Immunology and Oncology Unit, Calvary Mater Newcastle Hospital, Newcastle, New South Wales, Australia.
Cell Death Dis. 2010 Sep 2;1(9):e69. doi: 10.1038/cddis.2010.48.
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, Bim(S), remains largely unappreciated. Here, we show that inhibition of the mutant B-RAF(V600E) triggers preferential splicing to produce Bim(S), which is particularly important in induction of apoptosis in B-RAF(V600E) melanoma cells. Although the specific B-RAF(V600E) inhibitor PLX4720 upregulates all three major isoforms of Bim, Bim(EL), Bim(L), and Bim(S), at the protein and mRNA levels in B-RAF(V600E) melanoma cells, the increase in the ratios of Bim(S) mRNA to Bim(EL) and Bim(L) mRNA indicates that it favours Bim(S) splicing. Consistently, enforced expression of B-RAF(V600E) in wild-type B-RAF melanoma cells and melanocytes inhibits Bim(S) expression. The splicing factor SRp55 appears necessary for the increase in Bim(S) splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of Bim(S) and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in Bim(S) splicing is also mirrored in freshly isolated B-RAF(V600E) melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAF(V600E) inhibitors.
Bim 被认为通过抑制 RAF/MEK/ERK 通路在杀死黑色素瘤细胞中起关键作用。然而，Bim 的最有效凋亡诱导同工型 Bim(S) 的潜在作用在很大程度上仍未得到认识。在这里，我们表明抑制突变型 B-RAF(V600E)触发优先剪接以产生 Bim(S)，这在 B-RAF(V600E)黑色素瘤细胞中诱导凋亡特别重要。虽然特定的 B-RAF(V600E)抑制剂 PLX4720 在 B-RAF(V600E)黑色素瘤细胞中上调所有三种主要同工型 Bim(EL)、Bim(L)和 Bim(S)的蛋白和 mRNA 水平，但 Bim(S)mRNA 与 Bim(EL)和 Bim(L)mRNA 的比值增加表明它有利于 Bim(S)剪接。一致地，在野生型 B-RAF 黑色素瘤细胞和黑素细胞中强制表达 B-RAF(V600E)抑制 Bim(S)表达。剪接因子 SRp55 似乎是增加 Bim(S)剪接所必需的，因为 SRp55 上调，并且其通过小干扰 RNA 的抑制阻止了 PLX4720 诱导的 Bim(S)和凋亡的诱导。PLX4720 诱导的、SRp55 介导的 Bim(S)剪接增加也反映在新分离的 B-RAF(V600E)黑色素瘤细胞中。这些结果确定了 PLX4720 诱导凋亡的关键机制，并且对于使黑色素瘤细胞对 B-RAF(V600E)抑制剂敏感具有指导意义。
