Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. RASSFs are a family of tumor suppressors that are frequently inactivated by promoter hypermethylation in various cancers. We studied CpG island promoter hypermethylation in MCC of RASSF2, RASSF5A, RASSF5C and RASSF10 by combined bisulfite restriction analysis (COBRA) in MCC samples and control tissue. We found RASSF2 to be methylated in three out of 43 (7%), RASSF5A in 17 out of 39 (44%, but also 43% in normal tissue), RASSF5C in two out of 26 (8%) and RASSF10 in 19 out of 84 (23%) of the cancer samples. No correlation between the methylation status of the analyzed RASSFs or between RASSF methylation and MCC characteristics (primary versus metastatic, Merkel cell polyoma virus infection, age, sex) was found. Our results show that RASSF2, RASSF5C and RASSF10 are aberrantly hypermethylated in MCC to a varying degree and this might contribute to Merkel cell carcinogenesis.
Grabowski, J.; Saltzstein, S.L.; Sadler, G.R.; Tahir, Z.; Blair, S. A comparison of Merkel cell carcinoma and melanoma: Results from the california cancer registry. Clin. Med. Oncol. 2008, 2, 327–333.
[3]
Spurgeon, M.E.; Lambert, P.F. Merkel cell polyomavirus: A newly discovered human virus with oncogenic potential. Virology 2013, 435, 118–130, doi:10.1016/j.virol.2012.09.029.
[4]
Kassem, A.; Schopflin, A.; Diaz, C.; Weyers, W.; Stickeler, E.; Werner, M.; Zur Hausen, A. Frequent detection of Merkel cell polyomavirus in human Merkel cell carcinomas and identification of a unique deletion in the VP1 gene. Cancer Res. 2008, 68, 5009–5013, doi:10.1158/0008-5472.CAN-08-0949.
[5]
Feng, H.; Shuda, M.; Chang, Y.; Moore, P.S. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science 2008, 319, 1096–1100, doi:10.1126/science.1152586.
[6]
Becker, J.C.; Houben, R.; Ugurel, S.; Trefzer, U.; Pfohler, C.; Schrama, D. Mc polyomavirus is frequently present in Merkel cell carcinoma of european patients. J. Invest. Dermatol. 2009, 129, 248–250, doi:10.1038/jid.2008.198.
Martel-Jantin, C.; Filippone, C.; Cassar, O.; Peter, M.; Tomasic, G.; Vielh, P.; Briere, J.; Petrella, T.; Aubriot-Lorton, M.H.; Mortier, L.; et al. Genetic variability and integration of Merkel cell polyomavirus in Merkel cell carcinoma. Virology 2012, 426, 134–142, doi:10.1016/j.virol.2012.01.018.
[9]
Martel-Jantin, C.; Pedergnana, V.; Nicol, J.T.; Leblond, V.; Tregouet, D.A.; Tortevoye, P.; Plancoulaine, S.; Coursaget, P.; Touze, A.; Abel, L.; et al. Merkel cell polyomavirus infection occurs during early childhood and is transmitted between siblings. J. Clin. Virol. 2013, 58, 288–291, doi:10.1016/j.jcv.2013.06.004.
[10]
Helmbold, P.; Lahtz, C.; Herpel, E.; Schnabel, P.A.; Dammann, R.H. Frequent hypermethylation of RASSF1A tumour suppressor gene promoter and presence of Merkel cell polyomavirus in small cell lung cancer. Eur. J. Cancer 2009, 45, 2207–2211, doi:10.1016/j.ejca.2009.04.038.
[11]
Lasithiotaki, I.; Antoniou, K.M.; Derdas, S.P.; Sarchianaki, E.; Symvoulakis, E.K.; Psaraki, A.; Spandidos, D.A.; Stathopoulos, E.N.; Siafakas, N.M.; Sourvinos, G. The presence of Merkel cell polyomavirus is associated with deregulated expression of Braf and Bcl-2 genes in non-small cell lung cancer. Int. J. Cancer 2013, 133, 604–611, doi:10.1002/ijc.28062.
[12]
Houben, R.; Michel, B.; Vetter-Kauczok, C.S.; Pfohler, C.; Laetsch, B.; Wolter, M.D.; Leonard, J.H.; Trefzer, U.; Ugurel, S.; Schrama, D.; et al. Absence of classical map kinase pathway signalling in Merkel cell carcinoma. J. Invest. Dermatol. 2006, 126, 1135–1142, doi:10.1038/sj.jid.5700170.
[13]
Van Gele, M.; Kaghad, M.; Leonard, J.H.; van Roy, N.; Naeyaert, J.M.; Geerts, M.L.; van Belle, S.; Cocquyt, V.; Bridge, J.; Sciot, R.; et al. Mutation analysis of P73 and TP53 in Merkel cell carcinoma. Br. J. Cancer 2000, 82, 823–826, doi:10.1054/bjoc.1999.1006.
[14]
Lassacher, A.; Heitzer, E.; Kerl, H.; Wolf, P. p14ARF hypermethylation is common but INK4a-ARF locus or p53 mutations are rare in Merkel cell carcinoma. J. Invest. Dermatol. 2008, 128, 1788–1796, doi:10.1038/sj.jid.5701256.
[15]
Taberlay, P.C.; Jones, P.A. DNA methylation and cancer. Fortschr. Arzneimittelforsch. 2011, 67, 1–23.
[16]
Bergman, Y.; Cedar, H. DNA methylation dynamics in health and disease. Nat. Struct. Mol. Biol. 2013, 20, 274–281, doi:10.1038/nsmb.2518.
[17]
Richter, A.M.; Pfeifer, G.P.; Dammann, R.H. The RASSF proteins in cancer; from epigenetic silencing to functional characterization. Biochim. Biophys. Acta 2009, 1796, 114–128.
[18]
Endoh, M.; Tamura, G.; Honda, T.; Homma, N.; Terashima, M.; Nishizuka, S.; Motoyama, T. RASSF2, a potential tumour suppressor, is silenced by CpG island hypermethylation in gastric cancer. Br. J. Cancer 2005, 93, 1395–1399, doi:10.1038/sj.bjc.6602854.
[19]
Schagdarsurengin, U.; Richter, A.M.; Hornung, J.; Lange, C.; Steinmann, K.; Dammann, R.H. Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences. Mol. Cancer 2010, 9, e264, doi:10.1186/1476-4598-9-264.
[20]
Zhang, Z.; Sun, D.; van do, N.; Tang, A.; Hu, L.; Huang, G. Inactivation of RASSF2A by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma. Int. J. Cancer 2007, 120, 32–38, doi:10.1002/ijc.22185.
[21]
Hesson, L.; Dallol, A.; Minna, J.D.; Maher, E.R.; Latif, F. NORE1A, a homologue of RASSF1A tumour suppressor gene is inactivated in human cancers. Oncogene 2003, 22, 947–954, doi:10.1038/sj.onc.1206191.
[22]
Macheiner, D.; Heller, G.; Kappel, S.; Bichler, C.; Stattner, S.; Ziegler, B.; Kandioler, D.; Wrba, F.; Schulte-Hermann, R.; Zochbauer-Muller, S.; et al. NORE1B, a candidate tumor suppressor, is epigenetically silenced in human hepatocellular carcinoma. J. Hepatol. 2006, 45, 81–89, doi:10.1016/j.jhep.2005.12.017.
Richter, A.M.; Walesch, S.K.; Würl, P.; Taubert, H.; Dammann, R.H. The tumor suppressor RASSF10 is upregulated upon contact inhibition and frequently epigenetically silenced in cancer. Oncogenesis 2012, 1, e18, doi:10.1038/oncsis.2012.18.
[25]
Helmbold, P.; Richter, A.M.; Walesch, S.K.; Skorokhod, A.; Marsch, W.C.; Enk, A.; Dammann, R.H. RASSF10 promoter hypermethylation is frequent in malignant melanoma of the skin but uncommon in nevus cell nevi. J. Invest. Dermatol. 2012, 132, 687–694, doi:10.1038/jid.2011.380.
[26]
Hill, V.K.; Underhill-Day, N.; Krex, D.; Robel, K.; Sangan, C.B.; Summersgill, H.R.; Morris, M.; Gentle, D.; Chalmers, A.D.; Maher, E.R.; et al. Epigenetic inactivation of the RASSF10 candidate tumor suppressor gene is a frequent and an early event in gliomagenesis. Oncogene 2010, 30, 978–989.
[27]
Hesson, L.B.; Dunwell, T.L.; Cooper, W.N.; Catchpoole, D.; Brini, A.T.; Chiaramonte, R.; Griffiths, M.; Chalmers, A.D.; Maher, E.R.; Latif, F. The novel RASSF6 and RASSF10 candidate tumour suppressor genes are frequently epigenetically inactivated in childhood leukaemias. Mol. Cancer 2009, 8, e42, doi:10.1186/1476-4598-8-42.
[28]
Shinawi, T.; Hill, V.; Dagklis, A.; Baliakas, P.; Stamatopoulos, K.; Agathanggelou, A.; Stankovic, T.; Maher, E.R.; Ghia, P.; Latif, F. KIBRA gene methylation is associated with unfavorable biological prognostic parameters in chronic lymphocytic leukemia. Epigenetics 2012, 7, 211–215, doi:10.4161/epi.7.3.19222.
[29]
Rice, P.; Longden, I.; Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite. Available online: http://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/ (accessed on 30 April 2013).
[30]
Underhill-Day, N.; Hill, V.; Latif, F. N-terminal RASSF family: RASSF7–RASSF10. Epigenetics 2011, 6, 284–292, doi:10.4161/epi.6.3.14108.
[31]
Vos, M.D.; Ellis, C.A.; Elam, C.; Ulku, A.S.; Taylor, B.J.; Clark, G.J. RASSF2 is a novel K-Ras-specific effector and potential tumor suppressor. J. Biol. Chem. 2003, 278, 28045–28051.
[32]
Hesson, L.B.; Wilson, R.; Morton, D.; Adams, C.; Walker, M.; Maher, E.R.; Latif, F. CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations. Oncogene 2005, 24, 3987–3994, doi:10.1038/sj.onc.1208566.
[33]
Akino, K.; Toyota, M.; Suzuki, H.; Mita, H.; Sasaki, Y.; Ohe-Toyota, M.; Issa, J.P.; Hinoda, Y.; Imai, K.; Tokino, T. The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer. Gastroenterology 2005, 129, 156–169, doi:10.1053/j.gastro.2005.03.051.
[34]
Park, H.W.; Kang, H.C.; Kim, I.J.; Jang, S.G.; Kim, K.; Yoon, H.J.; Jeong, S.Y.; Park, J.G. Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers. Int. J. Cancer 2007, 120, 7–12, doi:10.1002/ijc.22276.
[35]
Tommasi, S.; Dammann, R.; Jin, S.G.; Zhang, X.F.; Avruch, J.; Pfeifer, G.P. RASSF3 and NORE1: Identification and cloning of two human homologues of the putative tumor suppressor gene RASSF1. Oncogene 2002, 21, 2713–2720, doi:10.1038/sj.onc.1205365.
[36]
Chen, J.; Lui, W.O.; Vos, M.D.; Clark, G.J.; Takahashi, M.; Schoumans, J.; Khoo, S.K.; Petillo, D.; Lavery, T.; Sugimura, J.; et al. The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas. Cancer Cell 2003, 4, 405–413, doi:10.1016/S1535-6108(03)00269-1.
[37]
Aoyama, Y.; Avruch, J.; Zhang, X.F. Nore1 inhibits tumor cell growth independent of Ras or the MST1/2 kinases. Oncogene 2004, 23, 3426–3433, doi:10.1038/sj.onc.1207486.
[38]
Vos, M.D.; Martinez, A.; Ellis, C.A.; Vallecorsa, T.; Clark, G.J. The pro-apoptotic Ras effector Nore1 may serve as a Ras-regulated tumor suppressor in the lung. J. Biol. Chem. 2003, 278, 21938–21943.
[39]
Song, H.; Kim, H.; Lee, K.; Lee, D.H.; Kim, T.S.; Song, J.Y.; Lee, D.; Choi, D.; Ko, C.Y.; Kim, H.S.; et al. Ablation of RASSF2 induces bone defects and subsequent haematopoietic anomalies in mice. EMBO J. 2012, 31, 1147–1159, doi:10.1038/emboj.2011.480.
[40]
Moshnikova, A.; Frye, J.; Shay, J.W.; Minna, J.D.; Khokhlatchev, A.V. The growth and tumor suppressor NORE1A is a cytoskeletal protein that suppresses growth by inhibition of the ERK pathway. J. Biol. Chem. 2006, 281, 8143–8152.
[41]
Katagiri, K.; Ohnishi, N.; Kabashima, K.; Iyoda, T.; Takeda, N.; Shinkai, Y.; Inaba, K.; Kinashi, T. Crucial functions of the Rap1 effector molecule RAPL in lymphocyte and dendritic cell trafficking. Nat. Immunol. 2004, 5, 1045–1051, doi:10.1038/ni1111.
[42]
Wei, Z.; Chen, X.; Chen, J.; Wang, W.; Xu, X.; Cai, Q. RASSF10 is epigenetically silenced and functions as a tumor suppressor in gastric cancer. Biochem. Biophys. Res. Commun. 2013, 432, 632–637, doi:10.1016/j.bbrc.2013.02.033.
[43]
Dansranjavin, T.; Wagenlehner, F.; Gattenloehner, S.; Steger, K.; Weidner, W.; Dammann, R.; Schagdarsurengin, U. Epigenetic down regulation of RASSF10 and its possible clinical implication in prostate carcinoma. Prostate 2012, doi:10.1002/pros.22510.
[44]
Dumitrescu, R.G. Epigenetic markers of early tumor development. Methods Mol. Biol. 2012, 863, 3–14, doi:10.1007/978-1-61779-612-8_1.
