Oncogenic BRAF induces chronic ER stress condition resulting in increased basal autophagy and apoptotic resistance of cutaneous melanoma

Abstract

The notorious unresponsiveness of metastatic cutaneous melanoma to current treatment strategies coupled with its increasing incidence constitutes a serious worldwide clinical problem. Moreover, despite recent advances in targeted therapies for patients with BRAF(V600E) mutant melanomas, acquired resistance remains a limiting factor and hence emphasises the acute need for comprehensive pre-clinical studies to increase the biological understanding of such tumours in order to develop novel effective and longlasting therapeutic strategies. Autophagy and ER stress both have a role in melanoma development/progression and chemoresistance although their real impact is still unclear. Here, we show that BRAF(V600E) induces a chronic ER stress status directly increasing basal cell autophagy. BRAF(V600E)-mediated p38 activation stimulates both the IRE1/ASK1/JNK and TRB3 pathways. Bcl-XL/Bcl-2 phosphorylation by active JNK releases Beclin1 whereas TRB3 inhibits the Akt/mTor axes, together resulting in an increase in basal autophagy. Furthermore, we demonstrate chemical chaperones relieve the BRAF(V600E)-mediated chronic ER stress status, consequently reducing basal autophagic activity and increasing the sensitivity of melanoma cells to apoptosis. Taken together, these results suggest enhanced basal autophagy, typically observed in BRAF(V600E) melanomas, is a consequence of a chronic ER stress status, which ultimately results in the chemoresistance of such tumours. Targeted therapies that attenuate ER stress may therefore represent a novel and more effective therapeutic strategy for BRAF mutant melanoma.

Figure 1

Enhanced basal autophagy and less apoptotic responsiveness of BRAF^V600E melanoma cells. BRAF wt (CHL-1) and BRAF^V600E (A375) melanoma cells were exposed 3 and 6 h (a) to bafilomycin A (Baf) and basal autophagy was evaluated by monitoring both the LC3 conversion by western blotting analysis (a; Gapdh was used as the loading control) and p62 accumulation by immunofluorescence (b; bar=10 μm). Basal autophagy was also evaluated in a panel of BRAF^WT or BRAF^V600E melanoma cell lines, in presence or absence of Baf (4 h), by monitoring LC3 conversion by western blotting analysis (c, left panels; Gapdh was used as the loading control). Densitometric analysis of LC3-II bands is shown in each cell lines, comparing treated with untreated cells (with Baf); mean±S.D. of LC3-II bands in BRAF^WT and BRAF^V600E cell lines is also reported (c, right panel). Autophagic flux was quantitated in all melanoma cell lines stably expressing a p62-GFP recombinant protein, treated with Baf in a time-dependent manner, by cytofluorimetric analysis (d; n=3). Apoptosis induction was evaluated by cytofluorimetric analysis of propidium iodide-stained CHL-1 and A375 cells exposed 24 h to thapsigargin (TG, 10 μg/ml; (e; n=3; P=0.004)

Figure 2

BRAF induces a chronic ER stress status. (a) BRAF-mediated ER stress was evaluated in both BRAF^WT and BRAF^V600E melanoma cell lines by measuring the mRNA levels of ER stress markers ERdj5, ERp57, ATF4 and Xbp-1 (spliced, mature form) by qRT-PCR (n=3). BRAF^V600E and GFP were transduced in BRAF wt SK-Mell-110 melanoma cells. BRAF and P-ERK1/2 (ERK1/2 was used as the loading control) protein levels were evaluated by western blotting analysis (c). ER stress status was monitored in GFP or BRAF^V600E expressing cells comparing: (b) mRNA levels of ERp57, ATF4, ERdj5 and Xbp-1 (spliced, mature form) by qRT-PCR (n=3); (d) protein level of Calnexin (CLX), ERp57 and eIF2a-P (eIF2a was used as loading control) by western blotting analysis and (e) Xbp-1 mRNA splicing by RT-PCR

Figure 3

BRAF^V600E expression and basal autophagy. BRAF^V600E and GFP or empty vector (EV) were transduced in BRAF wt SK-Mell-110 melanoma cells. Cells were incubated with bafilomycin A as indicated, and basal autophagy was evaluated by both confocal analysis of p62 puncta (a; bar=10 μm) or LC3 conversion by western blotting (b; Gapdh was used as the loading control)

Figure 4

Autophagy modulation by the IRE1/TRAF2/JNK axis. JNK activation (P-JNK) was evaluated in primary melanocytes and in a panel of BRAF^WT or BRAF^V600E melanoma cell lines (a), or in SK-Mell-110-expressing GFP or BRAF^V600E (c) cells by western blotting analysis (JNK was used as loading control). The recruitment of TRAF2 by activated IRE1 on ER was monitored by western blotting analysis of subcellular fractions from CHL-1 and A375 (b) or SK-Mell-110-expressing GFP or BRAF^V600E (d) cells, by using specific anti-TRAF2 and anti-CLX antibodies; arrows have been used to highlight the different distribution of TRAF2 onto ER membranes, between compared cell lines. (e) A375 cells were infected with indicated shIRE1- or shCtrl-carrying lentiviruses and IRE1 levels were evaluated by qRT-PCR (right panel; P=0.0005; n=3); P-JNK was evaluated by western blotting analysis (JNK was used as the loading control; upper panel) and LC3 conversion was monitored by western blotting analysis in the presence or absence of bafilomycin (Baf, 3 h; Gapdh was used as the loading control; bottom panel)

Figure 5

JNK and basal autophagy. A375 cells were exposed to SP600125, and JNK activation (P-JNK) was evaluated by western blotting analysis (a; JNK was used as the loading control). A375 cells expressing a p62-GFP recombinant protein were treated or untreated with bafilomycin A (Baf, 4 h) and SP600125 (6 h) alone or in combination, and the occurrence of autophagy was analysed by measuring p62-GFP levels by citofluorimetric analysis (b; n=3), LC3 conversion by western blotting analysis (c; Gapdh was used as the loading control), and by evaluating the presence of p62-GFP cytosolic puncta by confocal analysis (d; bar=10 μm). A Flag-tagged JNK dominant negative (JNK-DN) was ectopically expressed in A375 cells by transient transfection and expression levels of JNK-DN protein and LC3 conversion, and accumulation was evaluated in presence or absence of Baf by western blotting (e). GFP or BRAFV^600E expressing SK-Mel-110 cells were transiently transfected with expression plasmids encoding Flag-tagged Beclin 1 and protein extracts were subjected to IP using an anti-Flag antibody. Purified complexes were analysed together with the corresponding total extracts by western blotting using anti-Flag (f, top), anti-Bcl-X_L (f, middle) and anti-Mcl-1 (bottom) antibodies. A375 cells were transiently transfected with expression plasmids encoding wild-type or a T69A/S70A/S87A mutant Bcl-2. Cells were treated or untreated with bafilomycin, as indicated, and total Bcl-2 protein expression together with LC3 conversion were evaluated by western blotting analysis (g; Gapdh was used as the loading control)

Figure 6

Modulation of TRB3 expression by BRAF. TRB3 expression was evaluated in CHL-1, A375 and SK-Mel-110 cells expressing GFP or BRAF^V600E cells by qRT-PCR (a) and western blotting (b). A375 cells were transiently transfected with specific siRNA oligos and TRB3 downregulation was evaluated by both qRT-PCR and western blotting (c). Cells were then treated or untreated with bafilomycin and LC3 conversion or p62 puncta accumulation were evaluated by western blotting (d; Gapdh was used as the loading control) or confocal (e) analysis, respectively. Cells were also treated in the presence or absence of both bafilomycin and SP600125 as indicated, and autophagic rate was evaluated by measuring the LC3 conversion by western blotting (f; Gapdh was used as the loading control) or by p62 puncta accumulation by confocal microscopy (e; bar=10 μm). A375 cells expressing a p62-GFP recombinant protein were transiently transfected with specific siRNA oligos (siCtrl or siTRB3), treated as in (b) and autophagic flux was evaluated by measuring the levels of p62-GFP by flow cytometry (g; *P=0.0002; **P=0.003; n=3)

Figure 7

p38 activation and ER stress induction in BRAF-mutated cells. p38 activation (P-p38) was evaluated by western blotting analysis in both CHL-1 and A375 (a, upper panel; p38 was used as the loading control). A375 cells were treated or untreated with SB202190 inhibitor as indicated and JNK activation (P-JNK), Calnexin (CLX) and ERp57 expression were evaluated by western blotting analysis (a, bottom panel, and b; JNK or Gapdh were used as the loading control). LC3 conversion was evaluated in A375 exposed to SB202190 (8 h) in presence or absence of bafilomycin A by western blotting analysis (c; Gapdh was used as the loading control). A375 cells were transiently transfected with specific siRNA oligos and p38, P-JNK, TRB3 and p62 protein levels were evaluated by western blotting analysis (d; Gapdh was used as the loading control). The expression of TRB3 and ER stress markers (ATF4, ERp57), and LC3 conversion were evaluated in SK-Mel-110 BRAF^V600E cells continuously exposed to 4-PBA (3 mM), by qRT-PCR or western blotting (e and f; Gapdh was used as the loading control; *P=0.012; **P=0.025; ***P=0.002; n=3), in the presence or absence of bafilomycin A, as indicated. Apoptotic rates were compared in SK-Mel-110 BRAF^V600E cells in the presence or absence of 4-PBA, treated or untreated 24 h with thapsigargin (TG), staurosporine (STS) or doxurubicin (DoxR), by cytofluorimetric analysis of PI-stained cells (g). Schematic representation of BRAF-induced ER stress and basal autophagy modulation (h)