Mutations in BRCA1 lead to hereditary ovarian cancer in about 5% of ovarian cancer patients. However 60- 90% of epithelial ovarian cancers have markedly decreased levels of BRCA1 protein resulting from various perturbations in BRCA1 gene function spanning acquired mutation, gene methylation, and protein instability. Gene transfer of BRCA1 into ovarian cancer cells inhibits tumorigenesis in cell lines and animal models and lead to an FDA sponsor-investigator IND for our Phase I and Phase II trials for BRCA1 retroviral gene therapy. Problems with immune response and concerns about potential malignant transformation ultimately terminated these trials, but generally supported the concept that elevating BRCA1 expression inhibits ovarian cancer. We have continued to study BRCA1 regulation and have recently discovered that 1) estrogen and progesterone lead to an akt-mediated phosphorylation of BRCA1 presumably at Thr509 which markedly, post-translationally, stabilizes BRCA1 protein, and 2) that BRCA1 is rapidly degraded by the proteasome in the absence of these hormones. Based on these data we hypothesize that phosphorylation of BRCA1 at Thr509 inhibits proteasomal degradation and propose that this explains why some ovarian cancers respond to Bortezomib therapy. Since decreased BRCA1 expression is a clear and important mechanism for ovarian cancer and BRCA1 protein is rapidly degraded by the proteasome, we propose that BRCA1 protein degradation may represent a biomarker for Bortezomib response in ovarian cancer. We hypothesize that ovarian cancers with moderate to high levels of BRCA1 mRNA but low levels of BRCA1 protein will respond to Bortezomib therapy. Phase I Milestones: 1) Treat SKOV3 and ES-2 ovarian cancer cell lines and xenografts (low level BRCA1 protein but moderate mRNA levels) with the proteasomal inhibitors Bortezomib and MG132 and quantitate BRCA1 mRNA and protein levels and extent of tumor inhibition. 2) Construct BRCA1 mutant expression vectors expressing distinct phosphorylation mutants at threonine 509: specifically T509A (non-phosphorylatable); T509E (constitutively charged, presumed active), disease-associated patient mutation R504H (non-phosphorylatable), and wildtype T509T. 3) Transfect SKOV3 cells with Aim 2 expression vectors; analyze phosphorylation and protein degradation in vitro and in vivo and analyze tumor inhibition. These cell lines will permit Phase II Proof- Of-Concept studies to test if decreased BRCA1 protein stability predicts response to proteasomal agents.
Public Health Relevance: This Phase I study will develop the reagents and cell lines for a definitive Proof-of-Principle Phase II Study testing the hypothesis that S3291 BRCA2 phosphorylation predicts responders to PARP inhibitors and that an IHC test using this antibody can predict which patients should receive this therapy. This proposal will create engineered cancer cell lines with either absent or increased S3291 BRCA2 phosphorylation to allow direct testing of PARP inhibitor effects in vitro and in animal tumors. If the hypothesis is correct then an IHC test will be developed and marketed to predict responders to this targeted PARP therapy.
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