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 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 study, we are focusing on NSCLC models for examining distal signaling mechanisms that modulate the chemotherapy sensitivity, generation, and maintenance of NSCLC cells/tumors. Specifically, the grant application focuses on the cell signaling pathways regulating both hnRNP L function and the alternative splicing of caspase 9. The expression of caspase 9 is regulated by alternative splicing via the inclusion or exclusion of a four exon cassette (exons 3, 4, 5, and 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 caspase 9b. Studies from our laboratory have demonstrated that NSCLC tumors present with a dysregulated ratio of caspase 9/caspase 9b analogous to an anti-apoptotic/chemotherapy resistance phenotype. Subsequent studies by our laboratory demonstrated that the alternative splicing of caspase 9 had important functions in anchorage-independent growth (AIG) in NSCLC cells, AIG induced by EGFR mutation in non-transformed human bronchial epithelial cells, and erlotinib sensitivity. Mechanistically, our laboratory identified an exonic splicing silencer (C9/E3-ESS) in exon 3 that regulates the inclusion of the exon 3, 4, 5, and 6 cassette of caspase 9 pre-mRNA. hnRNP L was shown to associate with this RNA cis-element, and repress the inclusion of the exon cassette. 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. The overall goal of this study is to determine the mechanisms and cell signaling pathways regulating the definition of exon 3, and thus the alternative splicing of caspase 9. Furthermore, we are proposing pre-clinical studies to determine whether these mechanisms as well as specific targeting of caspase 9b are effective targets for treating NSCLC as well as enhancing the effectiveness of current chemotherapeutic agents used in the clinic.

Public Health Relevance

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 to be 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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA154314-02
Application #
8305473
Study Section
Special Emphasis Panel (ZRG1-OBT-B (02))
Program Officer
Strasburger, Jennifer
Project Start
2011-07-21
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
2
Fiscal Year
2012
Total Cost
$310,213
Indirect Cost
$102,713
Name
Virginia Commonwealth University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
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Kolawole, Elizabeth Motunrayo; McLeod, Jamie Josephine Avila; Ndaw, Victor et al. (2016) Fluvastatin Suppresses Mast Cell and Basophil IgE Responses: Genotype-Dependent Effects. J Immunol 196:1461-70
Vu, Ngoc T; Park, Margaret A; Shultz, Michael D et al. (2016) Caspase-9b Interacts Directly with cIAP1 to Drive Agonist-Independent Activation of NF-κB and Lung Tumorigenesis. Cancer Res 76:2977-89
Johnson, Ryan M; Vu, Ngoc T; Griffin, Brian P et al. (2015) The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer. J Biol Chem 290:25717-27
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Barbour, Suzanne E; Nguyen, Phuong T; Park, Margaret et al. (2015) Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5'-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-Cells. J Biol Chem 290:11021-31
Wijesinghe, Dayanjan S; Brentnall, Matthew; Mietla, Jennifer A et al. (2014) Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts. J Lipid Res 55:1298-309
West, N Winston; Garcia-Vargas, Aileen; Chalfant, Charles E et al. (2013) OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2. BMC Cancer 13:256
Vu, Ngoc T; Park, Margaret A; Shultz, Jacqueline C et al. (2013) hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L. J Biol Chem 288:8575-84

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