Constitutively active androgen receptor variants upregulate expression of mesenchymal markers in prostate cancer cells

Abstract

Androgen receptor (AR) signaling pathway remains the foremost target of novel therapeutics for castration-resistant prostate cancer (CRPC). However, the expression of constitutively active AR variants lacking the carboxy-terminal region in CRPC may lead to therapy inefficacy. These AR variants are supposed to support PCa cell growth in an androgen-depleted environment, but their mode of action still remains unresolved. Moreover, recent studies indicate that constitutively active AR variants are expressed in primary prostate tumors and may contribute to tumor progression. The aim of this study was to investigate the impact of constitutively active AR variants on the expression of tumor progression markers. N-cadherin expression was analyzed in LNCaP cells overexpressing the wild type AR or a constitutively active AR variant by qRT-PCR, Western blot and immunofluorescence. We showed here for the first time that N-cadherin expression was increased in the presence of constitutively active AR variants. These results were confirmed in C4-2B cells overexpressing these AR variants. Although N-cadherin expression is often associated with a downregulation of E-cadherin, this phenomenon was not observed in our model. Nevertheless, in addition to the increased expression of N-cadherin, an upregulation of other mesenchymal markers expression such as VIMENTIN, SNAIL and ZEB1 was observed in the presence of constitutively active variants. In conclusion, our findings highlight novel consequences of constitutively active AR variants on the regulation of mesenchymal markers in prostate cancer.

Figure 1. N-cadherin expression is upregulated in the presence of constitutively active androgen receptor variants.

A). N-cadherin expression was assessed by qRT-PCR in LNCaP cells overexpressing the constitutively active AR Q640X or AR-V7, or the AR-WT and in cells transfected with the empty plasmid (C3). Cells were grown in complete medium for 9 days after transfection. Parental LNCaP cells were used as control. y-Axis represents the relative fold change compared with control (parental LNCaP cells). β-ACTIN was used as the endogenous normalization control. Relative expression is presented as the mean ± SEM from three independent experiments. Each sample is compared one by one by two tail unpaired t test. NS: Not significant * P<0.05, **P<0.01 and ***P<0.001. B). Western Blot showing AR expression in transfected and non transfected LNCaP cells. C). Immunoblot analysis of N-cadherin expression in transfected LNCaP cells 4 and 9 days after transfection. D). Kinetic analysis of N-cadherin expression by Western Blot in LNCaP cells overexpressing AR Q640X from 2 to 9 days after transfection. β-actin was used as loading control.

Figure 2. N-cadherin upregulation was restricted to LNCaP cells expressing constitutively active androgen receptor variants.

LNCaP cells were transiently transfected with pEGFP-ARWT or pEGFP-ARQ640X plasmid and were sorted 4 days after. A). N-CADHERIN expression was analyzed by qRT-PCR in EGFP positive (transfected) and EGFP negative (non-transfected) fractions. B). Androgen receptor (AR) level was analyzed by Western Blot to verify the purity of each fraction after cell sorting. C). Immunofluorescence analysis of N-cadherin (red fluorescence) expression in LNCaP cells transfected with EGFP-tagged (green fluorescence) AR-WT, AR Q640X or AR Q670X expression plasmid. Magnification: ×20.

Figure 3. Androgens abrogate N-cadherin upregulation induced by constitutively active androgen receptor variants in LNCaP cells.

A). LNCaP cells were grown in RPMI-1640 containing 5% DCC-FCS and 100 nM of DHT or vehicle (EtOH). N-cadherin expression was analyzed by qRT-PCR in LNCaP cells 4 days after transfection with AR-WT or the constitutively active AR Q640X or AR-V7 expression plasmid. B). N-cadherin expression level in LNCaP cells was investigated by qRT-PCR 4 days after transfection with AR Q640X expression plasmid in the presence of different DHT concentrations (10 nM, 25 nM and 50 nM) or vehicle. C). N-cadherin expression induced by constitutively active AR variants (AR variants) was negatively regulated when LNCaP cells were grown in the presence of DHT. We hypothesize that endogenous AR-FL present in LNCaP cells and AR variants could act differently. In this model, DHT-stimulated endogenous AR-FL represses N-cadherin expression whereas AR variants upregulate its expression. D). LNCaP cells overexpressing AR-WT, AR Q640X and AR-V7 were cultured in DCC-FCS medium supplemented with 100 nM of DHT and in the presence of 100 nM of MDV3100 or DMSO as control during 3 days. N-cadherin expression was analyzed by qRT-PCR 4 days after transfection, and was normalized to β-ACTIN. The ΔΔCt method was used to calculate relative expression and each value was reported as the mean of ΔΔCt ± SEM. NS: Not Significant * P<0.05, **P<0.01 and ***P<0.001.

Figure 4. Upregulation of mesenchymal markers by constitutively active androgen variants in prostate cancer cells.

LNCaP cells were transfected with the ARWT, ARQ640X or AR-V7 expression plasmid or the empty plasmid (C3). A). E-CADHERIN, B). VIMENTIN, C). TWIST1, D). SNAIL and E). ZEB1 expression levels were analyzed by qRT-PCR at day-9 after transfection. For each sample, expression levels were normalized to PBGD or β-ACTIN and reported as relative value to LNCaP parental cell line. Values are presented as the mean of ΔΔCt ± SEM. NS: Not Significant * P<0.05, **P<0.01 and ***P<0.001. F). Western Blot showing evolution of ZEB1 expression in LNCaP cells overexpressing constitutively active AR variants. Immunoblot from 100 µg of total protein extracts. β-actin was used as loading control.