This research project has two goals - to understand how nuclear Abl tyrosine kinase stimulates apoptosis and to apply this knowledge to increase the efficacy of ovarian cancer therapy. The Cancer Genome Atlas (TCGA) project has found that p53 is mutated in 96% of high-grade serous ovarian cancer (SOVC). SOVC is routinely treated with cisplatin (or carboplatin), which can kill p53-deficient cells because it activates th nuclear Abl tyrosine kinase to stimulate apoptosis. The TCGA data show that Abl remains wild type in greater than 99% of SOVC. We have created an Abl-NLS (; mutated for nuclear localization signals) allele in mice by knock-in mutations to block Abl nuclear import. With the Abl/ mice, we have demonstrated that nuclear Abl is indeed required for cisplatin to induce apoptosis in vivo. Importantly, we have uncovered two novel nuclear Abl functions: (a) stimulation of PUMA translation to sustain its accumulation, and (b) stimulation of primary microRNA-34c (pri-mir-34c) processing to promote its upregulation. Interestingly, both PUMA and pri-mir-34c are transcriptional targets of p53. Together, our previous and recent results show that nuclear Abl functions upstream and downstream of the p53-family to stimulate the expression of pro-apoptotic effectors. Because activation of nuclear Abl can stimulate the apoptotic response to cisplatin, we propose to understand and to exploit the pro-apoptotic functions of nuclear Abl in ovarian cancer therapy by focusing on three specific aims:
Aim -1. To investigate Abl-dependent regulation of PUMA protein expression: We will delineate the cis- sequence that is required for Abl to stimulate PUMA translation and identify Abl-regulated trans-acting factors. We will screen a human shRNA library to identify proteins besides Abl that can stimulate HA-PUMA translation. This un-biased screen will facilitate the discovery of additional players in the translational regulation of PUMA.
Aim 2. To investigate Abl-dependent microRNA (miR) regulation in cisplatin-induced apoptosis: We will test the hypothesis that Abl stimulates the interaction of DGCR8 with CTD tyrosine-phosphorylated RNA polymerase II to promote DGCR8 recruitment to the nascent pre-mir hairpin for efficient processing by Drosha. We will apply microRNA-seq to identify miRs that show reduced expression in the Abl/ kidney tissues and renal tubule epithelial cells, which are both dependent on nuclear Abl for cisplatin-induced apoptosis. We will screen a Lenti-miR minigene library to identify additional human pri-mir's that can be stimulated by Abl for processing.
Aim -3. To examine the role of nuclear Abl in cisplatin-induced apoptosis of ovarian cancer cells: To pave the way for translating the knowledge from Aims 1 and 2 to ovarian cancer therapy, we will examine the nuclear ABL functions in the induction of apoptosis in a panel of ovarian cancer cell lines. We will establish tumorigenic ovarian surface epithelial cells from the wild-type, the Abl/, the p53-/- and the double mutant (Abl/; p53-/-) mice and apply this genetic model of ovarian epithelial cancer to test reagents, to be generated from Aims 1 and 2, as novel drivers of nuclear Abl-dependent apoptosis in ovarian cancer cells.
Chemo-radiation and oncogene-targeted therapies are most effective when they trigger apoptosis, which is a normal cellular stress response that is often compromised in malignant cancer cells. Our research has identified an apoptosis activator that is not mutated in the high-grade serous ovarian cancer. The goal of this research project is to understand and exploit that apoptosis activation mechanism to increase the efficacy of ovarian cancer therapy.
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