Abstract Introduction: MUC16 is a heavily glycosylated, type I transmembrane mucin, which is over expressed in different cancers. We have previously shown that significant overexpression of MUC16 in human PDAC tissues with disease progression compared to normal pancreas. However, the functional consequences of MUC16 and their role in PDAC is poorly understood. Based on this our hypothesis is that MUC16 can drive pancreatic cancer metastasis through FAK-mediated Akt and ERK/MAPK signaling activation and altering EMT markers. Methods: We have developed MUC16 knockdown Capan1 and Colo-357 PDAC cells to study the functional impacts. Congenic cell survival, soft-agar colony formation, and trans-well chamber assays were performed to determine the in vitro tumorigenicity. Orthotopic implantation was carried out using capan-1 and colo-357 PDAC cells to determine the oncogenic and metastatic potential of MUC16. Binding assay was performed to determine the cell adhesion property of MUC16 in colo-357 cells. The physical interaction between MUC16 and mesothelin, galectin-3 and FAK were evaluated by confocal and immunoprecipitation analysis. Immunoblot analyses were performed to determine the downstream signaling in MUC16 knockdown cells. Results: MUC16 knockdown in capan-1 and colo-357 PDAC cell lines resulted in significantly decreased cell proliferation (P<0.05), colony formation (P<0.01), and migration (P<0.01) in vitro. Further, MUC16 knockdown capan-1 and colo-357 cells significantly decreases the tumor formation (P<0.05) and metastasis (liver P<0.05, spleen P<0.001, intestinal wall P<0.01, diaphragm P<0.01 and peritoneum P<0.001) in orthotopic xenograft mouse model. Adhesion assay displays decreased cell attachment of MUC16 knockdown cells with recombinant galectin-1 and galectin-3 proteins. Immunoprecipitation and immunofluorescence studies confirmed that MUC16 interaction with mesothelin and galectin-3 in PDAC cells. Co-immunoprecipitation revealed a novel interaction between MUC16 and FAK in PDAC cells. Interestingly, we observed decreased expression of mesenchymal markers (N-cadherin and Zeb1) and increased expression of epithelial markers (E-cadherin and CK18) in MUC16 silenced PDAC cells, correlating with the decrease in metastasis. Moreover, MUC16 knockdown show decreased FAK-mediated Akt and ERK/MAPK activation in PDAC cells. Conclusion: Overall our study concludes that MUC16 interacts with FAK leads to the activation of EMT markers for enhancing pancreatic cancer metastasis. Citation Format: Sakthivel Muniyan, Dhanya Haridas, Satyanarayana Rachagani, Imayavaramban Lakshmanan, Suprit Gupta, Seema Chugh, Parthasarathy Seshacharyulu, Moorthy P. Ponnusamy, Surinder K. Batra. Novel interaction of MUC16 with FAK activate EMT process and metastasis of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1629.