Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non-small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser 52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four-exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathologic classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, epidermal growth factor receptor (EGFR) overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of phosphoinositide 3-kinase or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227, and 234. Taken together, our findings show that oncogenic factors activating the phosphoinositide 3-kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a. Cancer Res; 70(22); 9185-96. ©2010 AACR.

Background: Two splice variants of caspase-9 can be generated by the inclusion/exclusion of the exon 3,4,5,6 cassette. Results: hnRNP U is an enhancer of this exonic cassette and is opposed by phosphorylation of hnRNP L via the AKT pathway. Conclusion: hnRNP U promotes the exon cassette inclusion to form caspase-9a. Significance: Understanding the regulation of caspase-9 alternative splicing is important for the treatment of lung cancer.

Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology with the alternative pre-mRNA processing of caspase 9 as one example. In this study, we delve into the underlying molecular mechanisms that regulate the alternative splicing of caspase 9. Specifically, the pre-mRNA sequence of caspase 9 was analyzed for RNA cis-elements known to interact with SRSF1, a required enhancer for caspase 9 RNA splicing. This analysis revealed thirteen possible RNA cis-elements for interaction with SRSF1 with mutagenesis of these RNA cis-elements identifying a strong intronic splicing enhancer located in intron 6 (C9-I6/ISE). SRSF1 specifically interacted with this sequence, which was required for SRSF1 to act as a splicing enhancer of the inclusion of the four exon cassette. To further determine the biological importance of this mechanism, we employed RNA oligonucleotides to redirect caspase 9 pre-mRNA splicing in favor of caspase 9b expression, which resulted in an increase in the IC50 of non-small cell lung cancer (NSCLC) cells to daunorubicin, cisplatinum, and paclitaxel. In contrast, downregulation of caspase 9b induced a decrease in the the IC50 of these chemotherapeutic drugs. Lastly, these studies demonstrated that caspase 9 RNA splicing was a major mechanism for the synergistic effects of combination therapy with daunorubicin and erlotinib. Overall, we have identified a novel intronic splicing enhancer that regulates caspase 9 RNA splicing and specifically interacts with SRSF1. Furthermore, we demonstrate that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs.

Two splice variants derived from the BCL-x gene via alternative 5′ splice site selection (5′SS) are pro-apoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L). Previously, our laboratory demonstrated that apoptotic signaling pathways regulated the alternative 5′SS selection via protein phosphatase-1 and de novo ceramide. In this study, we examined the elusive pro-survival signaling pathways that regulate the 5′SS selection of Bcl-x pre-mRNA in cancer cells. Taking a broad-based approach by utilizing a number of small molecule inhibitors of various mitogenic/survival pathways, we found that only treatment of non-small cell lung cancer (NSCLC) cell lines with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (50 μM) or the pan-PKC inhibitor GÖ6983 (25 μM) decreased the Bcl-x(L)/Bcl-x(s) mRNA ratio. Pan-PKC inhibitors that did not target the atypical PKCs, PKCι and PKCζ, had no effect on the Bcl-x(L)/Bcl-x(s) mRNA ratio. Additional studies demonstrated that downregulation of the proto-oncogene, PKCι, in contrast to PKCζ, also resulted in a decrease in the Bcl-x(L)/Bcl-x(s) mRNA ratio. Furthermore, downregulation of PKCι correlated with a dramatic decrease in the expression of SAP155, an RNA trans-acting factor that regulates the 5′SS selection of Bcl-x pre-mRNA. Inhibition of the PI3K or atypical PKC pathway induced a dramatic loss of SAP155 complex formation at ceramide-responsive RNA cis-element 1. Lastly, forced expression of Bcl-x(L) “rescued” the loss of cell survival induced by PKCι siRNA. In summary, the PI3K/PKCι regulates the alternative splicing of Bcl-x pre-mRNA with implications in the cell survival of NSCLC cells.

Background: ␤-Cell apoptosis, a critical contributor to T1D, involves iPLA 2 ␤ activation and is suppressed by Bcl-x(L). Results: iPLA 2 ␤-derived lipids activate an alternative 5Ј-splice site, reducing protective Bcl-x(L) protein. Conclusion:Modulation of Bcl-x splicing is another key mechanism by which iPLA 2 ␤-derived lipids promote ␤-cell apoptosis. Significance: Delineation of molecular mechanisms underlying iPLA 2 ␤-regulated splicing will elucidate novel strategies to counter ␤-cell death in T1D.

A number of studies have demonstrated that the overexpression of Bcl-x(L) in cells confers apoptosis resistance as well as cooperates with oncogenic factors (e.g. c-Myc) in tumorigenesis (1-10). The regulation of Bcl-x(L) expression can be complex at times, consisting of both transcriptional and post-transcriptional processes. In regard to post-transcriptional processing/ alternative splicing, the BCL-x gene, via alternative 5Ј splice site (5ЈSS) 5 selection within exon 2, produces either the Bcl-x(s) isoform through activation of an upstream/proximal 5ЈSS or the Bcl-x(L) isoform through activation of a downstream/distal 5ЈSS. A number of studies have demonstrated that Bcl-x(s), in contrast to Bcl-x(L), promotes apoptosis (9,(11)(12)(13)(14). Hence, the alternative 5ЈSS selection of Bcl-x pre-mRNA emerged as a potential target for anti-cancer therapeutics. For example, Taylor et al. (15) demonstrated that Bcl-x 5ЈSS selection can be specifically modulated using antisense oligonucleotides specific against the Bcl-x(L) 5Ј splice site. Treatment of cells with these oligonucleotides induced an increase in the expression of Bcl-x(s) and a decrease in the expression of Bcl-x(L), resulting in sensitization of NSCLC cells to chemotherapeutic agents (15). These findings were also demonstrated by Kole and co-* This work was supported by research grants from the Veteran's Administration (VA Merit Review, I BX001792 (to C. E. C.) and a Research Career Scientist Award, 13F-RCS-002 (to C. E. C.)); from the Vietnam Education Foundation (fellowship to N. T. V.); from the National Institutes of Health via Grants HL125353 (to C. E. C.), HD087198 (to C. E. C.), CA117950 (to C. E. C.), CA154314 (to C. E. C.), RR031535 (to C. E. C.), CA192613 (to P. D.), CA097318 (to P. B. F.), CA127641 (to P. B. F.), P01 CA104177 (to P. B. F.), and a T32 PostDoctoral Fellowship (Postdoctoral Training Program in Cancer Biology, CA085159) (to B. A. S.); from the United States-Israel Binational Science Foundation via Grant BSF#2011360 (to C. E. C.); and from the National Foundation for Cancer Research (to P. B. F.). The authors declare that they have no conflicts of interest with the contents of this article.

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