ZFHX3 is integral to androgen/AR signaling involving protein association with AR in prostate cancer cells.

The androgen receptor (AR) signaling drives prostatic development and carcinogenesis, whereas the zinc finger homeobox 3 (ZFHX3) transcription factor modulates these processes. AR upregulates ZFHX3 transcription, but whether and how ZFHX3 plays a role in the AR signaling is unknown. RNA-seq was used to identify AR target genes that were also affected by ZFHX3 loss. Gene expression changes were verified using western blotting and qPCR. Immunoprecipitation, luciferase promoter-reporter assay, and western blotting were performed to assess ZFHX3's impact on AR transcriptional activity and ZFHX3 and AR protein interaction. Cell proliferation and colony formation assays were used to evaluate ZFHX3's impact on AR function. Kaplan-Meier analysis assessed the association of AR/ZFHX3 expression with patient survival. ZFHX3 loss in C4-2B/LNCaP cells downregulated classic AR target genes such as KLK3, FKBP5, and TMPRSS2 while upregulating some unclassical AR target genes (e.g., TNK1, ADAM7, and MAPRE2). ZFHX3 protein bound AR via multiple regions, particularly residues 1-223. ZFHX3 loss promoted cell proliferation/colony formation and attenuated enzalutamide's efficacy. Higher AR expression levels correlated with worse disease-free survival only in lower-ZFHX3 prostate cancer patients. Biochemically, ZFHX3's absence weakened AR's transactivity, including AR's binding to target gene promoters. These findings suggest that ZFHX3 is integral for AR signaling in prostate epithelial cells. ZFHX3 loss, which occurs in advanced prostate cancer, affects AR's function in gene transcription and could thus compromise PSA/KLK3 utility in prostate cancer detection.