A series of experiments have been undertaken to comprehensively examine miRNAs that contribute to initiation and progression of tobacco-induced human lung cancers. Briefly, array techniques were used to examine miRNA expression profiles in lung cancer lines established from smokers and non-smokers as well as normal respiratory epithelia cultured in the presence or absence of CSC. This analysis revealed that miRNA signatures coincided with human lung cancer progression. Furthermore, miRNA profiles distinguished lung cancers derived from smokers relative to nonsmokers. Under relevant exposure conditions, CSC consistently up-regulated miR-31 in cultured normal respiratory epithelia and lung cancer cells. qRT-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 suggesting reprogramming in these cells. qRT-PCR experiments revealed that 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 expression of Wnt-5a, a non-canonical Wnt ligand implicated in maintenance of cancer stem cells, which enhances the malignant phenotype of lung cancers in-vitro and in-vivo. Chromatin immunoprecipitation (ChIP) experiments demonstrated that 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. These experiments demonstrating that miR-31 functions as an oncomir during tobacco-induced human pulmonary carcinogenesis were published recently in PLoS One. More recent studies utilizing the same in-vitro model system revealed that CSC significantly repressed miR-487b in cultured normal respiratory epithelia and lung cancer cells. Interestingly, analogous to what was observed for miR-31, repression of miR-487b in cultured cells persisted following cessation of CSC exposure. qRT-PCR experiments demonstrated that miR-487b expression was significantly lower in primary lung cancers particularly those from smokers relative to adjacent normal lung parenchyma. Software-guided analysis revealed numerous potential targets for miR-487b including Wnt5a, SUZ12, BMI1, c-MYC and K-ras-mediators of stem cell pluripotency. Constitutive over-expression of miR-487b inhibited, whereas depletion of endogenous miR-487b enhanced expression of Wnt5a, BMI1, SUZ12, c-MYC and K-ras in SAEC and Calu-6 cells. ChIP analysis revealed that repression of miR-487b coincided with increased recruitment of SUZ12 and BMI1 to Dkk-1, SFRP1, SFRP4, and WIF-1 promoter regions, and down-regulation of these genes in normal respiratory epithelia and lung cancer cells. CLIP and 3'UTR experiments confirmed direct interference of miR-487b with Wnt5a, BMI1, SUZ12, c-MYC, and K-ras transcripts. Sodium bisulfite sequencing, methylated DNA precipitation (MeDIP) and ChIP, and nucleosome positioning experiments demonstrated that repression of miR-487b coincided with DNA methylation, de-novo nucleosome occupancy, and recruitment of SUZ12 and BMI1with decreased H2AZ and TCF1 levels within the miR-487b promoter region. Deoxyazacytidine induced miR-487b expression, and attenuated CSC-mediated repression of miR-487b. TGF-beta1 recapitulated CSC-mediated effects on miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling;inhibited lung cancer invasion mediated by CSC, or over-expression of c-MYC, k-ras, or TGF-beta1;and decreased proliferation, and tumorigenicity of lung cancer cells. These findings indicate that epigenetic silencing of miR-487b cooperates with up-regulation of miR-31to activate critical pathways mediating pluripotency during tobacco-induced human pulmonary carcinogenesis, and suggest that DNA demethylating agents may be useful for restoring miR-487b expression for lung cancer therapy. A manuscript pertaining to these studies, which are the first to directly implicate repression of miR-487b in the pathogenesis of human lung cancers, has been submitted for publication. Additional experiments have been performed to further examine mechanisms by which cigarette smoke increases the malignant phenotype of lung and esophageal cancer cells. Briefly, lung and esophageal cancer cells (A549, Calu-6, NCI-SB-ESC1 and NCI-SB-ESC2), were cultured in normal media (NM) with or without CSC under clinically relevant exposure conditions. Microarray analysis revealed that five day CSC exposure significantly up-regulated ABCG2, encoding a xenobiotic pump highly expressed in cancer stem cells. Quantitative reverse transcription-PCR (qRT-PCR), western blot, and immunohistochemistry experiments confirmed up-regulation of ABCG2 in cultured cancer lines, but not normal small airway epithelial cells (SAEC) or immortalized esophageal squamous cells (HET1A) exposed to CSC. Flow cytometry experiments demonstrated that CSC increased the side population (SP) of cultured cancer cells. Transient transfection experiments using ABCG2 promoter reporter constructs revealed that deletion of xenobiotic response elements as well as SP-1 sites markedly attenuated ABCG2 induction by CSC. ChIP experiments revealed that CSC-mediated induction of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor (AHR), SP-1, and Nrf2, as well as increased levels of RNA pol II and H3K9Ac within the ABCG2 promoter. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AHR, SP-1, and Nrf2 levels within the ABCG2 promoter, markedly down-regulated ABCG2 expression, decreased SP, and dramatically inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Micro-array analysis revealed that mithramycin treatment significantly repressed cell cycle and cancer-related genes. Approximately 1260 genes were commonly altered in Calu-6 and A549 cells at two mithramycin doses;the vast majority of these genes were repressed by drug treatment. For example, 37 of 38 cancer transformation genes were down-regulated by mithramycin. Additional analysis revealed significant inhibition of critical cancer pathways including AKT, TGF-beta, and Wnt following mithramycicn exposure. Collectively, these findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical evaluation of mithramycin for targeting cancer stem cell signaling in thoracic malignancies. A manuscript pertaining to these studies is nearing completion, and a novel clinical protocol utilizing mithramycin to eradicate cancer stem cells in patients with thoracic malignancies will be initiated in the next several months.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011418-01
Application #
8349541
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2011
Total Cost
$514,430
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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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
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