Regulation of caspase 9 RNA splicing in NSCLC cancer

Summary

Principal Investigator: Charles E Chalfant
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 2011-07-21 - 2015-04-30
more information: NIH RePORT

Top Publications

  1. pmc SRSF1 regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells
    Jacqueline C Shultz
    Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, VA 23298 0614, USA
    Mol Cancer Res 9:889-900. 2011
  2. pmc OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2
    N Winston West
    University of Richmond, 28 Westhampton Way, Richmond, VA 23173, USA
    BMC Cancer 13:256. 2013
  3. pmc Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size
    Savita Khanna
    Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA
    J Cereb Blood Flow Metab 33:1197-206. 2013
  4. pmc Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase
    Jennifer A Mietla
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA
    J Lipid Res 54:1834-47. 2013
  5. pmc hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L
    Ngoc T Vu
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298, USA
    J Biol Chem 288:8575-84. 2013
  6. pmc The molecular basis of ceramide-1-phosphate recognition by C2 domains
    Katherine E Ward
    Department of Chemistry and Biochemistry and the Mike and Josie Harper Center for Cancer Research, University of Notre Dame, Notre Dame, IN, USA
    J Lipid Res 54:636-48. 2013
  7. pmc Metabolic gene remodeling and mitochondrial dysfunction in failing right ventricular hypertrophy secondary to pulmonary arterial hypertension
    Jose Gomez-Arroyo
    Victoria Johnson Center for Lung Obstructive Disease Research, Virginia Commonwealth University, Richmond, VA 23298, USA
    Circ Heart Fail 6:136-44. 2013
  8. pmc Metallofullerene-nanoplatform-delivered interstitial brachytherapy improved survival in a murine model of glioblastoma multiforme
    John D Wilson
    Departments of Radiology, Virginia Commonwealth University, 1101 East Marshall Street, Richmond, Virginia 23298, USA
    Bioconjug Chem 23:1873-80. 2012
  9. pmc The Proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA
    Jacqueline C Shultz
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Room 2 016, Sanger Hall, 1101 East Marshall Street, P O Box 980614, Richmond, VA 23298, USA
    Mol Cancer Res 10:660-9. 2012
  10. pmc The flip-flop HuR: part of the problem or the solution in fighting cancer?
    Jacqueline C Shultz
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298 0614, USA
    J Clin Invest 122:16-9. 2012

Detail Information

Publications11

  1. pmc SRSF1 regulates the alternative splicing of caspase 9 via a novel intronic splicing enhancer affecting the chemotherapeutic sensitivity of non-small cell lung cancer cells
    Jacqueline C Shultz
    Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, VA 23298 0614, USA
    Mol Cancer Res 9:889-900. 2011
    ..Furthermore, we showed that the alternative splicing of caspase 9 is an important molecular mechanism with therapeutic relevance to NSCLCs...
  2. pmc OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2
    N Winston West
    University of Richmond, 28 Westhampton Way, Richmond, VA 23173, USA
    BMC Cancer 13:256. 2013
    ..In this study, we examined mechanisms associated with enhancing the activity of lapatinib via combination with other therapies...
  3. pmc Loss of miR-29b following acute ischemic stroke contributes to neural cell death and infarct size
    Savita Khanna
    Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA
    J Cereb Blood Flow Metab 33:1197-206. 2013
    ..Such loss is contributed by activity of the 12-lipoxygenase pathway providing maiden evidence linking arachidonic acid metabolism to miR-dependent mechanisms in stroke. ..
  4. pmc Characterization of eicosanoid synthesis in a genetic ablation model of ceramide kinase
    Jennifer A Mietla
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA
    J Lipid Res 54:1834-47. 2013
    ....
  5. pmc hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L
    Ngoc T Vu
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298, USA
    J Biol Chem 288:8575-84. 2013
    ....
  6. pmc The molecular basis of ceramide-1-phosphate recognition by C2 domains
    Katherine E Ward
    Department of Chemistry and Biochemistry and the Mike and Josie Harper Center for Cancer Research, University of Notre Dame, Notre Dame, IN, USA
    J Lipid Res 54:636-48. 2013
    ..Taken together, this is the first study demonstrating the molecular origin of C1P recognition...
  7. pmc Metabolic gene remodeling and mitochondrial dysfunction in failing right ventricular hypertrophy secondary to pulmonary arterial hypertension
    Jose Gomez-Arroyo
    Victoria Johnson Center for Lung Obstructive Disease Research, Virginia Commonwealth University, Richmond, VA 23298, USA
    Circ Heart Fail 6:136-44. 2013
    ..Thus, we sought to characterize metabolic gene expression changes and mitochondrial dysfunction in functional and dysfunctional RV hypertrophy...
  8. pmc Metallofullerene-nanoplatform-delivered interstitial brachytherapy improved survival in a murine model of glioblastoma multiforme
    John D Wilson
    Departments of Radiology, Virginia Commonwealth University, 1101 East Marshall Street, Richmond, Virginia 23298, USA
    Bioconjug Chem 23:1873-80. 2012
    ..Overall, these results provide evidence that nanomaterial platforms can be used to deliver effective interstitial brachytherapy...
  9. pmc The Proto-oncogene PKCι regulates the alternative splicing of Bcl-x pre-mRNA
    Jacqueline C Shultz
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Room 2 016, Sanger Hall, 1101 East Marshall Street, P O Box 980614, Richmond, VA 23298, USA
    Mol Cancer Res 10:660-9. 2012
    ..Finally, 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...
  10. pmc The flip-flop HuR: part of the problem or the solution in fighting cancer?
    Jacqueline C Shultz
    Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, Virginia 23298 0614, USA
    J Clin Invest 122:16-9. 2012
    ..These observations highlight the need to understand the roles of HuR in distinct cell populations in vivo and suggest that enhancing HuR activity may be of clinical benefit in protecting against pathologic inflammation and cancer...
  11. pmc Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids
    Dhirendra K Simanshu
    Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA
    Nature 500:463-7. 2013
    ..The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation. ..

Research Grants30

  1. The Role of Caspase 9b Expression in NSCLC Survival/Chemotherapeutic Sensitivity
    Charles E Chalfant; Fiscal Year: 2013
    ..g. cisplatinum and paclitaxel). ..