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Background <p>Urothelial carcinoma (UC) is a prevalent malignancy that shows higher incidence in males but reportedly poorer outcomes in females. Among molecular subtypes, the basal/squamous (Ba/Sq) subtype is associated with poor prognosis and therapeutic resistance. Androgen receptor (AR) signaling has been implicated in UC pathogenesis, but its precise role in the Ba/Sq subtype remains unclear.</p> Methods <p>J82 cells stably expressing AR (J82-FLAG-AR) were established by retroviral transduction. Gene and protein expression were assessed by quantitative reverse transcription PCR (RT-qPCR) and Western blotting following dihydrotestosterone (DHT) treatment. To test AR-dependency, J82-FLAG-AR cells were co-treated with DHT and the AR antagonist enzalutamide (10 µM) for 24&#xa0;h. To test p21 dependency of DHT-induced growth suppression, J82-FLAG-AR cells were transfected with siControl or siP21 (50 nM) followed by DHT treatment, with proliferation assessed by WST-8 assay. Co-immunoprecipitation was used to examine AR–enhancer of zeste homolog 2 (EZH2) interaction.</p> Results <p>AR expression in J82 cells was associated with reduced CLDN1 and CLDN4 (epithelial markers), increased GATA3 (luminal marker), and increased CDKN1A (p21) expression at both mRNA and protein levels following DHT stimulation. DHT treatment suppressed proliferation of J82-FLAG-AR cells. Co-immunoprecipitation demonstrated AR–EZH2 interaction. Combined DHT and EZH2 inhibitor (GSK126) treatment further increased p21 mRNA but did not produce additional antiproliferative effect. Pharmacological AR antagonism with enzalutamide attenuated DHT-induced p21 mRNA induction in a DHT-dose-dependent manner, consistent with competitive AR antagonism. siP21-mediated p21 knockdown abolished the DHT-induced suppression of proliferation observed in siControl-transfected cells (siRNA × DHT interaction, <i>P</i> = 0.047).</p> Conclusions <p>In the Ba/Sq UC cell line J82, AR-dependent p21 induction contributes to DHT-induced suppression of proliferation, together with altered epithelial and luminal marker transcription. These observations support a p21-mediated, AR-dependent growth-restraining role for AR signaling in this subtype.</p>

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Investigation of the significance and role of androgen receptor expression in basal/squamous type urothelial carcinoma cell line J82 cells

  • 期刊论文

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  • 发表日期：

    2026年7月28日

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  • Ryotaro Sakai,
  • Hiroki Shimada,
  • Shuko Hata,
  • Atsushi Yokoyama,
  • Yasuhiro Nakamura

摘要

Background

Urothelial carcinoma (UC) is a prevalent malignancy that shows higher incidence in males but reportedly poorer outcomes in females. Among molecular subtypes, the basal/squamous (Ba/Sq) subtype is associated with poor prognosis and therapeutic resistance. Androgen receptor (AR) signaling has been implicated in UC pathogenesis, but its precise role in the Ba/Sq subtype remains unclear.

Methods

J82 cells stably expressing AR (J82-FLAG-AR) were established by retroviral transduction. Gene and protein expression were assessed by quantitative reverse transcription PCR (RT-qPCR) and Western blotting following dihydrotestosterone (DHT) treatment. To test AR-dependency, J82-FLAG-AR cells were co-treated with DHT and the AR antagonist enzalutamide (10 µM) for 24 h. To test p21 dependency of DHT-induced growth suppression, J82-FLAG-AR cells were transfected with siControl or siP21 (50 nM) followed by DHT treatment, with proliferation assessed by WST-8 assay. Co-immunoprecipitation was used to examine AR–enhancer of zeste homolog 2 (EZH2) interaction.

Results

AR expression in J82 cells was associated with reduced CLDN1 and CLDN4 (epithelial markers), increased GATA3 (luminal marker), and increased CDKN1A (p21) expression at both mRNA and protein levels following DHT stimulation. DHT treatment suppressed proliferation of J82-FLAG-AR cells. Co-immunoprecipitation demonstrated AR–EZH2 interaction. Combined DHT and EZH2 inhibitor (GSK126) treatment further increased p21 mRNA but did not produce additional antiproliferative effect. Pharmacological AR antagonism with enzalutamide attenuated DHT-induced p21 mRNA induction in a DHT-dose-dependent manner, consistent with competitive AR antagonism. siP21-mediated p21 knockdown abolished the DHT-induced suppression of proliferation observed in siControl-transfected cells (siRNA × DHT interaction, P = 0.047).

Conclusions

In the Ba/Sq UC cell line J82, AR-dependent p21 induction contributes to DHT-induced suppression of proliferation, together with altered epithelial and luminal marker transcription. These observations support a p21-mediated, AR-dependent growth-restraining role for AR signaling in this subtype.