ZIA BC 011115 (ZIA) | |||
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Title | Epigenetic Mechanisms of Gene Expression in Thoracic Malignancies | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Schrump, David | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $830,853 | Project Dates | 10/01/2007 - 00/00/0000 |
Fiscal Year | 2014 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) |
Esophagus (20.0%) Lung (80.0%) |
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Research Type | |||
Cancer Initiation: Alterations in Chromosomes Systemic Therapies - Discovery and Development |
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Abstract | |||
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 c-Myc and K-ras 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 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 submitted for publication. 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 heterozygou" |