To evaluate SUB1-mediated effects in prostate cancer progression, we performed gene expression analysis using RNA from SUB1 knockdown prostate cell lines. We identified multiple genes that were modulated upon SUB1 knockdown including PLK1, C-MYC, BUB1B and CDKN1B, among others (Figure 4a). PLK1 and C-MYC are known to have a role in cell proliferation, invasion and metastasis.41, 42 We validated the activation of CDKN1B and the downregulation of PLK1, C-MYC and BUB1B, both at the mRNA and protein levels upon SUB1 knockdowns (Supplementary Figures S5a–h, Figure 4b and Supplementary Figures S6a–c) and observed induction of these genes upon SUB1 overexpression in RWPE cells (Figure 4c). TCGA data show that PLK1 and BUB1B are overexpressed in metastatic prostate adenocarcinoma (Supplementary Figures S7a and b). Additionally, we validated both PLK1 mRNA and protein levels across benign, prostrate carcinoma and metastatic prostate cancer tissues by qPCR and immunoblot analysis, respectively (Supplementary Figures S7c and d), which confirmed the direct correlation between SUB1 and PLK1. Additionally, SUB1 and PLK1 mRNA levels are correlated in prostate cancer cell lines (Supplementary Figures S7e and f). Further, we generated stable SUB1-expressing RWPE cells using lentivirus (Supplementary Figure S8). Overexpression of SUB1 resulted in increased cell proliferation (Supplementary Figure S8 and Figure 4d) and invasion (Figure 4e). Furthermore, SUB1 overexpression elevated PLK1 and C-MYC expression, and reduced CDKN1B expression (Figure 4c and Supplementary Figure S6c), showing SUB1 dysregulation triggers alterations in critical oncogenes and tumor suppressors in prostate cancer. To verify the role of PLK1 in cell proliferation and invasion, we treated stable SUB1-overexpressing RWPE cells with PLK1 siRNA SMARTpool or PLK1 inhibitor (volasertib (BI6727)) and analyzed Myc-DDK-tagged SUB1 and PLK1 (Figure 4d inset). Both PLK1 knockdown and PLK1 inhibitor decreased cell proliferation (Figure 4d) and Matrigel invasion (Figure 4e). Thus, these consolidated observations underscore a downstream role for PLK1, C-MYC and CDKN1B in SUB1-mediated prostate cancer cell proliferation and invasion.