Increase in the sensitivity to PLX4720 through inhibition of transcription factor EB-dependent autophagy in BRAF inhibitor-resistant cells.

Long-term treatment with oncogenic BRAF inhibitors confers resistance to BRAF inhibitor monotherapy. In this study, a combination treatment strategy with autophagy inhibitors was proposed to increase the sensitivity of BRAF mutant containing A375P melanoma cells that have developed resistance to BRAF inhibitors. We found that the A375P/Multi-drug resistance (A375P/Mdr) cells, which are resistant to both BRAF inhibitors and MEK inhibitors, exhibited a higher basal autophagic flux compared to their parental A375P cells, as determined by tandem mRFP-GFP-tagged LC3 imaging assay and LC3 conversion. In addition, transcription factor EB (TFEB), which acts as a transcription factor regulating the transcription of autophagy-related genes, was much more localized in the nucleus in A375P/Mdr cells than in A375P cells, indicating that the increase in basal autophagic flux was TFEB-dependent. In particular, the overexpression of an activated form of TFEB (TFEBAA) caused a modest increase in PLX4720 resistance in A375P/Mdr cells. Interestingly, treatment with early stage autophagy inhibitors reversed BRAF inhibitor-induced resistance, whereas late autophagy inhibition did not. In contrast, inhibition of ER stress by 4-phenylbutyric acid suppressed basal autophagic flux. Moreover, ER stress inhibition significantly remarkably inhibited the nuclear localization of TFEB, resulting in an increase in the sensitivity of A375P/Mdr cells to PLX4720. Taken together, these results suggest that autophagy may be an important mechanism of acquired resistance to BRAF inhibitors. Thus, targeting autophagy may be suitable for the treatment of tumors resistant to BRAF inhibitor.

Differential Sensitivity of Wild-Type and BRAF-Mutated Cells to Combined BRAF and Autophagy Inhibition.

BRAF inhibitors are insufficient monotherapies for BRAF-mutated cancer; therefore, we investigated which inhibitory pathway would yield the most effective therapeutic approach when targeted in combination with BRAF inhibition. The oncogenic BRAF inhibitor, PLX4720, increased basal autophagic flux in BRAF-mutated cells compared to wild-type (WT) BRAF cells. Interestingly, early autophagy inhibition improved the effectiveness of PLX4720 regardless of BRAF mutation, whereas late autophagy inhibition did not. Although ATG5 knockout led to PLX4720 resistance in both WT and BRAF-mutated cells, the MEK inhibitor trametinib exhibited a synergistic effect on PLX4720 sensitivity in WT BRAF cells but not in BRAF-mutated cells. Conversely, the prolonged inhibition of endoplasmic reticulum (ER) stress reduced basal autophagy in BRAF-mutated cells, thereby increasing PLX4720 sensitivity. Taken together, our results suggest that the combined inhibition of ER stress and BRAF may simultaneously suppress both pro-survival ER stress and autophagy, and may therefore be suitable for treatment of BRAF-mutated tumors whose autophagy is increased by chronic ER stress. Similarly, for WT BRAF tumors, therapies targeting MEK signaling may be a more effective treatment strategy. Together, this study presents a rational combination treatment strategy to improve the efficacy of BRAF inhibitors depending on BRAF mutation status.

Predicting Carcinogenic Mechanisms of Non-Genotoxic Carcinogens via Combined Analysis of Global DNA Methylation and In Vitro Cell Transformation.

An in vitro cell transformation assay (CTA) is useful for the detection of non-genotoxic carcinogens (NGTXCs); however, it does not provide information on their modes of action. In this study, to pursue a mechanism-based approach in the risk assessment of NGTXCs, we aimed to develop an integrated strategy comprising an in vitro Bhas 42 CTA and global DNA methylation analysis. For this purpose, 10 NGTXCs, which were also predicted to be negative through Derek/Sarah structure-activity relationship analysis, were first tested for transforming activity in Bhas 42 cells. Methylation profiles using reduced representation bisulfite sequencing were generated for seven NGTXCs that were positive in CTAs. In general, the differentially methylated regions (DMRs) within promoter regions showed slightly more bias toward hypermethylation than the DMRs across the whole genome. We also identified 13 genes associated with overlapping DMRs within the promoter regions in four NGTXCs, of which seven were hypermethylated and six were hypomethylated. Using ingenuity pathway analysis, the genes with DMRs at the CpG sites were found to be enriched in cancer-related categories, including "cell-to-cell signaling and interaction" as well as "cell death and survival". Moreover, the networks related to "cell death and survival", which were considered to be associated with carcinogenesis, were identified in six NGTXCs. These results suggest that epigenetic changes supporting cell transformation processes occur during non-genotoxic carcinogenesis. Taken together, our combined system can become an attractive component for an integrated approach for the testing and assessment of NGTXCs.

ATG5 knockout promotes paclitaxel sensitivity in drug-resistant cells via induction of necrotic cell death.

Autophagy regulators are often effective as potential cancer therapeutic agents. Here, we investigated paclitaxel sensitivity in cells with knockout (KO) of ATG5 gene. The ATG5 KO in multidrug resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr) was generated using the CRISPR/Cas9 technology. The qPCR and LC3 immunoblot confirmed knockout of the gene and protein of ATG5, respectively. The ATG5 KO restored the sensitivity of Ras-NIH 3T3/Mdr cells to paclitaxel. Interestingly, ATG5 overexpression restored autophagy function in ATG5 KO cells, but failed to rescue paclitaxel resistance. These results raise the possibility that low level of resistance to paclitaxel in ATG5 KO cells may be related to other roles of ATG5 independent of its function in autophagy. The ATG5 KO significantly induced a G2/M arrest in cell cycle progression. Additionally, ATG5 KO caused necrosis of a high proportion of cells after paclitaxel treatment. These data suggest that the difference in sensitivity to paclitaxel between ATG5 KO and their parental MDR cells may result from the disparity in the proportions of necrotic cells in both populations. Thus, our results demonstrate that the ATG5 KO in paclitaxel resistant cells leads to a marked G2/M arrest and sensitizes cells to paclitaxel-induced necrosis.

An ATG5 knockout promotes paclitaxel resistance in v-Ha-ras-transformed NIH 3T3 cells.

Autophagy plays a contradictory role in cell survival and death. Here, we investigated changes in paclitaxel sensitivity of cells with an ATG5 gene-knockout (KO), incapable of synthesizing an E3 ubiquitin ligase necessary for autophagy. The ATG5 KO in v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3) was established using the CRISPR/Cas9 system. An LC3 immunoblot and a qRT-PCR assay were used to confirm the KO of functional ATG5. We found that the ATG5 KO led to paclitaxel resistance in Ras-NIH 3T3 cells through an ATP-binding cassette (ABC) transporter-independent mechanism. Flow cytometric analyses revealed that paclitaxel induced a remarkable significant G2/M arrest in parental cells, whereas it was relatively less effective in ATG5 KO cells. Additionally, the proportion of early apoptotic cells significantly decreased in ATG5 KO cells treated with paclitaxel than in parental cells. Interestingly, overexpression of ATG5 N-terminal cleavage product in ATG5 KO cells restored their sensitivity to paclitaxel. Taken together, our results suggest that ATG5 KO cells are resistant to paclitaxel due to the inability to produce tATG5.

Genome-wide DNA methylation changes in transformed foci induced by nongenotoxic carcinogens.

In vitro cell transformation assays (CTA) have been proposed as a method to identify possible nongenotoxic carcinogens. However, the current protocols do not provide information on the mechanism of action of the test articles. In this study, we combined an in vitro Bhas 42 CTA and sequencing-based DNA methylation profiling analysis to elucidate the carcinogenic mechanism associated with nongenotoxic carcinogens. Three nongenotoxic carcinogens were evaluated: cadmium chloride, methyl carbamate, and lithocholic acid. Methylation profiles were generated for the two nongenotoxic carcinogens (cadmium chloride and lithocholic acid) that were positive in Bhas 42 CTA. Methyl carbamate did not exhibit any promoter activity. Approximately 9.8% of all differentially methylated regions (DMRs) identified in cadmium chloride-induced transformed foci overlapped with DMRs in lithocholic acid-induced transformed foci. Interestingly, overlapping DMRs showed more hypermethylation than individual DMRs. In addition, the DMRs in CpG island elements common to both nongenotoxic carcinogens showed considerably more bias toward hypermethylated DMRs than those unique to either cadmium chloride or lithocholic acid. Pathway enrichment analysis revealed that genes harboring hypermethylated DMRs were significantly enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including pathways in cancer, basal cell carcinoma, and Wnt signaling. The genes harboring hypomethylated DMRs were significantly related to mRNA surveillance pathway, RNA transport, and autophagy. Taken together, our preliminary results on genome-wide methylation analysis of cell clones from nongenotoxic carcinogen-induced foci could be exploited for CTAs improvement, but further research will be required to standardize and assess the specificity and sensitivity of this combined approach. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.