GLPG0187 is currently in phase Ib clinical trial for patients with a variety of solid tumors. The effect of blocking integrin receptors by GLPG0187 were similar to effects of knockdown of ITGAV in prostate cancer cells. These data indicate that ITGAV is functionally involved in the migratory, mesenchymal cellular phenotype of prostate cancer cells. Moreover, ITGAV is important for the acquisition of prostate cancer cells with a metastasis-initiating capacity. Inhibition of av integrin might also have therapeutic potential in bladder cancer, since ITGAV is significantly overexpressed in bladder tumors compared to normal urothelium and a trend is observed of stage and grade-dependent increase in ITGAV expression. In the present study we determined the effect of functional inactivation of ITGAV on migration, EMT and stemness in bladder cancer using the human bladder carcinoma cell line UM-UC-3 and the human papilloma cell line RT-4. Functional inactivation of ITGAV in bladder cancer leads to a less malignant phenotype as illustrated by significantly impaired migration, EMT response, clonogenicity and a reduction in the size of the stem/progenitor pool. In line with these in vitro observations, knockdown of ITGAV or LY2109761 TGF-beta inhibitor treatment with GLPG0187 significantly inhibited metastasis and secondary tumor growth. These data indicate that ITGAV inhibition represents a novel, promising strategy for the prevention and/or treatment of bladder cancer growth and metastasis. Low survival rates of metastatic bladder cancer emphasize the need for a drug that can prevent and/or treat metastatic cancer. A promising approach that has been explored for these means in breast, melanoma and prostate cancer is the targeting of av integrins, which has been shown to reduce tumor growth, metastasis and angiogenesis. In this study, the role of ITGAV, which are highly expressed in bladder carcinomas, and its potential as a drug target in bladder cancer were investigated both by treatment with the ITGAV integrin-inhibitor GLPG0187 and knockdown of ITGAV. RT-4 and UM-UC-3luc2 cells were investigated to determine whether av integrin targeted therapies could be beneficial for these different grades of bladder tumors. The rationale for targeting of ITGAV is their involvement in cell proliferation, migration, invasion, survival and angiogenesis, which are essential processes for primary tumor growth and metastasis formation. These processes are induced via activation of focal adhesion kinase and srcfamily kinases, that activate the ERK/MAPK, NF-kB and AKT/PKB pathways. Modulating adherence to the extracellular matrix by changing the affinity of integrins for their ECM ligands, is important for the motility of cancer cells. Both GLPG0187 treatment and knockdown of ITGAV resulted in loss of adhesion, resembling loss of adherence to the ECM, which might have an inhibitory effect on cell motility and migration.