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1.  Integrating Murine and Clinical Trials with Cabozantinib to Understand Roles of MET and VEGFR-2 as Targets for Growth Inhibition of Prostate Cancer 
We performed parallel investigations in cabozantinib-treated patients in a Phase 2 trial and simultaneously in Patient-derived Xenograft (PDX) models to better understand the roles of MET and VEGFR-2 as targets for prostate cancer therapy.
Experimental Design
In the clinical trial, radiographic imaging and serum markers were examined, as well as molecular markers in tumors from bone biopsies. In mice harboring PDX intrafemurally or subcutaneously, cabozantinib effects on tumor growth, MET, PDX in which MET was silenced, VEGFR-2, bone turnover, angiogenesis and resistance were examined.
In responsive patients and PDX, islets of viable, p-MET-positive tumor cells persisted, which rapidly regrew after drug withdrawal. Knockdown of MET in PDX did not affect tumor growth in mice, nor did it affect cabozantinib-induced growth inhibition, but did lead to induction of FGFR-1. Inhibition of VEGFR-2 and MET in endothelial cells reduced the vasculature, leading to necrosis. However, each islet of viable cells surrounded a VEGFR-2-negative vessel. Reduction of bone turnover was observed in both cohorts.
Our studies demonstrate that MET in tumor cells is not a persistent therapeutic target for metastatic CRPC, but inhibition of VEGF-R2 and MET in endothelial cells and direct effects on osteoblasts are responsible for cabozantinib-induced tumor inhibition. However, vascular heterogeneity represents one source of primary therapy resistance, whereas induction of FGFR-1 in tumor cells suggests a potential mechanism of acquired resistance. Thus, integrated cross-species investigations demonstrate the power of combining preclinical models with clinical trials to understand mechanisms of activity and resistance of investigational agents.
PMCID: PMC4703437  PMID: 26272062
2.  Long-term Safety and Antitumor Activity in the Phase 1–2 Study of Enzalutamide in Pre- and Post-docetaxel Castration-Resistant Prostate Cancer 
European urology  2015;68(5):795-801.
Given that some patients with castration-resistant prostate cancer (CRPC) have shown extended responses to the androgen receptor inhibitor enzalutamide, long-term safety of this drug is of interest.
To evaluate the long-term safety and antitumor activity of enzalutamide in CRPC patients.
Design, setting, and participants
This phase 1–2 study evaluated enzalutamide in 140 CRPC patients with and without prior chemotherapy. Initial findings were published in 2010. We report updated results from an additional 17-mo follow-up for antitumor activity and >4 yr for safety.
Patients received 30–600 mg/d oral enzalutamide. During long-term dosing, all patients were switched first to the maximum tolerated dose of 240 mg/d and then to the phase 3 dose of 160 mg/d.
Outcome measurements and statistical analysis
Safety was assessed regularly. The Kaplan-Meier method was used to estimate the distributions of time to prostate-specific antigen (PSA) progression and time to radiographic progression.
Results and limitations
The safety profile of enzalutamide was consistent over time, with little change in the rates of commonly reported adverse events (AEs) or the incidence of grade 3/4 AEs. Fatigue of any grade was the most common dose-dependent AE, experienced by 70% of patients, with 14% of patients reporting grade 3/4 fatigue. The median time to PSA progression was not reached for chemotherapy-naive patients and was 45 wk for postchemotherapy patients; the corresponding median time to radiographic progression was 56 wk and 25 wk.
Enzalutamide showed durable antitumor activity in chemotherapy-naive and postchemotherapy patients, and was well tolerated, even in patients treated for 4 yr.
Patient summary
Enzalutamide was active against prostate cancer and was well tolerated, even for up to 4 yr of treatment, supporting its potential for long-term use in men with prostate cancer. Fatigue was the most common side effect, occurring at varying degrees of severity in most patients.
PMCID: PMC5013546  PMID: 25698064
Androgen receptor inhibitor; Castration-resistant prostate cancer; Enzalutamide; Long-term follow-up; MDV3100; Tolerability
3.  Targeting of CYP17A1 Lyase by VT-464 Inhibits Adrenal and Intratumoral Androgen Biosynthesis and Tumor Growth of Castration Resistant Prostate Cancer 
Scientific Reports  2016;6:35354.
Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a validated treatment target for the treatment of metastatic castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA) inhibits both 17α-hydroxylase (hydroxylase) and 17,20-lyase (lyase) reactions catalyzed by CYP17A1 and thus depletes androgen biosynthesis. However, coadministration of prednisone is required to suppress the mineralocorticoid excess and cortisol depletion that result from hydroxylase inhibition. VT-464, a nonsteroidal small molecule, selectively inhibits CYP17A1 lyase and therefore does not require prednisone supplementation. Administration of VT-464 in a metastatic CRPC patient presenting with high tumoral expression of both androgen receptor (AR) and CYP17A1, showed significant reduction in the level of both dehydroepiandrosterone (DHEA) and serum PSA. Treatment of a CRPC patient-derived xenograft, MDA-PCa-133 expressing H874Y AR mutant with VT-464, reduced the increase in tumor volume in castrate male mice more than twice as much as the vehicle (P < 0.05). Mass spectrometry analysis of post-treatment xenograft tumor tissues showed that VT-464 significantly decreased intratumoral androgens but not cortisol. VT-464 also reduced AR signaling more effectively than abiraterone in cultured PCa cells expressing T877A AR mutant. Collectively, this study suggests that VT-464 therapy can effectively treat CRPC and be used in precision medicine based on androgen receptor mutation status.
PMCID: PMC5066251  PMID: 27748439
4.  Hedgehog signaling inhibition by the small molecule Smoothened inhibitor GDC-0449 in the bone forming prostate cancer xenograft MDA PCa 118b 
The Prostate  2012;72(15):1638-1647.
Hedgehog signaling is a stromal-mesenchymal pathway central to the development and homeostasis of both the prostate and the bone. Aberrant Hedgehog signaling activation has been associated with prostate cancer aggressiveness. We hypothesize that Hedgehog pathway is a candidate therapeutic target in advanced prostate cancer. We report aberrant Hedgehog signaling in bone metastatic castrate resistant prostate cancer and we examine the pharmacodynamic effect of Smoothened inhibition in an experimental prostate cancer model.
Hedgehog signaling was assessed in tissue microarrays of high grade locally advanced and bone metastatic disease. Male SCID mice subcutaneously injected with the bone forming xenograft MDA PCa 118b were treated with GDC-0449. Hedgehog signaling in the tumor microenvironment was assessed by proteomic and species specific RNA expression and compared between GDC-0449 treated and untreated animals.
We observe Hedgehog signaling activation in high grade locally advanced and bone marrow infiltrating disease. Evidence of paracrine activation of Hedgehog signaling in the tumor xenograft, was provided by increased Sonic Hedgehog expression in human tumor epithelial cells, coupled with increased Gli1 and Patched1 expression in the murine stromal compartment, while normal murine stroma didn’t exhibit Hh signaling expression. GDC-0449 treatment attenuated Hh signaling as evidenced by reduced expression of Gli1 and Ptch1. Reduction in proliferation (Ki67) was observed with no change in tumor volume.
GDC-0449 treatment is pharmacodynamically effective as evidenced by paracrine Hedgehog signaling inhibition and results in tumor cell proliferation reduction. Understanding these observations will inform the clinical development of therapy based on Hedgehog signaling inhibition.
PMCID: PMC4977841  PMID: 22457212
Hh signaling; prostate cancer; MDA PCa 118b
5.  Targeting the Interleukin-11 Receptor α in Metastatic Prostate Cancer: A First-in-man Study 
Cancer  2015;121(14):2411-2421.
Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. We have worked systematically to map human endothelial receptors (“vascular ZIP codes”) within tumors through direct peptide library selection in cancer patients. Previously, we selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature.
We generated a ligand-directed peptidomimetic drug (Bone Metastasis Targeting Peptidomimetic-11; BMTP-11) for IL-11Rα-based human tumor vascular targeting. Pre-clinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effect, PK/PD, and dose-range determination including formal (GLP) toxicity across rodent and non-human primate species. We also report the initial BMTP-11 clinical development: A single-institution, open-label, first-in-class, first-in-man trial (NCT00872157) in metastatic castrate-resistant prostate cancer patients.
BMTP-11 was pre-clinically promising and was therefore chosen for clinical development in patients. We enrolled a limited number of castrate-resistant prostate cancer patients with osteoblastic bone metastases into a phase zero trial with biology-driven endpoints. We demonstrate biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, we identified the MTD on a weekly schedule (20-30 mg/m2). Finally, we determined a renal DLT, namely, dose-dependent reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine.
These biological endpoints establish BMTP-11 as a targeted drug candidate in metastatic castrate-resistant prostate cancer. Within a larger discovery context, our findings indicate functional tumor vascular ligand-receptor targeting systems may be found through direct combinatorial selection of peptide libraries in cancer patients.
PMCID: PMC4490036  PMID: 25832466
BMTP-11; clinical trial; IL-11 receptor α; prostate cancer; vascular targeting
6.  Expression of hedgehog pathway components in prostate carcinoma microenvironment: shifting the balance towards autocrine signalling 
Histopathology  2011;58(7):1037-1047.
The hedgehog (Hh) signalling pathway has been implicated in the pathogenesis and aggressiveness of prostate cancer through epithelial–mesenchymal interactions. The aim of this study was to elucidate the cell-type partitioned expression of the Hh pathway biomarkers in the non-neoplastic and tumour microenvironments and to correlate it with the grade and stage of prostate cancer.
Methods and results
Expression of the Hh pathway components (Shh, Smo, Ptch, Gli1) in the microenvironment of non-neoplastic peripheral zone (n = 119), hormone-naive primary prostate carcinoma (n = 141) and castrate-resistant bone marrow metastases (n = 53) was analysed using immunohistochemistry in tissue microarrays and bone marrow sections. Results showed that epithelial Shh, Smo and Ptch expression was up-regulated, whereas stromal Smo, Ptch, and Gli1 expression was down-regulated in prostate carcinomas compared to non-neoplastic peripheral zone tissue. Ptch expression was modulated further in high-grade and high-stage primary tumours and in bone marrow metastases. Hh signalling correlated with ki67 and vascular endothelial growth factor (VEGF) but not with CD31 expression.
Our results highlight the importance of Hh-mediated epithelial–mesenchymal interactions in the non-neoplastic prostate and imply that shifting the balance from paracrine towards autocrine signalling is important in the pathogenesis and progression of prostate carcinoma.
PMCID: PMC4934422  PMID: 21707705
hedgehog signalling; microenvironment; prostate carcinoma
7.  Tissue Effects in a Randomized Controlled Trial of Short-term Finasteride in Early Prostate Cancer☆ 
EBioMedicine  2016;7:85-93.
In the Prostate Cancer Prevention Trial, finasteride selectively suppressed low-grade prostate cancer and significantly reduced the incidence of prostate cancer in men treated with finasteride compared with placebo. However, an apparent increase in high-grade disease was also observed among men randomized to finasteride. We aimed to determine why and hypothesized that there is a grade-dependent response to finasteride.
From 2007 to 2012, we randomized dynamically by intranet-accessible software 183 men with localized prostate cancer to receive 5 mg finasteride or placebo daily in a double-blind study during the 4–6 weeks preceding prostatectomy. As the primary end point, the expression of a predefined molecular signature (ERβ, UBE2C, SRD5A2, and VEGF) differentiating high- and low-grade tumors in Gleason grade (GG) 3 areas of finasteride-exposed tumors from those in GG3 areas of placebo-exposed tumors, adjusted for Gleason score (GS) at prostatectomy, was compared. We also determined androgen receptor (AR) levels, Ki-67, and cleaved caspase 3 to evaluate the effects of finasteride on the expression of its downstream target, cell proliferation, and apoptosis, respectively. The expression of these markers was also compared across grades between and within treatment groups. Logistic regression was used to assess the expression of markers.
We found that the predetermined molecular signature did not distinguish GG3 from GG4 areas in the placebo group. However, AR expression was significantly lower in the GG4 areas of the finasteride group than in those of the placebo group. Within the finasteride group, AR expression was also lower in GG4 than in GG3 areas, but not significantly. Expression of cleaved caspase 3 was significantly increased in both GG3 and GG4 areas in the finasteride group compared to the placebo group, although it was lower in GG4 than in GG3 areas in both groups.
We showed that finasteride's effect on apoptosis and AR expression is tumor grade dependent after short-term intervention. This may explain finasteride's selective suppression of low-grade tumors observed in the PCPT.
•We studied 183 men with early prostate cancer receiving 5 mg finasteride or placebo daily for 4–6 weeks preprostatectomy.•Expression of androgen receptor and cleaved caspase 3 was dependent on tumor grade.•Finasteride's demonstrated effects in tissue may explain earlier seminal findings in the Prostate Cancer Prevention Trial.
This study advances the fields of prostate cancer biology and prostate cancer treatment because it demonstrates effects of finasteride in prostatectomy tissues that offer an explanation for the mixed results of the 18,882-subject randomized and controlled Prostate Cancer Prevention Trial (PCPT). Initial results showing a 25% reduction in prevalence in prostate cancer in men in the finasteride group while revealing an apparent increase in high-grade disease among those in the same group suggested a grade-associated response to finasteride. The results of this present study indicate finasteride's effects are grade dependent, which may explain the split findings of the PCPT.
PMCID: PMC4909608  PMID: 27322462
GG, Gleason grade; AR, androgen receptor; PCPT, Prostate Cancer Prevention Trial; REDUCE, Reduction by Dutasteride of Prostate Cancer Events; REDEEM, Reduction by Dutasteride of Clinical Progression Events in Expectant Management; DHT, dihydrotestosterone; CRFs, case report forms; DCP, Division of Cancer Prevention; HE, hematoxylin and eosin; ERβ, estrogen receptor β; UBE2C, ubiquitin-conjugating enzyme E2C; SRD5A2, 3-oxo-5α-steroid 4-dehydrogenase 2; VEGF, vascular epithelial growth factor; GS, Gleason score; PZ, peripheral zone; TZ, transition zone; CZ, central zone; Prostate cancer; 5α-reductase inhibitors; Finasteride; Cancer prevention; Biomarkers
8.  Molecular Characterization of Enzalutamide-treated Bone Metastatic Castration-resistant Prostate Cancer 
European urology  2014;67(1):53-60.
Enzalutamide is a novel antiandrogen with proven efficacy in metastatic castration-resistant prostate cancer (mCRPC).
To evaluate enzalutamide’s effects on cancer and on androgens in blood and bone marrow, and associate these with clinical observations.
Design, setting, and participants
In this prospective phase 2 study, 60 patients with bone mCRPC received enzalutamide 160 mg orally daily and had transilial bone marrow biopsies before treatment and at 8 wk of treatment.
Outcome measurements and statistical analysis
Androgen signaling components (androgen receptor [AR], ARV7, v-ets avian erythroblastosis virus E26 oncogene homolog [ERG], cytochrome P450, family 17, subfamily A, polypeptide 1 [CYP17]) and molecules implicated in mCRPC progression (phospho-Met, phospho-Src, glucocorticoid receptor, Ki67) were assessed by immunohistochemistry; testosterone, cortisol, and androstenedione concentrations were assessed by liquid chromatography–tandem mass spectrometry; and AR copy number was assessed by real-time polymerase chain reaction. Descriptive statistics were applied.
Results and limitations
Median time to treatment discontinuation was 22 wk (95% confidence interval, 19.9–29.6). Twenty-two (37%) patients exhibited primary resistance to enzalutamide, discontinuing treatment within 4 mo. Maximal prostate-specific antigen (PSA) decline ≥50% and ≥90% occurred in 27 (45%) and 13 (22%) patients, respectively. Following 8 wk of treatment, bone marrow and circulating testosterone levels increased. Pretreatment tumor nuclear AR overexpression (>75%) and CYP17 (>10%) expression were associated with benefit (p = 0.018). AR subcellular localization shift from the nucleus was confirmed in eight paired samples (with PSA decline) of 23 evaluable paired samples. Presence of an ARV7 variant was associated with primary resistance to enzalutamide (p = 0.018). Limited patient numbers warrant further validation.
The observed subcellular shift of AR from the nucleus and increased testosterone concentration provide the first evidence in humans that enzalutamide suppresses AR signaling while inducing an adaptive feedback. Persistent androgen signaling in mCRPC was predictive of benefit and ARV7 was associated with primary resistance.
Patient summary
We report a first bone biopsy study in metastatic prostate cancer in humans that searched for predictors of outcome of enzalutamide therapy. Benefit is linked to a pretreatment androgen-signaling signature.
PMCID: PMC4247811  PMID: 24882673
Enzalutamide; Antiandrogens; Castration-resistant prostate cancer; Predictors of outcome; Bone metastasis; Bone tumor microenvironment; Adaptive feedback mechanism; Androgen; Androgen receptor; Primary resistance to enzalutamide; Androgen signaling inhibition; Tissue-based research; Bone marrow biopsy
9.  Targeting the interleukin-11 receptor α in metastatic prostate cancer: A first-in-man study 
Cancer  2015;121(14):2411-2421.
Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors (“vascular zip codes”) within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature.
The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα–based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer.
BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m2). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine.
These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients. Cancer 2015;121:2411–2421. © 2015 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.
The authors report on the development of a new ligand-directed peptidomimetic (termed bone metastasis-targeting peptidomimetic-11) for interleukin-11 receptor-based human vascular targeting, including the translation from preclinical studies to a first-in-class, first-in-man clinical trial in patients with metastatic, castrate-resistant prostate cancer.
PMCID: PMC4490036  PMID: 25832466
bone metastasis-targeting peptidomimetic-11; clinical trial; interleukin-11 receptor α; prostate cancer; vascular targeting
10.  Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis 
Oncogene  2014;34(19):2450-2460.
Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, while induction of autophagy decreases HIF2α. The E3 ligase von Hippel Lindau (VHL) and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, while proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear cell renal cell carcinoma (ccRCC) is frequently associated with mono-allelic loss and/or mutation of autophagy related gene ATG7, and low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy plays an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.
PMCID: PMC4286517  PMID: 24998849
RCC; autophagy; proteasome; HIF2α; VHL; p62
11.  Updated Interim Efficacy Analysis and Long-term Safety of Abiraterone Acetate in Metastatic Castration-resistant Prostate Cancer Patients Without Prior Chemotherapy (COU-AA-302) 
European urology  2014;66(5):815-825.
Abiraterone acetate (an androgen biosynthesis inhibitor) plus prednisone is approved for treating patients with metastatic castration-resistant prostate cancer (mCRPC). Study COU-AA-302 evaluated abiraterone acetate plus prednisone versus prednisone alone in mildly symptomatic or asymptomatic patients with progressive mCRPC without prior chemotherapy.
Report the prespecified third interim analysis (IA) of efficacy and safety outcomes in study COU-AA-302.
Design, setting, and participants
Study COU-AA-302, a double-blind placebo-controlled study, enrolled patients with mCRPC from April 2009 to June 2010. A total of 1088 patients were stratified by Eastern Cooperative Oncology Group performance status (0 vs 1).
Patients were randomised 1:1 to abiraterone 1000 mg plus prednisone 5 mg twice daily by mouth versus prednisone.
Outcome measurements and statistical analysis
Co–primary end points were radiographic progression-free survival (rPFS) and overall survival (OS). Median times to event outcomes were estimated using the Kaplan-Meier method. Hazard ratios (HRs) and 95% confidence intervals (CIs) were derived using the Cox model, and treatment comparison used the log-rank test. The O’Brien-Fleming Lan-DeMets α-spending function was used for OS. Adverse events were summarised descriptively.
Results and limitations
With a median follow-up duration of 27.1 mo, improvement in rPFS was statistically significant with abiraterone treatment versus prednisone (median: 16.5 vs 8.2 mo; HR: 0.52 [95% CI, 0.45–0.61]; p < 0.0001). Abiraterone improved OS (median: 35.3 vs 30.1 mo; HR: 0.79 [95% CI, 0.66–0.95]; p = 0.0151) but did not reach the prespecified statistical efficacy boundary (α-level: 0.0035). A post hoc multivariate analysis for OS using known prognostic factors supported the primary results (HR: 0.74 [95% CI, 0.61–0.89]; p = 0.0017), and all clinically relevant secondary end points and patient-reported outcomes improved. While the post hoc nature of the long-term safety analysis is a limitation, the safety profile with longer treatment exposure was consistent with prior reports.
The updated IA of study COU-AA-302 in patients with mCRPC without prior chemotherapy confirms that abiraterone delays disease progression, pain, and functional deterioration and has clinical benefit with a favourable safety profile, including in patients treated for ≥24 mo.
Trial registration
Study COU-AA-302, number, NCT00887198.
PMCID: PMC4418928  PMID: 24647231
Abiraterone acetate; Chemotherapy-naive; Efficacy; Metastatic castration-resistant prostate cancer; Safety
12.  Prostate Cancer Cell–Stromal Cell Cross-Talk via FGFR1 Mediates Antitumor Activity of Dovitinib in Bone Metastases 
Science translational medicine  2014;6(252):252ra122.
Bone is the most common site of prostate cancer (PCa) progression to a therapy-resistant, lethal phenotype. We found that blockade of fibroblast growth factor receptors (FGFRs) with the receptor tyrosine kinase inhibitor dovitinib has clinical activity in a subset of men with castration-resistant PCa and bone metastases. Our integrated analyses suggest that FGF signaling mediates a positive feedback loop between PCa cells and bone cells and that blockade of FGFR1 in osteoblasts partially mediates the antitumor activity of dovitinib by improving bone quality and by blocking PCa cell–bone cell interaction. These findings account for clinical observations such as reductions in lesion size and intensity on bone scans, lymph node size, and tumor-specific symptoms without proportional declines in prostate-specific antigen concentration. Our findings suggest that targeting FGFR has therapeutic activity in advanced PCa and provide direction for the development of therapies with FGFR inhibitors.
PMCID: PMC4407499  PMID: 25186177
13.  Targeting Poly (ADP-Ribose) Polymerase and the c-Myb-TopBP1-ATR-Chk1 Signaling Pathway in Castration-Resistant Prostate Cancer 
Science signaling  2014;7(326):ra47.
Androgen deprivation is the standard systemic treatment for advanced prostate cancer (PCa), but most patients ultimately develop castration-resistance. We show here that MYB is transcriptionally activated by androgen deprivation or impairment of androgen receptor (AR) signaling. MYB gene silencing significantly inhibited PCa growth in vitro and in vivo. Microarray data revealed that c-Myb shares a substantial subset of DNA damage response (DDR) target genes with AR, suggesting that c-Myb may replace AR for the dominant role in the regulation of their common DDR target genes in AR inhibition-resistant or AR-negative PCa. Gene signatures comprising AR, MYB, and their common DDR target genes are significantly correlated with metastasis, castration-resistance, recurrence, and shorter overall survival in PCa patients. We demonstrated in vitro that silencing of MYB, BRCA1 or TOPBP1 synergized with poly (ADP-ribose) polymerase (PARP) inhibitor olaparib (OLA) to increase cytotoxicity to PCa cells. We further demonstrated that targeting the c-Myb-TopBP1-ATR-Chk1 pathway by using the Chk1 inhibitor AZD7762 synergizes with OLA to increase PCa cytotoxicity. Our results reveal new mechanism-based therapeutic approaches for PCa by targeting PARP and the c-Myb-TopBP1-ATR-Chk1 pathway.
PMCID: PMC4135429  PMID: 24847116
14.  Androgenic Biomarker Profiling in Human Matrices and Cell Culture Samples Using High Through put, Electrospray Tandem Mass Spectrometry 
The Prostate  2014;74(7):722-731.
A high throughput, high pressure liquid chromatographic (HPLC) method with triple quadrupole mass spectral detection (LC/MS/MS) was validated for the measurement of 5 endogenous androgens in human plasma and serum and applied to various in vivo and in vitro study samples to pursue a better understanding of the interrelationship of the androgen axis, intracrine metabolism, and castration-recurrent prostate cancer (CaP).
A Shimadzu HPLC system interfaced with a Sciex QTRAP 5500 mass spectrometer with electrospray ionization was used with inline column-switching. Samples were liquid/liquid extracted and chromatographed on a Luna C18(2) column at 60°C with a biphasic gradient using a 15-min run time.
The method was validated for five androgens in human plasma and serum, and applied to four sets of samples. Plasma (n = 188) and bone marrow aspirate (n = 129) samples from patients with CaP, who received abiraterone acetate plus prednisone for up to 945 days (135 weeks), had undetectable androgens after 8 weeks of treatment. Plasma dehydroepiandrosterone (DHEA) concentrations were higher in African Americans than Caucasian Americans with newly diagnosed CaP. Analysis of prostate tumor tissue homogenates demonstrated reproducible testosterone (T) and dihydrotestosterone (DHT) concentrations with a minimal sample size of ~1.0–2.0 mg of tissue. Finally, cell pellet and media samples from the LNCaP C4-2 cell line showed conversion of T to DHT.
The proposed LC/MS/MS method was validated for quantitation of five endogenous androgens in human plasma and serum, and effectively profiles androgens in clinical specimens and cell culture samples.
PMCID: PMC4335642  PMID: 24847527
androgen axis; prostate cancer; testosterone; dihydrotestosterone; steroid 5α-reductase; LC/MS/MS
15.  Phase III, Randomized, Double-Blind, Multicenter Trial Comparing Orteronel (TAK-700) Plus Prednisone With Placebo Plus Prednisone in Patients With Metastatic Castration-Resistant Prostate Cancer That Has Progressed During or After Docetaxel-Based Therapy: ELM-PC 5 
Journal of Clinical Oncology  2015;33(7):723-731.
Orteronel (TAK-700) is an investigational, nonsteroidal, reversible, selective 17,20-lyase inhibitor. This study examined orteronel in patients with metastatic castration-resistant prostate cancer that progressed after docetaxel therapy.
Patients and Methods
In our study, 1,099 men were randomly assigned in a 2:1 schedule to receive orteronel 400 mg plus prednisone 5 mg twice daily or placebo plus prednisone 5 mg twice daily, stratified by region (Europe, North America [NA], and non-Europe/NA) and Brief Pain Inventory–Short Form worst pain score. Primary end point was overall survival (OS). Key secondary end points (radiographic progression-free survival [rPFS], ≥ 50% decrease of prostate-specific antigen [PSA50], and pain response at 12 weeks) were to undergo statistical testing only if the primary end point analysis was significant.
The study was unblinded after crossing a prespecified OS futility boundary. The median OS was 17.0 months versus 15.2 months with orteronel-prednisone versus placebo-prednisone (hazard ratio [HR], 0.886; 95% CI, 0.739 to 1.062; P = .190). Improved rPFS was observed with orteronel-prednisone (median, 8.3 v 5.7 months; HR, 0.760; 95% CI, 0.653 to 0.885; P < .001). Orteronel-prednisone showed advantages over placebo-prednisone in PSA50 rate (25% v 10%, P < .001) and time to PSA progression (median, 5.5 v 2.9 months, P < .001) but not pain response rate (12% v 9%; P = .128). Adverse events (all grades) were generally more frequent with orteronel-prednisone, including nausea (42% v 26%), vomiting (36% v 17%), fatigue (29% v 23%), and increased amylase (14% v 2%).
Our study did not meet the primary end point of OS. Longer rPFS and a higher PSA50 rate with orteronel-prednisone indicate antitumor activity.
PMCID: PMC4879718  PMID: 25624429
16.  Molecular Classification of Prostate Cancer Progression: Foundation for Marker driven-Treatment of Prostate Cancer 
Cancer discovery  2013;3(8):849-861.
Recently, many therapeutic agents for prostate cancer (PCa) have been approved that target the androgen receptor and/or the prostate tumor microenvironment. Each of these therapies has modestly increased patient survival. However, if a better understanding as to when in the course of PCa progression specific therapies should be applied, and what biomarkers would indicate when resistance arises, survival due to these therapies would almost certainly improve. Thus, applying the armamentarium of therapeutic agents in the right sequences in the right combination at the right time is a major goal in prostate cancer treatment. For this to occur, an understanding of prostate cancer evolution during progression is required. In this review, we discuss the current understanding of PCa progression, but challenge the prevailing view by proposing a new model of PCa progression, with the goal of improving biologic classification and treatment strategies. We use this model to discuss how integrating clinical and basic understanding of PCa will lead to better implementation of molecularly-targeted therapeutics and improve patient survival.
PMCID: PMC3926428  PMID: 23811619
17.  Glucocorticoid Receptor Confers Resistance to Anti-Androgens by Bypassing Androgen Receptor Blockade 
Cell  2013;155(6):1309-1322.
The treatment of advanced prostate cancer has been transformed by novel antiandrogen therapies such as enzalutamide. Here we identify induction of glucocorticoid receptor (GR) expression as a common feature of drug resistant tumors in a credentialed preclinical model, a finding also confirmed in patient samples. GR substituted for the androgen receptor (AR) to activate a similar but distinguishable set of target genes and was necessary for maintenance of the resistant phenotype. The GR agonist dexamethasone was sufficient to confer enzalutamide resistance whereas a GR antagonist restored sensitivity. Acute AR inhibition resulted in GR upregulation in a subset of prostate cancer cells due to relief of AR-mediated feedback repression of GR expression. These findings establish a novel mechanism of escape from AR blockade through expansion of cells primed to drive AR target genes via an alternative nuclear receptor upon drug exposure.
PMCID: PMC3932525  PMID: 24315100
18.  Modeling A Lethal Prostate Cancer Variant with Small Cell Carcinoma Features 
Small-cell prostate carcinoma (SCPC) morphology predicts for a distinct clinical behavior, resistance to androgen ablation, and frequent but short responses to chemotherapy. We sought to develop model systems that reflect human SCPC and can improve our understanding of its biology.
Experimental Design
We developed a set of CRPC xenografts and examined their fidelity to their human tumors of origin. We compared the expression and genomic profiles of SCPC and large cell neuroendocrine carcinoma (LCNEC) xenografts to those of typical prostate adenocarcinoma xenografts. Results were validated immunohistochemically in a panel of 60 human tumors.
The reported SCPC and LCNEC xenografts retain high fidelity to their human tumors of origin and are characterized by a marked upregulation of UBE2C and other mitotic genes in the absence of AR, retinoblastoma (RB1) and cyclin D1 (CCND1) expression. We confirmed these findings in a panel of CRPC patients' samples. In addition, array comparative genomic hybridization of the xenografts showed that the SCPC/LCNEC tumors display more copy number variations than the adenocarcinoma counterparts. Amplification of the UBE2C locus and microdeletions of RB1 were present in a subset, but none displayed AR nor CCND1 deletions. The AR, RB1, and CCND1 promoters showed no CpG methylation in the SCPC xenografts.
Modeling human prostate carcinoma with xenografts allows in-depth and detailed studies of its underlying biology. The detailed clinical annotation of the donor tumors enables associations of anticipated relevance to be made. Futures studies in the xenografts will address the functional significance of the findings.
PMCID: PMC3923417  PMID: 22156612
castration resistance prostate cancer; small cell neuroendocrine carcinoma; retinoblastoma; UBE2C; cyclin D1
19.  A New Therapy Paradigm for Prostate Cancer Founded on Clinical Observations 
Efficacy equivalent to that reported in other common adult solid tumors considered to be chemotherapy-sensitive has been reported with Docetaxel in patients with castrate-resistant prostate cancer. However, in contrast to other cancers, the expected increase in efficacy with the use of chemotherapy in earlier disease states has not been reported to date in prostate cancer. On the basis of these observations, we speculated that the therapy development paradigm used successfully in other cancers may not apply to the majority of prostate cancers. Several lines of supporting clinical and experimental observations implicate the tumor microenvironment in prostate carcinogenesis and resistance to therapy.
We conclude that a foundation to guide the development of therapy for prostate cancer is required. The therapy paradigm we propose accounts for the central role of the tumor microenvironment in bone and, if correct, will lead to microenvironment-targeted therapy.
PMCID: PMC3891669  PMID: 20145177
20.  Randomized Phase 3 Trial of Abiraterone Acetate in Men with Metastatic Castration-Resistant Prostate Cancer and No Prior Chemotherapy 
The New England journal of medicine  2012;368(2):138-148.
Abiraterone acetate, an androgen biosynthesis inhibitor, improves overall survival (OS) in metastatic castration-resistant prostate cancer (mCRPC) post-chemotherapy. Many mCRPC patients never receive chemotherapy and thus cannot benefit from abiraterone acetate; we evaluated this agent in mCRPC patients who had not received chemotherapy.
In this double-blind study, 1088 patients were randomized 1:1 to abiraterone acetate (1000 mg) plus prednisone (5 mg twice daily) or placebo plus prednisone. Co-primary end points were radiographic progression-free survival (rPFS) and OS. Secondary end points measured clinically relevant landmarks of mCRPC progression. Patient-reported outcomes included pain progression and quality of life.
The study was unblinded after a planned interim analysis (IA) at 43% of OS events. Treatment with abiraterone acetate-prednisone resulted in a 57% reduction in the risk of radiographic progression or death (hazard ratio [HR], 0.43; 95% confidence interval [CI]: 0.35 to 0.52; P<0.001; 13% OS events IA) and an estimated 25% decrease in the risk of death (HR, 0.75; 95% CI: 0.61 to 0.93; P=0.009; 43% OS events IA). Secondary end points supported superiority of abiraterone acetate-prednisone: time to cytotoxic chemotherapy initiation, opiate use for cancer-related pain, prostate-specific antigen progression (all P<0.001) and performance status deterioration (P=0.005). Self-reported time to pain progression and patient functional status degradation favored abiraterone acetate-prednisone (P=0.05 and P=0.003). Grade 3/4 mineralocorticoid-related adverse events and liver function test abnormalities were more common with abiraterone acetate-prednisone.
Abiraterone acetate produces OS and rPFS benefits, as well as significant delays in clinical deterioration and initiation of chemotherapy, in mCRPC.
PMCID: PMC3683570  PMID: 23228172
Abiraterone acetate; prednisone; metastatic castration-resistant prostate cancer; androgen; CYP17
21.  Integrated Hedgehog signaling is induced following castration in human and murine prostate cancers 
The Prostate  2012;73(2):10.1002/pros.22550.
The interplay between androgen and Hedgehog (Hh) signaling pathways may be associated with prostate cancer progression and resistance to therapy.
Tissue microarrays from prostatectomy specimens were derived from 53 patients treated preoperatively with androgen ablation (AA) with or without chemotherapy, and from 26 stage- and grade-matched controls. A previously characterized androgen-regulated human prostate cancer xenograft was used to conduct parallel murine studies. Expression of markers of interest was determined on both untreated and castrated tumors.
Four-month exposure to AA or AA with chemotherapy led to a uniform increase in Hh signaling as compared to controls, paired with an inverse trend of androgen receptor (AR) and CYP17 expression in clinically derived specimens. Changes in the expression profiles of Hh signaling were observed in the epithelium and stroma, in response to genotoxic stress of androgen ablation and chemotherapy. A reduced expression of KI67and increased bcl2 expression was observed in the malignant epithelial compartment.
To our knowledge, this is the first clinical evidence that Hh signaling is induced by AA or the combination of AA and chemotherapy and, by inference, contributes to castrate-resistant progression of prostate cancer as supported by parallel human and murine studies. These data are in agreement with previous reports that implicate Hh signaling in castrate-resistant progression of prostate cancer. Based on these findings, we are pursuing parallel clinical and murine investigations to determine if Hh signaling inhibition combined with AA will be more effective than AA alone.
PMCID: PMC3878994  PMID: 22753310
Prostate cancer; preoperative treatment; Hedgehog signaling; resistance to treatment; androgen ablation
22.  Recurrent seminomas: Clinical features and biologic implications 
Urologic oncology  2010;30(4):10.1016/j.urolonc.2010.05.011.
Certain patients with seminoma and clinically atypical phenotypes—visceral metastases, elevated levels of βhuman chorionic gonadotropin (βHCG), and/or recurrent disease— have a poor prognosis. The primary goal of this pilot study was to characterize the clinical characteristics and treatment profile of these rare patients. We also wished to test whether these tumors expressed any specific biomarkers that might distinguish them as a unique subtype of seminoma.
Materials and methods
We retrospectively identified 25 patients with a history of seminoma plus visceral metastases, βHCG levels >200 mU/ml, and/or recurrent disease. We reviewed these patients’ histories for treatment efficacy and clinical outcome. Tissue samples were available from 6 of those patients, and we studied them for expression of the markers OCT 3/4, PLAP, CD30, TRA-1-60, c-kit, and gp200. We compared our results with the expression of those markers in tissue samples from mixed seminoma/embryonal carcinomas and classic seminomas.
Our analysis suggested that certain chemotherapeutic regimens (such as ifosfamide, paclitaxel, and cisplatin) are efficacious for the treatment of patients with these atypical seminomas. Further, specimens from the atypical seminomas generally had staining profiles that resembled those of classic seminomas and the seminoma components in mixed germ-cell tumors, but the profiles differed from those of the embryonal carcinoma components in the same mixed germ-cell tumors.
Although these atypical seminomas tend to be resistant to chemotherapy, they may still respond to certain chemotherapeutic regimens. Our pilot immunohistochemical study also suggested that the unique phenotypes associated with these atypical seminomas do not result from any relationship with embryonal carcinomas. More study is needed to confirm these initial findings.
PMCID: PMC3856193  PMID: 20822932
Seminoma; Testicular neoplasms; Embryonal carcinoma; TRA-1-60; gp200
23.  Phase II Study of Abiraterone Acetate Plus Prednisone in Chemotherapy-Naïve Metastatic Castration-Resistant Prostate Cancer Demonstrating Radiographic Flare Discordant With Serologic Measures of Response 
Abiraterone is an oral inhibitor of CYP17, essential for androgen biosynthesis. This multicenter study assessed its efficacy in patients with CRPC without prior exposure to chemotherapy or CYP17 targeted therapy, and assessed the frequency of interpretation of bone scans discordant with PSA and clinical response.
Patients and Methods
33 patients received abiraterone acetate 1000 mg daily with prednisone 5 mg twice daily in continuous 28-day cycles. Patients were evaluated monthly for efficacy and safety. Bone scan flare was defined as the combination, after 3 months of therapy, of an interpreting radiologist's report indicating “disease progression” in the context of a ≥50% decline in PSA, with scan improvement 3 months later.
A ≥ 50% PSA decline at week 12 was confirmed in 22/33 (67%) patients. PSA declines of ≥ 50% were seen in 26 (79%) patients. Undetectable PSA levels (≤ 0.1 ng/mL) occurred in 2 patients. Median time on therapy and time to PSA progression are 63 and 71 weeks, respectively. Twenty three patients were evaluable for bone scan flare. Progression was indicated in the radiologist's report in 12/23 (52 %), and 10/12 subsequently showed improvement. As prospectively defined, bone scan flare was observed in 10/23 (43.5%) evaluable patients or 10/33 (30%) enrolled patients. Adverse events were typically grade 1/2 and consistent with prior published abiraterone reports.
Clinical response to abiraterone acetate plus prednisone was frequent and durable in men with metastatic CRPC progressing on hormonal therapy with over half of patients on therapy > 1 year. Further investigation is needed to clarify the potential confounding effect of the frequently occurring bone scan flare phenomena on patient management and interpretation of clinical trial results.
PMCID: PMC3657705  PMID: 21632851
abiraterone acetate; castration-resistant prostate cancer; CRPC; hormone-resistant prostate cancer; therapy; efficacy
24.  Effects of Abiraterone Acetate on Androgen Signaling in Castrate-Resistant Prostate Cancer in Bone 
Journal of Clinical Oncology  2011;30(6):637-643.
Persistent androgen signaling is implicated in castrate-resistant prostate cancer (CRPC) progression. This study aimed to evaluate androgen signaling in bone marrow–infiltrating cancer and testosterone in blood and bone marrow and to correlate with clinical observations.
Patients and Methods
This was an open-label, observational study of 57 patients with bone-metastatic CRPC who underwent transiliac bone marrow biopsy between October 2007 and March 2010. Patients received oral abiraterone acetate (1 g) once daily and prednisone (5 mg) twice daily. Androgen receptor (AR) and CYP17 expression were assessed by immunohistochemistry, testosterone concentration by mass spectrometry, AR copy number by polymerase chain reaction, and TMPRSS2-ERG status by fluorescent in situ hybridization in available tissues.
Median overall survival was 555 days (95% CI, 440 to 965+ days). Maximal prostate-specific antigen decline ≥ 50% occurred in 28 (50%) of 56 patients. Homogeneous, intense nuclear expression of AR, combined with ≥ 10% CYP17 tumor expression, was correlated with longer time to treatment discontinuation (> 4 months) in 25 patients with tumor-infiltrated bone marrow samples. Pretreatment CYP17 tumor expression ≥ 10% was correlated with increased bone marrow aspirate testosterone. Blood and bone marrow aspirate testosterone concentrations declined to less than picograms-per-milliliter levels and remained suppressed at progression.
The observed pretreatment androgen-signaling signature is consistent with persistent androgen signaling in CRPC bone metastases. This is the first evidence that abiraterone acetate achieves sustained suppression of testosterone in both blood and bone marrow aspirate to less than picograms-per-milliliter levels. Potential admixture of blood with bone marrow aspirate limits our ability to determine the origin of measured testosterone.
PMCID: PMC3295561  PMID: 22184395
25.  Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells 
Clinical Cancer Research  2012;18(3):726-736.
To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction.
Experimental Design
We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs.
Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling.
We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients.
PMCID: PMC3271798  PMID: 22298898
beta-catenin; prostate cancer; androgen receptor; hyaluronan synthase

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