Polycomb repressor complex-2 (PRC-2) - a critical mediator of stem cell pluripotency, is aberrantly expressed in a variety of human malignancies including lung cancers. PRC-2 contains three core proteins, enhancer of zeste 2 (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED). The SET (suppressor of variegation-enhancer of zeste-trithorax) domain of EZH2 induces trimethylation of lysine 27 on histone H3 (H3K27Me3), a repressive histone mark observed in bivalent chromatin, as well as gene promoters normally inactivated during differentiation, or aberrantly silenced during malignant transformation. Previously we demonstrated that following in-vitro exposure to cigarette smoke condensate (CSC), lung cancer cells exhibit significantly increased tumorigenicity in nude mice. This phenomenon coincided with PRC-2 mediated repression of Dkk-1, encoding an antagonist of Wnt signaling implicated in maintenance of cancer stem cells. Knockdown of Dkk-1 recapitulated the pro-tumorigenic effects of cigarette smoke in lung cancer cells. The present study was undertaken to examine the effects of the S-adenosylhomocysteine hydrolase inhibitor, DZNep in lung cancer cells. Immunoblot analysis revealed that DZNep mediated dose-dependent depletion of EZH2, EED and SUZ12, with concomitant decreases in global H3K27Me3 levels in cultured human lung cancer cells. Microarray and qRT-PCR experiments revealed that DZNep induced Dkk-1 expression in A549, H841, and H322 lung cancer cells, a phenomenon coinciding with growth inhibition and apoptosis in these cells. Promoter reporter assays, ELISA and immunoblot experiments confirmed that DZNep significantly increased Dkk-1 promoter activity, and enhanced Dkk-1 protein expression/secretion in a dose-dependent manner in these cells. Chromatin immunoprecipitation (ChIP) experiments demonstrated that DZNep-mediated activation of Dkk-1 coincided with decreased levels of H3K27Me3 within the Dkk-1 promoter. qRT-PCR, ELISA and immunoblot experiments revealed that exposure of lung cancer cells to DZNep either before or concurrent with CSC, significantly abrogated tobacco-mediated repression of Dkk-1 in vitro. Additional experiments utilizing a novel in-vivo model system revealed that daily intra-peritoneal (IP) injections of CSC for 21 days significantly decreased Dkk-1 gene expression in subcutaneous A549 xenografts, and enhanced growth of these tumors in nude mice. Simultaneous IP administration of DZNep (2.5 mg/kg BID q4 days every 7 days) significantly attenuated CSC-mediated repression of Dkk-1, and abrogated growth of tumor xenografts induced by CSC. Collectively, these findings suggest that DZNep inhibits polycomb-mediated repression of tumor suppressor genes in lung cancer cells exposed to cigarette smoke in-vitro and in-vivo, and support evaluation of DZNep for epigenetic treatment and prevention of tobacco-induced lung cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A161.