LAUSR

Introduction: Advanced bladder cancer is a deadly and costly disease with locally invasive tumors carrying the worst prognosis for progression and mortality. The ADAM15 membrane disintegrin is key regulator in the invasive processes. Our previous studies demonstrated that increased expression of ADAM15 mRNA and protein is significantly associated with the local submucosal and lymphovascular invasion (LVI) and metastatic progression of human bladder cancer. Additionally, ADAM15 mediates integrin binding and has specifically been linked to the tumor-associated β1 integrins. This study aims to understand the ADAM15 disintegrin function and its potential as target in invasive bladder cancer. Methods: Collagen I represents the bulk of the submucosal matrix in the bladder and likely contributes to bladder cancer progression. Here, we developed a 3-dimensional (3D) culture system in collagen I to evaluate the phenotype of a panel of invasive bladder cancer cells. Furthermore, we developed a cyclic hexapeptide based on ADAM15 structure (adamatide), which can bind to the β1 integrin binding site; a rabbit polyclonal antibody against ADAM15 disintegrin domain (anti-A15DD); and, human recombinant ADAM15 disintegrin domain (hrA15DD) to compete with the endogenous protein. The effect of these inhibitors was evaluated on our 3D systems and compared to previously stablished ADAM15 knockdowns (shADAM15) in bladder cancer cell lines. Results: We assessed ADAM15 expression by Western blotting and immunofluorescence imaging in a panel of invasive bladder cancer cells. ADAM15 was highly expressed in the invasive cell lines, including UM-UC6 and UM-UC9. However, when cells were grown on plastic, ADAM15 was exclusively localized in cytoplasmic vesicles. On the contrary, when using 3D collagen cultures, ADAM15 translocated to filopodium-like protrusions (FLPs) on the cell membrane. Confocal imaging showed both co-expression and localization of ADAM15 and integrin β1, at the cellular level in collagen systems. These findings were validated by IHC of novel orthotopic models of bladder cancer, showing that both molecules localized at the muscle invasive front of the tumors as well as tumor emboli (LVI). Cells grown in collagen also presented increased FLP formation, invasion and proliferation rate when compared with those in plastic. Pharmacological inhibition of ADAM15 disintegrin activity or shADAM15 decreased cell attachment to collagen, invasion and FLPs in our 3D systems. Cells proliferation was also inhibited. Decreased cell viability was observed after treatment with the inhibitors on the 3D cultures. Early intracellular signaling events associated with these observations included inhibition of the FAK-Akt pathway. Conslusions: Our results show that ADAM15 has a key role in the invasive phenotype of bladder cancer cells via integrin β1 and FAK-Akt pathway. Thus, inhibition of ADAM15 activity represents a novel in vitro targeting strategy for bladder cancer Citation Format: Guadalupe Lorenzatti Hiles, Angelica L. Cates, John R. Rubin, Matthew C. Winkler, Hannah L. Briggs, Mark L. Day. Validation of ADAM15 as a therapeutic target in invasive bladder cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B08.