The RASSF1A tumor suppressor gene is frequently inactivated by promoter methylation in human tumors. The RASSF1A protein forms an endogenous complex with tubulin and promotes the stabilization of microtubules. Loss of RASSF1A expression sensitizes cells to microtubule destabilizing stimuli. We have observed a strong correlation between the loss of RASSF1A expression and the development of Taxol resistance in primary ovarian cancer samples. Thus, we sought to determine if RASSF1A levels could dictate the response to Taxol and whether an epigenetic therapy approach might be able to reverse the Taxol resistant phenotype of RASSF1A negative ovarian tumor cells. We found that knocking down RASSF1A expression in an ovarian cancer cell line inhibited Taxol-mediated apoptosis and promoted cell survival during Taxol treatment. Moreover, using a combination of small molecule inhibitors of DNA Methyl Transferase enzymes, we were able restore RASSF1A expression and Taxol sensitivity. This identifies a role for RASSF1A in modulating the tumor response to Taxol and provides proof of principal for the use of epigenetic therapy to overcome Taxol resistance. 1. Introduction RASSF1A is a poorly understood tumor suppressor that can modulate the cell cycle, tubulin dynamics and apoptosis [1–3]. It is subjected to epigenetic inactivation at high frequency in a broad range of human tumors, including approximately 50% of ovarian tumors [1, 4, 5]. Overexpression of RASSF1A promotes hyperstabilization of microtubules reminiscent of Taxol [6, 7], and previous investigations have shown that loss of RASSF1A sensitizes cells to microtubule destabilizing drugs such as nocodazole [7]. Thus, RASSF1A appears to play an important role in modulating microtubule stabilization. This implies that the RASSF1A levels in a tumor cell may impact how the cell responds to Taxol treatment. The development of resistance to Taxol remains a serious problem in the treatment of ovarian cancer. The most frequent mechanism by which RASSF1A is inactivated in tumors is by hypermethylation promoter leading to transcriptional silencing [1, 4, 5]. Thus, the gene remains intact, just dormant. Over recent years, a series of small molecules have been identified that can inhibit the DNA methylation system and restore expression of genes that have suffered aberrant promoter methylation [8]. This has given rise to the concept of epigenetic therapy, whereby a tumor would be treated with drugs to restore the expression and function of RASSF1A or some other epigenetically inactivated target. If RASSF1A plays a key
References
[1]
H. Donninger, M. D. Vos, and G. J. Clark, “The RASSF1A tumor suppressor,” Journal of Cell Science, vol. 120, no. 18, pp. 3163–3172, 2007.
[2]
R. Dammann, U. Schagdarsurengin, C. Seidel et al., “The tumor suppressor RASSF1A in human carcinogenesis: an update,” Histology and Histopathology, vol. 20, no. 2, pp. 645–663, 2005.
[3]
G. P. Pfeifer, R. Dammann, and S. Tommasi, “RASSF proteins,” Current Biology, vol. 20, no. 8, pp. R344–R345, 2010.
[4]
L. B. Hesson, W. N. Cooper, and F. Latif, “The role of RASSF1A methylation in cancer,” Disease Markers, vol. 23, no. 1-2, pp. 73–87, 2007.
[5]
A. Agathanggelou, W. N. Cooper, and F. Latif, “Role of the Ras-association domain family 1 tumor suppressor gene in human cancers,” Cancer Research, vol. 65, no. 9, pp. 3497–3508, 2005.
[6]
M. D. Vos, A. Martinez, C. Elam et al., “A role for the RASSF1A tumor suppressor in the regulation of tubulin polymerization and genomic stability,” Cancer Research, vol. 64, no. 12, pp. 4244–4250, 2004.
[7]
L. Liu, S. Tommasi, D. H. Lee, R. Dammann, and G. P. Pfeifer, “Control of microtubule stability by the RASSF1A tumor suppressor,” Oncogene, vol. 22, no. 50, pp. 8125–8136, 2003.
[8]
J. Ren, B. N. Singh, Q. Huang et al., “DNA hypermethylation as a chemotherapy target,” Cellular Signalling, vol. 23, no. 7, pp. 1082–1093, 2011.
[9]
M. D. Vos, A. Dallol, K. Eckfeld et al., “The RASSF1A tumor suppressor activates bax via MOAP-1,” The Journal of Biological Chemistry, vol. 281, no. 8, pp. 4557–4563, 2006.
[10]
V. E. Marquez, J. A. Kelley, R. Agbaria et al., “Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy.,” Annals of the New York Academy of Sciences, vol. 1058, pp. 246–254, 2005.
[11]
C. Stresemann, B. Brueckner, T. Musch, H. Stopper, and F. Lyko, “Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines,” Cancer Research, vol. 66, no. 5, pp. 2794–2800, 2006.
[12]
H. Donninger, T. Bonome, M. Radonovich et al., “Whole genome expression profiling of advance stage papillary serous ovarian cancer reveals activated pathways,” Oncogene, vol. 23, no. 49, pp. 8065–8077, 2004.
[13]
K. A. Kreuzer, U. Lass, A. Bohn, O. Landt, and C. A. Schmidt, “Lightcycler technology for the quantitation of bcr/abl fusion transcripts,” Cancer Research, vol. 59, no. 13, pp. 3171–3174, 1999.
[14]
H. Donninger, N. P. Allen, A. Henson et al., “Salvador protein is a tumor suppressor effector of RASSF1A with hippo pathway-independent functions,” The Journal of Biological Chemistry, vol. 286, no. 21, pp. 18483–18491, 2011.
[15]
G. Gamboa, P. M. Carpenter, Y. D. Podnos et al., “Characterization and development of UCl 107, a primary human ovarian carcinoma cell line,” Gynecologic Oncology, vol. 58, no. 3, pp. 336–343, 1995.
[16]
A. Dallol, A. Agathanggelou, S. L. Fenton et al., “RASSF1A interacts with microtubule-associated proteins and modulates microtubule dynamics,” Cancer Research, vol. 64, no. 12, pp. 4112–4116, 2004.
[17]
A. Matsuyama, T. Shimazu, Y. Sumida et al., “In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation,” The EMBO Journal, vol. 21, no. 24, pp. 6820–6831, 2002.
[18]
C. B. Yoo, J. C. Cheng, and P. A. Jones, “Zebularine: a new drug for epigenetic therapy,” Biochemical Society Transactions, vol. 32, no. 6, pp. 910–912, 2004.
[19]
B. Brueckner, R. G. Boy, P. Siedlecki et al., “Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases,” Cancer Research, vol. 65, no. 14, pp. 6305–6311, 2005.
[20]
C. Balch, P. Yan, T. Craft et al., “Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer,” Molecular Cancer Therapeutics, vol. 4, no. 10, pp. 1505–1514, 2005.
[21]
D. Kuck, T. Caulfield, F. Lyko, and J. L. Medina-Franco, “Nanaomycin A selectively inhibits DNMT3B and reactivates silenced tumor suppressor genes in human cancer cells,” Molecular Cancer Therapeutics, vol. 9, no. 11, pp. 3015–3023, 2010.
[22]
M. El-Kalla, C. Onyskiw, and S. Baksh, “Functional importance of RASSF1A microtubule localization and polymorphisms,” Oncogene, vol. 29, no. 42, pp. 5729–5740, 2010.
[23]
H. Donninger, T. Barnoud, N. Nelson, et al., “RASSF1A and the rs2073498 cancer associated SNP,” Frontiers in Cancer Genetics, vol. 1, article 54, 2011.
