estimated molecular weight of 11774 Reprimo was found in less than 40% of tested cases. Da. Reprimo is a highly glycosylated protein which has two sites in amino acids 7 and 18. The protein has a Gastric cancer potential transmembrane site covering amino acids 56 Disease to 76. Aberrant hypermethylation of Reprimo is frequently Expression found in primary gastric cancer as well as in pair plasma samples. In plasma from asymptomatic The expression of Reprimo is induced by tumor protein controls, Reprimo is infrequently methylated. p53 following X-ray irradiation. Therefore, plasmatic detection of Reprimo is a putative Localisation biomarker for early detection of gastric cancer. When Reprimo is ectopically expressed, it is localized

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 223 RPRM (reprimo, TP53 dependent G2 arrest mediator candidate) Corvalan AH, TorresVA

The above histogram represents the percentage of positive cases for Reprimo and other genes (APC, SHP1, CDH-1, ER, SEMA3B and 3OST2) in 43 prospectively collected gastric cancer cases and 31 asymptomatic age- and gender-matched controls. Only Reprimo shows a significant difference in plasma between gastric cancer and asymptomatic controls (Bernal et al., Clin Cancer Res. 2008;14:6264-9).

Pancreatic cancer Takahashi T, Suzuki M, Shigematsu H, Shivapurkar N, Echebiri C, Nomura M, Stastny V, Augustus M, Wu CW, Disease Wistuba II, Meltzer SJ, Gazdar AF. Aberrant methylation of Aberrant hypermethylation of Reprimo is also common Reprimo in human malignancies. Int J Cancer. 2005 Jul 1;115(4):503-10 in pancreatic cell lines (91%) and in pancreatic adenocarcinomas (66%). Reprimo methylation is Hamilton JP, Sato F, Jin Z, Greenwald BD, Ito T, Mori Y, Paun correlated with poor prognosis in a large series of BC, Kan T, Cheng Y, Wang S, Yang J, Abraham JM, Meltzer SJ. Reprimo methylation is a potential biomarker of Barrett's- resected pancreatic cancers. This fact raises the Associated esophageal neoplastic progression. Clin Cancer possibility that aberrant methylation of Reprimo is an Res. 2006 Nov 15;12(22):6637-42 epigenetic event that may have a mechanistic role in Sato N, Fukushima N, Matsubayashi H, Iacobuzio-Donahue pancreatic cancer. CA, Yeo CJ, Goggins M. Aberrant methylation of Reprimo correlates with genetic instability and predicts poor prognosis References in pancreatic ductal adenocarcinoma. Cancer. 2006 Jul 15;107(2):251-7 Ohki R, Nemoto J, Murasawa H, Oda E, Inazawa J, Tanaka N, Bernal C, Aguayo F, Villarroel C, Vargas M, Díaz I, Ossandon Taniguchi T. Reprimo, a new candidate mediator of the p53- FJ, Santibáñez E, Palma M, Aravena E, Barrientos C, mediated cell cycle arrest at the G2 phase. J Biol Chem. 2000 Corvalan AH. Reprimo as a potential biomarker for early Jul 28;275(30):22627-30 detection in gastric cancer. Clin Cancer Res. 2008 Oct Sato N, Fukushima N, Maitra A, Matsubayashi H, Yeo CJ, 1;14(19):6264-9 Cameron JL, Hruban RH, Goggins M. Discovery of novel targets for aberrant methylation in pancreatic carcinoma using This article should be referenced as such: high-throughput microarrays. Cancer Res. 2003 Jul Corvalan AH, TorresVA. RPRM (reprimo, TP53 dependent G2 1;63(13):3735-42 arrest mediator candidate). Atlas Genet Cytogenet Oncol Suzuki M, Shigematsu H, Takahashi T, Shivapurkar N, Haematol. 2012; 16(3):223-224. Sathyanarayana UG, Iizasa T, Fujisawa T, Gazdar AF. Aberrant methylation of Reprimo in lung cancer. Lung Cancer. 2005 Mar;47(3):309-14

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Gene Section Mini Review

VMP1 (vacuole membrane protein 1) Alejandro Ropolo, Andrea Lo Ré, María Inés Vaccaro Molecular Pathophysiology Lab, School of Pharmacie and Biochemistry, University of Buenos Aires, Argentina (AR, AL, MIV)

Published in Atlas Database: November 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/VMP1ID50079ch17q23.html DOI: 10.4267/2042/47284 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

amino-acid length containing 6 putative transmembrane Identity domains and with no known homologues in yeast. Other names: DKFZp566I133, EPG3, TMEM49 Expression HGNC (Hugo): VMP1 VMP1 was characterized because is not constitutively Location: 17q23.1 expressed in pancreatic acinar cells and it is highly activated early during experimental acute pancreatitis DNA/RNA in acinar cells. Description Localisation 12 exons, spans approximately 133 kb of genomic Autophagosomal membrane. DNA in the centromere-to-telomere orientation. The Function translation initiation codon is located to exon 2, and the VMP1 is an autophagy-related membrane protein. stop codon to exon 12. VMP1 expression triggers autophagy, even under Transcription nutrient-replete conditions. VMP1 is required for mRNA of 2,17 kb. autophagosome development through interaction with Beclin1. Recently, it has been demonstrated that Protein participate in a novel selective form of autophagy, called zymophagy, mediated by VMP1-USP9x-p62 Description pathway during acute pancreatitis. The pancreatitis-associated protein vacuole membrane protein 1 (VMP1) is a transmembrane protein of 406

Genomic organization of the VMP1/TMEM49 gene.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 225 VMP1 (vacuole membrane protein 1) Ropolo A, et al.

Schematic representation of VMP1 protein and localization of transmembrane domains.

Upon CCK-R hyperstimulation, wild type mice Implicated in developed acute pancreatitis with high amylase and Pancreatic cancer lipase serum levels. On the contrary, enzymatic levels in cerulein-treated Disease ElaI-VMP1 mice were significantly lower compared to Pancreatic ductal adenocarcinoma is one of the most wild type mice. Consistently, ElaI-VMP1 mouse aggressive human malignancies with a 2-3% 5-year pancreata showed remarkably less macroscopic survival rate. This is due to both the aggressive nature evidence of acute pancreatitis compared to wild type of the disease and the lack of specific symptoms and animals, which showed marked edema and early-detection tools. It is relatively refractory to hemorrhage. Histological analyses displayed a high traditional cytotoxic agents and radiotherapy. degree of necrosis as well as infiltration in wild type Gemcitabine, the standard chemotherapy agent for the pancreata with acute pancreatitis. In contrast, neither treatment of pancreatic cancer, induces autophagy of necrosis nor significant inflammation was seen in cancer cells and that this process mediates the cell cerulein-treated ElaI-VMP1 mice. ElaIVMP1 mice death-promoting activity of this compound. Early showed secretory granules with normal ultrastructural induction of autophagy by gemcitabine leads to cancer characteristics CCK-R hyperstimulation in wild type cell death and this cellular process is mediated by the animals induced a markedly altered distribution pattern activation of VMP1 expression. In PANC-1 and of the secretory granules. Acinar cells lose their MIAPaCa-2 cells the inhibition of autophagy polarity, which results in the relocation of zymogen significantly reduced the percentage of dead cells in granules to the basolateral membrane. These alterations response to gemcitabine. In addition, gemcitabine in vesicular traffic are known to occur in acinar cells promoted early VMP1 expression, and downregulation during acute pancreatitis and upon hyperstimulation of of VMP1 expression significantly reduced cell death. their CCK-R with cerulein. ElaI-VMP1 mice subjected Acute pancreatitis to CCK-R hyperstimulation revealed that acinar cells Disease preserve their structure and polarity with negligible or no alteration in vesicular transport. Surprisingly, in VMP1 was characterized because is not constitutively pancreata from cerulein-treated ElaI-VMP1 mice, we expressed in pancreatic acinar cells and it is highly observed autophagosomes containing zymogen activated early during experimental acute pancreatitis granules displaying a distinct localization to the apical in acinar cells. VMP1 is an autophagy-related area of the acinar cell. VMP1, the ubiquitin-protease membrane protein involved in the initial steps of the USP9x, and the ubiquitin-binding protein p62 mediate mammalian cell autophagic process. VMP1 is a this process. Moreover, VMP1 interacts with USP9x, transmembrane protein that co-localizes with LC3, a indicating that there is a close cooperation between the marker of the autophagosomes, in pancreas tissue autophagy pathway and the ubiquitin recognition undergoing pancreatitis-induced autophagy. VMP1 machinery required for selective autophagosome interacts with with Beclin1, a mammalian autophagy formation. We have coined the term "zymophagy" to initiator, to start autophagosome formation. We refer to this process. Zymophagy is activated by developed the ElaI-VMP1 mouse in which acinar cell- experimental pancreatitis and by acute pancreatitis in specific constitutive expression of a VMP1-EGFP humans. Furthermore, zymophagy has chimera induces the formation of autophagosomes.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 226 VMP1 (vacuole membrane protein 1) Ropolo A, et al.

pathophysiological relevance by controlling and autophagy during the early cellular events in pancreatitis-induced intracellular zymogen activation response to experimental diabetes. and helping to prevent cell death. This new selective autophagy is activated in pancreatic acinar cells during References pancreatitis-induced vesicular transport alteration to Dusetti NJ, Jiang Y, Vaccaro MI, Tomasini R, Azizi Samir A, sequester and degrade potentially deleterious activated Calvo EL, Ropolo A, Fiedler F, Mallo GV, Dagorn JC, Iovanna zymogen granules. JL. Cloning and expression of the rat vacuole membrane protein 1 (VMP1), a new gene activated in pancreas with acute pancreatitis, which promotes vacuole formation. Biochem Biophys Res Commun. 2002 Jan 18;290(2):641-9 Vaccaro MI, Grasso D, Ropolo A, Iovanna JL, Cerquetti MC. VMP1 expression correlates with acinar cell cytoplasmic vacuolization in arginine-induced acute pancreatitis. Pancreatology. 2003;3(1):69-74 Jiang PH, Motoo Y, Vaccaro MI, Iovanna JL, Okada G, Sawabu N. Expression of vacuole membrane protein 1 (VMP1) in spontaneous chronic pancreatitis in the WBN/Kob rat. Pancreas. 2004 Oct;29(3):225-30 Ropolo A, Grasso D, Pardo R, Sacchetti ML, Archange C, Lo Re A, Seux M, Nowak J, Gonzalez CD, Iovanna JL, Vaccaro MI. The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells. J Biol Chem. 2007 Dec 21;282(51):37124-33 Vaccaro MI. Autophagy and pancreas disease. Pancreatology. 2008;8(4-5):425-9 Vaccaro MI, Ropolo A, Grasso D, Iovanna JL. A novel mammalian trans-membrane protein reveals an alternative initiation pathway for autophagy. Autophagy. 2008 Confocal microscopy of AR42J cell transfected with Apr;4(3):388-90 pEGFP-VMP1. Grasso D, Sacchetti ML, Bruno L, Lo Ré A, Iovanna JL, Diabetes Gonzalez CD, Vaccaro MI. Autophagy and VMP1 expression are early cellular events in experimental diabetes. Disease Pancreatology. 2009;9(1-2):81-8 Experimental diabetes activates VMP1 expression and Pardo R, Lo Ré A, Archange C, Ropolo A, Papademetrio DL, autophagy in pancreas beta cells as a direct response to Gonzalez CD, Alvarez EM, Iovanna JL, Vaccaro MI. streptozotocin (STZ). VMP1 mRNA expression is Gemcitabine induces the VMP1-mediated autophagy pathway activated after STZ treatment by islet beta cells. to promote apoptotic death in human pancreatic cancer cells. Electron microscopy shows chromatin aggregation and Pancreatology. 2010;10(1):19-26 autophagy morphology that was confirmed by LC3 Grasso D, Ropolo A, Lo Ré A, Boggio V, Molejón MI, Iovanna expression and LC3-VMP1 co-localization. Apoptotic JL, Gonzalez CD, Urrutia R, Vaccaro MI. Zymophagy, a novel selective autophagy pathway mediated by VMP1-USP9x-p62, cell death and the reduction of beta cell pool are prevents pancreatic cell death. J Biol Chem. 2011 Mar evident after 24h treatment, while VMP1 is still 11;286(10):8308-24 expressed in the remaining cells. VMP1-Beclin1 colocalization in pancreas tissue from STZ-treated rats This article should be referenced as such: suggests that VMP1-Beclin1 interaction is involved in Ropolo A, Lo Ré A, Vaccaro MI. VMP1 (vacuole membrane the autophagic process activation during experimental protein 1). Atlas Genet Cytogenet Oncol Haematol. 2012; diabetes. Pancreas beta cells trigger VMP1 expression 16(3):225-227.

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Gene Section Review

XPO1 (exportin 1 (CRM1 homolog, yeast)) Alessandra Ruggiero, Maria Giubettini, Patrizia Lavia CNR (National Research Council), Institute of Molecular Biology and Pathology, c/o Sapienza University of Rome, via degli Apuli 4, 00185 Rome, Italy (AR, MG, PL)

Published in Atlas Database: November 2011 Online updated version : http://AtlasGeneticsOncology.org/Genes/XPO1ID44168ch2p15.html DOI: 10.4267/2042/47285 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

which case the t(6;9) yielded a SET-CAN fusion. Wild- Identity type CAN is identical to the nucleoporin NUP214. Other names: CRM1, DKFZp686B1823, emb CC112 was capable of interacting with both wild-type HGNC (Hugo): XPO1 CAN/NUP214 and with both its fusion proteins, DEK- CAN and SET-CAN, suggesting potential roles in Location: 2p15 proliferation of cancer cells (Fornerod et al., 1996). Note Description The human XPO1/hCRM1 gene is localized on the 2p16 region (Fornerod et al., 1997a). The human XPO1/CRM1 protein is composed of 1071 aminoacidic residues with a molecular weight of 112 DNA/RNA kDa (Fornerod et al., 1997b). It is a modular protein composed of several fuctional domains: Transcription - The N-terminal region shares sequence similarity with The human XPO1/hCRM1 gene is transcribed in a cell importin β in a region called the CRIME domain cycle-dependent manner, with the onset of mRNA (acronym for CR M1, im portin beta etc.). This domain transcription taking place in late G1 phase and peaking interacts with the GTPase RAN. In the GTP-bound in the G2/M phases of the cell cycle (Kudo et al., form, RAN stabilizes export complex formed by CRM1 1997). NFY/CBP, Sp1 and p53 transcription factors are and NES-containing proteins. reported to interact with the XPO1/hCRM1 gene - Most of the XPO1/CRM1 protein is composed of 19 promoter and play an important role in XPO1/hCRM1 HEAT repeat motifs. HEAT repeat 8 contains an acidic promoter activity in transformed and cancer cells (van loop which cooperates with the CRIME domain in der Watt and Leaner, 2011). RANGTP binding. - The central region of XPO1/CRM1 is involved in Protein NES binding. Cys528, lying in this region, is specifically blocked by the inhibitor leptomycin B Note (LMB), which therefore blocks the export activity of A human protein, originally named CC112 based on its XPO1/CRM1 (Wolff et al., 1997). apparent molecular weight, was identified in a search - The C-terminal region is thought to modulate the for interacting partners of CAN/NUP214, a nucleoporin affinity of XPO1/CRM1 for its cargoes. regarded as a proto-oncogenic factor. CAN was Structures implicated in acute myeloid leukemia and in The structure of the region corresponding to residues myelodysplastic syndrome (von Lindern et al., 1992) as 707-1034 (C-terminal region) was elucidated by X-ray part of the DEK-CAN fusion gene generated in the crystallography (Petosa et al., 2004). translocation t(6;9)(p23;q34). Another potentially The structure of XPO1/CRM1 complexed to various oncogenic fusion protein involving CAN was identified NESs and to RANGTP has been solved (Güttler et al., in a patient with acute undifferentiated leukemia, in 2010).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 228 XPO1 (exportin 1 (CRM1 homolog, yeast)) Ruggiero A, et al.

The plates show the subcellular localisation of CRM1 (detected by indirect immunofluorescence) in interphase and mitotic human HeLa cells. Upper row: an interphase cell showing CRM1 (in red in the left panel) within the nucleus and especially around the nuclear envelope, where it concentrates with a regular, punctuated pattern typical of the association with nuclear pore complexes. The nuclear shape is depicted in the upper right panel by staining the DNA with the fluorochrome 4',6-diamidino-2-phenylindole (DAPI, in blue). Lower row: a metaphase cell showing CRM1 (in red in the left panel) concentrating at the kinetochores (compare with the middle panel, where kinetochore proteins are stained using CREST antiserum and a blue-emitting secondary antibody). A CRM1 fraction is also visible at spindle poles (compare with the staining of the mitotic spindle microtubules using an antibody against alpha-tubulin, in green). The merged picture shows a 3.5x magnification of the overlay of all three images: CRM1 (red) lies at the interface between the kinetochores (blue) and the microtubules (green) projecting from opposite spindle poles.

Expression Function XPO1/CRM1 protein levels remain constant hCRM1 was found to interact stably in complexes throughout the cell cycle (Kudo et al., 1997). containing not only NUP214/CAN (or its derivatives), Localisation but also another component of nuclear pores, the nucleoporin NUP88 (Fornerod et al., 1997b). These Due to its function as a shuttling nuclear transport interactions hinted at a possible role of hCRM1 in receptor between the nucleus and cytoplasm, the human nucleocytoplasmic transport. Further studies indeed XPO1/CRM1 protein is preferentially localized at the demonstrated that hCRM1 acts as a nuclear export nuclear envelope in interphase cells (Kudo et al., 1997; factor (reviewed by Fried and Kutaj, 2003; Hutten and Fornerod et al., 1997b). In the nucleus it can be Kehlenbach, 2007): it interacts with various classes of detected in specific bodies called CRM1 nucleolar RNAs and with proteins carrying nuclear export signals bodies (CNoBs). CNoBs depend on RNA polymerase I (NES) (Fornerod et al., 1997c; Fukuda et al., 1997; activity, suggesting a role in ribosome biogenesis Ossareh-Nazari et al., 1997), short aminoacidic (Ernoult-Lange et al., 2009). stretches harbouring hydrophobic residues (general In mitotic cells, a fraction of XPO1 is found at consensus LX(2-3) ΦX(2-3)LX Φ, where can be L, I, M centrosomes (Forgues et al., 2003; Wang et al., 2005) or F), present in many shuttling proteins of cellular or and a substantial fraction localizes to the kinetochores viral origin, and transports these molecules out of the (Arnaoutov et al., 2005). nucleus through nuclear pore complexes in a manner

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 229 XPO1 (exportin 1 (CRM1 homolog, yeast)) Ruggiero A, et al.

dependent on the GTPase RAN. The protein is carcinomas compared with borderline tumors and therefore alternatively called either exportin-1 or benign lesions. Cytoplasmic CRM1 expression XPO1, based on its function, or hCRM1, based on significantly correlated with advanced tumor stage (P= evolutionary conservation. 0.043), poorly differentiated carcinomas (P= 0.011) and Regulated export of some shuttling proteins (e.g., p53, high mitotic rate (P= 0.008). Nuclear CRM1 was p27, STAT, NF-kB and many viral proteins) out of the significantly associated with high cyclooxygenase-2 nucleus is essential for regulated cell cycle and cell (COX-2) expression (P= 0.002) and poor overall proliferation (reviewed by Fabbro and Henderson, survival (P= 0.01). CRM1 was previously directly 2003; Rensen et al., 2008). This has lead some authors implicated in nuclear export of COX-2 (Jang et al., to view nuclear export as a promising target process in 2003). The study by Noske et al. (2008) suggests that cancer therapy (reviewed by Yashiroda and Yoshida, elevated expression of CRM1 may be causal to COX-2 2003; Turner and Sullivan, 2008). up-regulation, with direct clinical relevance. Recent findings have revealed additional roles of Oncogenesis XPO1/CRM1 in mitosis: first, an XPO1/CRM1 fraction CRM1 is highly expressed in ovarian carcinomas regulates the localisation of nucleophosmin tissues and regulates export of COX-2. (NPM/B23), a regulator of centrosome duplication. XPO1/CRM1 is required to prevent centrosome Osteosarcoma (Yao et al., 2009) overduplication and the formation of multipolar Prognosis spindles (reviewed by Budhu and Wang, 2005; The CRM1 protein is reported to be expressed with Ciciarello and Lavia, 2005). Second, a kinetochore- increased abundance in osteosarcoma compared to non- associated fraction of XPO1/CRM1 regulates the tumour tissues (P= 0.037, 57 patients). High levels of assembly of the so-called k-fibers, bundles of CRM1 were significantly associated with increased microtubules that stably connect the spindle poles to serum levels of alkaline phosphatase (ALP, P= 0.001). the kinetochores of mitotic chromosomes to ensure In univariate analysis, a significant association between proper chromosome segregation (reviewed by CRM1 expression and tumor size (P= 0.014), as well as Arnaoutov and Dasso, 2005; Ciciarello and Lavia, histological grade (P= 0.003) was observed. In Kaplan- 2005; Dasso, 2006). Third, XPO1/CRM1 regulates Meier survival analysis, high CRM1 expression was a survivin, a member of the chromosomal passenger significant prognostic indicator for poor progression- complex with roles in chromosome segregation and free survival (P= 0.016) as well as overall survival (P= apoptosis (reviewed by Knauer et al., 2007). 0.008). Multivariate analysis demonstrated that high In synthesis, XPO1/CRM1 acts in control of cell expression of CRM1 was significantly related to proliferation, and affects loss of proliferation control in shorter survival (95% CI, 1.27-5.39). cancer cells, through several pathways: 1. as a nuclear Oncogenesis export factor, it directly regulates the subcellular CRM1 is significantly increased in osteosarcoma localisation, and hence the activity, of oncogenes and compared with normal tissue. tumour suppressor proteins that contain nuclear export sequences; 2. it acts in control of the mitotic apparatus Cervical cancer (van der Watt et al., 2009) and chromosome segregation; 3. it influences the Oncogenesis maintenance of nuclear and chromosome structure. CRM1 protein abundance is significantly increased in Homology cervical cancer cells compared with normal tissue (P< The human protein originally named CC112 showed 0.05). Inhibition of CRM1 by RNA interference homology to the Schizosaccharomyces pombe CRM1 resulted in increased cell death, associated with nuclear protein, first identified for being implicated in the retention of p53, likely protecting p53 from degradation control of higher order chromosome structure: mutation as the latter predominantly occurs in the cytoplasm. of the coding gene was associated with the appearance Pancreas cancer (Huang et al., 2009) of "deformed nuclear chromosome domains" in fission Prognosis yeast conditional mutant strains. The gene product was Increased expression abundance of CRM1 protein was therefore named CRM1 ( chromosome region detected in pancreatic cancer tissues (P= 0.0013, 69 maintenance 1; Adachi and Yanagida, 1989). Based on patients at stages I and II). CRM1 expression correlates this homology, the human protein name of CC112 was with increased levels of serum CEA (P= 0.002) and abandoned and the name hCRM1 was used. CA19.9 (P= 0.005), tumour size (P= 0.011), lymphadenopathy (P= 0.004) and metastasis (P= Implicated in 0.0041). High CRM1 expression was a prognostic Ovarian cancer (Noske et al., 2008) indicator for progression-free survival (PFS) (P= 0.0011) as well as overall survival (OS) (P= 0.004). Prognosis The authors proposed that CRM1 be used as a Increased nuclear (52.7%) and cytoplasmic (56.8%) prognostic parameter for poor PFS and OS (95% CI, expression of CRM1 were reported observed in 1.27-5.39).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 230 XPO1 (exportin 1 (CRM1 homolog, yeast)) Ruggiero A, et al.

Glioma (Shen et al., 2009) Kudo N, Khochbin S, Nishi K, Kitano K, Yanagida M, Yoshida M, Horinouchi S. Molecular cloning and cell cycle-dependent Prognosis expression of mammalian CRM1, a protein involved in nuclear CRM1 overexpression is significantly associated with export of proteins. J Biol Chem. 1997 Nov 21;272(47):29742- 51 the pathological state (P= 0.001, 56 patients), with glioma tumour grade, with high expression of phospho- Ossareh-Nazari B, Bachelerie F, Dargemont C. Evidence for a ser10p27 and with reduced overall abundance of p27. role of CRM1 in signal-mediated nuclear protein export. Science. 1997 Oct 3;278(5335):141-4 Oncogenesis Wolff B, Sanglier JJ, Wang Y. Leptomycin B is an inhibitor of Given the direct implication of CRM1 in nuclear export nuclear export: inhibition of nucleo-cytoplasmic translocation of of p27, the data in this study suggest that increased the human immunodeficiency virus type 1 (HIV-1) Rev protein CRM1 abundance yields increased cytoplasmic and Rev-dependent mRNA. Chem Biol. 1997 Feb;4(2):139-47 localisation of p27, which is probably targeted to Fabbro M, Henderson BR. Regulation of tumor suppressors by degradation, leading to uncontrolled tumour growth. nuclear-cytoplasmic shuttling. Exp Cell Res. 2003 Jan Phospho-ser10p27 may be resistant to CRM1-mediated 15;282(2):59-69 nuclear export. High CRM1 and low p27 expression Forgues M, Difilippantonio MJ, Linke SP, Ried T, Nagashima are associated with high grade glioma and high CRM1 K, Feden J, Valerie K, Fukasawa K, Wang XW. Involvement of protein expression is proposed as a prognostic factor of Crm1 in hepatitis B virus X protein-induced aberrant centriole replication and abnormal mitotic spindles. Mol Cell Biol. 2003 overall survival and poor outcome. Aug;23(15):5282-92 Fried H, Kutay U. Nucleocytoplasmic transport: taking an To be noted inventory. Cell Mol Life Sci. 2003 Aug;60(8):1659-88 Note Jang BC, Muñoz-Najar U, Paik JH, Claffey K, Yoshida M, Hla CRM1 protein levels are abnormally high in several T. Leptomycin B, an inhibitor of the nuclear export receptor CRM1, inhibits COX-2 expression. J Biol Chem. 2003 Jan cancers, with high levels of CRM1 being associated 31;278(5):2773-6 with poor patient survival (van der Watt and Leaner, 2011). Yashiroda Y, Yoshida M. Nucleo-cytoplasmic transport of proteins as a target for therapeutic drugs. Curr Med Chem. References 2003 May;10(9):741-8 Petosa C, Schoehn G, Askjaer P, Bauer U, Moulin M, Adachi Y, Yanagida M. Higher order chromosome structure is Steuerwald U, Soler-López M, Baudin F, Mattaj IW, Müller CW. affected by cold-sensitive mutations in a Schizosaccharomyces Architecture of CRM1/Exportin1 suggests how cooperativity is pombe gene crm1+ which encodes a 115-kD protein achieved during formation of a nuclear export complex. Mol preferentially localized in the nucleus and its periphery. J Cell Cell. 2004 Dec 3;16(5):761-75 Biol. 1989 Apr;108(4):1195-207 Arnaoutov A, Azuma Y, Ribbeck K, Joseph J, Boyarchuk Y, von Lindern M, Fornerod M, van Baal S, Jaegle M, de Wit T, Karpova T, McNally J, Dasso M. Crm1 is a mitotic effector of Buijs A, Grosveld G. The translocation (6;9), associated with a Ran-GTP in somatic cells. Nat Cell Biol. 2005 Jun;7(6):626-32 specific subtype of acute myeloid leukemia, results in the Arnaoutov A, Dasso M. Ran-GTP regulates kinetochore fusion of two genes, dek and can, and the expression of a attachment in somatic cells. Cell Cycle. 2005 Sep;4(9):1161-5 chimeric, leukemia-specific dek-can mRNA. Mol Cell Biol. 1992 Apr;12(4):1687-97 Budhu AS, Wang XW. Loading and unloading: orchestrating centrosome duplication and spindle assembly by Ran/Crm1. Fornerod M, Boer J, van Baal S, Morreau H, Grosveld G. Cell Cycle. 2005 Nov;4(11):1510-4 Interaction of cellular proteins with the leukemia specific fusion proteins DEK-CAN and SET-CAN and their normal Ciciarello M, Lavia P. New CRIME plots. Ran and transport counterpart, the nucleoporin CAN. Oncogene. 1996 Oct factors regulate mitosis. EMBO Rep. 2005 Aug;6(8):714-6 17;13(8):1801-8 Wang W, Budhu A, Forgues M, Wang XW. Temporal and Fornerod M, van Baal S, Valentine V, Shapiro DN, Grosveld G. spatial control of nucleophosmin by the Ran-Crm1 complex in Chromosomal localization of genes encoding CAN/Nup214- centrosome duplication. Nat Cell Biol. 2005 Aug;7(8):823-30 interacting proteins--human CRM1 localizes to 2p16, whereas Nup88 localizes to 17p13 and is physically linked to SF2p32. Dasso M. Ran at kinetochores. Biochem Soc Trans. 2006 Genomics. 1997a Jun 15;42(3):538-40 Nov;34(Pt 5):711-5 Fornerod M, van Deursen J, van Baal S, Reynolds A, Davis D, Hutten S, Kehlenbach RH. CRM1-mediated nuclear export: to Murti KG, Fransen J, Grosveld G. The human homologue of the pore and beyond. Trends Cell Biol. 2007 Apr;17(4):193-201 yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 Knauer SK, Mann W, Stauber RH. Survivin's dual role: an and a novel nuclear pore component Nup88. EMBO J. 1997b export's view. Cell Cycle. 2007 Mar 1;6(5):518-21 Feb 17;16(4):807-16 Noske A, Weichert W, Niesporek S, Röske A, Buckendahl AC, Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an Koch I, Sehouli J, Dietel M, Denkert C. Expression of the export receptor for leucine-rich nuclear export signals. Cell. nuclear export protein chromosomal region 1997c Sep 19;90(6):1051-60 maintenance/exportin 1/Xpo1 is a prognostic factor in human Fukuda M, Asano S, Nakamura T, Adachi M, Yoshida M, ovarian cancer. Cancer. 2008 Apr 15;112(8):1733-43 Yanagida M, Nishida E. CRM1 is responsible for intracellular Rensen WM, Mangiacasale R, Ciciarello M, Lavia P. The transport mediated by the nuclear export signal. Nature. 1997 GTPase Ran: regulation of cell life and potential roles in cell Nov 20;390(6657):308-11 transformation. Front Biosci. 2008 May 1;13:4097-121

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Turner JG, Sullivan DM. CRM1-mediated nuclear export of Yao Y, Dong Y, Lin F, Zhao H, Shen Z, Chen P, Sun YJ, Tang proteins and drug resistance in cancer. Curr Med Chem. LN, Zheng SE. The expression of CRM1 is associated with 2008;15(26):2648-55 prognosis in human osteosarcoma. Oncol Rep. 2009 Jan;21(1):229-35 Ernoult-Lange M, Wilczynska A, Harper M, Aigueperse C, Dautry F, Kress M, Weil D. Nucleocytoplasmic traffic of CPEB1 Güttler T, Madl T, Neumann P, Deichsel D, Corsini L, Monecke and accumulation in Crm1 nucleolar bodies. Mol Biol Cell. T, Ficner R, Sattler M, Görlich D. NES consensus redefined by 2009 Jan;20(1):176-87 structures of PKI-type and Rev-type nuclear export signals bound to CRM1. Nat Struct Mol Biol. 2010 Nov;17(11):1367-76 Huang WY, Yue L, Qiu WS, Wang LW, Zhou XH, Sun YJ. Prognostic value of CRM1 in pancreas cancer. Clin Invest van der Watt PJ, Leaner VD. The nuclear exporter, Crm1, is Med. 2009 Dec 1;32(6):E315 regulated by NFY and Sp1 in cancer cells and repressed by p53 in response to DNA damage. Biochim Biophys Acta. 2011 Shen A, Wang Y, Zhao Y, Zou L, Sun L, Cheng C. Expression Jul;1809(7):316-26 of CRM1 in human gliomas and its significance in p27 expression and clinical prognosis. Neurosurgery. 2009 This article should be referenced as such: Jul;65(1):153-9; discussion 159-60 Ruggiero A, Giubettini M, Lavia P. XPO1 (exportin 1 (CRM1 van der Watt PJ, Maske CP, Hendricks DT, Parker MI, Denny homolog, yeast)). Atlas Genet Cytogenet Oncol Haematol. L, Govender D, Birrer MJ, Leaner VD. The Karyopherin 2012; 16(3):228-232. proteins, Crm1 and Karyopherin beta1, are overexpressed in cervical cancer and are critical for cancer cell survival and proliferation. Int J Cancer. 2009 Apr 15;124(8):1829-40

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Leukaemia Section Short Communication t(11;18)(p15;q12) Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t1118p15q12ID1466.html DOI: 10.4267/2042/47286 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

phosphatase 2A). SETBP1 impairs PP2A activity via Clinics and pathology SET and promotes proliferation of acute myeloid Disease leukemia cells (Cristóbal et al., 2010). T-cell acute lymphoid leukemia (T-ALL) Germinal mutations In Schinzel-Giedion midface retraction syndrome. Epidemiology One case to date, a 9-year-old boy. Result of the chromosomal Evolution anomaly Remission was obtained and the patient remains in complete remission 28 months after diagnosis. Hybrid gene Description Cytogenetics 5' NUP98 - 3' SETBP1 Cytogenetics morphological Transcript Exon 12 of NUP98 (nucleotide (nt) 1552) fused in- The translocation was accompanied with a del(12p). frame with exon 5 of SETBP1 (nt 4015). Genes involved and proteins References NUP98 Panagopoulos I, Kerndrup G, Carlsen N, Strömbeck B, Isaksson M, Johansson B. Fusion of NUP98 and the SET Location binding protein 1 (SETBP1) gene in a paediatric acute T cell 11p15.4 lymphoblastic leukaemia with t(11;18)(p15;q12). Br J Protein Haematol. 2007 Jan;136(2):294-6 Nucleoporin: associated with the nuclear pore complex. Cristóbal I, Blanco FJ, Garcia-Orti L, Marcotegui N, Vicente C, Role in nucleocytoplasmic transport processes. Rifon J, Novo FJ, Bandres E, Calasanz MJ, Bernabeu C, Odero MD. SETBP1 overexpression is a novel leukemogenic SETBP1 mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia. Blood. 2010 Jan 21;115(3):615- Location 25 18q12.3 This article should be referenced as such: Protein Contains 3 DNA binding domains (A.T hooks). Huret JL. t(11;18)(p15;q12). Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3): 233. SETBP1 protects SET from protease cleavage. SETBP1 forms a complex with SET and PP2A (protein

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Leukaemia Section Short Communication t(11;21)(q21;q22) Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t1121q21q22ID1592.html DOI: 10.4267/2042/47287 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

inhibited cell adhesion. Strongly expressed in Identity hematopoietic cells. LPXN is involved in bone Note resorption and stimulates prostate cancer cell migration This translocation is different from the (Chen and Kroog, 2010). t(11;21)(q12;q22) with MACROD1/RUNX1 RUNX1 involvement. Location Clinics and pathology 21q22.3 Protein Disease Transcription factor (activator) for various Acute myeloid leukemia (AML) hematopoietic-specific genes. Epidemiology Result of the chromosomal One case to date, a 65-year-old male patient with M2- AML (Dai et al., 2007). anomaly Evolution Hybrid gene The patient died 10 months after diagnosis. Description 5' RUNX1 - 3' LPXN Genes involved and proteins Transcript LPXN Two in frame fusion transcripts -fusion of exon 5 or 6 of RUNX1 to LPXN exon 8. Protein LPXN contains two types of protein-protein interaction Fusion protein domains: leucine-aspartate (LD) repeats in N-term, and Description LIM (Lin-11 Isl-1 Mec-3) domains at the C-term. The two variant fusion proteins RUNX1-LPXN Belongs to the paxillin family (PXN, LPXN, localized in the nucleus and inhibited RUNX1 TGFB1I1). Protein involved in focal adhesion. LPXN transactivation (Dai et al., 2009). It is hypothesized that and paxillin had opposite roles in adhesion to collagen the reciprocal LPXN-RUNX1 may also play a role in LPXN siRNA stimulated whereas paxillin siRNA leukemogenesis.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 234 t(11;21)(q21;q22) Huret JL

Chen PW, Kroog GS.. Leupaxin is similar to paxillin in focal References adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading. Cell Adh Migr. Dai H, Xue Y, Pan J, Wu Y, Wang Y, Shen J, Zhang J.. Two 2010 Oct-Dec;4(4):527-40. novel translocations disrupt the RUNX1 gene in acute myeloid leukemia. Cancer Genet Cytogenet. 2007 Sep;177(2):120-4. This article should be referenced as such: Dai HP, Xue YQ, Zhou JW, Li AP, Wu YF, Pan JL, Wang Y, Huret JL. t(11;21)(q21;q22). Atlas Genet Cytogenet Oncol Zhang J.. LPXN, a member of the paxillin superfamily, is fused Haematol. 2012; 16(3):234-235. to RUNX1 in an acute myeloid leukemia patient with a t(11;21)(q12;q22) translocation. Genes Chromosomes Cancer. 2009 Dec;48(12):1027-36.

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Leukaemia Section Short Communication t(8;17)(q24;q22) ???BCL3/MYC Jean-Loup Huret Genetics, Dept Medical Information, University of Poitiers, CHU Poitiers Hospital, F-86021 Poitiers, France (JLH)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0817q24q22ID1494.html DOI: 10.4267/2042/47288 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

apoptosis and self-renewal, and protein synthesis Identity through ribosome biogenesis (van Riggelen et al., Note 2010). It is unlikely that the BCL3 gene (HGNC official BCL3 name) is involved in this translocation with a Location breakpoint in 17q22, since BCL3 sits in 19q13.32 (coordonates: starts at 45251978 and ends at 45263301 19q13.32 bp from 19pter); the alternative would be a cryptic Protein translocation, involving a cryptic inserted fragment of BCL3 is mainly found in the nucleus. Protein which 19q13.32, including BCL3, within 17q22. contains seven ankyrin repeats. Ankyrin repeats are found in IkB family members, including IkBa, IkBb, Clinics and pathology and IkBe. BCL3 is a member of the IkappaB family, whose proteins regulate the NFkappaB family of Disease transcription factors. Component of a complex with a Aggresive prolymphocytic leukemia NF-kB p52-p52 homodimer Down-regulates inflammatory responses through limiting the Epidemiology transcription of NF-kB-dependent genes. Binds to NF- Only one case to date, with no clinical data. kB p50 and p52, Jab1, Pirin, Tip60 and Bard1. Bcl-3 is an adaptor protein (Dechend et al., 1999; Kreisel et al., Cytogenetics 2011). Regulates genes involved in cell proliferation and apoptosis. NFkappaB plays a major role in B-cell Cytogenetics morphological development. The karyotype also showed the classical t(14;18)(q32;q21), usually found in follicular Result of the chromosomal lymphoma, a 12q+ and a Xp+, not otherwise described. anomaly Genes involved and proteins Hybrid gene Note Description As said above, it is unprobable that the MYC partner is Disruption of MYC close to the first intron, with the BCL3. decapitation of the first intron, replaced by a sequence MYC of 1.7 kb, that the authors have called "BCL3". Protein References MYC regulates the transcription of genes required to coordinate a range of cellular processes, including Gauwerky CE, Huebner K, Isobe M, Nowell PC, Croce CM. Activation of MYC in a masked t(8;17) translocation results in those essential for proliferation, growth, differentiation, an aggressive B-cell leukemia. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8867-71

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Dechend R, Hirano F, Lehmann K, Heissmeyer V, Ansieau S, Kreisel D, Sugimoto S, Tietjens J, Zhu J, Yamamoto S, Wulczyn FG, Scheidereit C, Leutz A. The Bcl-3 oncoprotein Krupnick AS, Carmody RJ, Gelman AE. Bcl3 prevents acute acts as a bridging factor between NF-kappaB/Rel and nuclear inflammatory lung injury in mice by restraining emergency co-regulators. Oncogene. 1999 Jun 3;18(22):3316-23 granulopoiesis. J Clin Invest. 2011 Jan 4;121(1):265-76 van Riggelen J, Yetil A, Felsher DW. MYC as a regulator of This article should be referenced as such: ribosome biogenesis and protein synthesis. Nat Rev Cancer. 2010 Apr;10(4):301-9 Huret JL. t(8;17)(q24;q22) ???BCL3/MYC. Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):236-237.

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Deep Insight Section

Plasticity and Tumorigenicity Elena Campos-Sanchez, Isidro Sanchez-Garcia, Cesar Cobaleda Centro de Biologia Molecular "Severo Ochoa", CSIC/Universidad Autonoma de Madrid, C/Nicolas Cabrera 1, Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain (ECS, CC), Experimental Therapeutics and Translational Oncology Program, Instituto de Biologia Molecular y Celular del Cancer, CSIC/ Universidad de Salamanca, Campus M de Unamuno s/n, 37007-Salamanca, Spain (ISG)

Published in Atlas Database: September 2011 Online updated version : http://AtlasGeneticsOncology.org/Deep/PluripotencyID20103.html DOI: 10.4267/2042/47289 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Summary The research fields of developmental biology and oncology have always been tightly linked, since the times of Rudolf Virchow's cellular theory (" omnis cellula e cellula ") and embryonal rest hypothesis. On the other side, for many years, contemporary cancer research has been mainly focused on the altered controls of proliferation in tumoral cells. This has been reflected in the therapeutic approaches employed in the clinic to treat the patients: with very few exceptions, anticancer treatments are targeted at the mechanisms of abnormal tumoral growth. Such therapies, however, are very unspecific, highly toxic and, ultimately, inefficient in most cases. In the last years, a new recognition of the role of aberrant differentiation at the root of cancer has arisen, mainly driven by the coming of age of the "cancer stem cell" (CSC) theory. From this point of view, the comprehensive knowledge of the developmental mechanisms by which normal cells acquire their identity is essential to understand how these controls are deregulated in tumours. New insights into the mechanisms that maintain the molecular boundaries of cell identity have been gained from the study of induced pluripotency, showing that cell fate can be much more susceptible to change than previously thought. Applied to cancer, these findings imply that the oncogenic events that take place in an otherwise healthy cell lead to a reprogramming of the normal cellular fate and establish a new pathologic developmental program. Therefore, cancer reprogramming and cellular plasticity are closely related, since only some cells possess the plasticity required to allow reprogramming to occur, and only some oncogenic events can, in the right plastic cell, induce this change. Here we discuss the latest findings in the fields of cellular plasticity and reprogramming and we consider their consequences for our understanding of cancer development and treatment.

Enlightenment, regeneration and plasticity will become Historical perspective the matter of scientific research. In 1712, Réaumur The search for the capacity of regenerating disease- describes the regeneration of the limbs and claws of affected organs is probably as old as mankind crayfish (Réaumur, 1712); in 1744, Trembley discovers (Odelberg, 2004). The examples are abundant in that a part of the Hydra polyp can regenerate the ancient religions, from the Egyptian god Osiris, who complete organism (Trembley, 1744); in 1769, resurrected and recomposed his maimed body from the Spallanzani reports that tadpoles can regenerate their pieces that had been thrown into the Nile, to the tails and salamanders their amputated jaws, limbs and legendary Hydra that could regenerate its severed tails (Spallanzani, 1769). The research performed heads. Or the mythological Prometheus, who had his during most of the 19 th and first half of the 20 th viscera eaten by an eagle every day, only to regenerate centuries showed that, for regeneration to occur, the them again. But also from a more scientific point of cells that are normally forming part of the organs are view, it was already noticed by Aristotle (384-322 BC) not sufficient, and a special type of cells are required: that lizards can regenerate their tails after amputation. the progenitor cells (Odelberg, 2004; Birnbaum and th But until the 18 century this knowledge was mainly Sánchez Alvarado, 2008). The origin of these cells was anecdotic, and only with the arrival of the Age of not very clear (and it is still a matter of debate and

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intense research, in fact, see (Sánchez Alvarado, 2000; controlled eye development and led to the development Kragl et al., 2009; Rinkevich et al., 2011)); for some of ectopic eyes in the fly's legs (Gehring, 1996). In tissues, like skin, blood, muscles or bones, progenitors mammals, the first master regulator factor to be were shown to exist in the tissues in small numbers, identified was MyoD, which was shown to be capable and to become activated as a consequence of the of transdifferentiating fibroblasts into the myogenic lesions. In other cases, the progenitors seemed to arise lineage (Davis et al., 1987). Other examples of factors from mature cells that become dedifferentiated. The with fate-reprogramming capacity are C/EBP α, capable best example supporting this possibility has been of mediating the transdifferentiation of mouse B cells described in primitive vertebrates like the urodeles (e.g. into macrophages (Xie et al., 2004) or Pax5, whose loss salamanders and axolotls). In these animals, after a leads to the dedifferentiation of committed B cells wound harms the organism, the cells from the normal (Nutt et al., 1999; Cobaleda et al., 2007a; Cobaleda and tissues form a group of cells known as the regenerative Busslinger, 2008). All these data proved that the lack or blastema, which will generate all the tissues in the new excess of just one factor could lead to a radical limb/tail (Chalkey, 1954; Bodemer and Everett, 1959; alteration of the transcriptional profile and could cause Hay and Fischman, 1961). It has long been held that the stable fate changes. This evidence, together with the blastema was the result of cellular dedifferentiation to one coming from reprogramming by nuclear progenitors. However, the most recent findings seem to transplantation, paved the way to the search for the indicate that there is no cellular dedifferentiation to factors capable of reprogramming to full pluripotency progenitors involved in this process, and the that led, in 2006, to the identification of the four regeneration is always due to the action of resident transcription factors capable of inducing pluripotency tissue-specific stem cells and progenitors, thus in virtually every kind of terminally differentiated cells questioning the role of mature cellular plasticity in (Takahashi and Yamanaka, 2006). We will discuss this tissue regeneration (Kragl et al., 2009; Rinkevich et al., aspect with more detail afterwards. 2011). We have therefore seen how the study of On the other side, cancer has also been recognized as a "naturally" occurring regeneration opened the way to a distinct pathological entity since the origins of new understanding of the stem cell-based architecture mankind. The first references are the Edwin Smith and of the organs and tissues, especially with the study of Ebers papyri from the 1600 BC and 1500 BC, primitive vertebrates. In 1952, amphibians also approximately (Hajdu, 2004). The Edwin Smith provided the first animal model of experimentally- papyrus contains the first mention and description of induced reprogramming when Briggs and King breast cancer, and it concludes that there is no generated Xenopus tadpoles by transplanting the treatment for the disease. Cancer was not so common in nucleus of cells from the blastula into oocytes, ancient times, mainly because life span was much therefore reverting the cellular differentiation program shorter, but it was already clearly recognized. (Briggs and King, 1952). Afterwards, it was shown that Hippocrates (460-375 BC) realized that growing more differentiated cells, like those from the intestinal tumors occurred typically in adults and they reminded epithelia, could also be reprogrammed by nuclear him of a moving crab, which led to the terms carcinos transfer (Gurdon, 1962). These landmark findings and cancer . Celsus (25 BC-AD 50) also compared undoubtedly showed that the genetic potential of cells cancer with a crab, because it penetrates the was not lost during differentiation, and that surrounding organs like if it had claws; Celsus development did not imply genetic changes. This introduced the first classification for breast cancer and principle was extended to mammals with the cloning of advocated for surgical therapy. Furthermore, he already Dolly the sheep in 1997 (Wilmut et al., 1997). This was realized that tumors could only be cured if they were the ultimate proof showing that the changes that occur removed in their early stages and that, even after during differentiation are totally reversible, and removal and wound healing, breast carcinomas tended demonstrated that the fate restrictions that take place to recur causing swelling in the armpit and, finally, during development are the result of epigenetic death by spreading throughout the body. Galen (131- modifications. These studies also showed that there AD 200) already recommended surgery by cutting a were factors in the oocyte cytoplasm capable of wide margin of healthy tissue around the edges of the inducing a reprogramming that led to the appearance of tumor (Hajdu, 2004). If we jump now to our days, it a totipotent phenotype. seems disappointing to see how little those old critical In a parallel way, the search for the molecular findings have been overcome by modern medicine, regulators responsible for establishing and controlling 2000 years later. Indeed, still today, clean surgical cellular identity led finally to the identification of the margins and lack of lymph node invasion are the most factors capable of reprogramming cellular fate. In important prognostic markers for the successful 1987, it was shown that ectopic expression of the eradication of solid tumors, and only if tumors are Antennapedia homeotic gene lead to changes in the completely resected before they metastasize (something body plan of Drosophila , that got extra legs instead of that it is anyhow impossible to determine with current antennae (Schneuwly et al., 1987). Also, Gehring et al. technologies) can curation be guaranteed. However, in showed that the ectopic expression of eyeless the last thirty years we have gained an enormous

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knowledge about the molecular biology of the disease. As we have seen, evidences about cellular plasticity In 1979, it was shown that the phenotype of had being accumulating for decades (Hochedlinger and transformed cells could be transferred to normal Jaenisch, 2006; Gurdon and Melton, 2008; Graf and fibroblasts by DNA transfection (Shih et al., 1979), a Enver, 2009; Vicente-Dueñas et al., 2009a), but the finding that lead to the rapid molecular cloning of the latest findings in the field of reprogramming have first human oncogene (the RAS gene), simultaneously definitively shown how switching to a different by several groups (Goldfarb et al., 1982; Lane et al., phenotype can be a lot easier than previously expected, 1982; Parada et al., 1982; Santos et al., 1982). Since and can have real physiological relevance, beyond then, many genes have been described as being either basic research. Cancer is a perfect example of oncogenes or tumor suppressors, and the molecular pathological reprogramming in which, from a normal mechanisms of their transforming capabilities have tissue, a whole new differentiation lineage is opened been analyzed to great detail, in close relationship with with its own hierarchy and structure (Reya et al., 2001; their functions in "normal" conditions. This is a field Sánchez-García et al., 2007). So, without forgetting the that has expanded tremendously in the last decades, and so well-studied aberrant proliferation, reprogramming a comprehensive study of the topic falls out of the is an essential part of the tumorigenesis process, and it scope of this revision. However, there are some aspects is closely dependent on the cellular plasticity of the that must be taken into account for posterior debate. A cancer-initiating cells. The term plasticity, as we will very important one is the fact that, for many types of use it here, refers to the ability of cells (stem or tumors, specific genetic mutations have been shown to differentiated) to adopt the biological properties (gene correlate closely with the phenotype of the tumors, expression profile, phenotype, etc.) of other suggesting that the oncogenic alterations might be differentiated types of cells (belonging to the same or acting as new specification factors that determine the different lineages). This definition comprises also the tumor appearance and/or phenotype. This association is property of competence, i.e. the ability of stem cells especially evident in the case of mesenchymal tumors and progenitors to give rise to their different caused by chromosomal aberrations (Sánchez-García, descendant lineages during normal development. We 1997; Cobaleda et al., 1998). In 2000, Hanahan and use such an ample definition of the term precisely to Weinberg summarized the main features that had to be reflect the fact that the molecular mechanisms that are disrupted in normal cellular behavior in order for allow important for progenitors' competence during normal a tumor to appear and progress (Hanahan and development are the same ones responsible for the Weinberg, 2000), and this list has expanded with the plasticity changes of more differentiated types of cells, years (Hanahan and Weinberg, 2011). These main both in pathological processes and in experimentally- aspects are related with the survival and proliferation of induced reprogramming. Here we will discuss the vital cancer cells, but it must be noted that most of them are role of cellular plasticity in the origin and maintenance equally shared by non-malignant tumors (Lazebnik, of tumoral cells. We will first revise the latest research 2010). However, all the aspects related to the discoveries in the fields of normal developmental and alterations of the normal developmental regulatory experimentally-induced plasticity, and afterwards will mechanisms in tumorigenesis have received much less link these findings with what we know about cancer attention. But in fact, if cellular fate was carved into biology. stone, cancer would be impossible, since no new Lineage commitment and cellular lineages could be generated other than the normal, physiologic ones. Here is where the normal identity Adult stem cells are the responsible of generating all mechanisms regulating cellular identity and plasticity the different specialized cellular types forming the play an essential role in allowing cancers to arise and organism. The majority of them perform this job hopefully, as we will discuss, they might be the key to throughout the whole life of the organism, thanks to its eradication. their self-renewal capacity. This property allows them The specification of cellular identity during to divide asymmetrically, therefore given rise to a new development and differentiation is a dynamic process identical daughter stem cell and to a multipotential that starts with stem and progenitor cells and ends with progenitor, lacking self-renewal capacity, which will terminal differentiation into each specialized cellular give rise to all the differentiated tissue cells. Although type. In this progression there can be many cellular it is known that there are some specific factors that are intermediates; some of them are transient, and some essential for the specification and maintenance of stem can be long-lasting, but the maintenance of cellular cell identity (Boyer et al., 2005), the molecular bases of identity at each stage is determined by the signals from the choice that stem cells have to make between the environment and, in an intrinsic manner, by specific maintaining competence (i.e. plasticity) or entering into transcription factors and epigenetic modifiers that the differentiation programs are not yet completely establish a defined chromatin architecture and a understood (Niakan et al., 2010). In this context, a first specific gene expression profile. important aspect to consider is the fact that the stem

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cell population itself is intrinsically heterogeneous. spermatogonial stem cells), adult stem cells are usually This means that the "stemness" is not a static condition multipotent, and they can give rise to a wide range of defined by stable, constant levels of expression of differentiated cell types. In the first instance, stem cells intrinsic stem factors and surface stem markers, but it is lose their self-renewal potential (their stemness) and more of a continuum that moves within certain start the differentiation process by becoming margins. For example, in a clonal population of multipotential progenitors. We have seen that the haematopoietic progenitor cells, there is a Gaussian differentiation program can be pre-set already by the distribution of the levels of expression of Sca-1, one of oscillatory patterns of gene expression at the stem cell the most classical stem cell markers (Chang et al., population level, and that cells lying at the different 2008). Furthermore, cells at both the low- or high-end ends of specific gradients of gene expression can have levels of expression can, when isolated, regenerate the opposite differentiation preferences (Chang et al., whole population with all the range of expression 2008). So, once they leave the stem cell state, the cells levels. However, every one of these sub-populations, start making lineage choices that are usually mutually defined by their levels of a surface marker, also excluding and are normally conceptualized in a expresses different transcriptomes, and has therefore branching pattern. These alternative options are usually distinct intrinsic differentiation tendencies towards controlled by the cross-antagonism between different lineages. Therefore, each individual cell in the transcription factors with competing, opposing stem population represents a metastable transitional functions (Swiers et al., 2006; Loose et al., 2007). A point in a continuum of constantly changing very well characterized developmental system is transcriptomes. In fact, this is most probably the hematopoietic differentiation where several models of mechanism at the basis of the stochastic choice of lineage-specification have been identified which seem lineage, when some cells approach too much to the to be based on the aforementioned mechanism. For "edges" of the normal distribution and the example, the choice between erythroid/megakaryocyte transcriptome changes become irreversible (Chang et or myeloid-monocytic fates at the level of al., 2008). erythromyeloid progenitors is controlled by the In 1957 Waddington conceptualized the irreversible reciprocal inhibition between the transcription factors process of cellular differentiation as marbles falling GATA-1 and PU.1, therefore creating a binary decision down a slope (Waddington, 1957). This metaphorical for the progenitor (Laiosa et al., 2006; Enver et al., concept has regained new momentum with the 2009). The bipotent progenitor itself would therefore be mathematical interpretation of transcriptional cellular this intermediate state created and maintained by the states as Gene Regulatory Networks (GRNs). In this equilibrium between the both factors. This fact helps type of analysis, pluripotency is represented as a understanding the phenomenon of multilineage gene mathematical attractor (a condition towards which a priming, in which uncommitted progenitors present low dynamical system tends to progress over time), in such levels of simultaneous expression of multiple a way that the points (cells) that get close enough to the transcription factors corresponding to different mature attractor remain close even if slightly disturbed. This cell types and possessing antagonistic functions (Hu et attractor is surrounded by a "differentiation landscape" al., 1997; Enver et al., 2009). In general, there seems to where other stable cellular fates are represented by be a progressive loss of developmental potential in a stable "valleys" and differentiation routes towards them hierarchical process that moves through sequential are "channels" through which the cells move (Enver et differentiation options and in which, at any given point, al., 2009; Huang, 2009). Under this light, pluripotency a progenitor would only have to choose between two can be considered as a dynamic state of controlled mutually exclusive options (Brown et al., 2007; heterogeneity within a population, where small Ceredig et al., 2009). Additionally, in the process of individual fluctuations in the levels of expression of maturation into a given lineage, the progenitors will transcription factors and epigenetic regulators maintain receive (and react to) the necessary extrinsic signals a global status of apparent stability. The cells that (for example, cytokines) that, according to this model, approach the limits of the attractor (those who, in their would be more permissive than instructive. random fluctuations, go too far from the middle point Maintenance of the cellular identity of the Gaussian curve) are therefore more prone to differentiate, suggesting that commitment, although of mature differentiated cells rare, is an spontaneous phenomenon (unless it is Plasticity, in normal development, is a property that is specifically triggered by an external signal that "intended" to be restricted to stem cells and unbalances the dynamic equilibrium) (Huang, 2009). progenitors. In general, the final differentiated cellular Maintenance of cellular identity types of any given organ or tissue possesses stable identities, in consequence with the fact that they throughout the differenciation usually are highly specialized cells with very specific process physiological functions. Therefore, it would not make Although in some rare cases they are unipotent (e.g. sense, from the biological point of view, that a

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specialized cell would be the source of other most favourable conditions. This fact clearly indicates differentiated cell types. This, as we have mentioned, is that, independently on how many cells of the the role of stem cells, with their physiological plasticity population are initially responsive to the (i.e., normal competence) that we have previously reprogramming factors, very few of them can complete discussed. However, the concept of the stability of the path towards full reprogramming (Yamanaka, differentiated cell types has been shaken by the 2009). Also, this is a gradual process in which several discovery of the fact that the 4 Yamanaka transcription non-physiological cellular intermediates can be isolated factors (4Y TFs) Oct4, Sox2, c-Myc and Klf4 (Mikkelsen et al., 2008; Stadtfeld et al., 2008). The (Takahashi and Yamanaka, 2006) are enough for the study of these incompletely reprogrammed reprogramming of most differentiated cells types into intermediates has revealed that they have re-activated induced pluripotent stem cells (iPSCs). This finding has the self-renewal and maintenance stem cell genes, but altered our notion of the latent developmental potential not yet those of pluripotency; also, these stages of hidden in differentiated cells, showing how it can be aborted reprogramming have not been able to "awakened" by experimental manipulations in the completely repress the expression of lineage-specific laboratory. This, as we have described, was already transcription factors and retain persistent DNA known to a certain extent from the nuclear hypermethylation marks as a proof of their failure in reprogramming experiments performed in amphibians achieving complete epigenetic remodelling (Mikkelsen more than 50 years ago (Briggs and King, 1952; et al., 2008). But perhaps the most patent proof of the Gurdon, 1962). Nevertheless, although those difficulty of the process of full reprogramming to experiments already proved that the cell nucleus could pluripotency is the persistence of an epigenetic memory be reprogrammed from a differentiated cell type into a in the iPCs that makes them more prone to re- pluripotent progenitor, Yamanaka's experiments differentiate into the lineages from which they were showed that only 4 factors were actually enough to initially derived, indicating that a complete elimination make the whole process possible. We have seen that, a of the initial epigenetic program cannot yet be achieved more modest level, it had already been proven that the (Kim et al., 2010; Bar-Nur et al., 2011). overexpression or loss of individual transcription factors could induce fate changes in differentiated cells Tumoral reprogramming and (MyoD, C/EBPa, Pax5, etc). Although these were induction of pluripotency: examples of transdifferentiation taking place between similarities closely related cell types, they already pointed the way The role of transcription factors in the control of for the search of the factors capable of reprogramming tumoral reprogramming and induction of pluripotency to full pluripotency. Since the differentiated state is the We have seen in the initial section of this review that more stable one (indicating that the GRNs are less both cancer research and developmental biology have subject to fluctuation), where the cells have reached been the focus of intense attention since ancient times. after "rolling down" the differentiation pathway in the What's more, they have always been closely related normal process of development, therefore an from the conceptual point of view. The cellular theory "activation energy" is required to move the cells of Rudolf Virchow is clearly essential for the "uphill" to become again pluripotent. Conceptually, understanding of both development and there are at least two main possible scenarios to explain tumorigenenesis. But he went further, since he already the population dynamics in the process of proposed the embryonal rest hypothesis of tumour reprogramming to pluripotency (Yamanaka, 2009): one origin, after realising the histological similarities possibility (the so-called elite model) is that only some between tumours and embryonic tissues (Virchow, cells can be reprogrammed, and these are the ones that 1855). This concept was afterwards expanded by Julius are selected among the entire population, since they are Conheim, who suggested that tumours arise from the only ones that are receptive to the action of the residual embryonic remnants "lost" during normal reprogramming factors. Alternatively, it might happen development (Cohnheim, 1867). This hypothesis that all the differentiated cells are equally capable of actually connects with the current theory of the cancer undergoing reprogramming, and it is only due to stem cells (CSCs) in which progenitors are situated at technical or methodological reasons that we are not the root of cancer maintenance (see below). Another able to reveal this potential in all of them (stochastic example of the influence of cancer research in the model). According to the accumulating evidences, it progress of the fields of stem cell biology and would seem that the stochastic model is the one that is developmental biology is the fact that embryonic stem closer to reality and that, given the right combination of (ES) cells were identified in a search that has been factors; any cell could be reprogrammed to initiated in the study of teratocarcinomas (Solter, 2006; pluripotency (Yamanaka, 2009). However, as we have Morange, 2007; Hochedlinger and Plath, 2009). mentioned, this is a developmentally and energetically In the field of cancer research it has traditionally been unfavourable process, a fact that is evidenced by postulated that more than one molecular hit is required several details. The most obvious one is the very low to generate a tumour cell, because several aspects of efficiency of the reprogramming process, even in the

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cellular biology must be altered in the progress towards before the activation of the regulators of the pluripotent a full-blown tumour (Hanahan and Weinberg, 2000). state and, consequently, ectopic expression of c-Myc is Therefore, in order to achieve tumoral reprogramming required only during the first few days of the (although this was not the terminology traditionally reprogramming process (Sridharan et al., 2009). In fact, used), more than one single molecular alteration had to c-Myc is dispensable for reprogramming, but in its happen. We have mentioned before that for a "simple" absence there is an enormous drop in the efficiency of transformation, like a lineage switch, the change in the the procedure (Nakagawa et al., 2008; Wernig et al., levels of expression of a single transcription factor 2008). The other three factors, Oct4, Sox2, and Klf4, could be enough (Davis et al., 1987; Nutt et al., 1999; need to act together to achieve the entry into the Xie et al., 2004; Cobaleda et al., 2007a). Similarly, a pluripotent condition, as evidenced by the fact that, single initial oncogenic lesion may contribute to just a when they are used individually, they cannot bind their part of the tumoral phenotype, by causing a block in pluripotent target genes in cells that are sill differentiation, or an alteration in the control of cell incompletely reprogrammed, most likely because the cycle. In oncogenesis, many factors and routes have pattern of epigenetic modifications at these loci is not been shown to be altered, and their individual permissive for their binding (Sridharan et al., 2009). contributions to the tumoral phenotype are clear, Indeed, Oct4, Sox2, and also Nanog co-bind to a although their synergy and interactions are less known. plethora of genes in overlapping genomic sites (Boyer In the case of reprogramming to pluripotency, the et al., 2005; Loh et al., 2006), in such a way that the discovery of Takahashi and Yamanaka (Takahashi and transcriptional program required for pluripotency is Yamanaka, 2006) revealed the nature of these factors. maintained by the coordinated action of these key Before, reprogramming to pluripotency was only genes. possible by the use of nuclear transplantation, but it In general, for the reprogramming of almost every cell was not known which of the factors present in the type to pluripotency, the 4 Yamanaka transcription zygote possessed the required reprogramming capacity. factors are enough. However, there are some Interestingly enough, the 4 Yamanaka factors are exceptional cases in which additional alterations are known to be involved in tumorigenesis in different required. For example, in the case of mature B cells it contexts, and both c-Myc and Klf4 are well-known is necessary to interfere with the activity of the oncogenes (Rowland et al., 2005; Okita et al., 2007; transcription factor Pax5, which is the master regulator Tanaka et al., 2007; Chen et al., 2008), thus further of B cell identity (Cobaleda et al., 2007a; Hanna et al., linking reprogramming to tumorigenesis. 2008). Previous experiments had revealed that the In summary, the experimental results show that the elimination of Pax5, in the absence of any other genetic maintenance of cellular identity is essential for manipulation, allowed mature B cells to dedifferentiate differentiated cells, and that only strong transcriptional to early haematopoietic multipotential progenitors or epigenetic regulators can subvert it. In this way, the (Cobaleda et al., 2007b). These findings again correlate multistep nature of tumorigenesis is paralleled by reprogramming with cancer development, since it has reprogramming to pluripotency in the series of "uphill" also been shown that the elimination of Pax5 function steps required and in the need for the sum of the effects in mature B cells induces a process of pathological of several factors to overcome the built-in safety dedifferentiation that gives rise to progenitor cell mechanisms designed to protect cells from lymphomas (Cobaleda et al., 2007a). Therefore, the transformation or, in other words, to prevent cells from loss of a transcription factor that is required for the changing their identity. In the case of the maintenance of cellular identity can be a tumour- reprogramming factors, the precise role of each of them inducing lesion. However, and contrary to mature B is not yet clear, but their experimental introduction at cells, earlier stages of B cell development can be different times during the process of reprogramming is reprogrammed to pluripotency in the presence of shedding some light on this issue (Sridharan et al., functional Pax5, just with the 4 Yamanaka transcription 2009), by identifying distinct contributions of the factors (Hanna et al., 2008), thus supporting the different factors along the reprogramming progression. intuitive idea that the degree of differentiation of the In the early stages of reprogramming, the most target cell has an effect on the final efficiency of important process happening is the silencing of the reprogramming (see below). gene expression programs of the differentiated cells. In the genetic landscape, the oncogenic mutations alter This aspect is previous to the induction of the ES-like the architecture of the whole gene regulatory network, expression program, and the main molecular since it modifies one of the nodes. This leads to an responsible for this function seems to be c-Myc. alteration in the landscape that gives rise to new However, it has also been shown that treatment with abnormal attractors (new "valleys") where cancer cells histone deacetylase inhibitors like valproic acid (VPA) reside (Huang et al., 2009). Furthermore, this alteration can substitute for c-Myc, because of their capacity for in the landscape gives the cell a new momentum to repressing the gene expression programs of move towards new directions, and this effect can differentiated cells (Huangfu et al., 2008). Therefore, it persist even when the initial stimulus has disappeared. would seem that the action of c-Myc takes place mainly From the point of view of tumoral reprogramming, this

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implies that the expression of a tumour-promoting the pluripotencial state as the progenitors exit to the gene, even if it is transient, can by itself trigger a differentiation process (Feldman et al., 2006; Epsztejn- durable malignant phenotype that does not require Litman et al., 2008). This is achieved by its histone anymore of the initial mutation for its maintenance methylation activity, that prevents the reactivation of its (Huang et al., 2009). target genes (for example embryonic genes like Oct4) The role of epigenetic factors in the control of tumoral when their transcriptional repressors are no longer reprogramming and induction of pluripotency present (Feldman et al., 2006). Also, at the same time, In the previous section we have seen that either the gain G9a promotes DNA methylation, that stops reversion or the loss of function of transcription factors plays an towards the undifferentiated state (Feldman et al., essential role in reprogramming to pluripotency, in the 2006; Epsztejn-Litman et al., 2008). Therefore, same way as how oncogene overexpression or loss of genome-wide epigenetic changes affecting many still tumour suppressors promote tumorigenesis. Also, unknown loci, are essential in the late stages of similarly to tumour progression, large-scale epigenetic direct reprogramming, and inhibition of the proteins changes are required for full reprogramming to happen. responsible for generating or maintaining these marks Today, it is clearly established that not only genetic lowers the "activation energy" required for the alterations are responsible for cancer development, but transition to pluripotency. Therefore, it makes sense there is also an important role of epigenetic alterations that several of the chemical inhibitors that we have just (Esteller and Herman, 2002; Esteller, 2007; Esteller, mentioned are in fact already in use, or in clinical trials 2008) that lead to the specification of an heritable, to be used as therapeutic agents against cancer. AZA abnormal pattern of gene expression that plays an was approved by the FDA in 2004 for the treatment of essential role in cancer initiation and progression (Ting myelodysplastic syndromes, being the first drug into et al., 2006). All the relevant epigenetic marks, from the new class of demethylating agents (Kaminskas et DNA methylation to histone modifications, are al., 2005). Its mechanism of action is very unspecific, perturbed in tumour progression. The subsequent aimed at the restoration of the normal levels of changes in gene expression patterns are especially expression of genes whose expression has been lost due relevant when they affect the levels of expression of to promoter hypermethylation during tumoral specific oncogenes or tumour suppressors, but they progression, and that might be necessary for the control affect in fact the whole epigenome, and therefore of proliferation and differentiation. Like in the case of condition all cellular identity. All these epigenetic most antitumoral drugs, AZA is expected to affect alterations are usually secondary, and they can be just primarily the tumoral cells and leave non-proliferative due to tumour progression and therefore independent cells unaffected (Sacchi et al., 1999; Kaminskas et al., from (i.e., not directly caused by) the initiating 2005). Something similar happens for HDAC inhibitors oncogenic mutation, but they can also be directly (Dey, 2006; Lane and Chabner, 2009). All these induced by the first oncogenic event, like it happens findings underscore once more the concept of cancer as when chromosomal aberrations deregulate histone a reprogramming disease and a case of wrong modification genes (Esteller, 2008). In the process of differentiation. reprogramming to pluripotency, epigenetic Instructive and permissive factors in the progression modifications are intrinsically required for the process and selection of the processes of tumoral to take place, and they have to occur all throughout the reprogramming and induction of pluripotency genome, not being just restricted to the activation or We have seen how both genome-wide changes in repression of individual genes, something that is epigenetic marks and the loss and/or gain of already achieved by the transcription factors. This transcriptional regulators are essential components of explains why the efficiency of reprogramming is the processes of tumour generation and reprogramming significantly superior in the presence of chemicals that to pluripotency. However, it is clear that these changes can globally interfere with epigenetic marks. For are clearly unwanted from the points of view of normal example, the DNA methyltransferase inhibitor 5-aza- development and cellular function. Therefore, cells cytidine (AZA) causes a rapid and stable transition to a have developed many built-in protection mechanisms fully reprogrammed iPS state (Huangfu et al., 2008; to maintain their identity against these transcriptional, Mikkelsen et al., 2008). Similarly, treatment with genetic and epigenetic changes. Nevertheless, all these valproic acid (VPA), a histone deacetylase (HDAC) mechanisms are bypassed, in one way or another inhibitor, considerably improves the induction to (Hanahan and Weinberg, 2000; Hanahan and pluripotency (Huangfu et al., 2008). Other example is Weinberg, 2011), and cancer appears. How this provided by the use of the compound BIX-01294, an happens in "progression to pluripotency" (in analogy to inhibitor of G9a methyltransferase that makes it tumoral progression) is still to be discovered. However, possible to achieve reprogramming to pluripotency it has recently been shown by several groups (Zhao et using only Oct4 and Klf4 transcription factors, with an al., 2008; Banito et al., 2009; Hong et al., 2009; efficiency comparable to the one obtained when using Kawamura et al., 2009; Krizhanovsky and Lowe, 2009; the four factors (Shi et al., 2008). In normal Li et al., 2009; Marión et al., 2009; Utikal et al., 2009) development, the biological role of G9a is to terminate that, exactly as it happens in cancer progression, the

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elimination of the DNA damage control checkpoint like in any other stem-cell based tissue, the majority of (p53-p21) greatly improves the efficiency of the cells composing the tumour mass lack this capacity. reprogramming process, making it possible that many Hence, if tumours are maintained by aberrant cells of the starting cells become successfully possessing stem cell characteristics, then what is the reprogrammed. This is done at the expense of an origin of these cells? This cancer cell-of-origin (not to increased level of genetic instability, and most of the be confused with the CSC, which would be the cancer- iPSCs obtained in the absence of a functional p53-p21 maintaining cell of the already developed tumour) is axis carry genetic aberrations of different kinds. This is initially a normal cell (not necessarily a stem cell) that in connection with what we have mentioned before will be reprogrammed by the oncogenic events in order about reprogramming being an "uphill", unfavourable to finally originate (or convert into) a tumoral cell with process, which most of the cells fail to complete stem properties. There are two main mechanisms that (Mikkelsen et al., 2008). Therefore, eliminating the could be invoked in this scenario. One option is that the DNA damage checkpoint diminishes the selection and cell-of-origin suffering the oncogenic mutation(s) is allows a larger number of cells to survive until already a stem cell, which therefore becomes pluripotency. These results support the idea of cancer reprogrammed to give rise to a new pathological tissue as a disease of cellular differentiation and, furthermore, instead of the normal one. In the case of CML, it has reinforce the idea that suggests that the driving forces recently been demonstrated, using genetically modified behind the tumoral process are aberrantly expressed mice, that the restricted expression of the oncogenic transcription factors, epigenetic regulators and alteration in the stem cell/progenitor compartment is signalling molecules, while the role of many of the enough to generate a human-like tumour with all the other alterations found in tumours (for example, the variety of differentiated tumour cells (Pérez-Caro et al., loss of p53) is mainly permissive. 2009; Vicente-Dueñas et al., 2009b). In mouse models Role of the cell of origin in tumoral reprogramming of intestinal cancer it has also been found that tumours and induction of pluripotency originate in the crypt stem cell, since when the In the study of oncogenesis, it has traditionally been oncogenic stimulus (activation of the Wnt signalling assumed that the phenotype of the tumour cells was a pathway) is targeted to the stem cell compartment, reflection of that of the normal cell that gave rise to the intestinal adenomas develop in which a developmental tumour in the first place. There were some classical hierarchy is maintained. On the contrary, when the examples in which this what not the case like, for oncogenic lesions are targeted at the non-stem example, chronic myelogenous leukaemia (CML), intestinal epithelial cells, they only generate short- where the t(9;22) chromosomal translocation could be lived, small microadenomas (Barker et al., 2008; Zhu et found in most types of differentiated haematopoietic al., 2008). In the nervous system, targeting cells, therefore indicating that a common, earlier astrocytoma-associated oncogenic lesions to progenitor, should be the cell of origin (Melo and progenitors (in this case in the subventricular zone) Barnes, 2007). But, in general, since most cancerous results in tumour development, while targeting them to cells are reminiscent of some differentiated cell type, the differentiated cells of the adult parenchyma does for every type of tumour, the cell of origin was not result in tumours, only in local astrogliosis postulated to be the corresponding normal (Alcantara Llaguno et al., 2009). Therefore, there are differentiated cell. However, the cancer stem cell many examples (Dirks, 2008; Joseph et al., 2008; (CSC) theory has led to a change in our perspective Zheng et al., 2008) where it has been proven that the (Cobaleda and Sánchez-García, 2009; Vicente-Dueñas initiating event takes place in a normal stem cell, even et al., 2009a; Vicente-Dueñas et al., 2009b). The CSC if the mature tumour is composed by differentiated theory proposes that tumours are stem cell-based cells, indicating a true tumoral reprogramming tissues just like any other, and this has several radical mediated by the oncogenic lesions (Vicente-Dueñas et consequences for our understanding of cancer. The al., 2009b). most important one is the fact that not all the tumoral The other alternative is that the cancer cell-of-origin cells are equally capable of regenerating the tumour. can be a differentiated cell that regains stem cell This means that, when tumoral cells are experimentally characteristics in the process of tumoral transplanted into a new host, or when some tumour reprogramming. This option relies on two cells remain in the patient after incomplete tumour requirements: first, the oncogenic alteration must be excision, the reappearance of the tumour is caused by capable of conferring or programming these just a certain tumoral cellular subpopulation. Only characteristics in the target cell and, second, the cell those cells, possessing stem cell characteristics, can must be plastic enough so as to be reprogrammed by give rise to the whole tumour with all its cellular this precise oncogenic alteration. It has been shown that heterogeneity. Although there can be a big range of some oncogenes, like MOZ-TIF2 (Huntly et al., 2004), variability in the percentage of CSCs within a tumour, MLL-AF9 (Krivtsov et al., 2006; Somervaille and from very few to 25% (Quintana et al., 2008; Cobaleda Cleary, 2006), MLL-ENL (Cozzio et al., 2003), MLL- and Sánchez-García, 2009; Vicente-Dueñas et al., GAS (So et al., 2003) or PML-RAR α (Guibal et al., 2009a; Vicente-Dueñas et al., 2009b), the fact is that, 2009; Wojiski et al., 2009) can generate CSCs when

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they are introduced into committed target cells. Gene characteristics of the CSC population in a certain expression arrays have revealed that MLL-AF9 can moment may not relate at all any more to those of the activate a stem cell-like program in committed initial cancer cell-of-origin (Barabé et al., 2007). granulocyte-macrophage progenitors, therefore As we already mentioned when we described the view conferring them the property of self-renewal (Krivtsov of reprogramming to pluripotency from the perspective et al., 2006). Also c-Myc can induce a transcriptional of the GRNs, the inducing factors are not required program reminiscent of that of embryonic stem cells in anymore once the cells have reached the pluripotent differentiated epithelial cells, and originate epithelial condition and the new identity (however plastic this is) CSCs (Wong et al., 2008). However, other oncogenes has been established. If cancer stem cells are generated are unable of conferring self-renewal properties, like by a tumoral reprogramming process, then maybe the for example BCR-ABLp190 (Huntly et al., 2004). In oncogenes that initiate tumour formation might be not these cases the oncogene, since it cannot immediately be required for tumour progression (Krizhanovsky and confer stem cell properties, could give rise to a Lowe, 2009). If this were the case, it would explain the precancerous cell that can afterwards, with the presence aforementioned examples in which a pre-cancerous of additional alterations conferring "stemness", give lesion exists stably in an aberrant cell population that rise to the cancer stem cell (Chen et al., 2007). In any does only evolve to an open tumour when secondary case, the cellular origin where the cancer-initiating mutations occur. In this scenario, the initiating lesion lesions take place is difficult to determine since, in would be the driving force in the reprogramming many cases, the functional impact of the oncogenic process, but once this has been completed, it would lesion (i.e. the tumour clonal expansion) can present only be a passenger mutation, or could even perform a with phenotypes mimicking differentiation stages that different role that would be independent from its can be either upstream or downstream of the initiating reprogramming capacity, like for example in tumour cell. For example, the translocations that are the expansion/proliferation. A mechanism of this kind initiating lesions of many childhood B acute would explain why some targeted therapies fail in spite lymphoblastic leukaemias (ALL) originate in utero of their initial apparent efficacy: for example, imatinib, during embryonic haematopoiesis and promote the a drug targeted against the deregulated kinase activity conversion of partially committed cells into of BCR-ABL, successfully eliminates differentiated preleukaemic cells with altered self-renewal and tumour cells, but it fails to kill the BCR-ABL + CSCs, survival properties, that will require a second postnatal since it does not seem to interfere with the function of hit to develop into full leukemias (Hong et al., 2008). the chimeric oncogene in this cellular context (Graham Also, in leukemias carrying the AML1-ETO et al., 2002; Barnes and Melo, 2006). translocation, this aberration can be detected in stem The fact that CSCs can originate from differentiated cells in patients in remission. These stem cells behave cells represents the last and most patent similarity apparently normal during the remission phase, between tumorigenesis and reprogramming to indicating that they can remain dormant and, with time, pluripotency. Also in iPSCs generation, the nature of some of their descendants can become tumorigenic and the cell of origin is key in determining the global originate the relapse (Miyamoto et al., 2000). We have success. In this way, it has been described that, in the described previously that, in mice, the loss of Pax5 in haematopoietic system, the capacity of reprogramming mature B cells leads to the dedifferentiation to cells decreases as they differentiate, since HSC are 300 multipotent progenitors and the appearance of times more likely to be reprogrammed than B or T cells progenitor B cell lymphomas (Cobaleda et al., 2007a). (Eminli et al., 2009). In the case of the nervous system, In human Hodking lymphomas, the overexpression of when the starting cells are adult neural stem cells specific antagonists leads to the functional inactivation (NSCs), then pluripotency can be achieved using only of the B cell factor E2A, which in turn causes the loss Oct4 (Kim et al., 2009), probably because of the high of B cell markers and induces the expression of similarity of NSCs transcriptional profile to that of ES lineage-inappropriate genes characteristic of the Reed- cells. Similarly, in a liver model of transdetermination Sternberg Hodking lymphoma cells (Mathas et al., it has been demonstrated that Neurogenin3 can convert 2006). Also in children's B-ALLs, the CSCs can hepatic progenitor cells into neo-islets but it cannot present with the phenotypes of different stages of early transdifferentiate mature hepatocytes (Yechoor et al., B cell development that, on top of that, can apparently 2009). interconvert among them, therefore complicating even Outlook more the task of identifying the cancer-cell of origin (le Viseur et al., 2008). A genomic analysis of samples The knowledge obtained in the research of the from relapsed ALL patients, when compared with the molecular and cellular mechanisms that control cellular samples at diagnosis, has shown that the same ancestral plasticity, pluripotency and reprogramming will also clone can be found at both stages of the disease have a profound impact in our understanding of (Mullighan et al., 2008). So, clearly in many cases the tumorigenesis and, in a more distant future, in the cancer-maintaining cell evolves over time and adapts to treatment of cancer. It is clear that the two fields of treatment to finally lead to relapse, and therefore the research will continue being mutually interdependent.

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By way of example, the main obstacle for the future cells can be reprogrammed by nuclear transplantation use of iPSCs in the clinic is precisely the generation of (Hochedlinger et al., 2004). Also, embryonal carcinoma tumours as a result of uncontrolled growth or cells or mouse brain tumours have been used as a valid differentiation of the cells, once they are in the patient. starting material for nuclear cloning experiments (Li et Therefore, the knowledge and control of the narrow al., 2003; Blelloch et al., 2004). Therefore, maybe in a limits of gene expression that mark the difference not so distant future we might have the knowledge and between normal and tumoral differentiation and tools to manipulate tumoral cell identity to force cancer reprogramming will be required before this problem cells to differentiate, or to make them vulnerable to can be overcome. therapy. Assuming the role that reprogramming plays in cancer Acknowledgments generation makes it possible to initiate the development of new therapeutic strategies aimed at re-directing the Research in C.C. lab was partially supported by wrong differentiation program towards a new outcome FEDER, Fondo de Investigaciones Sanitarias (ideally, in most cases, terminal non-tumoral (PI080164), CSIC P.I.E. 200920I055 and 201120E060, differentiation and cellular death). Differentiation from the ARIMMORA project (FP7-ENV-2011, therapies are already in use in some cases, like the European Union Seventh Framework Program) and administration of retinoic acid to differentiate tumoral from an institutional grant from the "Fundación Ramón cells in PML-RAR α+ positive acute promyelocytic Areces". Research in ISG group was partially leukemias. We have described how reprogramming to supported by FEDER and by MICINN (SAF2009- pluripotency, due to its inefficiency, can get caught up 08803 to ISG), by Junta de Castilla y León (REF. at several points before reaching the iPSC state CSI007A11-2 and Proyecto Biomedicina 2009-2010), (Mikkelsen et al., 2008). Tumoral cells are probably by MEC OncoBIO Consolider-Ingenio 2010 (Ref. very close to these incompletely reprogrammed CSD2007-0017), by NIH grant (R01 CA109335- intermediates, and the study of the latter should help us 04A1), by Sandra Ibarra Foundation, by Group of in understanding how to get the former ones out of their Excellence Grant (GR15) from Junta de Castilla y pathologic block. In fact, epigenetic therapies are most Leon, and the ARIMMORA project (FP7-ENV-2011, probably going to be on the rise in the coming years for European Union Seventh Framework Program) and by the treatment of many types of tumours, since our "Proyecto en Red de Investigación en Celulas Madre knowledge about the molecular mechanisms Tumorales en Cancer de Mama", supported by Obra controlling the epigenetic marks and their role in self- Social Kutxa y Conserjería de Sanidad de la Junta de renewal, differentiation and maintenance is increasing Castilla y León. All Spanish funding is co-sponsored very quickly, and this should help us to obtain more by the European Union FEDER program. ISG is an and better (more specific) epigenetic drugs (Jones, API lab of the EuroSyStem project. ECS is the 2007; Shen et al., 2009). recipient of a JAE-predoc Fellowship from CSIC and a The discovery of reprogramming to pluripotency has "Residencia de Estudiantes" Fellowship. The authors transfigured the research in the field of cellular declare no conflict of interest. plasticity. It is nowadays possible, using just three ectopic factors, to reprogram fibroblasts into functional References neurons (Vierbuchen et al., 2010), to convert in vivo Reaumur RAF.. Sur les diverses reproductions qui se font pancreatic exocrine cells to β cells (Zhou et al., 2008) dans les ecrevisses, les omars, les crabes, etc. et entr'autres or to directly transdifferentiate mouse mesoderm into sur celles de leurs jambes et de leurs ecailles. Mem Acad Roy. heart tissue (Takeuchi and Bruneau, 2009). One of the 1712;Sci 223-245. most remarkable examples in this context is the Trembley A.. Memoires Pour Servir A L'Histoire D'Un Genre de phenotype caused by the deletion of a single gene, Polypes D'Eau Douce, a Bras En Forme de Cornes. Leiden, Foxl2 , in adult ovarian follicles. 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Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function Xiaodong Lu, Wenxin Qin School of Medical Science and Laboratory Medicine, Jiangsu University, China (XL), State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, China (WQ)

Published in Atlas Database: September 2011 Online updated version : http://AtlasGeneticsOncology.org/Deep/V-ATPaseInCancerID20104.html DOI: 10.4267/2042/47290 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Vacuolar H+-ATPase (V-ATPase) is a highly arrangement of alternating A and B subunits, which evolutionarily conserved enzyme, which is distributed participate in ATP binding and hydrolysis. Other within the plasma membranes and the membranes of subunits of V1 include three copies of E and G subunits some organelles such as endosome, lysosome and which are the stator, one copy of the regulatory C and secretory vesicle. The mayor function of V-ATPase is H subunits, one copy of subunits D and F which form a to pump protons across the cell membrane to central rotor axle. The V0 section includes a ring of extracellular milieu or across the organelle membrane proteolipid subunits (c, c' and c") that are adjacent to to intracellular compartments. V-ATPases located in subunits a and e. Subunits D and F of V1 and subunit a cell surface act as important proton transporters that of V0 form the central stalk, whereas the multiple regulate the cytosolic pH to ~7.0 which is essential for peripheral stalks are composed of subunits C, E, G, H most physiological processes, whereas V-ATPases and the N-terminal domain of subunit a. V1 and V0 is within intracellular membrane are involved in cellular connected by both stalks. Several subunits like a, d, e, processes as receptor-mediated endocytosis, membrane C, G, H, D and F contain slice variants as to spatial and trafficking, protein processing or degradation, and temporal expression pattern in different cell types nutrients uptake (Nishi et al., 2002; Forgac et al., 2007; (Forgac et al., 2007; Miranda et al., 2010). As for Toei et al., 2010; Cruciat et al., 2010). Malfunctioned tumor cells, especially those with high metastatic V-ATPase is closely related to several diseases potential, the V-ATPases are usually excessively including tumor. More and more evidences indicate agitated. The altered structures of V-ATPase of tumor that V-ATPase is an enhancer for carcinogenesis and cells may include the increased level of subunit cancer progression, such as malignant transformation, expressions and unique spliced variants of some growth and proliferation, invasion and metastasis, subunits. acquirement of multi-drug resistance, etc., which The level of the subunit c expression was found to be strongly supports that V-ATPase should be an effective related to the metastasis potentials in tumors. One of target of anticancer strategy (Fais et al., 2007). the studies is the comparison of subunit c expression The structure of V-ATPases and its between normal and pancreatic carcinoma tissues and between invasive and non-invasive pancreatic cancers, expression in tumor cells which immunohistochemical data showed the notable The molecular structure of normal V-ATPase of yeast difference - 92% invasive ductal cancers (42/46) were and mammalian cells has been well studied. V-ATPase mild to marked subunit c positive in the cytoplasm, is a delicate complex which is composed of a cytosolic whereas neither non-invasive ductal cancers nor benign catalytic domain V1 and an integral domain V0, the cystic neoplasms expressed detectable immunoreactive former responsible for ATP hydrolysis and the latter proteins (Ohta et al., 1996). Subunit c seems to be one providing transmembraneous proton channel (Nishi et of the V-ATPase subunit which significantly influence al., 2002; Yokoyama et al., 2005; Wang al., 2007). The the proliferation and metastasis of tumor cells. The core of the V1 section is composed of a hexameric inhibition of the V-ATPase subunit c via siRNA

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resulted in the suppression of growth and metastasis of (Nishihara et al., 1995; De Milito et al., 2007) and a hepatocellular carcinoma cell line in vitro and in mice breast cancer (McHenry et al., 2010).The deficiency of model (Lu et al., 2005), which is according to another V-ATPase will decrease cytosol pH and increased result of the suppression of subunit c in Hela cell via lysosome pH, both of which might influence lysosome antisense oligonucleotides (Zhan et al., 2003). But in function. The apoptosis induced by V-ATPase oral squamous cell carcinoma cells, subunit C1 was the inhibitors were in either lysosome-mediated or non- most strongly over-expressed gene at the mRNA level lysosome-mediated manner. In the first case, when compared to other genes of the V-ATPase complex lysosomal V-ATPase was defected, lysosomal pH and (Otero-Rey et al., 2008). permeability will be increased, resulted in the release of Specific spliced variants of subunit have been observed cathepsin D and activation of caspase, with no in tumors. A study of expression of subunit a of V- significant impact on mitochondrial transmembrane ATPase in breast cancer cell lines displayed the potential (Nakashima et al., 2003). In the other case, metastasis-specific subunit a isoform expression mitochondria and lysosome might be together involved profile. In highly metastatic breast cancer cell line in V-ATPase-inhibitor-induced apoptosis via capsase compared with its lowly metastatic parallel, levels of a3 pathway or ROS-dependant manner (Ishisaki et al., and a4 were much higher although all the four a 1999; De Milito et al., 2007). The inhibition of V- isoforms - a1-4 can be detectable. They distribute ATPase could also induce apoptosis by suppressing differently, and especially, a4-containing v-ATPases anti-apoptotic Bcl-2 or Bcl-xL and facilitate the were located mainly in the plasma membrane of higher caspase-independent apoptotic pathway (Sasazawa et metastatic breast cancer cell, seeming to be involved in al., 2009). In order to survive from the apoptosis the formation of the leading surface of the cells due to induced by acidosis resulted from glycolysis, tumor the combination with F-actin and closely correlated to cells needs to extrude excessive acid, in which the potency of invasion. a3-containing V-ATPases processes V-ATPase plays a crucial role. It is were located in intracellular compartment membrane, reasonable to postulate that the inhibition of proton which regulated the pH of the cytosol and intracellular extrusion may be more susceptible or vulnerable to cell compartments and also involved in invasion (Hinton et death of cancer cells than normal cells. al., 2009). In accordance with this data, the strongly Moreover, the slightly alkalized cytosolic pH favors the expressed a3 isoform were observed in high-metastatic growth and proliferation of the cells. Some glycolysis- melanoma cells and in bone metastases (Nishisho et al., related enzymes or oncogenes are sensitive the narrow 2011). Other tumor-relevant spliced variants are yet to range of pH alteration. Alkalization of cytosol, which be found. mainly regulated by V-ATPase in tumor cells, could activate glycolysis whereas repress oxidative The roles of the v-ATPase in the phosphorylation, meanwhile also promote the growth, proliferation or apoptosis in transcription of oncogenes like HIF-1, akt, myc, ras, etc tumor cells (Gillies et al., 2008; López-Lázaro, 2008). The cytosol One of cancer hallmarks is the shift in energy pH of tumor cells was found to be higher than in production from oxidative phosphorylation to aerobic untransformed controls (Busa et al.,1984; Casey et al., glycolysis, ie "Warburg effect", which produces excess 2010) and increasing cytosol pH was sufficient to intracellular acidosis (Gillies et al., 2008). However, confer tumourigenicity to cultured fibroblasts (Perona cancer cells usually have neutral to alkaline et al., 1988). On the contrast, p53, the important tumor intracellular pH in the acidized extracellular suppressor could be inactivated in the condition of microenvironment. The V-ATPase is among the four alkalization (Xiao et al., 2003). It is much likely that major types of pH regulators (the other three are: the glucose metabolism shift and mutant V-ATPase Na+/H+ exchangers, bicarbonate transporters, may be the co-selectors in selecting those "adaptive proton/lactate symporters). Much data implies proton phenotype", which may take the advantages for pump is essential in tumors and cells seem to render V- survival and proliferation during the initial stage of ATPases more than any other three transporters to carcinogenesis. regulate pH in cytosol (Torigoe The functions of the v-ATPase in cellular signals processing et al., 2002). The ability to extrude intracellular protons and maintain the cytosol pH is critical for V-ATPase is the important factor that regulates the cancer cell survival from a cascade of self-digestion process of internization and activation of cellular triggered by acidosis. signals. It is mainly due that the V-ATPase is the main The inhibition of v-ATPase may induce apoptotic cell contributor of low intracellular vesicles pH, which is death in several human cancer cell lines including essential for various membrane traffic processes. V- pancreatic cancer (Ohta et al., 1998; Hayash et al., ATPase activity influence endocytosis and degradation 2006), liver cancer (Morimura et al., 2008), gastric of molecule-receptor complex, recycling of the released cancer (Nakashima et al., 2003), B-cell hybridoma cells receptor, recruitment of signal molecules, and their

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proper spatial intracellular distributions (Hurtado- gene of IGF-I (insulin-like growth factor) and having Lorenzo et al., 2006; Marshansky et al., 2008), an interaction with subunit c. HRG-1 could promote therefore exerts a profound effect on cell behavior such endosomal acidification and receptor trafficking, as growth, proliferation or metastasis via the modulated enhance the proliferative and invasive phenotype of signals and their pathways. It has been reported that cancer cells. It was implied that the increased active V- tumor-associated m-TOR (mammalian target of ATPase by HRG-1 not only regulate the endocytosis rapamycin) (O'Callaghan et al., 2009), Notch (Fortini and degradation of receptors that promote signaling for and Bilder, 2009; Vaccari et al., 2010) or Wnt (Cruciat survival, growth, and migration of cancer tumor, but et al., 2010; Buechling et al., 2010) could be regulated also facilitate micronutrient uptake necessary for tumor by V-ATPase. cellular metabolism (O'Callaghan et al., 2009). Early endosomes are important sites for signal The contributions of the V-ATPase molecules internalization and activation in mammalian cells. Studies of the effects of V-ATPases inhibitors on in cancer metastasis isolated rat hepatocytes and rat sinusoidal endothelial Invasion and metastasis is the relatively late event of cells suggested that the pH gradient between the development of malignant cells, which is the endocytic compartments and the cytoplasm was continuous process of breaking through the basement necessary for the receptor-mediated endocytosis membrane, degrading extracellular matrix, (Harada et al., 1996; Harada et al., 1997). Inhibition of angiogenesis, invading vascular system and V-ATPases can retard recycling of transferrin receptor redistributing in the distinct host sites. The activation of (Presley et al., 1997), impair the formation of the proteases which break down extracellular matrix is endosomal carrier vesicle (Clague et al., 1994), and required during the procedure. The invasive phenotype inhibit late endosome-lysosome fusion (van Weert et is closely related to its highly active V-ATPase. It has al., 1995). Although the significance of active V- been reported that the improper activated V-ATPases ATPase in signal molecules endocytosis and processing correlates with an invasive phenotype of several types on the behavior of tumor cells is not yet full elucidated of tumors, including breast cancer (Sennoune et al., for most data was gained from yeast or normal 2004; Hinton et al., 2009), pancreatic cancer (Chung et mammalian cells, it could be hypothesized that V- al., 2011) and melanoma (Nishisho et al., 2011). The ATPase might regulate some signal pathways via tumor metastasis can be suppressed in vitro or in modulating the recycling rate of receptor, which would animal model by the inhibition of V-ATPase inhibitors be responsible for the sensitivity of tumor cells to some or siRNA (Lu et al., 2005; Hinton et al., 2009; Supino signal molecules, ie, the faster rate at which the et al., 2008). Subunit a isoform and c seem to be receptor cycling in a V-ATPase-regulated membrane important factors in regulating the metastasis of cancer. trafficking, the more efficiently the cells render the The main mechanisms by which overly active V- receptors, the more signal molecules could be recruited, ATPases enhance the tumor invasion and metastasis and the stronger or more lasting response to the may be that the extracellular milieu is acidized and it is stimulation by the signal molecules could be expected. suitable for optimal pH of proteases that degenerate For example, the activation of Notch, a common extracellular matrix (ECM). The plasma membrane V- hallmark of an increasing number of cancers (Miele et ATPases is responsible for pumping cytosol protons to al., 2006; Roy et al., 2007), is involved in V-ATPase- the extracellular space resulting in a low extracelluar associated endosomal system (Yan et al., 2009; Vaccari pH, which is required for the activation of several types et al., 2010). V-ATPase activity is required for Notch of proteases including cathepsins, metalloproteases, signaling. In V-ATPase mutant cells, Notch and its and gelatinases. V-ATPase may influence the receptors are trapped in an expanded lysosome-like expression of proteases directly independent of the compartment, where they accumulate rather than being whole enzyme V-ATPase function. For example, degraded and a substantial reduction expression in transfectants which over express V-ATPase subunit c at downstream gene of notch. V-ATPase regulates Notch the mRNA level showed an enhance invasiveness in via: i) endocytosis of Notch, for acidification of earlier vitro with a concomitant increases in secretion of endosomal compartments is required in this process matrix metalloproteinase-2 (Kubota et al., 2000). V- and a reduced rate of Notch endocytosis was found in ATPase may also regulate metastasis by enhancing V-ATPase mutant cells ii) endosomal cleavage patterns proteases activation. Cathepsin is an example, which is of the protease that degrade the Notch in the secreted by several types of tumor cells and related to accordingly forms, each of which process exerting its invasion. Once the extracellular cathepsin is activated, own activating potency (Vaccari et al., 2010) iii) it can both degrade extracellular matrix proteins and regulating endosome-lysosome fusion and Notch activate other secreted proteases involved in invasion, intracellular re-distribution or the targeting to cell such as matrix metalloprotease (Joyce et al., 2004; surface. Gocheva et al., 2007) and gelatinases (Martínez- The V-ATPase-associated signal molecules processing Zaguilá et al., 1996). The plasma membrane V-ATPase itself may also be regulated by endosomal protein, for appeared to be recruited at the proceeding edge of the example, HRG-1(heme-regulated genes), a downstream cancer cell by the interaction with F-actin so as to give

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rise an acidic microenvironment by the edge (Hinton et evolutionarily conserved family of the ATP binding al., 2009). Moreover, intracellular V-ATPases, the cassette (ABC) proteins pg, yet it is documented that major contributor of acidity of intracellular V-ATPase plays a role in MDR in a pg-independent compartment and membrane trafficking regulator, also manner, and the inhibition of V-ATPase could not only facilitate in the invasion and metastasis, which is due to suppress tumor cells directly, but also sensitize the possible modulating proteolytic activation of cathepsins tumor cells to the chemical therapy (De Milito et al., or matrix metalloproteases within lysosomes or 2005). It was documented that proton pump inhibitor secretory vesicles and targeting the proteases- (PPI) pretreatment sensitized tumor cell lines to the containing secretory vesicles to the cell surface to be effects of cisplatin, 5-fluorouracil, and vinblastine extracytosed (Hinton et al., 2009). The accumulation of significantly. PPI treatment will increases both acidity, concentration of plasma membrane V-ATPase extracellular pH and the pH of lysosomal organelles, and activated protease crown the proceeding surface of which induced a marked increase in the cytoplasmic a metastatic cell, conferring the tumor cell a "cutting retention of the cytotoxic drugs, with clear targeting to edge". the nucleus in the case of doxorubicin. In vivo Mobility is crucial for spread of tumor cells to the experiments, oral pretreatment with omeprazole was distant sites. NiK-12192, one of V-ATPase inhibitor able to induce sensitivity of human solid tumors to was shown able to reduce the migration/invasion of cisplatin (Lucian et al., 2004). human lung cancer cells in vitro and significantly V-ATPase renders several mechanisms of multidrug reduce the number of spontaneous metastases in the resistance including: neutralized drug extracellularly or lung of nude mice implanted with a human lung intracellularly, decreased drug internalization, altered carcinoma. After the treatment of NiK-12192, the lung DNA repair and inhibition of apoptosis. The pH of the cancer cells in vitro showed that actin fibers were tumor microenvironment may influence the uptake of broken, spots of aggregation were evident and no anticancer drugs. Molecules diffuse passively across pseudopodia and regular structure for actin filaments the cell membrane most efficiently in the uncharged could be seen, comparing to the control cells with long form. Because the extracellular pH in tumors is low and and regular fibers of tubulin in the cell cytoplasm and the intracellular pH of tumor cells is neutral to alkaline, filaments of actin forming pseudopodia. NiK-12192- weakly basic drugs that have an acid dissociation treated cells also demonstrate a reduction in the constant of 7.5-9.5, such as doxorubicin, mitoxantrone, experiment of wound healing assay due to the retard of vincristine, and vinblastine, are protonated and display migration (Supino et al., 2008). V-ATPase subunit B decreased cellular uptake (Raghunand et al., 1999; and C appear to contain the binding sites to the actin Gerweck et al., 2006; McCarty and Whitaker, 2010). cytoskeleton (Vitavska et al., 2003; Vitavska et al., The data in vitro or in animal models indicates that 2005; Zuo et al., 2006). The interactions between V- extracellular alkalinization leads to substantial ATPase and cytoskeleton implicate their involvement improvement in the therapeutic effectiveness of and regulation of cell mobility and membrane antitumor drugs via enhanced the cellular drug trafficking (Sun-Wada et al., 2009). uptake and cytotoxicity (Gerweck et al., 2006; Trédan Angiogenesis, a consequence of the mutual interaction et al., 2007).The reduced intracellular accumulation of between cancer cells and the stoma cells of anticancer drugs may also be due that V-ATPase has a extracellular microenvironments, is another important role as cooperating factor of ATP-dependent membrane step during metastasis, during which process, proteins that function as drug efflux pumps endothelial cells is mainly involved. It was documented (Raghunand et al., 1999). Interestingly, the levels of V- that V- ATPases play a crucial role in growth and ATPase subunit expressions can be up-regulated by phenotypic modulation of myofibroblasts that anticancer drug. The treatment of cisplatin on human contribute to neointimal formation in cultured human epidermoid cancer KB cells increased the protein levels saphenous vein (Otani et al., 2000) The microvascular of the majority of the subunits such as c, c", D, a, A, C endothelial cells in tumor tissue also incline to render and E, which indicates it may stimulate the expression plasma membrane V-ATPase to cope with the acidic of the V-ATPase complex as a whole. It is suggested extracellular environment. The ability of migration of that the V-ATPase expression may be a defensive endothelial cell toward the adjacent tissue is required response to the anticancer drug (Murakami et al., 2001; during angiogenesis, in which process V-ATPase plays Torigoe et al., 2002). Still, there are also some a role, shown in the result that the penetration of controversial results on the relationship between the basement membrane of endothelial cell was suppressed cationic drugs uptake and V-ATPase - the inhibition of by bafilomycin treatment (Rojas et al., 2006). V-ATPase decreased the uptake of the cationic drugs The relations of V-ATPase and drug (Morissette et al., 2009; Marceau et al., 2009), which might be explained that the influence of V-ATPase on resistance in cancer the drug uptake may also be depend upon the Acquired multidrug resistance (MDR) can limit characteristics of the drugs and its relation to therapeutic potential and one of the reasons of relapse. membrane trafficking. It is well known that MDR is correlate to the

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The roles of V-ATPase in cancer cells. 1) Protons produced by glycolysis are pumped by plasma membrane V-ATPase (green circle: V0; blue circle: V1) which prevents the cell from acidosis-induced apoptosis and the slightly basic of cytosolic pH enhanced cell growth and proliferation; 2) Acidification of secretary vesicle, which is maintained by intracellular V-ATPase, is essential for protease secretion and activation (orange bars: active form; orange-red bars: inactive forms of protease). The interaction between V-ATPase and actin (green wave line) may contribute the recruitment of V-ATPase on plasma membrane. The accumulation of V-ATPase on the plasma membrane, the extracellular acidic-microenvironment and activated-protease appear to crown the tumor cell, conferring it a "cutting edge" at the proceeding surface which facilitates invasion and metastasis. Moreover, in acidic microenvironment, angiogenesis is enhanced; 3) V-ATPases might regulate signal pathway via controlling international of signal molecules (red circle), releasing and recycling the receptors, and processing signal molecules. Therefore, V-ATPases may exert effects on cell behavior via signal pathway; 4) V-ATPases contributes to acquirement of resistance of anticancer drug (green square) supported by the data that inhibition of V-ATPase sensitize the tumor cells to chemical therapy, which is partly due to the increased influx of anticancer drug when in a basic extracellular condition.

That the defects of V-ATPase increase the sensitivity to these aspects: enhanced proliferation and growth, drugs may be partly due to the decreased cytosolic pH, evading apoptosis, facilitating metastasis and which were observed in the influence of cisplatin on angiogenesis, and acquirement of the drug resistance. the V-ATPase mutant yeast Saccharomyces cerevisiae V-ATPase will be a prospective candidate for cancer (Liao et al., 2006) or increased toxicity of combined diagnosis and treatment. treatment of V-ATPase inhibition and anticancer drug on lung cancer cell, breast cancer or liver cancer cell References lines (Wong et al., 2005; Farina et al., 2006; You et al., Busa WB, Nuccitelli R. Metabolic regulation via intracellular 2009). At low cytosolic pH, sensitivity to DNA pH. Am J Physiol. 1984 Apr;246(4 Pt 2):R409-38 damaging drugs or UV irradiation in V-ATPase mutants may be associated with altered DNA Perona R, Serrano R. Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump. Nature. 1988 Aug conformation or defective DNA damage repair 4;334(6181):438-40 mechanisms, rendering DNA more prone to damage Robinson H, van der Marel GA, van Boom JH, Wang AH. (Robinson et al., 1992; Petrangolini et al., 2006; Liao et Unusual DNA conformation at low pH revealed by NMR: al., 2006). parallel-stranded DNA duplex with homo base pairs. Conclusions Biochemistry. 1992 Nov 3;31(43):10510-7 Clague MJ, Urbé S, Aniento F, Gruenberg J. Vacuolar ATPase According to the roles V-ATPase in tumor cells, we activity is required for endosomal carrier vesicle formation. J conclude that alteration of V-ATPase is much likely the Biol Chem. 1994 Jan 7;269(1):21-4 necessary initial step of transformation of the malignant Nishihara T, Akifusa S, Koseki T, Kato S, Muro M, Hanada N. cells and the malfunctional V-ATPase acts as a Specific inhibitors of vacuolar type H(+)-ATPases induce continual enhancer of carcinogenesis and tumor apoptotic cell death. Biochem Biophys Res Commun. 1995 Jul progression. Tumor cells take the advantages of 6;212(1):255-62 disfunctioned plasma and intracellular V-ATPase in

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Ausiello DA, Brown D, Marshansky V. V-ATPase interacts with Morimura T, Fujita K, Akita M, Nagashima M, Satomi A. The ARNO and Arf6 in early endosomes and regulates the protein proton pump inhibitor inhibits cell growth and induces degradative pathway. Nat Cell Biol. 2006 Feb;8(2):124-36 apoptosis in human hepatoblastoma. Pediatr Surg Int. 2008 Oct;24(10):1087-94 Miele L, Golde T, Osborne B. Notch signaling in cancer. Curr Mol Med. 2006 Dec;6(8):905-18 Supino R, Petrangolini G, Pratesi G, Tortoreto M, Favini E, Bo LD, Casalini P, Radaelli E, Croce AC, Bottiroli G, Misiano P, Petrangolini G, Supino R, Pratesi G, Dal Bo L, Tortoreto M, Farina C, Zunino F. Antimetastatic effect of a small-molecule Croce AC, Misiano P, Belfiore P, Farina C, Zunino F. Effect of vacuolar H+-ATPase inhibitor in in vitro and in vivo preclinical a novel vacuolar-H+-ATPase inhibitor on cell and tumor studies. J Pharmacol Exp Ther. 2008 Jan;324(1):15-22 response to camptothecins. J Pharmacol Exp Ther. 2006 Sep;318(3):939-46 Fortini ME, Bilder D. Endocytic regulation of Notch signaling. Curr Opin Genet Dev. 2009 Aug;19(4):323-8 Rojas JD, Sennoune SR, Maiti D, Bakunts K, Reuveni M, Sanka SC, Martinez GM, Seftor EA, Meininger CJ, Wu G, Hinton A, Sennoune SR, Bond S, Fang M, Reuveni M, Wesson DE, Hendrix MJ, Martínez-Zaguilán R. Vacuolar-type Sahagian GG, Jay D, Martinez-Zaguilan R, Forgac M. Function H+-ATPases at the plasma membrane regulate pH and cell of a subunit isoforms of the V-ATPase in pH homeostasis and migration in microvascular endothelial cells. Am J Physiol in vitro invasion of MDA-MB231 human breast cancer cells. J Heart Circ Physiol. 2006 Sep;291(3):H1147-57 Biol Chem. 2009 Jun 12;284(24):16400-8 Zuo J, Jiang J, Chen SH, Vergara S, Gong Y, Xue J, Huang H, Marceau F, Bawolak MT, Bouthillier J, Morissette G. Vacuolar Kaku M, Holliday LS. Actin binding activity of subunit B of ATPase-mediated cellular concentration and retention of vacuolar H+-ATPase is involved in its targeting to ruffled quinacrine: a model for the distribution of lipophilic cationic membranes of osteoclasts. J Bone Miner Res. 2006 drugs to autophagic vacuoles. Drug Metab Dispos. 2009 May;21(5):714-21 Dec;37(12):2271-4 De Milito A, Iessi E, Logozzi M, Lozupone F, Spada M, Marino Morissette G, Ammoury A, Rusu D, Marguery MC, Lodge R, ML, Federici C, Perdicchio M, Matarrese P, Lugini L, Nilsson A, Poubelle PE, Marceau F. Intracellular sequestration of Fais S. Proton pump inhibitors induce apoptosis of human B- amiodarone: role of vacuolar ATPase and macroautophagic cell tumors through a caspase-independent mechanism transition of the resulting vacuolar cytopathology. Br J involving reactive oxygen species. Cancer Res. 2007 Jun Pharmacol. 2009 Aug;157(8):1531-40 1;67(11):5408-17 Sasazawa Y, Futamura Y, Tashiro E, Imoto M. Vacuolar H+- Fais S, De Milito A, You H, Qin W. Targeting vacuolar H+- ATPase inhibitors overcome Bcl-xL-mediated chemoresistance ATPases as a new strategy against cancer. Cancer Res. 2007 through restoration of a caspase-independent apoptotic Nov 15;67(22):10627-30 pathway. Cancer Sci. 2009 Aug;100(8):1460-7 Forgac M. Vacuolar ATPases: rotary proton pumps in Sun-Wada GH, Tabata H, Kawamura N, Aoyama M, Wada Y. physiology and pathophysiology. Nat Rev Mol Cell Biol. 2007 Direct recruitment of H+-ATPase from lysosomes for Nov;8(11):917-29 phagosomal acidification. J Cell Sci. 2009 Jul 15;122(Pt 14):2504-13 Gocheva V, Joyce JA. Cysteine cathepsins and the cutting edge of cancer invasion. Cell Cycle. 2007 Jan 1;6(1):60-4 Yan Y, Denef N, Schüpbach T. The vacuolar proton pump, V- ATPase, is required for notch signaling and endosomal Liao C, Hu B, Arno MJ, Panaretou B. Genomic screening in trafficking in Drosophila. Dev Cell. 2009 Sep;17(3):387-402 vivo reveals the role played by vacuolar H+ ATPase and cytosolic acidification in sensitivity to DNA-damaging agents You H, Jin J, Shu H, Yu B, De Milito A, Lozupone F, Deng Y, such as cisplatin. Mol Pharmacol. 2007 Feb;71(2):416-25 Tang N, Yao G, Fais S, Gu J, Qin W. Small interfering RNA targeting the subunit ATP6L of proton pump V-ATPase Roy M, Pear WS, Aster JC. The multifaceted role of Notch in overcomes chemoresistance of breast cancer cells. Cancer cancer. Curr Opin Genet Dev. 2007 Feb;17(1):52-9 Lett. 2009 Jul 18;280(1):110-9 Trédan O, Galmarini CM, Patel K, Tannock IF. Drug resistance Buechling T, Bartscherer K, Ohkawara B, Chaudhary V, and the solid tumor microenvironment. J Natl Cancer Inst. Spirohn K, Niehrs C, Boutros M. Wnt/Frizzled signaling 2007 Oct 3;99(19):1441-54 requires dPRR, the Drosophila homolog of the prorenin Wang Y, Cipriano DJ, Forgac M. Arrangement of subunits in receptor. Curr Biol. 2010 Jul 27;20(14):1263-8 the proteolipid ring of the V-ATPase. J Biol Chem. 2007 Nov Casey JR, Grinstein S, Orlowski J. Sensors and regulators of 23;282(47):34058-65 intracellular pH. Nat Rev Mol Cell Biol. 2010 Jan;11(1):50-61 Otero-Rey EM, Somoza-Martín M, Barros-Angueira F, García- Cruciat CM, Ohkawara B, Acebron SP, Karaulanov E, García A. Intracellular pH regulation in oral squamous cell Reinhard C, Ingelfinger D, Boutros M, Niehrs C. Requirement carcinoma is mediated by increased V-ATPase activity via of prorenin receptor and vacuolar H+-ATPase-mediated over-expression of the ATP6V1C1 gene. Oral Oncol. 2008 acidification for Wnt signaling. Science. 2010 Jan Feb;44(2):193-9 22;327(5964):459-63 Gillies RJ, Robey I, Gatenby RA. Causes and consequences of McCarty MF, Whitaker J. Manipulating tumor acidification as a increased glucose metabolism of cancers. J Nucl Med. 2008 cancer treatment strategy. Altern Med Rev. 2010 Jun;49 Suppl 2:24S-42S Sep;15(3):264-72 López-Lázaro M. The warburg effect: why and how do cancer McHenry P, Wang WL, Devitt E, Kluesner N, Davisson VJ, cells activate glycolysis in the presence of oxygen? Anticancer McKee E, Schweitzer D, Helquist P, Tenniswood M. Iejimalides Agents Med Chem. 2008 Apr;8(3):305-12 A and B inhibit lysosomal vacuolar H+-ATPase (V-ATPase) Marshansky V, Futai M. The V-type H+-ATPase in vesicular activity and induce S-phase arrest and apoptosis in MCF-7 trafficking: targeting, regulation and function. Curr Opin Cell cells. J Cell Biochem. 2010 Mar 1;109(4):634-42 Biol. 2008 Aug;20(4):415-26

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 258 Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function Lu X, Qin W

Miranda KC, Karet FE, Brown D. An extended nomenclature Chung C, Mader CC, Schmitz JC, Atladottir J, Fitchev P, for mammalian V-ATPase subunit genes and splice variants. Cornwell ML, Koleske AJ, Crawford SE, Gorelick F. The PLoS One. 2010 Mar 10;5(3):e9531 vacuolar-ATPase modulates matrix metalloproteinase isoforms in human pancreatic cancer. Lab Invest. 2011 May;91(5):732- O'Callaghan KM, Ayllon V, O'Keeffe J, Wang Y, Cox OT, 43 Loughran G, Forgac M, O'Connor R. Heme-binding protein HRG-1 is induced by insulin-like growth factor I and associates Nishisho T, Hata K, Nakanishi M, Morita Y, Sun-Wada GH, with the vacuolar H+-ATPase to control endosomal pH and Wada Y, Yasui N, Yoneda T. The a3 isoform vacuolar type receptor trafficking. J Biol Chem. 2010 Jan 1;285(1):381-91 H⁺ -ATPase promotes distant metastasis in the mouse B16 melanoma cells. Mol Cancer Res. 2011 Jul;9(7):845-55 Toei M, Saum R, Forgac M. Regulation and isoform function of the V-ATPases. Biochemistry. 2010 Jun 15;49(23):4715-23 This article should be referenced as such: Vaccari T, Duchi S, Cortese K, Tacchetti C, Bilder D. The Lu X, Qin W. Vacuolar H(+)-ATPase in Cancer Cells: Structure vacuolar ATPase is required for physiological as well as and Function. Atlas Genet Cytogenet Oncol Haematol. 2012; pathological activation of the Notch receptor. Development. 16(3):252-259. 2010 Jun;137(11):1825-32

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A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) Jill D Kremer, Anwar N Mohamed Cytogenetics Laboratory, Pathology Department, Wayne State University School of Medicine, Detroit Medical Center, Detroit MI, USA (JDK, ANM)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Reports/t1122q23q13MohamedID100059.html DOI: 10.4267/2042/47291 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Clinics Pathology Bone marrow aspirate appeared hypocellular with 95% Age and sex lymphoblasts of L1 morphology, 2% myeloid series, 14 months old male patient. and 3% erythroid series. Previous history Electron microscopy No preleukemia, no previous malignancy, inborn Not performed. condition of note. Patient has hemoglobin S trait. Diagnosis Organomegaly CD34 negative B-precursor ALL. Hepatomegaly, splenomegaly, enlarged lymph nodes, central nervous system involvement. Survival Blood Date of diagnosis: 01-2011 Treatment: Methotrexate, Cytarabine, Vincristine, 9 WBC : 33 X 10 /l Dexamethasone, PEG-aspargase HB : 2.6g/dl Complete remission : no 9 Platelets : 1 X 10 /l Treatment related death : no Blasts : 72% Relapse : no Bone marrow : 100 bone marrow blast replacement. Status: Alive. Last follow up: 10-2011 Cyto-Pathology Survival: 9 months Classification Karyotype Cytology Sample: Bone marrow aspirate Acute lymphoblastic leukemia (ALL) with L1 Culture time: 24hr without stimulant and 48hr with morphology 10% conditioned medium. Immunophenotype Banding: GTG Flow cytometry of bone marrow aspirate identified a Results dim CD45 lymphoblast population (85%) expressing HLA-DR, CD19 and partially expressing CD10, CD22, 46,Y,der(X)t(X;9)(p11.1;q11),add(9)(q11),t(11;22)(q23 CD9 and CD40. ;q13)[20] (see Figure 1). Post induction bone marrow study demonstrated a normal 46,XY karyotype. Rearranged Ig Tcr Not performed.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 260 A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) Kremer JD, Mohamed AN

Figure 1: G-banded karyotype showing 46,Y,der(X)t(X;9)(p11.1;q11),add(9)(q11),t(11;22)(q23;q13). Arrows pointed to t(11;22).

Figure 2: FISH. A. Interphase hybridized with LSI MLL dual-color break apart probe showed a split signal pattern of MLL (1O1G1F). B. Metaphase hybridized with BCR/ABL dual-fusion probe showed 2O2G signaling. C. For identification of chromosome 22, the same metaphase subsequently hybridized with LSI MLL probe showing relocation of the telomeric side (orange signal) of MLL to 22q confirming t(11;22)(q23;q13) (arrows). Note: G= green; O= orange; F= fusion. (Figure 2). The hybridization with the BCR/ABL probe Other Molecular Studies showed two signals each Technics: (unfused), however on a previously G-banded Fluorescence in situ hybridization (FISH) using the metaphase it appeared that the BCR signals remained ALL panel DNA probes including CEP 4, 10, and 17 on chromosome 22 while one ABL signal was alpha satellite probes, LSI MLL dual-color break apart translocated to der(X). The remaining probes produced probe, BCR/ABL and TEL/AML1 dual-fusion a normal hybridization pattern. translocation probes was performed (Abbott Molecular, Downers Grove, IL). Comments Results: The patient described here is a 14 month-old-male Hybridization with MLL probe produced a presented with an upper respiratory tract infection split/translocation pattern in 61% of interphase cells. unresponsive to antibiotics. Subsequently he was Metaphase FISH showed that the telomeric region of diagnosed with high risk B-precursor ALL due to the MLL gene was translocated to 22q13 distal to BCR positivity of MLL/11q23 rearrangement. The patient

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 261 A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) Kremer JD, Mohamed AN

Table 1: AML cases with t(11;22)(q23;q13) reported in literature. Primary Patient Leukemia Karyotype Gene Malignancy Non-Hodgkin 4 Y/M [2] AML M1 48,XY,+8,+8,t(11;22)(q23;q13) MLL-EP300 Lymphoma 5 Y/F [3] Neuroblastoma AML M2 46,XX,t(1;22;11)(q44;q13;q23),t(10;17)(q22;q21) MLL-EP300 65 Y/M [4] AML with MDS AMML 46,XY,t(11;22)(q23;q13)[15]/47,idem,+8[2] MLL-EP300 was started on a Children's Oncology Group induction markedly heterogeneous, it remains unclear whether chemotherapy protocol. Secondary to his high risk EP300 or other gene is involved in the present case status, the patient is being evaluated for a bone marrow which may be responsible for the different phenotype transplant. At time of diagnosis chromosome analysis of this leukemia. revealed the presence t(11;22)(q23;q13) in all 20 metaphases and rearrangement of the MLL gene. References Translocations involving the MLL/11q23 region are the Ida K, Kitabayashi I, Taki T, Taniwaki M, Noro K, Yamamoto most common genomic aberrations in infant ALL seen M, Ohki M, Hayashi Y. Adenoviral E1A-associated protein in ~80% of cases (Raimondi, 2004). Generally p300 is involved in acute myeloid leukemia with leukemia harboring MLL translocation is clinically t(11;22)(q23;q13). Blood. 1997 Dec 15;90(12):4699-704 aggressive and associated with poor prognosis. The Raimondi SC.. 11q23 rearrangements in childhood acute most common chromosomes involved in 11q23 lymphoblastic leukemia. Atlas Genet Cytogenet Oncol translocations are t(4;11) followed by t(11;19) and Haematol. February 2004. URL : t(9;11). Additionally, leukemia with MLL/11q23 http://AtlasGeneticsOncology.org/Anomalies/11q23ChildALLID 1321.html . translocations are frequently associated with over expression of FLT3, therefore, targeted therapy Ohnishi H, Taki T, Yoshino H, Takita J, Ida K, Ishii M, Nishida K, Hayashi Y, Taniwaki M, Bessho F, Watanabe T.. A complex inhibitors of FLT3 (a tyrosine kinase) may be t(1;22;11)(q44;q13;q23) translocation causing MLL-p300 fusion beneficial for those patients. Currently there are only gene in therapy-related acute myeloid leukemia. Eur J three reported cases in the literature with Haematol. 2008 Dec;81(6):475-80. Epub 2008 Sep 6. t(11;22)(q23;q13), unlike our case all having secondary Duhoux FP, De Wilde S, Ameye G, Bahloula K, Medves S, acute myeloid leukemia with prior therapy of Lege G, Libouton JM, Demoulin JB, A Poirel H.. Novel variant topoisomerase II inhibitor (table 1). Moreover, form of t(11;22)(q23;q13)/MLL-EP300 fusion transcript in the rearrangement of the MLL gene and MLL-EP300 evolution of an acute myeloid leukemia with myelodysplasia- related changes. Leuk Res. 2011 Mar;35(3):e18-20. Epub fusion gene were demonstrated in those three cases (Ida 2010 Oct 25. et al., 1997; Ohnishi et al., 2008; Duhoux et al., 2011). The clinical presentation of our case is quit different This article should be referenced as such: from these three cases. Although our case had a Kremer JD, Mohamed AN. A case of Acute Lymphoblastic rearrangement of the MLL/11q23 gene, the MLL- Leukemia with rare t(11;22)(q23;q13). Atlas Genet Cytogenet EP300 fusion gene was not tested. Because the partner Oncol Haematol. 2012; 16(3):260-262. genes involved in MLL/11q23 translocations are

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Case Report Section Paper co-edited with the European LeukemiaNet

Insertion as an alternative mechanism of CBFB- MYH11 gene fusion in a new case of acute myeloid leukemia with an abnormal chromosome 16

Yaser Hussein, Vandana Kulkarni, Anwar N Mohamed Cytogenetics Laboratory, Pathology Department, Wayne State University School of Medicine, Detroit Medical Center, Detroit MI, USA (YH, VK, ANM)

Published in Atlas Database: October 2011 Online updated version : http://AtlasGeneticsOncology.org/Reports/ins16q22p13p13MohamID100058.html DOI: 10.4267/2042/47292 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2012 Atlas of Genetics and Cytogenetics in Oncology and Haematology

CD34, HLA-DR, CD9, CD13, CD33, CD117 and Clinics partially expressing CD15, CD11b, and CD64. A Age and sex second population of monocytes is also identified 17 years old female patient. (37%) expressing CD4, CD14, CD15, CD36 and CD64. Previous history No preleukemia, no previous malignancy, inborn Rearranged Ig Tcr: Not performed. condition of note. Thalassemia trait carrier. Pathology Organomegaly Bone marrow aspirate revealed myeloblasts, Hepatomegaly, splenomegaly, enlarged lymph nodes, monoblasts, monocytes, and increased eosinophils no central nervous system involvement. many of which had abnormal granules (FAB AML- M4eos). Blood Electron microscopy: Not performed. WBC : 138.7 X 10 9/l Diagnosis HB : 6.9g/dl Acute myelomonocytic leukemia with abnormal Platelets : 51 X 10 9/l eosinophils (AML-M4eos) and CBFB/16q22 Blasts : 76% rearrangement. Bone marrow : 100 Bone marrow biopsy was hypercellular (100%) and replaced by myeloblasts and Survival monoblasts. Normal hematopoiesis was greatly Date of diagnosis: 03-2011 decreased and there was prominent hemophagocytosis. The majority of the blasts were myeloperoxidase Treatment: Intrathecal methotrexate, hydrocortisone, positive however another smaller component of blasts and cytarabine. was nonspecific esterase positive. Treatment related death : no Relapse : no Cyto-Pathology Status: Alive. Last follow up: 09-2011 Classification Survival: 6 months Cytology: Acute myeloid leukemia with abnormal Karyotype eosinophils (AML-M4eos). Sample: Bone marrow Immunophenotype Culture time: 24 hrs without stimulating agents and 48 Flow cytometry of bone marrow aspirate identified a hrs with 10% conditioned medium. significant population of myeloblasts (49%) expressing

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 263 A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) Kremer JD, Mohamed AN

Banding: GTG Results: Results The hybridization with the CBFB break-apart probe At time of diagnosis abnormal metaphase cells with the produced a split pattern in 62% of interphase cells. On following karyotype was found; metaphase cells, the 5'CBFB (SepctrumRed) and 46,XX,ins(16)(q22p13p13)[20] (see Figure 1). 3'CBFB (SepctrumGreen) signals stayed on the 16q, Remission bone marrow on 4/20/2011 and 9/13/2011 instead of 5'CBFB being relocated to 16p as seen in the revealed a normal female karyotype; 46,XX[20]. standard inv(16). The CBFB signals were separated but maintained the orientation pattern of the 5' and 3' Other Molecular Studies probe, suggesting they were split by an insertion (Figure 2A). Subsequently, using the CBFB-MYH11 Technics: probe on metaphases showed that MYH11 signal on Fluorescence in situ hybridization (FISH) using LSI 16p moved and juxtaposed to CBFB on 16q, CBFB dual color break-apart rearrangement DNA confirming the insertion of MYH11 into CBFB (Figure probes (Abbott Molecular IL, USA), and 2B). CBFB/MYH11 dual fusion translocation DNA probe (Cytocell Inc. Cambridge, UK) were performed.

Figure 1. G-Banded karyotype from the diagnostic bone marrow sample demonstrating the ins(16)(q22p13p13) (arrowed).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 264 A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) Kremer JD, Mohamed AN

Figure 2. A. Metaphase FISH using LSI CBFB/q22 breakapart rearrangement probe showing one normal fusion signal and split signals (red and green) on 16q (arrow). B. Metaphase hybridized with CBFB/MYH11 probe showing insertion of MYH11 green signal (appearing yellow) within CBFB/16q22 red signal (arrow).

represents a variant rare rearrangement for the Comments formation of this fusion. FISH is highly recommended The patient described here is a 17 year old female to characterize unusual abnormalities of chromosome presented with upper respiratory tract infection and 16 and to confirm the CBFB-MYH11 fusion. bruises for 2 weeks. Subsequently she was diagnosed with AML (FAB M4 eos). Cytogenetics, performed on References bone marrow aspirate revealed a unique structural Le Beau MM, Larson RA, Bitter MA, Vardiman JW, Golomb abnormality of chromosome 16 which was interpreted HM, Rowley JD. Association of an inversion of chromosome 16 as insertion; 46, XX, ins(16)(q22p13p13). FISH with abnormal marrow eosinophils in acute myelomonocytic confirmed that the MYH11/p13 gene was inserted into leukemia. A unique cytogenetic-clinicopathological association. the CBFB/16q22 gene region (Figure 2B). The result of N Engl J Med. 1983 Sep 15;309(11):630-6 this unusual structural rearrangement was the fusion of Tobal K, Johnson PR, Saunders MJ, Harrison CJ, Liu Yin JA. CBFB /MYH11 genes commonly seen in Detection of CBFB/MYH11 transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and inv(16)(p13q22) bearing leukemia. remission. Br J Haematol. 1995 Sep;91(1):104-8 The CBFB/MYH11 gene fusion is strongly associated with AML-M4 with abnormal eosinophils. Generally, O'Reilly J, Chipper L, Springall F, Herrmann R. A unique structural abnormality of chromosome 16 resulting in a CBF the fusion is generated from inv(16)(p13q22) or beta-MYH11 fusion transcript in a patient with acute myeloid t(16;16) with the inversion being much more common leukemia, FAB M4. Cancer Genet Cytogenet. 2000 than translocation (Le Beau et al., 1983; Tobal et al., Aug;121(1):52-5 1995). The case presented here demonstrates that This article should be referenced as such: insertion is another mechanism in producing CBFB/MYH11 gene fusion in AML-M4eos. To our Hussein Y, Kulkarni V, Mohamed AN. Insertion as an alternative mechanism of CBFB-MYH11 gene fusion in a new best knowledge, there is only one reported case of case of acute myeloid leukemia with an abnormal chromosome AML-M4 having similar structural abnormality of 16. Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):263- chromosome 16 and CBFB/MYH11 fusion (O'Reilly et 265. al., 2000). These two cases suggest that insertion

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A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) Kremer JD, Mohamed AN