Interleukin-6 increases prostate cancer cells resistance to bicalutamide via TIF2

Abstract

The standard treatment for advanced, androgen-responsive prostate cancer is androgen deprivation therapy with or without a nonsteroidal antiandrogen, such as bicalutamide. Although maximal androgen blockade exhibits favorable responses in the majority of patients, prostate cancer eventually progresses to an androgen-refractory stage. The mechanism underlying bicalutamide resistance in the course of prostate cancer progression is incompletely understood. However, interleukin-6 (IL-6) plays a critical role in the development and progression of CRPC. Herein, we explored an association between IL-6 and bicalutamide resistance. To study this, series of lower and higher passages of LNCaP cell sublines generated by long-term exposure to IL-6 were used. The cells from higher passages of LNCaP treated with IL-6 developed resistance to bicalutamide treatment compared with parental LNCaP cells. The levels of transcriptional intermediary factor 2 (TIF2) in IL-6-treated LNCaP cells were found to be significantly higher than parental LNCaP cells. Down-regulation of TIF2 expression via short hairpin RNA in IL-6-treated LNCaP cells sensitized these cells to bicalutamide treatment, whereas overexpression of TIF2 in the parental LNCaP cells increased resistance to bicalutamide. Furthermore, overexpression of IL-6 attenuated bicalutamide-mediated blockage of androgen-induced androgen receptor nuclear translocation and recruitment. These results show that overexpression of IL-6 increases the resistance of prostate cancer cells to bicalutamide via TIF2. Overexpression of IL-6 not only plays an important role in prostate cancer progression but also contributes to bicalutamide resistance. Our studies suggest that bicalutamide-IL-6-targeted adjunctive therapy may lead to a more effective intervention than bicalutamide alone.

Figure 1

Comparison of cell growth between LNCaP and LNCaP-IL-6+ cells in androgen-deprived condition in vitro. Cells were cultured in RPMI 1640 supplemented with 10% FBS. After 24 h, cells were switched to either 10% FBS or 10% CS-FBS. Cell number was counted after incubating another 72 h. Cell numbers in the complete FBS were expressed as 100%, and cell numbers in the CS-FBS were expressed as percentage relative to those in complete FBS. Mean ± SD of triplicate samples. Three independent experiments were done. *, P < 0.05.

Figure 2

Effect of bicalutamide on cell growth and AR nuclear translocation. A, LNCaP-IL-6+ cells are more resistant to bicalutamide treatment compared with parental LNCaP cells. Cells were seeded in 12-well plates at 1 × 10⁵ per well. After 24 h, increasing doses of bicalutamide (0–5 µmol/L) were administered to the cells. Cell number was counted after 48 h. Cell numbers in the control (without bicalutamide) were expressed as 100%, and cell numbers in the treated groups were expressed as percentage relative to the control without bicalutamide. Mean ± SD of triplicate samples. Three independent experiments were done. *, P < 0.05, compared with LNCaP cells at each point. B, knockdown of IL-6 signaling resensitized LNCaP-IL-6+ cells to bicalutamide treatment. LNCaP-IL-6+ cells were transfected with shRNA specific to IL-6 receptor and gp130 and treated with 5 µmol/L bicalutamide for 48 h. Cell number was determined, and gp130 and IL-6 receptor mRNA expression was determined by reverse transcription-PCR analysis (C). D, effect of IL-6 on AR nuclear translocation. LNCaP and LNCaP-IL-6+ cells were cultured in CS-FBS condition in the presence of 1 nmol/L DHT and treated with or without 5 µmol/L bicalutamide. Nuclear proteins were isolated after overnight treatment and subjected to Western blot analysis using AR antibody. Pol II was used as protein loading control. E, Chromatin immunoprecipitation assay of AR recruitment to the ARE site in the presence and absence of bicalutamide (Bic) treatment. LNCaP and LNCaP-IL-6+ cells were cultured in CS-FBS for 3 days, and 1 nmol/L DHT was then added to the cells overnight in the presence and absence of 5 µmol/L bicalutamide. Cell extracts were subjected to chromatin immunoprecipitation assay as described in Materials and Methods.

Figure 3

TIF2 expression in LNCaP and LNCaP-IL-6+ cells. Total RNA and protein were extracted from LNCaP and LNCaP-IL-6+ cells. TIF2 mRNA expression was analyzed by Northern blot (NB), and TIF2 protein expression was examined by Western blot (WB) using antibody against TIF2. GAPDH and actin were used as loading controls for RNA and protein, respectively. Levels of TIF2 protein were normalized to those of actin and expressed as fold change relative to LNCaP cells.

Figure 4

Effect of TIF2 expression on the response of cells to bicalutamide. A, LNCaP-IL-6+ cells were transfected with increasing doses of TIF2 specific shRNA (0–0.2 µg). Cells were then treated with 5 µmol/L bicalutamide for 48 h. Cells without bicalutamide treatment were used as control. Cell number was counted. Bottom, TIF2 protein expression by Western blot analysis. B, LNCaP cells were transfected with increasing doses of TIF2 expression plasmid (0–0.2 µg). Cells were then treated with 5 µmol/L bicalutamide for 48 h. The controls are the untreated cells. Cell number was counted. Bottom, TIF2 protein expression by Western blot analysis. Levels of TIF2 protein were normalized to those of actin and expressed as fold induction relative to the controls. Mean ± SD of triplicate samples. *, P < 0.05.