Intratumoral Steroidogenesis Research Articles

Semaphorin 3C promotes de novo steroidogenesis in prostate cancer cells.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Abstract 3444: Role of AKR1C3 in converting reservoirs of DHEA-S into potent androgens that drive prostate cancer cell growth

Abstract Castration resistant prostate cancer (CRPC) treatment aims to stop a resurgence of androgen receptor (AR) signaling with a class of therapeutics known as androgen receptor signaling inhibitors (ARSI). Through inhibition of CYP17A1, Abiraterone (Abi) prevents an upstream step in androgen biosynthesis, while Enzalutamide (ENZ) acts as an AR antagonist. Despite the early success of ARSI, a subset of patients develop resistance and succumb to the disease. One major mode of resistance is thought to occur when steroidogenic enzymes that are upregulated in CRPC convert weaker circulating precursors to testosterone (T) and dihydrotestosterone (DHT) to restore AR signaling. In this study, we identify a mechanism driving intratumoral steroidogenesis that involves AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase type 5). AKR1C3 is a major steroidogenic enzyme in the prostate that is involved in all androgen biosynthetic pathways to T and DHT, upregulated in CRPC patients, and is a component of Abi and ENZ drug resistance. Previously with Taplin et al. we observed in patients on Leuprolide that T was reduced to 5 ng/dL, and the addition of Abi lowered DHEA-S by 90% (from 230,000 ng/dL to 22,000 ng/dL). However, the DHEA-S that remained was 4,000x greater than circulating T, making it a reservoir for intratumoral steroidogenesis post Abi treatment. Here, we demonstrate that post-Abi concentrations of DHEA-S, and castrate levels of DHEA-S (post-leuprolide but pre-Abi treatment), which also corresponds to DHEA-S levels patients on ENZ are 1) converted to T in an AKR1C3-dependent manner in prostate cancer (PCa) cells, and 2) sufficient to stimulate AKR1C3-dependent cell growth. Our observations are consistent across both primary and metastatic PCa cell lines, CWR22PC and DuCaP, respectively. Androgen measurements were made using our highly sensitive and specific stable isotope dilution liquid chromatography tandem mass spectrometry (SID-LC-MS/MS) method. Additionally, we demonstrate AKR1C3-dependence using genetic and pharmacological approaches with an AKR1C3 stable knockdown for each cell line and two AKR1C3 competitive inhibitors. Taken together, we have identified a mechanism of resistance to ARSIs common to both primary and metastatic cell lines that operates through AKR1C3’s intratumoral conversion of DHEA-S to potent androgens that drive prostate cancer growth. Citation Format: Andrea J. Detlefsen, Ling Duan, Clementina Mesaros, Trevor M. Penning. Role of AKR1C3 in converting reservoirs of DHEA-S into potent androgens that drive prostate cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3444.

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

The roles of RUNX2 and osteoclasts in regulating expression of steroidogenic enzymes in castration-resistant prostate cancer cells

Intratumoral steroidogenesis is involved in development of castration-resistant prostate cancer (CRPC) as bone metastases. The osteoblast transcription factor RUNX2 influences steroidogenesis and is induced in CRPC cells by osteoblasts. This study investigates osteoclastic influence on RUNX2 in intratumoral steroidogenesis.Steroidogenic enzymes and steroid receptors were detected with immunohistochemistry in xenograft intratibial tumors from CRPC cells. In vitro, expression of RUNX2 was increased by osteoclasts in osteoblastic LNCaP-19 cells, but not in osteolytic PC-3. Silencing of RUNX2 downregulates expression of CYP11A1, CYP17A1 and HSD3B1 in LNCaP-19 cells co-cultured with osteoclasts, leading to inhibition of KLK3 expression. Osteoclasts promoted CYP11A1 and RUNX2 promoted AKR1C3, HSD17B3 and CYP19A1, but suppressed ESR2 in PC-3 cells.This study shows that osteoclasts promote RUNX2 regulated induction of key steroidogenic enzymes, influencing activation of androgen receptor in CRPC cells. The potential of RUNX2 as a target to inhibit progression of skeletal metastases of CRPC needs further investigation.

Endothelin-1 induces changes in the expression levels of steroidogenic enzymes and increases androgen receptor and testosterone production in the PC3 prostate cancer cell line.

Endothelin-1 (ET-1) is involved in the regulation of steroidogenesis. Additionally, patients with castration-resistant prostate cancer (PCa) have a higher ET-1 plasma concentration than those with localized PCa and healthy individuals. The aim of the present study was to evaluate the effect of ET-1 on steroidogenesis enzymes, androgen receptor (AR) and testosterone (T) production in PCa cells. The expression levels of endothelin receptors in prostate tissue from patients with localized PCa by immunohistochemistry, and those in LNCaP and PC3 cells were determined reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Furthermore, the expression levels of ET-1 were determined in LNCaP and PC3 cells by RT-qPCR and western blotting. The ET-1 receptor activation was evaluated by intracellular calcium measurement, the expression levels of AR and enzymes participating in steroidogenesis [cytochrome P450 family 11 subfamily A member 1 (CyP11A1), cytochrome P450 family 17 subfamily A member 1, aldo-keto reductase family member C2 and 3β-hydroxysteroid dehydrogenase/isomerase 2 (3β HSD2)] were determined by western blotting and T concentration was determined by ELISA using PC3 cells. The present results revealed higher expression levels of endothelin A receptor (ETAR) in tissues obtained from samples of patients with PCa with a low Gleason Score. No changes were identified for endothelin B receptor (ETBR). PC3 cells expressed higher levels of ET-1 and ETAR, while LNCaP cells exhibited higher expression levels of ETBR. Blocking of ETAR and endothelin B receptor decreased the expression levels of CyP11A1 and 3β HSD2 enzymes and AR in PC3 cells, as well as T secretion. These findings suggested that ET-1 has a potential role in modulating the intratumoral steroidogenesis pathway and might have relevance as a possible therapeutic target.

Abstract 776: Posttransriptional control of steroid biosynthesis pathway in prostate cancer

Abstract Prostate cancer (PCa) depends on androgens for growth and maintenance. Despite curative therapeutics of the androgen-sensitive PCa, one-third of patients relapse to a more aggressive form of the disease known as castration-resistant PCa (CRPC). Androgen signaling mediated via Androgen receptor (AR) continues to promote CRPC and therapeutic resistance. The synthesis of testosterone (T) and dihydrotestosterone (DHT) via Intratumoral steroidogenesis appears to play a crucial role in CRPC. It appears that PCa cells synthesize de-novo androgen from cholesterol from acetyl-coA via Terpenoid backbone and Steroid biosynthesis pathways. We found that several enzymes of the intracellular cholesterol synthesis pathways including HMGS1, HMGCR, SREBP1, FASN, SCARB1 are potential targets of hsa-miR-149 5p. Currently, we are characterizing miR-149-5p mediated posttranscriptional regulation of these genes and determining its potential therapeutic application in CRPC. Citation Format: Asmita Bhattarai, Girish C. Shukla. Posttransriptional control of steroid biosynthesis pathway in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 776.

MRNA expression of CYP17A1, CYP11A1, CYP19A1 , HSD3B1 and AKR1C2 in metastatic and non-metastatic prostate cancer patients

The progression of prostate cancer (PCa) mainly occurs caused by androgens. There is a link between intratumoral steroidogenesis and castration-resistant prostate cancer. This study aimed to determine the mRNA expression of various steroidogenic enzymes (CYP17A1, CYP11A1, CYP19A1, HSD3B1, and AKR1C2) in metastatic and non-metastatic prostate cancer patients. This study was conducted at the Anatomical Pathology Laboratory and Urologi Division, Department of Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito General Hospital, Yogyakarta from September- November 2017. Samples were taken from 30 paraffin blocks with adenocarcinoma of prostate, stained with hematoxylin-eosin (HE) and then classified into metastatic and nonmetastatic groups. Samples then underwent deparaffinization procedure and examination of mRNA expression of CYP17A1, CYP11A1, CYP19A1, HSD3B1, AKR1C2 genes using Real-Time PCR. The mean mRNA expressions of CYP11A1, CYP17A1, CYP19A1, HSD3B1, and AKR1C2 genes in the metastatic adenocarcinoma prostate group were 7.08, 10.11, 3.94, 4.84 and 3.58, respectively. In the non-metastatic group, the mean mRNA expressions of CYP11A1, CYP17A1, CYP19A1, HSD3B1, and AKR1C2 genes were 4.62, 9.45, 3.46, 2.68 and 4.92, respectively. The mean of mRNA expression of CYP11A1, CYP17A1, CYP19A1, and HSD3B1 genes were higher in the metastatic group than nonmetastatic adenocarcinoma prostate group. However, it was not statistically significant (p>0.05). The highest mRNA expression of steroidogenic enzymes was the CYP17A1 gene. In conclusion, the mRNA expressions of CYP17A1, CYP11A1, CYP19A1, HSD3B1 were higher in the metastatic prostate cancer patients compared to that in non-metastatic prostate cancer patients but statistically not significant.

ACSL3 promotes intratumoral steroidogenesis in prostate cancer cells.

Long‐chain acyl‐coenzyme A (CoA) synthetase 3 (ACSL3) is an androgen‐responsive gene involved in the generation of fatty acyl‐CoA esters. ACSL3 is expressed in both androgen‐sensitive and castration‐resistant prostate cancer (CRPC). However, its role in prostate cancer remains elusive. We overexpressed ACSL3 in androgen‐dependent LNCaP cells and examined the downstream effectors of ACSL3. Furthermore, we examined the role of ACSL3 in the androgen metabolism of prostate cancer. ACSL3 overexpression led to upregulation of several genes such as aldo‐keto reductase 1C3 (AKR1C3) involved in steroidogenesis, which utilizes adrenal androgen dehydroepiandrosterone sulfate (DHEAS) as substrate, and downregulated androgen‐inactivating enzyme UDP‐glucuronosyltransferase 2 (UGT2B). Exposure to DHEAS significantly increased testosterone levels and cell proliferative response in ACSL3‐overexpressing cells when compared to that in control cells. A public database showed that ACSL3 level was higher in CRPC than in hormone‐sensitive prostate cancer. CRPC cells showed an increased expression of ACSL3 and an expression pattern of AKR1C3 and UGT2B similar to ACSL3‐overexpressing cells. DHEAS stimulation significantly promoted the proliferation of CRPC cells when compared to that of LNCaP cells. These findings suggest that ACSL3 contributes to the growth of CRPC through intratumoral steroidogenesis (i.e. promoting androgen synthesis from DHEAS and preventing the catabolism of active androgens).

Intracrine androgen biosynthesis in renal cell carcinoma

Background:Renal cell carcinoma (RCC) is one of the most lethal genitourinary cancers. The presence of androgen receptor (AR) in RCC has recently been shown to be associated with higher tumour stage irrespective of gender. Because the clinical context of androgens in female RCC patients is similar to that of prostate cancer patients undergoing androgen-deprivation therapy, mechanisms underlying the emergence of castration-resistant prostate cancer (CRPC) may be at play in AR-positive RCC cells. Therefore, we hypothesized that AR-positive RCC has intratumoral steroidogenesis and that anti-androgen therapy may result in tumour suppression.Methods:Mice were injected with an AR-positive RCC cell line. When tumours became palpable, surgical castration was performed and tumour volume was measured. Using ELISA, the levels of intracellular testosterone and dihydrotesterone were measured in AR-positive human RCC cell lines. Lastly, male mice containing xenografts were treated with enzalutamide or abiraterone acetate (AA) for 3 weeks to measure tumour volume.Results:We first observed in vivo that castration retards the growth of AR-positive RCC tumour xenograft in mice. Next, AR-positive human RCC cell lines and tissues were found to have elevated levels of testosterone and dihydrotestosterone and express key enzymes required for intracellular androgen biosynthesis. A mouse xenograft study with AR-positive RCC cell line using the commonly used anti-androgen therapies showed significant tumour suppression (P<0.01).Conclusions:Intracrine androgen biosynthesis is a potential source of androgen in AR-positive RCC and that the androgen signaling axis is a potential target of intervention in RCC.

Paracrine sonic hedgehog signaling contributes significantly to acquired steroidogenesis in the prostate tumor microenvironment.

Despite the substantial benefit of androgen deprivation therapy (ADT) for metastatic prostate cancer, patients often progress to castration-resistant disease (CRPC) that is more difficult to treat. CRPC is associated with renewed androgen receptor activity in tumor cells and restoration of tumor androgen levels through acquired intratumoral steroidogenesis (AIS). Although prostate cancer (PCa) cells have been shown to have steroidogenic capability in vitro, we previously found that benign prostate stromal cells (PrSCs) can also synthesize testosterone (T) from an adrenal precursor, DHEA, when stimulated with a hedgehog (Hh) pathway agonist, SAG. Here, we show exposure of PrSCs to a different Smoothened (Smo) agonist, Ag1.5, or to conditioned medium from sonic hedgehog overexpressing LNCaP cells induces steroidogenic enzyme expression in PrSCs and significantly increases production of T and its precursor steroids in a Smo-dependent manner from 22-OH-cholesterol substrate. Hh agonist-/ligand-treated PrSCs produced androgens at a rate similar to or greater than that of PCa cell lines. Likewise, primary bone marrow stromal cells became more steroidogenic and produced T under the influence of Smo agonist. Treatment of mice bearing LNCaP xenografts with a Smo antagonist, TAK-441, delayed the onset of CRPC after castration and substantially reduced androgen levels in residual tumors. These outcomes support the idea that stromal cells in ADT-treated primary or metastatic prostate tumors can contribute to AIS as a consequence of a paracrine Hh signaling microenvironment. As such, Smo antagonists may be useful for targeting prostate tumor stromal cell-derived AIS and delaying the onset of CRPC after ADT.

Osteoblasts promote castration-resistant prostate cancer by altering intratumoral steroidogenesis

The skeleton is the preferred site for prostate cancer (PC) metastasis leading to incurable castration-resistant disease. The increased expression of genes encoding steroidogenic enzymes found in bone metastatic tissue from patients suggests that up-regulated steroidogenesis might contribute to tumor growth at the metastatic site. Because of the overall sclerotic phenotype, we hypothesize that osteoblasts regulate the intratumoral steroidogenesis of castration resistant prostate cancer (CRPC) in bone. We here show that osteoblasts alter the steroidogenic transcription program in CRPC cells, closely mimicking the gene expression pattern described in CRPC. Osteoblast-stimulated LNCaP-19 cells displayed an increased expression of genes encoding for steroidogenic enzymes (CYP11A1, HSD3B1, and AKR1C3), estrogen signaling-related genes (CYP19A1, and ESR2), and genes for DHT-inactivating enzymes (UGT2B7, UGT2B15, and UGT2B17). The observed osteoblast-induced effect was exclusive to osteogenic CRPC cells (LNCaP-19) in contrast to osteolytic PC-3 and androgen-dependent LNCaP cells. The altered steroid enzymatic pattern was specific for the intratibial tumors and verified by immunohistochemistry in tissue specimens from LNCaP-19 xenograft tumors. Additionally, the overall steroidogenic effect was reflected by corresponding levels of progesterone and testosterone in serum from castrated mice with intratibial xenografts. A bi-directional interplay was demonstrated since both proliferation and Esr2 expression of osteoblasts were induced by CRPC cells in steroid-depleted conditions. Together, our results demonstrate that osteoblasts are important mediators of the intratumoral steroidogenesis of CRPC and for castration-resistant growth in bone. Targeting osteoblasts may therefore be important in the development of new therapeutic approaches.

Oral simvastatin administration delays castration-resistant progression and reduces intratumoral steroidogenesis of LNCaP prostate cancer xenografts.

Growing evidence supports the idea that de novo steroidogenesis has an important role in prostate cancer's progression to the castration-resistant state following androgen deprivation therapy. Therefore, reducing the availability of cholesterol for use as a precursor in androgen synthesis may reduce proliferation and disease progression. LNCaP xenograft-bearing mice were castrated and administered simvastatin via diet, and tumor volume and PSA concentration were monitored for 8 weeks post castration. Levels of serum and intratumoral androgens along with serum simvastatin and common toxicity markers were measured at end point. Reduced post-castration tumor growth rate in simvastatin-treated mice correlated with delayed time to castration-resistant progression, determined by two serum PSA doublings from post-castration nadir, when compared with xenografts in mice on control diet. At 8 weeks post castration, serum simvastatin levels were comparable to clinically relevant human doses with no evidence of overt muscle or liver toxicity. This suppressed post-castration tumor growth in the simvastatin diet group was correlated with reduced intratumoral testosterone and dihydrotestosterone levels. Reduced tumor growth and intratumoral androgen levels observed in simvastatin-treated, castrated mice harboring LNCaP xenograft suggests that suppressing de novo steroidogenesis can delay castration-resistant progression of this tumor model.

Intratumoral steroidogenesis in castration-resistant prostate cancer: a target for therapy.

Development of castration-resistant prostate cancer (CRPC) in a low androgen environment, arising from androgen deprivation therapy (ADT), is a major problem in patients with advanced prostate cancer (PCa). Several mechanisms have been hypothesized to explain the progression of PCa to CRPC during ADT, one of them is so called persistent intratumoral steroidogenesis. The existence of intratumoral steroidogenesis was hinted based on the residual levels of intraprostatic testosterone (T) and dihydrotestosterone (DHT) after ADT. Accumulating evidence has shown that the intraprostatic androgen levels after ADT are sufficient to induce cancer progression. Several studies now have demonstrated that PCa cells are able to produce T and DHT from different androgen precursors, such as cholesterol and the adrenal androgen, dehydroepiandrosterone (DHEA). Furthermore, up-regulation of genes encoding key steroidogenic enzymes in PCa cells seems to be an indicator for active intratumoral steroidogenesis in CRPC cells. Currently, several drugs are being developed targeting those steroidogenic enzymes, some of which are now in clinical trials or are being used as standard care for CRPC patients. In the future, novel agents that target steroidogenesis may add to the arsenal of drugs for CRPC therapy.

AKR1C3 Inhibition Therapy in Castration-Resistant Prostate Cancer and Breast Cancer: Lessons from Responses to SN33638.

In the current issue of Frontiers in Oncology – Cancer Endocrinology Section, Yin et al. report that the aldo-keto reductase family 1 member C3 (AKR1C3) inhibitor SN33638 was able to reduce testosterone production, PSA expression, and cell proliferation only in some castration-resistant prostate cancer (CRPC) cells, overexpressing the enzyme, but not the majority of a panel of prostate and breast cell lines investigated, and revealing a novel caveat in developing AKR1C3 inhibitors for cancer therapy. Prostate and breast cancers are the most common cancer and the second-leading cause of cancer-related deaths in males and females, respectively (1, 2). The mainstay treatment for advanced or metastatic prostate or ER-positive breast carcinoma is androgen-deprivation therapy or anti-estrogen therapy. Although initially these treatments are beneficial and reduce tumor burden, tumors ultimately become resistant to these therapies. There are few treatment options for this stage of cancers (termed CRPC and tamoxifen-resistant breast cancer). Thus, new approaches to treat or prevent these hormone-insensitive cancers or their progression are in great need. The AKR1C3, also known as 17β-hydroxysteroid dehydrogenase type 5, is capable to produce intratumorally testosterone and 17β-estradiol by reducing the androgen precursors and estrone, respectively. The local conversion of less potent hormones to more potent ones will lead to nuclear receptor activation and tumor progression. Therefore, AKR1C3 has recently been regarded as a potential anti-cancer drug target in both CRPC and ER-positive breast cancer (3, 4). The AKR1C3 inhibitor SN33638 is a lead compound newly synthesized by Dr. Jamieson’s group (5). It had low nanomolar potency against AKR1C3 and >300-fold selectivity for AKR1C3 over the other AKR1C isoforms. Equipped by this powerful compound, Yin et al. (6) treated a panel of CRPC and ER-positive breast cancer cell lines, in the presence of hormone or prostaglandin precursors, prior to evaluation of cell proliferation and levels of 11β-prostaglandin F2α (11β-PGF2α), testosterone production, PSA expression, and cell proliferation (6). They observed that AKR1C3 mRNA was upregulated in CRPC in a meta-analysis of patient samples. 11β-PGF2α and testosterone levels in the cell line panel correlated with AKR1C3 protein expression. SN33638 prevented 11β-PGF2α formation in cell lines that expressed AKR1C3, but only partially inhibited testosterone formation and subsequent cell proliferation and/or PSA expression only in high (LAPC4 AKR1C3) or moderate (22RV1) AKR1C3-expressing cell lines. This may be explained by that there is AKR1C3-independent steroid hormone production in most cells, and the data suggest that AKR1C3 is not an effective drug target in most CRPC cancer patients. For ER-positive breast cancers, their finding on AKR1C3 inhibition is even less promising. They observed AKR1C3 mRNA was downregulated in patient samples, a poor correlation between AKR1C3 protein expression and 17β-estradiol production, while SN33638 was largely ineffective at preventing estrone reduction or cell proliferation. These results call into question of the possibility of using AKR1C3 inhibitors in treating breast cancer patients. In summary, Yin et al. (6) revealed a novel finding that AKR1C3 inhibitor therapy is unlikely to be beneficial for the treatment of most CRPC and ER-positive breast cancers. Nevertheless, given the role of AKR1C3 in intratumoral steroidogenesis, we still hold enthusiastically the hope that a subpopulation of CRPC patients with high AKR1C3 might benefit from AKR1C3 inhibitor therapy, whose tumors are “addicted” from AKR1C3 to produce testosterone. The study by Yin et al. (6) provides a valuable framework for future preclinical or clinical studies aiming at verifying this hypothesis that AKR1C3 inhibition suppresses tumor formation in a selected population of AKR1C3-high CRPC patients.

Characterization of Prostate Cancer Bone Metastases According to Expression Levels of Steroidogenic Enzymes and Androgen Receptor Splice Variants

BackgroundIntra-tumoral steroidogenesis and constitutive androgen receptor (AR) activity have been associated with castration-resistant prostate cancer (CRPC). This study aimed to examine if CRPC bone metastases expressed higher levels of steroid-converting enzymes than untreated bone metastases. Steroidogenic enzyme levels were also analyzed in relation to expression of constitutively active AR variants (AR-Vs) and to clinical and pathological variables.Methodology/Principal FindingsUntreated, hormone-naïve (HN, n = 9) and CRPC bone metastases samples (n = 45) were obtained from 54 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomy specimens. Transcript and protein levels were analyzed by real-time RT-PCR, immunohistochemistry and immunoblotting. No differences in steroidogenic enzyme levels were detected between CRPC and HN bone metastases. Significantly higher levels of SRD5A1, AKR1C2, AKR1C3, and HSD17B10 mRNA were however found in bone metastases than in non-malignant and/or malignant prostate tissue, while the CYP11A1, CYP17A1, HSD3B2, SRD5A2, and HSD17B6 mRNA levels in metastases were significantly lower. A sub-group of metastases expressed very high levels of AKR1C3, which was not due to gene amplification as examined by copy number variation assay. No association was found between AKR1C3 expression and nuclear AR staining, tumor cell proliferation or patient outcome after metastases surgery. With only one exception, high AR-V protein levels were found in bone metastases with low AKR1C3 levels, while metastases with high AKR1C3 levels primarily contained low AR-V levels, indicating distinct mechanisms behind castration-resistance in individual bone metastases.Conclusions/SignificanceInduced capacity of converting adrenal-gland derived steroids into more potent androgens was indicated in a sub-group of PC bone metastases. This was not associated with CRPC but merely with the advanced stage of metastasis. Sub-groups of bone metastases could be identified according to their expression levels of AKR1C3 and AR-Vs, which might be of relevance for patient response to 2nd line androgen-deprivation therapy.

Abstract 1632: Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease.

Abstract Androgen deprivation therapy (ADT) is frontline treatment for metastatic prostate cancer (PCa). ADT initially blocks disease progression but eventually allows the outgrowth of castration resistant disease (CRPC) that is refractory to the diminished systemic androgen levels in ADT-treated patients. CRPC is associated with acquired intratumoral steroidogenesis (IS), a process that increases the androgen levels in the local microenvironment of prostate tumors and enables further tumor growth and progression. IS has mainly been attributed to a steroidogenic process linked to CRPC cells and here we present evidence that benign tumor-associated prostate stromal cells (PrSCs) can also contribute to IS through a paracrine Hedgehog (HH) tumor microenvironment associated with increased production and release ofHedgehog ligands (Hhs) from prostate cancer cells in response to ADT (Chen, et al., Mol Cancer, 9: 89, 2010). Primary human PrSCs upregulate expression of a large repertoire of steroidogenic genes upon treatment with HH chemical agonists, SAG (100 nM) or Hg 1.5 (1 nM). These effects are Gli2-dependent (Levina, et al., Prostate, 72: 817, 2012) and they can be inhibited by the HH antagonist, KAAD-cyclopamine. Previously we showed that this upregulation significantly increased output of testosterone (T) in PrSCs fed DHEA precursor. More comprehensive analysis (via LC/MS/MS) of androgen outputs from PrSCs fed 22 OH-cholesterol showed that Hg1.5 significantly increased the outputs of Progesterone, DHEA, androsterone, T and dihydrotestosterone (DHT) showing that HH signaling activity effects outputs throughout the androgen biosynthetic pathway. Upregulation of the RDH5 gene by Hg1.5 suggests that the effects on production of DHT from PrSCs might be generated by increased synthesis through both classical and back-door pathways. On a weight-to-weight basis, Hg1.5-stimulated PrSCs produce higher T and DHT outputs than LNCaP cells or an androgen growth-independent variant, LNCaP-AI. Finally, we found that treatment of Sonic Hh-producing LNCaP cells with simvastatin reduced the ability of conditioned medium from these cells to induce steroidogenic gene expression from PrSCs. In summary, this work provides evidence for a stromal cell contribution to IS in CRPC mediated by a paracrine-active HH signaling tumor microenvironment induced by ADT-treatment and identifies potential treatment strategies to interfere with this process. Citation Format: Amy A. Lubik, Hans Adomat, Emma S. Guns, Ralph Buttyan. Paracrine Hedgehog as a mediator of stromal cell steroidogenesis that contributes to progression of prostate cancer to castration resistant disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1632. doi:10.1158/1538-7445.AM2013-1632

Abstract 3698: Inhibition of cholesterol uptake with ezetimibe reduces intra-tumoral testosterone levels and slows tumor growth in transgenic mouse models of prostate cancer.

Abstract Introduction: Western countries have 6 fold higher incidence of prostate cancer (PC). Dietary composition, in addition to other factors, may contribute to this geographic disparity. Cholesterol, the precursor for de novo steroid hormone synthesis, has been hypothesized to promote PC growth via multiple pathways including intratumoral steroidogenesis. While lowering serum cholesterol slows xenograft PC growth, this has not been tested in transgenic models, which allow testing of PC prevention as well as modulating PC growth in an orthotopic position. We determined the effect of cholesterol uptake inhibition, using ezetimibe, on PC growth in Hi-Myc and PTEN transgenic mice. Methods: We randomized 50 c-myc/wt (Hi-Myc) mice and 30 PTENloxP/loxP-Cre+ (PTEN) to one of two diets: high fat high cholesterol (HFHC; 40% fat, 17% protein, 43% carbohydrate, 1.25% cholesterol), or HFHC with ezetimibe (30 mg/kg food) (HFHC+Z). Mice were fed ad libitum starting right after weaning and body weights measured twice weekly. Prostate, liver, spleen, adipose tissue and serum were harvested from Hi-Myc and PTEN mice at age 6 and 4 months, respectively. Serum cholesterol and testosterone (T) and tumor T were measured in Hi-Myc mice and are ongoing in PTEN mice. Results: In Hi-Myc mice, body weights were significantly greater in the HFHC+Z group at sacrifice, relative to the HFHC group (p=0.04), but there were no differences in prostate weight between groups with adjustment for body weight (p=0.84). In PTEN mice, there was no difference in body weights between groups, but prostate weight was significantly lower in the HFHC+Z group, relative to the HFHC group (p=0.046). In Hi-Myc mice, while serum T levels did not vary between groups (p=0.93), tumor T, dihydrotestosterone (DHT) and androstenedione concentrations were significantly reduced in the HFHC+Z group, relative to the HFHC group (p=0.01, p=0.01, p=0.006, respectively). Discussion: Reduction of serum cholesterol levels using cholesterol uptake inhibitor, ezetimibe, significantly reduced prostate weight in PTEN mice consuming a HFHC diet. Furthermore, supplementation of the HFHC diet with ezetimibe significantly reduced tumor concentrations of T, DHT and androstenedione in Hi-Myc mice, although there was no effect on prostate weight. These findings highlight a potentially critical role for cholesterol in PC progression by providing evidence that reduction of hypercholesterolemia may reduce intratumoral de novo steroidogenesis and slow PC growth. This study may provide new insight into the use of cholesterol-lowering drugs for PC prevention and treatment. Citation Format: Emma H. Allott, Elizabeth M. Masko, Alexa Choy, Alexis R. Gaines, Keith R. Solomon, Salvatore V. Pizzo, Stephen J. Freedland. Inhibition of cholesterol uptake with ezetimibe reduces intra-tumoral testosterone levels and slows tumor growth in transgenic mouse models of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3698. doi:10.1158/1538-7445.AM2013-3698

992 A PARACRINE HEDGEHOG SIGNALING MICROENVIRONMENT IN ADT-TREATED PROSTATE TUMORS INDUCES STEROIDOGENESIS FROM BENIGN PROSTATE STROMAL CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research (V)1 Apr 2013992 A PARACRINE HEDGEHOG SIGNALING MICROENVIRONMENT IN ADT-TREATED PROSTATE TUMORS INDUCES STEROIDOGENESIS FROM BENIGN PROSTATE STROMAL CELLS Amy Lubik, Emma Guns, Hans Adomat, and Ralph Buttyan Amy LubikAmy Lubik Vancouver, Canada More articles by this author , Emma GunsEmma Guns Vancouver, Canada More articles by this author , Hans AdomatHans Adomat Vancouver, Canada More articles by this author , and Ralph ButtyanRalph Buttyan Vancouver, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.574AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen deprivation therapy (ADT) induces the expression and release of Hedgehog ligands (Hhs) from prostate cancer (PCa) cells. Previously we showed that treatment of primary human prostate stromal cells (PrSCs) with an HH agonist (SAG) increased the expression of steroidogenic genes and enabled them to significantly increase production of testosterone (T) when fed with DHEA, an adrenal precursor. We proposed that a paracrine HH microenvironment in ADT-treated PCas contribute to intratumoral steroidogenesis through this process. Here we show that steroidogenic activity of PrSCs is induced by alternate HH agonists and better describe the steroidogenic process leading to androgen production in PrSCs induced by paracrine HH. METHODS Human primary PrSCs (Clonetics) were grown in recommended medium. PrSCs were pre-starved of serum to induce primary cilia formation, then treated with 1 nM Ag1.5 (a HH agonist) for 48 hrs. Expression of steroidogenic genes was measured by quantitative PCR. Outputs of steroids from vehicle- or agonist-treated PrSC cells fed 22 OH-cholesterol were measured by LC/MS/MS. The HH antagonist, KAAD-cyclopamine (100 nM) or an HMGR inhibitor (simvastatin) was used to block the PrSC response to Ag1.5 or to inhibit active Hh release from Sonic Hh producing LNCaP cells. RESULTS Ag1.5 is as effective as 100 nM SAG in inducing expression of steroidogenic genes in PrSCs. Outputs of progesterone, DHEA, androsterone, T, or dihydrotestosterone (DHT) from 22 OH-cholesterol was significantly increased by Ag1.5. The effects of Ag1.5 on the expression of these genes was blocked by KAAD-cyclopamine. The ability of Ag1.5 to induce RDH5 expression and androsterone production show that the DHT output from PrSCs was a consequence of increased classical and backdoor pathway activities. Steroid outputs from HH-induced PrSCs was significantly higher (on a weight-per-weight basis) than production from parental LNCaP or the androgen growth-independent LNCaP-AI cells. Simvastatin reduced the release of active Hhs from LNCaP-Sonic Hh producing cells as measured by the reduced ability of conditioned medium from treated cells to induce steroidogenic genes in PrSCs. CONCLUSIONS Our results further confirm the potential of a paracrine HH signaling microenvironment of ADT-treated prostate tumor to induce steroidogenesis and androgen production from benign PrSCs. Outcomes show that a HH antagonist or cholesterol synthesis inhibitor might block the effects of paracrine HH in an ADT-treated tumor and delay progression to CRPC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e407 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Amy Lubik Vancouver, Canada More articles by this author Emma Guns Vancouver, Canada More articles by this author Hans Adomat Vancouver, Canada More articles by this author Ralph Buttyan Vancouver, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Aldo-keto Reductase Family 1 Member C3 (AKR1C3) Is a Biomarker and Therapeutic Target for Castration-Resistant Prostate Cancer

Current endocrine treatment for advanced prostate cancer does not result in a complete ablation of adrenal androgens. Adrenal androgens can be metabolized by prostate cancer cells, which is one of the mechanisms associated with progression to castration-resistant prostate cancer (CRPC). Aldo-keto reductase family 1 member C3 (AKR1C3) is a steroidogenic enzyme that plays a crucial role in the conversion of adrenal androgen dehydroepiandrosterone (DHEA) into high-affinity ligands for the androgen receptor (testosterone [T] and dihydrotestosterone [DHT]). The aim of this study was to examine whether AKR1C3 could be used as a marker and therapeutic target for CRPC. AKR1C3 mRNA and protein levels were upregulated in CRPC tissue, compared with benign prostate and primary prostate cancer tissue. High AKR1C3 levels were found only in a subset of CRPC patients. AKR1C3 can be used as a biomarker for active intratumoral steroidogenesis and can be measured in biopsy or transurethral resection of the prostate specimens. DuCaP (a CRPC cell line that has high AKR1C3 expression levels) used and converted DHEA under hormone-depleted conditions into T and DHT. The DHEA-induced growth of DuCaP could be antagonized by indomethacine, an inhibitor of AKR1C3. This study indicates that AKR1C3 can be considered a therapeutic target in a subgroup of patients with high AKR1C3 expression.

Activin A Stimulates AKR1C3 Expression and Growth in Human Prostate Cancer

Local androgen synthesis in prostate cancer (PC) may contribute to the development of castration-resistant PC (CRPC), but pathways controlling intratumoral steroidogenic enzyme expression in PC are unknown. We investigated the effects of activin, a factor involved in the regulation of PC growth and steroidogenic enzyme expression in other steroidogenic tissues, on intratumoral steroidogenesis in PC. Activin A effects and regulation of the activin-signaling pathway molecules were studied in the PC cell lines LNCaP, VCaP, and PC-3 and in 13 individual PC xenograft models. Also, expression levels of inhibin βA- and βB-subunits (INHBA and INHBB) and of the activin antagonist follistatin were quantitated in patient PC tissues. Activin A induced the expression and enzyme activity of 17β-hydroxysteroid dehydrogenase enzyme AKR1C3 in LNCaP and VCaP cells. Inhibition of endogenous activin A action in the PC-3 cell line decreased AKR1C3 levels and consequently testosterone synthesis. In return, androgens suppressed INHBA expression in both VCaP cells and the PC xenograft models. The antiproliferative effects of activin A were opposed by physiological concentrations of androstenedione in LNCaP cells. In patient PC tissues, expression levels of INHBA were increased in CRPC samples and correlated with AKR1C3 levels. Moreover, a high ratio of activin subunits to follistatin was associated with a worse metastasis-free survival in patients. In conclusion, activin A is controlled by androgens in PC models and regulates local androgen production. Activin A thus seems to mediate (residual) intratumoral androgen levels and could form a novel therapeutic target in CRPC.