Fig. 6

TWIST1 upregulates SOX2 transcription through the E-box element, contributing to stemness. A PC3 cells were transfected for 48 h with TWIST1 expression vector or siRNA specific to TWIST1, and then lysed for immunoblot analysis. Arrow indicates endogenous TWIST1. B PC3 cells were co-transfected for 48 h with TWIST1 expression vector and siRNA specific to TWIST1 or BMI1, and then lysed for immunoblot analysis. Anti-flag antibody was used to detect flag-tagged TWIST1. C Cells were transfected for 48 h with siRNA specific to TWIST1 or TWIST1-expression vector and then lysed for real-time qPCR analysis. D PC3 cells were co-transfected with a TWIST1 expression vector and a SOX2 promoter (− 2546/+ 544) reporter construct in the pGL3 vector. Values represent mean ± SD. ***P < 0.001. E An E-box site mutant reporter construct was generated from the SOX2 promoter (− 484/+ 537) reporter construct in the pGL3 vector and used in reporter assay. Values represent mean ± SD. ***P < 0.001 compared with vector + WT; ^§§§P < 0.001 compared with TWIST1 + WT. F ChIP analysis of the interaction of TWIST1 with the SOX2 promoter. Upper, Chromatin fragments from PC3 cells transfected for 48 h with a TWIST1 expression vector or an empty vector were immunoprecipitated with control mouse IgG or anti-TWIST1 and analyzed by PCR using primers specific for the SOX2 promoter (− 62/+ 45). Lower, ChIP assay using PC3 cells transfected with TMPRSS4 expression vector for 48 h. An irrelevant region (− 450/− 345) was analyzed in parallel. The input control (1%) is in lane 3. G PC3 cells were transfected with a SLUG or TWIST1 expression vector and siRNA specific to SOX2 for 48 h and then subjected to ALDH assay and immunoblot analysis. Anti-flag antibody was used to detect flag-tagged TWIST1. Values represent mean ± SD. *P < 0.05; ***P < 0.001 compared with vector + control siRNA; ^§§§P < 0.001 compared with SLUG or TWIST1 + control siRNA. Densitometric quantification was performed on the immunoblot using GAPDH as a loading control. The mean relative density from three independent experiments is shown under the immunoblots (A, B, G). H PC3 cells were transfected with a SLUG or TWIST1 expression vector or siRNA specific to SLUG or TWIST1 for 48 h. Transfected cells were incubated under suspension culture conditions. Cell viability was determined by colorimetric WST assay. Values represent mean ± SD. ***P < 0.001. I A schematic representation illustrating the pathways underlying TMPRSS4-induced cancer stem–like properties in human cancer cells. We previously reported that TMPRSS4 activates the transcription factors AP-1 and SP1 in a manner dependent on MAPKs including JNK and ERK1/2 [26]. Our previous [22] and present studies show that TMPRSS4 upregulates SLUG and TWIST1 in an AP-1- and SP1-dependent manner. This study demonstrates that TMPRSS4 upregulates SOX2 at the transcriptional and protein stability levels through TWIST1 and SLUG, respectively, leading to acquisition of cancer stem–like features, including ALDH activation, thereby contributing to tumor growth and metastatic seeding. Of note, expression levels of SLUG and TWIST1 are interdependent. BMI1 suppression enhances SOX2 expression, indicating that BMI1 negatively regulates SOX2 expression in our system