Maintenance of intratumoral androgens in metastatic prostate cancer: a mechanism for castration-resistant tumor growth.
Journal: 2008/August - Cancer Research
ISSN: 1538-7445
Abstract:
Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor (AR). Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative reverse transcription-PCR, we evaluated androgen levels and transcripts encoding steroidogenic enzymes in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer, and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men [0.74 ng/g; 95% confidence interval (95% CI), 0.59-0.89] were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/g; 95% CI, 0.03-0.44; P < 0.0001). Compared with primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including up-regulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1, and UGT2B17 and down-regulated expression of SRD5A2 (P < 0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared with eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment.
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Cancer Res 68(11): 4447-4454

Maintenance of Intratumoral Androgens in Metastatic Prostate Cancer: A Mechanism for Castration-Resistant Tumor Growth

Introduction

Androgens and the androgen receptor (AR) signaling pathway are intimately associated with prostate carcinogenesis, and testosterone suppression remains the most effective therapy for metastatic prostate cancer (1). However, despite initial clinical responses to castrate or ‘anorchid’ serum androgen levels, progression to castration-resistant disease is nearly universal. Of importance, recurrent tumors frequently re-express AR-target genes such as prostate specific antigen (PSA), and nearly 30% of patients with progressive disease respond to additional hormonal manipulations (2). These findings suggest that many recurrent prostate cancers are neither ‘hormone-refractory’ nor ‘androgen-independent’, but maintain a clinically-relevant reliance on the AR signaling axis. The observation that androgen-regulated genes are frequently re-expressed in castration-resistant prostate tumors has prompted a search for processes contributing to AR activation in the anorchid environment. Proposed mechanisms include amplification and over-expression of the AR, AR gene mutations leading to promiscuous ligand interaction, enhanced AR signal transduction through alterations in coactivators/corepressors, and activation of the AR or downstream regulatory molecules by “cross talk” with other signaling pathways (3-5). Importantly, many mechanisms proposed to confer a castration-resistant phenotype either still require or are enhanced by the presence of AR ligands.

The most parsimonious explanation for persistent AR signaling in the setting of anorchid serum testosterone concentrations is the continued presence of intracellular androgens at levels adequate to activate wild-type AR. Studies of primary prostate tissues following medical or surgical castration have measured intraprostatic testosterone or the active metabolite dihydrotestosterone (DHT) in quantities sufficient to stimulate AR-mediated gene expression and protein synthesis (6-8). While the source of residual tissue androgens in the setting of anorchid serum testosterone levels has not been established, intracrine androgen production (the local synthesis or conversion of androgens within a tissue), may play a critical role in maintaining tumoral androgen levels (9). In this regard, the increased expression of genes mediating the conversion of adrenal androgens to testosterone has been reported in bone marrow metastases from men with castration-resistant prostate cancer (10).

In this study we sought to determine if physiologically-relevant androgen levels are present in metastatic soft-tissue tumor deposits from patients with castration-resistant prostate cancer. To ascertain the potential for intracrine androgen synthesis, we comprehensively evaluated the expression of genes encoding each enzyme in the steroidogenic pathway leading from cholesterol to testosterone, DHT and their metabolites. We demonstrate that metastatic human prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and sustain intratumoral androgens at concentrations capable of activating AR-target genes and maintaining tumor cell survival.

Materials and Methods

Tissue Acquisition and Immunohistochemical Staining

All procedures involving human subjects were approved by the Institutional Review Board of the University of Washington Medical Center, and all subjects signed written informed consent. Under a University of Washington institutional protocol for the use of excess tissue following surgery, 8 matched samples of benign and tumor prostate tissue were obtained from eugonadal patients undergoing radical prostatectomy for localized prostate cancer. In addition, benign prostate tissue was obtained from two patients undergoing cystoprostatectomy unrelated to prostate cancer. Patients with metastatic prostate cancer underwent rapid autopsies under the aegis of the University of Washington Prostate Cancer Donor Autopsy Program as previously described (11). Autopsies were performed within 4-10 hours of death. Samples of all gross metastatic tumor sites were obtained under sterile conditions and the site and volume of osseous and non-osseous metastases were recorded. Fresh tissue was snap frozen in liquid nitrogen immediately after harvesting and maintained at −80°C. Tissues utilized in this study were distant or local soft tissue metastases acquired from 8 surgically or medically castrated patients (the latter with documented anorchid serum testosterone levels (<50 ng/dL)), and included 2-4 metastatic lymph node, liver, bladder or lung tumor deposits per patient. For three patients, non-osseous control tissues not involved with prostate cancer (including skin, liver and muscle) were simultaneously harvested during the autopsy procedure and were used for analyses of androgen levels. A Hematoxylin and Eosin stained section of each primary and metastatic tumor sample was examined microscopically for the presence of adenocarcinoma, and areas consisting of >85% tumor tissue were grossly dissected for further use. Benign samples were examined to ensure absence of tumor tissue. Samples were subdivided into pieces ranging from 20-50 mg of which three from each tumor sample were used for determination of androgen levels and one was used for RNA isolation. Androgen levels were determined in 4 sets of the matched tumor and benign prostate specimens, two benign prostate specimens, and in all control and metastatic autopsy tissues. Immunohistochemical staining for AR and PSA was carried out as we have previously described (12), using a polyclonal anti-PSA antibody (DAKO, Inc) and a monoclonal anti-AR antibody (clone F36.4.1, Biogenex). Negative control immunostains, substituting pre-immune immunoglobulin of the same species as that in which the antibody was generated, showed no reaction product.

LuCaP Human Prostate Cancer Xenografts

The establishment and maintenance of the LuCaP 23 and 35 xenografts from the lymph node metastases of two individuals with castration-resistant prostate cancer was performed as previously described (13, 14). The LuCaP 96 xenograft was generated from a specimen obtained by transurethral resection of the prostate from a patient one month prior to documentation of castration-resistant prostate cancer. All lines were established as a component of the University of Washington Rapid Autopsy program, and all lines contain wild type AR, produce serum PSA and respond to castration with the subsequent development of castration-resistant or ‘androgen-independent’ growth. All experiments involving animals were performed in accordance with protocols approved by the University of Washington Institutional Animal Care Use Committee. Castration-sensitive and castration-resistant strains of each xenograft were grown, respectively, in 3-5 intact (non-castrate) and 3-5 castrate male C.B-17 SCID mice (Charles River Laboratories, Wilmington MA). Prior terminology describes these tumor phenotypes as androgen dependent (AD) and androgen independent (AI), but our data indicate that these terms are misnomers and thus we use the castration-sensitive (CS) and castration-resistant (CR) nomenclature. When tumors reached approximately 500mg in size, the animals were euthanized according to institutional protocol and the xenografts harvested and flash frozen for determination of tissue androgens and extraction of total RNA. Samples of normal kidney and liver were simultaneously obtained from each intact and castrate animal for determination of tissue androgen levels in non-tumor tissue. In addition, a set of LuCaP 35 xenografts were harvested from 5 mice in which castration-sensitive tumors had been subjected to castration-induced regression with subsequent ‘androgen independent’ or castration-resistant re-growth (CS→CR).

Steroid Measurements

Androgen levels were determined by mass spectrometry (MS) using methods we have recently described (15). In brief, frozen tissue samples were individually thawed, weighed, and homogenized in PBS. The homogenates were extracted with 8 ml of diethyl ether and the organic phase decanted after freezing the aqueous phase in a dry ice/ethanol bath. The organic phase was dried and concentrated with 2 × 0.5 ml ether washes under a stream of purified air. Each individual concentrated extract was dissolved in 1.0 ml redistilled ethanol and stored at −20°C until MS analysis. Samples were spiked with internal standards: 50 pg of deuterated (D3)-DHT and D3-testosterone, vortexed briefly, and evaporated to dryness. The residue was then reconstituted in 0.5 mL of water prior to extraction with methylene chloride. The organic phase was removed under nitrogen and the sample was dissolved in 0.1 M hydroxylamine hydrochloride in 50% MeOH/water, vortexed, and heated at 60° for 1 hour. Standards for DHT and testosterone were prepared in parallel. The resulting oximes were analyzed by LC-MS-MS using a Waters Aquity HPLC and Premier XE mass spectrometer (Milford, MA). Ions monitored were 350>309 and 347>306 for DHT-IS and DHT respectively, and 307>124 and 304>124 for testosterone-IS and testosterone respectively. This procedure resulted in a lower limit of quantitation of 100 and 500 attoMoles on column for testosterone and DHT respectively. Intra-assay coefficients of variation generated using human serum for high, mid and low-range samples were 3.5, 3.1 and 3.8% for testosterone and 6.3, 4.3 and 15.8% for DHT respectively.

RNA Isolation and Quantitative RT-PCR

Samples were individually homogenized in Trizol (Invitrogen, Carlsbad CA) and total RNA was isolated using the RNeasy Kit (Qiagen Inc, Valencia, CA), followed by treatment with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc). RNA was quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and RNA integrity evaluated using gel electrophoresis. cDNA was generated from each sample using 2-5 μg of total RNA in an oligo dT-primed reverse transcription reaction. Quantitative PCR (qRT-PCR) reactions were performed in triplicate using an Applied Biosystems 7700 sequence detector with approximately 5 ng of cDNA, 1 μM of each primer pair and SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). Primers specific for genes of interest were designed using the Web-based primer design service Primer3 www.cgihttp://jura.wi.mit.edu/rozen/papers/rozen-and-skaletsky-2000-primer3.pdf provided by the Whitehead Institute for Biomedical Research, except for AKR1C1, AKR1C2 and AKR1C3, (16) and 17BHSD10 (17) for which previously published primer sequences were used. Sequences are provided in Supplementary Table 1.

Statistical Analyses

To account for having multiple samples (i.e. 2-4 metastatic deposits) with replicate measurements from the same patient, statistical comparison of androgen levels in the human prostate and metastatic autopsy samples was performed using the following linear mixed effects model: [yij = β0 + bi + β1xij + εij] where yij is the androgen level (T, DHT), bi is a random intercept with distribution N(0, σb), xij indicates tissue type, and εij is an individual-specific error term with distribution N(0, σε) (here i indexes patients and j indexes patient-specific observations). Further, we assume that bi and εij are independent. This model accounts for within-individual correlations, which are assumed to be the same for each individual, and was used to derive P values for the comparison of mean tissue androgen levels among sample types. For each of the three xenograft lines, differences in androgen levels between the castration-sensitive and castration-resistant tumors were assessed by unpaired two sample t-tests. P values < 0.05 were considered significant.

For analysis of the qRT-PCR data, the mean cycle threshold (Ct) obtained for each gene was normalized to the expression of the housekeeping gene RPL13A in the same sample (the delta Ct). Reactions with Ct's>35 were considered undetectable for that transcript, and the specificity of amplification in each reaction was assessed based on the melting point of the dissociation curve. Unpaired two sample t-tests were used to compare the mean delta Ct's for each gene between the primary prostate cancers (n=8) and metastatic autopsy samples (n=16-22). Welch's modification of the t-test was utilized if the F test to compare sample variances was significant (but was only applicable to one gene, UGT2B15). P values < 0.05 were considered significant. The fold change was calculated by unlogging the difference in mean delta Ct's between the sample groups. Similarities among the human prostate and metastatic autopsy samples based on expression of steroidogenic gene transcripts were assessed by unsupervised, hierarchical, average linkage clustering using Cluster 3.0 software (http://bonsai.ims.u-tokyo. ac.jp/mdehoon/software/cluster/software.htm) and plotted using TreeView version 1.6 (http://rana.lbl.gov/EisenSoftware.htm). This program organizes genes and samples into a tree structure based on their similarity, in which items are joined by short branches if they are similar to each other and by increasingly longer branches as their similarity decreases. In average linkage clustering, the distance between 2 items x and y is the mean of all pairwise distances between items contained in x and y, and therefore provides a visual estimate of the similarity among different items in a sample.

Tissue Acquisition and Immunohistochemical Staining

All procedures involving human subjects were approved by the Institutional Review Board of the University of Washington Medical Center, and all subjects signed written informed consent. Under a University of Washington institutional protocol for the use of excess tissue following surgery, 8 matched samples of benign and tumor prostate tissue were obtained from eugonadal patients undergoing radical prostatectomy for localized prostate cancer. In addition, benign prostate tissue was obtained from two patients undergoing cystoprostatectomy unrelated to prostate cancer. Patients with metastatic prostate cancer underwent rapid autopsies under the aegis of the University of Washington Prostate Cancer Donor Autopsy Program as previously described (11). Autopsies were performed within 4-10 hours of death. Samples of all gross metastatic tumor sites were obtained under sterile conditions and the site and volume of osseous and non-osseous metastases were recorded. Fresh tissue was snap frozen in liquid nitrogen immediately after harvesting and maintained at −80°C. Tissues utilized in this study were distant or local soft tissue metastases acquired from 8 surgically or medically castrated patients (the latter with documented anorchid serum testosterone levels (<50 ng/dL)), and included 2-4 metastatic lymph node, liver, bladder or lung tumor deposits per patient. For three patients, non-osseous control tissues not involved with prostate cancer (including skin, liver and muscle) were simultaneously harvested during the autopsy procedure and were used for analyses of androgen levels. A Hematoxylin and Eosin stained section of each primary and metastatic tumor sample was examined microscopically for the presence of adenocarcinoma, and areas consisting of >85% tumor tissue were grossly dissected for further use. Benign samples were examined to ensure absence of tumor tissue. Samples were subdivided into pieces ranging from 20-50 mg of which three from each tumor sample were used for determination of androgen levels and one was used for RNA isolation. Androgen levels were determined in 4 sets of the matched tumor and benign prostate specimens, two benign prostate specimens, and in all control and metastatic autopsy tissues. Immunohistochemical staining for AR and PSA was carried out as we have previously described (12), using a polyclonal anti-PSA antibody (DAKO, Inc) and a monoclonal anti-AR antibody (clone F36.4.1, Biogenex). Negative control immunostains, substituting pre-immune immunoglobulin of the same species as that in which the antibody was generated, showed no reaction product.

LuCaP Human Prostate Cancer Xenografts

The establishment and maintenance of the LuCaP 23 and 35 xenografts from the lymph node metastases of two individuals with castration-resistant prostate cancer was performed as previously described (13, 14). The LuCaP 96 xenograft was generated from a specimen obtained by transurethral resection of the prostate from a patient one month prior to documentation of castration-resistant prostate cancer. All lines were established as a component of the University of Washington Rapid Autopsy program, and all lines contain wild type AR, produce serum PSA and respond to castration with the subsequent development of castration-resistant or ‘androgen-independent’ growth. All experiments involving animals were performed in accordance with protocols approved by the University of Washington Institutional Animal Care Use Committee. Castration-sensitive and castration-resistant strains of each xenograft were grown, respectively, in 3-5 intact (non-castrate) and 3-5 castrate male C.B-17 SCID mice (Charles River Laboratories, Wilmington MA). Prior terminology describes these tumor phenotypes as androgen dependent (AD) and androgen independent (AI), but our data indicate that these terms are misnomers and thus we use the castration-sensitive (CS) and castration-resistant (CR) nomenclature. When tumors reached approximately 500mg in size, the animals were euthanized according to institutional protocol and the xenografts harvested and flash frozen for determination of tissue androgens and extraction of total RNA. Samples of normal kidney and liver were simultaneously obtained from each intact and castrate animal for determination of tissue androgen levels in non-tumor tissue. In addition, a set of LuCaP 35 xenografts were harvested from 5 mice in which castration-sensitive tumors had been subjected to castration-induced regression with subsequent ‘androgen independent’ or castration-resistant re-growth (CS→CR).

Steroid Measurements

Androgen levels were determined by mass spectrometry (MS) using methods we have recently described (15). In brief, frozen tissue samples were individually thawed, weighed, and homogenized in PBS. The homogenates were extracted with 8 ml of diethyl ether and the organic phase decanted after freezing the aqueous phase in a dry ice/ethanol bath. The organic phase was dried and concentrated with 2 × 0.5 ml ether washes under a stream of purified air. Each individual concentrated extract was dissolved in 1.0 ml redistilled ethanol and stored at −20°C until MS analysis. Samples were spiked with internal standards: 50 pg of deuterated (D3)-DHT and D3-testosterone, vortexed briefly, and evaporated to dryness. The residue was then reconstituted in 0.5 mL of water prior to extraction with methylene chloride. The organic phase was removed under nitrogen and the sample was dissolved in 0.1 M hydroxylamine hydrochloride in 50% MeOH/water, vortexed, and heated at 60° for 1 hour. Standards for DHT and testosterone were prepared in parallel. The resulting oximes were analyzed by LC-MS-MS using a Waters Aquity HPLC and Premier XE mass spectrometer (Milford, MA). Ions monitored were 350>309 and 347>306 for DHT-IS and DHT respectively, and 307>124 and 304>124 for testosterone-IS and testosterone respectively. This procedure resulted in a lower limit of quantitation of 100 and 500 attoMoles on column for testosterone and DHT respectively. Intra-assay coefficients of variation generated using human serum for high, mid and low-range samples were 3.5, 3.1 and 3.8% for testosterone and 6.3, 4.3 and 15.8% for DHT respectively.

RNA Isolation and Quantitative RT-PCR

Samples were individually homogenized in Trizol (Invitrogen, Carlsbad CA) and total RNA was isolated using the RNeasy Kit (Qiagen Inc, Valencia, CA), followed by treatment with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc). RNA was quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and RNA integrity evaluated using gel electrophoresis. cDNA was generated from each sample using 2-5 μg of total RNA in an oligo dT-primed reverse transcription reaction. Quantitative PCR (qRT-PCR) reactions were performed in triplicate using an Applied Biosystems 7700 sequence detector with approximately 5 ng of cDNA, 1 μM of each primer pair and SYBR Green PCR master mix (Applied Biosystems, Foster City, CA). Primers specific for genes of interest were designed using the Web-based primer design service Primer3 www.cgihttp://jura.wi.mit.edu/rozen/papers/rozen-and-skaletsky-2000-primer3.pdf provided by the Whitehead Institute for Biomedical Research, except for AKR1C1, AKR1C2 and AKR1C3, (16) and 17BHSD10 (17) for which previously published primer sequences were used. Sequences are provided in Supplementary Table 1.

Statistical Analyses

To account for having multiple samples (i.e. 2-4 metastatic deposits) with replicate measurements from the same patient, statistical comparison of androgen levels in the human prostate and metastatic autopsy samples was performed using the following linear mixed effects model: [yij = β0 + bi + β1xij + εij] where yij is the androgen level (T, DHT), bi is a random intercept with distribution N(0, σb), xij indicates tissue type, and εij is an individual-specific error term with distribution N(0, σε) (here i indexes patients and j indexes patient-specific observations). Further, we assume that bi and εij are independent. This model accounts for within-individual correlations, which are assumed to be the same for each individual, and was used to derive P values for the comparison of mean tissue androgen levels among sample types. For each of the three xenograft lines, differences in androgen levels between the castration-sensitive and castration-resistant tumors were assessed by unpaired two sample t-tests. P values < 0.05 were considered significant.

For analysis of the qRT-PCR data, the mean cycle threshold (Ct) obtained for each gene was normalized to the expression of the housekeeping gene RPL13A in the same sample (the delta Ct). Reactions with Ct's>35 were considered undetectable for that transcript, and the specificity of amplification in each reaction was assessed based on the melting point of the dissociation curve. Unpaired two sample t-tests were used to compare the mean delta Ct's for each gene between the primary prostate cancers (n=8) and metastatic autopsy samples (n=16-22). Welch's modification of the t-test was utilized if the F test to compare sample variances was significant (but was only applicable to one gene, UGT2B15). P values < 0.05 were considered significant. The fold change was calculated by unlogging the difference in mean delta Ct's between the sample groups. Similarities among the human prostate and metastatic autopsy samples based on expression of steroidogenic gene transcripts were assessed by unsupervised, hierarchical, average linkage clustering using Cluster 3.0 software (http://bonsai.ims.u-tokyo. ac.jp/mdehoon/software/cluster/software.htm) and plotted using TreeView version 1.6 (http://rana.lbl.gov/EisenSoftware.htm). This program organizes genes and samples into a tree structure based on their similarity, in which items are joined by short branches if they are similar to each other and by increasingly longer branches as their similarity decreases. In average linkage clustering, the distance between 2 items x and y is the mean of all pairwise distances between items contained in x and y, and therefore provides a visual estimate of the similarity among different items in a sample.

Results

Expression of the Androgen-AR Signaling Axis in Castration-Resistant Prostate Cancer Metastases

To study mechanisms responsible for prostate cancer progression in the setting of anorchid serum testosterone levels, we first sought to evaluate the integrity of the AR signaling axis within tumor metastases by examining the expression of AR and the androgen-regulated genes PSA and FKBP5. Using a rapid post-mortem tissue collection protocol (11), we obtained metastatic tumor samples from patients with progressive disease despite either surgical castration, or medical castration with clinically-documented anorchid serum testosterone concentrations (T≤50 ng/dL). Castration-resistant tumors generally demonstrated intense nuclear staining for AR as well as strong cytoplasmic PSA reactivity (Fig. 1A), although heterogeneity in expression was found in a subset of tumors as we and others have previously described (11, 18). Quantification of AR, PSA, and FKBP5 gene expression by qRT-PCR demonstrated increased expression of AR and equivalent levels of PSA and FKBP5 in the castration-resistant metastases compared to benign prostate tissue and primary prostate cancers (Fig. 1B). These data are consistent with prior reports (19-22), and demonstrate the continued activity of the AR signaling axis in most castration-resistant tumors despite anorchid serum androgen levels.

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Object name is nihms-58302-f0001.jpg

Expression of androgen receptor and PSA in castration-resistant metastases. A) Immunohistochemical analysis of AR and PSA expression in metastatic lymph node foci of prostate adenocarcinoma. Protein expression is reflected as brown chromogen reactivity: (i) hematoxylin and eosin staining demonstrating characteristics of adenocarcinoma; (ii) AR staining of the same metastasis as in (i) with abundant nuclear AR expression; (iii) PSA staining of the same metastasis as in (i) with abundant cytoplasmic PSA expression (all images at 10x magnification). B) Transcript levels for AR, PSA and FKBP5 in the benign prostate (BP), cancer prostate (CP) and castration-resistant metastatic tumor (Mets) samples. Cycle thresholds (Ct) for each gene were normalized to the housekeeping gene RPL13A in the same sample. The y-axis is the RPL13A-normalized Ct, more positive numbers reflect higher transcript abundance. Unpaired two sample t-tests were used to compare the mean Ct's for each gene between the cancer prostate (CP) and metastatic tumor (Mets) samples. P values < 0.05 were considered significant.

Testosterone Levels in Castration-Resistant Metastases and Primary Prostate Tumors

To investigate whether the AR-signaling activity observed in the castration-resistant prostate cancer metastases could be due to the presence of intratumoral androgens, we obtained multiple soft-tissue metastatic deposits from patients with progressive disease and anorchid serum testosterone concentrations (using the rapid post-mortem tissue collection protocol described above (11)). Benign samples (control tissues not involved with tumor, including skin, muscle and liver samples) were simultaneously obtained from a subset of patients. We also evaluated paired cancer and benign prostate tissues from untreated eugonadal patients undergoing prostatectomy, and prostate tissue from patients without prostate cancer undergoing cystoprostatectomy for bladder cancer. Multiple samples from each tissue were separately processed and androgen concentrations were quantified by mass spectrometry.

Testosterone concentrations in metastatic prostate tumors from anorchid patients ranged from 0.2-1.78 ng/gram (Fig. 2A), up to four-fold higher than levels in non-cancerous control tissues acquired at autopsy, or in primary prostate cancer samples from eugonadal patients (Table 1, p<0.0001). Higher levels of testosterone and DHT were observed in primary prostate cancers compared to paired benign prostate tissues (p=0.01 for testosterone and p<0.0001 for DHT; Fig. 2A and B, and Table 1). Primary prostate tumors from eugonadal patients retained the 10:1 to 20:1 ratio of DHT to testosterone observed in benign prostate tissues. In contrast, this ratio was markedly reversed in metastatic tumors, which displayed a DHT:testosterone ratio of approximately 0.25:1, consistent with a prior report evaluating locally-recurrent, castration-resistant primary prostate tumors (7). Importantly, the testosterone concentrations measured in the prostate cancer metastases are higher than those in the non-prostatic control tissues, exceed mass-equivalent concentrations in the serum of these anorchid men, and are well within a range known to stimulate the AR and support prostate cancer cell proliferation (22-24).

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Object name is nihms-58302-f0002.jpg

Quantitation of tissue androgens in primary and castration-resistant metastatic prostate tumors. A) Testosterone and B) DHT levels were evaluated by mass spectrometry in paired benign and cancer prostate tissues from 4 eugonadal patients undergoing prostatectomy (P1-P4); in benign prostate tissue from 2 patients undergoing cystoprostatectomy for bladder cancer (P5-P6); and in multiple metastatic tumor deposits obtained at autopsy from each of 8 patients with castration-resistant prostate cancer (M1-M8). Control tissues not involved by tumor were simultaneously obtained from a subset of patients during the autopsy procedure (C1-C3). Each tissue sample was subdivided into triplicate samples that were separately processed; the data points represent the mean of each triplicate.

Table 1

Mean Tissue Androgen Levels in Castration-Resistant Metastases from Anorchid Patients vs. Primary Prostate Tissues from Eugonadal Men.

Tissue SourceTestosterone
ng/gm (95% CI)*
DHT
ng/gm (95% CI)*
Benign Prostate (BP)0.04 (0.00 – 0.24)1.92 (1.63 – 2.21)
Cancer Prostate (CP)0.23 (0.03 – 0.44)2.75 (2.45 – 3.04)
Control Tissue (Ctrl)**0.10 (0.00 – 0.26)0.05 (0.00 – 0.30)
Metastatic Tissue (Mets)0.74 (0.59 – 0.89)0.25 (0.00 – 0.50)
P<0.0001 for comparison among BP vs. CP, Ctrl vs. Mets, and CP vs. Mets for both T and DHT (except p=0.01 for difference in T between BP vs. CP). Mean values, confidence intervals and p-values were calculated using a linear mixed-effects model to account for multiple observations and intra-individual correlations.
Non-tumor tissues obtained concurrently with tumor metastases from men with castration-resistant prostate cancer.

Alterations in Transcripts Encoding Steroidogenic Enzymes in Castration-Resistant Metastases

To determine whether prostate cancer metastases may be capable of synthesizing androgens de novo, we quantified transcripts encoding each enzyme involved in the sequential biosynthesis of testosterone and DHT from cholesterol precursors (Fig. 3A). Compared to untreated primary prostate tumors, castration-resistant metastases demonstrated significant increases in the expression of FASN, HSD3B1, HSD3B2, CYP17A1, AKR1C3 and HSD17B3, key enzymes required for metabolism of progestins to adrenal androgens and their subsequent conversion to testosterone. Representative results are shown in Fig. 3B, and the data for all genes are summarized in Table 2. Consistent with the marked reversal of the DHT:testosterone ratio in the metastatic samples, they expressed significantly lower levels of SRD5A2, which catalyses the conversion of testosterone to DHT, and higher levels of UGT2B15 and UGT2B17, which mediate the irreversible glucuronidation of DHT metabolites (Fig. 3B and Table 2). Interestingly, marked upregulation of CYP19A1, which mediates the aromatization of testosterone to estradiol, was also observed in the metastases, and is consistent with prior reports demonstrating upregulated expression of aromatase in malignant vs. benign prostate epithelium (25).

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Object name is nihms-58302-f0003.jpg

Expression of steroidogenic enzyme transcripts in primary and metastatic prostate tumors. A) The enzymatic pathways mediating the sequential biosynthesis and metabolism of Testosterone and DHT from cholesterol and progestin precursors were evaluated by quantitative RT-PCR. Bold arrows in A denote several of the key metabolic steps (colored enzymes) for which transcript levels were significantly altered in the castration-resistant prostate cancer (CRPC) metastases versus primary prostate tumors. B) Representative dot plots for the key metabolic enzymes highlighted in panel A (HSD3B1, CYP17A1, AKR1C3, SRD5A2 and UGT2B17). Transcript levels for the indicated enzymes were evaluated in the benign prostate (BP), cancer prostate (CP) and metastatic tumor (Mets) samples; cycle thresholds (Ct) for each gene were normalized to expression of the housekeeping gene RPL13A in the same sample. The y-axis is the RPL13A-normalized Ct, where more positive numbers reflect higher transcript abundance. Unpaired two sample t-tests were used to compare the mean Ct's for each gene between the cancer prostate (CP) and metastatic tumor (Mets) samples. P values < 0.05 were considered significant.

Table 2

Relative Expression of Steroidogenic Enzymes in Castration-Resistant Metastases vs. Primary Prostate Tumors

GeneFold Change*p-Value
STAR5.10.0105
FASN9.60.0003
CYP11A−1.10.8362
CYP17A16.90.0005
3BHSD18.5<0.0001
3BHSD27.50.0091
17BSHD28.20.0137
17BHSD38.7<0.0001
17BHSD44.80.0019
AKR1C12.70.0601
AKR1C21.10.7895
AKR1C3**8.00.0026
SRD5A12.630.0050
SRD5A2−9.40.0005
CYP19A130.3<0.0001
UGT2B1510.00.0779
UGT2B1734.70.0013
Fold change calculated by unlogging the difference in mean cycle threshold between the sample groups. P values derived from unpaired two sample t-tests.
also termed 17BHSD5

Hierarchical clustering of tumors based on expression of steroidogenic enzyme transcripts clearly distinguished primary prostate cancers and benign prostate tissue from castration-resistant metastases, with metastatic samples generally clustering by patient of origin (Fig. 4A). This observation suggests that adaptive modulation of steroidogenic pathways to the castrate environment may occur within the tumor prior to initiation of the metastatic cascade. Importantly, transcripts encoding the full complement of enzymes comprising the steroidogenic pathway were detectable in the majority of primary and metastatic prostate tumors examined (Fig. 4B).

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Unsupervised hierarchical clustering of primary prostate tissues and castration-resistant metastases based on expression of steroidogenic enzyme transcripts. A) The dendrogram depicts the relationship of the different tissue samples based on relative expression of the indicated genes listed in panel b. Metastatic tumor deposits are color coded according to patient of origin. B) The heatmap depicts the mean-centered expression of each gene relative to the average RPL13A-normalized cycle threshold for each gene across all samples. The scale is from bright green (lowest expression) to black (equivalent expression) to bright red (highest expression). Gray squares denote samples for which no transcript was detectable.

Intratumoral Androgen Levels in Prostate Cancer Xenografts Grown in Intact and Castrate Mice

We next evaluated androgen levels in a series of prostate cancer xenografts grown in non-castrate (intact) and castrate male SCID mice. Xenografts were derived from castration-resistant lymph node metastases (LuCap 23.1 and 35) or primary prostate tumor (LuCap 96). Castration-sensitive xenografts were passaged in intact mice, while isogenic castration-resistant variants were maintained in castrate hosts. Remarkably, testosterone levels in the LuCap 23.1 and LuCap35 xenografts derived from lymph node metastases were equivalent whether tumors were grown in castrate or intact mice (Fig 5, and Supplemental Table 2). Testosterone levels in the prostate-derived LuCap 96 were higher in tumors from intact eugonadal mice, but remained easily detectable in the castration resistant tumors from the castrate hosts. Similarly, while more heterogeneous among replicates, DHT levels in the majority of castration resistant xenograft samples from castrate mice were equivalent or higher than those measured in the isogenic castration sensitive xenografts grown in intact mice. Testosterone and DHT levels in normal tissue samples (kidney and muscle) from either the intact or castrate host animals did not approach those in tumor tissue.

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Androgen Levels In LuCaP Prostate Cancer Xenografts Grown in Castrate and Intact Mice. A) Testosterone and B) DHT levels were measured by mass spectrometry in castration-sensitive (CS) and castration-resistant (CR) variants of the indicated xenografts. Two to five CS and CR tumors of each line were passaged in non-castrate (intact) or castrate male SCID mice respectively, as indicated. The LuCaP 35 CS→CR samples are xenografts initially passaged as castration-sensitive in intact mice, which then responded to castration with castration-resistant growth. Each data point represents the mean value for an individual xenograft harvested from one mouse; samples were subdivided and assayed in duplicate. Androgen levels were also evaluated in normal kidney and muscle tissues simultaneously obtained from each set of castrate and intact animals. Mean androgen levels for each CS and CR xenografts are presented in Supplemental Table 2.

Expression of the Androgen-AR Signaling Axis in Castration-Resistant Prostate Cancer Metastases

To study mechanisms responsible for prostate cancer progression in the setting of anorchid serum testosterone levels, we first sought to evaluate the integrity of the AR signaling axis within tumor metastases by examining the expression of AR and the androgen-regulated genes PSA and FKBP5. Using a rapid post-mortem tissue collection protocol (11), we obtained metastatic tumor samples from patients with progressive disease despite either surgical castration, or medical castration with clinically-documented anorchid serum testosterone concentrations (T≤50 ng/dL). Castration-resistant tumors generally demonstrated intense nuclear staining for AR as well as strong cytoplasmic PSA reactivity (Fig. 1A), although heterogeneity in expression was found in a subset of tumors as we and others have previously described (11, 18). Quantification of AR, PSA, and FKBP5 gene expression by qRT-PCR demonstrated increased expression of AR and equivalent levels of PSA and FKBP5 in the castration-resistant metastases compared to benign prostate tissue and primary prostate cancers (Fig. 1B). These data are consistent with prior reports (19-22), and demonstrate the continued activity of the AR signaling axis in most castration-resistant tumors despite anorchid serum androgen levels.

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Expression of androgen receptor and PSA in castration-resistant metastases. A) Immunohistochemical analysis of AR and PSA expression in metastatic lymph node foci of prostate adenocarcinoma. Protein expression is reflected as brown chromogen reactivity: (i) hematoxylin and eosin staining demonstrating characteristics of adenocarcinoma; (ii) AR staining of the same metastasis as in (i) with abundant nuclear AR expression; (iii) PSA staining of the same metastasis as in (i) with abundant cytoplasmic PSA expression (all images at 10x magnification). B) Transcript levels for AR, PSA and FKBP5 in the benign prostate (BP), cancer prostate (CP) and castration-resistant metastatic tumor (Mets) samples. Cycle thresholds (Ct) for each gene were normalized to the housekeeping gene RPL13A in the same sample. The y-axis is the RPL13A-normalized Ct, more positive numbers reflect higher transcript abundance. Unpaired two sample t-tests were used to compare the mean Ct's for each gene between the cancer prostate (CP) and metastatic tumor (Mets) samples. P values < 0.05 were considered significant.

Testosterone Levels in Castration-Resistant Metastases and Primary Prostate Tumors

To investigate whether the AR-signaling activity observed in the castration-resistant prostate cancer metastases could be due to the presence of intratumoral androgens, we obtained multiple soft-tissue metastatic deposits from patients with progressive disease and anorchid serum testosterone concentrations (using the rapid post-mortem tissue collection protocol described above (11)). Benign samples (control tissues not involved with tumor, including skin, muscle and liver samples) were simultaneously obtained from a subset of patients. We also evaluated paired cancer and benign prostate tissues from untreated eugonadal patients undergoing prostatectomy, and prostate tissue from patients without prostate cancer undergoing cystoprostatectomy for bladder cancer. Multiple samples from each tissue were separately processed and androgen concentrations were quantified by mass spectrometry.

Testosterone concentrations in metastatic prostate tumors from anorchid patients ranged from 0.2-1.78 ng/gram (Fig. 2A), up to four-fold higher than levels in non-cancerous control tissues acquired at autopsy, or in primary prostate cancer samples from eugonadal patients (Table 1, p<0.0001). Higher levels of testosterone and DHT were observed in primary prostate cancers compared to paired benign prostate tissues (p=0.01 for testosterone and p<0.0001 for DHT; Fig. 2A and B, and Table 1). Primary prostate tumors from eugonadal patients retained the 10:1 to 20:1 ratio of DHT to testosterone observed in benign prostate tissues. In contrast, this ratio was markedly reversed in metastatic tumors, which displayed a DHT:testosterone ratio of approximately 0.25:1, consistent with a prior report evaluating locally-recurrent, castration-resistant primary prostate tumors (7). Importantly, the testosterone concentrations measured in the prostate cancer metastases are higher than those in the non-prostatic control tissues, exceed mass-equivalent concentrations in the serum of these anorchid men, and are well within a range known to stimulate the AR and support prostate cancer cell proliferation (22-24).

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Quantitation of tissue androgens in primary and castration-resistant metastatic prostate tumors. A) Testosterone and B) DHT levels were evaluated by mass spectrometry in paired benign and cancer prostate tissues from 4 eugonadal patients undergoing prostatectomy (P1-P4); in benign prostate tissue from 2 patients undergoing cystoprostatectomy for bladder cancer (P5-P6); and in multiple metastatic tumor deposits obtained at autopsy from each of 8 patients with castration-resistant prostate cancer (M1-M8). Control tissues not involved by tumor were simultaneously obtained from a subset of patients during the autopsy procedure (C1-C3). Each tissue sample was subdivided into triplicate samples that were separately processed; the data points represent the mean of each triplicate.

Table 1

Mean Tissue Androgen Levels in Castration-Resistant Metastases from Anorchid Patients vs. Primary Prostate Tissues from Eugonadal Men.

Tissue SourceTestosterone
ng/gm (95% CI)*
DHT
ng/gm (95% CI)*
Benign Prostate (BP)0.04 (0.00 – 0.24)1.92 (1.63 – 2.21)
Cancer Prostate (CP)0.23 (0.03 – 0.44)2.75 (2.45 – 3.04)
Control Tissue (Ctrl)**0.10 (0.00 – 0.26)0.05 (0.00 – 0.30)
Metastatic Tissue (Mets)0.74 (0.59 – 0.89)0.25 (0.00 – 0.50)
P<0.0001 for comparison among BP vs. CP, Ctrl vs. Mets, and CP vs. Mets for both T and DHT (except p=0.01 for difference in T between BP vs. CP). Mean values, confidence intervals and p-values were calculated using a linear mixed-effects model to account for multiple observations and intra-individual correlations.
Non-tumor tissues obtained concurrently with tumor metastases from men with castration-resistant prostate cancer.

Alterations in Transcripts Encoding Steroidogenic Enzymes in Castration-Resistant Metastases

To determine whether prostate cancer metastases may be capable of synthesizing androgens de novo, we quantified transcripts encoding each enzyme involved in the sequential biosynthesis of testosterone and DHT from cholesterol precursors (Fig. 3A). Compared to untreated primary prostate tumors, castration-resistant metastases demonstrated significant increases in the expression of FASN, HSD3B1, HSD3B2, CYP17A1, AKR1C3 and HSD17B3, key enzymes required for metabolism of progestins to adrenal androgens and their subsequent conversion to testosterone. Representative results are shown in Fig. 3B, and the data for all genes are summarized in Table 2. Consistent with the marked reversal of the DHT:testosterone ratio in the metastatic samples, they expressed significantly lower levels of SRD5A2, which catalyses the conversion of testosterone to DHT, and higher levels of UGT2B15 and UGT2B17, which mediate the irreversible glucuronidation of DHT metabolites (Fig. 3B and Table 2). Interestingly, marked upregulation of CYP19A1, which mediates the aromatization of testosterone to estradiol, was also observed in the metastases, and is consistent with prior reports demonstrating upregulated expression of aromatase in malignant vs. benign prostate epithelium (25).

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Expression of steroidogenic enzyme transcripts in primary and metastatic prostate tumors. A) The enzymatic pathways mediating the sequential biosynthesis and metabolism of Testosterone and DHT from cholesterol and progestin precursors were evaluated by quantitative RT-PCR. Bold arrows in A denote several of the key metabolic steps (colored enzymes) for which transcript levels were significantly altered in the castration-resistant prostate cancer (CRPC) metastases versus primary prostate tumors. B) Representative dot plots for the key metabolic enzymes highlighted in panel A (HSD3B1, CYP17A1, AKR1C3, SRD5A2 and UGT2B17). Transcript levels for the indicated enzymes were evaluated in the benign prostate (BP), cancer prostate (CP) and metastatic tumor (Mets) samples; cycle thresholds (Ct) for each gene were normalized to expression of the housekeeping gene RPL13A in the same sample. The y-axis is the RPL13A-normalized Ct, where more positive numbers reflect higher transcript abundance. Unpaired two sample t-tests were used to compare the mean Ct's for each gene between the cancer prostate (CP) and metastatic tumor (Mets) samples. P values < 0.05 were considered significant.

Table 2

Relative Expression of Steroidogenic Enzymes in Castration-Resistant Metastases vs. Primary Prostate Tumors

GeneFold Change*p-Value
STAR5.10.0105
FASN9.60.0003
CYP11A−1.10.8362
CYP17A16.90.0005
3BHSD18.5<0.0001
3BHSD27.50.0091
17BSHD28.20.0137
17BHSD38.7<0.0001
17BHSD44.80.0019
AKR1C12.70.0601
AKR1C21.10.7895
AKR1C3**8.00.0026
SRD5A12.630.0050
SRD5A2−9.40.0005
CYP19A130.3<0.0001
UGT2B1510.00.0779
UGT2B1734.70.0013
Fold change calculated by unlogging the difference in mean cycle threshold between the sample groups. P values derived from unpaired two sample t-tests.
also termed 17BHSD5

Hierarchical clustering of tumors based on expression of steroidogenic enzyme transcripts clearly distinguished primary prostate cancers and benign prostate tissue from castration-resistant metastases, with metastatic samples generally clustering by patient of origin (Fig. 4A). This observation suggests that adaptive modulation of steroidogenic pathways to the castrate environment may occur within the tumor prior to initiation of the metastatic cascade. Importantly, transcripts encoding the full complement of enzymes comprising the steroidogenic pathway were detectable in the majority of primary and metastatic prostate tumors examined (Fig. 4B).

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Object name is nihms-58302-f0004.jpg

Unsupervised hierarchical clustering of primary prostate tissues and castration-resistant metastases based on expression of steroidogenic enzyme transcripts. A) The dendrogram depicts the relationship of the different tissue samples based on relative expression of the indicated genes listed in panel b. Metastatic tumor deposits are color coded according to patient of origin. B) The heatmap depicts the mean-centered expression of each gene relative to the average RPL13A-normalized cycle threshold for each gene across all samples. The scale is from bright green (lowest expression) to black (equivalent expression) to bright red (highest expression). Gray squares denote samples for which no transcript was detectable.

Intratumoral Androgen Levels in Prostate Cancer Xenografts Grown in Intact and Castrate Mice

We next evaluated androgen levels in a series of prostate cancer xenografts grown in non-castrate (intact) and castrate male SCID mice. Xenografts were derived from castration-resistant lymph node metastases (LuCap 23.1 and 35) or primary prostate tumor (LuCap 96). Castration-sensitive xenografts were passaged in intact mice, while isogenic castration-resistant variants were maintained in castrate hosts. Remarkably, testosterone levels in the LuCap 23.1 and LuCap35 xenografts derived from lymph node metastases were equivalent whether tumors were grown in castrate or intact mice (Fig 5, and Supplemental Table 2). Testosterone levels in the prostate-derived LuCap 96 were higher in tumors from intact eugonadal mice, but remained easily detectable in the castration resistant tumors from the castrate hosts. Similarly, while more heterogeneous among replicates, DHT levels in the majority of castration resistant xenograft samples from castrate mice were equivalent or higher than those measured in the isogenic castration sensitive xenografts grown in intact mice. Testosterone and DHT levels in normal tissue samples (kidney and muscle) from either the intact or castrate host animals did not approach those in tumor tissue.

An external file that holds a picture, illustration, etc.
Object name is nihms-58302-f0005.jpg

Androgen Levels In LuCaP Prostate Cancer Xenografts Grown in Castrate and Intact Mice. A) Testosterone and B) DHT levels were measured by mass spectrometry in castration-sensitive (CS) and castration-resistant (CR) variants of the indicated xenografts. Two to five CS and CR tumors of each line were passaged in non-castrate (intact) or castrate male SCID mice respectively, as indicated. The LuCaP 35 CS→CR samples are xenografts initially passaged as castration-sensitive in intact mice, which then responded to castration with castration-resistant growth. Each data point represents the mean value for an individual xenograft harvested from one mouse; samples were subdivided and assayed in duplicate. Androgen levels were also evaluated in normal kidney and muscle tissues simultaneously obtained from each set of castrate and intact animals. Mean androgen levels for each CS and CR xenografts are presented in Supplemental Table 2.

Discussion

The mechanisms by which advanced prostate cancers maintain AR-mediated gene expression following castration are poorly defined. In this study, we determined that soft-tissue metastases from castration-resistant prostate cancers exhibit elevated testosterone concentrations compared to untreated primary tumors. The processes responsible for sustaining intratumoral androgen levels in the setting of systemic testosterone suppression have yet to be determined. The potential contribution of adrenal androgens to prostate tumor growth is well-recognized, and inhibiting this androgen source is a major treatment focus for castration-resistant tumors. Drugs which were serendipitously found to target steroid synthesizing enzymes in the adrenal gland have demonstrated significant, albeit short-term responses (26, 27). Adrenalectomy and hypophysectomy also have efficacy in a limited number of patients (28). Besides the uptake and conversion of circulating adrenal androgens, prostate cancer metastases may also be capable of de novo androgen biosynthesis from cholesterol and/or progesterone precursors (19).

Our data demonstrate that transcripts encoding the full complement of enzymes involved in the sequential biosynthesis of testosterone and DHT from cholesterol precursors were expressed in the majority of castration-resistant metastatic tumors examined. Furthermore, the specific steroidogenic genes altered in our data confirm and extend previous studies of gene expression in castration-resistant bone marrow metastases (10) to include upregulated expression of CYP17A1, a critical enzyme mediating sequential steps in the production of adrenal androgens from progestins, as well as increased expression of HSD17B3, which mediates the same metabolic step as AKR1C3, the conversion of androstenedione to testosterone (29). A clear limitation of our study is the degree to which transcript alterations correlate with changes in biosynthetic enzyme activity. While demonstration of enzymatic function is beyond the scope of the present work, the presence of transcripts encoding each gene in the androgen biosynthetic pathway is a necessary prerequisite to steroidogenesis, and a comprehensive assessment of genes in this pathway has not been previously reported in prostate cancer metastases.

The increased expression of AR transcripts in the castration-resistant metastases may be of particular importance in promoting tumor cell growth at the androgen levels detected in these samples. Gregory et al have shown that prostate cancer cell lines derived from recurrent tumors demonstrate increased expression and stability of the AR, in association with an increased sensitivity to proliferation at low levels of DHT. Furthermore, Chen et al have demonstrated that increased AR expression is instrumental in the progression from androgen-dependent to castration resistant growth in a xenograft model, with the degree of AR upregulation observed sufficient to allow tumor cell proliferation in 80% lower androgen concentrations (3). Importantly, ligand binding was required for hormone refractory growth, and modest increases in AR expression were sufficient to support signaling in a low androgen environment. Conversely, AR protein expression was equivalent in a series of locally recurrent prostate tumors compared to benign prostate epithelium (7). As such, increased AR message may not necessarily result in uniformly higher AR protein expression, but may contribute to maintaining AR protein expression at levels required for sustaining tumor cell growth.

Our study found a marked reversal in the ratio of testosterone to DHT in the castration-resistant tumor metastases compared to the primary prostate tissues. This observation is consistent with the study of Mohler et al in which testosterone levels in locally-recurrent, castration-resistant primary prostate tumors were maintained at levels found in untreated BPH tissues while DHT levels were significantly decreased (7). Although DHT is approximately 10 fold more potent than testosterone in binding and activating the AR (30), kinetic experiments have demonstrated that testosterone at high concentrations interacts with the AR similarly to DHT (31). In studies evaluating androgen induced prostate regrowth in castrated rats, Wright et al found that 1.6-1.9 fold increases in testosterone compared to DHT were sufficient to achieve comparable measures of prostate regrowth (32). Conversely, Xu et al have shown that dutasteride treatment inhibited the growth of Dunning R-3327H rat prostate tumors and LNCaP human prostate xenografts, despite a concomitant two-fold rise in tissue testosterone levels. However, while dutasteride reduced tumor DHT levels to essentially the same nadir as castration, it did not produce the same magnitude of tumor growth inhibition as castration. Moreover, the combination of dutasteride plus castration produced greater growth inhibition of LNCaP xenografts than either castration or dutasteride alone, suggesting that while testosterone may not be as potent as DHT, it is capable of stimulating prostate tumor growth. In earlier studies evaluating the growth response of androgen dependent xenografts, Van Weerden et al found that androgen-induced increases in tumor cell growth only occurred when tissue androgen levels exceeded a critical threshold value, corresponding to ∼0.9 ng/gm of DHT (33). Although higher than the DHT levels observed in the tumor metastases in our study, this value may reflect the androgen dependence of the xenografts evaluated by van Weerden, and does not preclude the possibility of tumor cell stimulation at the lower androgen levels detected in the castration-resistant metastases.

The detection of testosterone as the primary androgen in the castration-resistant metastases may reflect the subtotal decrease in tumoral SRD5A activity that has been consistently observed in prostate tumors since its original demonstration in lymph node metastases and primary prostate cancers by Klein et al. (34) More recently, studies have demonstrated a decrease in SRD5A2 expression in neoplastic compared to benign prostate tissues, accompanied by a relative shift in expression and enzymatic activity to SRD5A1 in primary and recurrent prostate tumors (10, 35-39). These findings are consistent with our measurements of ∼9 fold decrease and ∼2.5 fold increase in SRD5A2 and SRD5A1 expression, respectively, in the tumor metastases. Alternatively, the concentrations of DHT detected in the tumor metastases we analyzed may actually be an underestimate of the in vivo tumor androgen levels, as several studies have demonstrated that DHT levels in autopsy samples may be factitiously low due to ongoing postmortem androgen metabolism or degradation (40, 41). We cannot exclude this possibility, which may also underlie the relatively higher levels of DHT observed in the prostate cancer xenografts as these tissues can be immediately frozen upon harvesting. Interestingly, the androgen levels detected in the castration-resistant xenografts suggests these tissues may be capable of de novo androgen biosynthesis, as some (albeit not all) studies have suggested that castrate rodents do not make adrenal androgens due to a lack of CYP17 expression in the rodent adrenal gland (42, 43).

In conclusion, we propose that metastatic prostate cancers may adapt to low systemic testosterone levels by maintaining intratumoral androgens through the modulation of enzymes involved in intracrine steroidogenesis and androgen catabolism. Our data suggest that secondary hormonal manipulations and pharmacological inhibitors of androgen biosynthesis derive a component of their activity by directly targeting intratumoral androgen production. This mechanism may explain the relatively high response rates observed in recent clinical studies evaluating specific CYP17A1 inhibitors for castration-resistant prostate cancer, when compared to historical rates observed with adrenalectomy (28, 44-46). Moreover, elevated tumoral androgen levels may underlie the lack of substantial survival benefit associated with the use of androgen receptor antagonists, as agents such as bicalutamide have an affinity for the AR which is 30-fold lower than the endogenous ligand (47, 48) These observations strongly suggest that improving clinical outcomes in castration-resistant prostate cancer will require combinatorial treatment strategies designed to abrogate intracrine as well as systemic contributions to the tumoral androgen axis. Furthermore, the application of agents targeting intra-tumoral androgen production during the treatment of androgen-sensitive prostate cancer may delay or prevent the progression to castration-resistant disease.

Supplementary Material

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Acknowledgements

We thank the patients and their families who made this work possible through their altruistic donation of tissues. We thank Roger Coleman for technical assistance, Ilsa Coleman and Roman Gulati for providing assistance with statistical analyses, and members of University of Washington Urology Department and the Tissue Acquisition Necropsy team for assisting with sample collections.

Financial support: National Institutes of Health (Pacific Northwest Prostate Cancer SPORE P50CA97186 to R.V., L.D.T. and P.S.N.; K23CA122820 to E.A.M.; P30CA15704 to R.B.M. and R.V.;R01 DK65204 to P.S.N.; Oregon National Primate Research Center Core Grant RR00163, D.L.H.); Department of Defense (PC041158 to P.S.N.) and the Prostate Cancer Foundation (to E.A.M.).

Department of Medicine, University of Washington School of Medicine
Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA
Department of Urology, University of Washington School of Medicine
Oregon National Primate Research Center, Oregon Health and Sciences University, Beaverton, OR
Department of Pathology, University of Washington School of Medicine
Correspondence to: Peter S. Nelson, Fred Hutchinson Cancer Research Center,1100 Fairview Ave N, MS D4-100, Seattle, WA 91809-1024 (email: gro.crchf@noslenp)
These authors contributed equally to this work

Abstract

Therapy for advanced prostate cancer centers on suppressing systemic androgens and blocking activation of the androgen receptor. Despite anorchid serum androgen levels, nearly all patients develop castration-resistant disease. We hypothesized that ongoing steroidogenesis within prostate tumors and the maintenance of intratumoral androgens may contribute to castration-resistant growth. Using mass spectrometry and quantitative RT-PCR we evaluated androgen levels and transcripts encoding steroidogenic enzyme in benign prostate tissue, untreated primary prostate cancer, metastases from patients with castration-resistant prostate cancer and xenografts derived from castration-resistant metastases. Testosterone levels within metastases from anorchid men (0.74 ng/gm; 95% CI 0.59-0.89) were significantly higher than levels within primary prostate cancers from untreated eugonadal men (0.23 ng/gm; 95% CI 0.03 – 0.44, p<0.0001). Compared to primary prostate tumors, castration-resistant metastases displayed alterations in genes encoding steroidogenic enzymes, including upregulated expression of FASN, CYP17A1, HSD3B1, HSD17B3, CYP19A1 and UBT2B17, and down regulated expression of SRD5A2 (p≤0.001 for all). Prostate cancer xenografts derived from castration-resistant tumors maintained similar intratumoral androgen levels when passaged in castrate compared to eugonadal animals. Metastatic prostate cancers from anorchid men express transcripts encoding androgen-synthesizing enzymes and maintain intratumoral androgens at concentrations capable of activating AR-target genes and maintaining tumor cell survival. We conclude that intracrine steroidogenesis may permit tumors to circumvent low levels of circulating androgens. Maximal therapeutic efficacy in the treatment of castration-resistant prostate cancer will require novel agents capable of inhibiting intracrine steroidogenic pathways within the prostate tumor microenvironment.

Keywords: prostate cancer, castration-resistant, hormone refractory, intracrine, steroidogenesis
Abstract

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