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）是卵巢癌中最常见、侵袭性和致命的形式。 上皮性卵巢癌，大多数患者的5年生存率为30-40%。发展精准 HGSOC的实验模型是必要的，以阐明疾病的发病机制，并评估新的 治疗。对癌症基因组图谱（TCGA）数据的分析显示， 编码LATS 1和LATS 2蛋白激酶，以及它们的下游效应激酶DYRK 1A，占65%， 分别为59%和38%的HGSOC病例。LATS 1和LATS 2是真正的肿瘤抑制因子， 进化保守的Hippo信号通路的组成部分。当激活时，LATS激酶 磷酸化并抑制致癌转录激活因子雅普。除了这个典型的功能，LATS 激酶与其他肿瘤抑制因子如pRb和DREAM转录调节复合物协同作用， 介导E2 F靶基因的阻遏。DYRK 1A是DREAM复杂装配所必需的， 作为LATS激酶信号传导至DREAM的介质。尽管基因编码经常丢失， LATS激酶和DYRK 1A，其抑制在HGSOC中的意义尚不清楚。初步 使用SKOV 3卵巢癌细胞的研究显示，DYRK 1A单独或LATS 1和LATS 2的缺失 （LATS 1/2），导致CKD 4/6激活，pRb和DREAM阻遏物功能丧失，细胞增殖增加， 体外增殖。有趣的是，与DYRK 1A缺失相反，LATS 1/2的缺失也导致了 体内实验表明，雅普能明显抑制肿瘤生长。这些发现使我们假设， 雅普和CDK 4/6的激活可能是促进体内卵巢肿瘤形成所必需的， 这些致癌途径的药物抑制剂可能对卵巢癌的一个子集有效 LATS 1/2下调。为了验证这一假设，我们将首先生成并表征HGSOC电池 使用表达荧光素酶的SKOV 3和Caov-3细胞，对LATS 1/2或DYRK 1A激酶进行耗竭的细胞系， 体内成像。这些细胞系将使用一组体外细胞增殖测定来表征， qPCR测定和蛋白质印迹以确定雅普和DREAM的功能状态，以及体内 原位肿瘤异种移植物测定以评估致瘤性。为了确定单独激活雅普是否可以 增加HGSOC细胞致瘤性，或是否需要额外的CDK 4/6活化（由缺失引起 我们将组成型活性YAPS 6A引入对照或DYRK 1A缺失的SKOV 3中， Caov-3细胞，并如上所述表征细胞。此外，我们将进行体外药物剂量 反应研究，以确定LATS 1/2的缺失是否可以使HGSOC细胞对药物抑制剂敏感， CDK 4/6（帕博西尼）或雅普（维替泊芬）或其组合。这项研究将促进我们对 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.