Abstract
Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, BimS, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAFV600E triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFV600E melanoma cells. Although the specific B-RAFV600E inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFV600E melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing. Consistently, enforced expression of B-RAFV600E in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression. The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFV600E melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFV600E inhibitors.
Highlights
It is well established that blockade of the RAF/MEK/ERK pathway inhibits melanoma cell growth.[5]
This was shown by its inhibitory effect on ERK activation in B-RAFV600E melanoma cell lines, but not in those carrying the wild-type B-RAF even when it was used at 10 mM (Figure 1a and Supplementary Figure 1A)
We examined whether induction of apoptosis was involved in PLX4720-mediated inhibition of cell growth in Mel-RMu (B-RAFV600E) and Mel-RM cells
Summary
It is well established that blockade of the RAF/MEK/ERK pathway inhibits melanoma cell growth.[5]. Several mechanisms have been reported to contribute to apoptosis induced by inhibition of the RAF/MEK/ERK pathway These include dephosphorylation of Bad, translocation of Bmf, upregulation of BimEL, and downregulation of Mcl-1.7–11 Among them, upregulation of BimEL via inhibition of its phosphorylation and subsequent proteasomal degradation may be the best documented[7,8] and is of particular interest, in that Bim, unlike other more selective Bcl-2 homology 3 (BH3)-only proteins such as Bad and Bmf, can bind with high affinity to and inhibit all prosurvival Bcl-2 family proteins.[12] In addition, Bim can directly bind to and activate Bax.[12] It is of note that besides posttranslational changes, inhibition of the RAF/MEK/ERK pathway has been shown to cause upregulation of Bim mRNA.[13]. BimS is not subject to any known posttranslational regulation and is the most potent apoptosis inducer among the three isofoms.[13,16,17]
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