Dissecting FAK-regulated oncogenic signaling programs in ovarian cancer

标题：剖析卵巢癌中FAK调节的致癌信号程序 高级别浆液性卵巢癌(HGSOC)在60个月内导致五分之四的女性死亡。HGSOC是 基因复杂，这减缓了过去临床前模型的发展。为此，我们在分子水平上 描述了一种新的体内进化的、侵袭性的、可移植的和同基因的小鼠卵巢癌模型。 这些细胞表现出许多自发的获得性拷贝数变化，包括K-RAS、Myc和FAK/PTK2 基因(这里称为KMF细胞)以及其他与HGSOC惊人的相似之处。粘着斑激酶(FAK)是 一种典型的支持整合素信号、运动性和机械感觉的酪氨酸激酶。我们的集体 在HGSOC和KMF细胞中的研究方法，包括药物抑制、FAK基因敲除、FAK重新表达、 互补性和生物信息学分析表明，非规范黏附非依赖性FAK信号转导 维持对铂类化疗的内在耐药，部分是通过b-连环蛋白的激活和 支持茎和DNA修复基因的转录因子。FAK在存活的肿瘤患者中被激活 化疗和获得性铂耐药可促进卵巢肿瘤对FAK的依赖 成长。我们确定了一个与FAK表达相关的基因集和一个与固有FAK活性相关的子集 在3D类器官细胞培养中。外源性活化的b-连环蛋白表达足以挽救FAK丢失或 在3D培养中的失活表型，但b-连环蛋白并不促进FAK缺失的小鼠肿瘤生长。因此，FAK 在体内选择性地促进致癌信号和FAK感知肿瘤微环境。我们的建议 将测试一种假设，即应激诱导在小鼠腹膜内存活的肿瘤球体中FAK的激活 环境触发特定的细胞重新编程，培养高致瘤性的干状状态。在……里面 AIM-1，我们独特的基因编辑的人和小鼠卵巢肿瘤系统将与诱导剂一起使用 FAK表达系统用于表征驱动恶性肿瘤的FAK定位和依赖于激酶的信号。在……里面 Aim-2，将从荷瘤小鼠分离的细胞进行全细胞和单细胞rna-seq，以确定 FAK在体内调节靶点。结合单细胞RNA-seq和atac-seq将决定亚群如何 对依赖于时间的FAK激活的反应以及基因标志物之间的相互关系 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 用于识别FAK抑制和患者预后的样本和生物标记物。我们的建议 包括细胞生物学、高级RNA测序、表观基因组图谱以及单细胞测序 进行生物信息学聚类分析。这些方法，以及对临床试验患者的评估 样本，将识别可重新测试其重要性的依赖于FAK的细胞生物标记物基因签名 在KMF和HGSOOC肿瘤模型中。这些研究将为有针对性的 支持HGSOC恶性的信号通路。

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.