To demonstrate the role of SUB1 on tumor growth in vivo, we used a chick chorioallantoic membrane (CAM) assay and measured spontaneous metastasis, including local invasion, intravasation and metastasis to distant organs. CAM assay was performed as described previously,37, 48 using prostate cancer PC3-SUB1 knockdown cells. Depletion of SUB1 resulted in significantly reduced tumor weight compared with non-target short hairpin RNA (shRNA)-transfected control cells (Figure 6a). SUB1 knockdown in PC3 cells impaired their ability to invade the CAM basement membrane and resulted in a significantly decreased number of intravasated cells in the lower CAM compared with control cells (Figure 6b). Furthermore, there was attenuation of tumor metastasis in the SUB1 knockdown group compared with the control group (Figure 6c). Next, we examined SUB1-mediated tumorigenesis in a murine PC3 xenograft model using Non-T shRNA or two independent SUB1 stable knockdown PC3 cells. Both SUB1-shRNA 1 and 2 cells showed significantly reduced tumor growth and tumor weight in mice (Figures 6d and e) relative to control animals, demonstrating that SUB1 inhibition attenuates tumor growth (CAM assay and murine xenografts) and metastasis (CAM assay) in vivo. These observations show that SUB1 has a role in prostate tumor growth in vivo.