Intracrine Androgen Synthesis Research Articles

Mesoporous silica nanoparticles combined with AKR1C3 siRNA inhibited the growth of castration-resistant prostate cancer by suppressing androgen synthesis in vitro and in vivo

Intracrine androgen synthesis plays a critical role in the development of castration-resistant prostate cancer (CRPC). Aldo-keto reductase family 1 member C3 (AKR1C3) is a vital enzyme in the intracrine androgen synthesis pathway. In this study, mesoporous silica nanoparticles (MSNs) were employed to deliver small interfering RNA targeting AKR1C3 (siAKR1C3) to downregulate AKR1C3 expression in CPRC cells. The optimal weight ratio of MSNs/siAKR1C3 was determined by a gel retardation assay. Prostate cancer cells such as VCaP cells, which intracrinally express AKR1C3, and LNCaP-AKR1C3 cells stably transfected with AKR1C3 were used to investigate the antitumour effect of MSNs-siAKR1C3. Fluorescence detection and Western blot analyses were applied to confirm the entrance of MSNs-siAKR1C3 into the cells. A SRB (Sulforhodamine B) assay was employed to assess the cell viability, and a radioimmunoassay was used to measure the androgen concentration. Moreover, real-time PCR (RT-PCR), Western blot analysis and ELISA were used to determine the transcription and expression of prostate-specific antigen (PSA), AKR1C3 and androgen receptor (AR). Meanwhile, a reporter gene assay was performed to determine the AR activity. Additionally, a castrated nude mouse xenograft tumour model was produced to verify the inhibitory effect of MSNs-siAKR1C3 in vivo. The results showed that the optimal weight ratio of MSNs/siAKR1C3 was 140:1, and the complex could effectively enter cells, downregulate AKR1C3 expression, reduce the androgen concentration, inhibit AR activation, and inhibit CRPC development both in vitro and in vivo. These results indicate that decreasing intracrine androgen synthesis and inactivating AR signals by MSNs-siAKR1C3 may be a potential effective method for CRPC treatment.

Steroid Sulfatase Stimulates Intracrine Androgen Synthesis and is a Therapeutic Target for Advanced Prostate Cancer.

Most patients with prostate cancer receiving enzalutamide or abiraterone develop resistance. Clinical evidence indicates that serum levels of dehydroepiandrosterone sulfate (DHEAS) and biologically active DHEA remain in the high range despite antiandrogen treatment. The conversion of DHEAS into DHEA by steroid sulfatase (STS) may contribute to sustained intracrine androgen synthesis. Here, we determine the contribution of STS to treatment resistance and explore the potential of targeting STS to overcome resistance in prostate cancer. STS expression was examined in patients and cell lines. In vitro, STS activity and expression were modulated using STS-specific siRNA or novel STS inhibitors (STSi). Cell growth, colony formation, androgen production, and gene expression were examined. RNA-sequencing analysis was conducted on VCaP cells treated with STSi. Mice were treated with STSis with or without enzalutamide to determine their effects in vivo. STS is overexpressed in patients with castration-resistant prostate cancer (CRPC) and resistant cells. STS overexpression increases intracrine androgen synthesis, cell proliferation, and confers resistance to enzalutamide and abiraterone. Inhibition of STS using siRNA suppresses prostate cancer cell growth. Targeting STS activity using STSi inhibits STS activity, suppresses androgen receptor transcriptional activity, and reduces the growth of resistant C4-2B and VCaP prostate cancer cells. STSis significantly suppress resistant VCaP tumor growth, decrease serum PSA levels, and enhance enzalutamide treatment in vitro and in vivo. These studies suggest that STS drives intracrine androgen synthesis and prostate cancer proliferation. Targeting STS represents a therapeutic strategy to treat CRPC and improve second-generation antiandrogen therapy.

Genistein Combined Polysaccharide (GCP) Can Inhibit Intracrine Androgen Synthesis in Prostate Cancer Cells.

Our group and others have previously shown that genistein combined polysaccharide (GCP), an aglycone isoflavone-rich extract with high bioavailability and low toxicity, can inhibit prostate cancer (CaP) cell growth and survival as well as androgen receptor (AR) activity. We now elucidate the mechanism by which this may occur using LNCaP and PC-346C CaP cell lines; GCP can inhibit intracrine androgen synthesis in CaP cells. UPLC-MS/MS and qPCR analyses demonstrated that GCP can mediate a ~3-fold decrease in testosterone levels (p < 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3βHSD (p < 0.001), 17βHSD (p < 0.001), CYP17A (p < 0.01), SRB1 (p < 0.0001), and StAR (p < 0.01)), respectively. Reverse-phase HPLC fractionation and bioassay identified three active GCP fractions. Subsequent NMR and LC-MS analysis of the fraction with the highest level of activity, fraction 40, identified genistein as the primary active component of GCP responsible for its anti-proliferative, pro-apoptotic, and anti-AR activity. GCP, fraction 40, and genistein all mediated at least a ~2-fold change in these biological activities relative to vehicle control (p < 0.001). Genistein caused similar decreases in the expression of 17βHSD and CYP17A (2.5-fold (p < 0.001) and 1.5-fold decrease (p < 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes; 3βHSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.

The value of AKR1C3 in predicting the therapeutic efficacy of abiraterone for patients with metastatic castration-resistant prostate cancer.

313 Background: AKR1C3 is a multifunctional enzyme playing a significant role in androgen synthesis and metabolism. Previous studies have proved that the activation of AKR1C3 was associated with resistance to abiraterone through increasing the intracrine androgen synthesis. However, clinical validation is still lacking as to the prognostic value of AKR1C3 in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone. Methods: Data of 117 patients with mCRPC between 2016-2018 in our center were retrospectively analyzed. AKR1C3 was detected by the immunohistochemical staining from the 12-core prostate biopsy. Kaplan-Meier curves and COX regression were used to analyze the association between AKR1C3 and the treatment outcomes of abiraterone. The endpoints of this study were PSA progression-free survival (PSA-PFS) and radiograph progression-free survival (rPFS). Results: In total, AKR1V3 was detected in 40/117 (34.2%) cases. The positive AKR1C3 was significantly associated with shorter PSA-PFS for mCRPC patients treated with abiraterone (median PSA-PFS: 6.2 Mo vs. 11.1 Mo, p &lt; 0.001). Those with positive AKR1C3 were also accompanied with obviously poorer rPFS compared to those with negative AKR1C3 staining, despite that the difference was not statistically significant (median rPFS: 11.1 Mo vs. 21.8 Mo, p = 0.250). Multivariate COX regression indicated that, AKR1C3 was an independent prognosticator of rapid PSA progression for the abiraterone treatment (HR,95%CI: 2.612, 1.54-4.44, p &lt; 0.001). Conclusions: This is the first study verifying the adverse prognostic significance of AKR1C3 for mCRPC patients receiving abiraterone treatment. Our results suggested that, AKR1C3 was closely related to early treatment failure of abiraterone, and thus, was worthwhile to be routinely described in pathological report.

The Promise of Circulating Tumor Cells in Metastatic CRPC

The Promise of Circulating Tumor Cells in Metastatic CRPC

Modulation of AKR1C2 by curcumin decreases testosterone production in prostate cancer.

Intratumoral androgen biosynthesis has been recognized as an essential factor of castration‐resistant prostate cancer. The present study investigated the effects of curcumin on the inhibition of intracrine androgen synthesis in prostate cancer. Human prostate cancer cell lines, LNCaP and 22Rv1 cells were incubated with or without curcumin after which cell proliferation was measured at 0, 24, 48 and 72 hours, respectively. Prostate tissues from the transgenic adenocarcinoma of the mouse prostate (TRAMP) model were obtained after 1‐month oral administration of 200 mg/kg/d curcumin. Testosterone and dihydrotestosterone concentrations in LNCaP prostate cancer cells were determined through LC‐MS/MS assay. Curcumin inhibited cell proliferation and induced apoptosis of prostate cancer cells in a dose‐dependent manner. Curcumin decreased the expression of steroidogenic acute regulatory proteins, CYP11A1 and HSD3B2 in prostate cancer cell lines, supporting the decrease of testosterone production. After 1‐month oral administration of curcumin, Aldo‐Keto reductase 1C2 (AKR1C2) expression was elevated. Simultaneously, decreased testosterone levels in the prostate tissues were observed in the TRAMP mice. Meanwhile, curcumin treatments considerably increased the expression of AKR1C2 in prostate cancer cell lines, supporting the decrease of dihydrotestosterone. Taken together, these results suggest that curcumin's natural bioactive compounds could have potent anticancer properties due to suppression of androgen production, and this could have therapeutic effects on prostate cancer.

Inhibition of AKR1C3 Activation Overcomes Resistance to Abiraterone in Advanced Prostate Cancer.

Abiraterone suppresses intracrine androgen synthesis via inhibition of CYP17A1. However, clinical evidence suggests that androgen synthesis is not fully inhibited by abiraterone and the sustained androgen production may lead to disease relapse. In the present study, we identified AKR1C3, an important enzyme in the steroidogenesis pathway, as a critical mechanism driving resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. We found that overexpression of AKR1C3 confers resistance to abiraterone while downregulation of AKR1C3 resensitizes resistant cells to abiraterone treatment. In abiraterone-resistant prostate cancer cells, AKR1C3 is overexpressed and the levels of intracrine androgens are elevated. In addition, AKR1C3 activation increases intracrine androgen synthesis and enhances androgen receptor (AR) signaling via activating AR transcriptional activity. Treatment of abiraterone-resistant cells with indomethacin, an AKR1C3 inhibitor, overcomes resistance and enhances abiraterone therapy both in vitro and in vivo by reducing the levels of intracrine androgens and diminishing AR transcriptional activity. These results demonstrate that AKR1C3 activation is a critical mechanism of resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. Furthermore, this study provides a preclinical proof-of-principle for clinical trials investigating the combination of targeting AKR1C3 using indomethacin with abiraterone for advanced prostate cancer. Mol Cancer Ther; 16(1); 35-44. ©2016 AACR.

Steroid hormone synthetic pathways in prostate cancer.

While androgen deprivation therapy (ADT) remains the primary treatment for metastatic prostate cancer (PCa) since the seminal recognition of the disease as androgen-dependent by Huggins and Hodges in 1941, therapy is uniformly marked by progression to castration-resistant prostate cancer (CRPC) over a period of about 18 months, with an ensuing median survival of 1 to 2 years. Importantly, castration does not eliminate androgens from the prostate tumor microenvironment. Castration resistant tumors are characterized by elevated tumor androgens that are well within the range capable of activating the AR and AR-mediated gene expression, and by steroid enzyme alterations which may potentiate de novo androgen synthesis or utilization of circulating adrenal androgens. The dependence of CRPC on intratumoral androgen metabolism has been modeled in vitro and in vivo, and residual intratumoral androgens are implicated in nearly every mechanism by which AR-mediated signaling promotes castration-resistant disease.These observations suggest that tissue based alterations in steroid metabolism contribute to the development of CRPC and underscore these metabolic pathways as critical targets of therapy. Herein, we review the accumulated body of evidence which strongly supports intracrine (tumoral) androgen synthesis as an important mechanism underlying PCa progression. We first discuss the presence and significance of residual prostate tumor androgens in the progression of CRPC. We review the classical and non-classical pathways of androgen metabolism, and how dysregulated expression of these enzymes is likely to potentiate tumor androgen production in the progression to CRPC. Next we review the in vitro and in vivo data in human tumors, xenografts, and cell line models which demonstrate the capacity of prostate tumors to utilize cholesterol and adrenal androgens in the production of testosterone (T) and dihydrotestosterone (DHT), and briefly review the potential role of exogenous influences on this process. Finally, we discuss the emerging data regarding mechanisms of response and resistance to potent ligand synthesis inhibitors entering clinical practice, and conclude by discussing the implications of these findings for future therapy.

Abstract 4554: Regulation of intracrine androgen synthesis by NF-kappaB2/p52

Abstract Introduction: Benign prostatic hyperplasia and the initial stages of prostate cancer (CaP) exhibit androgen dependence, but androgen ablation results only in temporary regression of CaP, with progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) signaling remains active in CRPC due to aberrant activation of the AR. Synthesis of intracrine androgens has emerged as one of the mechanisms by which AR is activated in CRPC after androgen ablation. Even though levels of androgen biosynthetic enzymes have been shown to be elevated in CRPC, their regulation is not completely understood. In this study, we examined the role of NF-kappaB2/p52 in intracrine androgen synthesis and castration-resistant progression of CaP. Methods: Expression levels of androgen biosynthetic enzymes were measured in CaP cells with or without expression of NF-kappaB2/p52 using real-time RT-PCR and western blotting. Regulation by NF-kappaB2/p52 was examined using luciferase reporter assays and plasmids containing regulatory elements of androgen biosynthetic enzymes. Intracrine levels of androgens were measured using EIA in tumors obtained from castrated mice. Results: Expression levels of androgen biosynthetic enzymes including AKR1C3, CYP17A1, HSD3B2, and SRD5A1 were found to be elevated in CaP cells expressing NF-kappaB2/p52. Luciferase assays showed that NF-kappaB2/p52 regulates their expression directly by binding to their promoters and inducing transcription. The levels of total testosterone in CaP cells expressing NF-kappaB2/p52 were approximately 3-fold higher than control cells as measured using EIA. Intraprostatic androgen levels were found to be at 1002 ± 232 pg/g tissue, compared to 377.8 ± 105 pg/g tissue in control tumors obtained by orthotopic implantation of CaP cells expressing NF-kappaB2/p52. These data suggest that CaP cells synthesize detectable levels of testosterone in the absence of exogenous steroid precursors and overexpression of NF-kappaB2/p52 can increase this process, possibly by enhancing the expression of genes encoding steroidogenic enzymes. Conclusions: Intraprostatic androgen synthesis in recurrent prostate tumors contributes significantly to resistance to androgen ablation and development of CRPC. NF-kappaB2/p52 regulates the expression levels of steroidogenic enzymes and thereby enhances synthesis of intracrine androgens and aberrant activation of the AR. Coupled with our previous studies, these data suggest that antagonizing NF-kappaB2/p52 signaling may prove beneficial in CRPC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4554. doi:10.1158/1538-7445.AM2011-4554

Abstract 359: Neuropeptides promote castration-resistant prostate cancer through intracrine androgen biosynthesis

Abstract Introduction and Objectives: Neuroendocrine differentiation is associated with castration resistant prostate cancer (CRPC) with detectable increased serum levels of chromogranin A in patients. Neuropeptides secreted from the neuroendocrine cells are believed to activate androgen receptor (AR) in low or no androgen environments. We hypothesize that neuropeptides sustain CRPC by promoting intracrine androgen synthesis. Methods: We engineered LNCaP cells overexpressing gastrin-releasing peptide (GRP) and examined the level of androgen biosynthetic enzymes by RT-PCR and Western blots. Inhibition of androgen-free growth of LNCaP-GRP cells was performed with siRNA to AKR1C3 or CYP17A1 with or without Src inhibitor saracatinib. PSA and total testosterone levels were assessed in LNCaP-GRP cells, serum from tumor-bearing animals and xenografts. Results: The androgen biosynthetic enzymes were up-regulated in LNCaP-GRP cells, with AKR1C3, CYP17A1 and HSD17B2 displaying over 40 fold increase compared to the control line by real-time RT-PCR analysis. Transfection of siRNA against AKR1C3 and CYP17A1 inhibited LNCaP-GRP cell growth in androgen-free medium. Specific Src family kinase inhibitor saracatinib inhibits androgen-independent LNCaP-GRP cell growth in vitro and tumor metastasis in vivo. Saracatinib also reduced the expression of AKR1C3, AKR1C1/2 and HSD3B1/2 in GRP cells at both RNA and protein levels. When combined with siRNA for AKR1C3 and CYP17A1, saracatinib further inhibited androgen-independent LNCaP-GRP cell growth. Testosterone was detectable by EIA assay in LNCaP-GRP cell lysates (109.1 and 44.5 pg/mg protein vs. 18.3 in control), serum from castrated mice bearing GRP tumors (2235.5 pg/ml serum) and xenograft tissue (59.2 pg/mg tumor). These enhanced T levels were greatly reduced when treated with saracatinib, to 594 pg/ml serum and 12 pg/mg tumor, respectively. Conclusions: Secretion of neuropeptides promotes CRPC through up-regulation of intracrine androgen biosynthesis. Saracatinib not only targets neuropeptide-mediated AR activation but also blocks steroidogenesis in CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 359. doi:10.1158/1538-7445.AM2011-359

Antiandrogene Strategien im Prostatakarzinom

In advanced prostate cancer, albeit castration resistant, an active androgen receptor is still pivotal for growth and cell survival. Recent therapies involving more effective antiandrogens such as MDV3100 proved to be successful. Furthermore, blocking de novo intracrine androgen synthesis, e.g. with abiraterone acetate, provides additional benefit. Besides these antiandrogen measures, compounds which enable the reconstitution of the oestrogen receptor beta as a tumour suppressor restrain aberrant androgen receptor signalling.

Abstract 1727: Interleukin-6 and intracrine androgens in prostate cancer cells

Abstract INTRODUCTION AND OBJECTIVES: The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor (AR) and stimulate tumor growth, suggesting that prostate cancer cells may survive androgen deprivation therapies by increasing intracrine androgen synthesis within the prostate. However, factors that regulate de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of AR activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 regulates intraprostatic androgen synthesis in prostate cancer cells. METHODS: Quantitative reverse transcription-PCR and western blot were performed to detect expression levels of steroidogenic enzymes. AKR1C3 promoter reporter was constructed and analyzed for IL-6-mediated AKR1C3 transcriptional activity. IL-6-mediated signaling was knocked down using siRNAs specific to IL-6R and gp130 and the effect on AKR1C3 expression was examined. To measure intraprostatic androgen levels, IL-6 overexpressing LNCaP-IL6+ cells were inoculated orthotopically into the prostate of castrated male nude mice and generated tumors. Intraprostatic androgen levels were measured by an enzyme immunoassay (Testosterone EIA kit) based on the competition between testosterone and a testosterone-acetylcholinesterase (AChE) conjugate (testosterone tracer) for a limited amount of testosterone in the sample. RESULTS: We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes including HSD3B2 and AKR1C3 involved in androgen biosynthesis. Down regulation of IL-6 receptor and gp130 expression using specific siRNA abolished IL-6 mediated AKR1C3 expression, suggesting that IL-6 signaling is responsible for AKR1C3 expression. IL-6 increases AKR1C3 promoter activity, indicating that the increase in IL-6-mediated AKR1C3 expression is in part at the transcriptional level. Treatment of IL-6 increased testosterone level in LNCaP cells. The tumor testosterone levels were detected at 378 pg/g in tumors generated from IL-6 overexpressing LNCaP-IL6+ cells inoculated orthotopically into the prostates of castrated male nude mice. CONCLUSIONS: These results suggest that IL-6 increases levels of intracrine androgens through enhanced expression of genes mediating androgen metabolism in prostate cancer cells during androgen deprivation therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1727.

Interleukin-6 Regulates Androgen Synthesis in Prostate Cancer Cells

The standard systemic treatment for prostate cancer patients is androgen deprivation therapy. Although serum testosterone concentrations were significantly reduced after androgen deprivation therapy, levels of intraprostatic androgens are reproducibly measured at concentrations sufficient to activate androgen receptor and stimulate tumor growth, suggesting that prostate cancer cells may survive androgen deprivation therapies by increasing intracrine androgen synthesis within the prostate. However, factors that regulate de novo intracrine androgen synthesis have not been identified. Interleukin-6 (IL-6) has been implicated in the modulation of androgen receptor activation and growth and differentiation in prostate cancer. In this study, we investigate whether IL-6 regulates intraprostatic androgen synthesis in prostate cancer cells. Quantitative reverse transcription-PCR and Western blotting were done to detect expression levels of steroidogenic enzymes. AKR1C3 promoter reporter was constructed and analyzed for IL-6-mediated AKR1C3 transcriptional activity. IL-6-mediated signaling was knocked down using small interfering RNA specific to IL-6 receptor and gp130, and the effect on AKR1C3 expression was examined. Intraprostatic androgen levels in prostate cancer cells in culture and in tumors were measured by an enzyme immunoassay (Testosterone EIA kit). We found that IL-6 increases the expression of genes encoding many steroidogenic enzymes, including HSD3B2 and AKR1C3, involved in androgen biosynthesis. Down-regulation of IL-6 receptor and gp130 expression using specific small interfering RNA abolished IL-6-mediated AKR1C3 expression, suggesting that IL-6 signaling is responsible for AKR1C3 expression. IL-6 increases AKR1C3 promoter activity, indicating that the increase in IL-6-mediated AKR1C3 expression is in part at the transcriptional level. Treatment of IL-6 increased testosterone level in LNCaP cells. The tumor testosterone levels were detected at 378 pg/g in tumors generated from IL-6-overexpressing LNCaP-IL6(+) cells inoculated orthotopically into the prostates of castrated male nude mice. These results suggest that IL-6 increases levels of intracrine androgens through enhanced expression of genes mediating androgen metabolism in prostate cancer cells.