The relative impact of the Hippo and RB tumor suppressor pathways inhibition on the ovarian cancer progression

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Project Summary-Abstract High Grade Serous Ovarian Carcinoma (HGSOC) is the most common, aggressive and lethal form of epithelial ovarian cancer, with 5-year survival rate of 30-40% for most patients. Development of accurate experimental models of HGSOC is necessary to elucidate the disease pathogenesis and to evaluate new treatments. Analysis of The Cancer Genome Atlas (TCGA) data revealed copy number losses of the genes encoding LATS1 and LATS2 protein kinases, as well as their downstream effector kinase DYRK1A, in 65%, 59% and 38% of HGSOC cases, respectively. LATS1 and LATS2 are bona fide tumor suppressors and core components of the evolutionary conserved Hippo signaling pathway. When active, LATS kinases phosphorylate and inhibit oncogenic transcriptional activator YAP. Apart from this canonical function, LATS kinases cooperate with other tumor suppressors such as pRb and the DREAM transcription regulatory complex, to mediate repression of E2F target genes. DYRK1A is required for DREAM complex assembly, and could serve as a mediator of LATS kinases signaling to DREAM. Despite frequent losses of the genes encoding LATS kinases and DYRK1A, the significance of their inhibition in HGSOC is not well known. Preliminary studies using SKOV3 ovarian cancer cells revealed that loss of DYRK1A alone, or both LATS1 and LATS2 (LATS1/2), resulted in activation of CKD4/6, loss of pRb and DREAM repressor function and increased cell proliferation in vitro. Intriguingly, loss of LATS1/2, in contrast to DYRK1A depletion, also resulted in activation of YAP, and increased the tumor growth in vivo. These findings lead us to hypothesize that a combined activation of YAP and CDK4/6 could be required to promote ovarian tumor formation in vivo, and that pharmacological inhibitors of these oncogenic pathways could be efficient against a subset of ovarian cancers with downregulation of LATS1/2. To test this hypothesis, we will first generate and characterize HGSOC cell lines with depletion of LATS1/2 or DYRK1A kinases using SKOV3 and Caov-3 cells expressing luciferase, for in vivo imaging. These cell lines will be characterized using a panel of the in vitro cell proliferation assays, qPCR assays and Western blotting to determine functional status of YAP and DREAM, as well as in vivo orthotopic tumor xenograft assays to assess tumorigenicity. To determine if activation of YAP alone could increase HGSOC cell tumorigenicity, or whether it requires an additional activation of CDK4/6 (caused by loss of DYRK1A), we will introduce constitutively active YAPS6A into the control or DYRK1A-depleted SKOV3 and Caov-3 cells, and characterize the cells as described above. Furthermore, we will perform in vitro drug dose response studies to determine if loss of LATS1/2 can sensitize HGSOC cells to pharmacological inhibitors of CDK4/6 (palbociclib), or YAP (verteporfin), or their combination. This study will advance our understanding of HGSOC pathogenesis, and set the stage for developing new treatments for ovarian cancer.