Dissecting FAK-regulated oncogenic signaling programs in ovarian cancer

剖析卵巢癌中 FAK 调节的致癌信号传导程序

基本信息

批准号：
10403441
负责人：
David D Schlaepfer
金额：
$ 46.06万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10403441
关键词：
3-Dimensional ATAC-seq Adhesions Automobile Driving Back Bioinformatics Biological Assay Biological Markers Biopsy Cancer Etiology Cancer Model Cancer Patient Cancer Relapse Cause of Death Cell Adhesion Cell Culture Techniques Cells Cellular biology Cessation of life Chemoresistance Chromosomes Clinical Trials Clinical trial protocol document Cluster Analysis Clustered Regularly Interspaced Short Palindromic Repeats Collection Complement Complex DDR1 gene DNA Repair DNA Repair Gene Databases Defect Dependence Disease Drug Targeting Environment Exhibits FOXM1 gene Focal Adhesion Kinase 1 Fostering Genes Genetic Genomics Goals Growth Human Hypoxia In Vitro Integrins KRAS2 gene Knock-out Knowledge Link Malignant Female Reproductive System Neoplasm Malignant Neoplasms Malignant neoplasm of ovary Modeling Molecular Molecular Conformation Mus Neoadjuvant Therapy Nuclear Oncogenes Oncogenic Organoids Ovarian PTK2 gene Paclitaxel Patient-Focused Outcomes Patients Peritoneal Pharmacology Phenotype Phosphotransferases Platinum Pre-Clinical Model Protein Tyrosine Kinase RNA Recurrence Resistance Role Sampling Serous Signal Pathway Signal Transduction Signaling Molecule Sleeping Beauty Stress Supporting Cell System TP53 gene Testing The Cancer Genome Atlas Time Transplantation Tyrosine Phosphorylation Validation Woman base bench to bedside beta catenin cell motility chemotherapy clinical trial analysis combinatorial epigenome genetic signature in vivo insight mechanotransduction model development mutant neoplastic cell novel ovarian neoplasm prognostic programs reconstitution research clinical testing response single cell sequencing single-cell RNA sequencing standard of care stem stemness three dimensional cell culture transcription factor transcriptome sequencing transcriptomics treatment response tumor tumor growth tumor heterogeneity tumor microenvironment

项目摘要

Title: Dissecting FAK-regulated oncogenic signaling programs in ovarian cancer High-Grade Serous Ovarian Cancer (HGSOC) kills four of five women within sixty months. HGSOC is genetically complex, which has slowed past preclinical model development. To this end, we have molecularly characterized a new and in vivo evolved, aggressive, implantable, and syngeneic murine ovarian cancer model. These cells display many spontaneous acquired copy number changes, including K-Ras, Myc, and FAK/PTK2 genes (herein termed KMF cells) among other striking similarities to HGSOC. FAK (focal adhesion kinase) is a tyrosine kinase canonically supporting integrin signaling, motility and mechano-sensing. Our collective approaches in HGSOC and KMF cells, including pharmacological inhibition, FAK knockout, FAK re-expression, complementation, and bioinformatic analyses reveal that non-canonical adhesion-independent FAK signaling sustains intrinsic resistance to platinum chemotherapy in part via b-catenin activation and the elevation of transcription factors supporting stemness and DNA repair genes. FAK is activated in patient tumors surviving chemotherapy and acquired platinum resistance can facilitate ovarian tumorsphere dependence on FAK for growth. We identified a gene set associated with FAK expression and a subset linked to intrinsic FAK activity in 3D organoid cell culture. Exogenous activated b-catenin expression was sufficient to rescue FAK loss or inactivation phenotypes in 3D culture, but b-catenin did not promote FAK-null tumor growth in mice. Thus, FAK selectively promotes oncogenic signaling in vivo and FAK senses the tumor microenvironment. Our proposal will test the hypothesis that stress-induced FAK activation in tumorspheres surviving within a mouse peritoneal environment triggers specific cellular reprogramming, fostering stem-like state of heightened oncogenicity. In Aim-1, our unique gene-edited human and murine ovarian tumor systems will be used together with an inducible FAK expression system to characterize FAK localization- and kinase-dependent signals driving malignancy. In Aim-2, total and single cell RNA-seq will be performed on cells isolated from tumor-bearing mice to determine FAK regulated targets in vivo. Combined single cell RNA-seq and ATAC-seq will determine how subpopulations of cells are derived in response to time-dependent FAK activation and the interrelationship of gene markers in cell subpopulations. In Aim-3, we will use molecular and immunohistochemical analyses of patient clinical trial samples to identify and biomarkers associated with FAK inhibition and patient outcome. Our proposal encompasses cell biology, advanced RNA sequencing, epigenome mapping as well as single cell sequencing with bioinformatic clustering analysis. These approaches, together with the evaluation of clinical trial patient samples, will identify a “FAK-dependent” cell biomarker gene signature that can be re-tested for significance within KMF and HGSOOC tumor models. These studies will provide important insights into a targetable signaling pathway sustaining HGSOC malignancy.

标题：剖析卵巢癌中FAK调节的致癌信号计划高级浆液卵巢癌（HGSOC）在60个月内杀死了五名女性中的四名。 HGSOC是遗传复杂，这已经放慢了过去的临床前模型发展。为此，我们有分子表征了一种新的和体内的进化，侵略性，可植入和同性鼠卵巢癌模型。这些单元格显示了许多赞助获得的拷贝数更改，包括K-RAS，MYC和FAK/PTK2 基因（本文称为KMF细胞）以及与HGSOC的其他相似之处。 FAK（焦点粘合剂激酶）是酪氨酸激酶在规范上支持整联蛋白信号传导，运动性和机械感应。我们的集体 HGSOC和KMF细胞的方法，包括药物抑制，FAK敲除，FAK重新表达，互补和生物信息学分析表明，非传统依赖性的FAK信号传导通过B-catenin激活部分持续对铂化疗的固有抗药性支持干性和DNA修复基因的转录因子。在存活的患者肿瘤中激活FAK 化学疗法和获得的铂耐药性可以促进卵巢肿瘤的依赖对FAK的依赖生长。我们确定了与FAK表达相关的基因集和与固有FAK活性相关的子集在3D器官细胞培养中。外源激活的B-catenin表达足以挽救FAK损失或 3D培养中的灭活表型，但B-catenin并未促进小鼠的FAK-null肿瘤生长。那，fak 有选择地促进体内的致癌信号传导和肿瘤微环境的FAK感觉。我们的建议将检验以下假设，即应激诱导的肿瘤球中的FAK激活在小鼠腹膜内存活环境会触发特定的细胞重编程，从而促进了茎状致癌性的状态。在 AIM-1，我们独特的基因编辑的人和鼠卵巢肿瘤系统将与可诱导一起使用 FAK表达系统以表征FAK定位和激酶依赖性信号驱动恶性肿瘤。在 AIM-2，总细胞和单细胞RNA-seq将在从肿瘤小鼠中分离的细胞上进行，以确定 FAK在体内调节靶标。联合单细胞RNA-SEQ和ATAC-SEQ将决定如何亚群细胞的响应于时间依赖性的FAK激活和基因标记中的相互关系细胞亚群。在AIM-3中，我们将使用患者临床试验的分子和免疫组织化学分析样品以识别与FAK抑制和患者结局相关的生物标志物。我们的建议包括细胞生物学，高级RNA测序，表观基因组映射以及单细胞测序通过生物信息学分析。这些方法，以及临床试验患者的评估样本将识别一个“依赖FAK”的细胞生物标志物基因特征，可以重新测试以提高意义在KMF和HGSOOC肿瘤模型中。这些研究将为目标提供重要的见解信号通路维持HGSOC恶性肿瘤。