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.

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.

Epr is a minor extracellular protease secreted by Bacillus subtilis 168. In this study, we show that epr is transcribed by E D , the RNA polymerase associated with transcription of genes involved in chemotaxis and motility. Disruption of epr abolished swarming of Bacillus subtilis, suggesting its involvement in motility.At the onset of stationary phase, Bacillus subtilis secretes at least seven extracellular proteases of which subtilisin (aprE) and a neutral metalloprotease E (nprE) are the most abundant (6). Of the remaining five minor extracellular proteases, Epr (epr) contributes 2 to 4% to this pool (18). The expression of these enzymes is stringently regulated, as best exemplified by the aprE gene (23). However, these proteases appear to be dispensable for growth (11,19), and their relevance in the physiology of the cell remains to be determined.The transition state is also characterized by the acquisition of specific functions such as competence and motility (23). The expression of genes related to motility, chemotaxis, and flagellar assembly is governed by the alternate sigma factor D (14). D complexed to the core RNA polymerase (E D ) recognizes and binds the bipartite promoter sequences 5Ј-CTAAA-3Ј (Ϫ35) and 5Ј-CCGATAT-3Ј (Ϫ10) (14). We present here evidence that epr is transcribed from a D -dependent promoter and that this gene is involved in the swarming of B. subtilis.Although epr has been cloned and sequenced (4, 18), the identity of its promoter and the possible physiological role(s) of this gene have so far not been elucidated. To identify the promoter for epr, we examined the DNA sequence upstream of the ribosome-binding site (RBS) for a putative bipartite promoter sequence that may be related to known promoter sequences for the different sigma factors in B. subtilis. A D -type promoter with a Ϫ35 (CTATT) and a Ϫ10 (CCGATAT) element was identified (Fig. 1) that showed matches of 3 of 5 and 7 of 7, respectively, with the consensus D promoter (14) and an optimal spacing of 17 bp between the two elements. To determine that indeed this promoter was utilized, primer extension analysis was employed to identify the epr transcription start site. Total RNA was isolated from B. subtilis 168/pIC56-4 and 1A716/pIC56-4 carrying the epr gene in a multicopy plasmid, pIC56-4 (Table 1). Since the levels of Epr are very low (4, 18), we were unable to detect epr mRNA from a single-copy gene and hence we have used a multicopy vector. A polyacrylamide gel electrophoresis-purified primer, KKR50 (5Ј-CGAG GATCCTGTACAACAAGTTTGCA-3Ј) (Fig. 1), end labeled with [␥-32 P]ATP (5,000 Ci/mM) and T4 polynucleotide kinase, was annealed with 10 g of RNA, and the primer was extended with Moloney murine leukemia virus reverse transcriptase (1, 17). The extended product(s) was analyzed on a 7 M urea-8% polyacrylamide denaturing gel. To obtain the template for the sequencing reactions, a DNA fragment of 360 bp that overlapped the putative start site was obtained from pIC56-4 by PCR amplification with the primers KKR76 (5Ј-CGAGATCT CTG...