Cigarette smoke mediates epigenetic repression of miR-487b during pulmonary carcinogenesis.

MicroRNAs are critical mediators of stem cell pluripotency, differentiation, and malignancy. Limited information exists regarding microRNA alterations that facilitate initiation and progression of human lung cancers. In this study, array techniques were used to evaluate microRNA expression in normal human respiratory epithelia and lung cancer cells cultured in the presence or absence of cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly repressed miR-487b. Subsequent experiments demonstrated that miR-487b directly targeted SUZ12, BMI1, WNT5A, MYC, and KRAS. Repression of miR-487b correlated with overexpression of these targets in primary lung cancers and coincided with DNA methylation, de novo nucleosome occupancy, and decreased H2AZ and TCF1 levels within the miR-487b genomic locus. Deoxy-azacytidine derepressed miR-487b and attenuated CSC-mediated silencing of miR-487b. Constitutive expression of miR-487b abrogated Wnt signaling, inhibited in vitro proliferation and invasion of lung cancer cells mediated by CSC or overexpression of miR-487b targets, and decreased growth and metastatic potential of lung cancer cells in vivo. Collectively, these findings indicate that miR-487b is a tumor suppressor microRNA silenced by epigenetic mechanisms during tobacco-induced pulmonary carcinogenesis and suggest that DNA demethylating agents may be useful for activating miR-487b for lung cancer therapy.

Cigarette smoke induces C/EBP-ß-mediated activation of miR-31 in normal human respiratory epithelia and lung cancer cells.

BACKGROUND: Limited information is available regarding mechanisms by which miRNAs contribute to pulmonary carcinogenesis. The present study was undertaken to examine expression and function of miRNAs induced by cigarette smoke condensate (CSC) in normal human respiratory epithelia and lung cancer cells. METHODOLOGY: Micro-array and quantitative RT-PCR (qRT-PCR) techniques were used to assess miRNA and host gene expression in cultured cells, and surgical specimens. Software-guided analysis, RNA cross-link immunoprecipitation (CLIP), 3' UTR luciferase reporter assays, qRT-PCR, focused super-arrays and western blot techniques were used to identify and confirm targets of miR-31. Chromatin immunoprecipitation (ChIP) techniques were used to evaluate histone marks and transcription factors within the LOC554202 promoter. Cell count and xenograft experiments were used to assess effects of miR-31 on proliferation and tumorigenicity of lung cancer cells. RESULTS: CSC significantly increased miR-31 expression and activated LOC554202 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. CLIP and 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 SFRP1, SFRP4, and WIF-1, and increased Wnt-5a expression. CSC increased H3K4Me3, H3K9/14Ac and C/EBP-ß levels within the LOC554202 promoter. Knock-down of C/EBP-ß 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. CONCLUSIONS: Cigarette smoke induces expression of miR-31 targeting several antagonists of cancer stem cell signaling in normal respiratory epithelia and lung cancer cells. miR-31 functions as an oncomir during human pulmonary carcinogenesis.

Thoracic metastasectomy for adoptive immunotherapy of melanoma: a single-institution experience.

OBJECTIVES: Although refractory to chemotherapy, metastatic melanoma may respond to adoptive immunotherapy. As novel treatments evolve, surgeons may be asked to perform metastasectomy not only for palliation or potential cure but also for isolation of tumor-infiltrating lymphocytes. This study was undertaken to examine outcomes of patients with melanoma undergoing thoracic metastasectomy in preparation for investigational immunotherapy. METHODS: A retrospective review identified 107 consecutive patients who underwent 116 thoracic metastasectomy procedures from April 1998 to July 2009. Indications for surgical intervention included procurement of tumor-infiltrating lymphocytes, rendering of patients to no evaluable disease status, palliation, and diagnosis. Response Evaluation Criteria in Solid Tumors criteria were used to assess tumor response. RESULTS: Thoracotomy, lobectomy, and video-assisted thoracoscopic surgery with nonanatomic resection were the most common procedures. Major complications included 1 death and 1 coagulopathy-induced hemothorax. Seventeen patients were rendered to no evaluable disease status. Virtually all patients with residual disease had tumor specimens cultured for tumor-infiltrating lymphocytes; approximately 70% of tumor-infiltrating lymphocyte cultures exhibited antitumor reactivity. Of the 91 patients with residual or recurrent disease, 24 (26%) underwent adoptive cell transfer of tumor-infiltrating lymphocytes, of whom 7 exhibited objective responses (29% response rate and 8% based on intent to treat). Rapid disease progression precluded tumor-infiltrating lymphocyte therapy in most cases. Actuarial 1- and 5-year survival rates for patients rendered to no evaluable disease status or receiving or not receiving tumor-infiltrating lymphocytes were 93% and 76%, 64% and 33%, and 43% and 0%, respectively. CONCLUSIONS: Relatively few patients currently having thoracic metastasectomy undergo adoptive cell transfer. Continued refinement of tumor-infiltrating lymphocyte expansion protocols and improved patient selection might increase the number of patients with melanoma benefiting from these interventions.