PurposeIntrinsic and acquired resistance limit the therapeutic benefits of inhibitors of oncogenic BRAF in melanoma. To identify microRNAs (miRNAs) associated with resistance to a BRAF inhibitor, we compared miRNA expression levels in three cell lines with different BRAF inhibitor sensitivity.Materials and MethodsmiRNA microarray analysis was conducted to compare miRNA expression levels. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to confirm the expression of differentially expressed miRNAs. The cellular effects of miR-1246 were further examined by MTT assay, immunoblotting analysis, cell cycle analysis, flow cytometric assay of apoptosis, and autophagy assay.ResultsThe miRNA microarray analysis and qRT-PCR identified five miRNAs (miR-3617, miR-92a-1, miR-1246, miR-193b-3p, and miR-17-3p) with expression that was consistently altered in two BRAF inhibitor-resistant cell lines. Among the five miRNAs, a miR-1246 mimic significantly reduced the antiproliferative effects of the BRAF inhibitor PLX4720 in BRAF inhibitor–resistant A375P (A375P/Mdr) cells, suggesting that miR-1246 upregulation confers acquired resistance to BRAF inhibition. In particular, apoptosis was identified as a major type of cell death in miR-1246–transfected cells; however, necrosis predominated in mimic-control-transfected cells, indicating that the resistance to PLX4720 in miR-1246 mimic-transfected cells is predominantly due to a reduction in necrosis. Furthermore, we found that miR-1246 promoted G2/M arrest through autophagy as a way to escape cell death by necrosis and apoptosis in response to PLX4720. The promotion of BRAF inhibitor resistance by miR-1246 was associated with lowered levels of p-ERK.ConclusionThese results suggest that miR-1246 may be a potential therapeutic target in melanoma with acquired resistance to BRAF inhibitors.

Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/ Cas9 system. In ATG5 KO cells, RT-PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild-type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto-ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G 2 /M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G 2 /M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.Autophagy is a crucial adaptive response that functions as a primary route of degradation for long-lived proteins and cytoplasmic organelles during normal cellular homeostasis (Mizushima & Komatsu, 2011). Nonetheless, there are conflicting data on the involvement of autophagy in the regulation of cell death or survival. In fact, autophagy can either promote or inhibit cell death in different cellular contexts (Maycotte & Thorburn, 2011; White et al., 2011). Our previous studies also showed that autophagy can serve either pro-survival or -death functions (Ahn, Lee, Ahn, & Lee, 2014;Kim & Lee, 2014;Kim, Han, & Lee, 2016). We recently reported a cytoprotective role of autophagy against BH3 mimetic gossypol in melanoma cells and glioma cells (Kim & Lee, 2014;Kim et al., 2016). Conversely, we found that an inhibitor (UAI-201) of oncogenic protein BRAF induces cell cycle arrest and autophagy in BRAF-mutant glioma cells (Ahn et al., 2014). In particular, deletion of ATG5 in mice results in the development of a benign tumor in the liver (Takamura et al., 2011), suggesting that autophagy has tumor-suppressive effects.Src family kinases (SFKs) are kept in an inactive state but can be switched to an active state by abnormal events such as a mutation (Okada, 2012). Active forms of SFKs are central mediators in multiple signaling pathways that are important in oncogenesis (Kim, Song, & Haura, 2009). Thus, Src inhibition has been proposed recently as a strategy for cancer therapy (Kim et al., 2...

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