Tumor Biol. (2015) 36:489–494 DOI 10.1007/s13277-014-3019-1

REVIEW

The tumor suppressor protein p150Sal2 in carcinogenesis Chang Kyoo Sung & Hyungshin Yim

Received: 5 November 2014 / Accepted: 23 December 2014 / Published online: 22 January 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract The transcription factor p150Sal2, the product of the SALL2 gene, was first identified as a binding target of the oncogenic mouse polyomavirus T antigen. However, mouse polyomavirus is not the only oncogenic virus that targets p150Sal2; the human papillomavirus E6 protein also targets this cellular protein in order to overcome p150Sal2-mediated growth arrest. Studies have demonstrated that p150Sal2 recognizes GC-rich regions of the promoter and transcriptionally induces p21Cip1/Waf1 and BAX in human ovarian epithelial cancer cells, resulting in cell growth arrest and apoptosis. Although the p150Sal2 protein is strongly expressed in surface epithelial cells of the ovary, immunostaining experiments showed that expression of p150Sal2 was lost in 90 % of 210 human ovarian carcinomas, supporting an important tumor suppressive role for p150Sal2 in the human ovary. Mechanisms of silencing SALL2 in OVCA cell lines and primary tumors and possible therapeutic approaches for ovarian carcinoma are discussed in this review. Keywords Mouse polyomavirus . Human papillomavirus . SALL2 . Transcriptional targets . Ovarian carcinoma . Promoter methylation

The members of the SALL family Four members of the SALL (Spalt-like) gene family are evolutionarily conserved in species ranging from Caenorhabditis C. K. Sung Department of Biological and Health Sciences, Texas A&M University-Kingsville, Kingsville, TX 78363, USA H. Yim (*) Department of Pharmacy, College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi-do 426-791, South Korea e-mail: [email protected]

elegans to human and encode multi-zinc finger transcription factors that play roles in embryonic development and genetic disorders [1, 2]. Mutations in SALL1 lead to Townes-Brocks syndrome, which is associated with a variety of developmental defects [3], whereas mutations in SALL4 result in Okihiro syndrome with defects in heart and kidney development [4, 5]. SALL4 is a critical transcription factor for pluripotency in embryonic stem cells [6]. Conditional inactivation of SALL4 during primordial germ cells specification led to a reduction in the number of mouse primordial germ cells in embryonic gonads, indicating that SALL4 is a critical factor in stem differentiation. Supporting this, SALL4 works together with Sox2 to maintain pluripotency in embryonic stem cells [7]. Furthermore, upregulation of SALL4 plays crucial roles in carcinogenesis such as gliomas and gastric cancer [8, 9]. SALL3 is another human SALL gene for which no germ line mutations have been reported. SALL3 homozygous mutant mice died after birth with abnormalities in cranial nerves [10]. It has been also reported that SALL3 regulated the development of cone photoreceptor, especially terminal differentiation of it [11]. Compared to SALL4 and SALL2, SALL3 has not been studied well. The transcription factor p150Sal2, the product of the SALL2 gene, was first identified as a binding target of the oncogenic mouse polyomavirus (Py). Mutation of SALL2 induced recessive ocular coloboma in embryonic eye development [12]. Analysis of SALL2-deficient mouse embryos revealed delayed apposition of the optic fissure margins and the persistence of an anterior retinal coloboma phenotype after birth. SALL2 is important in eye morphogenesis and that loss of function of the gene causes ocular coloboma in humans and mice [12]. During cell cycle, SALL2 functions in growth arrest and programmed cell death by transcriptionally activating p21Cip1/ Waf1 and BAX, a mechanism that implicates a possible role as a tumor suppressor [13–15]. Additionally, p150Sal2 is also required for cell cycle exit in response to growth factor

490

Tumor Biol. (2015) 36:489–494

deprivation in human foreskin fibroblasts, suggesting a role as a cellular quiescence factor [16]. Furthermore, loss of SALL2 expression is observed in many cases of human ovarian carcinoma, whereas normal ovarian epithelial cells maintain high levels of the p150Sal2 protein [17]. Together, this evidence supports the hypothesis that p150Sal2 is involved in the suppression of cancers. Here, we discuss SALL2 as a viral target, a proapoptotic regulator, and a tumor suppressor in order to elucidate possible target pathways for therapeutic approaches to suppress human ovarian carcinoma.

Polyoma large T binding protein p150Sal2 Studies of oncogenic viruses in animal and cell culture systems have yielded findings of relevance to human cancer. This relevance derives from the similarities between virus-altered host pathways and those altered in various human cancers, whether virally induced or not [18]. The polyoma mouse virus system is widely considered to be a robust and tractable mouse model of cancer [19, 20]. Decades of research in many laboratories worldwide have revealed the genetic and biochemical characteristics of this virus, most especially with regard to how they affect tumorigenesis [20, 21]. The protein networks that are changed in Py-induced tumors share many characteristics with those affected in a multitude of human cancers and highlight the broad applicability of the Py system to oncology [22–25]. A “tumor host range” selection procedure using mutant viruses and cultured normal and cancer cells employed to identify possible tumor suppressor genes yielded p150Sal2 as a binding target of the Py large T (tumor) antigen (Py LT antigen) [13]. Studies have revealed that the transcription factor p150Sal2 has proapoptotic and growth arrest functions and that the virus must override these inhibitory functions to allow viral DNA replication and cell proliferation [13, 14, 16, 26]. p150Sal2 proteins in vertebrates have four single or clustered zinc finger domains (Z1–Z4; Fig. 1). Z1 is a putative single C3H finger, whereas Z2 is a C2H2 zinc finger pair with a currently unknown cellular function. Z3 is a triple C2H2 finger cluster constituting the DNA binding domain essential for

Z1

Z2

Zinc finger

Z3

DNA binding

Z4

LT binding

Fig. 1 The p150 transcriptional factor has four zinc finger domains Z1, Z2, Z3, and Z4. The Z3 domain constitutes DNA binding for transcriptional regulation while Z4 is known to be targeted by the mouse polyomavirus large T antigen

transcriptional regulation [14, 26]. Z4 was identified as the region targeted by the Py LT antigen [13], although the functional roles of this domain in protein/gene regulation and its normal cellular targets are presently unknown. Since other SALL proteins in vertebrates lack a zinc finger domain homologous to Z4 and only the p150Sal2-Z4 domain is targeted by the oncogenic virus [2, 13], this domain may have unique partners and functions in cellular pathways that would be inhibitory to the viral life cycle, including growth arrest and proapoptotic pathways. Another small DNA tumor virus, human papillomavirus type 16, was also shown to target p150Sal2 with viral protein E6, and this interaction leads to cell cycle deregulation. Interestingly, the level of p150Sal2 is high in cells with expression of papilloma E6; however, this accumulated p150Sal2 appears to be non-functional and fails to induce its cellular target p21Cip1/Waf1 [27]. It would be worth investigating whether p150Sal2 is targeted by other viruses including Merkel cell polyomavirus and human polyomaviruses BK and JC, as this may reveal possible suppressive roles of p150Sal2 in human Merkel cell carcinogenesis and polyoma viral replication in general.

Downstream targets of SALL2 In order to understand p150Sal2-mediated transcriptional regulation, Gu et al. investigated the DNA sequence specificity of p150Sal2 binding using various molecular biological and biochemical assays [26]. These approaches revealed that the cons e ns u s b i n d i ng s eq u e n c e is th e h e p t a n u c l e o t i d e GGG(T/C)GGG and that these GC-rich elements are found in the promoter regions of p21Cip1/Waf1 and BAX and are positively regulated by the p150Sal2 protein in normal ovarian surface epithelial cells [26]. This study also determined that the triple-zinc finger motif Z3 is essential for DNA binding (Fig. 1) [26]. The consensus p150Sal2 binding site is also found in the promoter region of the c-MYC oncogene, which is frequently overexpressed in many different types of cancer. The p150Sal2 protein negatively regulates c-MYC by binding to its promoter [28]. This seems to be a general function for p150Sal2; inverse correlations in the expression of SALL2 and c-MYC in glioblastoma, lung, breast, and ovarian carcinoma have also been reported [28]. Genome-wide microarray analyses were performed to identify genes essential for the induction and maintenance of cellular quiescence in human foreskin fibroblasts [16]. Their study showed that genes involved in growth arrest depend on the SALL2 gene for their expression, although it is unclear whether these genes are direct transcriptional targets of p150Sal2 [16]. Nevertheless, it would be worth investigating whether the consensus sequence for p150Sal2 binding exists in their promoter regions and whether they are directly targeted

Tumor Biol. (2015) 36:489–494

by p150Sal2 under growth inhibitory conditions. For a complete list of the downstream targets of p150Sal2 and for a better understanding of p150Sal2-mediated gene regulation, genomewide expression profiling and chromatin immunoprecipitation sequencing and functional analyses should be performed.

SALL2 and oncogenesis It has been suggested that p150Sal2 is a tumor suppressor because the expression of SALL2 is markedly reduced in ovarian carcinomas, lung carcinoma, colorectal adenocarcinoma, prostate carcinoma, and acute myeloid leukemia [14, 15, 17, 29], and overexpression of SALL2 reduces the tumorigenicity of ovarian cancer cells in nude mice with increased expression of p21Cip1/Waf1 CDK inhibitory protein and BAX proapoptotic protein [14]. SALL2 increases p21Cip1/Waf1 promoter activity and p21Cip1/Waf1 protein expression in ovarian cells and neuronal cells [26, 30]. In addition, p150Sal2 is an essential factor for cell cycle exit when growth factors are depleted in human foreskin fibroblasts [16]. More importantly, SALL2 expression is lost in human ovarian cancer cells but not in normal ovarian cells [17]. The low level of p150Sal2 in ovarian carcinoma is explained by DNA methylation [17]; the promoter of SALL2 is hypermethylated, resulting in silencing of SALL2 and low expression level of p150Sal2 in the majority of the tumor sections tested [17]. This is similar to other tumor suppressors including p16INK4a, p15INK4b, PTEN, p53, and RUNX3 that are silenced through hypermethylation in carcinoma cells [31–34]. These data support a role of p150Sal2 in the suppression of ovarian carcinogenesis. Controversially, the expression level of SALL2 was found to be high in some cancer types when tested by microarray analyses. SALL2 was highly expressed in synovial sarcoma analyzed by tissue microarray [35], in squamous cell carcinoma of the tongue detected by oligonucleotide microarray [36], and in testicular germ cell tumor studied by transcriptome analysis [37]. Detailed analyses and follow-up investigations of these systems were not performed, and it is not clear why the SALL2 level is high in these malignant tumors. Further studies are obviously needed to understand the role of SALL2 in these tumors. Overexpression of p150Sal2 might exert different cellular effects through interactions with various proteins involved in cell growth or by abnormally regulating transcriptional targets. We reported that the interacting proteins of p150Sal2 in normal human ovarian epithelial cells are diverse and include factors involved in transcription, chromatin remodeling, DNA replication, RNA splicing, DNA repair, and transportation (Fig. 2) [38]. Thus, it is plausible that the overexpressed p150Sal2 protein in some tumors interacts with several of the above factors to cause abnormal cell growth. However, many questions remain as to why SALL2 is highly expressed in synovial sarcoma cancers, squamous cell

491

carcinoma of the tongue, and testicular germ cell tumor and how the p150Sal2 protein regulates proliferation of these cancer cells. It would also be worth studying whether (and if so, why) the promoter region of SALL2 is unmethylated in these cancers. Additional questions include whether the DNA/ protein sequences of SALL2 in these types of cancer cells are normal and whether the protein still functions in the induction of target genes such as p21Cip1cWaf1 and BAX. Studies addressing these issues will shed light on the general function of SALL2 in tumorigenesis. Interestingly, the tumor suppressor protein p53 appeared to transcriptionally repress SALL2 under genotoxic stress including doxorubicin treatment and gamma radiation [39]. The putative p53 binding site was also identified in the human SALL2 promoter region (Fig. 3b) [39]. Although the underlying mechanism of the downregulation of SALL2 by the tumor suppressor p53 is not fully understood, this negative regulation appears to be tissue specific [39]. Since p150Sal2 and p53 proteins independently induce the expression of p21Cip1/Waf1 in response to DNA damage [14] and p150Sal2 is negatively controlled by p53 [39], we may speculate that p150Sal2 would be a backup player while p53 is the major cellular factor to govern growth arrest and apoptotic pathways under certain conditions. This notion would be backed by evidence that loss of both p53 and Sall2 in mice (Sall2−/−or+/−/p53−/−) displayed significantly increased tumorigenesis and mortality rates as well as more advanced tumor metastasis compared with those found in p53 single mutant mice (Sall2+/+/p53−/−) [29].

SALL2 and human ovarian carcinoma Immunoblotting assays with various mouse tissues revealed that the p150Sal2 protein is expressed in the brain, kidney, and lung, but the highest level of expression is observed in ovarian tissue [13]. Further immunohistochemical staining showed that p150Sal2 is highly expressed in surface epithelial cells of the normal human ovary. In contrast, results of a histological survey of 210 cases of human ovarian carcinoma showed that expression of p150Sal2 was lost in 90 % of cases, suggesting that p150Sal2 may function as a tumor suppressor in the human ovary [17]. Restoration of p150Sal2 expression in human ovarian carcinoma cells that have lost p150Sal2 expression suppressed tumor growth in SCID mice [14]. Moreover, it has been reported that the SALL2 gene is located in a chromosomal region (14q12) that is related to ovarian carcinoma [40]. SALL2 is expressed from alternate promoters P1 and P2, giving rise to the E1 and E1A splice variants, respectively (Fig. 3a). It has been shown that the E1A variant produced by the P2 promoter is the major form expressed in normal ovarian surface epithelial cells [17]. Many CpG dinucleotides are present in the P2 promoter, suggesting susceptibility to methylation silencing (Fig. 3b). Indeed, pyrosequencing

492

Tumor Biol. (2015) 36:489–494

Fig. 2 The p150Sal2-interacting proteins in HOSE cells were categorized by their functional roles in cellular pathways. HOSE cells were transfected with GSTp150Sal2 and harvested at 36 h post-transfection. The p150Sal2interacting proteins were pulled down with glutathione beads and analyzed by mass spectrometry [38]. The functions of interacting proteins were categorized with the main function of each protein

analyses using bisulfite-converted DNA samples from human ovarian tumor sections and cultured ovarian carcinoma cells showed that loss of p150Sal2 in ovarian carcinoma is due to methylation of the SALL2 promoter [17]. Ovarian cancer is the deadliest of all gynecologic malignancies, estimated to be the cause of death in more than 125, 000 women annually, and long-term survival rates have not improved [41]. High-grade serous tumors are both the most frequent type and are associated with the highest morbidity and mortality [42]. Epigenetic modifications are emerging as important early premalignant events in human cancers [43]. As epigenetic changes can be reversible, genes that are inactivated by promoter methylation may be good therapeutic targets. The argument is that, if the methylated cytosines in CpG islands of the promoter regions could be demethylated, progression of ovarian cancer might be halted. Since the SALL2 gene is silenced by promoter methylation in the majority of human ovarian carcinomas [17] and its forced expression leads to growth arrest and apoptosis in OVCA cells [14], demethylation of the SALL2 promoter in ovarian cancer tissues could result in re-expression of p150Sal2, which would Fig. 3 The human SALL2 promoter regions. a The SALL2 gene has alternative exon E1 and E1A that are regulated by P1 and P2 promoter, respectively. The long common exon 2 (E2) is also shown. b Many CpG dinucleotides (shaded in yellow) are found in the P2 promoter region. Underlined sequences indicate putative AP4-binding sites while translational start site for E1A is shown in bold. The putative p53 binding site (CCTG CCC) is shown in red

suppress the growth of cancer cells. Nucleoside analogs including azacytidine have been clinically used as inhibitors of promoter methylation, but their lack of target specificity and short molecule life limit their use in cancer therapy. DNA demethylases that recognize the silenced SALL2 promoter and reactivate the gene through promoter demethylation would be optimal candidates for suppressing ovarian cancer cells. However, such enzymes have not been identified. The transcriptional factor AP4 is known to target the SALL2 promoter for positive regulation (Fig. 3b) [38]. An AP4demethylase fusion construct might serve as a possible therapeutic molecule that is specifically recruited to the silenced SALL2 promoter, allowing re-expression of the SALL2 gene in patients with ovarian cancer. Although the ovary is the major site of SALL2 expression, this protein is also found in other tissues including the lung and kidney, which serve as major sites of polyomavirus infection and replication [13]. The molecular and biological functions of p150Sal2 in these cell types require further investigation and may support an important role for SALL2 as a general tumor suppressor or in other human diseases.

Tumor Biol. (2015) 36:489–494

Our understanding of SALL2 function is still incomplete, but its regulatory mechanism in ovarian cancer through methylation has recently been uncovered. The P2 promoter of SALL2 contains abundant CpG islands. Pyrosequencing analyses using bisulfite-converted DNA samples from human ovarian tumor sections and cultured ovarian carcinoma cells revealed that SALL2 is silenced in ovarian carcinoma as a result of methylation of its promoter. This study goes one step further in understanding SALL2 as a regulator of tumorigenesis. The existence of many p150Sal2-interacting proteins confers great versatility to the protein and indicates that it may have additional roles in other cellular mechanisms including translation, DNA replication, and DNA repair. Further investigation of the function of p150Sal2 by studying its binding partners and transcriptional downstream targets would contribute to a fundamental understanding of the cellular pathways regulated by p150Sal2. Examination of the expression of p150Sal2 in other human cancers, its upstream regulators, and its downstream targets for transcriptional regulation might reveal pathways that could be targeted in novel therapeutic approaches to suppress human carcinogenesis. Acknowledgments This work was supported by a Texas A&M University-Kingsville Research Award (to CKS; 160330–00016) and the research fund of Hanyang University (HY-2014-N) to HY.

References 1. de Celis JF, Barrio R. Regulation and function of Spalt proteins during animal development. Int J Dev Biol. 2009;53:1385–98. 2. Sweetman D, Munsterberg A. The vertebrate spalt genes in development and disease. Dev Biol. 2006;293:285–93. 3. Kohlhase J, Taschner PE, Burfeind P, Pasche B, Newman B, Blanck C, et al. Molecular analysis of SALL1 mutations in Townes-Brocks syndrome. Am J Hum Genet. 1999;64:435–45. 4. Al-Baradie R, Yamada K, St Hilaire C, Chan WM, Andrews C, McIntosh N, et al. Duane radial ray syndrome (Okihiro syndrome) maps to 20q13 and results from mutations in SALL4, a new member of the SAL family. Am J Hum Genet. 2002;71:1195–9. 5. Kohlhase J, Chitayat D, Kotzot D, Ceylaner S, Froster UG, Fuchs S, et al. SALL4 mutations in Okihiro syndrome (Duane-radial ray syndrome), acro-renal-ocular syndrome, and related disorders. Hum Mutat. 2005;26:176–83. 6. Yamaguchi YL, Tanaka SS, Kumagai M, Fujimoto Y, Terabayashi T, Matsui Y, Nishinakamura R: Sall4 is essential for mouse primordial germ cell specification by suppressing somatic cell program genes. Stem cells 2014 7. Tanimura N, Saito M, Ebisuya M, Nishida E, Ishikawa F. Stemnessrelated factor Sall4 interacts with transcription factors Oct-3/4 and Sox2 and occupies Oct-Sox elements in mouse embryonic stem cells. J Biol Chem. 2013;288:5027–38. 8. Liu J, Wang L, Yang A, Jiang P, Wang M. Up-regulation of SALL4 a s s o c i a t e d w i t h p o o r p r o g n o s i s i n g a s t r i c c a n c e r. Hepatogastroenterology. 2014;61:1459–64. 9. Zhang J, Tam WL, Tong GQ, Wu Q, Chan HY, Soh BS, et al. Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1. Nat Cell Biol. 2006;8:1114–23.

493 10. Parrish M, Ott T, Lance-Jones C, Schuetz G, Schwaeger-Nickolenko A, Monaghan AP. Loss of the Sall3 gene leads to palate deficiency, abnormalities in cranial nerves, and perinatal lethality. Mol Cell Biol. 2004;24:7102–12. 11. de Melo J, Peng GH, Chen S, Blackshaw S. The Spalt family transcription factor Sall3 regulates the development of cone photoreceptors and retinal horizontal interneurons. Development. 2011;138: 2325–36. 12. Kelberman D, Islam L, Lakowski J, Bacchelli C, Chanudet E, Lescai F, et al. Mutation of Sall2 causes recessive ocular coloboma in humans and mice. Hum Mol Genet. 2014;23:2511–26. 13. Li D, Dower K, Ma Y, Tian Y, Benjamin TL. A tumor host range selection procedure identifies p150(sal2) as a target of polyoma virus large t antigen. Proc Natl Acad Sci U S A. 2001;98:14619–24. 14. Li D, Tian Y, Ma Y, Benjamin T. p150(SAL2) is a p53-independent regulator of p21(WAF1/CIP). Mol Cell Biol. 2004;24:3885–93. 15. Ma Y, Li D, Chai L, Luciani AM, Ford D, Morgan J, et al. Cloning and characterization of two promoters for the human HSAL2 gene and their transcriptional repression by the Wilms tumor suppressor gene product. J Biol Chem. 2001;276:48223–30. 16. Liu H, Adler AS, Segal E, Chang HY. A transcriptional program mediating entry into cellular quiescence. PLoS Genet. 2007;3:e91. 17. Sung CK, Li D, Andrews E, Drapkin R, Benjamin T. Promoter methylation of the SALL2 tumor suppressor gene in ovarian cancers. Mol Oncol. 2013;7:419–27. 18. Howley PM, Livingston DM. Small DNA tumor viruses: large contributors to biomedical sciences. Virology. 2009;384:256–9. 19. Benjamin TL. Polyoma virus: old findings and new challenges. Virology. 2001;289:167–73. 20. Benjamin TL. Polyoma viruses. In: Fox JG, Davission MT, Quimby FW, Barthold SW, Newcomer CE, Smith AL, editors. In the mouse in biomedical research. Waltham: Academic; 2007. p. 105–39. 21. Imperiale MJ, Major, E.O.: Polyomaviruses; in Knipe DM, Howley, P.M. (ed) Fields virology. Philadelphia, PA, Lippincott Williams & Wilkins, 2007, vol 2, pp 2263–2298. 22. Cheng J, DeCaprio JA, Fluck MM, Schaffhausen BS. Cellular transformation by simian virus 40 and murine polyoma virus t antigens. Semin Cancer Biol. 2009;19:218–28. 23. Fluck MM, Schaffhausen BS. Lessons in signaling and tumorigenesis from polyomavirus middle T antigen. Microbiol Mol Biol Rev. 2009;73:542–63. Table of Contents. 24. Freund R, Bauer PH, Crissman HA, Bradbury EM, Benjamin TL. Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding. J Virol. 1994;68:7227– 34. 25. Rozenblatt-Rosen O, Deo RC, Padi M, Adelmant G, Calderwood MA, Rolland T, et al. Interpreting cancer genomes using systematic host network perturbations by tumour virus proteins. Nature. 2012;487:491–5. 26. Gu H, Li D, Sung CK, Yim H, Troke P, Benjamin T: DNA-binding and regulatory properties of the transcription factor and putative tumor suppressor p150(sal2). Biochim Biophys Acta 2011 27. Parroche P, Touka M, Mansour M, Bouvard V, Thepot A, Accardi R, et al. Human papillomavirus type 16 E6 inhibits p21(WAF1) transcription independently of p53 by inactivating p150(Sal2). Virology. 2011;417:443–8. 28. Sung CK, Yim H, Gu H, Li D, Andrews E, Duraisamy S, et al. The polyoma virus large T binding protein p150 is a transcriptional repressor of c-MYC. PLoS One. 2012;7:e46486. 29. Chai L. The role of HSAL (SALL) genes in proliferation and differentiation in normal hematopoiesis and leukemogenesis. Transfusion. 2011;51 Suppl 4:87S–93. 30. Pincheira R, Baerwald M, Dunbar JD, Donner DB. Sall2 is a novel p75NTR-interacting protein that links NGF signalling to cell cycle progression and neurite outgrowth. EMBO J. 2009;28:261–73.

494 31. Bae SC, Choi JK. Tumor suppressor activity of RUNX3. Oncogene. 2004;23:4336–40. 32. Chmelarova M, Krepinska E, Spacek J, Laco J, Beranek M, Palicka V. Methylation in the p53 promoter in epithelial ovarian cancer. Clin Transl Oncol. 2013;15:160–3. 33. Jha AK, Nikbakht M, Jain V, Capalash N, Kaur J. p16(INK4A) and p15(INK4B) gene promoter methylation in cervical cancer patients. Oncol Lett. 2012;3:1331–5. 34. Kang YH, Lee HS, Kim WH. Promoter methylation and silencing of PTEN in gastric carcinoma. Lab Invest. 2002;82:285–91. 35. Nielsen TO, Hsu FD, O’Connell JX, Gilks CB, Sorensen PH, Linn S, et al. Tissue microarray validation of epidermal growth factor receptor and SALL2 in synovial sarcoma with comparison to tumors of similar histology. Am J Pathol. 2003;163:1449–56. 36. Estilo CL, Oc P, Talbot S, Socci ND, Carlson DL, Ghossein R, et al. Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis. BMC Cancer. 2009;9:11.

Tumor Biol. (2015) 36:489–494 37. Alagaratnam S, Lind GE, Kraggerud SM, Lothe RA, Skotheim RI. The testicular germ cell tumour transcriptome. Int J Androl. 2011;34: e133–50. discussion e150-131. 38. Sung CK, Dahl J, Yim H, Rodig S, Benjamin TL. Transcriptional and post-translational regulation of the quiescence factor and putative tumor suppressor p150(Sal2). FASEB J. 2011;25:1275–83. 39. Farkas C, Martins CP, Escobar D, Hepp MI, Castro AF, Evan G, et al. Wild type p53 transcriptionally represses the SALL2 transcription factor under genotoxic stress. PLoS One. 2013;8:e73817. 40. Bandera CA, Takahashi H, Behbakht K, Liu PC, LiVolsi VA, Benjamin I, et al. Deletion mapping of two potential chromosome 14 tumor suppressor gene loci in ovarian carcinoma. Cancer Res. 1997;57:513–5. 41. Siegel R, DeSantis C, Virgo K, Stein K, Mariotto A, Smith T, et al. Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin. 2012;62:220–41. 42. Cho KR, Shih Ie M. Ovarian cancer. Annu Rev Pathol. 2009;4:287–313. 43. Tycko B. Epigenetic gene silencing in cancer. J Clin Invest. 2000;105:401–7.