Ovarian cancer (OC) is the leading cause of death among gynecological malignancies and metastasis to abdominal organs is the most common cause of OC mortality. Work in our laboratory demonstrated that tissue transglutaminase (TG2), an enzyme involved in Ca2+-dependent protein post-translational modifications and cross-linking, is highly expressed in the majority of ovarian tumors and critically regulates peritoneal metastasis. Mechanistic studies performed by our group during the tenure of this grant identified the interaction between TG2 and fibronectin (FN) as a critical player in the process of peritoneal metastasis. Our published work demonstrated that the TG2/FN complex is implicated in: a) adhesion to the extracellular matrix through interaction with ?ntegrins, b) induction of epithelial to mesenchymal transition, and c) regulation of ?atenin signaling, which in turn promotes cancer cell proliferation and maintenance of a cancer stem cell profile. These preliminary data provide a strong rationale to pursue further mechanistic studies to understand how the protein complex regulates oncogenic signaling leading to cancer dissemination and to initiate a search for inhibitors of this complex. To this end, we completed high throughput screening of a large library of compounds and discovered new diamino-pyrimidines as potent inhibitors for the TG2/FN complex. We demonstrated that several of these small molecules potently blocked ovarian cancer cell adhesion and migration and selected TG53 as the lead compound to be further evaluated. Based on our discoveries during the previous funding period, we hypothesized that the TG2/FN complex is critical to the development of OC metastasis and that its disruption will block tumor dissemination. Here we propose to elucidate the mechanism by which TG2/FN complex initiates oncogenic signaling leading to metastasis and to characterize pre-clinically the top hit identified during the screening process. A step wise approach utilizing biochemical and biological assays in OC models will address three objectives: Aim 1: To characterize the mechanisms by which formation of the TG2/FN/integrin complex activates EMT. Aim 2: To define mechanisms engaged by TG2/FN/integrin complexes to promote stem cell signaling. Aim 3: To characterize the effects of the lead TG2/FN inhibitor in an OC metastasis model. Successful characterization of the newly discovered TG2/FN inhibitor in the preclinical assays proposed will provide the basis for future clinical applications. In the long term, this line of innovative research taking advantage of our unique expertise and resources will develop new agents to prevent metastasis in a difficult to treat, fatal cancer.