Number 3 March 2012

VolumeVolume 16 1 -- NumberNumber 31 May March- Sept ember2012 1997

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Scope

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

Editorial correspondance

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

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

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

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

http://AtlasGeneticsOncology.org

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

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Editor

Jean-Loup Huret (Poitiers, France) Editorial Board

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

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

OPEN ACCESS JOURNAL AT INIST-CNRS

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 185 Jean-Loup Huret

Gene Section

CUX1 (cut-like homeobox 1) 189 Benjamin Kühnemuth, Patrick Michl DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) 194 June L Traicoff, Stephen M Hewitt, Joon-Yong Chung MYEOV (myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas)) 203 Jérôme Moreaux PCNA (proliferating cell nuclear antigen) 206 Ivaylo Stoimenov, Thomas Helleday RASSF5 (Ras association (RalGDS/AF-6) domain family member 5) 210 Lee Schmidt, Geoffrey J Clark RGS17 (regulator of G-protein signaling 17) 214 Chenguang Li, Lei Wang, Yihua Sun, Haiquan Chen SLC39A1 (solute carrier family 39 (zinc transporter), member 1) 216 Renty B Franklin, Leslie C Costello CBX7 (chromobox homolog 7) 218 Ana O'Loghlen, Jesus Gil RPRM (reprimo, TP53 dependent G2 arrest mediator candidate) 221 Alejandro H Corvalan, Veronica A Torres VMP1 (vacuole membrane protein 1) 223 Alejandro Ropolo, Andrea Lo Ré, María Inés Vaccaro XPO1 (exportin 1 (CRM1 homolog, yeast)) 226 Alessandra Ruggiero, Maria Giubettini, Patrizia Lavia

Leukaemia Section t(11;18)(p15;q12) 231 Jean-Loup Huret t(11;21)(q21;q22) 232 Jean-Loup Huret t(8;17)(q24;q22) ???BCL3/MYC 234 Jean-Loup Huret

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) Atlast(11;14)(q 13;q32)of Genetics in multiple myeloma and Cytogenetics Huret JL, Laï JL in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Deep Insight Section

Plasticity and Tumorigenicity 236 Elena Campos-Sanchez, Isidro Sanchez-Garcia, Cesar Cobaleda Vacuolar H(+)-ATPase in Cancer Cells: Structure and Function 251 Xiaodong Lu, Wenxin Qin

Case Report Section

A case of Acute Lymphoblastic Leukemia with rare t(11;22)(q23;q13) 259 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 16 262 Yaser Hussein, Vandana Kulkarni, Anwar N Mohamed

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 185

Why breast cancer and prostate cancer are so frequent? A Huret JL new genetic mechanism, involving hormones and viruses

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) 186

Why breast cancer and prostate cancer are so frequent? A Huret JL new genetic mechanism, involving hormones and viruses

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 187

Why breast cancer and prostate cancer are so frequent? A Huret JL new genetic mechanism, involving hormones and viruses

end transcriptome sequencing. Proc Natl Acad Sci USA Greenman CD, Jia M, Latimer C, Teague JW, Lau KW, 2009b; 106:12353-12358. Burton J, Quail MA, Swerdlow H, Churcher C, Natrajan R, Sieuwerts AM, Martens JW, Silver DP, Langerød A, Mani RS, Tomlins SA, Callahan K, Ghosh A, Nyati MK, Russnes HE, Foekens JA, Reis-Filho JS, van 't Veer L, Varambally S, Palanisamy N, Chinnaiyan AM. Induced Richardson AL, Børresen-Dale AL, Campbell PJ, Futreal chromosomal proximity and gene fusions in prostate PA, Stratton MR. Complex landscapes of somatic cancer. Science 2009; 326:1230-1232. rearrangement in human breast cancer genomes. Nature Mitelman F, Johansson B. and Mertens F. (Eds.). 2011. 2009; 462:1005-1010. Mitelman Database of Chromosome Aberrations and Gene Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Fusions in Cancer. Mehra R, Sun XW, Varambally S, Cao X, Tchinda J, Paprotka T, Delviks-Frankenberry KA, Cingöz O, Martinez Kuefer R, Lee C, Montie JE, Shah RB, Pienta KJ, Rubin A, Kung HJ, Tepper CG, Hu WS, Fivash MJ Jr, Coffin JM, MA, Chinnaiyan AM. Recurrent fusion of TMPRSS2 and Pathak VK. Recombinant Origin of the Retrovirus XMRV. ETS transcription factor genes in prostate cancer. Science Science 2011; 333:97-101. 2005; 310:644-648. Pogo BG, Holland JF, Levine PH. Human mammary tumor Williamson LM, Lees-Miller SP. Estrogen receptor α- virus in inflammatory breast cancer. Cancer 2010; mediated transcription induces cell cycle-dependent DNA 116:2741-2744. double-strand breaks. Carcinogenesis 2011; 32:279-285. Rickman DS, Pflueger D, Moss B, VanDoren VE, Chen This article should be referenced as such: CX, de la Taille A, Kuefer R, Tewari AK, Setlur SR, Demichelis F, Rubin MA. SLC45A3-ELK4 is a novel and Huret JL. Why breast cancer and prostate cancer are so frequent erythroblast transformation-specific fusion frequent? A new genetic mechanism, involving hormones transcript in prostate cancer. Cancer Res 2009; 69: 2734- and viruses. Atlas Genet Cytogenet Oncol Haematol. 2738. 2012; 16(3):185-188. Stephens PJ, McBride DJ, Lin ML,Varela I, Pleasance ED, Simpson JT, Stebbings LA, Leroy C, Edkins S, Mudie LJ,

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 188

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI CUX1ID403ch7q22.txt

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 189

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 dephosphorylation leads to translocation into the phosphorylate serine or threonine residues within nucleus (Sansregret et al., 2010). the cut repeats (Coqueret et al., 1998b; Li et al., Function 2007). Protein kinase A and cyclin A/Cdk1 phosphorylate specific serine residues in a region The vast majority of studies describes CUX1 as a between the Cut repeat 3 and the homeodomain transcriptional repressor (Lievens et al., 1995; Ai et (Michl et al., 2006; Santaguida et al., 2001). PCAF al., 1999; Catt et al., 1999a; Catt et al., 1999b; Ueda acetyl-transferase is able to acetylate CUX1 on a et al., 2007). The repressor activity can be mediated lysine residue in the homeodomain (Li et al., 2000). by competition for DNA binding sites with Both, phosphorylation and acetylation have been transcriptional activators (Kim et al., 1997; Stünkel shown to inhibit CUX1 DNA binding (Sansregret et et al., 2000), by recruitment of histone deacetylases al., 2010; Li et al., 2000). Consistent with this, (Li et al., 1999) or by recruitment of histone lysine dephosphorylation by Cdc 25A phosphatase is able methyltransferases (Nishio and Walsh, 2004). to increase DNA binding of CUX1 (Coqueret et al, CUX1 may also negatively regulate gene 1998a). expression by binding to matrix attachment regions and by modulating their association with the Expression nuclear scaffold (Banan et al., 1997; Stünkel et al., Early studies suggested that in mammalian cells, 2000; Goebel et al., 2002; Kaul-Ghanekar et al., CUX1 represses genes that are upregulated in 2004). In contrast, the mechanisms underlying its differentiated tissues. Furthermore, the expression effects on transcriptional activation are less well of CUX1 might be restricted to proliferating and understood. undifferentiated cells and is inversely related to the CUX1 is involved in at least three cellular degree of differentiation (vanden Heuvel et al., processes important for cancer progression: cell 1996; Pattison et al., 1997; van Gurp et al., 1999). proliferation, cell motility/invasiveness and More recently however, studies in mice revealed apoptosis. that CUX1 is also expressed in terminally Proliferation differentiated cells of many tissues (Khanna-Gupta Studies indicate that the pro-proliferative effects of et al., 2001; Ellis et al., 2001). CUX1 are mainly mediated by the p110 isoform. Increased CUX1 expression was found in various This isoform is produced by proteolytic cleavage of tumour types including multiple myelomas, acute the full length protein occuring during G1/S- lymphoblastic leukaemia, breast carcinoma and transition in the cell cycle (Goulet et al., 2004; pancreatic cancer (De Vos et al., 2002; Tsutsumi et Moon et al., 2001). Cells stably transfected with al., 2003; Michl et al., 2005; Ripka et al., 2007). p110 CUX1 showed increased proliferation due to a It has been shown that the cellular expression of shortened G1-phase whereas embryonic fibroblasts CUX1 mRNA and protein is elevated following obtained from CUX1 knockout mice showed TGF-beta stimulation in many cell types including elongated G1-phase and less proliferation compared fibroblasts, pancreatic cancer cells, breast cancer to cells isolated from wild-type mice (Sansregret et cells and malignant plasma cells (Fragiadaki et al., al., 2006). 2011; Michl et al., 2005; De Vos et al., 2002). This A genome-wide location array for p110 CUX1 regulation of CUX1 expression by TGF-beta is binding sites in transformed and non-transformed probably mediated by p38MAPK and Smad4 cell lines identified numerous CUX1 target genes signalling (Michl et al., 2005). that are related to proliferation and cell cycle Localisation progression (Harada et al., 2008). Most of these genes are activated by p110 CUX1 including DNA Studies indicate that phosphorylated CUX1 is polymerase-alpha, cyclin A2 and cyclin E2. In preferentially localized in the cytoplasm whereas

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 190

CUX1 (cut-like homeobox 1) Kühnemuth B, Michl P

contrast, other genes are repressed such as the CDK-inhibitor p21 (Truscott et al., 2003; Nishio Mutations and Walsh, 2004; Harada et al., 2008). Note Cell motility A missense mutation affecting the homeodomain First evidence that CUX1 plays a role in cell has been described in one patient suffering from motility originates from knockdown studies in acute myeloid leukaemia, the significance of which fibroblasts and a panel of human cancer cell lines remains to be elucidated (Thoennissen et al., 2011). that revealed that depletion of CUX1 leads to decreased cell migration and invasion (Michl et al., Implicated in 2005). In agreement with this, cells stably expressing p110 and p75 CUX1 show increased Pancreatic cancer cell migration and invasion (Kedinger et al., 2009; Note Cadieux et al., 2009). Additionally, tail vein In pancreatic cancer CUX1 expression is elevated injection of cells stably expressing shRNA against compared to normal pancreas tissue (Ripka et al., CUX1 resulted in reduced formation of lung 2010a). Furthermore, an increased expression in metastases, whereas injection of cells stably high-grade tumours compared to low grade tumours overexpressing CUX1 led to increased lung was described (Michl et al., 2005). metastases (Michl et al., 2005; Cadieux et al., The expression of CUX1 is accompanied by the 2009). overexpression of its downstream targets WNT5a The molecular basis for these effects on cell and GRIA3 that, at least in part, mediate the motility was in part elucidated in a genome-wide proinvasive and proproliferative effects of CUX1 location analysis in several cell lines (Kedinger et (Ripka et al., 2006; Ripka et al., 2010b). al., 2009). In this study, CUX1 was found to inhibit Antiapoptotic effects of CUX1 in pancreatic cancer, the expression of genes that repress cell migration that have been shown in in vitro studies and in (e.g. E-cadherin, occludin) and to turn on the xenograft models, are associated with a positive expression of genes that promote cell migration regulation of BCL2 and downregulation of tumour (e.g. FAK, N-cadherin, vimentin) (Kedinger et al., necrosis factor alpha and are, at least in part, 2009). The regulation of these genes seems to be mediated by the glutamate receptor GRIA3 (Ripka mediated both directly by binding of CUX1 to the et al., 2010a; Ripka et al., 2010b). gene promoters but also indirectly by modulation of transcription factors and signaling proteins involved Breast cancer in EMT (e.g. SNAI1, SNAI2, Src, Wnt5a) Note (Kedinger et al., 2009; Aleksic et al., 2007; Ripka In mammary carcinoma the CUX1 expression is et al., 2007). Additionally, several of the CUX1 increased in high-grade tumours compared to low target genes are known GTPases important for grade tumours and a reverse correlation between actin-cytoskeleton polymerization (Kedinger et al., CUX1 mRNA levels and the relapse free- and 2009). overall-survival was shown (Michl et al., 2005). Apoptosis Furthermore, is has been shown that the expression Studies in pancreatic cancer cell lines showed that levels of the intron 20-initiated mRNA, that leads to depletion of CUX1 by siRNA increases TNFalpha- the synthesis of the p75 CUX1 isoform, is and TRAIL-induced apoptosis whereas specifically expressed in breast cancer and overexpression of CUX1 rescues from apoptosis. positively correlated with a diffuse infiltrative Additionally, treatment of xenograft tumours with growth pattern (Goulet et al., 2002). Transgenic siRNA for CUX1 lead to retarded tumour growth mice expressing p75 and p110 CUX1 under the and increased apoptosis. These effects are at least in control of the mouse mammary tumour virus-long part explained by a positive regulation of the terminal repeat developed breast cancer after a long antiapoptotic protein BCL2 by CUX1 (Ripka et al., latency period. This tumour development was 2010a). Subsequently, the glutamate receptor accompanied by an increased activity of WNT-β- GRIA3 was identified as another downstream target catenin signalling (Cadieux et al., 2009). of CUX1 able to mediate its antiapoptotic effects (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 homeoprotein, cut. Nat Genet. 1992 evolution of metazoans. Homologues of the Apr;1(1):50-5 Drosophila melanogaster Cut protein have been described at least in human, dog and mouse Andres V, Nadal-Ginard B, Mahdavi V. Clox, a mammalian homeobox gene related to Drosophila cut, encodes DNA- (Neufeld et al., 1992; Andres et al., 1992; Valarché binding regulatory proteins differentially expressed during et al., 1993). In humans, a homologue gene, called development. Development. 1992 Oct;116(2):321-34 CUX2, was described (Jacobsen et al., 2001).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 191

CUX1 (cut-like homeobox 1) Kühnemuth B, Michl P

Scherer SW, Neufeld EJ, Lievens PM, Orkin SH, Kim J, homolog, is associated with histone deacetylation. J Biol Tsui LC. Regional localization of the CCAAT displacement Chem. 1999 Mar 19;274(12):7803-15 protein gene (CUTL1) to 7q22 by analysis of somatic cell hybrids. Genomics. 1993 Mar;15(3):695-6 van Gurp MF, Pratap J, Luong M, Javed A, Hoffmann H, Giordano A, Stein JL, Neufeld EJ, Lian JB, Stein GS, van Valarché I, Tissier-Seta JP, Hirsch MR, Martinez S, Goridis Wijnen AJ. The CCAAT displacement protein/cut C, Brunet JF. The mouse homeodomain protein Phox2 homeodomain protein represses osteocalcin gene regulates Ncam promoter activity in concert with Cux/CDP transcription and forms complexes with the retinoblastoma and is a putative determinant of neurotransmitter protein-related protein p107 and cyclin A. Cancer Res. phenotype. Development. 1993 Nov;119(3):881-96 1999 Dec 1;59(23):5980-8 Harada R, Dufort D, Denis-Larose C, Nepveu A. Li S, Aufiero B, Schiltz RL, Walsh MJ. Regulation of the Conserved cut repeats in the human cut homeodomain homeodomain CCAAT displacement/cut protein function protein function as DNA binding domains. J Biol Chem. by histone acetyltransferases p300/CREB-binding protein 1994 Jan 21;269(3):2062-7 (CBP)-associated factor and CBP. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7166-71 Lievens PM, Donady JJ, Tufarelli C, Neufeld EJ. Repressor activity of CCAAT displacement protein in HL- O'Connor MJ, Stünkel W, Koh CH, Zimmermann H, 60 myeloid leukemia cells. J Biol Chem. 1995 May Bernard HU. The differentiation-specific factor CDP/Cut 26;270(21):12745-50 represses transcription and replication of human papillomaviruses through a conserved silencing element. J Vanden Heuvel GB, Bodmer R, McConnell KR, Nagami Virol. 2000 Jan;74(1):401-10 GT, Igarashi P. Expression of a cut-related homeobox gene in developing and polycystic mouse kidney. Kidney Rong Zeng W, Soucie E, Sung Moon N, Martin-Soudant N, Int. 1996 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, 4;241(1):75-85 Kobayashi R, Webb CF, Gottlieb PD. Interaction of the nuclear matrix-associated region (MAR)-binding proteins, Stünkel W, Huang Z, Tan SH, O'Connor MJ, Bernard HU. SATB1 and CDP/Cux, with a MAR element (L2a) in an Nuclear matrix attachment regions of human upstream regulatory region of the mouse CD8a gene. J papillomavirus type 16 repress or activate the E6 Biol Chem. 1997 Jul 18;272(29):18440-52 promoter, depending on the 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 Ellis T, Gambardella L, Horcher M, Tschanz S, Capol J, promoter mediated by CCAAT binding transcription factors Bertram P, Jochum W, Barrandon Y, Busslinger M. The NF-Y/CBF, dbpA, and CDP/cut. Cell Growth Differ. 1997 transcriptional repressor CDP (Cutl1) is essential for Dec;8(12):1329-38 epithelial cell differentiation of the lung and the hair follicle. Genes Dev. 2001 Sep 1;15(17):2307-19 Lievens PM, Tufarelli C, Donady JJ, Stagg A, Neufeld EJ. CASP, a novel, highly conserved alternative-splicing Jacobsen NJ, Elvidge G, Franks EK, O'Donovan MC, product of the CDP/cut/cux gene, lacks cut-repeat and Craddock N, Owen MJ. CUX2, a potential regulator of homeo DNA-binding domains, and interacts with full-length NCAM expression: genomic characterization and analysis CDP in vitro. Gene. 1997 Sep 15;197(1-2):73-81 as a positional candidate susceptibility gene for bipolar disorder. Am J Med Genet. 2001 Apr 8;105(3):295-300 Pattison S, Skalnik DG, Roman A. CCAAT displacement protein, a regulator of differentiation-specific gene Khanna-Gupta A, Zibello T, Sun H, Lekstrom-Himes J, expression, binds a negative regulatory element within the Berliner N. C/EBP epsilon mediates myeloid differentiation 5' end of the human papillomavirus type 6 long control and is regulated by the CCAAT displacement protein region. J Virol. 1997 Mar;71(3):2013-22 (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, transcriptional repressor which downmodulates Nepveu A. S phase-specific proteolytic cleavage is p21WAF1/CIP1/SDI1 in S phase. EMBO J. 1998a Aug required to activate stable DNA binding by the CDP/Cut 17;17(16):4680-94 homeodomain protein. Mol Cell Biol. 2001 Sep;21(18):6332-45 Coqueret O, Martin N, Bérubé G, Rabbat M, Litchfield DW, Nepveu A. DNA binding by cut homeodomain proteins is Santaguida M, Ding Q, Bérubé G, Truscott M, Whyte P, down-modulated by casein kinase II. J Biol Chem. 1998b Nepveu A. Phosphorylation of the CCAAT displacement Jan 30;273(5):2561-6 protein (CDP)/Cux transcription factor by cyclin A-Cdk1 modulates its DNA binding activity in G(2). J Biol Chem. Ai W, Toussaint E, Roman A. CCAAT displacement protein 2001 Dec 7;276(49):45780-90 binds to and negatively regulates human papillomavirus type 6 E6, E7, and E1 promoters. J Virol. 1999 De Vos J, Thykjaer T, Tarte K, Ensslen M, Raynaud P, May;73(5):4220-9 Requirand G, Pellet F, Pantesco V, Rème T, Jourdan M, Rossi JF, Ørntoft T, Klein B. Comparison of gene Catt D, Hawkins S, Roman A, Luo W, Skalnik DG. expression profiling between malignant and normal plasma Overexpression of CCAAT displacement protein represses cells with oligonucleotide arrays. Oncogene. 2002 Oct the promiscuously active proximal gp91(phox) promoter. 3;21(44):6848-57 Blood. 1999a Nov 1;94(9):3151-60 Goebel P, Montalbano A, Ayers N, Kompfner E, Dickinson Catt D, Luo W, Skalnik DG. DNA-binding properties of L, Webb CF, Feeney AJ. High frequency of matrix CCAAT displacement protein cut repeats. Cell Mol Biol attachment regions and cut-like protein x/CCAAT- (Noisy-le-grand). 1999b Dec;45(8):1149-60 displacement protein and B cell regulator of IgH Li S, Moy L, Pittman N, Shue G, Aufiero B, Neufeld EJ, transcription binding sites flanking Ig V region genes. J LeLeiko NS, Walsh MJ. Transcriptional repression of the Immunol. 2002 Sep 1;169(5):2477-87 cystic fibrosis transmembrane conductance regulator Goulet B, Watson P, Poirier M, Leduy L, Bérubé G, gene, mediated by CCAAT displacement protein/cut Meterissian S, Jolicoeur P, Nepveu A. Characterization of

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 192

CUX1 (cut-like homeobox 1) Kühnemuth B, Michl P

a tissue-specific CDP/Cux isoform, p75, activated in breast Ripka S, König A, Buchholz M, Wagner M, Sipos B, tumor cells. Cancer Res. 2002 Nov 15;62(22):6625-33 Klöppel G, Downward J, Gress T, Michl P. WNT5A--target of CUTL1 and potent modulator of tumor cell migration and Truscott M, Raynal L, Premdas P, Goulet B, Leduy L, invasion in pancreatic cancer. Carcinogenesis. 2007 Bérubé G, Nepveu A. CDP/Cux stimulates transcription Jun;28(6):1178-87 from the DNA polymerase alpha gene promoter. Mol Cell Biol. 2003 Apr;23(8):3013-28 Truscott M, Denault JB, Goulet B, Leduy L, Salvesen GS, Nepveu A. Carboxyl-terminal proteolytic processing of Tsutsumi S, Taketani T, Nishimura K, Ge X, Taki T, Sugita CUX1 by a caspase enables transcriptional activation in K, Ishii E, Hanada R, Ohki M, Aburatani H, Hayashi Y. Two proliferating cells. J Biol Chem. 2007 Oct distinct gene expression signatures in pediatric acute 12;282(41):30216-26 lymphoblastic leukemia with MLL rearrangements. Cancer Res. 2003 Aug 15;63(16):4882-7 Ueda Y, Su Y, Richmond A. CCAAT displacement protein regulates nuclear factor-kappa beta-mediated chemokine Goulet B, Baruch A, Moon NS, Poirier M, Sansregret LL, transcription in melanoma cells. Melanoma Res. 2007 Erickson A, Bogyo M, Nepveu A. A cathepsin L isoform Apr;17(2):91-103 that is devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor. Harada R, Vadnais C, Sansregret L, Leduy L, Bérubé G, Mol Cell. 2004 Apr 23;14(2):207-19 Robert F, Nepveu A. Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the Kaul-Ghanekar R, Jalota A, Pavithra L, Tucker P, activation of DNA replication genes. Nucleic Acids Res. Chattopadhyay S. SMAR1 and Cux/CDP modulate 2008 Jan;36(1):189-202 chromatin and act as negative regulators of the TCRbeta enhancer (Ebeta). Nucleic Acids Res. 2004;32(16):4862- Cadieux C, Kedinger V, Yao L, Vadnais C, Drossos M, 75 Paquet M, Nepveu A. Mouse mammary tumor virus p75 and p110 CUX1 transgenic mice develop mammary Nishio H, Walsh MJ. CCAAT displacement protein/cut tumors of various histologic types. Cancer Res. 2009 Sep homolog recruits G9a histone lysine methyltransferase to 15;69(18):7188-97 repress transcription. Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):11257-62 Kedinger V, Sansregret L, Harada R, Vadnais C, Cadieux C, Fathers K, Park M, Nepveu A. p110 CUX1 Truscott M, Raynal L, Wang Y, Bérubé G, Leduy L, homeodomain protein stimulates cell migration and Nepveu A. The N-terminal region of the CCAAT invasion in part through a regulatory cascade culminating displacement protein (CDP)/Cux transcription factor in the repression of E-cadherin and occludin. J Biol Chem. functions as an autoinhibitory domain that modulates DNA 2009 Oct 2;284(40):27701-11 binding. J Biol Chem. 2004 Nov 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 Aug;59(8):1101-10 Cell. 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 cathepsin L. Biol Chem. 2006 Sep;387(9):1285-93 of tumor progression in pancreatic cancer. Neoplasia. Maitra U, Seo J, Lozano MM, Dudley JP. Differentiation- 2010b Aug;12(8):659-67 induced cleavage of Cutl1/CDP generates a novel Sansregret L, Gallo D, Santaguida M, Leduy L, Harada R, dominant-negative isoform that regulates mammary gene Nepveu A. Hyperphosphorylation by cyclin B/CDK1 in expression. Mol Cell Biol. 2006 Oct;26(20):7466-78 mitosis resets CUX1 DNA binding clock at each cell cycle. Michl P, Downward J. CUTL1: a key mediator of TGFbeta- J Biol Chem. 2010 Oct 22;285(43):32834-43 induced tumor invasion. Cell Cycle. 2006 Jan;5(2):132-4 Fragiadaki M, Ikeda T, Witherden A, Mason RM, Abraham Sansregret L, Goulet B, Harada R, Wilson B, Leduy L, D, Bou-Gharios G. High doses of TGF-β potently suppress Bertoglio J, Nepveu A. The p110 isoform of the CDP/Cux type I collagen via the transcription factor CUX1. Mol Biol transcription factor accelerates entry into S phase. Mol Cell Cell. 2011 Jun 1;22(11):1836-44 Biol. 2006 Mar;26(6):2441-55 Thoennissen NH, Lasho T, Thoennissen GB, Ogawa S, Aleksic T, Bechtel M, Krndija D, von Wichert G, Knobel B, Tefferi A, Koeffler HP. Novel CUX1 missense mutation in Giehl K, Gress TM, Michl P. CUTL1 promotes tumor cell association with 7q- at leukemic transformation of MPN. migration by decreasing proteasome-mediated Src Am J Hematol. 2011 Aug;86(8):703-5 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, Mukhopadhyay D. Protein kinase C-mediated modulation Kühnemuth B, Michl P. CUX1 (cut-like homeobox 1). Atlas of FIH-1 expression by the homeodomain protein Genet Cytogenet Oncol Haematol. 2012; 16(3):189-193. CDP/Cut/Cux. Mol Cell Biol. 2007 Oct;27(20):7345-53

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 193

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI DNAJA3ID40342ch16p13.txt

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 Location: 16p13.3 known DnaJ/Hsp40 proteins in the human genome Local order: According to NCBI Map Viewer, (Qiu et al., 2006). genes flanking DNAJA3 are COR07-PAM16, According to NCBI Gene, the DNAJA3 gene is NMRAL1, and HMOX2. conserved in human chimpanzee, cow, mouse, rat, Note chicken, zebrafish, fruit fly, mosquito, C. elegans, DNAJA3 was first identified by its ability to form S. pombe, S. cerevisiae, K. lactis, E. gossypii, M. complexes with the human papillomavirus E7 grisea, 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 The DNAJA3 gene is located on chromosome Drosophila tumor suppressor protein Tid56. 16p13.3 between markers D16S521 and D16S418. Furthermore, DNAJA3 contained a J-domain which This chromosomal region carries several loci is characteristic of the family of DnaJ proteins implicated in human proliferation disorders, which interact with and stimulate the ATPase including the tuberous sclerosis 2 gene (TSC2), activity of heat shock cognate 70 (hsc70) family polycystic kidney disease 1 gene (PKD1), and the members (Schilling et al., 1998). 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) 194

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 195

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

specifically with the carboxyl-terminal region Proteins encoded by the long and short splice forms (amino acids 94-185). The N-terminal end of of DNAJA3, DNAJA3L and DNAJA3S, DNAJA3 (amino acids 1-447) was required for this respectively, showed differential interactions with interaction. Furthermore, the DNAJA3S precursor heat shock proteins. Unassembled DNAJA3L (the co-sedimented with viral capsid-like particles long splice variant) was shown to interact with composed of the full-length core protein (Sohn et Hsc70 specifically in the cytosol (Lu et al., 2006). al., 2006). Interaction between DNAJA3 and the The unique carboxyl terminus of DNAJA3L was HBV core protein was confirmed in co- required for this interaction (Lu et al., 2006). Both immunoprecipitation experiments using transfected DNAJA3S and DNAJA3L could interact with hepatoma cells (Sohn et al., 2006). Hsp70 (Kim et al., 2004). Endogenous DNAJA3L Epstein-Barr virus-encoded BARF1 protein: and DNAJA3S coimmunoprecipitated with DNAJA3 (amino acids 149-320) associated with mitochondrial Hsp70, but not Hsc70, in U2OS the Epstein-Barr virus-encoded BARF1 protein osteosarcoma cells (Syken et al., 1999). (amino acids 21-221). Interaction between Tumor suppressor proteins DNAJA3 and BARF1 was confirmed in co- Adenomatous polyposis coli (APC): endogenous immunoprecipitation experiments using transfected cytosolic DNAJA3 proteins interacted with APC in HeLa cells (Wang et al., 2006). normal colon epithelium and colorectal cancer cell Herpes simplex virus type 1 UL9 protein: lines (HT-29, Caco-2, and HRT-18). The N- DNAJA3 associated with the herpes simplex virus terminal Armadillo domain of APC was sufficient type 1 (HSV-1) UL9 protein. UL9 protein is an for binding to DNAJA3. The DNAJA3 and APC origin-binding protein. Interaction between interaction comprised part of a larger multi- DNAJA3 and UL9 was confirmed by in vitro co- component complex that also contained Hsp70, immunoprecipitation (Eom and Lehman, 2002). Hsc70, Actin, Dvl, and Axin. This complex Human T cell leukemia virus type 1 (HTLV-1) functions independently of the known roles of APC Tax protein: DNAJA3 associated with HTLV-1 in beta-catenin degradation and proliferation Tax. The interaction occurred through a central mediated by Wg/Wnt signaling (Kurzik-Dumke and cysteine-rich zinc finger-like region of DNAJA3 Czaja, 2007). (amino acids 236 to 300). Interaction between Endogenous DNAJA3 proteins were shown to DNAJA3 and Tax was confirmed by co- interact with the caspase-cleaved N-terminus of immunoprecipitation experiments using transfected APC in HCT116 cells (Qian et al., 2010). The human embryonic kidney cells (HEK) (Cheng et caspase-cleaved APC protein has an important al., 2001). Furthermore, the DNAJA3 and Tax physiological role in mediating apoptosis (Qian et interaction occurred through a complex comprised al., 2010). of DNAJA3, Tax, and heat shock protein 70 Patched: endogenous human Patched interacted (Hsp70), in which the cysteine-rich region of with the cytosolic forms of the DNAJA3 proteins in DNAJA3 interacted with Tax, while the J domain human colon epithelium and colon tumor cells of DNAJA3 interacted with Hsp70 (Cheng et al., (Kurzik-Dumke and Czaja, 2007). The tumor- 2001). associated polymorphism in Patched (Ptch FVB Human papilloma virus-16 (HPV-16) E7 allele) was associated with poorer binding to oncoprotein: DNAJA3 was initially characterized DNAJA3 (Wakabayashi et al., 2007). through its interaction with the HPV-16 E7 INT6: endogenous human DNAJA3 interacted with oncoprotein. DNAJA3 amino acids 1 to 235 and INT6 (the p48 subunit of the eIF3 translation 297 to 342 independently interacted with HPV-16 initiation factor) in log phase, but not confluent, E7. Interaction between DNAJA3 and HPV-16 E7 Jurkat T-cells (Traicoff et al., 2007). was confirmed by in vitro binding assays and co- Von Hippel-Lindau protein (VHL): endogenous immunoprecipitation experiments using transfected pVHL co-immunoprecipitated with DNAJA3L human osteosarcoma (U2OS) cells (Schilling et al., protein in HEK293 cells (Bae et al., 2005). 1998). p53: DNAJA3 directly interacts with p53 through Heat shock proteins the DNAJA3 DNAJ domain. Either the N- or C- Hsp70 and Hsc70: endogenous DNAJA3 terminal domains of p53 was sufficient for the (specifically the cytosolic form) interaction (Trinh et al., 2010). immunoprecipitated with the heat shock proteins Receptors Hsp70 and Hsc70 in normal colon epithelium and Interferon-gamma receptor (IFN-gammaR) colon cancer cell lines (Kurzik-Dumke and Czaja, subunit IFN-gammaR2: DNAJA3 interacted with 2007). Endogenous DNAJA3 also interacted with IFN-gamma R2 in transfected COS cells. Hsp70/Hsc70 in HEp2 cells, and this interaction Furthermore, DNAJA3 bound more efficiently to a was reduced in cells treated with interferon-gamma IFN-gammaR2 chimera with an active kinase (Sarkar et al., 2001). The J domain of DNAJA3 was domain than to a similar construct with an inactive shown to be required for interaction with Hsp70 in kinase domain (Sarkar et al., 2001). HEK cells (Cheng et al., 2001).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 196

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

ErbB-2 (HER2/neu): endogenous ErbB-2 and Neural pathways DNAJA3 co-immunoprecipitated in SK-BR-3 Neuromuscular synaptogenesis: DNAJA3 is an breast cancer cells (Kim et al., 2004). The essential component of the agrin signaling pathway cytoplasmic domains of ErbB-2 and DNAJA3 were that is crucial for synaptic development. sufficient for this interaction (Kim et al., 2004). Motoneuron-derived agrin clusters nicotinic ErbB-2 co-immunoprecipiated with DNAJA3 and acetylcholine receptors (AChRs) in mammalian the carboxyl terminus of heat shock cognate 70 cells. DNAJA3 binds to the cytoplasmic domain of interacting protein (CHIP). This complex was muscle-specific kinase (MuSK), a component of the demonstrated in tissue extracts from breast tumor agrin receptor and colocalizes with AchRs at specimens as well as in transfected cell lines (Jan et developing, adult, and denvervated motor al., 2011). endplates. DNAJA3 transduces signals from MuSK Trk receptor tyrosine kinases: the carboxyl- activation to AchR clustering, culmintating in terminal end of DNAJA3 (residues 224-429) bound cross-linking to the subsynaptic cytoskeleton, as to Trk at its activation loop in a phosphotyrosine- demonstrated by knockdown and overexpression dependent manner (Liu et al., 2005). experiments. Muscle-specific kinase (MuSK) component of Knockdown of DNAJA3 in skeletal muscle fibers the agrin receptor: DNAJA3S, but not DNAJA3L, resulted in dispersed synaptic AchR clusters and associated with the cytoplasmic portion of MuSK in impaired neuromuscular transmission. Knockdown mouse skeletal muscle cells (Linnoila et al., 2008). of DNAJA3 in myotubes resulted in inhibition of Signaling proteins AchR clustering, inhibition of agrin-induced NF-kappaB: DNAJA3 strongly associated with the activation of the Rac and Rho small GTPases and cytoplasmic protein complex of NF-kappaB- tyrosine phosphorylation of AchR, and decreased IkappaB through direct interaction with Dok-7-induced clustering of AChRs. In contrast, IkappaBalpha/IkappaBbeta and the IKKalpha/beta overexpression of the N-terminal half of DNAJA3 subunits of the IkappaB kinase complex. The induced agrin-and MuSK-independent endogenous interaction was observed in Jurkat, phosphorylation and clustering of AChRs (Linnoila SAOS-2, and HEK293 cells (Cheng et al., 2005). et al., 2008; Song and Balice-Gordon, 2008). JAK/STAT: Jak2 interacted with DNAJA3S as Neurite outgrowth: DNAJA3 regulated nerve well as DNAJA3L as shown by growth factor (NGF)-induced neurite outgrowth in immunoprecipitation from transfected COS-1 cells PC12-derived nnr5 cells. DNAJA3 bound to Trk at expressing these proteins. Endogenous DNAJA3 the activation loop and DNAJA3 was tyrosine and Jak2 were shown to interact in HEp2 cells phosphorylated by Trk in yeast cells, transfected (Sarkar et al., 2001). cells, and in neurotophin-stimulated primary rat The carboxyl terminus of endogenous DNAJA3L, hippocampal neurons. Overexpression of DNAJA3 but not DNAJA3S, co-immunoprecipitated with led to NGF-induced neurite outgrowth in TrkA- STAT1 and with STAT3 in U2OS cells (Lu et al., expressing nnr5 cells. In contrast, knockdown of 2006). DNAJA3L remained associated with DNAJA3 resulted in reduced NGF-induced neurite activated phosphorylated STAT1 upon treatment growth in nnr5-TrkA cells (Liu et al., 2005). with interferon-gamma (Lu et al., 2006). Viral pathways The DNAJ domain of DNAJA3 interacted with the Hepatitis B virus replication: expression of transactivation domain of Stat5b in hematopoietic DNAJA3 suppressed replication of HBV in human cell lines (Dhennin-Duthille et al., 2011). hepatoma cells, while knockdown of DNAJA3 led p120 GTPase-activating protein (GAP): both the to increased HBV replication. The mechanism for cytoplasmic precursor and mitochondrial mature inhibited replication was through accelerated forms of murine DNAJA3 associated with GAP in degradation and destabilization of the viral core and vivo in rodent cells. GAP selectively bound to the HBx proteins (Sohn et al., 2006). unphosphorylated form of murine DNAJA3 Herpes simplex virus type 1 replication: the (Trentin et al., 2001). HSV-1 UL9 protein is an origin-binding protein DNA replication proteins that is essential for viral DNA replication. DNAJA3 DNA polymerase gamma (Polga) alpha subunit: modulates DNA replication by enhancing the endogenous DNAJA3 interacted with the alpha binding of UL9 protein to an HSV-1 origin and subunit of Polga in HEK293 cells. Polga is the only facilitating formation of the multimer from the mitochondrial DNA polymerase responsible for all dimeric UL9 protein, perhaps through a chaperone mitochondrial DNA synthetic reactions (Hayashi et function. However, DNAJA3 had no effect on the al., 2006). DNA-dependent ATPase or helicase activities II. Signaling pathways and cellular effects associated with the UL9 protein (Eom and Lehman, DNAJA3 modulates diverse signaling pathways 2002). and cellular effects that are vital for cell growth and Epstein-Barr virus secretion: the EBV BARF1 differentiation. gene encodes a secretory protein with transforming and mitogenic activities. Coexpression experiments

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 197

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

with BARF1 and DNAJA3 showed that DNAJA3 mutant of hTid-1S in a Th2 cell line strikingly could promote secretion of BARF1, perhaps enhanced activation of caspase 3 in response to through chaperone functions (Wang et al., 2006). CD3 stimulation, and caused the cells to become Motility and metastasis sensitive to AICD. Therefore, the accumulation of DNAJA3 was shown to negatively regulate the Tid-1S in Th2 cells following activation may motility and metastasis of breast cancer cells contribute to the induction of apoptosis resistance through attenuation of nuclear factor kappaB during the activation of Th2 cells (Syken et al., activity on the promoter of the IL8 gene (Kim et al., 2003). 2005). Reductions of DNAJA3 levels in MDA- DNAJA3 mediates apoptosis through the MB231 breast cancer cells increased their migration nuclear factor kappaB (NF-kappaB) pathway. as a result of increased interleukin-8 (IL-8) DNAJA3 repressed the activity of NF-kappaB secretion without affecting survival or growth rate. through physical and functional interactions with Furthermore, DNAJA3 was shown to negatively the IKK complex and IkappaB. Overexpression of modulate de novo synthesis of IL-8 through DNAJA3 led to inhibition of cell proliferation and regulating NFkappaB activity. Additionally, induction of apoptosis of human osteosarcoma cells DNAJA3 knockdown enhanced the metastasis of and human melanoma cells regardless of the p53 breast cancer cells in animals (Kim et al., 2005). expression status. In contrast, cells transduced with Other studies also indicate a potential role for a DNAJA3 mutant that has an N-terminal J domain DNAJA3 in inhibiting transformation and deletion and that lost suppressive activity on IKK, metastasis. Stable DNAJA3 knockdown cells continued to proliferate (Cheng et al., 2005). exhibited an enhanced ability for anchorage- DNAJA3 mediates apoptosis through the Bcl-2 independent growth, as measured by an increase in pathway. DNAJA3 induced apoptosis in SF767 soft-agar colony formation (Edwards and Münger, glioma cells that contained a tumor-associated 2004). In contrast, ectopic expression of DNAJA3 mutation at the DNAJA3 locus. Apoptosis resulted in HNSCC cells was shown to significantly inhibit from caspase activation and cytochrome c release cell proliferation, migration, invasion, anchorage- from mitochondria. However, Bcl-XL protected independent growth, and xenotransplantation cells from hTid-1S-induced cell death and tumorigenicity (Chen et al., 2009). Expression of cytochrome c release. However, hTid1S caused S DNAJA3 inhibited the transformation phenotype of and G2/M arrest in cells with wild type Tid1. two human lung adenocarcinoma cell lines (Cheng Interestingly, hTid1L had no effect on growth of et al., 2001). glioma cells (Trentin et al., 2004). Apoptosis Immature dnaja3(-/-) DN4 thymocytes exhibited DNAJA3 encodes two mitochondrial matrix significantly reduced expression of the localized splice variants: DNAJA3 (43 kD) and antiapoptotic bcl-2 gene (Lo et al., 2005). DNAJA3 (40 kD). DNAJA3 (43 kD) and DNAJA3 Expression of constitutively active AKT (pAKT) (40 kD) do not themselves induce apoptosis; counteracted and inhibited DNAJA3-induced instead they have opposing effects on apoptosis apoptosis in HNSCC cells (Chen et al., 2009). induced by exogenous stimuli. DNAJA3 mediates apoptosis through APC. Expression of DNAJA3 (43 kD) increases apoptosis DNAJA3 (40 kD) isoform inhibited apoptosis induced by both the DNA-damaging agent through antagonizing the apoptotic function of the mitomycin c and tumor necrosis factor-alpha. This N-terminal region of the APC protein (Qian et al., activity is J domain-dependent, since a J domain 2010). mutant of DNAJA3 (43 kD) suppressed apoptosis. DNAJA3 mediates apoptosis through p53. Conversely, expression of DNAJA3 (40 kD) Overexpression of DNAJA3 enhanced p53- suppressed apoptosis, while expression of a J dependent apoptosis, and restored pro-apoptotic domain mutant of DNAJA3 (40 kD) increased activity of mutant p53 in colon, breast, and glioma apoptosis (Syken et al., 1999). cell lines (Ahn et al., 2010). The mechanism is Cells lacking expression of DNAJA3 proteins were through direct interaction of the DNAJ domain of protected from cell death in response to multiple DNAJA3 and p53 (Trinh et al., 2010). In contrast, stimuli, including cisplatin, tumor necrosis factor depletion of DNAJA3 resulted in the inhibition of alpha/cycloheximide and mitomycin C (Edwards hypoxia or genotoxic stress-induced p53 and Münger, 2004). mitochondrial localization and apoptosis (Trinh et DNAJA3 regulates activation-induced cell death al., 2010). (AICD) in the Th2 subset of helper T cells. AICD is Mitochondrial functions an apoptotic process induced by stimulation Although DNAJA3 has many cellular functions, through the T-cell receptor and Th2 cells are DNAJA3 often localizes to the mitochondria and significantly less prone to AICD than Th1 cells are. also has important functions in the mitochondria. The antiapoptotic variant, Tid-1S was shown to be Epidermal growth factor (EGF) response: GAP selectively induced in murine Th2 cells following and DNAJA3 were shown to colocalize at activation. Expression of a dominant-negative

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 198

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

perinuclear mitochondrial membranes in response gamma treatment reduced the interaction between to EGF stimulation (Trentin et al., 2001). Hsp70/Hsc70 and DNAJA3 (Sarkar et al., 2001). p53 localization and apoptotic function: Oncogenic pathways depletion of DNAJA3 prevented p53 accumulation Erb-B2/HER2: DNAJA3 physically interacted at the mitochondria and resulted in resistance to with the signaling domain of ErbB-2 and ErbB-2 apoptosis under hypoxic or genotoxic stresses were shown to colocalize in mammary carcinoma (Trinh et al., 2010). DNAJA3 formed a complex cells (SK-BR-3). Overexpression of DNAJA3 with p53 under hypoxic conditions that directed induced growth arrest and apoptosis in ErbB-2- p53 translocation to the mitochondria and the overexpressing breast cancer cells; the DNAJ and subsequent initiation of apoptosis (Ahn et al., C-terminal domains of DNAJA3 were critical for 2010). Loss of DNAJA3 expression abrogated p53 mediating apoptosis. Downregulation of translocation to the mitochondria and inhibited ERK1/ERK2 and BMK1 MAPK pathways also apoptosis (Ahn et al., 2010). Conversely, contributed to apoptosis. DNAJA3S negatively overexpression of DNAJA3 promoted p53 regulated ErbB-2 signaling pathways by enhancing mitochondrial localization and apoptosis (Ahn et the degradation of ErbB-2. Finally, increased al., 2010). cellular DNAJA3 inhibited the growth of ErbB-2- Viral protein localization: in the absence of Tax, dependent tumors in mice (Kim et al., 2004). expression of the DNAJA3/Hsp70 molecular Mammary tumor tissue from breast cancer patients complex was targeted to perinuclear mitochondrial and transgenic mice carrying the rat HER-2/neu clusters. In the presence of Tax, DNAJA3 and its oncogene suggest that DNAJA3 suppresses ErbB-2 associated Hsp70 are sequestered within a in breast cancers (Kurzik-Dumke et al., 2010). cytoplasmic "hot spot" structure, a subcellular NF-kappaB: expression of DNAJA3 was distribution that is characteristic of Tax in HEK upregulated upon cellular senescence in rat and cells (Cheng et al., 2001). mouse embryo fibroblasts, as well as in premature APC interaction: the amino terminus of APC senescence of REF52 cells triggered by activated interacted with DNAJA3 at the mitochondria in ras. Conversely, spontaneous immortalization of rat vivo in colorectal cancer cell lines (Qian et al., embryo fibroblasts was suppressed upon ectopic 2010). expression of DNAJA3. Suppression of endogenous Chaperone function: DNAJA3 isoforms were also DNAJA3 activity alleviated the suppression of shown to exhibit a conserved mitochondrial DnaJ- tumor necrosis factor alpha-induced NF-kappaB like function substituting for the yeast activity by DNAJA3. These results suggest that mitochondrial DnaJ-like protein Mdj1p (Lu et al., DNAJA3 contributes to senescence by repressing 2006). NF-kappa B signaling (Tarunina et al., 2004). Mitochondrial biogenesis: DNAJA3 was shown to DNAJA3 repressed NF-kappaB activity induced by be crucial for mitochondrial biogenesis partly Tax, tumor necrosis factor alpha (TNFalpha), and through chaperone activity on DNA polymerase Bcl10. DNAJA3 specifically suppressed serine gamma (Hayashi et al., 2006). Mice deficient in phosphorylation of IkappaBalpha by activated Dnaja3 developed dilated cardiomyopathy (DCM) IkappaB kinase beta (IKKbeta). and died before 10 weeks of age (Hayashi et al., The suppressive activity of DNAJA3 on IKKbeta 2006). Progressive respiratory chain deficiency and required a functional J domain that mediates decreased copy number of mitochondrial DNA association with heat shock proteins and resulted in were observed in cardiomyocytes lacking Dnaja3 prolonging the half-life of the NF-kappaB inhibitors (Hayashi et al., 2006). IkappaBalpha and IkappaBbeta (Cheng et al., Tumor suppressor pathways 2002). APC: DNAJA3 directly bound to the APC tumor AKT: overexpression of DNAJA3 in HNSCC cells suppressor protein and promoted a physiological inhibited cell proliferation, migration, invasion, function for APC that was independent of APC's anchorage-independent growth, and involvement in beta-catenin degradation or xenotransplantation tumorigenicity. Overexpression regulation of the actin cytoskeleton (Kurzik-Dumke of DNAJA3 attenuated EGFR activity and blocked and Czaja, 2007). the activation of AKT in HNSCC cells, which are pVHL: TID1L directly interacted with von Hippel- known to be involved in the regulation of survival Lindau protein and enhanced the interaction in HNSCC cells. Conversely, ectopic expression of between HIF-1 alpha and pVHL. This resulted in constitutively active AKT greatly reduced apoptosis destabilization of HIF-1 alpha protein, decreased induced by DNAJA3 overexpression (Chen et al., vascular endothelial growth factor expression, and 2009). inhibition of angiogenesis (Bae et al., 2005). JAK2: DNAJA3L and DNAJA3S interacted with Interferon-gamma: DNAJA3L and DNAJA3S Jak2 in vivo in COS-1 cells. Interaction was interacted with the interferon-gamma receptor chain primarily in the cytoplasm and predominantly with IFN-gammaR2 and modulated IFN-gamma- the active kinase domain of Jak2 (Sarkar et al., mediated transcriptional activity. Furthermore, IFN- 2001).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 199

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

c-MET receptor tyrosine kinase (MetR): MetR The mutations appear to increase the steady-state interacted with DNAJA3L and DNAJA3S, but abundance of the mutant protein, resulting in showed preferential binding to DNAJA3S. aberrantly high levels of the DNAJA3 mutant Interaction occurred through the J domain. In RCC variant (Trentin et al., 2004). cells, overexpression of DNAJA3S enhanced HGF- mediated MetR autophosphorylation, while Implicated in DNAJA3L showed modest inhibition of MetR activity. Modulation of MetR phosphorylation Colon cancer levels was independent of pVHL. DNAJA3S Disease enhanced HGF-mediated cell migration and DNAJA3 and INT6 protein levels, as well as modulated HGF-mediated MAPK phosphorylation. DNAJA3 and Patched protein levels, were DNAJA3 knockdown inhibited MetR activation positively correlated in human colon tumor tissues and migration in response to HGF (Copeland et al., (Traicoff et al., 2007). However, there were no 2011). correlations between DNAJA3 and p53, c-Jun, or Signal transducers and activators of phospho-c-Jun protein levels (Traicoff et al., 2007). transcription (STAT) 5b: DNAJA3 specifically These results were demonstrated by multiplex interacted with STAT5b but not STAT5a in tissue immunoblotting of tissue microarrays hematopoietic cell lines. Interaction involved the (Traicoff et al., 2007). DNAJ domain. DNAJA3 negatively regulated the Progression of colorectal cancers correlated with expression and transcriptional activity of STAT5b overexpression and loss of polarization of and suppressed the growth of hematopoietic cells expression of DNAJA3. These changes were transformed by an oncogenic form of STAT5b associated with upregulation of Hsp70 and loss of (Dhennin-Duthille et al., 2011). compartmentalization of APC (Kurzik-Dumke et Cell Fate al., 2008). DNAJA3 was shown to be required for the T-cell transition from double-negative 3 to double- Breast cancer positive stages. Mice with dnaja3 specifically Disease deleted in T cells developed thymic atrophy, with DNAJA3 protein expression showed a strong dramatic reduction of double-positive and single- correlation with negative or weakly positive positive thymocytes in the dnaja3(-/-) thymus. expression of ErbB2 in human breast cancer tissue DNAJA3 deficiency inhibited cell proliferation and samples. High DNAJA3 levels were strongly enhanced cell death of DN4 cells. The expression correlated with high levels of CHIP (carboxyl profile of genes involved in cytokine receptor terminus of heat shock cognate 70 interacting signaling was altered in DN4 T-cells. Expression of protein). Lower expression of DNAJA3 had a human bcl-2 transgene restored T lymphocyte higher risk of unfavorable tumor grade, later proliferation and differentiation in the dnaja3 pathological stage, larger tumor size, and knockout mice. These results suggest that dnaja3 is microscopic features of a more malignant histology critical in early thymocyte development, especially (Jan et al., 2011). Higher expression of DNAJA3 during transition from the DN3 to double-positive correlated with increased 10-year overall and stages, possibly through its regulation of bcl-2 disease-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 Rat: Dnaja3 transformation. Furthermore, elevated DNAJA3L Cattle: DNAJA3 expression was associated with less aggressive Chimpanzee: DNAJA3 tumors (Kurzik-Dumke et al., 2010). Dog (domestic): DNAJA3 Immunohistochemical analysis demonstrated high levels of DNAJA3 protein in tumors of the luminal Mutations A subtype, but significantly lower levels of Note DNAJA3 protein in the luminal B subtype, triple negative tumors, and the HER-2 subtype which The SF767 glioma cell line exhibits an aberrant 52 overexpresses HER-2 (Kurzik-Dumke et al., 2010). kD molecular weight protein. Sequence analysis of Multiplex tissue immunoblotting of human breast cDNA generated from this line showed two tumor tissue microarrays was used to test mutations: an additional thymine at nucleotide correlations between DNAJA3 protein levels and a position 1438 and an additional cytosine at set of tumor suppressor proteins. DNAJA3 protein nucleotide position 1449. These mutations alter the levels showed strongly positive correlations with reading frame of the DNAJA3 sequence, p53, Patched, and INT6 proteins (Traicoff et al., introducing an additional 71 amino acids following 2007). Additionally, DNAJA3 protein levels the penultimate threonine residue at position 479.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 200

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

showed moderate positive correlations with c-Jun Cheng H, Cenciarelli C, Shao Z, Vidal M, Parks WP, and phospho-c-Jun proteins (Traicoff et al., 2007). Pagano M, Cheng-Mayer C. Human T cell leukemia virus type 1 Tax associates with a molecular chaperone Head and neck squamous cell complex containing hTid-1 and Hsp70. Curr Biol. 2001 Nov carcinoma (HNSCC) 13;11(22):1771-5 Sarkar S, Pollack BP, Lin KT, Kotenko SV, Cook JR, Lewis Disease A, Pestka S. hTid-1, a human DnaJ protein, modulates the The clinical association between DNAJA3 interferon signaling pathway. J Biol Chem. 2001 Dec expression and progression of HNSCC was 28;276(52):49034-42 explored using immunohistochemical analysis of Trentin GA, Yin X, Tahir S, Lhotak S, Farhang-Fallah J, Li primary HNSCC patient tumor tissue. DNAJA3 Y, Rozakis-Adcock M. A mouse homologue of the expression was negatively associated with tumor T Drosophila tumor suppressor l(2)tid gene defines a novel Ras GTPase-activating protein (RasGAP)-binding protein. stage, overall stage, survival, and recurrence. J Biol Chem. 2001 Apr 20;276(16):13087-95 Patients with higher expression of DNAJA3 were predicted to have better overall survival than those Yin X, Rozakis-Adcock M. Genomic organization and expression of the human tumorous imaginal disc (TID1) with low or undetectable expression of DNAJA3 gene. Gene. 2001 Oct 31;278(1-2):201-10 protein (Chen et al., 2009). Highly malignant Cheng H, Cenciarelli C, Tao M, Parks WP, Cheng-Mayer HNSCC cell lines also demonstrated low or C. HTLV-1 Tax-associated hTid-1, a human DnaJ protein, undetectable levels of DNAJA3, in contrast to less is a repressor of Ikappa B kinase beta subunit. J Biol aggressive lines where DNAJA3 protein was easily Chem. 2002 Jun 7;277(23):20605-10 detected (Chen et al., 2009). Eom CY, Lehman IR. The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus Ovarian cancer type 1 UL9 protein to oris, an origin of viral DNA Disease replication. Proc Natl Acad Sci U S A. 2002 Feb Multiplex tissue immunoblotting of ovarian tumor 19;99(4):1894-8 tissues demonstrated that DNAJA3 protein levels Sehgal PB. Plasma membrane rafts and chaperones in showed moderate positive correlations with INT6, cytokine/STAT signaling. Acta Biochim Pol. c-Jun, phospho-c-Jun, and p53. No correlations 2003;50(3):583-94 were observed between DNAJA3 and Patched Syken J, Macian F, Agarwal S, Rao A, Münger K. TID1, a (Traicoff et al., 2007). mammalian homologue of the drosophila tumor suppressor lethal(2) tumorous imaginal discs, regulates activation- Lung cancer induced cell death in Th2 cells. Oncogene. 2003 Jul 24;22(30):4636-41 Disease Multiplex tissue immunoblotting of lung tumor Edwards KM, Münger K. Depletion of physiological levels of the human TID1 protein renders cancer cell lines tissues demonstrated that DNAJA3 protein levels resistant to apoptosis mediated by multiple exogenous were strongly correlated with INT6. DNAJA3 stimuli. Oncogene. 2004 Nov 4;23(52):8419-31 protein levels were moderately correlated with Kim SW, Chao TH, Xiang R, Lo JF, Campbell MJ, Fearns Patched, c-Jun, and p53. However, DNAJA3 C, Lee JD. Tid1, the human homologue of a Drosophila proteins showed negative correlation with phospho- tumor suppressor, reduces the malignant activity of ErbB-2 c-Jun in these samples (Traicoff et al., 2007). in carcinoma cells. Cancer Res. 2004 Nov 1;64(21):7732-9 Tarunina M, Alger L, Chu G, Munger K, Gudkov A, Jat PS. Cardiomyopathy Functional genetic screen for genes involved in Note senescence: role of Tid1, a homologue of the Drosophila Mice deficient in Dnaja3 developed dilated tumor suppressor l(2)tid, in senescence and cell survival. Mol Cell Biol. 2004 Dec;24(24):10792-801 cardiomyopathy (DCM) and died before 10 weeks of age (Hayashi et al., 2006). Progressive Trentin GA, He Y, Wu DC, Tang D, Rozakis-Adcock M. Identification of a hTid-1 mutation which sensitizes gliomas respiratory chain deficiency and decreased copy to apoptosis. FEBS Lett. 2004 Dec 17;578(3):323-30 number of mitochondrial DNA were observed in cardiomyocytes lacking Dnaja3 (Hayashi et al., Bae MK, Jeong JW, Kim SH, Kim SY, Kang HJ, Kim DM, Bae SK, Yun I, Trentin GA, Rozakis-Adcock M, Kim KW. 2006). Tid-1 interacts with the von Hippel-Lindau protein and modulates angiogenesis by destabilization of HIF-1alpha. References Cancer Res. 2005 Apr 1;65(7):2520-5 Schilling B, De-Medina T, Syken J, Vidal M, Münger K. A Cheng H, Cenciarelli C, Nelkin G, Tsan R, Fan D, Cheng- novel human DnaJ protein, hTid-1, a homolog of the Mayer C, Fidler IJ. Molecular mechanism of hTid-1, the Drosophila tumor suppressor protein Tid56, can interact human homolog of Drosophila tumor suppressor l(2)Tid, in with the human papillomavirus type 16 E7 oncoprotein. the regulation of NF-kappaB activity and suppression of Virology. 1998 Jul 20;247(1):74-85 tumor growth. Mol Cell Biol. 2005 Jan;25(1):44-59 Syken J, De-Medina T, Münger K. TID1, a human homolog Kim SW, Hayashi M, Lo JF, Fearns C, Xiang R, Lazennec of the Drosophila tumor suppressor l(2)tid, encodes two G, Yang Y, Lee JD. Tid1 negatively regulates the migratory mitochondrial modulators of apoptosis with opposing potential of cancer cells by inhibiting the production of functions. Proc Natl Acad Sci U S A. 1999 Jul interleukin-8. Cancer Res. 2005 Oct 1;65(19):8784-91 20;96(15):8499-504 Kittler R, Pelletier L, Ma C, Poser I, Fischer S, Hyman AA, Buchholz F. RNA interference rescue by bacterial artificial

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 201

DNAJA3 (DnaJ (Hsp40) homolog, subfamily A, member 3) Traicoff JL, et al.

chromosome transgenesis in mammalian tissue culture Linnoila J, Wang Y, Yao Y, Wang ZZ. A mammalian cells. Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2396- homolog of Drosophila tumorous imaginal discs, Tid1, 401 mediates agrin signaling at the neuromuscular junction. Neuron. 2008 Nov 26;60(4):625-41 Liu HY, MacDonald JI, Hryciw T, Li C, Meakin SO. Human tumorous imaginal disc 1 (TID1) associates with Trk Song Y, Balice-Gordon R. New dogs in the dogma: Lrp4 receptor tyrosine kinases and regulates neurite outgrowth and Tid1 in neuromuscular synapse formation. Neuron. in nnr5-TrkA cells. J Biol Chem. 2005 May 2008 Nov 26;60(4):526-8 20;280(20):19461-71 Chen CY, Chiou SH, Huang CY, Jan CI, Lin SC, Hu WY, Lo JF, Zhou H, Fearns C, Reisfeld RA, Yang Y, Lee JD. Chou SH, Liu CJ, Lo JF. Tid1 functions as a tumour Tid1 is required for T cell transition from double-negative 3 suppressor in head and neck squamous cell carcinoma. J to double-positive stages. J Immunol. 2005 May Pathol. 2009 Nov;219(3):347-55 15;174(10):6105-12 Ahn BY, Trinh DL, Zajchowski LD, Lee B, Elwi AN, Kim Hayashi M, Imanaka-Yoshida K, Yoshida T, Wood M, SW. Tid1 is a new regulator of p53 mitochondrial Fearns C, Tatake RJ, Lee JD. A crucial role of translocation and apoptosis in cancer. Oncogene. 2010 mitochondrial Hsp40 in preventing dilated cardiomyopathy. Feb 25;29(8):1155-66 Nat Med. 2006 Jan;12(1):128-32 Kurzik-Dumke U, Hörner M, Nicotra MR, Koslowski M, Lu B, Garrido N, Spelbrink JN, Suzuki CK. Tid1 isoforms Natali PG. In vivo evidence of htid suppressive activity on are mitochondrial DnaJ-like chaperones with unique ErbB-2 in breast cancers over expressing the receptor. J carboxyl termini that determine cytosolic fate. J Biol Chem. Transl Med. 2010 Jun 17;8:58 2006 May 12;281(19):13150-8 Maselli RA, Arredondo J, Cagney O, Ng JJ, Anderson JA, Qiu XB, Shao YM, Miao S, Wang L. The diversity of the Williams C, Gerke BJ, Soliven B, Wollmann RL. Mutations DnaJ/Hsp40 family, the crucial partners for Hsp70 in MUSK causing congenital myasthenic syndrome impair chaperones. Cell Mol Life Sci. 2006 Nov;63(22):2560-70 MuSK-Dok-7 interaction. Hum Mol Genet. 2010 Jun 15;19(12):2370-9 Sohn SY, Kim SB, Kim J, Ahn BY. Negative regulation of hepatitis B virus replication by cellular Hsp40/DnaJ Qian J, Perchiniak EM, Sun K, Groden J. The proteins through destabilization of viral core and X mitochondrial protein hTID-1 partners with the caspase- proteins. J Gen Virol. 2006 Jul;87(Pt 7):1883-91 cleaved adenomatous polyposis cell tumor suppressor to facilitate apoptosis. Gastroenterology. 2010 Wang L, Tam JP, Liu DX. Biochemical and functional Apr;138(4):1418-28 characterization of Epstein-Barr virus-encoded BARF1 protein: interaction with human hTid1 protein facilitates its Trinh DL, Elwi AN, Kim SW. Direct interaction between p53 maturation and secretion. Oncogene. 2006 Jul and Tid1 proteins affects p53 mitochondrial localization 20;25(31):4320-31 and apoptosis. Oncotarget. 2010 Oct;1(6):396-404 Kurzik-Dumke U, Czaja J. Htid-1, the human homolog of Copeland E, Balgobin S, Lee CM, Rozakis-Adcock M. the Drosophila melanogaster l(2)tid tumor suppressor, hTID-1 defines a novel regulator of c-Met Receptor defines a novel physiological role of APC. Cell Signal. signaling in renal cell carcinomas. Oncogene. 2011 May 2007 Sep;19(9):1973-85 12;30(19):2252-63 Traicoff JL, Chung JY, Braunschweig T, Mazo I, Shu Y, Dhennin-Duthille I, Nyga R, Yahiaoui S, Gouilleux-Gruart Ramesh A, D'Amico MW, Galperin MM, Knezevic V, V, Régnier A, Lassoued K, Gouilleux F. The tumor Hewitt SM. Expression of EIF3-p48/INT6, TID1 and suppressor hTid1 inhibits STAT5b activity via functional Patched in cancer, a profiling of multiple tumor types and interaction. J Biol Chem. 2011 Feb 18;286(7):5034-42 correlation of expression. J Biomed Sci. 2007 May;14(3):395-405 Jan CI, Yu CC, Hung MC, Harn HJ, Nieh S, Lee HS, Lou MA, Wu YC, Chen CY, Huang CY, Chen FN, Lo JF. Tid1, Wakabayashi Y, Mao JH, Brown K, Girardi M, Balmain A. CHIP and ErbB2 interactions and their prognostic Promotion of Hras-induced squamous carcinomas by a implications for breast cancer patients. J Pathol. 2011 polymorphic variant of the Patched gene in FVB mice. Nov;225(3):424-37 Nature. 2007 Feb 15;445(7129):761-5 This article should be referenced as such: Kurzik-Dumke U, Hörner M, Czaja J, Nicotra MR, Simiantonaki N, Koslowski M, Natali PG. Progression of Traicoff JL, Hewitt SM, Chung JY. DNAJA3 (DnaJ (Hsp40) colorectal cancers correlates with overexpression and loss homolog, subfamily A, member 3). Atlas Genet Cytogenet of polarization of expression of the htid-1 tumor Oncol Haematol. 2012; 16(3):194-202. suppressor. Int J Mol Med. 2008 Jan;21(1):19-31

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 202

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI MYEOVID395.txt

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)

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 203

MYEOV (myeloma overexpressed (in a subset of t(11;14) Moreaux J positive multiple myelomas))

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 204

MYEOV (myeloma overexpressed (in a subset of t(11;14) Moreaux J positive multiple myelomas))

CCND1 in breast cancer. Int J Cancer. 2002 Dec Moreaux J, Hose D, Bonnefond A, Reme T, Robert N, 20;102(6):608-14 Goldschmidt H, Klein B. MYEOV is a prognostic factor in multiple myeloma. Exp Hematol. 2010 Dec;38(12):1189- Janssen JW, Imoto I, Inoue J, Shimada Y, Ueda M, 1198.e3 Imamura M, Bartram CR, Inazawa J. MYEOV, a gene at 11q13, is coamplified with CCND1, but epigenetically Sugahara K, Michikawa Y, Ishikawa K, Shoji Y, Iwakawa inactivated in a subset of esophageal squamous cell M, Shibahara T, Imai T. Combination effects of distinct carcinomas. J Hum Genet. 2002;47(9):460-4 cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma. Int J Oncol. Leyden J, Murray D, Moss A, Arumuguma M, Doyle E, 2011 Oct;39(4):761-9 McEntee G, O'Keane C, Doran P, MacMathuna P. Net1 and Myeov: computationally identified mediators of gastric Takita J, Chen Y, Okubo J, Sanada M, Adachi M, Ohki K, cancer. Br J Cancer. 2006 Apr 24;94(8):1204-12 Nishimura R, Hanada R, Igarashi T, Hayashi Y, Ogawa S. Aberrations of NEGR1 on 1p31 and MYEOV on 11q13 in Moss AC, Lawlor G, Murray D, Tighe D, Madden SF, neuroblastoma. Cancer Sci. 2011 Sep;102(9):1645-50 Mulligan AM, Keane CO, Brady HR, Doran PP, MacMathuna P. ETV4 and Myeov knockdown impairs This article should be referenced as such: colon cancer cell line proliferation and invasion. Biochem Biophys Res Commun. 2006 Jun 23;345(1):216-21 Moreaux J. MYEOV (myeloma overexpressed (in a subset of t(11;14) positive multiple myelomas)). Atlas Genet Lawlor G, Doran PP, MacMathuna P, Murray DW. MYEOV Cytogenet Oncol Haematol. 2012; 16(3):203-205. (myeloma overexpressed gene) drives colon cancer cell migration and is regulated by PGE2. J Exp Clin Cancer Res. 2010 Jun 22;29:81

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 205

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI PCNAID41670ch20p12.txt

Identity Transcription Other names: MGC8367 There are two reported gene transcripts, which encode 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). DNA/RNA The PCNA transcript variant 1 has seven exons, six of which are contributing to the protein sequence. Description The first intron is relatively large in comparison The PCNA gene is situated on human chromosome with the other PCNA transcript variant. Following 20 and it spans about 12 kb. It is a single-copy the splicing the length of the transcript is shortened gene, however, several pseudogenes have been to about 12% of that of the initial transcript. The noted. The precise localization of the PCNA gene is translation starts from the middle of the 2nd exon at the border of two histological G-bands (p12.3 and ends in the beginning of 7th exon. The product and p13) (Webb et al., 1990), thus it is reported in is a full length protein, designated as NP_002583.1 both locations depending on the probe used. The (NCBI), with 261 amino acids. human PCNA gene was first cloned and PCNA transcript variant 2 is 1319 bp long after the characterized in 1989 by Travali and co-workers completion of mRNA splicing. (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) 206

PCNA (proliferating cell nuclear antigen) Stoimenov I, Helleday T

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

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) 207

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 208

PCNA (proliferating cell nuclear antigen) Stoimenov I, Helleday T

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

Horiguchi J, Iino Y, Takei H, Maemura M, Takeyoshi I, This article should be referenced as such: Yokoe T, Ohwada S, Oyama T, Nakajima T, Morishita Y. Long-term prognostic value of PCNA labeling index in Stoimenov I, Helleday T. PCNA (proliferating cell nuclear primary operable breast cancer. Oncol Rep. 1998 May- antigen). Atlas Genet Cytogenet Oncol Haematol. 2012; Jun;5(3):641-4 16(3):206-209.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 209

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI RASSF5ID42059ch1q32.txt

Identity Description The human gene for RASSF5 is 81 kb in length and Other names: MGC10823, MGC17344, Maxp1, is located on chromosome 1(q32.1). The gene can NORE1, NORE1A, NORE1B, RAPL, RASSF3 produce 4 protein isoforms, two via differential HGNC (Hugo): RASSF5 exon usage, a third via differential promoter usage th Location: 1q32.1 and the genesis of the 4 (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 referred to as a zinc Note finger. Next is the Ras association domain and this Murine RASSF5 originally named Nore1a. Nore1B is followed by sequence containing the SARAH independently identified and designated RAPL. Rat 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 DNA/RNA 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) 210

RASSF5 (Ras association (RalGDS/AF-6) domain family Schmidt L, Clark GJ member 5)

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) 211

RASSF5 (Ras association (RalGDS/AF-6) domain family Schmidt L, Clark GJ member 5)

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 6;273(10):5439-42 47-kDa protein which contains a proline-rich region Khokhlatchev A, Rabizadeh S, Xavier R, Nedwidek M, at the N-terminus followed by a putative Chen T, Zhang XF, Seed B, Avruch J. Identification of a diacylglycerol/phorbol ester binding domain. This novel Ras-regulated proapoptotic pathway. Curr Biol. 2002 is followed by the Ras association (RA) domain and Feb 19;12(4):253-65 then the domain containing a SARAH motif. This Chen J, Lui WO, Vos MD, Clark GJ, Takahashi M, later is responsible for binding to the pro-apoptotic Schoumans J, Khoo SK, Petillo D, Lavery T, Sugimura J, kinases MST1 and MST2. Astuti D, Zhang C, Kagawa S, Maher ER, Larsson C, Alberts AS, Kanayama HO, Teh BT. The t(1;3) breakpoint- Expression spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas. Cancer Cell. 2003 Nov;4(5):405- Nore1a mRNA is expressed in the lung, kidney, 13 liver, brain, spleen, thymus and heart. Hesson L, Dallol A, Minna JD, Maher ER, Latif F. Localisation NORE1A, a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers. Oncogene. 2003 It can be detected on microtubules, in the Feb 13;22(6):947-54 centrosome, but appears most obvious in the Vos MD, Martinez A, Ellis CA, Vallecorsa T, Clark GJ. The nucleus. pro-apoptotic Ras effector Nore1 may serve as a Ras- regulated tumor suppressor in the lung. J Biol Chem. 2003 Function Jun 13;278(24):21938-43 RASSF5A is a pro-apoptotic Ras effector that can Aoyama Y, Avruch J, Zhang XF. Nore1 inhibits tumor cell bind and relocalize the pro-apoptotic MST kinases growth independent of Ras or the MST1/2 kinases. in the presence of activated Ras. It can also promote Oncogene. 2004 Apr 22;23(19):3426-33 cell 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 RASSF5A promotes cellular proliferation and soft the growth inhibitory proteins, RASSF1 and NORE1, and agar growth. Thus, RASSF5A appears to function by Ras. Biochem J. 2004 Jul 15;381(Pt 2):453-62 as a Ras regulated tumor suppressor. Analysis of Avruch J, Praskova M, Ortiz-Vega S, Liu M, Zhang XF. human tumors has found little evidence of somatic Nore1 and RASSF1 regulation of cell proliferation and of mutation but the gene is frequently inactivated by the MST1/2 kinases. Methods Enzymol. 2006;407:290-310 promoter methylation in a broad range of human Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee tumors. RASSF5C (also known as Nore1b or JS, Factor VM, Thorgeirsson SS. Ubiquitous activation of RAPL) has been reported to modulate cellular Ras and Jak/Stat pathways in human HCC. Gastroenterology. 2006 Apr;130(4):1117-28 adhesion and to be regulated by the Ras related protein Rap1a. RASSF5C has also been implicated Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci. 2007 Sep 15;120(Pt 18):3163-72 as serving as an adaptor protein to facilitate the interaction of Ras and CARMA1. Kumari G, Singhal PK, Rao MR, Mahalingam S. Nuclear transport of Ras-associated tumor suppressor proteins: different transport receptor binding specificities for Mutations arginine-rich nuclear targeting signals. J Mol Biol. 2007 Apr 13;367(5):1294-311 Note No tumor mutations yet reported. Calvisi DF, Donninger H, Vos MD, Birrer MJ, Gordon L, Leaner V, Clark GJ. NORE1A tumor suppressor candidate modulates p21CIP1 via p53. Cancer Res. 2009 Jun Implicated in 1;69(11):4629-37 Richter AM, Pfeifer GP, Dammann RH. The RASSF Clear cell renal carcinoma proteins in cancer; from epigenetic silencing to functional Note characterization. Biochim Biophys Acta. 2009 RASSF5 is frequently down-regulated by promoter Dec;1796(2):114-28 methylation in a variety of tumors including clear Bee C, Moshnikova A, Mellor CD, Molloy JE, Koryakina Y, cell renal carcinomas. Moreover, a rare hereditary Stieglitz B, Khokhlatchev A, Herrmann C. Growth and tumor suppressor NORE1A is a regulatory node between form of kidney cancer has been reported that maps Ras signaling and microtubule nucleation. J Biol Chem. with a translocation inactivating the RASSF5 gene. 2010 May 21;285(21):16258-66 Various cancers Kumari G, Singhal PK, Suryaraja R, Mahalingam S. Functional interaction of the Ras effector RASSF5 with the Note tyrosine kinase Lck: critical role in nucleocytoplasmic Nore1a is frequently inactivated by promoter transport and cell cycle regulation. J Mol Biol. 2010 Mar methylation in renal carcinoma, breast cancer, lung 19;397(1):89-109 cancer, liver cancer and neurological tumors. Park J, Kang SI, Lee SY, Zhang XF, Kim MS, Beers LF, Lim DS, Avruch J, Kim HS, Lee SB. Tumor suppressor ras

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 212

RASSF5 (Ras association (RalGDS/AF-6) domain family Schmidt L, Clark GJ member 5)

association domain family 5 (RASSF5/NORE1) mediates This article should be referenced as such: death 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 Overmeyer JH, Maltese WA. Death pathways triggered by Cytogenet Oncol Haematol. 2012; 16(3):210-213. activated Ras in cancer cells. Front Biosci. 2011 Jan 1;16:1693-713

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 213

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI RGS17ID47522ch6q25.txt

Identity Description Other names: RGS-17, RGSZ2, hRGS17 The RGS17 protein consists of 210 amino acid residues. This gene encodes a member of the HGNC (Hugo): RGS17 regulator of G-protein signaling family. This Location: 6q25.2 protein contains a 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 Localisation DNA including 4 coding exons and 1 non-coding Its cellular localization has not been formally exon (exon 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 proteins by binding to activated, GTP-bound G 633 bases. alpha subunits and acting as a GTPase activating Pseudogene protein (GAP), increasing the rate of conversion of RGS17P1 regulator of G-protein signaling 17 the GTP to GDP. RGS proteins are GTPase- pseudogene 1. activating proteins for Gi and Gq class G-alpha proteins. They accelerate transit through the cycle Protein of GTP binding and hydrolysis and thereby accelerate signaling kinetics and termination. This Note hydrolysis allows the G alpha subunits to bind G 210 amino acids; 24 kDa. beta/gamma subunit heterodimers, 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) 214

RGS17 (regulator of G-protein signaling 17) Li C, et al.

Homology The RGS17 gene is conserved in chimpanzee, dog, Ovarian cancer cow, mouse, rat, chicken, and zebrafish. Disease RGS2, RGS5, RGS10 and RGS17 transcripts are Mutations expressed at significantly lower levels in cells Germinal resistant to chemotherapy compared with parental, chemo-sensitive cells in ovarian cancer cells No germline mutations in this gene have been (Hooks et al., 2010). reported. Prognosis Somatic RGS17 loss of expression contributes to the A synonymous-coding somatic mutations of this development of chemoresistance in ovarian cancer gene is reported in pancreas cancer at codon 166, cells. P166P (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- Note PKA-CREB pathway. Cancer Res. 2009 Mar 1;69(5):2108- 16 Lung cancer, prostate cancer. You M, Wang D, Liu P, Vikis H, James M, Lu Y, Wang Y, Disease Wang M, Chen Q, Jia D, Liu Y, Wen W, Yang P, et al. Fine RSG17 is overexpressed in lung and prostate cancer mapping of chromosome 6q23-25 region in familial lung (James et al., 2009). Expression of RGS17 is up- cancer families reveals RGS17 as a likely candidate gene. regulated in 80% of lung tumors, and also up- Clin Cancer Res. 2009 Apr 15;15(8):2666-74 regulated in prostate tumors. Overexpression of Hooks SB, Callihan P, Altman MK, Hurst JH, Ali MW, RGS17 induce and maintain cell proliferation. Murph MM. Regulators of G-Protein signaling RGS10 and RGS17 regulate chemoresistance in ovarian cancer cells. Lung cancer Mol Cancer. 2010 Nov 2;9:289 Disease Sun Y, Fang R, Li C, Li L, Li F, Ye X, Chen H. Hsa-mir-182 suppresses lung tumorigenesis through down regulation of hsa-mir-182 is involved in the down regulation of RGS17 expression in vitro. Biochem Biophys Res RGS17 expression through two conserved sites Commun. 2010 May 28;396(2):501-7 located in its 3' UTR region (Sun et al., 2010). Two SNPs in the first intron of RGS17 (rs4083914 This article should be referenced as such: and rs9479510) were found associated with familial Li C, Wang L, Sun Y, Chen H. RGS17 (regulator of G- lung cancer susceptibility (You et al., 2009). protein signaling 17). Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):214-215.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 215

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI SLC39A1ID46571ch1q21.txt

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 216

SLC39A1 (solute carrier family 39 (zinc transporter), member Franklin RB, Costello LC 1)

SLC39A1 expression decreases cellular zinc and Golovine K, Makhov P, Uzzo RG, Shaw T, Kunkle D, increases growth of PC-3 cells (Franklin et al., Kolenko VM. Overexpression of the zinc uptake transporter hZIP1 inhibits nuclear factor-kappaB and 2003). Over expression of SLC39A1 inhibits reduces the malignant potential of prostate cancer cells in prostate tumor growth in a xenograft model vitro and in vivo. Clin Cancer Res. 2008 Sep (Golovine et al., 2008). 1;14(17):5376-84 Makhov P, Golovine K, Uzzo RG, Wuestefeld T, Scoll BJ, References Kolenko VM. Transcriptional regulation of the major zinc uptake protein hZip1 in prostate cancer cells. Gene. 2009 Gaither LA, Eide DJ. The human ZIP1 transporter Feb 15;431(1-2):39-46 mediates zinc uptake in human K562 erythroleukemia cells. J Biol Chem. 2001 Jun 22;276(25):22258-64 Milon BC, Agyapong A, Bautista R, Costello LC, Franklin RB. Ras responsive element binding protein-1 (RREB-1) Franklin RB, Ma J, Zou J, Guan Z, Kukoyi BI, Feng P, down-regulates hZIP1 expression in prostate cancer cells. Costello LC. Human ZIP1 is a major zinc uptake Prostate. 2010 Feb 15;70(3):288-96 transporter for the accumulation of zinc in prostate cells. J Inorg Biochem. 2003 Aug 1;96(2-3):435-42 This article should be referenced as such: Franklin RB, Feng P, Milon B, Desouki MM, Singh KK, Franklin RB, Costello LC. SLC39A1 (solute carrier family Kajdacsy-Balla A, Bagasra O, Costello LC. hZIP1 zinc 39 (zinc transporter), member 1). Atlas Genet Cytogenet uptake transporter down regulation and zinc depletion in Oncol Haematol. 2012; 16(3):216-217. prostate cancer. Mol Cancer. 2005 Sep 9;4:32

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 217

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI CBX7ID43845ch22q13.txt

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

Note

251 amino acids. Isoelectric point: 10,0228. Molecular weight of the protein: 28209 Da.

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) 218

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, Figure 4. 4a: Summary of Cbx7's mechanism in giving CBX7 a role in maintaining the repression of embryonic stem cells (ESC). Cbx7 is essential for ESC self-renewal. Loss of Cbx7, either by differentiating ESC or genes in the X chromosome. by an exogenous/endogenous induction of the microRNA Epigenetic regulation (miR) families miR-125 and miR-181, induces ESC CBX7, as part of the PRC1 complex, has a role in differentiation. This is accompanied by an increase in other maintaining the repressive state of its target genes. Cbxs as they are targets of Cbx7. On the other hand, overexpression of Cbx7 in ESC reinforces pluripotency and CBX7 binds to the long non-coding RNA ANRIL keeps the cells in an ESC-like state when forced to in order to represses the INK4a/ARF locus and this differentiate. 4b and 4c: Summary of Cbx7's mechanism interaction is essential for CBX7's function. Both in human primary fibroblasts (IMR-90). Ectopic CBX7 and ANRIL have been found to have high expression of the miR families miR-125 and miR-181 induces a degradation of Cbx7 mRNA in IMR-90. Depletion levels in prostate cancer tissues. of Cbx7 induces the cells to senesce. Thus, overexpression Stem cells self-renewal of miR-125 and miR-181 induces senescence through CBX7 has been recently implicated to be essential downregulation of Cbx7. for maintaining the pluripotency state of stem cells (ES cells). Overexpression of CBX7 in ESC Mutations impairs cell differentation. On the other hand, Note depletion of CBX7 from ESC induces spontaneous differentiation. Two different miR families (miR- Expression of CBX7 without the Pc box or with 125 and miR-181) were identified in a screening for point mutations in the chromodomain region CBX7 regulators and have been described to have a (F11A, K31A, W32A, W35A) does not extend the role in ESC differentiation by targeting the 3'UTR life span of human or mouse cells. The mutant of CBX7. R17Q, which affects the binding of CBX7 to RNA,

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 219

CBX7 (chromobox homolog 7) O'Loghlen A, Gil J

extended the lifespan of cells, but to a lesser extent Gil J, Bernard D, Peters G. Role of polycomb group than CBX7 wt. Point mutations in the Pc box as proteins in stem cell self-renewal and cancer. DNA Cell Biol. 2005 Feb;24(2):117-25 F234D or F244D result in loss or reduced interaction of CBX7 with RNF2. Bernstein E, Duncan EM, Masui O, Gil J, Heard E, Allis CD. Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in Implicated in facultative heterochromatin. Mol Cell Biol. 2006 Apr;26(7):2560-9 Various cancers Scott CL, Gil J, Hernando E, Teruya-Feldstein J, Narita M, Disease Martínez D, Visakorpi T, Mu D, Cordon-Cardo C, Peters G, CBX7 has been implicated in several tumors such Beach D, Lowe SW. Role of the chromobox protein CBX7 in lymphomagenesis. Proc Natl Acad Sci U S A. 2007 Mar as gastric cancer, follicular lymphoma, breast 27;104(13):5389-94 cancer, colon carcinoma, pancreatic cancer, tyroid cancer, glioma. Pallante P, Federico A, Berlingieri MT, Bianco M, Ferraro A, Forzati F, Iaccarino A, Russo M, Pierantoni GM, Leone Prognosis V, Sacchetti S, Troncone G, Santoro M, Fusco A. Loss of There is a controversy in the role of CBX7 in the CBX7 gene expression correlates with a highly malignant phenotype in thyroid cancer. Cancer Res. 2008 cancer, as some papers associate CBX7 Aug 15;68(16):6770-8 overexpression with poor prognosis and advanced estate of the tumor and aggressiveness, while others Yap KL, Li S, Muñoz-Cabello AM, Raguz S, Zeng L, Mujtaba S, Gil J, Walsh MJ, Zhou MM. Molecular interplay state that depletion of CBX7 from certain cancers of the noncoding RNA ANRIL and methylated histone H3 indicates the state of malignancy of the tumor. The lysine 27 by polycomb CBX7 in transcriptional silencing of ability of CBX7 to regulate multiple targets and the INK4a. Mol Cell. 2010 Jun 11;38(5):662-74 relevance of those targets in different tumor types Morey L, Pascual G, Cozzuto L, Roma G, Wutz A, Benitah and stages probably explain those paradoxical SA, Di Croce L. Nonoverlapping functions of the polycomb findings. group cbx family of proteins in embryonic stem cells. Cell Stem Cell. 2012 Jan 6;10(1):47-62 References O'Loghlen A, Muñoz-Cabello AM, Gaspar-Maia A, Wu HA, Banito A, Kunowska N, Racek T, Pemberton HN, Beolchi Gil J, Bernard D, Martínez D, Beach D. Polycomb CBX7 P, Lavial F, Masui O, Vermeulen M, Carroll T, Graumann has a unifying role in cellular lifespan. Nat Cell Biol. 2004 J, Heard E, Dillon N, Azuara V, Snijders AP, Peters G, Jan;6(1):67-72 Bernstein E, Gil J. MicroRNA Regulation of Cbx7 Mediates a Switch of Polycomb Orthologs during ESC Bernard D, Martinez-Leal JF, Rizzo S, Martinez D, Hudson Differentiation. Cell Stem Cell. 2012 Jan 6;10(1):33-46 D, Visakorpi T, Peters G, Carnero A, Beach D, Gil J. CBX7 controls the growth of normal and tumor-derived prostate This article should be referenced as such: cells by repressing the Ink4a/Arf locus. Oncogene. 2005 Aug 25;24(36):5543-51 O'Loghlen A, Gil J. CBX7 (chromobox homolog 7). Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):218-220.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 220

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI RPRMID42082ch2q23.txt

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 221

RPRM (reprimo, TP53 dependent G2 arrest mediator Corvalan AH, TorresVA candidate)

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 Reprimo in human malignancies. Int J Cancer. 2005 Jul 1;115(4):503-10 common in pancreatic cell lines (91%) and in pancreatic adenocarcinomas (66%). Reprimo Hamilton JP, Sato F, Jin Z, Greenwald BD, Ito T, Mori Y, methylation is correlated with poor prognosis in a Paun BC, Kan T, Cheng Y, Wang S, Yang J, Abraham JM, Meltzer SJ. Reprimo methylation is a potential biomarker large series of resected pancreatic cancers. This fact of Barrett's-Associated esophageal neoplastic progression. raises the possibility that aberrant methylation of Clin Cancer Res. 2006 Nov 15;12(22):6637-42 Reprimo is an epigenetic event that may have a Sato N, Fukushima N, Matsubayashi H, Iacobuzio- mechanistic role in pancreatic cancer. Donahue CA, Yeo CJ, Goggins M. Aberrant methylation of Reprimo correlates with genetic instability and predicts References poor prognosis in pancreatic ductal adenocarcinoma. Cancer. 2006 Jul 15;107(2):251-7 Ohki R, Nemoto J, Murasawa H, Oda E, Inazawa J, Bernal C, Aguayo F, Villarroel C, Vargas M, Díaz I, Tanaka N, Taniguchi T. Reprimo, a new candidate Ossandon FJ, Santibáñez E, Palma M, Aravena E, mediator of the p53-mediated cell cycle arrest at the G2 Barrientos C, Corvalan AH. Reprimo as a potential phase. J Biol Chem. 2000 Jul 28;275(30):22627-30 biomarker for early detection in gastric cancer. Clin Cancer Sato N, Fukushima N, Maitra A, Matsubayashi H, Yeo CJ, Res. 2008 Oct 1;14(19):6264-9 Cameron JL, Hruban RH, Goggins M. Discovery of novel targets for aberrant methylation in pancreatic carcinoma This article should be referenced as such: using high-throughput microarrays. Cancer Res. 2003 Jul Corvalan AH, TorresVA. RPRM (reprimo, TP53 dependent 1;63(13):3735-42 G2 arrest mediator candidate). Atlas Genet Cytogenet Suzuki M, Shigematsu H, Takahashi T, Shivapurkar N, Oncol Haematol. 2012; 16(3):221-222. Sathyanarayana UG, Iizasa T, Fujisawa T, Gazdar AF. Aberrant methylation of Reprimo in lung cancer. Lung Cancer. 2005 Mar;47(3):309-14

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 222

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI VMP1ID50079ch17q23.txt

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

Genomic organization of the VMP1/TMEM49 gene.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 223

VMP1 (vacuole membrane protein 1) Ropolo A, et al.

Schematic representation of VMP1 protein and localization of transmembrane domains.

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 224

VMP1 (vacuole membrane protein 1) Ropolo A, et al.

References Dusetti NJ, Jiang Y, Vaccaro MI, Tomasini R, Azizi Samir A, Calvo EL, Ropolo A, Fiedler F, Mallo GV, Dagorn JC, Iovanna 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, Confocal microscopy of AR42J cell transfected with Vaccaro MI. The pancreatitis-induced vacuole membrane pEGFP-VMP1. protein 1 triggers autophagy in mammalian cells. J Biol Diabetes Chem. 2007 Dec 21;282(51):37124-33 Disease Vaccaro MI. Autophagy and pancreas disease. Pancreatology. 2008;8(4-5):425-9 Experimental diabetes activates VMP1 expression and autophagy in pancreas beta cells as a direct Vaccaro MI, Ropolo A, Grasso D, Iovanna JL. A novel mammalian trans-membrane protein reveals an alternative response to streptozotocin (STZ). VMP1 mRNA initiation pathway for autophagy. Autophagy. 2008 expression is activated after STZ treatment by islet Apr;4(3):388-90 beta cells. Electron microscopy shows chromatin Grasso D, Sacchetti ML, Bruno L, Lo Ré A, Iovanna JL, aggregation and autophagy morphology that was Gonzalez CD, Vaccaro MI. Autophagy and VMP1 confirmed by LC3 expression and LC3-VMP1 coexpression are early cellular events in experimental localization. Apoptotic cell death and the reduction diabetes. Pancreatology. 2009;9(1-2):81-8 of beta cell pool are evident after 24h treatment, Pardo R, Lo Ré A, Archange C, Ropolo A, Papademetrio while VMP1 is still expressed in the remaining DL, Gonzalez CD, Alvarez EM, Iovanna JL, Vaccaro MI. cells. VMP1-Beclin1 colocalization in pancreas Gemcitabine induces the VMP1-mediated autophagy pathway to promote apoptotic death in human pancreatic tissue from STZ-treated rats suggests that VMP1- cancer cells. Pancreatology. 2010;10(1):19-26 Beclin1 interaction is involved in the autophagic process activation during experimental diabetes. Grasso D, Ropolo A, Lo Ré A, Boggio V, Molejón MI, Iovanna JL, Gonzalez CD, Urrutia R, Vaccaro MI. Pancreas beta cells trigger VMP1 expression and Zymophagy, a novel selective autophagy pathway autophagy during the early cellular events in mediated by VMP1-USP9x-p62, prevents pancreatic cell response to experimental diabetes. death. J Biol Chem. 2011 Mar 11;286(10):8308-24

This article should be referenced as such:

Ropolo A, Lo Ré A, Vaccaro MI. VMP1 (vacuole membrane protein 1). Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):223-225.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 225

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI XPO1ID44168ch2p15.txt

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 226

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 nuclear export factor (reviewed by Fried and Kutaj, 2003; Hutten and Kehlenbach, 2007): it interacts XPO1/CRM1 protein levels remain constant with various classes of RNAs and with proteins throughout the cell cycle (Kudo et al., 1997). carrying nuclear export signals (NES) (Fornerod et Localisation al., 1997c; Fukuda et al., 1997; Ossareh-Nazari et Due to its function as a shuttling nuclear transport al., 1997), short aminoacidic stretches harbouring receptor between the nucleus and cytoplasm, the hydrophobic residues (general consensus LX(2- human XPO1/CRM1 protein is preferentially 3)ΦX(2-3)LXΦ, where can be L, I, M or F), present localized at the nuclear envelope in interphase cells in many shuttling proteins of cellular or viral origin, (Kudo et al., 1997; Fornerod et al., 1997b). In the and transports these molecules out of the nucleus nucleus it can be detected in specific bodies called through nuclear pore complexes in a manner CRM1 nucleolar bodies (CNoBs). CNoBs depend dependent on the GTPase RAN. The protein is on RNA polymerase I activity, suggesting a role in therefore alternatively called either exportin-1 or ribosome biogenesis (Ernoult-Lange et al., 2009). XPO1, based on its function, or hCRM1, based on In mitotic cells, a fraction of XPO1 is found at evolutionary conservation. centrosomes (Forgues et al., 2003; Wang et al., Regulated export of some shuttling proteins (e.g., 2005) and a substantial fraction localizes to the p53, p27, STAT, NF-kB and many viral proteins) kinetochores (Arnaoutov et al., 2005). out of the nucleus is essential for regulated cell cycle and cell proliferation (reviewed by Fabbro Function and Henderson, 2003; Rensen et al., 2008). This has hCRM1 was found to interact stably in complexes lead some authors to view nuclear export as a containing not only NUP214/CAN (or its promising target process in cancer therapy derivatives), but also another component of nuclear (reviewed by Yashiroda and Yoshida, 2003; Turner pores, the nucleoporin NUP88 (Fornerod et al., and Sullivan, 2008). 1997b). These interactions hinted at a possible role Recent findings have revealed additional roles of of hCRM1 in nucleocytoplasmic transport. Further XPO1/CRM1 in mitosis: first, an XPO1/CRM1 studies indeed demonstrated that hCRM1 acts as a

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 227

XPO1 (exportin 1 (CRM1 homolog, yeast)) Ruggiero A, et al.

fraction regulates the localisation of nucleophosmin CRM1 may be causal to COX-2 up-regulation, with (NPM/B23), a regulator of centrosome duplication. direct clinical relevance. XPO1/CRM1 is required to prevent centrosome Oncogenesis overduplication and the formation of multipolar CRM1 is highly expressed in ovarian carcinomas spindles (reviewed by Budhu and Wang, 2005; tissues and regulates export of COX-2. Ciciarello and Lavia, 2005). Second, a kinetochore- associated fraction of XPO1/CRM1 regulates the Osteosarcoma (Yao et al., 2009) assembly of the so-called k-fibers, bundles of Prognosis microtubules that stably connect the spindle poles The CRM1 protein is reported to be expressed with to the kinetochores of mitotic chromosomes to increased abundance in osteosarcoma compared to ensure proper chromosome segregation (reviewed non-tumour tissues (P= 0.037, 57 patients). High by Arnaoutov and Dasso, 2005; Ciciarello and levels of CRM1 were significantly associated with Lavia, 2005; Dasso, 2006). Third, XPO1/CRM1 increased serum levels of alkaline phosphatase regulates survivin, a member of the chromosomal (ALP, P= 0.001). In univariate analysis, a passenger complex with roles in chromosome significant association between CRM1 expression segregation and apoptosis (reviewed by Knauer et and tumor size (P= 0.014), as well as histological al., 2007). grade (P= 0.003) was observed. In Kaplan-Meier In synthesis, XPO1/CRM1 acts in control of cell survival analysis, high CRM1 expression was a proliferation, and affects loss of proliferation significant prognostic indicator for poor control in cancer cells, through several pathways: 1. progression-free survival (P= 0.016) as well as as a nuclear export factor, it directly regulates the overall survival (P= 0.008). Multivariate analysis subcellular localisation, and hence the activity, of demonstrated that high expression of CRM1 was oncogenes and tumour suppressor proteins that significantly related to shorter survival (95% CI, contain nuclear export sequences; 2. it acts in 1.27-5.39). control of the mitotic apparatus and chromosome Oncogenesis segregation; 3. it influences the maintenance of CRM1 is significantly increased in osteosarcoma nuclear and chromosome structure. compared with normal tissue. Homology Cervical cancer (van der Watt et al., 2009) The human protein originally named CC112 Oncogenesis showed homology to the Schizosaccharomyces pombe CRM1 protein, first identified for being CRM1 protein abundance is significantly increased implicated in the control of higher order in cervical cancer cells compared with normal chromosome structure: mutation of the coding gene tissue (P< 0.05). Inhibition of CRM1 by RNA was associated with the appearance of "deformed interference resulted in increased cell death, nuclear chromosome domains" in fission yeast associated with nuclear retention of p53, likely conditional mutant strains. The gene product was protecting p53 from degradation as the latter therefore named CRM1 (chromosome region predominantly occurs in the cytoplasm. maintenance 1; Adachi and Yanagida, 1989). Based Pancreas cancer (Huang et al., 2009) on this homology, the human protein name of Prognosis CC112 was abandoned and the name hCRM1 was Increased expression abundance of CRM1 protein used. was detected in pancreatic cancer tissues (P= 0.0013, 69 patients at stages I and II). CRM1 Implicated in expression correlates with increased levels of serum Ovarian cancer (Noske et al., 2008) CEA (P= 0.002) and CA19.9 (P= 0.005), tumour size (P= 0.011), lymphadenopathy (P= 0.004) and Prognosis metastasis (P= 0.0041). High CRM1 expression Increased nuclear (52.7%) and cytoplasmic (56.8%) was a prognostic indicator for progression-free expression of CRM1 were reported observed in survival (PFS) (P= 0.0011) as well as overall carcinomas compared with borderline tumors and survival (OS) (P= 0.004). The authors proposed that benign lesions. Cytoplasmic CRM1 expression CRM1 be used as a prognostic parameter for poor significantly correlated with advanced tumor stage PFS and OS (95% CI, 1.27-5.39). (P= 0.043), poorly differentiated carcinomas (P= 0.011) and high mitotic rate (P= 0.008). Nuclear Glioma (Shen et al., 2009) CRM1 was significantly associated with high Prognosis cyclooxygenase-2 (COX-2) expression (P= 0.002) CRM1 overexpression is significantly associated and poor overall survival (P= 0.01). CRM1 was with the pathological state (P= 0.001, 56 patients), previously directly implicated in nuclear export of with glioma tumour grade, with high expression of COX-2 (Jang et al., 2003). The study by Noske et phospho-ser10p27 and with reduced overall al. (2008) suggests that elevated expression of abundance of p27.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 228

XPO1 (exportin 1 (CRM1 homolog, yeast)) Ruggiero A, et al.

Oncogenesis Wolff B, Sanglier JJ, Wang Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic Given the direct implication of CRM1 in nuclear translocation of the human immunodeficiency virus type 1 export of p27, the data in this study suggest that (HIV-1) Rev protein and Rev-dependent mRNA. Chem increased CRM1 abundance yields increased Biol. 1997 Feb;4(2):139-47 cytoplasmic localisation of p27, which is probably Fabbro M, Henderson BR. Regulation of tumor targeted to degradation, leading to uncontrolled suppressors by nuclear-cytoplasmic shuttling. Exp Cell tumour growth. Phospho-ser10p27 may be resistant Res. 2003 Jan 15;282(2):59-69 to CRM1-mediated nuclear export. High CRM1 Forgues M, Difilippantonio MJ, Linke SP, Ried T, and low p27 expression are associated with high Nagashima K, Feden J, Valerie K, Fukasawa K, Wang grade glioma and high CRM1 protein expression is XW. Involvement of Crm1 in hepatitis B virus X protein- proposed as a prognostic factor of overall survival induced aberrant centriole replication and abnormal mitotic spindles. Mol Cell Biol. 2003 Aug;23(15):5282-92 and poor outcome. Fried H, Kutay U. Nucleocytoplasmic transport: taking an To be noted inventory. Cell Mol Life Sci. 2003 Aug;60(8):1659-88 Jang BC, Muñoz-Najar U, Paik JH, Claffey K, Yoshida M, Note Hla T. Leptomycin B, an inhibitor of the nuclear export CRM1 protein levels are abnormally high in several receptor CRM1, inhibits COX-2 expression. J Biol Chem. 2003 Jan 31;278(5):2773-6 cancers, with high levels of CRM1 being associated with poor patient survival (van der Watt and Yashiroda Y, Yoshida M. Nucleo-cytoplasmic transport of Leaner, 2011). proteins as a target for therapeutic drugs. Curr Med Chem. 2003 May;10(9):741-8 References Petosa C, Schoehn G, Askjaer P, Bauer U, Moulin M, Steuerwald U, Soler-López M, Baudin F, Mattaj IW, Müller Adachi Y, Yanagida M. Higher order chromosome CW. Architecture of CRM1/Exportin1 suggests how structure is affected by cold-sensitive mutations in a cooperativity is achieved during formation of a nuclear Schizosaccharomyces pombe gene crm1+ which encodes export complex. Mol Cell. 2004 Dec 3;16(5):761-75 a 115-kD protein preferentially localized in the nucleus and Arnaoutov A, Azuma Y, Ribbeck K, Joseph J, Boyarchuk its periphery. J Cell Biol. 1989 Apr;108(4):1195-207 Y, Karpova T, McNally J, Dasso M. Crm1 is a mitotic von Lindern M, Fornerod M, van Baal S, Jaegle M, de Wit effector of Ran-GTP in somatic cells. Nat Cell Biol. 2005 T, Buijs A, Grosveld G. The translocation (6;9), associated Jun;7(6):626-32 with a specific subtype of acute myeloid leukemia, results Arnaoutov A, Dasso M. Ran-GTP regulates kinetochore in the fusion of two genes, dek and can, and the attachment in somatic cells. Cell Cycle. 2005 expression of a chimeric, leukemia-specific dek-can Sep;4(9):1161-5 mRNA. Mol Cell Biol. 1992 Apr;12(4):1687-97 Budhu AS, Wang XW. Loading and unloading: Fornerod M, Boer J, van Baal S, Morreau H, Grosveld G. orchestrating centrosome duplication and spindle Interaction of cellular proteins with the leukemia specific assembly by Ran/Crm1. Cell Cycle. 2005 Nov;4(11):1510- fusion proteins DEK-CAN and SET-CAN and their normal 4 counterpart, the nucleoporin CAN. Oncogene. 1996 Oct 17;13(8):1801-8 Ciciarello M, Lavia P. New CRIME plots. Ran and transport factors regulate mitosis. EMBO Rep. 2005 Aug;6(8):714-6 Fornerod M, van Baal S, Valentine V, Shapiro DN, Grosveld G. Chromosomal localization of genes encoding Wang W, Budhu A, Forgues M, Wang XW. Temporal and CAN/Nup214-interacting proteins--human CRM1 localizes spatial control of nucleophosmin by the Ran-Crm1 to 2p16, whereas Nup88 localizes to 17p13 and is complex in centrosome duplication. Nat Cell Biol. 2005 physically linked to SF2p32. Genomics. 1997a Jun Aug;7(8):823-30 15;42(3):538-40 Dasso M. Ran at kinetochores. Biochem Soc Trans. 2006 Fornerod M, van Deursen J, van Baal S, Reynolds A, Nov;34(Pt 5):711-5 Davis D, Murti KG, Fransen J, Grosveld G. The human homologue of yeast CRM1 is in a dynamic subcomplex Hutten S, Kehlenbach RH. CRM1-mediated nuclear with CAN/Nup214 and a novel nuclear pore component export: to the pore and beyond. Trends Cell Biol. 2007 Nup88. EMBO J. 1997b Feb 17;16(4):807-16 Apr;17(4):193-201 Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an Knauer SK, Mann W, Stauber RH. Survivin's dual role: an export receptor for leucine-rich nuclear export signals. Cell. export's view. Cell Cycle. 2007 Mar 1;6(5):518-21 1997c Sep 19;90(6):1051-60 Noske A, Weichert W, Niesporek S, Röske A, Buckendahl Fukuda M, Asano S, Nakamura T, Adachi M, Yoshida M, AC, Koch I, Sehouli J, Dietel M, Denkert C. Expression of Yanagida M, Nishida E. CRM1 is responsible for the nuclear export protein chromosomal region intracellular transport mediated by the nuclear export maintenance/exportin 1/Xpo1 is a prognostic factor in signal. Nature. 1997 Nov 20;390(6657):308-11 human ovarian cancer. Cancer. 2008 Apr 15;112(8):1733- 43 Kudo N, Khochbin S, Nishi K, Kitano K, Yanagida M, Yoshida M, Horinouchi S. Molecular cloning and cell cycle- Rensen WM, Mangiacasale R, Ciciarello M, Lavia P. The dependent expression of mammalian CRM1, a protein GTPase Ran: regulation of cell life and potential roles in involved in nuclear export of proteins. J Biol Chem. 1997 cell transformation. Front Biosci. 2008 May 1;13:4097-121 Nov 21;272(47):29742-51 Turner JG, Sullivan DM. CRM1-mediated nuclear export of Ossareh-Nazari B, Bachelerie F, Dargemont C. Evidence proteins and drug resistance in cancer. Curr Med Chem. for a role of CRM1 in signal-mediated nuclear protein 2008;15(26):2648-55 export. Science. 1997 Oct 3;278(5335):141-4

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 229

XPO1 (exportin 1 (CRM1 homolog, yeast)) Ruggiero A, et al.

Ernoult-Lange M, Wilczynska A, Harper M, Aigueperse C, Yao Y, Dong Y, Lin F, Zhao H, Shen Z, Chen P, Sun YJ, Dautry F, Kress M, Weil D. Nucleocytoplasmic traffic of Tang LN, Zheng SE. The expression of CRM1 is CPEB1 and accumulation in Crm1 nucleolar bodies. Mol associated with prognosis in human osteosarcoma. Oncol Biol Cell. 2009 Jan;20(1):176-87 Rep. 2009 Jan;21(1):229-35 Huang WY, Yue L, Qiu WS, Wang LW, Zhou XH, Sun YJ. Güttler T, Madl T, Neumann P, Deichsel D, Corsini L, Prognostic value of CRM1 in pancreas cancer. Clin Invest Monecke T, Ficner R, Sattler M, Görlich D. NES Med. 2009 Dec 1;32(6):E315 consensus redefined by structures of PKI-type and Rev- type nuclear export signals bound to CRM1. Nat Struct Mol Shen A, Wang Y, Zhao Y, Zou L, Sun L, Cheng C. Biol. 2010 Nov;17(11):1367-76 Expression of CRM1 in human gliomas and its significance in p27 expression and clinical prognosis. Neurosurgery. van der Watt PJ, Leaner VD. The nuclear exporter, Crm1, 2009 Jul;65(1):153-9; discussion 159-60 is regulated by NFY and Sp1 in cancer cells and repressed by p53 in response to DNA damage. Biochim Biophys van der Watt PJ, Maske CP, Hendricks DT, Parker MI, Acta. 2011 Jul;1809(7):316-26 Denny L, Govender D, Birrer MJ, Leaner VD. The Karyopherin proteins, Crm1 and Karyopherin beta1, are This article should be referenced as such: overexpressed in cervical cancer and are critical for cancer cell survival and proliferation. Int J Cancer. 2009 Apr Ruggiero A, Giubettini M, Lavia P. XPO1 (exportin 1 15;124(8):1829-40 (CRM1 homolog, yeast)). Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):226-230.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 230

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t1118p15q12ID1466.txt

SETBP1 forms a complex with SET and PP2A Clinics and pathology (protein phosphatase 2A). SETBP1 impairs PP2A Disease activity via SET and promotes proliferation of acute myeloid leukemia cells (Cristóbal et al., 2010). T-cell acute lymphoid leukemia (T-ALL) Germinal mutations Epidemiology In Schinzel-Giedion midface retraction syndrome. One case to date, a 9-year-old boy. Evolution Result of the chromosomal Remission was obtained and the patient remains in anomaly complete remission 28 months after diagnosis. Hybrid gene Cytogenetics Description 5' NUP98 - 3' SETBP1 Cytogenetics morphological Transcript The translocation was accompanied with a del(12p). Exon 12 of NUP98 (nucleotide (nt) 1552) fused in- Genes involved and frame with exon 5 of SETBP1 (nt 4015). proteins References Panagopoulos I, Kerndrup G, Carlsen N, Strömbeck B, NUP98 Isaksson M, Johansson B. Fusion of NUP98 and the SET Location binding protein 1 (SETBP1) gene in a paediatric acute T cell lymphoblastic leukaemia with t(11;18)(p15;q12). Br J 11p15.4 Haematol. 2007 Jan;136(2):294-6 Protein Cristóbal I, Blanco FJ, Garcia-Orti L, Marcotegui N, Nucleoporin: associated with the nuclear pore Vicente C, Rifon J, Novo FJ, Bandres E, Calasanz MJ, complex. Role in nucleocytoplasmic transport Bernabeu C, Odero MD. SETBP1 overexpression is a processes. novel leukemogenic mechanism that predicts adverse outcome in elderly patients with acute myeloid leukemia. SETBP1 Blood. 2010 Jan 21;115(3):615-25 Location This article should be referenced as such: 18q12.3 Huret JL. t(11;18)(p15;q12). Atlas Genet Cytogenet Oncol Protein Haematol. 2012; 16(3): 231. Contains 3 DNA binding domains (A.T hooks). SETBP1 protects SET from protease cleavage.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 231

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t1121q21q22ID1592.txt

whereas paxillin siRNA inhibited cell adhesion. Identity Strongly expressed in hematopoietic cells. LPXN is Note involved in bone resorption and stimulates prostate This translocation is different from the cancer cell migration (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 Transcript proteins Two in frame fusion transcripts -fusion of exon 5 or 6 of RUNX1 to LPXN exon 8. LPXN Fusion protein Protein Description LPXN contains two types of protein-protein The two variant fusion proteins RUNX1-LPXN interaction domains: leucine-aspartate (LD) repeats localized in the nucleus and inhibited RUNX1 in N-term, and LIM (Lin-11 Isl-1 Mec-3) domains transactivation (Dai et al., 2009). It is hypothesized at the C-term. Belongs to the paxillin family (PXN, that the reciprocal LPXN-RUNX1 may also play a LPXN, TGFB1I1). Protein involved in focal role in leukemogenesis. adhesion. LPXN and paxillin had opposite roles in adhesion to collagen LPXN siRNA stimulated

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 232 t(11;21)(q21;q22) Huret JL

Chen PW, Kroog GS.. Leupaxin is similar to paxillin in References focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading. Cell Adh Dai H, Xue Y, Pan J, Wu Y, Wang Y, Shen J, Zhang J.. Migr. 2010 Oct-Dec;4(4):527-40. Two novel translocations disrupt the RUNX1 gene in acute myeloid leukemia. Cancer Genet Cytogenet. 2007 This article should be referenced as such: Sep;177(2):120-4. Huret JL. t(11;21)(q21;q22). Atlas Genet Cytogenet Oncol Dai HP, Xue YQ, Zhou JW, Li AP, Wu YF, Pan JL, Wang Haematol. 2012; 16(3):232-233. Y, Zhang J.. LPXN, a member of the paxillin superfamily, is fused 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.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 233

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t0817q24q22ID1494.txt

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 234 t(8;17)(q24;q22) ???BCL3/MYC Huret JL

van Riggelen J, Yetil A, Felsher DW. MYC as a regulator References of ribosome biogenesis and protein synthesis. Nat Rev Cancer. 2010 Apr;10(4):301-9 Gauwerky CE, Huebner K, Isobe M, Nowell PC, Croce CM. Activation of MYC in a masked t(8;17) translocation Kreisel D, Sugimoto S, Tietjens J, Zhu J, Yamamoto S, results in an aggressive B-cell leukemia. Proc Natl Acad Krupnick AS, Carmody RJ, Gelman AE. Bcl3 prevents Sci U S A. 1989 Nov;86(22):8867-71 acute inflammatory lung injury in mice by restraining emergency granulopoiesis. J Clin Invest. 2011 Jan Dechend R, Hirano F, Lehmann K, Heissmeyer V, Ansieau 4;121(1):265-76 S, Wulczyn FG, Scheidereit C, Leutz A. The Bcl-3 oncoprotein acts as a bridging factor between NF- This article should be referenced as such: kappaB/Rel and nuclear co-regulators. Oncogene. 1999 Jun 3;18(22):3316-23 Huret JL. t(8;17)(q24;q22) ???BCL3/MYC. Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):234-235.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 235

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI PluripotencyID20103.txt

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, anti-cancer 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.

their tails after amputation. But until the 18th Historical perspective century this knowledge was mainly anecdotic, and The search for the capacity of regenerating disease- only with the arrival of the Age of Enlightenment, affected organs is probably as old as mankind regeneration and plasticity will become the matter (Odelberg, 2004). The examples are abundant in of scientific research. In 1712, Réaumur describes ancient religions, from the Egyptian god Osiris, the regeneration of the limbs and claws of crayfish who resurrected and recomposed his maimed body (Réaumur, 1712); in 1744, Trembley discovers that from the pieces that had been thrown into the Nile, a part of the Hydra polyp can regenerate the to the legendary Hydra that could regenerate its complete organism (Trembley, 1744); in 1769, severed heads. Or the mythological Prometheus, Spallanzani reports that tadpoles can regenerate who had his viscera eaten by an eagle every day, their tails and salamanders their amputated jaws, only to regenerate them again. But also from a more limbs and tails (Spallanzani, 1769). The research scientific point of view, it was already noticed by performed during most of the 19th and first half of Aristotle (384-322 BC) that lizards can regenerate

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 236

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

the 20th centuries showed that, for regeneration to In a parallel way, the search for the molecular occur, the cells that are normally forming part of regulators responsible for establishing and the organs are not sufficient, and a special type of controlling cellular identity led finally to the cells are required: the progenitor cells (Odelberg, identification of the factors capable of 2004; Birnbaum and Sánchez Alvarado, 2008). The reprogramming cellular fate. In 1987, it was shown origin of these cells was not very clear (and it is that ectopic expression of the Antennapedia still a matter of debate and intense research, in fact, homeotic gene lead to changes in the body plan of see (Sánchez Alvarado, 2000; Kragl et al., 2009; Drosophila, that got extra legs instead of antennae Rinkevich et al., 2011)); for some tissues, like skin, (Schneuwly et al., 1987). Also, Gehring et al. blood, muscles or bones, progenitors were shown to showed that the ectopic expression of eyeless exist in the tissues in small numbers, and to become controlled eye development and led to the activated as a consequence of the lesions. In other development of ectopic eyes in the fly's legs cases, the progenitors seemed to arise from mature (Gehring, 1996). In mammals, the first master cells that become dedifferentiated. The best regulator factor to be identified was MyoD, which example supporting this possibility has been was shown to be capable of transdifferentiating described in primitive vertebrates like the urodeles fibroblasts into the myogenic lineage (Davis et al., (e.g. salamanders and axolotls). In these animals, 1987). Other examples of factors with fate- after a wound harms the organism, the cells from reprogramming capacity are C/EBPα, capable of the normal tissues form a group of cells known as mediating the transdifferentiation of mouse B cells the regenerative blastema, which will generate all into macrophages (Xie et al., 2004) or Pax5, whose the tissues in the new limb/tail (Chalkey, 1954; loss leads to the dedifferentiation of committed B Bodemer and Everett, 1959; Hay and Fischman, cells (Nutt et al., 1999; Cobaleda et al., 2007a; 1961). It has long been held that the blastema was Cobaleda and Busslinger, 2008). All these data the result of cellular dedifferentiation to proved that the lack or excess of just one factor progenitors. However, the most recent findings could lead to a radical alteration of the seem to indicate that there is no cellular transcriptional profile and could cause stable fate dedifferentiation to progenitors involved in this changes. This evidence, together with the one process, and the regeneration is always due to the coming from reprogramming by nuclear action of resident tissue-specific stem cells and transplantation, paved the way to the search for the progenitors, thus questioning the role of mature factors capable of reprogramming to full cellular plasticity in tissue regeneration (Kragl et pluripotency that led, in 2006, to the identification al., 2009; Rinkevich et al., 2011). We have of the four transcription factors capable of inducing therefore seen how the study of "naturally" pluripotency in virtually every kind of terminally occurring regeneration opened the way to a new differentiated cells (Takahashi and Yamanaka, understanding of the stem cell-based architecture of 2006). We will discuss this aspect with more detail the organs and tissues, especially with the study of afterwards. primitive vertebrates. In 1952, amphibians also On the other side, cancer has also been recognized provided the first animal model of experimentally- as a distinct pathological entity since the origins of induced reprogramming when Briggs and King mankind. The first references are the Edwin Smith generated Xenopus tadpoles by transplanting the and Ebers papyri from the 1600 BC and 1500 BC, nucleus of cells from the blastula into oocytes, approximately (Hajdu, 2004). The Edwin Smith therefore reverting the cellular differentiation papyrus contains the first mention and description program (Briggs and King, 1952). Afterwards, it of breast cancer, and it concludes that there is no was shown that more differentiated cells, like those treatment for the disease. Cancer was not so from the intestinal epithelia, could also be common in ancient times, mainly because life span reprogrammed by nuclear transfer (Gurdon, 1962). was much shorter, but it was already clearly These landmark findings undoubtedly showed that recognized. Hippocrates (460-375 BC) realized that the genetic potential of cells was not lost during growing tumors occurred typically in adults and differentiation, and that development did not imply they reminded him of a moving crab, which led to genetic changes. This principle was extended to the terms carcinos and cancer. Celsus (25 BC-AD mammals with the cloning of Dolly the sheep in 50) also compared cancer with a crab, because it 1997 (Wilmut et al., 1997). This was the ultimate penetrates the surrounding organs like if it had proof showing that the changes that occur during claws; Celsus introduced the first classification for differentiation are totally reversible, and breast cancer and advocated for surgical therapy. demonstrated that the fate restrictions that take Furthermore, he already realized that tumors could place during development are the result of only be cured if they were removed in their early epigenetic modifications. These studies also stages and that, even after removal and wound showed that there were factors in the oocyte healing, breast carcinomas tended to recur causing cytoplasm capable of inducing a reprogramming swelling in the armpit and, finally, death by that led to the appearance of a totipotent phenotype. spreading throughout the body. Galen (131-AD

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 237

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

200) already recommended surgery by cutting a mechanisms regulating cellular identity and wide margin of healthy tissue around the edges of plasticity play an essential role in allowing cancers the tumor (Hajdu, 2004). If we jump now to our to arise and hopefully, as we will discuss, they days, it seems disappointing to see how little those might be the key to its eradication. old critical findings have been overcome by modern The specification of cellular identity during medicine, 2000 years later. Indeed, still today, clean development and differentiation is a dynamic surgical margins and lack of lymph node invasion process that starts with stem and progenitor cells are the most important prognostic markers for the and ends with terminal differentiation into each successful eradication of solid tumors, and only if specialized cellular type. In this progression there tumors are completely resected before they can be many cellular intermediates; some of them metastasize (something that it is anyhow impossible are transient, and some can be long-lasting, but the to determine with current technologies) can maintenance of cellular identity at each stage is curation be guaranteed. However, in the last thirty determined by the signals from the environment years we have gained an enormous knowledge and, in an intrinsic manner, by specific transcription about the molecular biology of the disease. In 1979, factors and epigenetic modifiers that establish a it was shown that the phenotype of transformed defined chromatin architecture and a specific gene cells could be transferred to normal fibroblasts by expression profile. DNA transfection (Shih et al., 1979), a finding that As we have seen, evidences about cellular plasticity lead to the rapid molecular cloning of the first had being accumulating for decades (Hochedlinger human oncogene (the RAS gene), simultaneously by and Jaenisch, 2006; Gurdon and Melton, 2008; Graf several groups (Goldfarb et al., 1982; Lane et al., and Enver, 2009; Vicente-Dueñas et al., 2009a), but 1982; Parada et al., 1982; Santos et al., 1982). Since the latest findings in the field of reprogramming then, many genes have been described as being have definitively shown how switching to a either oncogenes or tumor suppressors, and the different phenotype can be a lot easier than molecular mechanisms of their transforming previously expected, and can have real capabilities have been analyzed to great detail, in physiological relevance, beyond basic research. close relationship with their functions in "normal" Cancer is a perfect example of pathological conditions. This is a field that has expanded reprogramming in which, from a normal tissue, a tremendously in the last decades, and a whole new differentiation lineage is opened with its comprehensive study of the topic falls out of the own hierarchy and structure (Reya et al., 2001; scope of this revision. However, there are some Sánchez-García et al., 2007). So, without forgetting aspects that must be taken into account for posterior the so well-studied aberrant proliferation, debate. A very important one is the fact that, for reprogramming is an essential part of the many types of tumors, specific genetic mutations tumorigenesis process, and it is closely dependent have been shown to correlate closely with the on the cellular plasticity of the cancer-initiating phenotype of the tumors, suggesting that the cells. The term plasticity, as we will use it here, oncogenic alterations might be acting as new refers to the ability of cells (stem or differentiated) specification factors that determine the tumor to adopt the biological properties (gene expression appearance and/or phenotype. This association is profile, phenotype, etc.) of other differentiated especially evident in the case of mesenchymal types of cells (belonging to the same or different tumors caused by chromosomal aberrations lineages). This definition comprises also the (Sánchez-García, 1997; Cobaleda et al., 1998). In property of competence, i.e. the ability of stem cells 2000, Hanahan and Weinberg summarized the main and progenitors to give rise to their different features that had to be disrupted in normal cellular descendant lineages during normal development. behavior in order for allow a tumor to appear and We use such an ample definition of the term progress (Hanahan and Weinberg, 2000), and this precisely to reflect the fact that the molecular list has expanded with the years (Hanahan and mechanisms that are important for progenitors' Weinberg, 2011). These main aspects are related competence during normal development are the with the survival and proliferation of cancer cells, same ones responsible for the plasticity changes of but it must be noted that most of them are equally more differentiated types of cells, both in shared by non-malignant tumors (Lazebnik, 2010). pathological processes and in experimentally- However, all the aspects related to the alterations of induced reprogramming. Here we will discuss the the normal developmental regulatory mechanisms vital role of cellular plasticity in the origin and in tumorigenesis have received much less attention. maintenance of tumoral cells. We will first revise But in fact, if cellular fate was carved into stone, the latest research discoveries in the fields of cancer would be impossible, since no new lineages normal developmental and experimentally-induced could be generated other than the normal, plasticity, and afterwards will link these findings physiologic ones. Here is where the normal with what we know about cancer biology.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 238

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

Lineage commitment and cellular represented by stable "valleys" and differentiation routes towards them are "channels" through which identity the cells move (Enver et al., 2009; Huang, 2009). Adult stem cells are the responsible of generating Under this light, pluripotency can be considered as all the different specialized cellular types forming a dynamic state of controlled heterogeneity within a the organism. The majority of them perform this population, where small individual fluctuations in job throughout the whole life of the organism, the levels of expression of transcription factors and thanks to their self-renewal capacity. This property epigenetic regulators maintain a global status of allows them to divide asymmetrically, therefore apparent stability. The cells that approach the limits given rise to a new identical daughter stem cell and of the attractor (those who, in their random to a multipotential progenitor, lacking self-renewal fluctuations, go too far from the middle point of the capacity, which will give rise to all the Gaussian curve) are therefore more prone to differentiated tissue cells. Although it is known that differentiate, suggesting that commitment, although there are some specific factors that are essential for rare, is an spontaneous phenomenon (unless it is the specification and maintenance of stem cell specifically triggered by an external signal that identity (Boyer et al., 2005), the molecular bases of unbalances the dynamic equilibrium) (Huang, the choice that stem cells have to make between 2009). maintaining competence (i.e. plasticity) or entering into the differentiation programs are not yet Maintenance of cellular identity completely understood (Niakan et al., 2010). In this throughout the differenciation context, a first important aspect to consider is the process fact that the stem cell population itself is Although in some rare cases they are unipotent (e.g. intrinsically heterogeneous. This means that the spermatogonial stem cells), adult stem cells are "stemness" is not a static condition defined by usually multipotent, and they can give rise to a wide stable, constant levels of expression of intrinsic range of differentiated cell types. In the first stem factors and surface stem markers, but it is instance, stem cells lose their self-renewal potential more of a continuum that moves within certain (their stemness) and start the differentiation process margins. For example, in a clonal population of by becoming multipotential progenitors. We have haematopoietic progenitor cells, there is a Gaussian seen that the differentiation program can be pre-set distribution of the levels of expression of Sca-1, already by the oscillatory patterns of gene one of the most classical stem cell markers (Chang expression at the stem cell population level, and et al., 2008). Furthermore, cells at both the low- or that cells lying at the different ends of specific high-end levels of expression can, when isolated, gradients of gene expression can have opposite regenerate the whole population with all the range differentiation preferences (Chang et al., 2008). So, of expression levels. However, every one of these once they leave the stem cell state, the cells start sub-populations, defined by their levels of a surface making lineage choices that are usually mutually marker, also expresses different transcriptomes, and excluding and are normally conceptualized in a has therefore distinct intrinsic differentiation branching pattern. These alternative options are tendencies towards different lineages. Therefore, usually controlled by the cross-antagonism between each individual cell in the stem population transcription factors with competing, opposing represents a metastable transitional point in a functions (Swiers et al., 2006; Loose et al., 2007). continuum of constantly changing transcriptomes. A very well characterized developmental system is In fact, this is most probably the mechanism at the hematopoietic differentiation where several models basis of the stochastic choice of lineage, when some of lineage-specification have been identified which cells approach too much to the "edges" of the seem to be based on the aforementioned normal distribution and the transcriptome changes mechanism. For example, the choice between become irreversible (Chang et al., 2008). erythroid/megakaryocyte or myeloid-monocytic In 1957 Waddington conceptualized the irreversible fates at the level of erythromyeloid progenitors is process of cellular differentiation as marbles falling controlled by the reciprocal inhibition between the down a slope (Waddington, 1957). This transcription factors GATA-1 and PU.1, therefore metaphorical concept has regained new momentum creating a binary decision for the progenitor (Laiosa with the mathematical interpretation of et al., 2006; Enver et al., 2009). The bipotent transcriptional cellular states as Gene Regulatory progenitor itself would therefore be this Networks (GRNs). In this type of analysis, intermediate state created and maintained by the pluripotency is represented as a mathematical equilibrium between the both factors. This fact attractor (a condition towards which a dynamical helps understanding the phenomenon of system tends to progress over time), in such a way multilineage gene priming, in which uncommitted that the points (cells) that get close enough to the progenitors present low levels of simultaneous attractor remain close even if slightly disturbed. expression of multiple transcription factors This attractor is surrounded by a "differentiation corresponding to different mature cell types and landscape" where other stable cellular fates are

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 239

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

possessing antagonistic functions (Hu et al., 1997; the search of the factors capable of reprogramming Enver et al., 2009). In general, there seems to be a to full pluripotency. Since the differentiated state is progressive loss of developmental potential in a the more stable one (indicating that the GRNs are hierarchical process that moves through sequential less subject to fluctuation), where the cells have differentiation options and in which, at any given reached after "rolling down" the differentiation point, a progenitor would only have to choose pathway in the normal process of development, between two mutually exclusive options (Brown et therefore an "activation energy" is required to move al., 2007; Ceredig et al., 2009). Additionally, in the the cells "uphill" to become again pluripotent. process of maturation into a given lineage, the Conceptually, there are at least two main possible progenitors will receive (and react to) the necessary scenarios to explain the population dynamics in the extrinsic signals (for example, cytokines) that, process of reprogramming to pluripotency according to this model, would be more permissive (Yamanaka, 2009): one possibility (the so-called than instructive. elite model) is that only some cells can be reprogrammed, and these are the ones that are Maintenance of the cellular selected among the entire population, since they are identity of mature differentiated the only ones that are receptive to the action of the cells reprogramming factors. Alternatively, it might Plasticity, in normal development, is a property that happen that all the differentiated cells are equally is "intended" to be restricted to stem cells and capable of undergoing reprogramming, and it is progenitors. In general, the final differentiated only due to technical or methodological reasons cellular types of any given organ or tissue possesses that we are not able to reveal this potential in all of stable identities, in consequence with the fact that them (stochastic model). According to the they usually are highly specialized cells with very accumulating evidences, it would seem that the specific physiological functions. Therefore, it stochastic model is the one that is closer to reality would not make sense, from the biological point of and that, given the right combination of factors; any view, that a specialized cell would be the source of cell could be reprogrammed to pluripotency other differentiated cell types. This, as we have (Yamanaka, 2009). However, as we have mentioned, is the role of stem cells, with their mentioned, this is a developmentally and physiological plasticity (i.e., normal competence) energetically unfavourable process, a fact that is that we have previously discussed. However, the evidenced by several details. The most obvious one concept of the stability of differentiated cell types is the very low efficiency of the reprogramming has been shaken by the discovery of the fact that the process, even in the most favourable conditions. 4 Yamanaka transcription factors (4Y TFs) Oct4, This fact clearly indicates that, independently on Sox2, c-Myc and Klf4 (Takahashi and Yamanaka, how many cells of the population are initially 2006) are enough for the reprogramming of most responsive to the reprogramming factors, very few differentiated cells types into induced pluripotent of them can complete the path towards full stem cells (iPSCs). This finding has altered our reprogramming (Yamanaka, 2009). Also, this is a notion of the latent developmental potential hidden gradual process in which several non-physiological in differentiated cells, showing how it can be cellular intermediates can be isolated (Mikkelsen et "awakened" by experimental manipulations in the al., 2008; Stadtfeld et al., 2008). The study of these laboratory. This, as we have described, was already incompletely reprogrammed intermediates has known to a certain extent from the nuclear revealed that they have re-activated the self-renewal reprogramming experiments performed in and maintenance stem cell genes, but not yet those amphibians more than 50 years ago (Briggs and of pluripotency; also, these stages of aborted King, 1952; Gurdon, 1962). Nevertheless, although reprogramming have not been able to completely those experiments already proved that the cell repress the expression of lineage-specific nucleus could be reprogrammed from a transcription factors and retain persistent DNA differentiated cell type into a pluripotent progenitor, hypermethylation marks as a proof of their failure Yamanaka's experiments showed that only 4 factors in achieving complete epigenetic remodelling were actually enough to make the whole process (Mikkelsen et al., 2008). But perhaps the most possible. We have seen that, a more modest level, it patent proof of the difficulty of the process of full had already been proven that the overexpression or reprogramming to pluripotency is the persistence of loss of individual transcription factors could induce an epigenetic memory in the iPCs that makes them fate changes in differentiated cells (MyoD, C/EBPa, more prone to re-differentiate into the lineages from Pax5, etc). Although these were examples of which they were initially derived, indicating that a transdifferentiation taking place between closely complete elimination of the initial epigenetic related cell types, they already pointed the way for program cannot yet be achieved (Kim et al., 2010; Bar-Nur et al., 2011).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 240

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

Tumoral reprogramming and by the use of nuclear transplantation, but it was not known which of the factors present in the zygote induction of pluripotency: possessed the required reprogramming capacity. similarities Interestingly enough, the 4 Yamanaka factors are The role of transcription factors in the control of known to be involved in tumorigenesis in different tumoral reprogramming and induction of contexts, and both c-Myc and Klf4 are well-known pluripotency oncogenes (Rowland et al., 2005; Okita et al., 2007; We have seen in the initial section of this review Tanaka et al., 2007; Chen et al., 2008), thus further that both cancer research and developmental linking reprogramming to tumorigenesis. biology have been the focus of intense attention In summary, the experimental results show that the since ancient times. What's more, they have always maintenance of cellular identity is essential for been closely related from the conceptual point of differentiated cells, and that only strong view. The cellular theory of Rudolf Virchow is transcriptional or epigenetic regulators can subvert clearly essential for the understanding of both it. In this way, the multistep nature of tumorigenesis development and tumorigenenesis. But he went is paralleled by reprogramming to pluripotency in further, since he already proposed the embryonal the series of "uphill" steps required and in the need rest hypothesis of tumour origin, after realising the for the sum of the effects of several factors to histological similarities between tumours and overcome the built-in safety mechanisms designed embryonic tissues (Virchow, 1855). This concept to protect cells from transformation or, in other was afterwards expanded by Julius Conheim, who words, to prevent cells from changing their identity. suggested that tumours arise from residual In the case of the reprogramming factors, the embryonic remnants "lost" during normal precise role of each of them is not yet clear, but development (Cohnheim, 1867). This hypothesis their experimental introduction at different times actually connects with the current theory of the during the process of reprogramming is shedding cancer stem cells (CSCs) in which progenitors are some light on this issue (Sridharan et al., 2009), by situated at the root of cancer maintenance (see identifying distinct contributions of the different below). Another example of the influence of cancer factors along the reprogramming progression. In the research in the progress of the fields of stem cell early stages of reprogramming, the most important biology and developmental biology is the fact that process happening is the silencing of the gene embryonic stem (ES) cells were identified in a expression programs of the differentiated cells. This search that has been initiated in the study of aspect is previous to the induction of the ES-like teratocarcinomas (Solter, 2006; Morange, 2007; expression program, and the main molecular Hochedlinger and Plath, 2009). responsible for this function seems to be c-Myc. In the field of cancer research it has traditionally However, it has also been shown that treatment been postulated that more than one molecular hit is with histone deacetylase inhibitors like valproic required to generate a tumour cell, because several acid (VPA) can substitute for c-Myc, because of aspects of cellular biology must be altered in the their capacity for repressing the gene expression progress towards a full-blown tumour (Hanahan programs of differentiated cells (Huangfu et al., and Weinberg, 2000). Therefore, in order to achieve 2008). Therefore, it would seem that the action of c- tumoral reprogramming (although this was not the Myc takes place mainly before the activation of the terminology traditionally used), more than one regulators of the pluripotent state and, single molecular alteration had to happen. We have consequently, ectopic expression of c-Myc is mentioned before that for a "simple" required only during the first few days of the transformation, like a lineage switch, the change in reprogramming process (Sridharan et al., 2009). In the levels of expression of a single transcription fact, c-Myc is dispensable for reprogramming, but factor could be enough (Davis et al., 1987; Nutt et in its absence there is an enormous drop in the al., 1999; Xie et al., 2004; Cobaleda et al., 2007a). efficiency of the procedure (Nakagawa et al., 2008; Similarly, a single initial oncogenic lesion may Wernig et al., 2008). The other three factors, Oct4, contribute to just a part of the tumoral phenotype, Sox2, and Klf4, need to act together to achieve the by causing a block in differentiation, or an entry into the pluripotent condition, as evidenced by alteration in the control of cell cycle. In the fact that, when they are used individually, they oncogenesis, many factors and routes have been cannot bind their pluripotent target genes in cells shown to be altered, and their individual that are sill incompletely reprogrammed, most contributions to the tumoral phenotype are clear, likely because the pattern of epigenetic although their synergy and interactions are less modifications at these loci is not permissive for known. In the case of reprogramming to their binding (Sridharan et al., 2009). Indeed, Oct4, pluripotency, the discovery of Takahashi and Sox2, and also Nanog co-bind to a plethora of Yamanaka (Takahashi and Yamanaka, 2006) genes in overlapping genomic sites (Boyer et al., revealed the nature of these factors. Before, 2005; Loh et al., 2006), in such a way that the reprogramming to pluripotency was only possible transcriptional program required for pluripotency is

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 241

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

maintained by the coordinated action of these key alterations are responsible for cancer development, genes. but there is also an important role of epigenetic In general, for the reprogramming of almost every alterations (Esteller and Herman, 2002; Esteller, cell type to pluripotency, the 4 Yamanaka 2007; Esteller, 2008) that lead to the specification transcription factors are enough. However, there are of an heritable, abnormal pattern of gene expression some exceptional cases in which additional that plays an essential role in cancer initiation and alterations are required. For example, in the case of progression (Ting et al., 2006). All the relevant mature B cells it is necessary to interfere with the epigenetic marks, from DNA methylation to histone activity of the transcription factor Pax5, which is modifications, are perturbed in tumour progression. the master regulator of B cell identity (Cobaleda et The subsequent changes in gene expression patterns al., 2007a; Hanna et al., 2008). Previous are especially relevant when they affect the levels experiments had revealed that the elimination of of expression of specific oncogenes or tumour Pax5, in the absence of any other genetic suppressors, but they affect in fact the whole manipulation, allowed mature B cells to epigenome, and therefore condition all cellular dedifferentiate to early haematopoietic identity. All these epigenetic alterations are usually multipotential progenitors (Cobaleda et al., 2007b). secondary, and they can be just due to tumour These findings again correlate reprogramming with progression and therefore independent from (i.e., cancer development, since it has also been shown not directly caused by) the initiating oncogenic that the elimination of Pax5 function in mature B mutation, but they can also be directly induced by cells induces a process of pathological the first oncogenic event, like it happens when dedifferentiation that gives rise to progenitor cell chromosomal aberrations deregulate histone lymphomas (Cobaleda et al., 2007a). Therefore, the modification genes (Esteller, 2008). In the process loss of a transcription factor that is required for the of reprogramming to pluripotency, epigenetic maintenance of cellular identity can be a tumour- modifications are intrinsically required for the inducing lesion. However, and contrary to mature B process to take place, and they have to occur all cells, earlier stages of B cell development can be throughout the genome, not being just restricted to reprogrammed to pluripotency in the presence of the activation or repression of individual genes, functional Pax5, just with the 4 Yamanaka something that is already achieved by the transcription factors (Hanna et al., 2008), thus transcription factors. This explains why the supporting the intuitive idea that the degree of efficiency of reprogramming is significantly differentiation of the target cell has an effect on the superior in the presence of chemicals that can final efficiency of reprogramming (see below). globally interfere with epigenetic marks. For In the genetic landscape, the oncogenic mutations example, the DNA methyltransferase inhibitor 5- alter the architecture of the whole gene regulatory aza-cytidine (AZA) causes a rapid and stable network, since it modifies one of the nodes. This transition to a fully reprogrammed iPS state leads to an alteration in the landscape that gives rise (Huangfu et al., 2008; Mikkelsen et al., 2008). to new abnormal attractors (new "valleys") where Similarly, treatment with valproic acid (VPA), a cancer cells reside (Huang et al., 2009). histone deacetylase (HDAC) inhibitor, considerably Furthermore, this alteration in the landscape gives improves the induction to pluripotency (Huangfu et the cell a new momentum to move towards new al., 2008). Other example is provided by the use of directions, and this effect can persist even when the the compound BIX-01294, an inhibitor of G9a initial stimulus has disappeared. From the point of methyltransferase that makes it possible to achieve view of tumoral reprogramming, this implies that reprogramming to pluripotency using only Oct4 and the expression of a tumour-promoting gene, even if Klf4 transcription factors, with an efficiency it is transient, can by itself trigger a durable comparable to the one obtained when using the four malignant phenotype that does not require anymore factors (Shi et al., 2008). In normal development, of the initial mutation for its maintenance (Huang et the biological role of G9a is to terminate the al., 2009). pluripotencial state as the progenitors exit to the The role of epigenetic factors in the control of differentiation process (Feldman et al., 2006; tumoral reprogramming and induction of Epsztejn-Litman et al., 2008). This is achieved by pluripotency its histone methylation activity, that prevents the In the previous section we have seen that either the reactivation of its target genes (for example gain or the loss of function of transcription factors embryonic genes like Oct4) when their plays an essential role in reprogramming to transcriptional repressors are no longer present pluripotency, in the same way as how oncogene (Feldman et al., 2006). Also, at the same time, G9a overexpression or loss of tumour suppressors promotes DNA methylation, that stops reversion promote tumorigenesis. Also, similarly to tumour towards the undifferentiated state (Feldman et al., progression, large-scale epigenetic changes are 2006; Epsztejn-Litman et al., 2008). Therefore, required for full reprogramming to happen. Today, genome-wide epigenetic changes affecting many it is clearly established that not only genetic still unknown loci, are essential in the late stages of

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 242

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

direct reprogramming, and inhibition of the proteins kinds. This is in connection with what we have responsible for generating or maintaining these mentioned before about reprogramming being an marks lowers the "activation energy" required for "uphill", unfavourable process, which most of the the transition to pluripotency. Therefore, it makes cells fail to complete (Mikkelsen et al., 2008). sense that several of the chemical inhibitors that we Therefore, eliminating the DNA damage checkpoint have just mentioned are in fact already in use, or in diminishes the selection and allows a larger number clinical trials to be used as therapeutic agents of cells to survive until pluripotency. These results against cancer. AZA was approved by the FDA in support the idea of cancer as a disease of cellular 2004 for the treatment of myelodysplastic differentiation and, furthermore, reinforce the idea syndromes, being the first drug into the new class that suggests that the driving forces behind the of demethylating agents (Kaminskas et al., 2005). tumoral process are aberrantly expressed Its mechanism of action is very unspecific, aimed at transcription factors, epigenetic regulators and the restoration of the normal levels of expression of signalling molecules, while the role of many of the genes whose expression has been lost due to other alterations found in tumours (for example, the promoter hypermethylation during tumoral loss of p53) is mainly permissive. progression, and that might be necessary for the Role of the cell of origin in tumoral reprogramming control of proliferation and differentiation. Like in and induction of pluripotency the case of most antitumoral drugs, AZA is In the study of oncogenesis, it has traditionally been expected to affect primarily the tumoral cells and assumed that the phenotype of the tumour cells was leave non-proliferative cells unaffected (Sacchi et a reflection of that of the normal cell that gave rise al., 1999; Kaminskas et al., 2005). Something to the tumour in the first place. There were some similar happens for HDAC inhibitors (Dey, 2006; classical examples in which this what not the case Lane and Chabner, 2009). All these findings like, for example, chronic myelogenous leukaemia underscore once more the concept of cancer as a (CML), where the t(9;22) chromosomal reprogramming disease and a case of wrong translocation could be found in most types of differentiation. differentiated haematopoietic cells, therefore Instructive and permissive factors in the indicating that a common, earlier progenitor, should progression and selection of the processes of be the cell of origin (Melo and Barnes, 2007). But, tumoral reprogramming and induction of in general, since most cancerous cells are pluripotency reminiscent of some differentiated cell type, for We have seen how both genome-wide changes in every type of tumour, the cell of origin was epigenetic marks and the loss and/or gain of postulated to be the corresponding normal transcriptional regulators are essential components differentiated cell. However, the cancer stem cell of the processes of tumour generation and (CSC) theory has led to a change in our perspective reprogramming to pluripotency. However, it is clear (Cobaleda and Sánchez-García, 2009; Vicente- that these changes are clearly unwanted from the Dueñas et al., 2009a; Vicente-Dueñas et al., 2009b). points of view of normal development and cellular The CSC theory proposes that tumours are stem function. Therefore, cells have developed many cell-based tissues just like any other, and this has built-in protection mechanisms to maintain their several radical consequences for our understanding identity against these transcriptional, genetic and of cancer. The most important one is the fact that epigenetic changes. Nevertheless, all these not all the tumoral cells are equally capable of mechanisms are bypassed, in one way or another regenerating the tumour. This means that, when (Hanahan and Weinberg, 2000; Hanahan and tumoral cells are experimentally transplanted into a Weinberg, 2011), and cancer appears. How this new host, or when some tumour cells remain in the happens in "progression to pluripotency" (in patient after incomplete tumour excision, the analogy to tumoral progression) is still to be reappearance of the tumour is caused by just a discovered. However, it has recently been shown by certain tumoral cellular subpopulation. Only those several groups (Zhao et al., 2008; Banito et al., cells, possessing stem cell characteristics, can give 2009; Hong et al., 2009; Kawamura et al., 2009; rise to the whole tumour with all its cellular Krizhanovsky and Lowe, 2009; Li et al., 2009; heterogeneity. Although there can be a big range of Marión et al., 2009; Utikal et al., 2009) that, exactly variability in the percentage of CSCs within a as it happens in cancer progression, the elimination tumour, from very few to 25% (Quintana et al., of the DNA damage control checkpoint (p53-p21) 2008; Cobaleda and Sánchez-García, 2009; greatly improves the efficiency of the Vicente-Dueñas et al., 2009a; Vicente-Dueñas et reprogramming process, making it possible that al., 2009b), the fact is that, like in any other stem- many of the starting cells become successfully cell based tissue, the majority of cells composing reprogrammed. This is done at the expense of an the tumour mass lack this capacity. Hence, if increased level of genetic instability, and most of tumours are maintained by aberrant cells possessing the iPSCs obtained in the absence of a functional stem cell characteristics, then what is the origin of p53-p21 axis carry genetic aberrations of different these cells? This cancer cell-of-origin (not to be

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 243

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

confused with the CSC, which would be the cancer- macrophage progenitors, therefore conferring them maintaining cell of the already developed tumour) the property of self-renewal (Krivtsov et al., 2006). is initially a normal cell (not necessarily a stem cell) Also c-Myc can induce a transcriptional program that will be reprogrammed by the oncogenic events reminiscent of that of embryonic stem cells in in order to finally originate (or convert into) a differentiated epithelial cells, and originate tumoral cell with stem properties. There are two epithelial CSCs (Wong et al., 2008). However, main mechanisms that could be invoked in this other oncogenes are unable of conferring self- scenario. One option is that the cell-of-origin renewal properties, like for example BCR- suffering the oncogenic mutation(s) is already a ABLp190 (Huntly et al., 2004). In these cases the stem cell, which therefore becomes reprogrammed oncogene, since it cannot immediately confer stem to give rise to a new pathological tissue instead of cell properties, could give rise to a precancerous the normal one. In the case of CML, it has recently cell that can afterwards, with the presence of been demonstrated, using genetically modified additional alterations conferring "stemness", give mice, that the restricted expression of the oncogenic rise to the cancer stem cell (Chen et al., 2007). In alteration in the stem cell/progenitor compartment any case, the cellular origin where the cancer- is enough to generate a human-like tumour with all initiating lesions take place is difficult to determine the variety of differentiated tumour cells (Pérez- since, in many cases, the functional impact of the Caro et al., 2009; Vicente-Dueñas et al., 2009b). In oncogenic lesion (i.e. the tumour clonal expansion) mouse models of intestinal cancer it has also been can present with phenotypes mimicking found that tumours originate in the crypt stem cell, differentiation stages that can be either upstream or since when the oncogenic stimulus (activation of downstream of the initiating cell. For example, the the Wnt signalling pathway) is targeted to the stem translocations that are the initiating lesions of many cell compartment, intestinal adenomas develop in childhood B acute lymphoblastic leukaemias (ALL) which a developmental hierarchy is maintained. On originate in utero during embryonic haematopoiesis the contrary, when the oncogenic lesions are and promote the conversion of partially committed targeted at the non-stem intestinal epithelial cells, cells into preleukaemic cells with altered self- they only generate short-lived, small renewal and survival properties, that will require a microadenomas (Barker et al., 2008; Zhu et al., second postnatal hit to develop into full leukemias 2008). In the nervous system, targeting (Hong et al., 2008). Also, in leukemias carrying the astrocytoma-associated oncogenic lesions to AML1-ETO translocation, this aberration can be progenitors (in this case in the subventricular zone) detected in stem cells in patients in remission. results in tumour development, while targeting These stem cells behave apparently normal during them to the differentiated cells of the adult the remission phase, indicating that they can remain parenchyma does not result in tumours, only in dormant and, with time, some of their descendants local astrogliosis (Alcantara Llaguno et al., 2009). can become tumorigenic and originate the relapse Therefore, there are many examples (Dirks, 2008; (Miyamoto et al., 2000). We have described Joseph et al., 2008; Zheng et al., 2008) where it has previously that, in mice, the loss of Pax5 in mature been proven that the initiating event takes place in a B cells leads to the dedifferentiation to multipotent normal stem cell, even if the mature tumour is progenitors and the appearance of progenitor B cell composed by differentiated cells, indicating a true lymphomas (Cobaleda et al., 2007a). In human tumoral reprogramming mediated by the oncogenic Hodking lymphomas, the overexpression of specific lesions (Vicente-Dueñas et al., 2009b). antagonists leads to the functional inactivation of The other alternative is that the cancer cell-of-origin the B cell factor E2A, which in turn causes the loss can be a differentiated cell that regains stem cell of B cell markers and induces the expression of characteristics in the process of tumoral lineage-inappropriate genes characteristic of the reprogramming. This option relies on two Reed-Sternberg Hodking lymphoma cells (Mathas requirements: first, the oncogenic alteration must be et al., 2006). Also in children's B-ALLs, the CSCs capable of conferring or programming these can present with the phenotypes of different stages characteristics in the target cell and, second, the cell of early B cell development that, on top of that, can must be plastic enough so as to be reprogrammed apparently interconvert among them, therefore by this precise oncogenic alteration. It has been complicating even more the task of identifying the shown that some oncogenes, like MOZ-TIF2 cancer-cell of origin (le Viseur et al., 2008). A (Huntly et al., 2004), MLL-AF9 (Krivtsov et al., genomic analysis of samples from relapsed ALL 2006; Somervaille and Cleary, 2006), MLL-ENL patients, when compared with the samples at (Cozzio et al., 2003), MLL-GAS (So et al., 2003) or diagnosis, has shown that the same ancestral clone PML-RARα (Guibal et al., 2009; Wojiski et al., can be found at both stages of the disease 2009) can generate CSCs when they are introduced (Mullighan et al., 2008). So, clearly in many cases into committed target cells. Gene expression arrays the cancer-maintaining cell evolves over time and have revealed that MLL-AF9 can activate a stem adapts to treatment to finally lead to relapse, and cell-like program in committed granulocyte- therefore the characteristics of the CSC population

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 244

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

in a certain moment may not relate at all any more distant future, in the treatment of cancer. It is clear to those of the initial cancer cell-of-origin (Barabé that the two fields of research will continue being et al., 2007). mutually interdependent. By way of example, the As we already mentioned when we described the main obstacle for the future use of iPSCs in the view of reprogramming to pluripotency from the clinic is precisely the generation of tumours as a perspective of the GRNs, the inducing factors are result of uncontrolled growth or differentiation of not required anymore once the cells have reached the cells, once they are in the patient. Therefore, the the pluripotent condition and the new identity knowledge and control of the narrow limits of gene (however plastic this is) has been established. If expression that mark the difference between normal cancer stem cells are generated by a tumoral and tumoral differentiation and reprogramming will reprogramming process, then maybe the oncogenes be required before this problem can be overcome. that initiate tumour formation might be not be Assuming the role that reprogramming plays in required for tumour progression (Krizhanovsky and cancer generation makes it possible to initiate the Lowe, 2009). If this were the case, it would explain development of new therapeutic strategies aimed at the aforementioned examples in which a pre- re-directing the wrong differentiation program cancerous lesion exists stably in an aberrant cell towards a new outcome (ideally, in most cases, population that does only evolve to an open tumour terminal non-tumoral differentiation and cellular when secondary mutations occur. In this scenario, death). Differentiation therapies are already in use the initiating lesion would be the driving force in in some cases, like the administration of retinoic the reprogramming process, but once this has been acid to differentiate tumoral cells in PML-RARα+ completed, it would only be a passenger mutation, positive acute promyelocytic leukemias. We have or could even perform a different role that would be described how reprogramming to pluripotency, due independent from its reprogramming capacity, like to its inefficiency, can get caught up at several for example in tumour expansion/proliferation. A points before reaching the iPSC state (Mikkelsen et mechanism of this kind would explain why some al., 2008). Tumoral cells are probably very close to targeted therapies fail in spite of their initial these incompletely reprogrammed intermediates, apparent efficacy: for example, imatinib, a drug and the study of the latter should help us in targeted against the deregulated kinase activity of understanding how to get the former ones out of BCR-ABL, successfully eliminates differentiated their pathologic block. In fact, epigenetic therapies tumour cells, but it fails to kill the BCR-ABL+ are most probably going to be on the rise in the CSCs, since it does not seem to interfere with the coming years for the treatment of many types of function of the chimeric oncogene in this cellular tumours, since our knowledge about the molecular context (Graham et al., 2002; Barnes and Melo, mechanisms controlling the epigenetic marks and 2006). their role in self-renewal, differentiation and The fact that CSCs can originate from differentiated maintenance is increasing very quickly, and this cells represents the last and most patent similarity should help us to obtain more and better (more between tumorigenesis and reprogramming to specific) epigenetic drugs (Jones, 2007; Shen et al., pluripotency. Also in iPSCs generation, the nature 2009). of the cell of origin is key in determining the global The discovery of reprogramming to pluripotency success. In this way, it has been described that, in has transfigured the research in the field of cellular the haematopoietic system, the capacity of plasticity. It is nowadays possible, using just three reprogramming cells decreases as they differentiate, ectopic factors, to reprogram fibroblasts into since HSC are 300 times more likely to be functional neurons (Vierbuchen et al., 2010), to reprogrammed than B or T cells (Eminli et al., convert in vivo pancreatic exocrine cells to β cells 2009). In the case of the nervous system, when the (Zhou et al., 2008) or to directly transdifferentiate starting cells are adult neural stem cells (NSCs), mouse mesoderm into heart tissue (Takeuchi and then pluripotency can be achieved using only Oct4 Bruneau, 2009). One of the most remarkable (Kim et al., 2009), probably because of the high examples in this context is the phenotype caused by similarity of NSCs transcriptional profile to that of the deletion of a single gene, Foxl2, in adult ovarian ES cells. Similarly, in a liver model of follicles. This inactivation immediately upregulates transdetermination it has been demonstrated that testis-specific genes and leads to a full organ Neurogenin3 can convert hepatic progenitor cells reprogramming (Uhlenhaut et al., 2009) that shows into neo-islets but it cannot transdifferentiate that the maintenance of the identity of the ovarian mature hepatocytes (Yechoor et al., 2009). cells requires the active and constant presence of a specific gene. This is therefore an active process Outlook that resembles very much what we have described The knowledge obtained in the research of the for Pax5 and B cells, but affecting a whole organ molecular and cellular mechanisms that control with all its cellular diversity. cellular plasticity, pluripotency and reprogramming Our increasing knowledge and technical control will also have a profound impact in our over cellular identity should help us in the understanding of tumorigenesis and, in a more

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 245

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

development of strategies for the reprogramming of Chalkey DT.. A quantitative hystological analysis of tumoral cells. In fact, several experimental forelimb regeneration in Triturus viridescens. J Morphol. 1954;94:21-70. evidences seem to suggest that this is perfectly possible. For example, melanoma cells can be Waddington CH.. The Strategy of the Genes. London: reprogrammed by nuclear transplantation Allen and Unwin. 1957. (Hochedlinger et al., 2004). Also, embryonal Bodemer CW, Everett NB.. Localization of newly carcinoma cells or mouse brain tumours have been synthesized proteins in regenerating newt limbs as determined by radioautographic localization of injected used as a valid starting material for nuclear cloning methionine-S35. Dev Biol. 1959;1:327-342. experiments (Li et al., 2003; Blelloch et al., 2004). Therefore, maybe in a not so distant future we Hay ED, Fischman DA.. Origin of the blastema in regenerating limbs of the newt Triturus viridescens. An might have the knowledge and tools to manipulate autoradiographic study using tritiated thymidine to follow tumoral cell identity to force cancer cells to cell proliferation and migration. Dev Biol. 1961 Feb;3:26- differentiate, or to make them vulnerable to therapy. 59. Gurdon JB.. The developmental capacity of nuclei taken Acknowledgments from intestinal epithelium cells of feeding tadpoles. J Research in C.C. lab was partially supported by Embryol Exp Morphol. 1962 Dec;10:622-40. FEDER, Fondo de Investigaciones Sanitarias Shih C, Shilo BZ, Goldfarb MP, Dannenberg A, Weinberg (PI080164), CSIC P.I.E. 200920I055 and RA.. Passage of phenotypes of chemically transformed 201120E060, from the ARIMMORA project (FP7- cells via transfection of DNA and chromatin. Proc Natl ENV-2011, European Union Seventh Framework Acad Sci U S A. 1979 Nov;76(11):5714-8. Program) and from an institutional grant from the Goldfarb M, Shimizu K, Perucho M, Wigler M.. Isolation "Fundación Ramón Areces". Research in ISG group and preliminary characterization of a human transforming gene from T24 bladder carcinoma cells. Nature. 1982 Apr was partially supported by FEDER and by MICINN 1;296(5856):404-9. (SAF2009-08803 to ISG), by Junta de Castilla y León (REF. CSI007A11-2 and Proyecto Lane MA, Sainten A, Cooper GM.. Stage-specific transforming genes of human and mouse B- and T- Biomedicina 2009-2010), by MEC OncoBIO lymphocyte neoplasms. Cell. 1982 Apr;28(4):873-80. Consolider-Ingenio 2010 (Ref. CSD2007-0017), by NIH grant (R01 CA109335-04A1), by Sandra Parada LF, Tabin CJ, Shih C, Weinberg RA.. Human EJ bladder carcinoma oncogene is homologue of Harvey Ibarra Foundation, by Group of Excellence Grant sarcoma virus ras gene. Nature. 1982 Jun (GR15) from Junta de Castilla y Leon, and the 10;297(5866):474-8. ARIMMORA project (FP7-ENV-2011, European Santos E, Tronick SR, Aaronson SA, Pulciani S, Barbacid Union Seventh Framework Program) and by M.. T24 human bladder carcinoma oncogene is an "Proyecto en Red de Investigación en Celulas activated form of the normal human homologue of BALB- Madre Tumorales en Cancer de Mama", supported and Harvey-MSV transforming genes. Nature. 1982 Jul by Obra Social Kutxa y Conserjería de Sanidad de 22;298(5872):343-7. la Junta de Castilla y León. All Spanish funding is Davis RL, Weintraub H, Lassar AB.. Expression of a single co-sponsored by the European Union FEDER transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987-1000. program. ISG is an API lab of the EuroSyStem project. ECS is the recipient of a JAE-predoc Schneuwly S, Klemenz R, Gehring WJ.. Redesigning the body plan of Drosophila by ectopic expression of the Fellowship from CSIC and a "Residencia de homoeotic gene Antennapedia. Nature. 1987 Feb 26-Mar Estudiantes" Fellowship. The authors declare no 4;325(6107):816-8. conflict of interest. Gehring WJ.. The master control gene for morphogenesis and evolution of the eye. Genes Cells. 1996 Jan;1(1):11-5. References (REVIEW) Reaumur RAF.. Sur les diverses reproductions qui se font Hu M, Krause D, Greaves M, Sharkis S, Dexter M, dans les ecrevisses, les omars, les crabes, etc. et Heyworth C, Enver T.. Multilineage gene expression entr'autres sur celles de leurs jambes et de leurs ecailles. precedes commitment in the hemopoietic system. Genes Mem Acad Roy. 1712;Sci 223-245. Dev. 1997 Mar 15;11(6):774-85. Trembley A.. Memoires Pour Servir A L'Histoire D'Un Sanchez-Garcia I.. Consequences of chromosomal Genre de Polypes D'Eau Douce, a Bras En Forme de abnormalities in tumor development. Annu Rev Genet. Cornes. Leiden, Jean and Herman Verbeek. 1744. 1997;31:429-53. (REVIEW) Spallanzani L.. Prodromo di un opera da imprimersi sopra Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH.. la riproduzioni animali (An Essay on Animal Reproduction). Viable offspring derived from fetal and adult mammalian London: T. Becket and de Hondt. 1769. cells. Nature. 1997 Feb 27;385(6619):810-3. Virchow R.. Editorial. Virchows arch. Pathol Anat Physiol Cobaleda C, Perez-Losada J, Sanchez-Garcia I.. Klin Med. 1855. Chromosomal abnormalities and tumor development: from genes to therapeutic mechanisms. Bioessays. 1998 Cohnheim J.. Ueber entzundung und eiterung. Path Anat Nov;20(11):922-30. (REVIEW) Physiol Klin Med.1867; 40:1-79. Nutt SL, Heavey B, Rolink AG, Busslinger M.. Commitment Briggs R, King TJ.. Transplantation of Living Nuclei From to the B-lymphoid lineage depends on the transcription Blastula Cells into Enucleated Frogs' Eggs. Proc Natl Acad factor Pax5. Nature. 1999 Oct 7;401(6753):556-62. Sci U S A. 1952 May;38(5):455-63.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 246

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

Sacchi S, Kantarjian HM, O'Brien S, Cortes J, Rios MB, Kaminskas E, Farrell A, Abraham S, Baird A, Hsieh LS, Giles FJ, Beran M, Koller CA, Keating MJ, Talpaz M.. Lee SL, Leighton JK, Patel H, Rahman A, Sridhara R, Chronic myelogenous leukemia in nonlymphoid blastic Wang YC, Pazdur R; FDA.. Approval summary: azacitidine phase: analysis of the results of first salvage therapy with for treatment of myelodysplastic syndrome subtypes. Clin three different treatment approaches for 162 patients. Cancer Res. 2005 May 15;11(10):3604-8. Cancer. 1999 Dec 15;86(12):2632-41. Rowland BD, Bernards R, Peeper DS.. The KLF4 tumour Hanahan D, Weinberg RA.. The hallmarks of cancer. Cell. suppressor is a transcriptional repressor of p53 that acts 2000 Jan 7;100(1):57-70. (REVIEW) as a context-dependent oncogene. Nat Cell Biol. 2005 Nov;7(11):1074-82. Miyamoto T, Weissman IL, Akashi K.. AML1/ETO- expressing nonleukemic stem cells in acute myelogenous Barnes DJ, Melo JV.. Primitive, quiescent and difficult to leukemia with 8;21 chromosomal translocation. Proc Natl kill: the role of non-proliferating stem cells in chronic Acad Sci U S A. 2000 Jun 20;97(13):7521-6. myeloid leukemia. Cell Cycle. 2006 Dec;5(24):2862-6. Epub 2006 Dec 15. (REVIEW) Sanchez Alvarado A.. Regeneration in the metazoans: why does it happen? Bioessays. 2000 Jun;22(6):578-90. Dey P.. Chromatin remodeling, cancer and chemotherapy. (REVIEW) Curr Med Chem. 2006;13(24):2909-19. (REVIEW) Reya T, Morrison SJ, Clarke MF, Weissman IL.. Stem Feldman N, Gerson A, Fang J, Li E, Zhang Y, Shinkai Y, cells, cancer, and cancer stem cells. Nature. 2001 Nov Cedar H, Bergman Y.. G9a-mediated irreversible 1;414(6859):105-11. (REVIEW) epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat Cell Biol. 2006 Feb;8(2):188-94. Epub Esteller M, Herman JG.. Cancer as an epigenetic disease: 2006 Jan 15. DNA methylation and chromatin alterations in human tumours. J Pathol. 2002 Jan;196(1):1-7. (REVIEW) Hochedlinger K, Jaenisch R.. Nuclear reprogramming and pluripotency. Nature. 2006 Jun 29;441(7097):1061-7. Graham SM, Jorgensen HG, Allan E, Pearson C, Alcorn (REVIEW) MJ, Richmond L, Holyoake TL.. Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic Krivtsov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, myeloid leukemia are insensitive to STI571 in vitro. Blood. Faber J, Levine JE, Wang J, Hahn WC, Gilliland DG, 2002 Jan 1;99(1):319-25. Golub TR, Armstrong SA.. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Cozzio A, Passegue E, Ayton PM, Karsunky H, Cleary ML, Nature. 2006 Aug 17;442(7104):818-22. Epub 2006 Jul 16. Weissman IL.. Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid Laiosa CV, Stadtfeld M, Graf T.. Determinants of lymphoid- progenitors. Genes Dev. 2003 Dec 15;17(24):3029-35. myeloid lineage diversification. Annu Rev Immunol. 2006;24:705-38. (REVIEW) Li L, Connelly MC, Wetmore C, Curran T, Morgan JI.. Mouse embryos cloned from brain tumors. Cancer Res. Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, 2003 Jun 1;63(11):2733-6. Bourque G, George J, Leong B, Liu J, Wong KY, Sung KW, Lee CW, Zhao XD, Chiu KP, Lipovich L, Kuznetsov So CW, Karsunky H, Passegue E, Cozzio A, Weissman IL, VA, Robson P, Stanton LW, Wei CL, Ruan Y, Lim B, Ng Cleary ML.. MLL-GAS7 transforms multipotent HH.. The Oct4 and Nanog transcription network regulates hematopoietic progenitors and induces mixed lineage pluripotency in mouse embryonic stem cells. Nat Genet. leukemias in mice. Cancer Cell. 2003 Feb;3(2):161-71. 2006 Apr;38(4):431-40. Epub 2006 Mar 5. Blelloch RH, Hochedlinger K, Yamada Y, Brennan C, Kim Mathas S, Janz M, Hummel F, Hummel M, Wollert-Wulf B, M, Mintz B, Chin L, Jaenisch R.. Nuclear cloning of Lusatis S, Anagnostopoulos I, Lietz A, Sigvardsson M, embryonal carcinoma cells. Proc Natl Acad Sci U S A. Jundt F, Johrens K, Bommert K, Stein H, Dorken B.. 2004 Sep 28;101(39):13985-90. Epub 2004 Aug 11. Intrinsic inhibition of transcription factor E2A by HLH Hajdu SI.. Greco-Roman thought about cancer. Cancer. proteins ABF-1 and Id2 mediates reprogramming of 2004 May 15;100(10):2048-51. neoplastic B cells in Hodgkin lymphoma. Nat Immunol. 2006 Feb;7(2):207-15. Epub 2005 Dec 20. Hochedlinger K, Blelloch R, Brennan C, Yamada Y, Kim M, Chin L, Jaenisch R.. Reprogramming of a melanoma Solter D.. From teratocarcinomas to embryonic stem cells genome by nuclear transplantation. Genes Dev. 2004 Aug and beyond: a history of embryonic stem cell research. Nat 1;18(15):1875-85. Rev Genet. 2006 Apr;7(4):319-27. Huntly BJ, Shigematsu H, Deguchi K, Lee BH, Mizuno S, Somervaille TC, Cleary ML.. Identification and Duclos N, Rowan R, Amaral S, Curley D, Williams IR, characterization of leukemia stem cells in murine MLL-AF9 Akashi K, Gilliland DG.. MOZ-TIF2, but not BCR-ABL, acute myeloid leukemia. Cancer Cell. 2006 Oct;10(4):257- confers properties of leukemic stem cells to committed 68. murine hematopoietic progenitors. Cancer Cell. 2004 Swiers G, Patient R, Loose M.. Genetic regulatory Dec;6(6):587-96. networks programming hematopoietic stem cells and Odelberg SJ.. Unraveling the molecular basis for erythroid lineage specification. Dev Biol. 2006 Jun regenerative cellular plasticity. PLoS Biol. 2004 15;294(2):525-40. Epub 2006 Apr 19. Aug;2(8):E232. Epub 2004 Aug 17. (REVIEW) Takahashi K, Yamanaka S.. Induction of pluripotent stem Xie H, Ye M, Feng R, Graf T.. Stepwise reprogramming of cells from mouse embryonic and adult fibroblast cultures B cells into macrophages. Cell. 2004 May 28;117(5):663- by defined factors. Cell. 2006 Aug 25;126(4):663-76. Epub 76. 2006 Aug 10. Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Ting AH, McGarvey KM, Baylin SB.. The cancer Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner epigenome--components and functional correlates. Genes RG, Gifford DK, Melton DA, Jaenisch R, Young RA.. Core Dev. 2006 Dec 1;20(23):3215-31. (REVIEW) transcriptional regulatory circuitry in human embryonic Barabe F, Kennedy JA, Hope KJ, Dick JE.. Modeling the stem cells. Cell. 2005 Sep 23;122(6):947-56. initiation and progression of human acute leukemia in mice. Science. 2007 Apr 27;316(5824):600-4.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 247

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

Brown G, Hughes PJ, Michell RH, Rolink AG, Ceredig R.. genes. Nat Struct Mol Biol. 2008 Nov;15(11):1176-83. The sequential determination model of hematopoiesis. Epub 2008 Oct 26. Trends Immunol. 2007 Oct;28(10):442-8. Epub 2007 Sep 7. Esteller M.. Epigenetics in cancer. N Engl J Med. 2008 Mar 13;358(11):1148-59. (REVIEW) Cobaleda C, Jochum W, Busslinger M.. Conversion of mature B cells into T cells by dedifferentiation to Gurdon JB, Melton DA.. Nuclear reprogramming in cells. uncommitted progenitors. Nature. 2007a Sep Science. 2008 Dec 19;322(5909):1811-5. (REVIEW) 27;449(7161):473-7. Epub 2007 Sep 12. Hanna J, Markoulaki S, Schorderet P, Carey BW, Beard C, Cobaleda C, Schebesta A, Delogu A, Busslinger M.. Pax5: Wernig M, Creyghton MP, Steine EJ, Cassady JP, the guardian of B cell identity and function. Nat Immunol. Foreman R, Lengner CJ, Dausman JA, Jaenisch R.. Direct 2007b May;8(5):463-70. (REVIEW) reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell. 2008 Apr 18;133(2):250- Chen L, Shen R, Ye Y, Pu XA, Liu X, Duan W, Wen J, 64. Zimmerer J, Wang Y, Liu Y, Lasky LC, Heerema NA, Perrotti D, Ozato K, Kuramochi-Miyagawa S, Nakano T, Hong D, Gupta R, Ancliff P, Atzberger A, Brown J, Soneji Yates AJ, Carson WE 3rd, Lin H, Barsky SH, Gao JX.. S, Green J, Colman S, Piacibello W, Buckle V, Tsuzuki S, Precancerous stem cells have the potential for both benign Greaves M, Enver T.. Initiating and cancer-propagating and malignant differentiation. PLoS One. 2007 Mar cells in TEL-AML1-associated childhood leukemia. 14;2(3):e293. Science. 2008 Jan 18;319(5861):336-9. Esteller M.. Cancer epigenomics: DNA methylomes and Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, histone-modification maps. Nat Rev Genet. 2007 Chen AE, Melton DA.. Induction of pluripotent stem cells Apr;8(4):286-98. Epub 2007 Mar 6. (REVIEW) by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol. 2008 Jul;26(7):795-7. Epub Jones RS.. Epigenetics: reversing the 'irreversible'. Nature. 2008 Jun 22. 2007 Nov 15;450(7168):357-9. Joseph NM, Mosher JT, Buchstaller J, Snider P, McKeever Loose M, Swiers G, Patient R.. Transcriptional networks PE, Lim M, Conway SJ, Parada LF, Zhu Y, Morrison SJ.. regulating hematopoietic cell fate decisions. Curr Opin The loss of Nf1 transiently promotes self-renewal but not Hematol. 2007 Jul;14(4):307-14. (REVIEW) tumorigenesis by neural crest stem cells. Cancer Cell. 2008 Feb;13(2):129-40. Melo JV, Barnes DJ.. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev le Viseur C, Hotfilder M, Bomken S, Wilson K, Rottgers S, Cancer. 2007 Jun;7(6):441-53. (REVIEW) Schrauder A, Rosemann A, Irving J, Stam RW, Shultz LD, Harbott J, Jurgens H, Schrappe M, Pieters R, Vormoor J.. Morange M.. The field of cancer research: an indicator of In childhood acute lymphoblastic leukemia, blasts at present transformations in biology. Oncogene. 2007 Dec different stages of immunophenotypic maturation have 6;26(55):7607-10. Epub 2007 Jul 23. (REVIEW) stem cell properties. Cancer Cell. 2008 Jul 8;14(1):47-58. Okita K, Ichisaka T, Yamanaka S.. Generation of germline- Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, competent induced pluripotent stem cells. Nature. 2007 Jul Schorderet P, Bernstein BE, Jaenisch R, Lander ES, 19;448(7151):313-7. Epub 2007 Jun 6. Meissner A.. Dissecting direct reprogramming through Sanchez-Garcia I, Vicente-Duenas C, Cobaleda C.. The integrative genomic analysis. Nature. 2008 Jul theoretical basis of cancer-stem-cell-based therapeutics of 3;454(7200):49-55. Epub 2008 May 28. cancer: can it be put into practice? Bioessays. 2007 Mullighan CG, Phillips LA, Su X, Ma J, Miller CB, Shurtleff Dec;29(12):1269-80. (REVIEW) SA, Downing JR.. Genomic analysis of the clonal origins of Tanaka Y, Era T, Nishikawa S, Kawamata S.. Forced relapsed acute lymphoblastic leukemia. Science. 2008 Nov expression of Nanog in hematopoietic stem cells results in 28;322(5906):1377-80. a gammadeltaT-cell disorder. Blood. 2007 Jul Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, 1;110(1):107-15. Epub 2007 Mar 14. Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Birnbaum KD, Sanchez Alvarado A.. Slicing across Yamanaka S.. Generation of induced pluripotent stem cells kingdoms: regeneration in plants and animals. Cell. 2008 without Myc from mouse and human fibroblasts. Nat Feb 22;132(4):697-710. (REVIEW) Biotechnol. 2008 Jan;26(1):101-6. Epub 2007 Nov 30. Cobaleda C, Busslinger M.. Developmental plasticity of Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson lymphocytes. Curr Opin Immunol. 2008 Apr;20(2):139-48. TM, Morrison SJ.. Efficient tumour formation by single Epub 2008 May 9. (REVIEW) human melanoma cells. Nature. 2008 Dec 4;456(7222):593-8. Chang HH, Hemberg M, Barahona M, Ingber DE, Huang S.. Transcriptome-wide noise controls lineage choice in Shi Y, Do JT, Desponts C, Hahm HS, Scholer HR, Ding S.. mammalian progenitor cells. Nature. 2008 May A combined chemical and genetic approach for the 22;453(7194):544-7. generation of induced pluripotent stem cells. Cell Stem Cell. 2008 Jun 5;2(6):525-8. Chen Y, Shi L, Zhang L, Li R, Liang J, Yu W, Sun L, Yang X, Wang Y, Zhang Y, Shang Y.. The molecular mechanism Stadtfeld M, Maherali N, Breault DT, Hochedlinger K.. governing the oncogenic potential of SOX2 in breast Defining molecular cornerstones during fibroblast to iPS cancer. J Biol Chem. 2008 Jun 27;283(26):17969-78. Epub cell reprogramming in mouse. Cell Stem Cell. 2008 Mar 2008 May 2. 6;2(3):230-40. Epub 2008 Feb 14. Dirks PB.. Cancer's source in the peripheral nervous Wernig M, Meissner A, Cassady JP, Jaenisch R.. c-Myc is system. Nat Med. 2008 Apr;14(4):373-5. dispensable for direct reprogramming of mouse fibroblasts. Cell Stem Cell. 2008 Jan 10;2(1):10-2. Epub 2007 Dec 13. Epsztejn-Litman S, Feldman N, Abu-Remaileh M, Shufaro Y, Gerson A, Ueda J, Deplus R, Fuks F, Shinkai Y, Cedar Wong DJ, Liu H, Ridky TW, Cassarino D, Segal E, Chang H, Bergman Y.. De novo DNA methylation promoted by HY.. Module map of stem cell genes guides creation of G9a prevents reprogramming of embryonically silenced epithelial cancer stem cells. Cell Stem Cell. 2008 Apr 10;2(4):333-44.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 248

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

Zhao Y, Yin X, Qin H, Zhu F, Liu H, Yang W, Zhang Q, Kawamura T, Suzuki J, Wang YV, Menendez S, Morera Xiang C, Hou P, Song Z, Liu Y, Yong J, Zhang P, Cai J, LB, Raya A, Wahl GM, Belmonte JC.. Linking the p53 Liu M, Li H, Li Y, Qu X, Cui K, Zhang W, Xiang T, Wu Y, tumour suppressor pathway to somatic cell Zhao Y, Liu C, Yu C, Yuan K, Lou J, Ding M, Deng H.. Two reprogramming. Nature. 2009 Aug 27;460(7259):1140-4. supporting factors greatly improve the efficiency of human Epub 2009 Aug 9. iPSC generation. Cell Stem Cell. 2008 Nov 6;3(5):475-9. Kim JB, Sebastiano V, Wu G, Arauzo-Bravo MJ, Sasse P, Zheng H, Chang L, Patel N, Yang J, Lowe L, Burns DK, Gentile L, Ko K, Ruau D, Ehrich M, van den Boom D, Zhu Y.. Induction of abnormal proliferation by Meyer J, Hubner K, Bernemann C, Ortmeier C, Zenke M, nonmyelinating schwann cells triggers neurofibroma Fleischmann BK, Zaehres H, Scholer HR.. Oct4-induced formation. Cancer Cell. 2008 Feb;13(2):117-28. pluripotency in adult neural stem cells. Cell. 2009 Feb 6;136(3):411-9. Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA.. In vivo reprogramming of adult pancreatic exocrine cells to Kragl M, Knapp D, Nacu E, Khattak S, Maden M, Epperlein beta-cells. Nature. 2008 Oct 2;455(7213):627-32. Epub HH, Tanaka EM.. Cells keep a memory of their tissue 2008 Aug 27. origin during axolotl limb regeneration. Nature. 2009 Jul 2;460(7251):60-5. Alcantara Llaguno S, Chen J, Kwon CH, Jackson EL, Li Y, Burns DK, Alvarez-Buylla A, Parada LF.. Malignant Krizhanovsky V, Lowe SW.. Stem cells: The promises and astrocytomas originate from neural stem/progenitor cells in perils of p53. Nature. 2009 Aug 27;460(7259):1085-6. a somatic tumor suppressor mouse model. Cancer Cell. 2009 Jan 6;15(1):45-56. Lane AA, Chabner BA.. Histone deacetylase inhibitors in cancer therapy. J Clin Oncol. 2009 Nov 10;27(32):5459- Banito A, Rashid ST, Acosta JC, Li S, Pereira CF, Geti I, 68. Epub 2009 Oct 13. (REVIEW) Pinho S, Silva JC, Azuara V, Walsh M, Vallier L, Gil J.. Senescence impairs successful reprogramming to Li H, Collado M, Villasante A, Strati K, Ortega S, pluripotent stem cells. Genes Dev. 2009 Sep Canamero M, Blasco MA, Serrano M.. The Ink4/Arf locus 15;23(18):2134-9. Epub 2009 Aug 20. is a barrier for iPS cell reprogramming. Nature. 2009 Aug 27;460(7259):1136-9. Epub 2009 Aug 9. Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M, Danenberg E, Clarke AR, Marion RM, Strati K, Li H, Murga M, Blanco R, Ortega S, Sansom OJ, Clevers H.. Crypt stem cells as the cells-of- Fernandez-Capetillo O, Serrano M, Blasco MA.. A p53- origin of intestinal cancer. Nature. 2009 Jan mediated DNA damage response limits reprogramming to 29;457(7229):608-11. Epub 2008 Dec 17. ensure iPS cell genomic integrity. Nature. 2009 Aug 27;460(7259):1149-53. Epub 2009 Aug 9. Ceredig R, Rolink AG, Brown G.. Models of haematopoiesis: seeing the wood for the trees. Nat Rev Perez-Caro M, Cobaleda C, Gonzalez-Herrero I, Vicente- Immunol. 2009 Apr;9(4):293-300. Duenas C, Bermejo-Rodriguez C, Sanchez-Beato M, Orfao A, Pintado B, Flores T, Sanchez-Martin M, Jimenez Cobaleda C, Sanchez-Garcia I.. B-cell acute lymphoblastic R, Piris MA, Sanchez-Garcia I.. Cancer induction by leukaemia: towards understanding its cellular origin. restriction of oncogene expression to the stem cell Bioessays. 2009 Jun;31(6):600-9. (REVIEW) compartment. EMBO J. 2009 Jan 7;28(1):8-20. Epub 2008 Nov 27. Eminli S, Foudi A, Stadtfeld M, Maherali N, Ahfeldt T, Mostoslavsky G, Hock H, Hochedlinger K.. Differentiation Shen X, Kim W, Fujiwara Y, Simon MD, Liu Y, Mysliwiec stage determines potential of hematopoietic cells for MR, Yuan GC, Lee Y, Orkin SH.. Jumonji modulates reprogramming into induced pluripotent stem cells. Nat polycomb activity and self-renewal versus differentiation of Genet. 2009 Sep;41(9):968-76. Epub 2009 Aug 9. stem cells. Cell. 2009 Dec 24;139(7):1303-14. Enver T, Pera M, Peterson C, Andrews PW.. Stem cell Sridharan R, Tchieu J, Mason MJ, Yachechko R, Kuoy E, states, fates, and the rules of attraction. Cell Stem Cell. Horvath S, Zhou Q, Plath K.. Role of the murine 2009 May 8;4(5):387-97. (REVIEW) reprogramming factors in the induction of pluripotency. Cell. 2009 Jan 23;136(2):364-77. Graf T, Enver T.. Forcing cells to change lineages. Nature. 2009 Dec 3;462(7273):587-94. (REVIEW) Takeuchi JK, Bruneau BG.. Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors. Guibal FC, Alberich-Jorda M, Hirai H, Ebralidze A, Nature. 2009 Jun 4;459(7247):708-11. Epub 2009 Apr 26. Levantini E, Di Ruscio A, Zhang P, Santana-Lemos BA, Neuberg D, Wagers AJ, Rego EM, Tenen DG.. Uhlenhaut NH, Jakob S, Anlag K, Eisenberger T, Sekido Identification of a myeloid committed progenitor as the R, Kress J, Treier AC, Klugmann C, Klasen C, Holter NI, cancer-initiating cell in acute promyelocytic leukemia. Riethmacher D, Schutz G, Cooney AJ, Lovell-Badge R, Blood. 2009 Dec 24;114(27):5415-25. Epub 2009 Oct 1. Treier M.. Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell. 2009 Dec 11;139(6):1130- Hochedlinger K, Plath K.. Epigenetic reprogramming and 42. induced pluripotency. Development. 2009 Feb;136(4):509- 23. (REVIEW) Utikal J, Polo JM, Stadtfeld M, Maherali N, Kulalert W, Walsh RM, Khalil A, Rheinwald JG, Hochedlinger K.. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Immortalization eliminates a roadblock during cellular Nakagawa M, Okita K, Yamanaka S.. Suppression of reprogramming into iPS cells. Nature. 2009 Aug induced pluripotent stem cell generation by the p53-p21 27;460(7259):1145-8. Epub 2009 Aug 9. pathway. Nature. 2009 Aug 27;460(7259):1132-5. Epub 2009 Aug 9. Vicente-Duenas C, Gutierrez de Diego J, Rodriguez FD, Jimenez R, Cobaleda C.. The role of cellular plasticity in Huang S.. Reprogramming cell fates: reconciling rarity with cancer development. Curr Med Chem. 2009a;16(28):3676- robustness. Bioessays. 2009 May;31(5):546-60. 85. (REVIEW) Huang S, Ernberg I, Kauffman S.. Cancer attractors: a Vicente-Duenas C, Perez-Caro M, Abollo-Jimenez F, systems view of tumors from a gene network dynamics Cobaleda C, Sanchez-Garcia I.. Stem-cell driven cancer: and developmental perspective. Semin Cell Dev Biol. 2009 "hands-off" regulation of cancer development. Cell Cycle. Sep;20(7):869-76. Epub 2009 Jul 10. (REVIEW) 2009b May 1;8(9):1314-8. Epub 2009 May 18.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 249

Plasticity and Tumorigenicity Campos-Sanchez E, et al.

Wojiski S, Guibal FC, Kindler T, Lee BH, Jesneck JL, Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, Fabian A, Tenen DG, Gilliland DG.. PML-RARalpha Sherwood RI, Yamaki M, Dimos JT, Chen AE, Melton DA, initiates leukemia by conferring properties of self-renewal McMahon AP, Eggan K.. Sox17 promotes differentiation in to committed promyelocytic progenitors. Leukemia. 2009 mouse embryonic stem cells by directly regulating Aug;23(8):1462-71. Epub 2009 Mar 26. extraembryonic gene expression and indirectly antagonizing self-renewal. Genes Dev. 2010 Feb Yamanaka S.. Elite and stochastic models for induced 1;24(3):312-26. pluripotent stem cell generation. Nature. 2009 Jul 2;460(7251):49-52. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M.. Direct conversion of fibroblasts to Yechoor V, Liu V, Espiritu C, Paul A, Oka K, Kojima H, functional neurons by defined factors. Nature. 2010 Feb Chan L.. Neurogenin3 is sufficient for transdetermination of 25;463(7284):1035-41. Epub 2010 Jan 27. hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes. Dev Cell. 2009 Bar-Nur O, Russ HA, Efrat S, Benvenisty N.. Epigenetic Mar;16(3):358-73. memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human Zhu L, Gibson P, Currle DS, Tong Y, Richardson RJ, pancreatic islet Beta cells. Cell Stem Cell. 2011 Jul Bayazitov IT, Poppleton H, Zakharenko S, Ellison DW, 8;9(1):17-23. doi: 10.1016/j.stem.2011.06.007. Gilbertson RJ.. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature. 2009 Hanahan D, Weinberg RA.. Hallmarks of cancer: the next Jan 29;457(7229):603-7. Epub 2008 Dec 17. generation. Cell. 2011 Mar 4;144(5):646-74. (REVIEW) Kim K, Doi A, Wen B, Ng K, Zhao R, Cahan P, Kim J, Rinkevich Y, Lindau P, Ueno H, Longaker MT, Weissman Aryee MJ, Ji H, Ehrlich LI, Yabuuchi A, Takeuchi A, IL.. Germ-layer and lineage-restricted stem/progenitors Cunniff KC, Hongguang H, McKinney-Freeman S, regenerate the mouse digit tip. Nature. 2011 Aug Naveiras O, Yoon TJ, Irizarry RA, Jung N, Seita J, Hanna 24;476(7361):409-13. doi: 10.1038/nature10346. J, Murakami P, Jaenisch R, Weissleder R, Orkin SH, Weissman IL, Feinberg AP, Daley GQ.. Epigenetic This article should be referenced as such: memory in induced pluripotent stem cells. Nature. 2010 Sep 16;467(7313):285-90. Campos-Sanchez E, Sanchez-Garcia I, Cobaleda C. Plasticity and Tumorigenicity. Atlas Genet Cytogenet Lazebnik Y.. What are the hallmarks of cancer? Nat Rev Oncol Haematol. 2012; 16(3):236-250. Cancer. 2010 Apr;10(4):232-3.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 250

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Deep Insight Section

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI V-ATPaseInCancerID20104.txt

Vacuolar H+-ATPase (V-ATPase) is a highly hydrolysis and the latter providing evolutionarily conserved enzyme, which is transmembraneous proton channel (Nishi et al., distributed within the plasma membranes and the 2002; Yokoyama et al., 2005; Wang al., 2007). The membranes of some organelles such as endosome, core of the V1 section is composed of a hexameric lysosome and secretory vesicle. The mayor function arrangement of alternating A and B subunits, which of V-ATPase is to pump protons across the cell participate in ATP binding and hydrolysis. Other membrane to extracellular milieu or across the subunits of V1 include three copies of E and G organelle membrane to intracellular compartments. subunits which are the stator, one copy of the V-ATPases located in cell surface act as important regulatory C and H subunits, one copy of subunits proton transporters that regulate the cytosolic pH to D and F which form a central rotor axle. The V0 ~7.0 which is essential for most physiological section includes a ring of proteolipid subunits (c, c' processes, whereas V-ATPases within intracellular and c") that are adjacent to subunits a and e. membrane are involved in cellular processes as Subunits D and F of V1 and subunit a of V0 form receptor-mediated endocytosis, membrane the central stalk, whereas the multiple peripheral trafficking, protein processing or degradation, and stalks are composed of subunits C, E, G, H and the nutrients uptake (Nishi et al., 2002; Forgac et al., N-terminal domain of subunit a. V1 and V0 is 2007; Toei et al., 2010; Cruciat et al., 2010). connected by both stalks. Several subunits like a, d, Malfunctioned V-ATPase is closely related to e, C, G, H, D and F contain slice variants as to several diseases including tumor. More and more spatial and temporal expression pattern in different evidences indicate that V-ATPase is an enhancer cell types (Forgac et al., 2007; Miranda et al., for carcinogenesis and cancer progression, such as 2010). As for tumor cells, especially those with malignant transformation, growth and proliferation, high metastatic potential, the V-ATPases are invasion and metastasis, acquirement of multi-drug usually excessively agitated. The altered structures resistance, etc., which strongly supports that V- of V-ATPase of tumor cells may include the ATPase should be an effective target of anticancer increased level of subunit expressions and unique strategy (Fais et al., 2007). spliced variants of some subunits. The level of the subunit c expression was found to The structure of V-ATPases and be related to the metastasis potentials in tumors. its expression in tumor cells One of the studies is the comparison of subunit c The molecular structure of normal V-ATPase of expression between normal and pancreatic yeast and mammalian cells has been well studied. carcinoma tissues and between invasive and non- V-ATPase is a delicate complex which is composed invasive pancreatic cancers, which of a cytosolic catalytic domain V1 and an integral immunohistochemical data showed the notable domain V0, the former responsible for ATP difference - 92% invasive ductal cancers (42/46)

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 251

Vacuolar H(+)-ATPase in Cancer Cells: Structure and Lu X, Qin W Function were mild to marked subunit c positive in the et al., 2002). The ability to extrude intracellular cytoplasm, whereas neither non-invasive ductal protons and maintain the cytosol pH is critical for cancers nor benign cystic neoplasms expressed cancer cell survival from a cascade of self-digestion detectable immunoreactive proteins (Ohta et al., triggered by acidosis. 1996). Subunit c seems to be one of the V-ATPase The inhibition of v-ATPase may induce apoptotic subunit which significantly influence the cell death in several human cancer cell lines proliferation and metastasis of tumor cells. The including pancreatic cancer (Ohta et al., 1998; inhibition of the V-ATPase subunit c via siRNA Hayash et al., 2006), liver cancer (Morimura et al., resulted in the suppression of growth and metastasis 2008), gastric cancer (Nakashima et al., 2003), B- of a hepatocellular carcinoma cell line in vitro and cell hybridoma cells (Nishihara et al., 1995; De in mice model (Lu et al., 2005), which is according Milito et al., 2007) and breast cancer (McHenry et to another result of the suppression of subunit c in al., 2010).The deficiency of V-ATPase will Hela cell via antisense oligonucleotides (Zhan et al., decrease cytosol pH and increased lysosome pH, 2003). But in oral squamous cell carcinoma cells, both of which might influence lysosome function. subunit C1 was the most strongly over-expressed The apoptosis induced by V-ATPase inhibitors gene at the mRNA level compared to other genes of were in either lysosome-mediated or non-lysosome- the V-ATPase complex (Otero-Rey et al., 2008). mediated manner. In the first case, when lysosomal Specific spliced variants of subunit have been V-ATPase was defected, lysosomal pH and observed in tumors. A study of expression of permeability will be increased, resulted in the subunit a of V-ATPase in breast cancer cell lines release of cathepsin D and activation of caspase, displayed the metastasis-specific subunit a isoform with no significant impact on mitochondrial expression profile. In highly metastatic breast transmembrane potential (Nakashima et al., 2003). cancer cell line compared with its lowly metastatic In the other case, mitochondria and lysosome might parallel, levels of a3 and a4 were much higher be together involved in V-ATPase-inhibitor- although all the four a isoforms - a1-4 can be induced apoptosis via capsase pathway or ROS- detectable. They distribute differently, and dependant manner (Ishisaki et al., 1999; De Milito especially, a4-containing v-ATPases were located et al., 2007). The inhibition of V-ATPase could also mainly in the plasma membrane of higher induce apoptosis by suppressing anti-apoptotic Bcl- metastatic breast cancer cell, seeming to be 2 or Bcl-xL and facilitate the caspase-independent involved in the formation of the leading surface of apoptotic pathway (Sasazawa et al., 2009). In order the cells due to the combination with F-actin and to survive from the apoptosis induced by acidosis closely correlated to the potency of invasion. a3- resulted from glycolysis, tumor cells needs to containing V-ATPases were located in intracellular extrude excessive acid, in which processes V- compartment membrane, which regulated the pH of ATPase plays a crucial role. It is reasonable to the cytosol and intracellular compartments and also postulate that the inhibition of proton extrusion may involved in invasion (Hinton et al., 2009). In be more susceptible or vulnerable to cell death of accordance with this data, the strongly expressed a3 cancer cells than normal cells. isoform were observed in high-metastatic Moreover, the slightly alkalized cytosolic pH favors melanoma cells and in bone metastases (Nishisho et the growth and proliferation of the cells. Some al., 2011). Other tumor-relevant spliced variants are glycolysis-related enzymes or oncogenes are yet to be found. sensitive the narrow range of pH alteration. Alkalization of cytosol, which mainly regulated by The roles of the v-ATPase in the V-ATPase in tumor cells, could activate glycolysis growth, proliferation or apoptosis whereas repress oxidative phosphorylation, in tumor cells meanwhile also promote the transcription of One of cancer hallmarks is the shift in energy oncogenes like HIF-1, akt, myc, ras, etc (Gillies et production from oxidative phosphorylation to al., 2008; López-Lázaro, 2008). The cytosol pH of aerobic glycolysis, ie "Warburg effect", which tumor cells was found to be higher than in produces excess intracellular acidosis (Gillies et al., untransformed controls (Busa et al.,1984; Casey et 2008). However, cancer cells usually have neutral al., 2010) and increasing cytosol pH was sufficient to alkaline intracellular pH in the acidized to confer tumourigenicity to cultured fibroblasts extracellular microenvironment. The V-ATPase is (Perona et al., 1988). On the contrast, p53, the among the four major types of pH regulators (the important tumor suppressor could be inactivated in other three are: Na+/H+ exchangers, bicarbonate the condition of alkalization (Xiao et al., 2003). It is transporters, proton/lactate symporters). Much data much likely that the glucose metabolism shift and implies proton pump is essential in tumors and cells mutant V-ATPase may be the co-selectors in seem to render V-ATPases more than any other selecting those "adaptive phenotype", which may three transporters to regulate pH in cytosol (Torigoe take the advantages for survival and proliferation during the initial stage of carcinogenesis.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 252

Vacuolar H(+)-ATPase in Cancer Cells: Structure and Lu X, Qin W Function

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

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 253

Vacuolar H(+)-ATPase in Cancer Cells: Structure and Lu X, Qin W Function directly independent of the whole enzyme V- process, endothelial cells is mainly involved. It was ATPase function. For example, transfectants which documented that V- ATPases play a crucial role in over express V-ATPase subunit c at the mRNA growth and phenotypic modulation of level showed an enhance invasiveness in vitro with myofibroblasts that contribute to neointimal a concomitant increases in secretion of matrix formation in cultured human saphenous vein (Otani metalloproteinase-2 (Kubota et al., 2000). V- et al., 2000) The microvascular endothelial cells in ATPase may also regulate metastasis by enhancing tumor tissue also incline to render plasma proteases activation. Cathepsin is an example, membrane V-ATPase to cope with the acidic which is secreted by several types of tumor cells extracellular environment. The ability of migration and related to invasion. Once the extracellular of endothelial cell toward the adjacent tissue is cathepsin is activated, it can both degrade required during angiogenesis, in which process V- extracellular matrix proteins and activate other ATPase plays a role, shown in the result that the secreted proteases involved in invasion, such as penetration of basement membrane of endothelial matrix metalloprotease (Joyce et al., 2004; Gocheva cell was suppressed by bafilomycin treatment et al., 2007) and gelatinases (Martínez-Zaguilá et (Rojas et al., 2006). al., 1996). The plasma membrane V-ATPase appeared to be recruited at the proceeding edge of The relations of V-ATPase and the cancer cell by the interaction with F-actin so as drug resistance in cancer to give rise an acidic microenvironment by the edge Acquired multidrug resistance (MDR) can limit (Hinton et al., 2009). Moreover, intracellular V- therapeutic potential and one of the reasons of ATPases, the major contributor of acidity of relapse. It is well known that MDR is correlate to intracellular compartment and membrane the evolutionarily conserved family of the ATP trafficking regulator, also facilitate in the invasion binding cassette (ABC) proteins pg, yet it is and metastasis, which is due to possible modulating documented that V-ATPase plays a role in MDR in proteolytic activation of cathepsins or matrix a pg-independent manner, and the inhibition of V- metalloproteases within lysosomes or secretory ATPase could not only suppress tumor cells vesicles and targeting the proteases-containing directly, but also sensitize the tumor cells to the secretory vesicles to the cell surface to be chemical therapy (De Milito et al., 2005). It was extracytosed (Hinton et al., 2009). The documented that proton pump inhibitor (PPI) accumulation of acidity, concentration of plasma pretreatment sensitized tumor cell lines to the membrane V-ATPase and activated protease crown effects of cisplatin, 5-fluorouracil, and vinblastine the proceeding surface of a metastatic cell, significantly. PPI treatment will increases both conferring the tumor cell a "cutting edge". extracellular pH and the pH of lysosomal Mobility is crucial for spread of tumor cells to the organelles, which induced a marked increase in the distant sites. NiK-12192, one of V-ATPase cytoplasmic retention of the cytotoxic drugs, with inhibitor was shown able to reduce the clear targeting to the nucleus in the case of migration/invasion of human lung cancer cells in doxorubicin. In vivo experiments, oral pretreatment vitro and significantly reduce the number of with omeprazole was able to induce sensitivity of spontaneous metastases in the lung of nude mice human solid tumors to cisplatin (Lucian et al., implanted with a human lung carcinoma. After the 2004). treatment of NiK-12192, the lung cancer cells in V-ATPase renders several mechanisms of vitro showed that actin fibers were broken, spots of multidrug resistance including: neutralized drug aggregation were evident and no pseudopodia and extracellularly or intracellularly, decreased drug regular structure for actin filaments could be seen, internalization, altered DNA repair and inhibition of comparing to the control cells with long and regular apoptosis. The pH of the tumor microenvironment fibers of tubulin in the cell cytoplasm and filaments may influence the uptake of anticancer drugs. of actin forming pseudopodia. NiK-12192-treated Molecules diffuse passively across the cell cells also demonstrate a reduction in the experiment membrane most efficiently in the uncharged form. of wound healing assay due to the retard of Because the extracellular pH in tumors is low and migration (Supino et al., 2008). V-ATPase subunit the intracellular pH of tumor cells is neutral to B and C appear to contain the binding sites to the alkaline, weakly basic drugs that have an acid actin cytoskeleton (Vitavska et al., 2003; Vitavska dissociation constant of 7.5-9.5, such as et al., 2005; Zuo et al., 2006). The interactions doxorubicin, mitoxantrone, vincristine, and between V-ATPase and cytoskeleton implicate their vinblastine, are protonated and display decreased involvement and regulation of cell mobility and cellular uptake (Raghunand et al., 1999; Gerweck et membrane trafficking (Sun-Wada et al., 2009). al., 2006; McCarty and Whitaker, 2010). The data Angiogenesis, a consequence of the mutual in vitro or in animal models indicates that interaction between cancer cells and the stoma cells extracellular alkalinization leads to substantial of extracellular microenvironments, is another improvement in the therapeutic effectiveness of important step during metastasis, during which antitumor drugs via enhanced the cellular drug

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 254

Vacuolar H(+)-ATPase in Cancer Cells: Structure and Lu X, Qin W Function

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. uptake and cytotoxicity (Gerweck et al., 2006; That the defects of V-ATPase increase the Trédan et al., 2007).The reduced intracellular sensitivity to drugs may be partly due to the accumulation of anticancer drugs may also be due decreased cytosolic pH, which were observed in the that V-ATPase has a role as cooperating factor of influence of cisplatin on the V-ATPase mutant ATP-dependent membrane proteins that function as yeast Saccharomyces cerevisiae (Liao et al., 2006) drug efflux pumps (Raghunand et al., 1999). or increased toxicity of combined treatment of V- Interestingly, the levels of V-ATPase subunit ATPase inhibition and anticancer drug on lung expressions can be up-regulated by anticancer drug. cancer cell, breast cancer or liver cancer cell lines The treatment of cisplatin on human epidermoid (Wong et al., 2005; Farina et al., 2006; You et al., cancer KB cells increased the protein levels of the 2009). At low cytosolic pH, sensitivity to DNA majority of the subunits such as c, c", D, a, A, C damaging drugs or UV irradiation in V-ATPase and E, which indicates it may stimulate the mutants may be associated with altered DNA expression of the V-ATPase complex as a whole. It conformation or defective DNA damage repair is suggested that the V-ATPase expression may be mechanisms, rendering DNA more prone to a defensive response to the anticancer drug damage (Robinson et al., 1992; Petrangolini et al., (Murakami et al., 2001; Torigoe et al., 2002). Still, 2006; Liao et al., 2006). there are also some controversial results on the relationship between the cationic drugs uptake and Conclusions V-ATPase - the inhibition of V-ATPase decreased According to the roles V-ATPase in tumor cells, we the uptake of the cationic drugs (Morissette et al., conclude that alteration of V-ATPase is much likely 2009; Marceau et al., 2009), which might be the necessary initial step of transformation of the explained that the influence of V-ATPase on the malignant cells and the malfunctional V-ATPase drug uptake may also be depend upon the acts as a continual enhancer of carcinogenesis and characteristics of the drugs and its relation to tumor progression. Tumor cells take the advantages membrane trafficking. of disfunctioned plasma and intracellular V-ATPase in these aspects: enhanced proliferation and growth,

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 255

Vacuolar H(+)-ATPase in Cancer Cells: Structure and Lu X, Qin W Function evading apoptosis, facilitating metastasis and RJ. Enhancement of chemotherapy by manipulation of angiogenesis, and acquirement of the drug tumour pH. Br J Cancer. 1999 Jun;80(7):1005-11 resistance. V-ATPase will be a prospective Kubota S, Seyama Y. Overexpression of vacuolar ATPase candidate for cancer diagnosis and treatment. 16-kDa subunit in 10T1/2 fibroblasts enhances invasion with concomitant induction of matrix metalloproteinase-2. Biochem Biophys Res Commun. 2000 Nov 19;278(2):390- References 4 Busa WB, Nuccitelli R. Metabolic regulation via Otani H, Yamamura T, Nakao Y, Hattori R, Fujii H, intracellular pH. Am J Physiol. 1984 Apr;246(4 Pt 2):R409- Ninomiya H, Kido M, Kawaguchi H, Osako M, Imamura H, 38 Ohta T, Ohkuma S. Vacuolar H(+)-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts Perona R, Serrano R. Increased pH and tumorigenicity of in cultured human saphenous vein. Circulation. 2000 Nov fibroblasts expressing a yeast proton pump. Nature. 1988 7;102(19 Suppl 3):III269-74 Aug 4;334(6181):438-40 Murakami T, Shibuya I, Ise T, Chen ZS, Akiyama S, Robinson H, van der Marel GA, van Boom JH, Wang AH. Nakagawa M, Izumi H, Nakamura T, Matsuo K, Yamada Y, Unusual DNA conformation at low pH revealed by NMR: Kohno K. Elevated expression of vacuolar proton pump parallel-stranded DNA duplex with homo base pairs. genes and cellular PH in cisplatin resistance. Int J Cancer. Biochemistry. 1992 Nov 3;31(43):10510-7 2001 Sep;93(6):869-74 Clague MJ, Urbé S, Aniento F, Gruenberg J. Vacuolar Nishi T, Forgac M. The vacuolar (H+)-ATPases--nature's ATPase activity is required for endosomal carrier vesicle most versatile proton pumps. Nat Rev Mol Cell Biol. 2002 formation. J Biol Chem. 1994 Jan 7;269(1):21-4 Feb;3(2):94-103 Nishihara T, Akifusa S, Koseki T, Kato S, Muro M, Hanada Torigoe T, Izumi H, Ise T, Murakami T, Uramoto H, N. Specific inhibitors of vacuolar type H(+)-ATPases Ishiguchi H, Yoshida Y, Tanabe M, Nomoto M, Kohno K. induce apoptotic cell death. Biochem Biophys Res Vacuolar H(+)-ATPase: functional mechanisms and Commun. 1995 Jul 6;212(1):255-62 potential as a target for cancer chemotherapy. Anticancer van Weert AW, Dunn KW, Gueze HJ, Maxfield FR, Drugs. 2002 Mar;13(3):237-43 Stoorvogel W. Transport from late endosomes to Torigoe T, Izumi H, Ishiguchi H, Uramoto H, Murakami T, lysosomes, but not sorting of integral membrane proteins Ise T, Yoshida Y, Tanabe M, Nomoto M, Itoh H, Kohno K. in endosomes, depends on the vacuolar proton pump. J Enhanced expression of the human vacuolar H+-ATPase c Cell Biol. 1995 Aug;130(4):821-34 subunit gene (ATP6L) in response to anticancer agents. J Harada M, Sakisaka S, Yoshitake M, Kin M, Ohishi M, Biol Chem. 2002 Sep 27;277(39):36534-43 Shakado S, Mimura Y, Noguchi K, Sata M, Tanikawa K. Nakashima S, Hiraku Y, Tada-Oikawa S, Hishita T, Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)- Gabazza EC, Tamaki S, Imoto I, Adachi Y, Kawanishi S. ATPases, inhibits the receptor-mediated endocytosis of Vacuolar H+-ATPase inhibitor induces apoptosis via asialoglycoproteins in isolated rat hepatocytes. J Hepatol. lysosomal dysfunction in the human gastric cancer cell line 1996 May;24(5):594-603 MKN-1. J Biochem. 2003 Sep;134(3):359-64 Martínez-Zaguilán R, Seftor EA, Seftor RE, Chu YW, Vitavska O, Wieczorek H, Merzendorfer H. A novel role for Gillies RJ, Hendrix MJ. Acidic pH enhances the invasive subunit C in mediating binding of the H+-V-ATPase to the behavior of human melanoma cells. Clin Exp Metastasis. actin cytoskeleton. J Biol Chem. 2003 May 1996 Mar;14(2):176-86 16;278(20):18499-505 Ohta T, Numata M, Yagishita H, Futagami F, Tsukioka Y, Xiao H, Li TK, Yang JM, Liu LF. Acidic pH induces Kitagawa H, Kayahara M, Nagakawa T, Miyazaki I, topoisomerase II-mediated DNA damage. Proc Natl Acad Yamamoto M, Iseki S, Ohkuma S. Expression of 16 kDa Sci U S A. 2003 Apr 29;100(9):5205-10 proteolipid of vacuolar-type H(+)-ATPase in human pancreatic cancer. Br J Cancer. 1996 Jun;73(12):1511-7 Zhan H, Yokoyama K, Otani H, Tanigaki K, Shirota N, Takano S, Ohkuma S. Different roles of proteolipids and Harada M, Shakado S, Sakisaka S, Tamaki S, Ohishi M, 70-kDa subunits of V-ATPase in growth and death of Sasatomi K, Koga H, Sata M, Tanikawa K. Bafilomycin A1, cultured human cells. Genes Cells. 2003 Jun;8(6):501-13 a specific inhibitor of V-type H+-ATPases, inhibits the acidification of endocytic structures and inhibits Joyce JA, Hanahan D. Multiple roles for cysteine horseradish peroxidase uptake in isolated rat sinusoidal cathepsins in cancer. Cell Cycle. 2004 Dec;3(12):1516-619 endothelial cells. Liver. 1997 Oct;17(5):244-50 Luciani F, Spada M, De Milito A, Molinari A, Rivoltini L, Presley JF, Mayor S, McGraw TE, Dunn KW, Maxfield FR. Montinaro A, Marra M, Lugini L, Logozzi M, Lozupone F, Bafilomycin A1 treatment retards transferrin receptor Federici C, Iessi E, Parmiani G, Arancia G, Belardelli F, recycling more than bulk membrane recycling. J Biol Fais S. Effect of proton pump inhibitor pretreatment on Chem. 1997 May 23;272(21):13929-36 resistance of solid tumors to cytotoxic drugs. J Natl Cancer Inst. 2004 Nov 17;96(22):1702-13 Ohta T, Arakawa H, Futagami F, Fushida S, Kitagawa H, Kayahara M, Nagakawa T, Miwa K, Kurashima K, Numata Sennoune SR, Bakunts K, Martínez GM, Chua-Tuan JL, M, Kitamura Y, Terada T, Ohkuma S. Bafilomycin A1 Kebir Y, Attaya MN, Martínez-Zaguilán R. Vacuolar H+- induces apoptosis in the human pancreatic cancer cell line ATPase in human breast cancer cells with distinct Capan-1. J Pathol. 1998 Jul;185(3):324-30 metastatic potential: distribution and functional activity. Am J Physiol Cell Physiol. 2004 Jun;286(6):C1443-52 Ishisaki A, Hashimoto S, Amagasa T, Nishihara T. Caspase-3 activation during the process of apoptosis De Milito A, Fais S. Tumor acidity, chemoresistance and induced by a vacuolar type H(+)-ATPase inhibitor. Biol proton pump inhibitors. Future Oncol. 2005 Dec;1(6):779- Cell. 1999 Sep;91(7):507-13 86 Raghunand N, He X, van Sluis R, Mahoney B, Baggett B, Lu X, Qin W, Li J, Tan N, Pan D, Zhang H, Xie L, Yao G, Taylor CW, Paine-Murrieta G, Roe D, Bhujwalla ZM, Gillies Shu H, Yao M, Wan D, Gu J, Yang S. The growth and metastasis of human hepatocellular carcinoma xenografts

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 256

Vacuolar H(+)-ATPase in Cancer Cells: Structure and Lu X, Qin W Function

are inhibited by small interfering RNA targeting to the Roy M, Pear WS, Aster JC. The multifaceted role of Notch subunit ATP6L of proton pump. Cancer Res. 2005 Aug in cancer. Curr Opin Genet Dev. 2007 Feb;17(1):52-9 1;65(15):6843-9 Trédan O, Galmarini CM, Patel K, Tannock IF. Drug Vitavska O, Merzendorfer H, Wieczorek H. The V-ATPase resistance and the solid tumor microenvironment. J Natl subunit C binds to polymeric F-actin as well as to Cancer Inst. 2007 Oct 3;99(19):1441-54 monomeric G-actin and induces cross-linking of actin filaments. J Biol Chem. 2005 Jan 14;280(2):1070-6 Wang Y, Cipriano DJ, Forgac M. Arrangement of subunits in the proteolipid ring of the V-ATPase. J Biol Chem. 2007 Wong P, Lee C, Tannock IF. Reduction of intracellular pH Nov 23;282(47):34058-65 as a strategy to enhance the pH-dependent cytotoxic effects of melphalan for human breast cancer cells. Clin Otero-Rey EM, Somoza-Martín M, Barros-Angueira F, Cancer Res. 2005 May 1;11(9):3553-7 García-García A. Intracellular pH regulation in oral squamous cell carcinoma is mediated by increased V- Yokoyama K, Imamura H. Rotation, structure, and ATPase activity via over-expression of the ATP6V1C1 classification of prokaryotic V-ATPase. J Bioenerg gene. Oral Oncol. 2008 Feb;44(2):193-9 Biomembr. 2005 Dec;37(6):405-10 Gillies RJ, Robey I, Gatenby RA. Causes and Gerweck LE, Vijayappa S, Kozin S. Tumor pH controls the consequences of increased glucose metabolism of in vivo efficacy of weak acid and base chemotherapeutics. cancers. J Nucl Med. 2008 Jun;49 Suppl 2:24S-42S Mol Cancer Ther. 2006 May;5(5):1275-9 López-Lázaro M. The warburg effect: why and how do Hayashi Y, Katayama K, Togawa T, Kimura T, Yamaguchi cancer cells activate glycolysis in the presence of oxygen? A. Effects of bafilomycin A1, a vacuolar type H+ ATPase Anticancer Agents Med Chem. 2008 Apr;8(3):305-12 inhibitor, on the thermosensitivity of a human pancreatic cancer cell line. Int J Hyperthermia. 2006 Jun;22(4):275-85 Marshansky V, Futai M. The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function. Curr Hurtado-Lorenzo A, Skinner M, El Annan J, Futai M, Sun- Opin Cell Biol. 2008 Aug;20(4):415-26 Wada GH, Bourgoin S, Casanova J, Wildeman A, Bechoua S, Ausiello DA, Brown D, Marshansky V. V- Morimura T, Fujita K, Akita M, Nagashima M, Satomi A. ATPase interacts with ARNO and Arf6 in early endosomes The proton pump inhibitor inhibits cell growth and induces and regulates the protein degradative pathway. Nat Cell apoptosis in human hepatoblastoma. Pediatr Surg Int. Biol. 2006 Feb;8(2):124-36 2008 Oct;24(10):1087-94 Miele L, Golde T, Osborne B. Notch signaling in cancer. Supino R, Petrangolini G, Pratesi G, Tortoreto M, Favini E, Curr Mol Med. 2006 Dec;6(8):905-18 Bo LD, Casalini P, Radaelli E, Croce AC, Bottiroli G, Misiano P, Farina C, Zunino F. Antimetastatic effect of a Petrangolini G, Supino R, Pratesi G, Dal Bo L, Tortoreto M, small-molecule vacuolar H+-ATPase inhibitor in in vitro Croce AC, Misiano P, Belfiore P, Farina C, Zunino F. and in vivo preclinical studies. J Pharmacol Exp Ther. Effect of a novel vacuolar-H+-ATPase inhibitor on cell and 2008 Jan;324(1):15-22 tumor 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- Sahagian GG, Jay D, Martinez-Zaguilan R, Forgac M. type H+-ATPases at the plasma membrane regulate pH Function of a subunit isoforms of the V-ATPase in pH and cell migration in microvascular endothelial cells. Am J homeostasis and in vitro invasion of MDA-MB231 human Physiol Heart Circ Physiol. 2006 Sep;291(3):H1147-57 breast cancer cells. J Biol Chem. 2009 Jun 12;284(24):16400-8 Zuo J, Jiang J, Chen SH, Vergara S, Gong Y, Xue J, Huang H, Kaku M, Holliday LS. Actin binding activity of Marceau F, Bawolak MT, Bouthillier J, Morissette G. subunit B of vacuolar H+-ATPase is involved in its Vacuolar ATPase-mediated cellular concentration and targeting to ruffled membranes of osteoclasts. J Bone retention of quinacrine: a model for the distribution of Miner Res. 2006 May;21(5):714-21 lipophilic cationic drugs to autophagic vacuoles. Drug Metab Dispos. 2009 Dec;37(12):2271-4 De Milito A, Iessi E, Logozzi M, Lozupone F, Spada M, Marino ML, Federici C, Perdicchio M, Matarrese P, Lugini Morissette G, Ammoury A, Rusu D, Marguery MC, Lodge L, Nilsson A, Fais S. Proton pump inhibitors induce R, Poubelle PE, Marceau F. Intracellular sequestration of apoptosis of human B-cell tumors through a caspase- amiodarone: role of vacuolar ATPase and independent mechanism involving reactive oxygen macroautophagic transition of the resulting vacuolar species. Cancer Res. 2007 Jun 1;67(11):5408-17 cytopathology. Br J Pharmacol. 2009 Aug;157(8):1531-40 Fais S, De Milito A, You H, Qin W. Targeting vacuolar H+- Sasazawa Y, Futamura Y, Tashiro E, Imoto M. Vacuolar ATPases as a new strategy against cancer. Cancer Res. H+-ATPase inhibitors overcome Bcl-xL-mediated 2007 Nov 15;67(22):10627-30 chemoresistance through restoration of a caspase- independent apoptotic pathway. Cancer Sci. 2009 Forgac M. Vacuolar ATPases: rotary proton pumps in Aug;100(8):1460-7 physiology and pathophysiology. Nat Rev Mol Cell Biol. 2007 Nov;8(11):917-29 Sun-Wada GH, Tabata H, Kawamura N, Aoyama M, Wada Y. Direct recruitment of H+-ATPase from lysosomes for Gocheva V, Joyce JA. Cysteine cathepsins and the cutting phagosomal acidification. J Cell Sci. 2009 Jul 15;122(Pt edge of cancer invasion. Cell Cycle. 2007 Jan 1;6(1):60-4 14):2504-13 Liao C, Hu B, Arno MJ, Panaretou B. Genomic screening Yan Y, Denef N, Schüpbach T. The vacuolar proton pump, in vivo reveals the role played by vacuolar H+ ATPase and V-ATPase, is required for notch signaling and endosomal cytosolic acidification in sensitivity to DNA-damaging trafficking in Drosophila. Dev Cell. 2009 Sep;17(3):387-402 agents such as cisplatin. Mol Pharmacol. 2007 Feb;71(2):416-25 You H, Jin J, Shu H, Yu B, De Milito A, Lozupone F, Deng Y, Tang N, Yao G, Fais S, Gu J, Qin W. Small interfering RNA targeting the subunit ATP6L of proton pump V-

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 257

Vacuolar H(+)-ATPase in Cancer Cells: Structure and Lu X, Qin W Function

ATPase overcomes chemoresistance of breast cancer O'Callaghan KM, Ayllon V, O'Keeffe J, Wang Y, Cox OT, cells. Cancer Lett. 2009 Jul 18;280(1):110-9 Loughran G, Forgac M, O'Connor R. Heme-binding protein HRG-1 is induced by insulin-like growth factor I and Buechling T, Bartscherer K, Ohkawara B, Chaudhary V, associates with the vacuolar H+-ATPase to control Spirohn K, Niehrs C, Boutros M. Wnt/Frizzled signaling endosomal pH and receptor trafficking. J Biol Chem. 2010 requires dPRR, the Drosophila homolog of the prorenin Jan 1;285(1):381-91 receptor. Curr Biol. 2010 Jul 27;20(14):1263-8 Toei M, Saum R, Forgac M. Regulation and isoform Casey JR, Grinstein S, Orlowski J. Sensors and regulators function of the V-ATPases. Biochemistry. 2010 Jun of intracellular pH. Nat Rev Mol Cell Biol. 2010 15;49(23):4715-23 Jan;11(1):50-61 Vaccari T, Duchi S, Cortese K, Tacchetti C, Bilder D. The Cruciat CM, Ohkawara B, Acebron SP, Karaulanov E, vacuolar ATPase is required for physiological as well as Reinhard C, Ingelfinger D, Boutros M, Niehrs C. pathological activation of the Notch receptor. Requirement of prorenin receptor and vacuolar H+- Development. 2010 Jun;137(11):1825-32 ATPase-mediated acidification for Wnt signaling. Science. 2010 Jan 22;327(5964):459-63 Chung C, Mader CC, Schmitz JC, Atladottir J, Fitchev P, Cornwell ML, Koleske AJ, Crawford SE, Gorelick F. The McCarty MF, Whitaker J. Manipulating tumor acidification vacuolar-ATPase modulates matrix metalloproteinase as a cancer treatment strategy. Altern Med Rev. 2010 isoforms in human pancreatic cancer. Lab Invest. 2011 Sep;15(3):264-72 May;91(5):732-43 McHenry P, Wang WL, Devitt E, Kluesner N, Davisson VJ, Nishisho T, Hata K, Nakanishi M, Morita Y, Sun-Wada GH, McKee E, Schweitzer D, Helquist P, Tenniswood M. Wada Y, Yasui N, Yoneda T. The a3 isoform vacuolar type Iejimalides A and B inhibit lysosomal vacuolar H+-ATPase H⁺ -ATPase promotes distant metastasis in the mouse (V-ATPase) activity and induce S-phase arrest and B16 melanoma cells. Mol Cancer Res. 2011 Jul;9(7):845- apoptosis in MCF-7 cells. J Cell Biochem. 2010 Mar 55 1;109(4):634-42 Miranda KC, Karet FE, Brown D. An extended This article should be referenced as such: nomenclature for mammalian V-ATPase subunit genes Lu X, Qin W. Vacuolar H(+)-ATPase in Cancer Cells: and splice variants. PLoS One. 2010 Mar 10;5(3):e9531 Structure and Function. Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):251-258.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 258

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Case Report Section Paper co-edited with the European LeukemiaNet

A 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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI t1122q23q13MohamedID100059.txt

Clinics Pathology Bone marrow aspirate appeared hypocellular with Age and sex 95% lymphoblasts of L1 morphology, 2% myeloid 14 months old male patient. series, 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 9 Treatment: Methotrexate, Cytarabine, Vincristine, WBC : 33 X 10 /l Dexamethasone, PEG-aspargase HB : 2.6g/dl Complete remission : no Platelets : 1 X 109/l Treatment related death : no Blasts : 72% Relapse : no Bone marrow : 100 bone marrow blast replacement. Status: Alive. Last follow up: 10-2011 Survival: 9 months Cyto-Pathology Classification Karyotype Sample: Bone marrow aspirate Cytology Acute lymphoblastic leukemia (ALL) with L1 Culture time: 24hr without stimulant and 48hr morphology with 10% conditioned medium. Banding: GTG Immunophenotype Flow cytometry of bone marrow aspirate identified Results a dim CD45 lymphoblast population (85%) 46,Y,der(X)t(X;9)(p11.1;q11),add(9)(q11),t(11;22)( expressing HLA-DR, CD19 and partially q23;q13)[20] (see Figure 1). Post induction bone expressing CD10, CD22, CD9 and CD40. marrow study demonstrated a normal 46,XY karyotype. Rearranged Ig Tcr

Not performed.

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 259

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

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.

(unfused), however on a previously G-banded Other Molecular Studies metaphase it appeared that the BCR signals Technics: remained on chromosome 22 while one ABL signal Fluorescence in situ hybridization (FISH) using the was translocated to der(X). The remaining probes ALL panel DNA probes including CEP 4, 10, and produced a normal hybridization pattern. 17 alpha satellite probes, LSI MLL dual-color break apart probe, BCR/ABL and TEL/AML1 dual-fusion Comments translocation probes was performed (Abbott The patient described here is a 14 month-old-male Molecular, Downers Grove, IL). presented with an upper respiratory tract infection Results: unresponsive to antibiotics. Subsequently he was Hybridization with MLL probe produced a diagnosed with high risk B-precursor ALL due to split/translocation pattern in 61% of interphase the positivity of MLL/11q23 rearrangement. The cells. Metaphase FISH showed that the telomeric patient was started on a Children's Oncology Group region of MLL gene was translocated to 22q13 induction chemotherapy protocol. Secondary to his distal to BCR (Figure 2). The hybridization with the high risk status, the patient is being evaluated for a BCR/ABL probe showed two signals each bone marrow transplant. At time of diagnosis

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 260

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

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 chromosome analysis revealed the presence whether EP300 or other gene is involved in the t(11;22)(q23;q13) in all 20 metaphases and present case which may be responsible for the rearrangement of the MLL gene. different phenotype of this leukemia. Translocations involving the MLL/11q23 region are the most common genomic aberrations in infant References ALL seen in ~80% of cases (Raimondi, 2004). Ida K, Kitabayashi I, Taki T, Taniwaki M, Noro K, Generally leukemia harboring MLL translocation is Yamamoto M, Ohki M, Hayashi Y. Adenoviral E1A- clinically aggressive and associated with poor associated protein p300 is involved in acute myeloid prognosis. The most common chromosomes leukemia with t(11;22)(q23;q13). Blood. 1997 Dec involved in 11q23 translocations are t(4;11) 15;90(12):4699-704 followed by t(11;19) and t(9;11). Additionally, Raimondi SC.. 11q23 rearrangements in childhood acute leukemia with MLL/11q23 translocations are lymphoblastic leukemia. Atlas Genet Cytogenet Oncol frequently associated with over expression of Haematol. February 2004. URL : http://AtlasGeneticsOncology.org/Anomalies/11q23ChildAL FLT3, therefore, targeted therapy inhibitors of LID1321.html . FLT3 (a tyrosine kinase) may be beneficial for Ohnishi H, Taki T, Yoshino H, Takita J, Ida K, Ishii M, those patients. Currently there are only three Nishida K, Hayashi Y, Taniwaki M, Bessho F, Watanabe reported cases in the literature with T.. A complex t(1;22;11)(q44;q13;q23) translocation t(11;22)(q23;q13), unlike our case all having causing MLL-p300 fusion gene in therapy-related acute secondary acute myeloid leukemia with prior myeloid leukemia. Eur J Haematol. 2008 Dec;81(6):475- therapy of topoisomerase II inhibitor (table 1). 80. Epub 2008 Sep 6. Moreover, rearrangement of the MLL gene and Duhoux FP, De Wilde S, Ameye G, Bahloula K, Medves S, MLL-EP300 fusion gene were demonstrated in Lege G, Libouton JM, Demoulin JB, A Poirel H.. Novel variant form of t(11;22)(q23;q13)/MLL-EP300 fusion those three cases (Ida et al., 1997; Ohnishi et al., transcript in the evolution of an acute myeloid leukemia 2008; Duhoux et al., 2011). The clinical with myelodysplasia-related changes. Leuk Res. 2011 presentation of our case is quit different from these Mar;35(3):e18-20. Epub 2010 Oct 25. three cases. Although our case had a rearrangement This article should be referenced as such: of the MLL/11q23 gene, the MLL-EP300 fusion gene was not tested. Because the partner genes Kremer JD, Mohamed AN. A case of Acute Lymphoblastic involved in MLL/11q23 translocations are Leukemia with rare t(11;22)(q23;q13). Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3):259-261. markedly heterogeneous, it remains unclear

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 261

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

Case Report Section Paper co-edited with the European LeukemiaNet

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 Printable original version : http://documents.irevues.inist.fr/bitstream/DOI ins16q22p13p13MohamID100058.txt

Clinics Immunophenotype Flow cytometry of bone marrow aspirate identified Age and sex a significant population of myeloblasts (49%) 17 years old female patient. expressing CD34, HLA-DR, CD9, CD13, CD33, Previous history CD117 and partially expressing CD15, CD11b, and No preleukemia, no previous malignancy, inborn CD64. A second population of monocytes is also condition of note. Thalassemia trait carrier. identified (37%) expressing CD4, CD14, CD15, CD36 and CD64. Organomegaly Hepatomegaly, splenomegaly, enlarged lymph Rearranged Ig Tcr: Not performed. nodes, no central nervous system involvement. Pathology Bone marrow aspirate revealed myeloblasts, Blood monoblasts, monocytes, and increased eosinophils 9 many of which had abnormal granules (FAB AML- WBC : 138.7 X 10 /l M4eos). HB : 6.9g/dl Platelets : 51 X 109/l Electron microscopy: Not performed. Blasts : 76% Diagnosis Bone marrow : 100 Bone marrow biopsy was Acute myelomonocytic leukemia with abnormal hypercellular (100%) and replaced by myeloblasts eosinophils (AML-M4eos) and CBFB/16q22 and monoblasts. Normal hematopoiesis was greatly rearrangement. decreased and there was prominent hemophagocytosis. The majority of the blasts were Survival myeloperoxidase positive however another smaller component of blasts was nonspecific esterase Date of diagnosis: 03-2011 positive. Treatment: Intrathecal methotrexate, hydrocortisone, and cytarabine. Cyto-Pathology Treatment related death : no Classification Relapse : no Status: Alive. Last follow up: 09-2011 Cytology: Acute myeloid leukemia with abnormal Survival: 6 months eosinophils (AML-M4eos).

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 262

Insertion as an alternative mechanism of CBFB-MYH11 gene Hussein Y, et al. fusion in a new case of acute myeloid leukemia with an abnormal chromosome 16

probes (Abbott Molecular IL, USA), and Karyotype CBFB/MYH11 dual fusion translocation DNA Sample: Bone marrow probe (Cytocell Inc. Cambridge, UK) were Culture time: 24 hrs without stimulating agents performed. and 48 hrs with 10% conditioned medium. Results: Banding: GTG The hybridization with the CBFB break-apart probe produced a split pattern in 62% of interphase cells. Results On metaphase cells, the 5'CBFB (SepctrumRed) At time of diagnosis abnormal metaphase cells with and 3'CBFB (SepctrumGreen) signals stayed on the the following karyotype was found; 16q, instead of 5'CBFB being relocated to 16p as 46,XX,ins(16)(q22p13p13)[20] (see Figure 1). seen in the standard inv(16). The CBFB signals Remission bone marrow on 4/20/2011 and were separated but maintained the orientation 9/13/2011 revealed a normal female karyotype; pattern of the 5' and 3' probe, suggesting they were 46,XX[20]. split by an insertion (Figure 2A). Subsequently, using the CBFB-MYH11 probe on metaphases Other Molecular Studies showed that MYH11 signal on 16p moved and Technics: juxtaposed to CBFB on 16q, confirming the Fluorescence in situ hybridization (FISH) using LSI insertion of MYH11 into CBFB (Figure 2B). CBFB dual color break-apart rearrangement DNA

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) 263

Insertion as an alternative mechanism of CBFB-MYH11 gene Hussein Y, et al. fusion in a new case of acute myeloid leukemia with an abnormal chromosome 16

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).

the formation of this fusion. FISH is highly Comments recommended to characterize unusual abnormalities The patient described here is a 17 year old female of chromosome 16 and to confirm the CBFB- presented with upper respiratory tract infection and MYH11 fusion. bruises for 2 weeks. Subsequently she was diagnosed with AML (FAB M4 eos). Cytogenetics, References performed on bone marrow aspirate revealed a Le Beau MM, Larson RA, Bitter MA, Vardiman JW, unique structural abnormality of chromosome 16 Golomb HM, Rowley JD. Association of an inversion of which was interpreted as insertion; 46, XX, chromosome 16 with abnormal marrow eosinophils in ins(16)(q22p13p13). FISH confirmed that the acute myelomonocytic leukemia. A unique cytogenetic- clinicopathological association. N Engl J Med. 1983 Sep MYH11/p13 gene was inserted into the 15;309(11):630-6 CBFB/16q22 gene region (Figure 2B). The result of this unusual structural rearrangement was the fusion Tobal K, Johnson PR, Saunders MJ, Harrison CJ, Liu Yin JA. Detection of CBFB/MYH11 transcripts in patients with of CBFB /MYH11 genes commonly seen in inversion and other abnormalities of chromosome 16 at inv(16)(p13q22) bearing leukemia. presentation and remission. Br J Haematol. 1995 The CBFB/MYH11 gene fusion is strongly Sep;91(1):104-8 associated with AML-M4 with abnormal O'Reilly J, Chipper L, Springall F, Herrmann R. A unique eosinophils. Generally, the fusion is generated from structural abnormality of chromosome 16 resulting in a inv(16)(p13q22) or t(16;16) with the inversion CBF beta-MYH11 fusion transcript in a patient with acute being much more common than translocation (Le myeloid leukemia, FAB M4. Cancer Genet Cytogenet. 2000 Aug;121(1):52-5 Beau et al., 1983; Tobal et al., 1995). The case presented here demonstrates that insertion is This article should be referenced as such: another mechanism in producing CBFB/MYH11 Hussein Y, Kulkarni V, Mohamed AN. Insertion as an gene fusion in AML-M4eos. To our best alternative mechanism of CBFB-MYH11 gene fusion in a knowledge, there is only one reported case of new case of acute myeloid leukemia with an abnormal AML-M4 having similar structural abnormality of chromosome 16. Atlas Genet Cytogenet Oncol Haematol. chromosome 16 and CBFB/MYH11 fusion 2012; 16(3):262-264. (O'Reilly et al., 2000). These two cases suggest that insertion represents a variant rare rearrangement for

Atlas Genet Cytogenet Oncol Haematol. 2012; 16(3) 264

Atlas of Genetics and Cytogenetics

in Oncology and Haematology OPEN ACCESS JOURNAL AT INIST-CNRS

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

Subscription: The Atlas is FREE!

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

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

http://AtlasGeneticsOncology.org

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