Our published experiments have indicated that the DNA demethylating agent 5 aza 2 deoxycytidine (DAC) mediates dramatic induction of CT-X genes such as NY-ESO-1, MAGE-A1, and MAGE-A3 in cultured lung cancer cells, but not normal human bronchial epithelia (NHBE). Furthermore we have demonstrated that the HDAC inhibitor Depsipeptide FK228 (DP) enhances DAC-mediated induction of CT-X genes in lung cancer cells. Induction of CT-X gene expression by sequential DAC/DP treatment coincided with a dramatic increase in expression of several BORIS splice variants in lung cancer cells. Methylation specific PCR (MSP) and sodium bisulfite sequencing experiments demonstrated that activation of BORIS in cultured lung cancer cells coincided with demethylation of a CpG island within the BORIS promoter. Additional MSP experiments revealed de-repression of BORIS in 6 of 12 primary lung cancer specimens;in several patients, de-repression of BORIS was detected in lung cancers as well as histologically normal tissue adjacent to these neoplasms, raising the possibility that activation of BORIS is an early epigenetic event during pulmonary carcinogenesis. Subsequent electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that BORIS physically interacts with the NY-ESO-1 promoter, and that de-repression of NY-ESO-1 in lung cancer cells coincides with a CTCF-to-BORIS shift in occupancy and recruitment of Sp1 within the NY-ESO-1 promoter. A similar phenomenon was observed for MAGE-A1 activation in lung cancer cells. In more recent studies, pyrosequencing and chromatin immunoprecipitation (ChIP) techniques were used to comprehensively examine chromatin alterations associated with repression/activation of NY-ESO-1 in a panel of lung cancer lines. Repression of NY-ESO-1 in A549, Calu-6 and H841 cells as well as normal human bronchial epithelial cells coincides with CpG hypermethylation, recruitment of a variety of DNMTs, methyl CpG binding proteins, and histone demethylases including LSD1 and JARID1B, JARID1D, as well as deacetylation of histones H3/H4 and increased levels of H3K9me3 and H3K27me3 within the NY-ESO-1 promoter. In contrast, activation of NY-ESO-1 in untreated H1299 cells, or H841 cells following sequential DAC/DP exposure coincides with marked DNA demethylation, reduced levels of DNMTs, MBDs, and histone demethylases, accumulation of hyperacetylated core histones, as well as increased levels of H3K4me2, H3K4me3, and H3K9Ac within the NY-ESO-1 promoter. Knock-down of EZH2 or LSD1, JARID1B,JARID1D,or SIRT1 markedly enhanced DAC-mediated activation of NY-ESO-1. Comparable results were noted for MAGE-A1 and MAGE-A3 induction mediated by DAC. Additional studies have demonstrated that the polycomb inhibitor, DZNep also enhances DAC-mediated activation of CT-X gene expression in cancer cells. Furthermore, DZNep potentiates apoptosis mediated by DNA demethylating agents and HDAC inhibitors in lung cancer cells. Results of these studies may facilitate the development of novel epigenetic strategies to induce apoptosis and augment immunogenicity of thoracic malignancies. A series of experiments have been undertaken to ascertain if BORIS contributes to initiation and maintenance of the transformed phenotype of lung cancer cells, utilizing tet-inducible viral constructs to over-express or knock-down BORIS expression in SV40- or cdk4/h-tert- immortalized human bronchial epithelial cells (BEAS, and HBEC, respectively), which are deficient for BORIS and CT-X gene expression, as well as Calu-6 (p53 null) and A549 (p53 wt) lung cancer cells exhibiting extremely low level BORIS and CT-X gene expression, and H841 and H1299 lung cancer cells expressing very high levels of BORIS variants. Interestingly, BEAS cells transduced with a tet-inducible BORIS lentiviral construct exhibited loss of contact inhibition, increased proliferation and invasion, as well as enhanced clonogenic potential following tetracycline exposure relative to control cells. This phenomenon coincided with silencing of E-cadherin, a gene known to regulate epithelial-mesenchymal transition, which has been implicated in cancer metastasis. Additional studies suggest that knock-down of BORIS expression alters proliferation and tumorigenicity of lung cancer cells. Ongoing studies are focused on ascertaining if the effects of BORIS on E-cadherin expression are related to SV40 or HPV oncoprotein expression in immortalized or transformed lung cancer cells, and exploring the mechanisms by which BORIS mediates apparent EMT in cultured cells. Collectively, these studies may elucidate potential mechanisms and clinical relevance of BORIS activation during pulmonary carcinogenesis.
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