Novel in-vitro models and correlative experiments with primary tumor/normal tissue specimens have been utilized to identify microRNA (miR) alterations which contribute to initiation and progression of tobacco -associated thoracic malignancies. These experiments identified several micro-RNAs that are either up-regulated or silenced in normal respiratory epithelia and lung cancer cells by cigarette smoke. For example, cigarette smoke condensate (CSC) mediated epigenetic repression of miR-487b in normal respiratory epithelia and lung cancer cells. Basal levels of miR-487b were significantly lower in lung cancer cells relative to normal respiratory epithelia. qRT-PCR and RNA-CLIP experiments demonstrated that miR-487b directly targets transcripts encoding Wnt5a, polycomb group proteins SUZ12 and BMI1, as well as MYC and KRAS proto-oncogenes in SAEC and lung cancer cells. All of these targets have been implicated in the establishment of a stem-cell phenotype in pulmonary carcinomas. Expression levels of miR-487b were significantly lower in resected lung cancers (especially those from smokers) relative to adjacent normal lung tissues. Repression of mir-487b correlated significantly with over-expression of all five targets in primary lung cancers. Constitutive expression of miR-487b significantly decreased expression of the five targets, and decreased proliferation, tumorigenicity and in-vivo invasion/metastasis of lung cancer cells. Collectively these experiments were the first to demonstrate that mir-487b is a novel tumor suppressor silenced by epigenetic mechanisms during human pulmonary carcinogenesis. Results of these experiments were recently published in the Journal of Clinical Investigation. In more recent experiments, we observed that CSC mediated a 4-7 fold up-regulation of the long non-coding RNA (Lnc-RNA) BC070487, with a 4-8 fold down-regulation of ZNFX1 in cultured normal or immortalized human respiratory epithelial cells, as well as Calu-6 and H841 lung cancer cells. BC070487 expression correlated inversely with expression of ZNFX1 (BC070487: 1.38-10.31 fold up vs ZNFX1: 2.26-26.29 fold down) in primary lung cancers relative to adjacent normal lung parenchyma. Overexpression or depletion of BC070487 inhibited or enhanced expression of ZNFX1, respectively in normal respiratory epithelia and lung cancer cells. CSC as well as BC070487-mediated repression of ZNFX1 coincided with increased occupancy of EZH2, SUZ12, BMI1, and increased levels of H3K27Me3, with decreased H3K4Me3 within the ZNFX1 promoter region. CSC as well as BC070487 overexpression enhanced binding of BC070487 with EZH2, SUZ12 and BMI1 proteins in SAEC and Calu-6 cells. CSC exposure significantly increased DNA methylation in one of three CpG islands spanning the regulatory elements of ZNFX1. In addition, CSC enhanced binding of BC070487 with DNMT3A and DNMT3B with site-specific de novo DNA methylation of ZNFX1 in SAEC and Calu-6 cells. FAIRE assays identified an 800 bp regulatory sequence for BC070487; CSC-mediated activation of BC070487 coincided with increased levels of H3K4Me3, H3K27Ac, H3K36Me3, Sp1, and Ago proteins, with decreased levels of H3K27Me3 and H3K9Me3 within this regulatory region. miR-31, an oncomiR specifically induced by CSC, modulated the transcriptional activity of BC070487 via remodeling the distribution of histone modification marks (Up: H3K4Me3, H3K27Ac, H3K36Me3; Down: H3K27Me3 and H3K9Me3) in the regulatory region of BC070487. Overexpression or depletion of BC070487 increased or inhibited proliferation and invasion of lung cancer cells, and similarly modulated growth of SAEC and HBEC cells. In contrast, ZNFX1 overexpression or depletion decreased or enhanced growth of normal respiratory epithelial cells and lung cancer cells. BC070487 overexpression enhanced growth of lung cancer cells xenografts in athymic nude mice, while forced expression of ZNFX1 arrested growth of these xenografts. Collectively, these data suggest that CSC-mediated up-regulation of BC070487 represses ZNFX1 to promote pulmonary carcinogenesis. Results of these studies have been selected for oral presentation at the World Lung Cancer Conference in September 2015, and a manuscript pertaining to these experiments will be submitted for publication in the near future. 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 decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in EAC cells (EACC)/ primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. Subsequent experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Additional experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 (NF-1) within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression, and down-regulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and up-regulation of KLK7 in IEE and EACC. Over-expression of miR-217 significantly decreased, whereas over-expression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via up-regulation of KLK7, and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies. A manuscript summarizing these experiments has been published in Oncogene. In additional experiments, we sought to examine the frequency and potential clinical relevance of telomerase complex mutations in esophageal carcinomas after identifying a unique germ line telomerase RNA component (TERC) deletion in a patient undergoing surgery for Barrett's adenocarcinoma. Briefly, sequencing techniques were used to evaluate mutational status of telomerase reverse transcriptase (TERT) and TERC in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. These experiments identified one deletion involving TERC (TERC del 341-360), and two non-synonymous TERT variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p 0.01). Subsequent experiments demonstrated that relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-beta-catenin complex, markedly depleted beta-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. Fluorescence in-situ hybridization and spectral karyotyping experiments demonstrated that A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage. Collectively, these experiments were the first to identify a telomerase variant in a human malignancy that simultaneously disrupts canonical as well as non-canonical telomerase activities. Our findings support further analysis of variant/mutations involving telomerase complex in esophageal cancers and preneoplastic esophageal lesions. Results of these studies were recently published in PLoS One.

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Feingold, Paul L; Surman, Deborah R; Brown, Kate et al. (2018) Induction of Thioredoxin-Interacting Protein by a Histone Deacetylase Inhibitor, Entinostat, Is Associated with DNA Damage and Apoptosis in Esophageal Adenocarcinoma. Mol Cancer Ther 17:2013-2023
Xiong, Yin; Xi, Sichuan; Shan, Jigui et al. (2018) Epigenomic Alterations and Gene Expression Profiles in Human Respiratory Epithelial Cells Mediated by Hookah and Cigarette Smoke. Ann Am Thorac Soc 15:S124-S125
Taylor Ripley, R; Surman, Deborah R; Diggs, Laurence P et al. (2018) Metabolomic and BH3 profiling of esophageal cancers: novel assessment methods for precision therapy. BMC Gastroenterol 18:94
Xi, Sichuan; Payabyab, Eden C; Straughan, David M et al. (2018) Asbestos Induces Epigenetic Repression of Ras Association Domain-Containing Protein 1, p16 Kinase 4a Inhibitor, and p14 Alternative Reading Frame in Normal Human Mesothelial Cells. Ann Am Thorac Soc 15:S123
Song, Na-Young; Zhu, Feng; Wang, Zining et al. (2018) IKK? inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways. Proc Natl Acad Sci U S A 115:E812-E821
McLoughlin, Kaitlin C; Kaufman, Andrew S; Schrump, David S (2017) Targeting the epigenome in malignant pleural mesothelioma. Transl Lung Cancer Res 6:350-365
Xu, Yuan; Feingold, Paul L; Surman, Deborah R et al. (2017) Bile acid and cigarette smoke enhance the aggressive phenotype of esophageal adenocarcinoma cells by downregulation of the mitochondrial uncoupling protein-2. Oncotarget 8:101057-101071
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
Xi, S; Inchauste, S; Guo, H et al. (2015) Cigarette smoke mediates epigenetic repression of miR-217 during esophageal adenocarcinogenesis. Oncogene 34:5548-59
Zhang, Yuwei; Calado, Rodrigo; Rao, Mahadev et al. (2014) Telomerase variant A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal carcinomas. PLoS One 9:e101010

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