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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.

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.

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.

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.

Mutations in 3 cases with only 1 focus dissected

Somatic copy number alterations

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.

Unique somatic mutations

Supplementary Table S1. Clinicopathologic characteristics of microdissected cases; Supplementary Table S2. Genes up-regulated by 2-fold or more in the treated vs. untreated cases; Supplementary Table S3. Fold change in expression of AR coactivators in treated versus untreated cases; Supplementary Table S4. Genes down-regulated by 2-fold or more in the treated versus untreated cases; Supplementary Table S9. Number of genomic alterations in each focus.

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.

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.

The standard treatment for metastatic prostate cancer (PCa), 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 PCa 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 PCa 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.

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfal) consensus sequence (5'-AACCACA3') at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfal site was inhibited or supershifted by specific antibodies to Cbfal. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2(+/+) mice but not in cells from COX-2(-/-) mice. In vivo, BMP-2 (10 microg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (microCT), was decreased by 78% in COX-2(-/-) mice compared with COX-2(+/+) mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfal binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo.