Search

Fig S2. SLC35F2 Expression Determines Sensitivity to YM155

Correlation of tumor androgen levels with SLC35F2 transcript expression in PDX models

Figure S1: Additional analyses of NP and NPp53 GEM models Figure S2: Tolerance and efficacy of abiraterone in mouse prostate Figure S3: Additional analyses of tumor volumes in GEM models before and after treatment Figure S4: Analyses of abiraterone treatment of cell lines from NP and NPp53 tumors Figure S5: Examples of alternative histopathologies of NPp53 CRPC Figure S6: Additional cross-species computational analyses Figure S7: Additional analyses of expression of SOX11 and other NE genes Table S1: Description of human datasets used in this study Table S2: Summary of preclinical phenotypic analyses Table S3: Summary of histopathology of individual mouse tumors Table S4: List of master regulators Table S5: Quantitation of synaptophysin-positive cells in mouse CRPC Table S6: List of antibodies used in this study Table S7: List of primers and shRNA used in this study Detailed experimental procedures

Differentially expressed genes for mouse CRPC tumors

Androgen receptor (AR) signaling is fundamental to prostate cancer and is the dominant therapeutic target in metastatic disease. However, stringent androgen deprivation therapy regimens decrease quality of life and have been largely unsuccessful in curtailing mortality. Recent clinical and preclinical studies have taken advantage of the dichotomous ability of AR signaling to elicit growth-suppressive and differentiating effects by administering hyperphysiologic levels of testosterone. In this study, high-throughput drug screening identified a potent synergy between high-androgen therapy and YM155, a transcriptional inhibitor of survivin (BIRC5). This interaction was mediated by the direct transcriptional upregulation of the YM155 transporter SLC35F2 by the AR. Androgen-mediated YM155-induced cell death was completely blocked by the overexpression of multidrug resistance transporter ABCB1. SLC35F2 expression was significantly correlated with intratumor androgen levels in four distinct patient-derived xenograft models, and with AR activity score in a large gene expression dataset of castration-resistant metastases. A subset of tumors had significantly elevated SLC35F2 expression and, therefore, may identify patients who are highly responsive to YM155 treatment.Implications: The combination of androgen therapy with YM155 represents a novel drug synergy, and SLC35F2 may serve as a clinical biomarker of response to YM155. Mol Cancer Res; 15(5); 521–31. ©2017 AACR.

Figure S1: Assessment for neuroendocrine features by histology and IHC; Figure S2: Scatter plots depicting relationships between the 267-gene AR activity ssGSEA score and the 131-gene proliferation ssGSEA score for multiple genesets; Figure S3: Plots depicting correlations with AR activity ssGSEA score, and assessing GR expression; Figure S4: Segmented read depth array CGH-style plots for chromosomes showing alterations to cancer-related genes; Figure S5: Euler diagrams depicting shared and distinct somatic genomic events for cases 003, 007, 501, 503, 506, 507, 509, 512, and 520.

Color figure 3 for supplement.

The standard treatment for metastatic prostate cancer, androgen deprivation therapy (ADT), is designed to suppress androgen receptor (AR) activity. However, men invariably progress to castration-resistant prostate cancer (CRPC), and AR reactivation contributes to progression in most cases. To identify mechanisms that may drive CRPC, we examined a VCaP prostate cancer xenograft model as tumors progressed from initial androgen sensitivity prior to castration to castration resistance and then on to relapse after combined therapy with further AR-targeted drugs (abiraterone plus enzalutamide). AR activity persisted in castration-resistant and abiraterone/enzalutamide–resistant xenografts and was associated with increased expression of the AR gene and the AR-V7 splice variant. We then assessed expression of individual AR-regulated genes to identify those that persisted, thereby contributing to tumor growth, versus those that decreased and may therefore exhibit tumor suppressor activities. The most significantly decreased AR target gene was dipeptidyl peptidase 4 (DPP4), which encodes a membrane-anchored protein that cleaves dipeptides from multiple growth factors, resulting in their increased degradation. DPP4 mRNA and protein were also decreased in clinical CRPC cases, and inhibition of DPP4 with sitagliptin enhanced the growth of prostate cancer xenografts following castration. Significantly, DPP4 inhibitors are frequently used to treat type 2 diabetes as they increase insulin secretion. Together, these results implicate DPP4 as an AR-regulated tumor suppressor gene whose loss enhances growth factor activity and suggest that treatment with DPP4 inhibitors may accelerate emergence of resistance to ADT.Significance: These findings identify DPP4 as an AR-stimulated tumor suppressor gene that is downregulated during progression to castration-resistant prostate cancer, warning that treatment with DPP4 inhibitors, commonly used to treat type 2 diabetes, may accelerate prostate cancer progression following androgen deprivation therapy. Cancer Res; 78(22); 6354–62. ©2018 AACR.

Purpose: Prostate cancer is dependent on androgen receptor (AR) activation. Optimal AR antagonism may effectively cytoreduce local disease and suppress or eliminate micrometastases. We evaluated neoadjuvant therapy prior to prostatectomy with the potent AR antagonist enzalutamide (enza) either alone or in combination with dutasteride (dut) and leuprolide (enza/dut/luteinizing hormone-releasing hormone analogues [LHRHa]).Experimental Design: Forty-eight of 52 men with intermediate or high-risk localized prostate cancer proceeded to prostatectomy after neoadjuvant enzalutamide or enza/dut/LHRHa for 6 months. We assessed pathologic complete response (pCR), minimal residual disease (MRD; ≤3 mm maximum diameter of residual disease), residual cancer burden (RCB), and expression of PSA and serum and tissue androgen concentrations. We compared the proportion of patients with pCR in each treatment arm with a historical control rate of 5%, based on previous reports of flutamide with LHRHa.Results: In the enzalutamide arm, none of the 25 patients achieved pCR or MRD. In the enza/dut/LHRHa arm, one of 23 patients (4.3%) achieved pCR and 3 of 23 (13.0%) achieved MRD. Median RCB was higher in the enzalutamide arm than in the enza/dut/LHRHa arm (0.41 cm3 vs. 0.06 cm3, respectively). Tissue testosterone and dihydrotestosterone levels correlated with RCB. No adverse events leading to study drug discontinuation were reported.Conclusions: Combination therapy with enza/dut/LHRHa resulted in pCR and MRD rates comparable with historical controls. Evidence of continued AR activity in residual tumor suggests that AR signaling may contribute to survival. Strategies to more effectively ablate AR activity are warranted to determine whether more substantial antitumor effects are observed. Clin Cancer Res; 23(9); 2169–76. ©2016 AACR.

Purpose: Despite the efficacy of abiraterone, a CYP17A1 inhibitor, in metastatic castration-resistant prostate cancer (CRPC), nearly all patients develop resistance. The purpose of this phase II study was to evaluate mechanisms of resistance to more complete androgen synthesis inhibition with abiraterone and dutasteride.Experimental Design: Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy. Patients received abiraterone and prednisone for two 4-week cycles. After this time, high-dose dutasteride (3.5 mg daily) was added. Patients continued therapy until study withdrawal or radiographic progression. Repeat metastasis biopsy was obtained at progression. The primary endpoint was to assess mechanisms of resistance. Serum hormone and abiraterone levels were assessed. Tissue was assessed for androgen receptor (AR) and AR splice variant-7 (ARV7) expression.Results: Forty patients were enrolled. Sixty percent (n = 24) achieved a ≥50% reduction in prostate-specific antigen (PSA). The median time to radiographic progression was 11 months. Nearly all baseline (n = 29 of 31) and posttreatment (n = 16 of 16) tumors tested for AR nuclear expression were positive. Of those tested, ARV7 expression was present in 48% (n = 10 of 21) of baseline and 42% (n = 5 of 12) of treatment discontinuation specimens. Compared with patients with higher serum abiraterone levels at treatment discontinuation, patients with lower levels had higher circulating androgens.Conclusions: Despite increased androgen synthesis inhibition, we demonstrate that tumor AR axis remains important in disease progression. We highlight that abiraterone metabolism and pharmacokinetics may play a role in resistance. The noncomparative design limits conclusions on the efficacy of dual therapy with abiraterone and dutasteride, but the results support development of further multifaceted approaches toward AR inhibition. Clin Cancer Res; 23(4); 935–45. ©2016 AACR.

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes.Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes.Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05).Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592–601. ©2017 AACR.

Mutations in 3 cases with only 1 focus dissected

PDF file - 20K

Clean version of supplement for submission.

Purpose:Tumor androgens in castration-resistant prostate cancer (CRPC) reflect de novo intratumoral synthesis or adrenal androgens. We used C.B.-17 SCID mice in which we observed adrenal CYP17A activity to isolate the impact of adrenal steroids on CRPC tumors in vivo.Experimental Design:We evaluated tumor growth and androgens in LuCaP35CR and LuCaP96CR xenografts in response to adrenalectomy (ADX). We assessed protein expression of key steroidogenic enzymes in 185 CRPC metastases from 42 patients.Results:Adrenal glands of intact and castrated mice expressed CYP17A. Serum DHEA, androstenedione (AED), and testosterone (T) in castrated mice became undetectable after ADX (all P < 0.05). ADX prolonged median survival (days) in both CRPC models (33 vs. 179; 25 vs. 301) and suppressed tumor steroids versus castration alone (T 0.64 pg/mg vs. 0.03 pg/mg; DHT 2.3 pg/mg vs. 0.23 pg/mg; and T 0.81 pg/mg vs. 0.03 pg/mg, DHT 1.3 pg/mg vs. 0.04 pg/mg; all P ≤ 0.001). A subset of tumors recurred with increased steroid levels, and/or induction of androgen receptor (AR), truncated AR variants, and glucocorticoid receptor (GR). Metastases from 19 of 35 patients with AR positive tumors concurrently expressed enzymes for adrenal androgen utilization and nine expressed enzymes for de novo steroidogenesis (HSD3B1, CYP17A, AKR1C3, and HSD17B3).Conclusions:Mice are appropriate for evaluating adrenal impact of steroidogenesis inhibitors. A subset of ADX-resistant CRPC tumors demonstrate de novo androgen synthesis. Tumor growth and androgens were suppressed more strongly by surgical ADX than prior studies using abiraterone, suggesting reduction in adrenally-derived androgens beyond that achieved by abiraterone may have clinical benefit. Proof-of-concept studies with agents capable of achieving true “nonsurgical ADX” are warranted.

Supplementary Table 1 from Expression of SLCO Transport Genes in Castration-Resistant Prostate Cancer and Impact of Genetic Variation in SLCO1B3 and SLCO2B1 on Prostate Cancer Outcomes

Supplementary Figs S1-S6

Figure S1. The expressions of ERBB2 and ERBB3 are not increased by androgen deprivation; Figure S2. AR mRNA expression was not significantly decreased by lapatinib in LAPC4-CR cells; Figure S3. Lapatinib can block heregulin stimulated ErbB2 activation in C4-2 cells; Figure S4. Lapatinib decreased AR protein expression at 8h treatement; Figure S5. AR mRNA expression was not significantly decreased by lapatinib or AKT-x in C4-2 cells; Figure S6. AR protein nuclear expression was decreased by siRNA against ERBB2; Figure S7. AKT inhibitor treatment decreased AR protein expression and activity; Figure S8. Silencing PIK3CB expression decreased AR protein expression.

Prostate androgen levels after enza or enza/dut/LHRHa. Tissue androgens from prostatectomy specimens after 6 months of therapy were measured by mass spectroscopy to determine levels of (A) pregnenolone, (B) progesterone, (C) DHEA, (D) AED, (E) testosterone, and (F) DHT. P values were calculated using the nonparametric Mann-Whitney test. Limits of quantification were 0.015 pg/mg for AED, progesterone, and testosterone, 0.03 pg/mg for DHT and pregnenolone, and 0.49 pg/mg for DHEA.

PDF file - 97KB, Supplementary Table 1. List of genes up-regulated in abiraterone-resistant VCaP xenograft samples. Supplementary Table 2. List of genes down-regulated in abiraterone-resistant VCaP xenograft samples. Supplementary Table 3. AR mutation detected in RNA-Seq analysis.

Figure S3. Change in serum androgen levels after enza or enza/dut/LHRHa. Box plots of serum androgens for each cohort at initiation of therapy (day 0) and at prostatectomy (day 180) are depicted for each treatment arm. Levels of (A) pregnenolone, (B) progesterone, (C) DHEA, (D) AED, (E) testosterone, and (F) DHT were measured by mass spectroscopy. P values were calculated using the nonparametric Mann-Whitney test. Limits of quantification were 0.005 ng/mL for AED, progesterone and testosterone, 0.001 ng/mL for DHT and pregnenolone, and 0.15 ng/mL for DHEA.

Black and white figure for supplement.

Figure S4. Correlation of immunohistochemical expression of the glucocorticoid receptor (GR) after enza or enza/dut/LHRHa with residual cancer burden (RCB). A tissue microarray comprising cores of cancer tissue from each patient was analyzed for RCB and nuclear expression of GR expressed as total proportion of GR-positive tumor nuclei in (A) enza-treated and (B) enza/dut/LHRHa-treated patients. P values were calculated using the nonparametric Mann-Whitney test.

Background: Metastases from men with castration-resistant prostate cancer (CRPC) harbor increased tumoral androgens versus untreated prostate cancers. This may reflect steroid uptake by OATP (organic anion transporting polypeptide)/SLCO transporters. We evaluated SLCO gene expression in CRPC metastases and determined whether prostate cancer outcomes are associated with single nucleotide polymorphisms (SNP) in SLCO2B1 and SLCO1B3, transporters previously shown to mediate androgen uptake.Methods: Transcripts encoding eleven SLCO genes were analyzed in untreated prostate cancer and in metastatic CRPC tumors obtained by rapid autopsy. SNPs in SLCO2B1 and SLCO1B3 were genotyped in a population-based cohort of 1,309 Caucasian prostate cancer patients. Median survival follow-up was 7.0 years (0.77–16.4). The risk of prostate cancer recurrence/progression and prostate cancer–specific mortality (PCSM) was estimated with Cox proportional hazards analysis.Results: Six SLCO genes were highly expressed in CRPC metastases versus untreated prostate cancer, including SLCO1B3 (3.6-fold; P = 0.0517) and SLCO2B1 (5.5-fold; P = 0.0034). Carriers of the variant alleles SLCO2B1 SNP rs12422149 (HR: 1.99; 95% CI: 1.11–3.55) or SLCO1B3 SNP rs4149117 (HR: 1.76; 95% CI: 1.00–3.08) had an increased risk of PCSM.Conclusions: CRPC metastases show increased expression of SLCO genes versus primary prostate cancer. Genetic variants of SLCO1B3 and SLCO2B1 are associated with PCSM. Expression and genetic variation of SLCO genes which alter androgen uptake may be important in prostate cancer outcomes.Impact: OATP/SLCO genes may be potential biomarkers for assessing risk of PCSM. Expression and genetic variation in these genes may allow stratification of patients to more aggressive hormonal therapy or earlier incorporation of nonhormonal-based treatment strategies. Cancer Epidemiol Biomarkers Prev; 20(4); 619–27. ©2011 AACR.

Supplementary Data Supplementary Table S1 Characteristics of the patient-derived xenograft models and protein expression by immunohistochemistry Supplementary Table S2. Primer sequences used in real-time PCR analysis Supplementary Table S3. Microarray gene expression analysis of steroidogenic genes in AA- versus vehicle-treated tumors upon AAresistance (end of study, EOS) Supplementary Table S4. Castration effects (vs. intact) on LuCaP PDX AR gene expression Supplementary Table S5. Upregulated genes in both ultraresponsive LuCaP 136CR and minimal responder LuCaP 35CR: lack of involvement in AR/GR signaling Supplementary Fig. S1. Treatment scheme and survival curves for AA on CRPC PDXs. Castration-resistant tumors were developed by either A, implanting castration-resistant tumors into castrated SCID mice (LuCaP 70CR), or B, implanting androgen-sensitive tumors into intact SCID mice and castrating mice when the tumor reached 100 mm3 (LuCaP 86.2CR). Castrated mice with castration-resistant tumors were treated orally with either vehicle or AA (0.5 mmol/kg/day) on a weekly schedule of 5 days on, 2 days off. Tumors were measured twice weekly. Mice were sacrificed and tumors were harvested on D7 or when tumors reached 1,000 mm3 (EOS). Supplementary Fig. S2. Differential gene expression associated with secreted proteins in the AA ultraresponder LuCaP 136CR. A, Chart showing 44% (68 genes) of 156 genes that were differentially expressed in the ultraresponder LuCaP 136CR (vs. intermediate responder LuCaP 96CR and minimal responder LuCaP 35CR) and were mapped into gene family coding for secreted proteins. Supervised clustering analyses showing 68 genes coding for secreted proteins at B, D7 and C, EOS. Red: high gene expression; green: low gene expression. Supplementary Fig. S3. Heat map showing the top 10 upregulated and top 10 downregulated genes representing secreted proteins in the LuCaP models at D7 and EOS. Yellow: high gene expression; blue: low gene expression. The heat map showed a homogeneous upregulation of the top 10 upregulated genes in LuCaP 136CR and a heterogeneous downregulation of the top 10 downregulated genes in LuCaP 136CR. Supplementary Fig. S4. qPCR confirmation on the top 10 downregulated genes of secreted proteins identified in the AA ultraresponder LuCaP 136CR versus intermediate responder LuCaP 77CR and LuCaP 96CR, and minimal responder LuCaP 35CR. Each data point represented one animal. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Supplementary Fig. S5. GSEA showing the association between differentially expressed genes upon AA treatment (D7) and resistance (EOS) in the LuCaP models with biological functions. False discovery rate

Purpose: ErbB2 signaling appears to be increased and may enhance androgen receptor (AR) activity in a subset of patients with castration-resistant prostate cancer (CRPC), but agents targeting ErbB2 have not been effective. This study was undertaken to assess ErbB2 activity in abiraterone-resistant prostate cancer and to determine whether it may contribute to AR signaling in these tumors.Experimental Design: AR activity and ErbB2 signaling were examined in the radical prostatectomy specimens from a neoadjuvant clinical trial of leuprolide plus abiraterone and in the specimens from abiraterone-resistant CRPC xenograft models. The effect of ErbB2 signaling on AR activity was determined in two CRPC cell lines. Moreover, the effect of combination treatment with abiraterone and an ErbB2 inhibitor was assessed in a CRPC xenograft model.Results: We found that ErbB2 signaling was elevated in residual tumor following abiraterone treatment in a subset of patients and was associated with higher nuclear AR expression. In xenograft models, we similarly demonstrated that ErbB2 signaling was increased and associated with AR reactivation in abiraterone-resistant tumors. Mechanistically, we show that ErbB2 signaling and subsequent activation of the PI3K/AKT signaling stabilizes AR protein. Furthermore, concomitantly treating CRPC cells with abiraterone and an ErbB2 inhibitor, lapatinib, blocked AR reactivation and suppressed tumor progression.Conclusions: ErbB2 signaling is elevated in a subset of patients with abiraterone-resistant prostate cancer and stabilizes AR protein. Combination therapy with abiraterone and ErbB2 antagonists may be effective for treating the subset of CRPC with elevated ErbB2 activity. Clin Cancer Res; 22(14); 3672–82. ©2016 AACR.

Residual cancer burden (RCB) from each patient in the (A) enza and (B) enza/dut/LHRHa arms.

Purpose: Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer that progresses after castration (castration-resistant prostate cancer; CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear.Experimental Design: Prostate cancer xenografts were examined to identify mechanism of progression after castration and abiraterone.Results: AR reactivation in abiraterone-resistant VCaP xenografts was not associated with restoration of intratumoral androgens or alterations in AR coregulators. In contrast, mRNA encoding full-length AR (AR-FL) and a constitutively active splice variant (AR-V7) were increased compared with xenografts before castration, with an increase in AR-V7 relative to AR-FL. This shift toward AR-V7 was due to a feedback mechanism whereby the androgen-liganded AR stimulates expression of proteins that suppress generation of AR-V7 relative to AR-FL transcripts. However, despite the increases in AR-V7 mRNA, it remained a minor transcript (Conclusions: AR-V7 at these low levels is not adequate to restore AR activity, but its rapid induction after androgen deprivation allows tumors to retain basal AR activity that may be needed for survival until more potent mechanisms emerge to activate AR. Agents targeting AR splice variants may be most effective when used very early in conjunction with therapies targeting the AR ligand-binding domain. Clin Cancer Res; 20(6); 1590–600. ©2014 AACR.

Purpose: Abiraterone is a potent inhibitor of the steroidogenic enzyme CYP17A1 and suppresses tumor growth in patients with castration-resistant prostate cancer (CRPC). The effectiveness of abiraterone in reducing tumor androgens is not known, nor have mechanisms contributing to abiraterone resistance been established.Experimental Design: We treated human CRPC xenografts with abiraterone and measured tumor growth, tissue androgens, androgen receptor (AR) levels, and steroidogenic gene expression versus controls.Results: Abiraterone suppressed serum PSA levels and improved survival in two distinct CRPC xenografts: median survival of LuCaP35CR improved from 17 to 39 days (HR = 3.6, P = 0.0014) and LuCaP23CR from 14 to 24 days (HR = 2.5, P = 0.0048). Abiraterone strongly suppressed tumor androgens, with testosterone (T) decreasing from 0.49 ± 0.22 to 0.03 ± 0.01 pg/mg (P < 0.0001), and from 0.69 ± 0.36 to 0.03 ± 0.01 pg/mg (P = 0.002) in abiraterone-treated 23CR and 35CR, respectively, with comparable decreases in tissue DHT. Treatment was associated with increased expression of full-length AR (ARFL) and truncated AR variants (ARFL 2.3-fold, P = 0.008 and ARdel567es 2.7-fold, P = 0.036 in 23 CR; ARFL 3.4-fold, P = 0.001 and ARV7 3.1-fold, P = 0.0003 in 35CR), and increased expression of the abiraterone target CYP17A1 (∼2.1-fold, P = 0.0001 and P = 0.028 in 23CR and 35CR, respectively) and transcript changes in other enzymes modulating steroid metabolism.Conclusions: These studies indicate that abiraterone reduces CRPC growth via suppression of intratumoral androgens and that resistance to abiraterone may occur through mechanisms that include upregulation of CYP17A1, and/or induction of AR and AR splice variants that confer ligand-independent AR transactivation. Clin Cancer Res; 17(18); 5913–25. ©2011 AACR.

Supplemental Methods, Figure Legends, Table Legends, References

Figure S1. Targeted siRNA knockdown of DPP4 decreases DPP4 antibody positivity in western blots and IHC; Figure S2. Immunoscoring of AR, AR-V7, P-AR(S81), and PSA protein expression in serially biopsied VCaP xenografts; Figure S3. Ingenuity Pathway, Hierarchical Clustering, and Principal Component analysis of Pre-Cx, CRPC, and AER, serial biopsies; Figure S4. DPP4 is an AR-stimulated gene; Figure S5. DPP4 expression is decreased in Post-ADT therapy samples in several clinical and one preclinical data set compared to Pre-Cx; Figure S6. DPP4 expression is decreased in clinical CRPC sections, despite normal levels of PSA; Figure S7. CpG islands in the DPP4 promoter and exon 1 are unmethylated; Figure S8. lncRNA-OIS1 is expressed at similar levels in Pre-Cx and AER VCaP tumors; Figure S9. Androgen treatment of VCaP CRPC xenografts restores DPP4 expression; Figure S10. The volume ratio (End Volume/Starting Volume) of VCaP tumors shows a trend toward positive correlation with tumor DPP4 protein levels.

Supplementary Methods and Supplementary Tables 1-3

Supplementary Figures and Tables Supplementary Table S1. Baseline Patient Demographic and Clinical Characteristics Supplementary Table S2. Observed and Expected Hardy Weinberg (HW) Frequency of SLCO Genotypes Supplementary Table S3. Distribution of Men with Undetectable Tissue ABI Levels Supplementary Table S4. Characteristics of SLCO Genotypes and Potential Prostate Cancer Associations Supplementary Table S5. Association of SLCO Genotype with Tissue Abiraterone, Estimated Tumor Volume and Minimal Residual Disease (MRD) Supplementary Figure S1. Representative chromatograms showing retention time for abiraterone standard and tissue samples. Supplementary Figure S2. Serum steroid levels in samples with undetectable vs detectable prostate abiraterone levels. Supplementary Figure S3. Association of SLCO single nucleotide polymorphisms with serum or tissue steroid levels. Supplementary Figure S4. Absolute abiraterone levels measured in LNCaP cells overexpressing SLCO2B1 or SLCO1B3.

The file contains supplemental details related to real-time quantitative PCR, RNA sequencing data analysis, meta-analysis of DDP4 expression and AR score across multiple prostate cancer datasets, immunohistochemistry analysis (AR, AR-V7, pAR(S81), and PSA expression), and promoter methylation analysis. It also contains 9 references related to these methods.

Black and white figure for supplement.

Purpose: To identify the molecular signature associated with abiraterone acetate (AA) response and mechanisms underlying AA resistance in castration-resistant prostate cancer patient-derived xenografts (PDXs).Experimental Design: SCID mice bearing LuCaP 136CR, 77CR, 96CR, and 35CR PDXs were treated with AA. Tumor volume and prostate-specific antigen were monitored, and tumors were harvested 7 days after treatment or at end of study for gene expression and immunohistochemical studies.Results: Three phenotypic groups were observed based on AA response. An ultraresponsive phenotype was identified in LuCaP 136CR with significant inhibition of tumor progression and increased survival, intermediate responders LuCaP 77CR and LuCaP 96CR with a modest tumor inhibition and survival benefit, and LuCaP 35CR with minimal tumor inhibition and no survival benefit upon AA treatment. We identified a molecular signature of secreted proteins associated with the AA ultraresponsive phenotype. Upon resistance, AA ultraresponder LuCaP 136CR displayed reduced androgen receptor (AR) signaling and sustainably low nuclear glucocorticoid receptor (nGR) localization, accompanied by steroid metabolism alteration and epithelial–mesenchymal transition phenotype enrichment with increased expression of NF-κB–regulated genes; intermediate and minimal responders maintained sustained AR signaling and increased tumoral nGR localization.Conclusions: We identified a molecular signature of secreted proteins associated with AA ultraresponsiveness and sustained AR/GR signaling upon AA resistance in intermediate or minimal responders. These data will inform development of noninvasive biomarkers predicting AA response and suggest that further inhibition along the AR/GR signaling axis may be effective only in AA-resistant patients who are intermediate or minimal responders. These findings require verification in prospective clinical trials. Clin Cancer Res; 23(9); 2301–12. ©2016 AACR.

Primary prostate cancer can have extensive microheterogeneity, but its contribution to the later emergence of metastatic castration-resistant prostate cancer (mCRPC) remains unclear. In this study, we microdissected residual prostate cancer foci in radical prostatectomies from 18 men treated with neoadjuvant-intensive androgen deprivation therapy (leuprolide, abiraterone acetate, and prednisone) and analyzed them for resistance mechanisms. Transcriptome profiling showed reduced but persistent androgen receptor (AR) activity in residual tumors, with no increase in neuroendocrine differentiation. Proliferation correlated negatively with AR activity but positively with decreased RB1 expression, and whole-exome sequencing (WES) further showed enrichment for RB1 genomic loss. In 15 cases where 2 or 3 tumor foci were microdissected, WES confirmed a common clonal origin but identified multiple oncogenic alterations unique to each focus. These findings show that subclones with oncogenic alterations found in mCRPC are present in primary prostate cancer and are selected for by neoadjuvant-intense androgen deprivation therapy. In particular, this study indicates that subclonal RB1 loss may be more common than previously appreciated in intermediate- to high-risk primary prostate cancer and may be an early event, independent of neuroendocrine differentiation, in the development of mCRPC. Comprehensive molecular analyses of primary prostate cancer may detect aggressive subclones and possibly inform adjuvant strategies to prevent recurrence.Significance: Neoadjuvant androgen deprivation therapy for prostate cancer selects for tumor foci with subclonal genomic alterations, which may comprise the origin of metastatic castration-resistant prostate cancer. Cancer Res; 78(16); 4716–30. ©2018 AACR.

Supplementary Tables 1-2 from Intraprostatic Androgens and Androgen-Regulated Gene Expression Persist after Testosterone Suppression: Therapeutic Implications for Castration-Resistant Prostate Cancer

Supplementary Table 3 from Intraprostatic Androgens and Androgen-Regulated Gene Expression Persist after Testosterone Suppression: Therapeutic Implications for Castration-Resistant Prostate Cancer

Supplementary Table Legends 1-2 from Intraprostatic Androgens and Androgen-Regulated Gene Expression Persist after Testosterone Suppression: Therapeutic Implications for Castration-Resistant Prostate Cancer

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. [Cancer Res 2008;68(11):4447–54]

Androgen deprivation therapy (ADT) remains the primary treatment for advanced prostate cancer. The efficacy of ADT has not been rigorously evaluated by demonstrating suppression of prostatic androgen activity at the target tissue and molecular level. We determined the efficacy and consistency of medical castration in suppressing prostatic androgen levels and androgen-regulated gene expression. Androgen levels and androgen-regulated gene expression (by microarray profiling, quantitative reverse transcription-PCR, and immunohistochemistry) were measured in prostate samples from a clinical trial of short-term castration (1 month) using the gonadotropin-releasing hormone antagonist, Acyline, versus placebo in healthy men. To assess the effects of long-term ADT, gene expression measurements were evaluated at baseline and after 3, 6, and 9 months of neoadjuvant ADT in prostatectomy samples from men with localized prostate cancer. Medical castration reduced tissue androgens by 75% and reduced the expression of several androgen-regulated genes (NDRG1, FKBP5, and TMPRSS2). However, many androgen-responsive genes, including the androgen receptor (AR) and prostate-specific antigen (PSA), were not suppressed after short-term castration or after 9 months of neoadjuvant ADT. Significant heterogeneity in PSA and AR protein expression was observed in prostate cancer samples at each time point of ADT. Medical castration based on serum testosterone levels cannot be equated with androgen ablation in the prostate microenvironment. Standard androgen deprivation does not consistently suppress androgen-dependent gene expression. Suboptimal suppression of tumoral androgen activity may lead to adaptive cellular changes allowing prostate cancer cell survival in a low androgen environment. Optimal clinical efficacy will require testing of novel approaches targeting complete suppression of systemic and intracrine contributions to the prostatic androgen microenvironment. [Cancer Res 2007;67(10):5033–41]

PDF file - 100K, Supplemental Table 1. Expression correlation of AR and AR-V to selected AR target genes in LuCaP xenografts. Supplemental Table II. Protein expression levels in 67 CRPC TMA specimens

Supplementary Tables 1-2 from Maintenance of Intratumoral Androgens in Metastatic Prostate Cancer: A Mechanism for Castration-Resistant Tumor Growth

PDF file - 971K, Supplemental Figure 1. PSA promoter activity in LNCaP and LNCaP 95 cells Supplemental Figure 2-12. Analysis of AR-FL and AR-V protein and mRNA expression and the effect of their expression changes on target genes by gene set enrichment analysis. Supplemental Figure 13. Lack of AR-V7 response by androgen deprivation in LNCaP and CWR22Rv1 cells. Supplemental Figure 14. Nuclear staining for AR-FL, AR-V7, and UBE2C in CRPC specimens.

Supplementary Table 1 from Reduced Tumor Necrosis Factor Receptor–Associated Death Domain Expression Is Associated with Prostate Cancer Progression

Supplementary Figures 1-10 from Variability in the Androgen Response of Prostate Epithelium to 5α-Reductase Inhibition: Implications for Prostate Cancer Chemoprevention

Supplementary Methods, Figure Legends 1-2 from Reduced Tumor Necrosis Factor Receptor–Associated Death Domain Expression Is Associated with Prostate Cancer Progression

PDF file - 168K