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

Hippo 和 RB 肿瘤抑制途径抑制对卵巢癌进展的相对影响

• 批准号: 10087410
• 负责人: Fatmata Sesay
• 金额: $ 3.65万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-16 至 2022-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10087410
• 关键词: Adult; Agreement; Biological Assay; CDK4 gene; Cancer cell line; Cell Line; Cell Proliferation; Cells; Complex; Cyclin-Dependent Kinase Inhibitor 2A; Data; Development; Diagnosis; Disease; Dose; Down-Regulation; Embryonic Development; Epithelial ovarian cancer; Experimental Models; Future; G1/S Checkpoint Pathway; Genes; Genetic; Genetic Transcription; Homeostasis; In Vitro; LATS1 gene; LATS2 gene; Lead; Luciferases; Malignant Epithelial Cell; Malignant Female Reproductive System Neoplasm; Malignant Neoplasms; Malignant neoplasm of ovary; Mediating; Mediator of activation protein; Modeling; Oncogenes; Oncogenic; Ovarian Serous Adenocarcinoma; Pathogenesis; Pathway interactions; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphotransferases; Protein Family; Protein Kinase; Repression; Research Personnel; Retinoblastoma; Retinoblastoma Protein; Role; SKOV3 cells; Signal Pathway; Signal Transduction; Specificity; Survival Rate; Testing; The Cancer Genome Atlas; Time; Tissues; Transcription Coactivator; Tumor Suppressor Proteins; Tumorigenicity; Tyrosine; Verteporfin; Western Blotting; Woman; Work; Xenograft Model Antitumor Assays; base; bioluminescence imaging; cancer cell; functional status; imaging approach; improved; in vivo; in vivo bioluminescence imaging; in vivo imaging; inhibitor/antagonist; ovarian neoplasm; protein expression; response; treatment strategy; tumor growth; tumor progression; upstream kinase

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.

项目摘要提取 高级浆液卵巢癌（HGSOC）是最常见，侵略性和致命形式 上皮卵巢癌，大多数患者的5年生存率为30-40％。制定准确 HGSOC的实验模型是阐明疾病发病机理并评估新的必要 治疗。癌症基因组图集（TCGA）数据的分析揭示了基因的拷贝数损失 编码LATS1和LATS2蛋白激酶及其下游效应子激酶DYRK1A，分别为65％ HGSOC病例分别为59％和38％。 LATS1和LATS2是真正的肿瘤抑制剂和核心 进化保守的河马信号通路的成分。活跃时，纬度激酶 磷酸化并抑制致癌转录激活剂YAP。除了这种规范功能， 激酶与其他肿瘤抑制剂合作，例如PRB和DREAM转录调节络合物， 介导抑制E2F靶基因。 DYRK1A是Dream Complexssbly所必需的，并且可以 充当LATS激酶的调解人，以做梦。尽管经常损失编码的基因 LATS激酶和DYRK1A，它们在HGSOC中抑制的意义尚不清楚。初步的 使用SKOV3卵巢癌细胞的研究表明，单独的DYRK1A或LATS1和LATS2的丧失 （LATS1/2），导致CKD4/6的激活，PRB的损失和Dream Rebressor功能以及增加的单元格 体外增殖。有趣的是，与dyrk1a耗竭相比，LATS1/2的损失也导致激活 YAP的体内肿瘤生长。这些发现使我们假设一个结合了 可能需要YAP和CDK4/6的激活才能在体内促进卵巢肿瘤形成，并且 这些致癌途径的药理抑制剂可能有效地针对卵巢癌的一部分 lats1/2的下调。为了检验该假设，我们将首先生成并表征HGSOC单元 使用SKOV3和表达荧光素酶的CAOV-3细胞耗尽LATS1/2或DYRK1A激酶的线 体内成像。这些细胞系将使用一系列体外细胞增殖测定法进行表征 QPCR分析和蛋白质印迹以确定YAP和梦想的功能状态以及体内 原位肿瘤异种移植分析以评估肿瘤性。确定单独激活yap是否可以 增加HGSOC细胞肿瘤性，或是否需要额外激活CDK4/6（由损失引起 在dyrk1a）中，我们将介绍组成性活跃的YAPS6A到控制或DYRK1A耗尽的Skov3和 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 响应研究以确定LATS1/2的损失是否可以使HGSOC细胞对药理学抑制剂的敏感性敏感 CDK4/6（palbociclib）或yap（verteporfin）或它们的组合。这项研究将提高我们对 HGSOC发病机理，为开发卵巢癌的新疗法奠定了基础。