Novel in-vitro models and correlative experiments with primary tumor/normal tissue specimens have been utilized to identify epigenomic alterations which contribute to initiation and progression of tobacco -associated thoracic malignancies. Our recent experiments have identified several oncomirs and tumor suppressor micro-RNAs that are either up-regulated or silenced, respectively, in normal respiratory epithelia and lung cancer cells following exposure to cigarette smoke condensate (CSC). More recently we used micro-array, qRT-PCR, and immunoblot techniques were used to assess lncRNA and coding gene expression in normal respiratory epithelial cells (NREC) and lung cancer cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly increased expression of lncRNA BC070487 while decreasing expression of ZNFX1. Overexpression or depletion of BC070487 inhibited or increased ZNFX1 expression, respectively. BC070487 expression correlated inversely with ZNFX1 expression in primary lung cancers relative to paired normal lung tissues. CSC and BC070487-mediated repression of ZNFX1 coincided with DNA methylation and increased occupancy of EZH2, SUZ12, and BMI1 within the ZNFX1 promoter. CSC enhanced binding of BC070487 with DNMT3A, DNMT3B, EZH2, SUZ12 and BMI1 proteins. CSC-mediated induction of BC070487 could be recapitulated by over-expression of miR-31, an oncomir which exhibits sequence complementarity with the BC070487 regulatory region. CSC mediated activation of BC070487 also coincided with recruitment of SP1 to the BC070487 regulatory region; knock-down of SP1 significantly attenuated up-regulation of BC070487 by CSC. Overexpression or depletion of BC070487 increased or decreased, whereas ZNFX1 overexpression or depletion decreased or enhanced, respectively, in-vitro growth of NREC as well as growth and invasion of lung cancer cells in-vitro and in-vivo. Mithramycin repressed BC070487 and induced ZNFX1 expression in lung cancer cells in-vitro and in-vivo. Collectively, these data demonstrate a novel feed-forward noncoding RNA circuit that contributes to the pathogenesis of tobacco-induced lung cancers in part by suppression of ZNFX1, and suggest that mithramycin may be useful for restoring ZNFX1 expression for lung cancer therapy. Results of these studies have been presented in oral format at several international cancer epigenetics meetings, and a comprehensive manuscript pertaining to these experiments has been submitted for peer review. In additional experiments, array and qRT-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media (NM) with or without CSC. Under relevant exposure conditions, CSC significantly down-regulated miR-145 as well as miR-143 in immortalized esophageal epithelial cells (IEEC) and esophageal adenocarcinoma cells (EACC) in a time and dose dependent manner. miR-145 and miR-143 are intra-genic polycistronic microRNAs that have been implicated in inhibition of growth and metastasis of cancer cells in a cell specific and tissue context manner. CSC-mediated repression of miR-145 coincided with up-regulation of LOXL2 expression in IEEC and EACC. Software guided analysis, qRT-PCR and luciferase experiments demonstrated that miR-145 directly targets the 3'UTR of lysyl-like oxidase 2 (LOXL2), which encodes a secreted protein that activates multiple pathways promoting EMT, and also functions as a co-repressor with SNAIL to deaminate H3K4Me3 and silence differentiation related genes such as CDH. Endogenous levels of miR-145 and miR-143 were significantly lower in EACC/primary EAC compared to IEEC/normal esophageal mucosa (p0.003). Furthermore, down-regulation of mir-145 coincided with over-expression of LOXL2 in EAC specimens, particularly those from smokers. Constitutive over-expression of LOXL2 enhanced proliferation, invasionand migration of EAC in-vitro, and significantly increased growth of EAC xenografts in athymic nude mice. These findings were recapitulated following stable knock-down of miR-145 in EAC cells. Interestingly, additional mechanistic studies demonstrated that CSC exposure or LOXL2 over-expression repressed mir-145 as well as miR-143 and their host gene, suggesting that LOXL2 negatively regulates the miR-143/miR-145 cluster. qChIP experiments confirmed recruitment of LOXL2 to the promoter of the miR-143/miR-145 host gene in IEEC and EACC following CSC exposure. Collectively, these studies have identified a novel positive feed-back loop which activates a master regulator of EMT during esophageal carcinogenesis, and suggest that LOXL2 is a potential therapeutic target in EAC. Additional studies are in progress to further characterize the transcriptional mechanisms by which cigarette smoke up-regulates LOXL2, prior to submission of a manuscript for peer review. Previously we have demonstrated that EZH2, a core component of polycomb repressive complex 2 (PRC2) is over-expressed in malignant pleural mesothelioma and is a potential therapeutic target in these neoplasms. In additional studies, we have demonstrated that Mithramycin (MM) significantly inhibits proliferation, invasion soft agar clonogenicity, and tumorigenicity of MPM cells, in part via depletion of SP1 and activation of p53. In more recent experiments, we have sought to develop clinically relevant strategies to enhance the potential efficacy of MM using inhibitors of HDM2, which is a p53 target gene whose protein product associates with PRC-2 to enhance stemness in cancer cells. qRT-PCR and immunoblot experiments demonstrated that MM mediated significant dose dependent up-regulation of HDM2 in MPM cells in-vitro and in-vivo. Subsequent experiments were performed to examine if RITA (Reactivation of p53 and Induction of Tumor cell Apoptosis) as well as RG7388, an agent in early phase clinical trials, could enhance MM mediated cytotoxicity in MPM cells. RITA induces conformational changes in mutant (mt) as well as wt p53 that inhibit binding of p53 to HDM2, whereas RG7388 directly inhibits binding of wt but not mt p53 to HDM2. MES1 and MES7 cells (wt p53) as well as H2452 cells (mt p53) were cultured in the presence or absence of RITA or RG7388. Treatment with either RITA or RG7388 alone reduced viability of MES1 and MES7 cell lines in a dose and time dependent manner. As anticipated, RITA was more effective than RG7388 in arresting growth of H2452 cells which express mutant p53. Immunoblot experiments demonstrated dose dependent increases in p53 and p21 protein levels in MES1 and MES7 cells following treatment with RITA and RG7388. RITA down-regulated HDM2 levels in a dose dependent manner; in contrast, low dose RG7388 decreased HDM2 levels, whereas higher doses of RG7388 significantly increased HDM2 expression. Sequential MM/RITA or MM/RG7388 exposure mediated additive to supra-additive pro-apoptotic effects in cultured MPM cells. This phenomenon coincided with markedly decreased Sp1, EZH2 and H3K27Me3 levels in MPM cells. Sequential drug treatments induced in-vivo growth inhibitory effects which were comparable to single agent MM but with a 66% reduction in total MM exposure (1mg/kg qwk vs 1mg/kg qMWF). Collectively these data support evaluation of HDM2i in combination with MM or other activators of p53 signaling for mesothelioma therapy. A manuscript pertaining to these experiments is in the final phase of preparation for peer review.
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