MicroRNA (miRNA) dysregulation contributes widely to human cancer but has not been fully assessed in oral cancers. In this study, we conducted a global microarray analysis of miRNA expression in 40 pairs of betel quid-associated oral squamous cell carcinoma (OSCC) specimens and their matched non-tumorous epithelial counterparts. Eighty-four miRNAs were differentially expressed in the OSCC specimens compared to the matched tissue. Among these downregulated miRNAs, 19 miRNAs were found and mapped to the chromosome 14q32.2 miRNA cluster region, which resides within a parentally imprinted region designated Dlk-Dio3 and known to be important in development and growth. Bioinformatic analysis predicted two miRNAs from the cluster region, miR-329 and miR-410, which could potentially target Wnt-7b, an activator of the Wnt/β-catenin pathway, thereby attenuating the Wnt/β-catenin signaling pathway in OSCC. Stable ectopic expression of Wnt-7b in OSCC cells overexpressing miR-329 or miR-410 restored proliferation and invasion capabilities abolished by these miRNA. Combining a demethylation agent and a histone deacetylase inhibitor was sufficient to re-express miR-329, miR-410 and Meg3 consistent with epigenetic regulation of these miRNA in human OSCC. Specifically, arecoline, a major betel nut alkaloid, reduced miR-329, miR-410 and Meg3 gene expression. Overall, our results provide novel molecular insights into how betel quid contribute to oral carcinogenesis through epigenetic silencing of tumor suppressor miRNA which target Wnt/β-catenin signaling.

BRAFV600E is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAFV600E by oncogene targeting therapy increases overall survival of patients with melanoma, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAFV600E inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma, and the mechanisms involved in the antimetastatic efficacy of BRAFV600E inhibitors remain to be determined. To address this question, we developed a metastatic BRAFV600E-mutant melanoma cell line and demonstrated that the antimetastatic properties of BRAF inhibitor PLX4720 (a research analogue of vemurafenib) require host natural killer (NK) cells and perforin. Indeed, PLX4720 not only directly limited BRAFV600E-induced tumor cell proliferation, but also affected NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAFV600E lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFNγ release in the context of anti-NKp30 and IL2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAFV600E inhibitor therapy to promote more durable responses in melanoma. Cancer Res; 74(24); 1–11. ©2014 AACR.

BRAFV600E is a major oncogenic mutation found in approximately 50% of human melanoma that confers constitutive activation of the MAPK pathway and increased melanoma growth. Inhibition of BRAFV600E by oncogene targeting therapy increases overall survival of melanoma patients, but is unable to produce many durable responses. Adaptive drug resistance remains the main limitation to BRAFV600E inhibitor clinical efficacy and immune-based strategies could be useful to overcome disease relapse. Tumor microenvironment greatly differs between visceral metastasis and primary cutaneous melanoma and the mechanisms involved the anti-metastatic efficacy of BRAFV600E inhibitors remain to be determined. To address this question, we developed a metastatic BRAFV600E mutant melanoma cell line and demonstrated that the anti-metastatic properties of BRAF inhibitor PLX4720 (a research analogue of Vemurafenib) require host NK cells and perforin. Indeed, PLX4720 was not only directly limiting BRAFV600E-induced tumor cell proliferation, but was also impacting upon NK cell functions. We showed that PLX4720 increases the phosphorylation of ERK1/2, CD69 expression, and proliferation of mouse NK cells in vitro. NK cell frequencies were significantly enhanced by PLX4720 specifically in the lungs of mice with BRAFV600E lung metastases. Furthermore, PLX4720 also increased human NK cell pERK1/2, CD69 expression, and IFN-γ release in the context of anti-NKp30 and IL-2 stimulation. Overall, this study supports the idea that additional NK cell-based immunotherapy (by checkpoint blockade or agonists or cytokines) may combine well with BRAFV600E inhibitor therapy to promote more durable responses in melanoma.

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Misexpression of developmental transcription factors occurs often in human cancers, where embryonic programs may be reinstated in a context that promotes or sustains malignant development. In this study, we report the involvement of the kidney development transcription factor Six2 in the metastatic progression of human breast cancer. We found that Six2 promoted breast cancer metastasis by a novel mechanism involving both transcriptional and epigenetic regulation of E-cadherin. Downregulation of E-cadherin by Six2 was necessary for its ability to increase soft agar growth and in vivo metastasis in an immunocompetent mouse model of breast cancer. Mechanistic investigations showed that Six2 represses E-cadherin expression by upregulating Zeb2, in part, through a microRNA-mediated mechanism and by stimulating promoter methylation of the E-cadherin gene (Cdh1). Clinically, SIX2 expression correlated inversely with CDH1 expression in human breast cancer specimens, corroborating the disease relevance of their interaction. Our findings establish Six2 as a regulator of metastasis in human breast cancers and demonstrate an epigenetic function for SIX family transcription factors in metastatic progression through the regulation of E-cadherin. Cancer Res; 74(24); 1–14. ©2014 AACR.