Targeting TR4 nuclear receptor-mediated p21 downregulation to increase apoptosis for reverse of the Enzalutamide-resistance in prostate cancer

Background: Enzalutamide (enz), also known as MDV3100, is a crucial treatment for advanced Castration-Resistant Prostate Cancer (CRPC). However, many patients develop enz resistance, leading to disease progression and recurrence. Testicular Nuclear Receptor-4 (TR4) has been implicated in regulating prostate cancer initiation, progression, metastasis and radiosensitivity. p21, a suppressor of cell growth, is crucial in caspase-activated apoptosis through both p53-dependent and -independent pathways. This study aimed to explore how TR4 contributes to enz-resistance in CRPC and the involvement of p21. Methods: Enz-resistant cell lines were generated by culturing C4-2 cells with increasing enz doses from 10 µM to 80 µM for 100 days. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay (MTS) and Immunohistochemistry (IHC)-morphology assay confirmed the establishment of resistant cells. After TR4 knockdown and overexpression, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot (WB) were used to detect TR4 and p21 expression changes and flow cytometry was employed to assess apoptosis. Results: C4-2 MDV-R cells were spindle-shaped and had a lower apoptosis rate than parental C4-2 cells under enz treatment. TR4 expression was elevated in C4-2 MDV-R cells. Overexpressing TR4 decreased the apoptosis rate of C4-2 cells treated with 25 µM enz, while knocking down TR4 increased it. Simultaneously, TR4 negatively regulated p21 expression. Knocking down TR4 in C4-2 MDV-R cells enhanced apoptosis during enz treatment. Conclusions: TR4 could be a potential therapy target in combination with enz as the promising treatment for CRPC.