The BRAF-mutant melanoma cell line A375 was obtained from the American Type Culture Collection (Manassas, VA, USA). A375 was cultured in RPMI-1640 medium (HyClone) supplemented with 10% FBS and 1% penicillin/streptomycin.

ACY-1215 and Z-VAD-FMK were purchased from Medchem Express (Monmouth Junction, NJ, USA). The anti-PARP (#9542), anti-eIF2α (#5324), anti-p-eIF2α (#9721) and anti-CHOP (#2895) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-caspase-3 (EAP0893) antibody was from Elabscience (Wuhan, Hubei, China). The anti-p-ERK (ab76299) antibody was purchased from Abcam (Cambridge, UK). The anti-ERK (16443-1-AP) and anti-GFP (50430-2-AP) antibodies was obtained from Proteintech (Chicago, IL, USA). The anti-β-actin mouse monoclonal antibody (AM1021B) was purchased from Abgent (San Diego, CA, USA).

A375 cell proliferation was measured at the indicated times by Cell counting kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan Japan). A375 colony formation was measured by seeding cells in 6-well plates 1,000 per well and were cultured over a 14-day period. Colonies were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet.

Effects of plasmids or drugs on apoptosis of A375 cells were evaluated by Annexin V/7-aminoactinomycin D (7-AAD) assay. Flow cytometric analysis of A375 cells labeled with Annexin V-phycoerythrin (PE) and 7-AAD apoptosis detection kit (BD Biosciences, San Jose, CA, USA) was performed according to the manufacturer's instructions. The rates of cellular apoptosis were acquired immediately on a FACSArial flow cytometer (BD Biosciences).

The plasmid encoding HDAC6 was kindly provided by Professor Jun Zhou (16). The plasmids have been validated by sequencing, transfection and subsequent western blotting. The plasmids were transfected into cells with Lipofectamine 2000 transfection reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol.

A375 cells were lysed in RIPA lysis buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and 50 mM Tris-HCl, pH 7.4) with protease inhibitor cocktails and phosphatase inhibitor cocktails from Roche (Mannheim, Germany) on ice for 30 min and centrifuged at 12,000 rpm for 15 min to collect whole cell lysate. Protein extracts were subjected to electrophoresis on a 10% SDS-PAGE gel and transferred onto PVDF membrane (Roche). Then the membrane was then incubated in blocking buffer for 2 h before the addition of the primary antibodies. The secondary antibody used was the horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibody (Proteintech). Signals were detected using WesternBright ECL HRP substrate (Advansta, Menlo Park, CA, USA) and developed with Kodak film.

Statistical analysis was performed with Student's t-test. All analyses were realized by using the statistical software SPSS19.0 (IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation (SD). P<0.05 was considered a statistically significant difference.

HDAC6 can confer resistance to chemotherapy in several types of cancer (12–14), but the role of HDAC6 in melanoma chemotherapy resistance is largely unknown. HDAC6 has been shown to be overexpressed in melanoma cell lines and tissues (10). We speculate that overexpression of HDAC6 in melanoma might confer resistance to vemurafenib. As shown in Fig. 1A, overexpression of HDAC6 significantly improved the percentage of viable A375 cells after vemurafenib treatment. We also came to the same conclusion by the colony formation assay (Fig. 1B). Vemurafenib has been shown to cause apoptotic cell death at elevated concentrations, thus we investigated whether HDAC6 regulates vemurafenib mediated cell apoptosis in melanoma cells. Overexpression of HDAC6 significantly decreased the percentage of apoptotic cells after vemurafenib treatment (Fig. 1C). A decrease in apoptosis was further evidenced by detection of cleaved PARP and caspase-3 (Fig. 1D).

Because HDAC6 confers resistance to vemurafenib, we speculated that the inhibition of HDAC6 might contribute to an increase in the efficiency of vemurafenib in melanoma. To further assess this possibility, we tested the effect of HDAC6 inhibition on A375 using the selective inhibitor ACY-1215. ACY-1215 is the first oral, selective HDAC6 inhibitor in clinical trials and was well tolerated as monotherapy ≤360 mg/day (17,18). Unlike non-selective HDAC inhibitors, which are associated with severe fatigue, vomiting, diarrhea and myelosuppression (18). ACY-1215 offers a therapeutic advantage due to minimal toxicity. HDAC6 is required for the proliferation and metastasis of melanoma cells (10,11). However, few studies have focused on the function of ACY-1215 in melanoma cells. We first assessed growth inhibition in response to ACY-1215 treatment in A375. A dose-dependent decrease in cell proliferation was observed (Fig. 2A). The same conclusion was drawn from the results of the clone formation assay. We noted a significant decrease in clone numbers after ACY-1215 treatment (Fig. 2B). ACY-1215 also induces apoptosis in A375, as evidenced by increases in the number of Annexin V-positive cells and the cleavage of PARP and caspase-3 (Fig. 2C and E). Combination inhibition of apoptosis by pan caspase inhibitor Z-VAD-FMK with ACY-1215 remarkablely improved the percentage of cell viability compared to ACY-1215 alone (Fig. 2D). To examine whether a cooperative effect exists between ACY-1215 and vemurafenib in the chemotherapeutic treatment of melanoma, we treated A375 cells with ACY-1215 and vemurafenib either alone or in combination. In agreement with our hypothesis, the co-treatment of ACY-1215 and vemurafenib significantly reduced the cell viability compared to vemurafenib alone (Fig. 3A). We also observed a significant increase of the percentage of apoptotic cells after cotreatment of vemurafenib with ACY-1215 compared to vemurafenib alone (Fig. 3B). The protein levels of cleaved PARP and caspase-3 also increased after co-treatment (Fig. 3C).

Next, we investigated the mechanism by which ACY-1215 contribute to the vemurafenib-induced cell death of melanoma cells. HDAC6 plays a vital role in regulation of degrading misfolded protein through the ubiquitin proteasome system and autophagy (19,20). We found that both vemurafenib and ACY-1215 increased the accumulation of polyubiquitinated proteins (Fig. 4A and B). Moreover, the combination of ACY-1215 plus vemurafenib increased the accumulation of polyubiquitinated proteins compared with either agent alone (Fig. 4C). Accumulation of polyubiquitinated proteins can induce ER stress (21). Vemurafenib has been shown to induce endoplasmic reticulum (ER) stress-mediated apoptosis to kill BRAF^V600E melanoma cells (22). Upon ER stress, the sensor PERK is activated and phosphorylates eIF2a (23,24). During ER stress, the protein levels of CHOP are also elevated and CHOP functions to mediate programmed cell death (24,25). To determine the onset of ER stress in response to ACY-1215, we examined the phosphorylation of eIF2α by western blotting and found that it was rapidly phosphorylated after ACY-1215 treatment (Fig. 4D). Vemurafenib can also clearly induce ER stress as previously reported (Fig. 4C). Combination treatment of vemurafenib and ACY-1215 significantly increased the phosphorylation of eIF2α and protein levels of CHOP. Taken together, these results indicate that ACY-1215 induced ER stress plays a role in overcoming resistance to vemurafenib.

Hyperactivation of the RAF-MEK-ERK signaling pathway plays a vital role in tumorigenesis of BRAF-mutant melanoma (26). RAF kinase directly phosphorylates MEK, which in turn phosphorylates ERK to promote cell proliferation and to inhibit apoptosis (27). Vemurafenib blocks ERK phosphorylation to inhibit proliferation of BRAF-mutant cells (3,22), this was also confirmed in our experiment (Fig. 5A). HDAC6 can induce ERK hyper-activation in Madin-Darby canine kidney (MDCK) and HEK293T cells (28). Overexpression of HDAC6 also increased ERK activity in A375 cells (Fig. 5B). Conversely, ACY-1215 decreases the phosphorylation of ERK in a dose-dependent manner (Fig. 5C). Moreover, overexpression of HDAC6 increased ERK activity after vemurafenib treatment (Fig. 5D). We found that the activation of ERK was inhibited by the combination of ACY-1215 and vemurafenib compared with the administration of either inhibitor alone (Fig. 5E). Taken together, our results show that inhibition of HDAC6 sensitizes A375 cells to vemurafenib partly via inactivation of ERK.