Prostate cancer androgen biosynthesis relies solely on CYP17A1 downstream metabolites

Prostate cancer (PC) is dependent on androgen receptor (AR) activation by testosterone and 5α-dihydrotestosterone (DHT). Intratumoral androgen accumulation and activation despite systemic androgen deprivation therapy underlies the development of castration-resistant PC (CRPC), but the precise pathways involved remain controversial. Here we investigated the differential contributions of de novo androgen biosynthesis and androgen precursor conversion to androgen accumulation. Steroid flux analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on (CR)PC cell lines and fresh patient PC tissue slices after incubation with classic and alternative biosynthesis intermediates, alongside quantitative PCR analysis for steroidogenic enzyme expression. Activity of CYP17A1 was undetectable in all PC cell lines and patient PC tissue slices. Instead, steroid flux analysis confirmed the generation of testosterone and DHT from adrenal precursors and reactivation of androgen metabolites. Precursor steroids upstream of DHEA were converted down the first steps of the alternative DHT biosynthesis pathway, but did not proceed through to active androgen generation. Comprehensive steroid flux analysis of (CR)PC cells provides strong evidence against intratumoral de novo androgen biosynthesis and demonstrates that androgen precursor steroids downstream of CYP17A1 activities constitute the major source of intracrine androgen generation.</p

Activation of c-MET Induces a Stem-Like Phenotype in Human Prostate Cancer

Prostate cancer consists of secretory cells and a population of immature cells. The function of immature cells and their mutual relation with secretory cells are still poorly understood. Immature cells either have a hierarchical relation to secretory cells (stem cell model) or represent an inducible population emerging upon appropriate stimulation of differentiated cells. Hepatocyte Growth Factor (HGF) receptor c-MET is specifically expressed in immature prostate cells. Our objective is to determine the role of immature cells in prostate cancer by analysis of the HGF/c-MET pathway

A bypass mechanism of abiraterone-resistant prostate cancer: Accumulating CYP17A1 substrates activate androgen receptor signaling

Background: Intratumoral steroidogenesis and its potential relevance in castration‐ resistant prostate cancer (CRPC) and in cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1)‐inhibitor treated hormone‐naïve and patients with CRPC are not well established. In this study, we tested if substrates for de novo steroidogenesis accumulating during CYP17A1 inhibition may drive cell growth in relevant preclinical models. Methods: PCa cell lines and their respective CRPC sublines were used to model CRPC in vitro. Precursor steroids pregnenolone (Preg) and progesterone (Prog) served as substrate for de novo steroid synthesis. TAK700 (orteronel), abiraterone, and small interfering RNA (siRNA) against CYP17A1 were used to block CYP17A1 enzyme activity. The antiandrogen RD162 was used to assess androgen receptor (AR) involvement. Cell growth was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. AR‐ target gene expression was quantified by reverse transcription polymerase chain reaction (RT‐PCR). Nuclear import studies using cells with green fluorescent protein (GFP)‐tagged AR were performed to assess the potential of precursor steroids to directly activate AR. Results: Preg and Prog stimulated cell proliferation and AR target gene expression in VCaP, DuCaP, LNCaP, and their respective CRPC sublines. The antiandrogen RD162, but not CYP17A1 inhibition with TAK700, abiraterone or siRNA, was able to block Preg‐ and Prog‐induced proliferation. In contrast to TAK700, abiraterone also affected dihydrotestosterone‐induced cell growth, indicating direct AR binding. Furthermore, Prog‐induced AR translocation was not affected by treatment with TAK700 or abiraterone, while it was effectively blocked by the AR antagonist enzalutamide, further demonstrating the direct AR activation by Prog. Conclusion: Activation of the AR by clinically relevant levels of Preg and Prog accumulating in abiraterone‐treated patients may act as a driver for CRPC. These data provide a scientific rationale for combining CYP17A1 inhibitors with antiandrogens, particularly in patients with overexpressed or mutated‐AR

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

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

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

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

Cell Line Characteristics Predict Subsequent Resistance to Androgen Receptor-Targeted Agents (ARTA) in Preclinical Models of Prostate Cancer

Treatment of prostate cancer (PCa) has changed considerably in the last decade due to the introduction of novel androgen receptor (AR)-targeted agents (ARTAs) for patients progressing on androgen deprivation therapy (ADT). Preclinical research however still relies heavily on AR-negative cell line models. In order to investigate potential differences in castration-resistant PCa (CRPC) growth, we set out to create a comprehensive panel of ARTA-progressive models from 4 androgen-responsive AR wild-type PCa cell lines and analyzed its androgen response as opposed to its ADT-progressive counterparts. Parallel cultures of VCaP, DuCaP, PC346C, and LAPC4 were established in their respective culture media with steroid-stripped fetal calf serum (FCS) [dextran-coated charcoal-stripped FCS (DCC)] without androgen (ADT) or in DCC plus 1 μM of the ARTAs bicalutamide, OH-flutamide, or RD162 (an enzalutamide/apalutamide analog). Cell growth was monitored and compared to those of parental cell lines. Short-term androgen response was measured using cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. qRT-PCR was performed to assess the mRNA expression of markers for AR signaling, steroidogenesis, glucocorticoid receptor (GR) signaling, epithelial-mesenchymal transition (EMT), and WNT signaling. Out of 35 parallel cultures per cell line, a total of 24, 15, 34, and 16 CRPC sublines emerged for VCaP, DuCaP, PC346C, and LAPC4, respectively. The addition of bicalutamide or OH-flutamide significantly increased CRPC growth compared to ADT or RD162. VCaP, DuCaP, and PC346C CRPC clones retained an AR-responsive phenotype. The expression of AR and subsequent androgen response were completely lost in all LAPC4 CRPC lines. Markers for EMT and WNT signaling were found to be elevated in the resilient PC346C model and CRPC derivatives of VCaP, DuCaP, and LAPC4. Although the resistant phenotype is pluriform between models, it seems consistent within models, regardless of type of ARTA. These data suggest that the progression to and the phenotype of the CRPC state might already be determined early in carcinogenesis

Darolutamide added to docetaxel augments antitumor effect in models of prostate cancer through cell cycle arrest at the G1-S transition

Resistance to taxane chemotherapy is frequently observed in metastatic prostate cancer. The androgen receptor (AR) is a major driver of prostate cancer and a key regulator of the G1-S cell cycle checkpoint, promoting cancer cell proliferation by irreversible passage to the S-phase. We hypothesized that AR signaling inhibitor (ARSi) darolutamide in combination with docetaxel could augment antitumor effect by impeding the proliferation of taxane-resistant cancer cells. We monitored cell viability in organoids, tumor volume and PSA secretion in patient-derived xenografts (PDXs) and analyzed cell cycle and signaling pathway alterations. Combination treatment increased anti-tumor effect in androgen-sensitive, AR-positive prostate cancer organoids and PDXs. Equally beneficial effects of darolutamide added to docetaxel were observed in a castration-resistant model, progressive on docetaxel, enzalutamide and cabazitaxel. In vitro studies showed that docetaxel treatment with simultaneous darolutamide resulted in a reduction of cells entering the S-phase in contrast to only docetaxel. Molecular analysis in the prostate cancer cell line LNCaP revealed an upregulation of Cyclin Dependent Kinase inhibitor p21, supporting blockade of S-phase entry and cell proliferation. Our results provide a preclinical support for combining taxanes and darolutamide as a multimodal treatment strategy in metastatic prostate cancer patients progressive on ARSi and taxane chemotherapy.</p

Profiling of Antibody Production against Xenograft-released Proteins by Protein Microarrays Discovers Prostate Cancer Markers

This study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of serum markers for prostate cancer. By immunizing immuno-competent mice with serum from nude mice bearing prostate cancer xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several prostate cancer-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing prostate cancer xenografts and prevalidated in human serum samples of prostate cancer patients. Among the discovered and validated proteins were the members of the TAM receptor family (TYRO3, AXL, and MERTK), ACY1, and PSMA1. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons

Profiling of Antibody Production against Xenograft-released Proteins by Protein Microarrays Discovers Prostate Cancer Markers

This study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of serum markers for prostate cancer. By immunizing immuno-competent mice with serum from nude mice bearing prostate cancer xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several prostate cancer-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing prostate cancer xenografts and prevalidated in human serum samples of prostate cancer patients. Among the discovered and validated proteins were the members of the TAM receptor family (TYRO3, AXL, and MERTK), ACY1, and PSMA1. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons

Profiling of Antibody Production against Xenograft-released Proteins by Protein Microarrays Discovers Prostate Cancer Markers

This study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of serum markers for prostate cancer. By immunizing immuno-competent mice with serum from nude mice bearing prostate cancer xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several prostate cancer-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing prostate cancer xenografts and prevalidated in human serum samples of prostate cancer patients. Among the discovered and validated proteins were the members of the TAM receptor family (TYRO3, AXL, and MERTK), ACY1, and PSMA1. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons