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.
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.
