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. 2022 Apr;82(5):505-516.
doi: 10.1002/pros.24297. Epub 2022 Jan 17.

Abiraterone switches castration-resistant prostate cancer dependency from adrenal androgens towards androgen receptor variants and glucocorticoid receptor signalling

J Matthijs Moll  1 , Johannes Hofland  2   3 , Wilma J Teubel  1 , Corina M A de Ridder  1 , Angela E Taylor  3 , Ralph Graeser  4 , Wiebke Arlt  3 , Guido W Jenster  1 , Wytske M van Weerden  1
Affiliations

Abiraterone switches castration-resistant prostate cancer dependency from adrenal androgens towards androgen receptor variants and glucocorticoid receptor signalling

J Matthijs Moll et al. Prostate. 2022 Apr.

Abstract

Introduction: Castration-resistant prostate cancer (CRPC) remains dependent on androgen receptor (AR) signalling, which is largely driven by conversion of adrenal androgen precursors lasting after castration. Abiraterone, an inhibitor of the steroidogenic enzyme CYP17A1, has been demonstrated to reduce adrenal androgen synthesis and prolong CRPC patient survival. To study mechanisms of resistance to castration and abiraterone, we created coculture models using human prostate and adrenal tumours.

Materials and methods: Castration-naïve and CRPC clones of VCaP were incubated with steroid substrates or cocultured with human adrenal cells (H295R) and treated with abiraterone or the antiandrogen enzalutamide. Male mice bearing VCaP xenografts with and without concurrent H295R xenografts were castrated and treated with placebo or abiraterone. Response was assessed by tumour growth and PSA release. Plasma and tumour steroid levels were assessed by LC/MS-MS. Quantitative polymerase chain reaction determined steroidogenic enzyme, nuclear receptor and AR target gene expression.

Results: In vitro, adrenal androgens induced castration-naïve and CRPC cell growth, while precursors steroids for de novo synthesis did not. In a coculture system, abiraterone blocked H295R-induced growth of VCaP cells. In vivo, H295R promoted castration-resistant VCaP growth. Abiraterone only inhibited VCaP growth or PSA production in the presence of H295R. Plasma steroid levels demonstrated CYP17A1 inhibition by abiraterone, whilst CRPC tumour tissue steroid levels showed no evidence of de novo intratumoural androgen production. Castration-resistant and abiraterone-resistant VCaP tumours had increased levels of AR, AR variants and glucocorticoid receptor (GR) resulting in equal AR target gene expression levels compared to noncastrate tumours.

Conclusions: In our model, ligand-dependent AR-regulated regrowth of CRPC was predominantly supported via adrenal androgen precursor production while there was no evidence for intratumoural androgen synthesis. Abiraterone-resistant tumours relied on AR overexpression, expression of ligand-independent AR variants and GR signalling.

Keywords: AR variants; CYP17A1 inhibition; abiraterone; adrenal androgens; androgen synthesis; castration resistant prostate cancer; glucocorticoid receptor; xenograft.

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Conflict of interest statement

The authors declare that there are no conflict of interests.

Figures

Figure 1
Figure 1
Adrenal steroids and tissue stimulate both castration‐naïve and castration‐resistant prostate cancer. (A) Growth response of castration‐naïve VCaP and its CRPC derivatives (DCC‐E, BIC‐C, FLU‐D) to androgen precursor steroids. Cells were cultured with physiological levels of 1.5 nM pregnenolone and 0.5 nM progesterone combined (PREC), 10 nM DHEA or 2.5 nM androstenedione (ADIONE), the combination of DHEA and ADION (ADRENAL) or 0.1 nM DHT and concomitantly treated with vehicle (DMSO), abiraterone (0.1 μM) or enzalutamide (1 μM). Bars represent mean fold over control (DCC/DMSO) ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Note: VCaP parental cells did not divide in castrate medium without steroids, whereas VCaP DCC‐E, BIC‐B and FLU‐D divided by an average of threefold. (B) Human adrenal cells induce prostate cancer cell growth under castrate conditions in a two compartment cell culture system. Left: fold growth induction of VCaP parental cells by the human adrenal cell line H295R. Right: fold growth induction of VCaP DCC‐E by H295R cells. Abiraterone reduced H295R‐induced proliferation, but not basal proliferation in both prostate cancer cell lines. Bars represent mean ± SEM. VCaP parental n = 3, VCaP DCC‐E n = 1. DMSO, dimethyl sulfoxide [Color figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
In vivo prostate tumour growth is stimulated by the presence of a human adrenal xenograft, which can be reduced by CYP17A1 inhibition. (A) Flowchart of the in vivo experiment: VCaP tumours were inoculated with or without H295R tumours on the contralateral side. At progression after sham castration or castration, animals were randomised to receive daily oral gavage of abiraterone or placebo. (B) Mean relative growth ±95% CI of placebo treated animals from castration to humane endpoint. Growth rates were significantly lower in castrate animals compared to intact animals (pooled students t‐test p = 0.0012, trend p = 0.0012). Growth rates were significantly higher in animals co‐inoculated with H295R cells (+ADR→ORx) compared to animals without (ORx) (students t‐test p = 0.04). DT = tumour doubling time in days. (C) Waterfall plot of PSA response after operation, defined as % PSA change 1 week after sham castration or castration relative to serum PSA at time of operation. Each bar represents an individual animal. Kruskal–Wallis p < 0.0001. (D) Waterfall plot of PSA response at progression after operation, defined as % PSA change at progression after sham castration or castration relative to PSA nadir after operation. Each bar represents an individual animal. Kruskall–Wallis p = 0.81. (E) Waterfall plot of PSA response after abiraterone, defined as % PSA change after 1 week of abiraterone treatment relative to PSA at start of treatment. Each bar represents an individual animal. (F) Mean relative growth ±95% CI of placebo and abiraterone treated animals from start of treatment to humane endpoint, stratified by hormonal arm (intact, ORx, +ADR→ORx). Growth rates were only significantly different in the +ADR→ORx arm (students t‐test p = 0.02) *p < 0.05. DT = tumour doubling time in days. (G) PSA levels (mean + SEM) per treatment group, stratified by hormonal arm (intact, ORx, +ADR→ORx). Mann–Whitney test, *p < 0.05 **p < 0.01. CI, confidence interval [Color figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Endocrine profiles of castration‐ and abiraterone‐resistant tumours and hosts. (A) Overview of the classical and alternative steroid synthesis pathway from cholesterol to DHT. (B). Classical testosterone synthesis intermediates. Left: intratumoural levels of the CYP17A1 substrate pregnenolone, the CYP17A1 metabolites DHEA and the androgen testosterone in VCaP tumours and corresponding levels in corresponding H295R tumors for +ADR→ORx. Right: corresponding plasma levels of indicated steroids. (C) Steroidogenic enzymes expression. mRNA expression of CYP11A1, CYP17A1, AKR1C3, and HSD17B6 relative to the housekeeping gene PBGD in VCaP and corresponding H295R tumours. Differences between VCaP and H295R were calculated by 2ΔΔCT. (D) Serum levels of intermediates of the alternative steroid synthesis pathway. Steroids could not be detected in tumour homogenates (data not shown). (E) Plasma levels of the main rodent glucocorticoid corticosterone and the CYP17A1‐dependent 11‐deoxycortisol in castrate male mice at humane endpoint. Dots indicate each measurable datapoint. Unmeasurable samples (ct‐value >40 for qPCR or steroid below LOD) not shown in this graph. qPCR, quantitative polymerase chain reaction [Color figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Upregulation of AR, AR variants, GR and target genes in CRPC and abiraterone resistant tumours. (A) Increased expression of full length AR and AR variants. Clockwise from top, left: VCaP tissue mRNA expression of full length Androgen Receptor (ARfl), the AR target gene PSA and the ligand independent AR variants 1 and 7. (B) Summary table of mean fold expression changes in mRNA expression levels for AR and AR variants, the AR regulated genes PSA, FKBP5, TMPRSS2, TIPARP, NDRG1, the glucocorticoid receptor and SGK1 relative to control (intact/placebo). †Significant difference versus intact/placebo treated tumours; ‡siginificant difference versus ORx/placebo treated tumours; ⁰significant difference between abiraterone versus placebo treated tumours within arm. 1Refercence expression level. (C) Increased GR signalling in CRPC and abiraterone resistant tumours. Top: Tissue RNA expression of Glucocorticoid Receptor (GR) and the GR target gene serum/glucocorticoid regulated kinase 1 (SGK1). Bottom, left: expression of GR and SGK1 sorted per treatment arm. Dotted lines indicate the 95% CI of the mean expression of GR and SGK1 in the intact group. Although GR expression is also upregulated in the co‐inoculated arms, SGK1 expression is only increased in the presence of abiraterone. Bottom, right: correlation of GR and SGK1 expression. (D) Androgen Receptor protein expression. Left: representative images of VCaP tumours (magnification ×20). Right: quantification of AR staining score as defined by multiplying the fraction of AR positive cells with staining intensity (0 = no staining, 1 = nuclear, 2 = nuclear + cytoplasmic) scored at magnification ×20. Statistical test using one‐way ANOVA with Bonferroni posttest: ***p < 0.001. (E) Glucocorticoid Receptor protein expression. Left: representative images of VCaP tumours, (magnification ×20). Right: quantification of GR positive cells as percentage of positive cells per field at ×20 magnification. Statistical test using one‐way ANOVA. ANOVA, analysis of variance; CRPC, castration‐resistant prostate cancer; GR, glucocorticoid receptor; mRNA, messenger RNA [Color figure can be viewed at wileyonlinelibrary.com]
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