Today, lung cancer is the leading cause of death in both men and women in industrialized countries, accounting for an estimated 28% of all cancer deaths in the United States. Non-small cell lung cancers (NSCLC) represent the majority of lung cancers and carry a poor prognosis with a median survival of less than 12 months. Most patients present with unresectable disease, and the current treatment options of chemotherapy and radiation are palliative at best. Therefore, new strategies are needed in the treatment of NSCLC in order to impact this disease. In this regard, we are focusing on NSCLC models for examining distal signaling mechanisms that modulate the generation and maintenance of NSCLC cells/tumors. Specifically, this grant application begins with a focus on the consequence of expressing caspase 9b (C9b) in lung epithelium. The expression of C9b is regulated by alternative RNA splicing via the inclusion or exclusion of a four exon cassette (exons 3,4,5,6). Inclusion of this exon cassette into the mature transcript produces the pro-apoptotic caspase 9 (caspase 9a) while the exclusion produces the anti-apoptotic and survival/oncogenic signaling factor, caspase 9b. Studies from our laboratory have demonstrated that NSCLC tumors present with a dysregulated (e.g. low) ratio of caspase 9/caspase 9b analogous to an anti-apoptotic/chemotherapy resistance phenotype. Subsequent studies by our laboratory demonstrated that the expression of C9b had important functions in the anchorage-independent growth (AIG) of NSCLC cells, AIG induced by oncogenic mutation in non-transformed human bronchial epithelial cells, and chemotherapy sensitivity (e.g. cisplatinum and paclitaxel). Mechanistically, our laboratory identified an exonic splicing silencer (C9/E3-ESS) in exon 3 that regulates the inclusion of the exon 3,4,5,6 cassette of caspase 9 pre-mRNA. The RNA trans-factor, hnRNP L, was shown to associate with this RNA cis-element, repress the inclusion of the exon cassette, and induce caspase 9b expression. Importantly, phosphorylation of hnRNP L on ser52 (observed only in transformed cells) was required for repression of the exon 3,4,5,6 cassette. Lastly, ser52 phosphorylation of hnRNP L was shown as a required mediator of the tumorigenic capacity of NSCLC cells via the alternative splicing of caspase 9. These key mechanisms are specific to transformed cells, translatable to >70% of NSCLCs, and at an extreme distal point in oncogenic pathways. Therefore, these distal mechanisms are plausible and highly desired targets for the development of new anti-cancer therapeutics. Our proposed studies will dramatically extend these previous findings by first determining whether C9b is a key oncogenic signaling factor in the transformation of lung epithelial cells. The next set of proposed studies will determine how hnRNP L becomes activated to drive the expression of C9b. Our last set of proposed studies extend the role of hnRNP L in regard to NSCLC. In stark contrast with our findings on the phosphorylation of Ser52 in hnRNP L in transformed cells, downregulation of hnRNP L in non-transformed cells had no effect on RNA splicing events important in maintaining oncogenic phenotypes (e.g. AIG). Further investigations by our laboratory determined that the lack of effect on RNA splicing events in non-transformed cells was due to a lack of phosphorylation of Ser52 in hnRNP L. Thus, these findings suggest that the phosphorylation of hnRNP L (i.e. activation in transformed cells) mediates specific RNA splicing events important in cell survival, proliferation, AIG, and tumor formation versus constitutive functions of non- phosphorylated hnRNP L. Therefore, we hypothesize that the phosphorylation of hnRNP L on Ser52 is required for modulating a specific subset of splicing events, which are important for NSCLC cells to develop and maintain transformed phenotypes. Our proposed studies will serve to determine this specific ?cluster? of RNA splicing events and further investigate the biological relevance of these events in maintaining the oncogenic phenotypes of NSCLC cells.
Cancer is the second leading cause of death among US veterans, and lung cancer is associated with the greatest mortality in these patients. Non-small cell lung cancers (NSCLC) represent the majority of lung cancers and carry a poor prognosis with a median survival of less than 12 months. Most patients present with unresectable disease, and current treatment options of chemotherapy and radiation are palliative at best. Our laboratory has identified key mechanisms specific to cancer cells and translatable to >70% of NSCLCs, which are plausible and highly desired targets for the development of new anti-cancer therapeutics. This grant application explores these cellular mechanisms in depth with the goal of developing new therapeutics to combat NSCLC, and thereby, aid US Veterans.
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