Ovarian carcinoma is a leading cause of cancer death in women. Many patients present with advanced disease and diffuse intra-abdominal spread. Understanding the molecular mechanisms controlling tumor dissociation from primary sites and the coordination of cell movement is vital to developing targeted therapies preventing metastatic spread. Transmembrane integrin receptors bind extracellular matrix proteins to facilitate ovarian tumor spread in part by activating cytoplasmic tyrosine kinases. Focal adhesion kinase (FAK) is the major integrin-activated tyrosine kinase. FAK expression is elevated in advanced stage ovarian cancer and associated with metastatic disease and poor patient survival. Human trials of FAK inhibitors are in progress with early signs of clinical efficacy. However, there is a great need for the development of better biomarkers that may distinguish activated FAK signaling and to identify tumor cells of high metastatic potential. Our objective in this renewal proposal is to test the hypothesis that FAK controls ovarian tumor growth-survival or ovarian tumor motility-metastasis through distinct signaling pathways that can be selectively inactivated by point mutations disrupting specific FAK phosphorylation sites. Our focus is on elucidating the molecular mechanisms controlling the binding of FAK to talin, an integrin- and cytoskeletal-associated protein. Talin binding is not conserved in the FAK-related Pyk2 kinase and we hypothesize that this is a major molecular difference between FAK and Pyk2 in promoting cell motility. Herein, we have identified the minimal FAK sequence required for talin binding, introduced gain-of-function talin binding into Pyk2, and identified a new FAK tyrosine phosphorylation site that may control the cycle of FAK-talin binding needed for adhesion site turnover and cell movement. We will test the hypothesis that the regulation of FAK-talin binding constitutes a motility- and metastasis-specific signaling pathway for ovarian cancer. We will use the first FAK knockout ovarian tumor cell model that is amenable to reconstitution and gain-of-function studies. The proposal has three specific aims: 1) To define the role of intrinsic FAK activity and site-specific FAK phosphorylation in regulating cell adhesio dynamics, cell motility, integrin activation-fibronectin binding, and traction force generation. These studies will use reconstituted FAK-null (from CRISPR knockout and murine FAK-flox/flox) and newly-generated FAK knockin ovarian carcinoma cells.
Aim -2 will test whether new phosphospecific antibodies to FAK may be used to detect invasive or metastatic tumor cells from human tumor samples.
Aim -3 will determine the role of intrinsic FAK activity and site-specific FAK phosphorylation in ovarian tumor growth and metastasis using three complementary tumor models: human orthotopic, murine syngeneic, and a spontaneous mouse tumor model. Our results will provide new insights of mechanisms driving tumor spread and will characterize new targets distinguishing aggressive from indolent ovarian cancers.
Ovarian cancer is a leading cause of cancer deaths among women due to tumor spread. Knowledge gained from these molecular studies will yield important information on drug-targetable intracellular signaling pathways driving cell motility and tumor progression.
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