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MiR-196b is epigenetically silenced during the pre-malignant stage of lung carcinogenesis

ABSTRACT: MicroRNA silencing by promoter hypermethylation may represent a mechanism by which lung cancer develops and progresses, but the microRNAs involved during malignant transformation are unknown. We previously established a model of pre-malignant lung cancer wherein we treated human bronchial epithelial cells (HBEC) with low doses of tobacco carcinogens. Here, we demonstrate that next-generation sequencing of carcinogen-transformed HBECs treated with the demethylating agent 5-aza-2'deoxycytidine revealed miR-196b and miR-34c-5p to be epigenetic targets. Bisulfite sequencing confirmed dense promoter hypermethylation indicative of silencing in multiple malignant cell lines and primary tumors. Chromatin immunoprecipitation studies further demonstrated an enrichment in repressive histone marks on the miR-196b promoter during HBEC transformation. Restoration of miR-196b expression by transfecting transformed HBECs with specific mimics led to cell cycle arrest mediated in part through transcriptional regulation of the FOS oncogene, and miR-196b re-expression also significantly reduced the growth of tumor xenografts. Luciferase assays demonstrated that forced expression of miR-196b inhibited the FOS promoter and AP-1 reporter activity. Finally, a case-control study revealed that methylation of miR-196b in sputum was strongly associated with lung cancer (OR = 4.7, p<0.001). Collectively, these studies highlight miR-196b as a tumor suppressor whose silencing early in lung carcinogenesis may provide a selective growth advantage to pre-malignant cells. Targeted delivery of miR-196b could therefore serve as a preventive or therapeutic strategy for the management of lung cancer. HBEC2MBT and H1975 were transiently transfected with a miR-196b mimic and subjected to array-based genome-wide gene expression profiling 48 hours post transfection.

ORGANISM(S): Homo sapiens

PROVIDER: GSE79987 | GEO | 2016/09/09

SECONDARY ACCESSION(S): PRJNA317578

REPOSITORIES: GEO

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Project description:We have developed cdk4/hTERT-immortalized normal human bronchial epithelial cells (HBECs) to study lung cancer pathogenesis. By studying the oncogenic effect of common lung cancer alterations (p53, KRAS, and c-MYC) we demonstrate the ability of this model to characterize the stepwise transformation of bronchial epithelial cells to full malignancy. Using HBECs derived from multiple individuals we found: 1) the combination of five genetic alterations (p53, KRASV12, c-MYC, CDK4 and hTERT) is sufficient for full tumorigenic conversion of HBECs; 2) high levels of KRASV12 are required for full malignant transformation of HBECs, however these levels also stimulate oncogene-induced senescence; 3) RAS-induced senescence is largely bypassed with loss of p53 function; 4) over-expression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; 5) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; 6) serum-induced epithelial-to-mesenchymal transition (EMT) increases in vivo tumorigenicity; 7) genetically-identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation as well as sensitivity to standard platinum-based chemotherapies; and 8) an mRNA signature derived from tumorigenic and non-tumorigenic clones is predictive of outcome in lung cancer patients. Collectively, we demonstrate this HBEC model system can be used to study the effect of oncogenic mutations on malignant progression, oncogene-induced senescence, and EMT along with clinically translatable applications such as development of prognostic signatures and drug response phenotypes. Human bronchial epithelial cells (HBECs) immortalized with cdk4 and hTERT were transformed with p53 knockdown, KrasV12 and cMYC over-expression and profiled on Illumina HumanHT-12 V4.0 expression beadchips. Transformed HBECs were grown in two different growth media: KSFM (defined, serum-free medium) or R10 (RPMI with 10% FBS) as indicated. Clones were isolated from HBECs with sh-p53 + KrasV12 and sh-p53 + KrasV12 + cMYC.

Project description:BACKGROUND: Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets. METHODS AND FINDINGS: In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. CONCLUSIONS: By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention. Experiment Overall Design: We compared the gene expression changes HBEC and NSCLC cells before and after treatment with 5-aza-2'deoxycytidine.

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MiR-196b is epigenetically silenced during the pre-malignant stage of lung carcinogenesis

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