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

• 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
• 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

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 调节的致癌信号传导程序 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 遗传复杂，这减缓了过去的临床前模型开发。为此，我们从分子角度 表征了一种新的体内进化的、侵袭性的、可植入的、同基因的小鼠卵巢癌模型。 这些细胞表现出许多自发获得性拷贝数变化，包括 K-Ras、Myc 和 FAK/PTK2 基因（本文称为 KMF 细胞）与 HGSOC 具有其他惊人的相似之处。 FAK（粘着斑激酶）是 一种典型地支持整合素信号、运动和机械传感的酪氨酸激酶。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 重新表达、 互补和生物信息学分析表明，非典型的粘附独立的 FAK 信号传导 部分通过β-连环蛋白激活和升高来维持对铂类化疗的内在耐药性 支持干性和 DNA 修复基因的转录因子。 FAK 在存活的患者肿瘤中被激活 化疗和获得性铂类耐药可促进卵巢肿瘤球对 FAK 的依赖性 生长。我们鉴定了与 FAK 表达相关的基因集和与内在 FAK 活性相关的子集 在 3D 类器官细胞培养中。外源激活的 β-连环蛋白表达足以挽救 FAK 丢失或 3D 培养中的失活表型，但 b-catenin 不会促进小鼠中 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 将检验以下假设：小鼠腹膜内存活的肿瘤球中应激诱导的 FAK 激活 环境触发特定的细胞重编程，促进致癌性增强的干状状态。在 Aim-1，我们独特的基因编辑人类和小鼠卵巢肿瘤系统，将与诱导型卵巢肿瘤系统一起使用 FAK 表达系统可表征驱动恶性肿瘤的 FAK 定位和激酶依赖性信号。在 Aim-2、总细胞和单细胞 RNA 测序将对从荷瘤小鼠分离的细胞进行，以确定 FAK 体内调节靶标。结合单细胞 RNA-seq 和 ATAC-seq 将确定亚群如何 细胞的衍生响应于时间依赖性 FAK 激活和基因标记的相互关系 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 涵盖细胞生物学、高级 RNA 测序、表观基因组图谱以及单细胞测序 通过生物信息聚类分析。这些方法以及对临床试验患者的评估 样本，将识别“FAK依赖性”细胞生物标志物基因特征，可以重新测试其显着性 KMF 和 HGSOOC 肿瘤模型中。这些研究将为有针对性的研究提供重要见解 维持 HGSOC 恶性肿瘤的信号通路。