Cultured lung cancer cells were treated with cigarette smoke condensates (CSC) using a variety of concentrations and exposure durations. Under conditions mimicking 1 pack per day (ppd) exposures for five days, CSC mediated no appreciable changes regarding in-vitro proliferation rates, but instead dramatically enhanced tumorigenicity of A549 and Calu-6 cells in nude mice. Affimetrix arrays and quantitative RT-PCR experiments revealed that CSC markedly inhibited expression of Dkk-1, a secreted Wnt signaling antagonist and putative tumor suppressor. Subsequent ChIP, pyrosequencing, and methylation specific PCR (MSP) experiments demonstrated that inhibition of Dkk-1 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 Dkk-1 promoter despite continuous CSC exposures ranging from 5-60 days. Cessation of CSC exposure resulted in diminution of Dkk-1 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 Dkk-1 in these cells. Interestingly, CSC exposure or knock-down of Dkk-1 dramatically up-regulated expression of Wnt5A, a ligand which activates a non-canonical planar-cell-polarity network implicated in tumor metastasis. Knock-down of Dkk-1 markedly enhanced tumorigenicity of lung cancer cells. Results of these studies have been published in Cancer Research. Recently, a series of experiments were conducted to specifically examine the effects of Wnt5a in lung cancer cells. Briefly, Calu-6, H358, A549, and H841 lung cancer lines were transduced with lentiviral vectors expressing Wnt5a, shRNA targeting Wnt5a, or control sequences. Calu-6, H358, and H841- but not A549 cells constitutively expressing Wnt5a exhibited significantly increased proliferation, migration, and invasion relative to vector controls. This phenomenon, which was also observed following exposure of parental Calu-6 cells to rWnt5a, was partially abrogated by Wnt5a-derived hexapeptide, Box-5. Knock-down of Wnt5a diminished proliferation, migration and invasion of A549 cells. Relative to vector controls, Calu-6 and H358 cells constitutively expressing Wnt5a exhibited significantly increased tumorigenicity in nude mice. Western blot, promoter-reporter, and focused quantitative RT-PCR array experiments demonstrated that Wnt5a increased expression of the nuclear orphan receptor, Ror2, and activated non-canonical Wnt signaling. Microarray array analysis revealed common as well as differential gene expression profiles mediated by Wnt5a in H358 tumor xenografts relative to cultured H358 cells;a gene expression profile consistent with epithelial-to-mesenchymal transition (EMT) was mediated by Wnt5a in H358 cells in-vitro and in-vivo. A similar, albeit less pronounced EMT phenomenon was observed in Calu-6 cells expressing Wnt5a. Collectively, these data demonstrate that Wnt5a enhances the malignant phenotype of lung cancer cells, and support the development of therapies targeting Wnt5a-mediated signaling for lung cancer therapy. A manuscript pertaining to these experiments is currently under peer review. 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-beta 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. 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 published recently in Oncogene. Additional experiments have been undertaken to comprehensively examine micro-RNA expression profiles in untreated and CSC-exposed lung cancer cells, and cultured normal respiratory epithelia, as well as primary lung cancers and paired adjacent histologically normal lung parenchyma from patients undergoing potentially curative resections at the NCI. This analysis revealed that miRNA signatures coincided with human lung cancer progression. Furthermore, miRNA profiles distinguished lung cancers derived from smoker and non-smokers. In addition, cigarette smoke consistently up-regulated mir-31, while down-regulating mir-487b in cultured normal respiratory epithelia and lung cancer cells. Quantitative RT-PCR and western blot experiments confirmed that CSC significantly increased miR-31 expression and activated LOC554202 (the host gene for miR-31) in normal respiratory epithelia and lung cancer cells;miR-31 and LOC554202 expression persisted following discontinuation of CSC exposure. miR-31 and LOC554202 expression levels were significantly elevated in lung cancer specimens relative to adjacent normal lung tissues. RNA cross-link immunoprecipitation (CLIP) and 3'UTR reporter assays demonstrated direct interaction of miR-31 with Dickkopf-1 (Dkk-1) and DACT-3. Over-expression of miR-31 markedly diminished Dkk-1 and DACT3 expression levels in normal respiratory epithelia and lung cancer cells. Knock-down of miR-31 increased Dkk-1 and DACT3 levels, and abrogated CSC-mediated decreases in Dkk-1 and DACT-3 expression. Furthermore, over-expression of miR-31 diminished expression of several other Wnt antagonists including SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-beta levels within the LOC554202 promoter. Knock-down of C/EBP-beta abrogated CSC-mediated activation of LOC554202. Over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells;knock-down of miR-31 inhibited growth of these cells. Collectively, these experiments suggest that dual mechanisms (polycomb and micro-RNA) mediate repression of several antagonists of Wnt signaling in normal respiratory epithelia and lung cancer cells during human pulmonary carcinogenesis. Results of these latter studies have been tentatively accepted for publication in PLoS One.
| Shukla, Vivek; Rao, Mahadev; Zhang, Hongen et al. (2017) ASXL3 Is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer. Cancer Res 77:6267-6281 |
| 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 |
| Reardon, Emily S; Hong, Julie A; Straughan, David M et al. (2015) Pulmonary Metastases Exhibit Epigenetic Clonality: Implications for Precision Cancer Therapy. Ann Thorac Surg 100:1839-48; discussion 1848 |
| 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 |
| Liu, F; Killian, J K; Yang, M et al. (2010) Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate. Oncogene 29:3650-64 |
