Cultured lung cancer cells were treated with tobacco smoke condensates (TSC) using a variety of concentrations and exposure durations. Under conditions mimicking 1 pack per day (ppd) exposures for five days, TSC mediated no appreciable changes regarding in-vitro proliferation rates, but instead dramatically enhanced tumorigenicity of A549 and Calu-6 cells in nude mice. Affimetrix long-oligo array and quantitative RT-PCR experiments revealed that TSC markedly inhibited expression of DKK1, a secreted Wnt signaling antagonist and putative tumor suppressor. Subsequent ChIP, pyrosequencing, and methylation specific PCR (MSP) experiments demonstrated that inhibition of DKK1 expression by TSC coincided with a dose-dependent increase in H3K27 trimethylation, and recruitment of polycomb repressor complexes without an appreciable increase in DNA methylation within the DKK1 promoter despite continuous TSC exposures ranging from 5-60 days. Cessation of TSC exposure resulted in diminution of DKK1 promoter-associated polycomb proteins, and restoration of DKK1 expression in these cancer cells. Western blot and focused qRT-PCR array experiments indicated that TSC mediated dose-dependent increases in Wnt signaling in A549 and Calu-6 cells. Similar findings were noted following siRNA mediated knock-down of DKK1 in these cells. Interestingly, TSC exposure or knock-down of DKK-1 dramatically upregulated expression of Wnt5A, a ligand which activates a non-canonical planar-cell-polarity network implicated in tumor metastasis. Results of these studies were published several months ago. More recent studies have demonstrated that constitutive over-expression of Wnt5A activates downstream targets of the PCP pathway, and markedly enhances invasion and tumorigenicity of Calu-6 cells. Interestingly, lung cancer patient who smoke have significantly worse outcomes than former or never smokers, and Wnt5A expression is an independent determinant of treatment failure and poor outcome in lung cancer patients. Additional experiments are in progress to characterize the mechanisms by which Wnt5A enhances the malignant phenotype of lung cancer cells, and, to ascertain if expression of Wnt5A enhances metastatic potential of lung cancer cells in an organ specific manner. Results of these experiments may provide the rationale for targeting Wnt5A as a strategy to diminish cancer stem cell signaling and metastases in lung cancer patients. Whereas the vast majority of lung cancers are directly attributable to cigarette smoking, limited information is available regarding early epigenetic events mediating initiation and progression of these neoplasms. As such, we sought to establish an in-vitro system to examine sequential epigenetic effects of cigarette smoke in respiratory epithelia. Briefly, normal human small airway epithelial cells (SAEC) and cdk4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) for up to nine months under potentially relevant exposure conditions. Western blot analysis demonstrated that CSC mediated dose- and time-dependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3;these histone alterations coincided with decreased DNMT1 and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3 as well as activation of Wnt signaling;and, hypermethylation of tumor suppressor genes such as RASSF1A, RUNX3, and RAR-β which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft agar clones derived from CSC-exposed HBEC;a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft agar clonogenicity but not tumorigenicity of HBEC. Collectively these data indicate that cigarette smoke induces cancer-associated epigenomic alterations in cultured respiratory epithelia. This in-vitro model may prove useful for delineating early epigenetic mechanisms of gene regulation during pulmonary carcinogenesis. A manuscript pertaining to these studies has been submitted for publication. Additional experiments have been undertaken to examine micro-RNA expression profiles in untreated and TSC-exposed HBEC and SAEC relative to cancer lines or primary tumor specimens from lung cancer patient. Four sets of cell lines were chosen for this analysis including short term NHBE and SAEC, immortalized HBECs, lung cancer lines from non-smokers (H1650 and H1975), as well as lung cancer lines from smokers (H1299, H358, Calu-6, and A549). Briefly, small RNAs were isolated from untreated NHBE, SAEC, HBEC, A549, and Calu-6 cells treated with TSC for 72h, and A549 and Calu-6 cells exposed to DAC/DP using our standard treatment regimen. miRNA expression profiles were evaluated using NCode Multi-Species miRNA Microarray V2 chips containing probes that target all of the miRNA species in the Sanger mirBase 9.0 for human, mouse, rat, Drosophila, C. elegans, and zebrafish. Microarray data were processed and normalized, with statistic and clustering analyses performed using Genespring software. Two-way analysis of variance (ANOVA) techniques were used to identify statistically different miRNA expression levels in untreated and TSC-exposed cells. Similar techniques were used to compare miRNA expression in primary respiratory epithelia, HBEC, smoker-, and nonsmoker- derived lung cancer lines for cancer progression modeling. This analysis yeilded several interesting results including: 1. miRNA signature can classify human lung cancer progression. Based on the global miRNA expression clustering, primary respiratory epithelia, immortalized bronchial epithelial cells, and smoker/ non-smoker derived cancer lines could be readily distinguished from one another. Each group exhibited its own set of miRNAs that were over- or under-expressed relative to the primary cells. 2. MiRNA profiles distinguish lung cancers derived from smoker and non-smokers. Forty-two miRNAs were differentially expressed in tobacco- associated lung cancer lines A549, Calu-6, H1299, and H358 relative to H1650 and H1975 cells that were derived from non-smokers. Notably, mir-31, mir-21 and several let-7 family members were significantly over-expressed in tobacco- associated lung cancers. 3. tobacco smoke modulates miRNA expression in normal respiratory epithelia and lung cancer cells. Comparison of the miRNA expression profiles between TSC-treated and untreated SAEC, NHBE, HBEC, A549 and Calu-6 cells, revealed that tobacco smoke exposure significantly altered expression of mir-21, mir-31 and mir-487b. Of particular interest, our recent experiments revealed that mir-31 targets DKK-1 and several other transcripts encoding Wnt antagonists. The host gene encoding mir-31 has been identified, and experiments are in progress to ascertain the mechanisms by which TSC induces this gene. These studies provide a direct link between cigarette carcinogens and activation of Wnt signaling, which is critical for maintenance of cancer stem cells;manuscripts pertaining to these studies are nearing completion, and will be submitted for publication soon. Ongoing translational efforts are focused on confirming these findings in [summary truncated at 7800 characters]
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