Dissecting FAK-regulated oncogenic signaling programs in ovarian cancer

标题：卵巢癌中FAK调节的致癌信号传导程序的剖析 高级别浆液性卵巢癌（HGSOC）在60个月内杀死了五分之四的女性。HGSOC是 基因复杂，这已经减缓了过去的临床前模型开发。为此，我们在分子水平上 特征在于一种新的和体内进化的、侵袭性的、可植入的和同基因的鼠卵巢癌模型。 这些细胞显示许多自发获得的拷贝数变化，包括K-Ras、Myc和FAK/PTK 2 基因（本文称为KMF细胞）与HGSOC的其他惊人相似之处。FAK（黏着斑激酶）是 一种酪氨酸激酶，典型地支持整联蛋白信号传导、运动性和机械感测。我们的集体 HGSOC和KMF细胞中的方法，包括药理学抑制，FAK敲除，FAK再表达， 互补和生物信息学分析揭示，非典型的粘附-独立的FAK信号转导 维持对铂化疗的内在抗性，部分是通过β-连环蛋白活化和 支持干细胞的转录因子和DNA修复基因。FAK在存活的患者肿瘤中被激活 化疗和获得性铂类耐药可以促进卵巢肿瘤对FAK的依赖， 增长我们鉴定了一组与FAK表达相关的基因和一个与固有FAK活性相关的亚组 3D类器官细胞培养外源性激活的b-连环蛋白表达足以挽救FAK丢失或 在3D培养中，β-连环蛋白没有促进FAK-null肿瘤的生长。因此，FAK 选择性促进体内致癌信号传导，FAK感知肿瘤微环境。我们的建议 将检验在小鼠腹膜内存活的肿瘤球中应激诱导的FAK活化的假设。 环境触发特定的细胞重编程，促进致癌性升高的干细胞样状态。在 目的-1，我们独特的基因编辑的人类和小鼠卵巢肿瘤系统将与诱导型 FAK表达系统表征FAK定位和激酶依赖性信号驱动恶性肿瘤。在 目的-2，将对从荷瘤小鼠分离的细胞进行总和单细胞RNA-seq，以确定 FAK在体内调节靶点。组合的单细胞RNA-seq和ATAC-seq将确定亚群如何 的细胞来源于对时间依赖性FAK激活的响应，以及细胞中基因标记物的相互关系。 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化分析 用于鉴定与FAK抑制和患者结果相关的生物标志物的样本。我们的建议 包括细胞生物学、先进的RNA测序、表观基因组作图以及单细胞测序 生物信息学聚类分析。这些方法，连同临床试验患者的评价 样本，将确定一个“FAK依赖性”的细胞生物标志物基因签名，可以重新测试的意义 在KMF和HGSOOC肿瘤模型中。这些研究将提供重要的见解， 维持HGSOC恶性肿瘤的信号通路。

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.