Abstract Introduction: Extracellular and cell surface proteases play indispensible roles in embryonic development and normal tissue homeostasis. They also contribute to tumor pathology involving cellular invasion and metastasis. As the majority of cancer-related deaths are associated with metastasis, understanding contributors to this process is essential for developing improved treatment strategies. TMPRSS2 is a Type II transmembrane serine protease (TTSP) regulated by androgenic hormones and is expressed preferentially in the human prostate. Recent studies have indicated that TMPRSS2 is expressed highly in primary and metastatic prostate cancers, though functional activities are poorly defined. Methods: To determine the relevance and consequence of TMPRSS2 overexpression in prostate cancer, we generated four transgenic mouse models that overexpress wild-type or mutant variants of human and mouse TMPRSS2 under the control of the prostate-specific probasin promoter (PB-TMPRSS2). We further crossed PB-TMPRSS2 mice with the LADY model of prostate cancer, and evaluated tumor growth and the impact on metastasis. Results: We confirmed that the PB-TMPRSS2 transgenic mouse lines have significantly elevated levels of TMPRSS2 protease in prostate epithelium. Although enhanced TMPRSS2 expression did not affect mouse fertility, we observed disorganization and disruption of the basement membrane of prostate glands, regardless of whether the overexpressed enzyme is of human or mouse origin. Immunohistochemical analysis identified dramatically altered levels of β4-integrin and collagen IV in the prostate glands of TMPRSS2-overexpressing mice, with the most significant changes occurring in ventral and anterior lobes. Histological changes in cell differentiation, proliferation or apoptosis were not observed. While a strain of LADY transgenic mice expressing SV40T antigen under the control of the probasin promoter (PB-SV40T) develop prostate cancer without metastatic spread, crosses with PB-TMPRSS2 mice produced offspring with high rates of metastasis to liver, lung, lymph node and bone. Conclusions: Our study pinpoints a mechanistic pathway by which TMPRSS2 contributes to primary prostate cancer progression and promotes metastasis, paralleling the effects of Hepsin overexpression, a structurally similar protease known to promote metastasis. These results provide support for the development of therapeutic approaches to specifically limit TMPRSS2 proteolytic activity in prostate cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2404. doi:10.1158/1538-7445.AM2011-2404