Regulation of Ovarian Cancer Multidrug Resistance by MAP Kinases

Summary

Principal Investigator: Scott T Eblen
Abstract: Many ovarian tumors respond to initial drug therapy, but become refractory to drug treatment upon recurrence. Therefore, defining the mechanisms of drug resistance is important for existing and new cancer therapies. Alternative mRNA splicing affects 74% of the human genome. Alterations in alternative mRNA splicing factor expression occur in cancer and can strongly influence apoptotic responses. Over-expression of the splicing factor SPF45 in ovarian cancer cells results in a tamoxifen sensitive drug-resistant phenotype. The mechanism of SPF45-mediated drug resistance is unknown. Numerous chemotherapeutic agents and cellular stimuli induce activation the mitogen-activated protein kinases (MAP kinases) ERK, JNK and p38, all of which are associated with modulating cell survival responses through phosphorylation of their protein substrates. Currently, Sam68 is the only known splicing factor affected by MAP kinase phosphorylation. We have identified SPF45 as a novel substrate of all three MAP kinases and identified two phosphorylation sites on SPF45. We generated phospho-specific antibodies and have shown SPF45 phosphorylation in human ascites and in cell lines in response to chemotherapeutic drugs, oxidative stress, phorbol esters and UV irradiation. Most importantly, phosphorylation of SPF45 on Ser222 by MAP kinases increases its alternative splicing activity. These data strongly demonstrate a direct link between MAP kinases, SPF45 phosphorylation, alternative splicing activity and drug resistance. The overall objective of this application is to define the mechanism by which SPF45 mediates drug resistance and how resistance is regulated by MAP kinase phosphorylation of SPF45. These studies will enable the identification of novel targets for therapeutic intervention to combat drug resistance in SPF45 over-expressing tumors. We hypothesize that MAP kinase phosphorylation of SPF45 in response to chemotherapeutic agents enhances the drug resistant phenotype by stimulating SPF45 alternative splicing activity. We will test this hypothesis through the following Specific Aims: Specific Aim 1. Generate phospho-specific antibodies to SPF45. Identify the changes in MAP kinase- specific SPF45 phosphorylation patterns in response to chemotherapeutic agents and identify the kinase(s) involved. Specific Aim 2. Determine the effect of MAP kinase phosphorylation on SPF45 splicing activity using the ?fas minigene assay. Identify novel splicing targets of SPF45 by exon array analysis and identify the mechanism by which these targets contribute to SPF45-mediated drug resistance. Specific Aim 3. Elucidate the role of SPF45 and its phosphorylation in the response to chemotherapeutic agent treatment in pre-neoplastic and neoplastic ovarian cell lines, tumors in nude mice and in malignant human ascites. Define the role o estrogen receptors in SPF45-mediated drug resistance. PUBLIC HEALTH RELEVANCE: ovarian tumors often develop resistance to chemotherapeutics, but the mechanisms are poorly understood. We hypothesize that this phenomenon is regulated in part by the ability of an intracellular signaling pathway that is often hyper-activated in cancer cells to chemically modify a protein with a known role in drug resistance. Elucidation of this synergistic mechanism of drug resistance would provide important information for treating drug-resistant ovarian cancer.
Funding Period: 2009-05-01 - 2014-07-31
more information: NIH RePORT

Top Publications

  1. pmc Regulation of chemoresistance via alternative messenger RNA splicing
    Scott T Eblen
    Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, 29425, USA
    Biochem Pharmacol 83:1063-72. 2012
  2. pmc Mitogen-activated protein kinase phosphorylation of splicing factor 45 (SPF45) regulates SPF45 alternative splicing site utilization, proliferation, and cell adhesion
    Adnan M Al-Ayoubi
    Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA
    Mol Cell Biol 32:2880-93. 2012
  3. pmc Oncogenic K-Ras regulates bioactive sphingolipids in a sphingosine kinase 1-dependent manner
    Christopher R Gault
    Ralph H Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401, USA
    J Biol Chem 287:31794-803. 2012
  4. pmc Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion
    Yuying Liu
    Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425, USA
    Nucleic Acids Res 41:4949-62. 2013
  5. pmc Transcriptional suppression, DNA methylation, and histone deacetylation of the regulator of G-protein signaling 10 (RGS10) gene in ovarian cancer cells
    Mourad W Ali
    Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, United States of America
    PLoS ONE 8:e60185. 2013
  6. pmc EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer
    Amber Bradley
    Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA
    Carcinogenesis 35:1100-9. 2014

Research Grants

  1. CARP-1: A Potential Therapeutic Agent for Breast Cancer
    ARUN KUMAR RISHI; Fiscal Year: 2013
  2. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013

Detail Information

Publications6

  1. pmc Regulation of chemoresistance via alternative messenger RNA splicing
    Scott T Eblen
    Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, 29425, USA
    Biochem Pharmacol 83:1063-72. 2012
    ....
  2. pmc Mitogen-activated protein kinase phosphorylation of splicing factor 45 (SPF45) regulates SPF45 alternative splicing site utilization, proliferation, and cell adhesion
    Adnan M Al-Ayoubi
    Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina, USA
    Mol Cell Biol 32:2880-93. 2012
    ..These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation...
  3. pmc Oncogenic K-Ras regulates bioactive sphingolipids in a sphingosine kinase 1-dependent manner
    Christopher R Gault
    Ralph H Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401, USA
    J Biol Chem 287:31794-803. 2012
    ....
  4. pmc Phosphorylation of the alternative mRNA splicing factor 45 (SPF45) by Clk1 regulates its splice site utilization, cell migration and invasion
    Yuying Liu
    Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Ave, Charleston, SC 29425, USA
    Nucleic Acids Res 41:4949-62. 2013
    ..Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1...
  5. pmc Transcriptional suppression, DNA methylation, and histone deacetylation of the regulator of G-protein signaling 10 (RGS10) gene in ovarian cancer cells
    Mourad W Ali
    Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, United States of America
    PLoS ONE 8:e60185. 2013
    ..Markers for loss of RGS10 expression may identify cancer cells with unique response to therapeutics...
  6. pmc EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer
    Amber Bradley
    Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA
    Carcinogenesis 35:1100-9. 2014
    ..4% reduction, P = 0.035), with a trend in A2780ip2 (60.3% reduction, P = 0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest that EDD is a therapeutic target for ovarian cancer. ..

Research Grants30

  1. CARP-1: A Potential Therapeutic Agent for Breast Cancer
    ARUN KUMAR RISHI; Fiscal Year: 2013
    ..abstract_text> ..
  2. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013
    ..The ultimate goal of all the proposed studies is to define the molecular events that feature in the formation of human prions in order to develop therapeutics that cure the human prion diseases. ..