Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS

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

Alternative splicing enriched cDNA libraries identify breast cancer-associated transcripts

<p>Abstract</p> <p>Background</p> <p>Alternative splicing (AS) is a central mechanism in the generation of genomic complexity and is a major contributor to transcriptome and proteome diversity. Alterations of the splicing process can lead to deregulation of crucial cellular processes and have been associated with a large spectrum of human diseases. Cancer-associated transcripts are potential molecular markers and may contribute to the development of more accurate diagnostic and prognostic methods and also serve as therapeutic targets. Alternative splicing-enriched cDNA libraries have been used to explore the variability generated by alternative splicing. In this study, by combining the use of trapping heteroduplexes and RNA amplification, we developed a powerful approach that enables transcriptome-wide exploration of the AS repertoire for identifying AS variants associated with breast tumor cells modulated by <it>ERBB2</it> (<it>HER-2/neu</it>) oncogene expression.</p> <p>Results</p> <p>The human breast cell line (C5.2) and a pool of 5 ERBB2 over-expressing breast tumor samples were used independently for the construction of two AS-enriched libraries. In total, 2,048 partial cDNA sequences were obtained, revealing 214 alternative splicing sequence-enriched tags (ASSETs). A subset with 79 multiple exon ASSETs was compared to public databases and reported 138 different AS events. A high success rate of RT-PCR validation (94.5%) was obtained, and 2 novel AS events were identified. The influence of <it>ERBB2</it>-mediated expression on AS regulation was evaluated by capillary electrophoresis and probe-ligation approaches in two mammary cell lines (Hb4a and C5.2) expressing different levels of <it>ERBB2</it>. The relative expression balance between AS variants from 3 genes was differentially modulated by <it>ERBB2</it> in this model system.</p> <p>Conclusions</p> <p>In this study, we presented a method for exploring AS from any RNA source in a transcriptome-wide format, which can be directly easily adapted to next generation sequencers. We identified AS transcripts that were differently modulated by <it>ERBB2</it>-mediated expression and that can be tested as molecular markers for breast cancer. Such a methodology will be useful for completely deciphering the cancer cell transcriptome diversity resulting from AS and for finding more precise molecular markers.</p