Preliminary qRT-PCR experiments allowed us to select for further study four lung cancer lines (H1299, H841, A549, Calu-6) exhibiting heterogeneous patterns of NY-ESO-1, MAGE-A1, and MAGE-A3 CT-X gene expression, as well as normal human bronchial epithelia (NHBE) and human small airway epithelial cells (SAEC), which do not express any CT-X genes. Immunoblot analysis confirmed qRT-PCR results. Extensive pyrosequencing and chromatin immunoprecipitation (ChIP) experiments demonstrated that lack of NY-ESO-1, MAGE-A1 or MAGE-A3 expression in lung cancer cells was attributable to persistence of normal heterochromatin structure, rather than global activation of these genes via promoter hypomethylation, followed by selective silencing associated with bivalent chromatin. Additional experiments were performed to ascertain if modulation of histone lysine methylation altered NY-ESO-1, MAGE-A1, and MAGE-A3 expression in lung cancer cells. Briefly, lentiviral shRNA techniques were used to knock-down LSD-1(KDM1) and JARID1B (KDM5B) that mediate demethylation of mono, di-, and trimethylated H3K4 (histone activation marks, or the histone lysine methyltransferase EZH2 (KMT6) that mediates trimethylation of H3K27 (histone repressive mark) in H841 cells that do not express NY-ESO-1, MAGE-A1, or MAGE-A3. ChIP experiments revealed that global changes in activation and repression marks coincided with similar alterations, and decreased occupancy of the respective histone modifiers within the NY-ESO-1, MAGE-A1 and MAGE-A3 promoters in knock-downs relative to control cells. Whereas knock-down of EZH2, LSD1, or JARID1B alone was insufficient to activate NY-ESO-1, MAGE-A1, or MAGE-A3, inhibition of EZH2, LSD1, or JARID1B expression significantly enhanced DAC-mediated induction of these CT genes in lung cancer cells. The effects of targeted modulation of histone lysine methylation were more pronounced than those observed following knock-down of the class III histone deacetylase, SirT1. Additional qRT-PCR and immunoblot experiments demonstrated that 3-deazaneplanocin A (DZNep), a novel pharmacologic inhibitor of EZH2 expression, at a concentration one log lower than the cytotoxic dose of this agent in cancer cells, significantly enhanced DAC-mediated activation of NY-ESO-1, MAGE-A1 and MAGE-A3 in H841 cells (Figure 2). This phenomenon extended to other CT-X genes such as MAGE-A6 and MAGE-A12, and was observed across numerous other lung cancer lines. The magnitude of enhancement of DAC-mediated de-repression of CT-X genes in lung cancer cells by DZNep was markedly higher than that observed in SAEC or NHBE, and approximated or exceeded that observed following sequential DAC-DP or DAC-trichostatin A (TSA) treatment. Pyrosequencing and ChIP experiments demonstrated that this phenomenon was not attributable to enhanced demethylation, but instead coincided with decreased EZH2 and H3K27Me3 levels within the NY-ESO-1, MAGE-A1 and MAGE-A3 promoters. Consistent with these observations, constitutive expression of EZH2 significantly attenuated the enhancement effect of DZNep on DAC-mediated induction of NY-ESO-1, MAGE-A1 or MAGE-A3 in lung cancer cells. Subsequent cytokine and chromium release assays demonstrated that DZNep significantly enhanced DAC-mediated recognition and lysis of lung cancer cells by allogeneic PBL expressing recombinant T cell receptors specific for NY-ESO-1 or MAGE-A3 in the context of HLA-A*0201. No cytokine release or lysis was observed following co-culture of effector cells with drug treated HLA-A*0201-transduced normal airway epithelial cells or dermal fibroblasts. Full details of these experiments, which were the first to demonstrate that modulation of histone lysine methylation may be a novel epigenetic strategy for cancer immunotherapy, have been published in Cancer Research. In additional studies, Affymetrix microarrays were used to examine global gene expression profiles, and specifically identify genes encoding PcG proteins in a panel of malignant pleural mesothelioma (MPM) lines relative to cultured normal mesothelia. This analysis demonstrated over-expression of EZH2 (KMT6) and to a lesser extent, EED and SUZ12, which encode core components of polycomb repressor complex-2 (PRC-2), in MPM lines. Quantitative RT-PCR (qRT-PCR) experiments using primers recognizing both EZH2 splice variants, and immunoblot analysis confirmed over-expression of EZH2, but not EED or SUZ12 in MPM lines relative to cultured normal mesothelial cells;up-regulation of EZH2 coincided with a global increase in the PRC-2 mediated repressive chromatin mark, H3K27Me3 in MPM cells. Subsequent qRT-PCR and immunohistochemistry experiments confirmed over- expression of EZH2 in 80%-85% of primary MPM specimens relative to normal pleura. In general, EZH2 levels tended to coincide with mRNA copy numbers, although some variations were noted, suggesting that post-transcriptional mechanisms also contribute to EZH2 over-expression in MPM. Consistent with these findings, levels of miR-101 or miR-26, which normally target the 3'UTR of EZH2, were significantly decreased in MPM specimens compared to normal pleura. Subsequent studies demonstrated that EZH2 protein levels did not correlate with stage of disease;however, intratumoral expression of either of the two EZH2 variants assessed by Illumina array techniques correlated with survival in patients with locally advanced MPM undergoing potentially curative resections. Additional experiments were performed to examine if aberrant PRC-2 activity directly contributes to the malignant phenotype of pleural mesothelioma cells. Briefly, shRNA techniques were used to knock-down EZH2 in cultured MPM cells;the shRNA used for these experiments targeted both splice variants of EZH2. Similar experiments were undertaken to knock-down EED, which although not over-expressed in primary MPM, is critical for maintaining stability of PRC-2, and histone methyltransferase activity of EZH2. Specific knock-down of these PRC-2 components significantly inhibited proliferation, migration, soft agar clonogenicity, as well as tumorgenicity of MPM cells. The effects of EED knock-down on global H3K27Me3 levels, as well as proliferation, migration, clonogenicity and tumorgenicity were more pronounced than EZH2 knock-down in MPM cells;these results may have been due to relative knock-down efficacies of the shRNAs, compensation of EZH2 knock-down by EZH1, or more profound destabilization of PRC-2 by depletion of EED. Subsequent immunoblot and pyrosequencing experiments demonstrated that DZNep mediated time and dose-dependent depletion of EZH2 and EED, and decreased H3K27Me3 levels without inducing global DNA demethylation in cultured MPM cells;these effects coincided with significantly decreased proliferation, migration and soft agar clonogenicity of these cells. In addition, intraperitoneal (IP) administration of DZNep significantly inhibited growth of established MPM xenografts in athymic nude mice without obvious systemic toxicities. Micro-array, qRT-PCR, and ChIP experiments demonstrated that the growth inhibitory effects of targeted disruption of PRC-2 or DZNep treatment in MPM cells coincided with decreased promoter occupancy of H3K27Me3, and up-regulation of numerous tumor suppressors modulating pluripotency, cell cycle progression and apoptosis in cancer cells. Detailed results of these experiments have been published recently in Clinical Cancer Research.

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Rao, Mahadev; Atay, Scott M; Shukla, Vivek et al. (2016) Mithramycin Depletes Specificity Protein 1 and Activates p53 to Mediate Senescence and Apoptosis of Malignant Pleural Mesothelioma Cells. Clin Cancer Res 22:1197-210
Xi, S; Inchauste, S; Guo, H et al. (2015) Cigarette smoke mediates epigenetic repression of miR-217 during esophageal adenocarcinogenesis. Oncogene :
Zhang, Yuwei; Calado, Rodrigo; Rao, Mahadev et al. (2014) Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas. PLoS One 9:e101010
Xi, Sichuan; Xu, Hong; Shan, Jigui et al. (2013) Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis. J Clin Invest 123:1241-61
Kemp, Clinton D; Rao, Mahadev; Xi, Sichuan et al. (2012) Polycomb repressor complex-2 is a novel target for mesothelioma therapy. Clin Cancer Res 18:77-90
Rao, Mahadev; Chinnasamy, Nachimuthu; Hong, Julie A et al. (2011) Inhibition of histone lysine methylation enhances cancer-testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer. Cancer Res 71:4192-204