Abstract 1627: Fibroblast HGF elicits c-MET-mediated signaling and migration in ovarian cancer cells.

Abstract Background: Epithelial ovarian cancer (EOC) has the highest mortality rate of all gynecologic malignancies diagnosed in the U.S. due to its rapid progression without symptom. Stromal components in the ovary contribute to rapid progression of cancer and interruption of their interaction with cancer cells are being explored as important therapeutic target. Hepatocyte growth factor (HGF) is expressed primarily in mesenchymal cells and acts on epithelial cells. HGF is also a unique cytokine that induces pleiotropic biological responses, involving proliferation, invasion, and dissemination of cancer cells. We studied c-MET/HGF axis as a mediator of tumor-stromal interactions in ovarian cancer using human ovarian fibroblast and their derived extracellular matrices (ECM). Methods: Six EOC cells were each cultured alone or in the presence of three human ovarian fibroblast cultures and their derived ECM in Transwell plates and the number of migrated cells was quantitated. ELISA was used to measure the concentration of HGF secreted by cancer cells and fibroblasts or released from fibroblast-derived ECM. EOC cells were treated with conditioned media derived from fibroblast that secretes high level of HGF and c-MET mediated signalling was assessed by immunoblotting. Results: Five out of the six EOC cells tested highly expressed c-MET and none produced detectable levels of HGF. IHFNO-303, a human ovarian fibroblast line, secreted the highest level of HGF (∼3,800 pg/mL) and markedly enhanced migration (2 to 140-fold increase) of the EOC cells that express c-MET. Recombinant HGF and a HGF neutralizing antibody were used to validate HGF as a major migratory factor secreted by fibroblast. HGF sequestered within the fibroblast-derived ECM was also able to stimulate cell migration by 1.5 to 24-fold, when released via EOC-associated degradation of the ECM. In cells containing constitutive c-MET phosphorylation, recombinant HGF and fibroblast-derived HGF negligibly affect c-MET phosphorylation on Tyr1234 and Tyr1003. However, both sources of HGF increased the phosphorylation of c-MET on Tyr1349, the multisubstrate docking site, by more than 6-fold and led to activation of downstream signalling transducers, e.g., AKT and ERK, in all c-MET expressing EOC cells. Therefore, signalling mediated by c-MET and HGF likely occurs in a paracrine manner in ovarian cancer. Conclusion: Our study demonstrates the functional contribution of c-MET and HGF signalling in malignant progression driven by important tumor-stromal interactions. We demonstrate for the first time that phosphorylation of c-MET on Tyr1349 requires extraneous HGF and phosphorylated c-MET level on Try1349 might be a good indicator of c-MET activation in the microenvironment and likely responsive to c-MET targeted therapy. Citation Format: Youngjoo Kwon, Yan Zhou, Andrew K. Godwin. Fibroblast HGF elicits c-MET-mediated signaling and migration in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1627. doi:10.1158/1538-7445.AM2013-1627