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

项目摘要-摘要 高级别浆液性卵巢癌(HGSOC)是最常见、最具侵袭性和致命性的卵巢癌 卵巢上皮癌，大多数患者的5年生存率为30-40%。精确度的发展 HGSOC的实验模型对于阐明其发病机制和评价新的发病机制是必要的。 治疗。对癌症基因组图谱(TCGA)数据的分析揭示了基因的拷贝数丢失 编码LATS1和LATS2蛋白激酶及其下游效应蛋白激酶DYRK1A，在65%， 分别占HGSOC病例的59%和38%。LATS1和LATS2是真正的肿瘤抑制因子和核心 进化保守的河马信号通路的组成部分。处于活动状态时，LATS激活酶 磷酸化并抑制致癌转录激活剂YAP。除了这个正则函数，LATS 激酶与其他肿瘤抑制因子如pRB和DREAM转录调控复合体协同工作， 介导E2F靶基因的抑制。DYRK1A是Dream Complex组装所必需的，并且可能 作为LATS信号转导到梦想的中介者。尽管编码基因频繁丢失 LATS激酶和DYRK1A在HGSOC中的抑制作用还不是很清楚。初步 使用SKOV3卵巢癌细胞的研究表明，DYRK1A单独或同时丢失LATS1和LATS2 (LATS1/2)，导致CKD4/6激活，失去pRB和DREAM抑制功能，细胞数量增加 体外增殖。有趣的是，与DYRK1A缺失相反，LATS1/2的缺失也导致了激活 并促进体内肿瘤的生长。这些发现使我们假设， YAP和CDK4/6的激活可能需要促进体内卵巢肿瘤的形成，并且 这些致癌途径的药理抑制剂可能对卵巢癌的一部分有效 LATS1/2的下调。为了验证这一假设，我们将首先生成并表征HGSOC细胞 使用SKOV3和CAOV-3细胞表达荧光素酶的LATS1/2或DYRK1A激酶缺失的株系，用于 活体成像。这些细胞系将使用一组体外细胞增殖分析来表征， 定量聚合酶链式反应和Western blotting检测YAP和DREAM的功能状态 原位肿瘤异种移植试验评估致瘤性。要确定单独激活YAP是否可以 增加HGSOC细胞的致瘤性，或者是否需要CDK4/6的额外激活(由丢失引起 ，我们将把成分活性YAPS6A引入对照或DYRK1A缺失的SKOV3中，并 CAOV-3细胞，并如上所述对细胞进行鉴定。此外，我们将进行体外药物剂量 确定LATS1/2缺失是否能使HGSOC细胞对药物抑制剂敏感的反应研究 CDK4/6(Palbociclib)或YAP(维替普芬)，或它们的组合。这项研究将增进我们对 HGSOC的发病机制，并为开发卵巢癌的新疗法奠定了基础。

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.