Calcium Activation of the Androgen Receptor in Prostate Cells

Abstract

Background: Although prostate cancer patients initially respond to androgen deprivation therapy, most patients progress to a resistant phenotype. Castration resistance is due, in part, to intratumoral and/or adrenal synthesis of androgens, overexpression or mutation of the androgen receptor (AR), stabilization of AR by chaperones, and ligand-independent activation of AR. Increasing evidence also links disruption of calcium homeostasis to progression of prostate cancer. Our previous study shows that heavy metal cadmium activates the AR through a ligand-independent mechanism. Cadmium mimics calcium in biological systems due to their similar ionic charge and radius. This study determines whether calcium activates AR and whether first- and second-generation antiandrogens block the ability of calcium to activate the receptor.

Methods: The expression of androgen-responsive genes and calcium channels was measured in prostate cells using a quantitative real-time polymerase chain reaction assay. Cell growth was measured.

Results: To ask whether calcium activates AR, prostate cells were treated with calcium in the absence and presence of the first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, and the expression of androgen-responsive genes and cell growth was measured. In the normal PWR-1E cells and HEK293T cells transiently expressing AR, treatment with calcium increased the expression of androgen-responsive genes by approximately 3-fold. The increase was blocked by enzalutamide but was not consistently blocked by the first-generation antiandrogens. In LNCaP cells which contain a mutant AR, treatment with calcium also increased the expression of androgen-responsive genes by approximately 3-fold, and the increase was more effectively blocked by enzalutamide than by hydroxyflutamide or bicalutamide. Treatment with calcium also increased cell growth that was blocked by enzalutamide. To ask whether dysregulation of calcium channels is associated with castration resistance, calcium channels were measured in the normal PWR-1E prostate cells, the hormone-responsive LNCaP cells, and the castration-resistant VCaP and 22RV1 cells. Compared to normal prostate cells, the hormone-responsive and hormone-resistant cells overexpressed several calcium channels.

Conclusions: The results of this study show that calcium activates AR and increases cell growth and that calcium channels are overexpressed in hormone-responsive and hormone-resistant prostate cancer cells. Taken together, the results suggest a novel role of calcium in the castration-resistant phenotype.

Figure 1

Effects of calcium and first- and second-generation antiandrogens on the expression of androgen receptor-regulated genes in PWR-1E cells. PWR-1E cells were maintained in hormone-free media and treated for 24 hours with dihydroxytestosterone (DHT; 5 nM), R1881 (5 nM), or calcium (Ca⁺²; 1 mM and 3 mM) in the absence and the presence of the first-generation antiandrogens hydroxyflutamide (HF; 10 μM) and bicalutamide (BIC; 10 μM) and the second-generation antiandrogen enzalutamide (ENZ; 10 μM). RNA was isolated, and the amount of mRNA was measured using a qRT-PCR assay and normalized to the amount of 18S rRNA. Data are presented as fold change (mean ± SEM; biological replicates = 3; technical replicates = 3; P < 0.05; ^∗P <  0.05, ^∗∗P <  0.01, ^∗∗∗P <  0.001, and ^∗∗∗∗P <  0.0001). Effects of calcium on the expression of WNT7B (a, b), NKX3.1 (NKX3; (c, d)), TMPRSS2 (e, f), and AR (g, h) in the presence of first-generation antiandrogens hydroxyflutamide and bicalutamide or second-generation antiandrogen enzalutamide, respectively.

Figure 2

Effects of calcium in androgen receptor transfected HEK293T cells. HEK293T cells were plated in lipoic acid free and phenol red free IMEM containing 5% CCS for 24 hours and transiently transfected with the androgen receptor plasmid (2.5 μg/μl). After 24 hours, the medium was diluted to 1% CCS and the cells were treated with dihydroxytestosterone (DHT, 5 nM), R1881 (5 nM), or calcium (Ca₊₂, 1 mM and 3 mM) in the absence and the presence of the second-generation antiandrogen enzalutamide (ET, 10 μM). RNA was isolated, and the amount of WNT7B (a) and NKX3.1 (b) mRNA was measured using a qRT-PCR assay and normalized to the amount of 18S rRNA. Data are presented as fold change (mean ± SEM; biological replicates = 3; technical replicates = 3; and P <  0.05, ^∗P <  0.05, ^∗∗P <  0.01, ^∗∗∗P <  0.001, ^∗∗∗∗P <  0.0001).

Figure 3

Effects of calcium and first- and second-generation antiandrogens on the expression of androgen receptor-regulated genes in LNCaP cells. LNCaP cells were maintained in hormone-free media and treated for 24 hours with dihydroxytestosterone (DHT; 5 nM), R1881 (5 nM), or calcium (Ca⁺²; 1 mM and 3 mM) in the absence and the presence of the first-generation antiandrogens Casodex (BIC; 10 μM) or hydroxyflutamide (HF; 10 μM) and the second-generation antiandrogen enzalutamide (ENZ; 10 μM). RNA was isolated, and the amount of mRNA was measured using a qRT-PCR assay and normalized to the amount of 18S rRNA. Data are presented as fold change (mean ± SEM; biological replicates = 3; technical replicates = 3; ^∗P <  0.05, ^∗∗P <  0.01, ^∗∗∗P <  0.001, and ^∗∗∗∗P <  0.0001). Effects of calcium on the expression of WNT7B (a, b), NKX3.1 (NKX3; (c, d)), TMPRSS2 (e, f), FKBP5 (g, h), and PSA (i, j) in the presence of first-generation antiandrogens hydroxyflutamide and bicalutamide and the second-generation antiandrogen enzalutamide, respectively.

Figure 4

Calcium channels in hormone-independent and castration-resistant prostate cancer cells. PWR-1E (PWR), LNCaP, VCaP, and 22RV1 cells were plated in improved minimum essential media (IMEM) containing phenol red and 10% FBS serum. To determine the expression of calcium channels, the amount of CACNA1-C, -D, -G, and -H; TRPM-7; and TRPV-6 mRNA was measured using a quantitative real-time PCR assay and normalized to the amount of 18S rRNA. Data are presented as fold change compared to PWR cells (mean ± SEM; biological replicates = 3; technical replicates = 3; ^∗P <  0.05, ^∗∗P <  0.01, ^∗∗∗P <  0.001, and ^∗∗∗∗P <  0.0001).

Figure 5

Effects of calcium on the growth of LNCaP cells. LNCaP cells were maintained in hormone-free media and treated with dihydroxytestosterone (DHT; 5 nM) or calcium (Ca; 1 mM and 3 mM) in the absence and the presence of the antiandrogen enzalutamide (ENZ; 10 μM) and continuously monitored for 8 days. Data are presented as fold change on days 2, 4, 6, and 8 (mean ± SEM; biological replicates = 3; technical replicates = 3; P < 0.05; ^∗P <  0.05, ^∗∗P <  0.01, ^∗∗∗P <  0.001, and ^∗∗∗∗P <  0.0001).