BRAF V600E-dependent role of autophagy in uveal melanoma

Abstract

Background: Autophagy can function in a dual role in cancer development and progression: It can be cytoprotective or contribute to cell death. Therefore, determining the contextual role of autophagy between these two opposing effects is important. So far, little is known about the role of autophagy in uveal melanoma. In the present study, we looked to investigate the autophagic process, as well as its effect on cell survival in uveal melanoma cell lines under stressed conditions (starvation). The possible role of autophagy during BRAF inhibition in uveal melanoma was also sought.

Methods: Two human uveal melanoma cell lines, OCM1A, which harbors the BRAF mutation V600E and Mel 290, which is BRAF wild type, were studied. Autophagy levels were determined by Western blot assay with/without the addition of autophagic flux inhibitor (bafilomycin A1). Cell proliferation was assessed by an MTT assay.

Results: Starvation triggered autophagy in BRAF V600E-mutant OCM1A cells but not in BRAF wild-type Mel 290 cells. Enhanced autophagy helped the OCM1A cells survive under stressed conditions. The BRAF inhibitor vemurafenib upregulated autophagy through suppression of the PI3K/Akt/mTOR/p70S6 K pathway in BRAF V600E-mutant uveal melanoma cells. Autophagy inhibition impaired the treatment efficacy of vemurafenib in BRAF V600E-mutant uveal melanoma cells.

Conclusions: Our data demonstrate that starvation-trigged autophagy, which is BRAF V600E dependent, promotes cancer cell survival in uveal melanoma. Vemurafenib induces autophagic cell death rather than adaptive cell survival in BRAF V600E-mutant melanoma.

Keywords: Autophagy; BRAF inhibitor; BRAF mutation; Uveal melanoma.

Fig. 1

Immunoblots and gel density quantifications against autophagy marker (LC3) in uveal melanoma cells. Cells were exposed to either standard media or starvation media (serum and nutrient withdrawal) for 4 h, with and without the presence of 20 nM Baf A1 to inhibit the degradation of LC3. Intensity of LC3 II and GAPDH was determined by ImageJ densitometry analysis. Bar graphs shown represent normalized intensity levels of LC3 II/GAPDH relative to no treatment control (standard media). Error bars, SD from three independent replicates; #, p value <0.05

Fig. 2

Effects of autophagy inhibitor on cell growth in uveal melanoma cells OCM1A and Mel 290 cells were treated with HCQ at two different doses (2.5 μM and 10 μM). These treatments were performed in complete medium (10% FBS) and low-serum medium (1% FBS) conditions. After 72-h incubation, MTT assays were performed to assess cell proliferation. The histogram presents quantitative analysis of cell growth in different groups. #, p < 0.05, error bars, SD from three independent experiments

Fig. 3

Vemurafenib treatment in uveal melanoma cells. a Activity of vemurafenib detected using MTT assay in OCM1A and Mel 290 cells. IC 50 values are shown in brackets behind the name of each cell (nmol/L). Results shown are representative of at least three independent experiments. b Immunoblots and gel density quantifications against autophagy marker (LC3 II) in OCM1A and Mel 290 cells. Cells were treated with indicated dose of vemurafenib (PLX) for 24 h. The ratio of LC3 II to GAPDH and p62 to GAPDH were evaluated by immunoblot analysis. Intensity of LC3 II, p62 and GAPDH was determined by ImageJ densitometry analysis

Fig. 4

Vemurafenib treatment suppressed PI3K/Akt/mTOR/p70S6 K pathway in BRAF V600E-mutant uveal melanoma cells. a Vemurafenib (PLX) triggered autophagy independently of MAPK signaling pathway inactivation. OCM1A and Mel 290 cells were treated with indicated dose of PLX for 24 h, after which protein lysates were prepared and subjected to protein gel blot analysis using the indicated antibodies. p-phospho-, t total. b Vemurafenib treatment suppressed PI3K/Akt/mTOR/p70S6K pathway. OCM1A and Mel 290 cells were treated with indicated dose of PLX for 24 h. Western blot was analyzed by using the indicated antibodies. p-phospho-

Fig. 5

Autophagy inhibition impaired the anti-tumor effect of vemurafenib in uveal melanoma cells. OCM1A cells were treated with increasing doses of vemurafenib in medium with or without HCQ for 72 h, and MTT assays were performed to assess cell proliferation. #, p value <0.05