Emma S. Guns

Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer.

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

Alterations in cholesterol regulation contribute to the production of intratumoral androgens during progression to castration‐resistant prostate cancer in a mouse xenograft model

Emerging evidence suggests that androgens and the androgen receptor (AR) are important mediators of castration‐resistant prostate cancer (CRPC) progression. Increased expression of several enzymes responsible for cholesterol synthesis and conversion into downstream androgens has been documented in human CRPC tumors in comparison to primary tumors. Based on these observations it is hypothesized that cholesterol and its overall regulation within the cell are altered, thus modifying precursor levels for de novo androgen synthesis within the castrate tumoral environment.

A Phase I Study of OGX-011, a 2′-Methoxyethyl Phosphorothioate Antisense to Clusterin, in Combination with Docetaxel in Patients with Advanced Cancer

Purpose: Clusterin is a cytoprotective chaperone protein that promotes cell survival and confers broad-spectrum treatment resistance. OGX-011 is a 2′-methoxyethyl–modified phosphorothioate antisense oligonucleotide that is complementary to clusterin mRNA, has a prolonged tissue half life, enhances drug efficacy in xenograft models, and reduces clusterin expression in humans with a biologically effective dose of 640 mg. The objective of this study was to determine a recommended phase II dose of OGX-011 in combination with docetaxel. Experimental Design: Patients with cancers known from the literature to express clusterin were eligible. OGX-011 was given by 2-h i.v. infusion starting at 40 mg weekly after loading doses on days 1, 3, and 5. Docetaxel was given i.v. 30 mg/m2 weekly for 5 of 6 weeks (schedule A) or 75 mg/m2 every 3 weeks (schedule B). All patients had serial samples of peripheral blood mononuclear cells and serum assessed for clusterin expression. Results: Forty patients were enrolled to eight cohorts. OGX-011 could be given at the full biologically effective single-agent dose of 640 mg with both docetaxel schedules. Toxic effects were primarily myelosuppression, fatigue, hair loss, gastrointestinal effects (expected docetaxel effects), as well as dose-related chills and fever (expected OGX-011 effects). OGX-011 AUC and Cmax increased proportionally with no apparent effect on docetaxel pharmacokinetics. At the end of cycle 1, serum clusterin showed mean decreases of 34% and 38% (range, 15-99%) at the 640-mg dose levels. Conclusions: OGX-011 can be given at a biologically effective dose with standard doses of docetaxel. Phase II trials of combined OGX-011 and chemotherapy are ongoing in patients with prostate, breast, and lung cancers.

Exosomes as Biomarker Enriched Microvesicles: Characterization of Exosomal Proteins Derived from a Panel of Prostate Cell Lines with Distinct AR Phenotypes

Prostate cancer is the leading type of cancer diagnosed in men. In 2010, ∼217,730 new cases of prostate cancer were reported in the United States. Prompt diagnosis of the disease can substantially improve its clinical outcome. Improving capability for early detection, as well as developing new therapeutic targets in advanced disease are research priorities that will ultimately lead to better patient survival. Eukaryotic cells secrete proteins via distinct regulated mechanisms which are either ER/Golgi dependent or microvesicle mediated. The release of microvesicles has been shown to provide a novel mechanism for intercellular communication. Exosomes are nanometer sized cup-shaped membrane vesicles which are secreted from normal and cancerous cells. They are present in various biological fluids and are rich in characteristic proteins. Exosomes may thus have potential both in facilitating early diagnosis via less invasive procedures or be candidates for novel therapeutic approaches for castration resistance prostate cancer. Because exosomes have been shown previously to have a role in cell-cell communication in the local tumor microenvironment, conferring activation of numerous survival mechanisms, we characterized constitutive lipids, cholesterol and proteins from exosomes derived from six prostate cell lines and tracked their uptake in both cancerous and benign prostate cell lines respectively. Our comprehensive proteomic and lipidomic analysis of prostate derived exosomes could provide insight for future work on both biomarker and therapeutic targets for the treatment of prostate cancer.

Reproducibility and efficiency of serum-derived exosome extraction methods.

OBJECTIVES Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. DESIGN AND METHODS We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. RESULTS Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. CONCLUSION ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.

Effect of Noncompetitive Proteasome Inhibition on Bortezomib Resistance

BACKGROUND Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance. METHODS 5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ-proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided. RESULTS 5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [IC(50)] of AMC release in intact cells ranged from 0.57 to 5.03 microM), induced cell death in intact cells from leukemia and myeloma cell lines (mean IC(50) values for cell growth ranged from 0.94 to 3.85 microM), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding beta5 proteasome subunit (mean IC(50) for cell death in the absence of bortezomib, wild-type THP1: 3.7 microM, 95% confidence interval = 3.4 to 4.0 microM; THP1/BTZ500: 6.6 microM, 95% confidence interval = 5.9 to 7.5 microM). 5AHQ interacted with the alpha subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002). CONCLUSIONS 5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro.

Diet and Dietary Supplement Intervention Trials for the Prevention of Prostate Cancer Recurrence: A Review of the Randomized Controlled Trial Evidence

PURPOSE We review the effect of diet and dietary supplement interventions on prostate cancer progression, recurrence and survival. MATERIALS AND METHODS A literature search was conducted in MEDLINE, EMBASE and CINAHL to identify diet and dietary supplement intervention studies in men with prostate cancer using prostate specific antigen or prostate specific antigen doubling time as a surrogate serum biomarker of prostate cancer recurrence and/or survival. RESULTS Of the 32 studies identified 9 (28%) were randomized controlled trials and the focus of this review. In these studies men had confirmed prostate cancer and elevated or increasing prostate specific antigen. Only 1 trial included men with metastatic disease. When body mass index was reported, men were overweight or obese. A significant decrease in prostate specific antigen was observed in some studies using a low fat vegan diet, soy beverage or lycopene supplement. While not often reported as an end point, a significant increase in prostate specific antigen doubling time was observed in a study on lycopene supplementation. In only 1 randomized controlled trial in men undergoing orchiectomy was a survival end point of fewer deaths with lycopene supplementation reported. CONCLUSIONS A limited number of randomized controlled trials were identified in which diet and dietary supplement interventions appeared to slow disease progression in men with prostate cancer, although results vary. Studies were limited by reliance on the surrogate biomarker prostate specific antigen, sample size and study duration. Well designed trials are warranted to expand knowledge, replicate findings and further assess the impact of diet and dietary supplement interventions on recurrence and treatment associated morbidities.

Selectively Targeting the DNA-binding Domain of the Androgen Receptor as a Prospective Therapy for Prostate Cancer

Background: The androgen receptor (AR) is a transcription factor regulating progression of prostate cancer. Results: Developed compounds inhibit AR transcriptional activity in vitro and in vivo by selective targeting of the AR-DNA-binding domain (DBD). Conclusion: By targeting the DBD, the compounds differ from conventional anti-androgens. Significance: Anti-androgens with a novel mechanism of action have the potential to treat recurrent prostate cancer. The androgen receptor (AR) is a transcription factor that has a pivotal role in the occurrence and progression of prostate cancer. The AR is activated by androgens that bind to its ligand-binding domain (LBD), causing the transcription factor to enter the nucleus and interact with genes via its conserved DNA-binding domain (DBD). Treatment for prostate cancer involves reducing androgen production or using anti-androgen drugs to block the interaction of hormones with the AR-LBD. Eventually the disease changes into a castration-resistant form of PCa where LBD mutations render anti-androgens ineffective or where constitutively active AR splice variants, lacking the LBD, become overexpressed. Recently, we identified a surfaced exposed pocket on the AR-DBD as an alternative drug-target site for AR inhibition. Here, we demonstrate that small molecules designed to selectively bind the pocket effectively block transcriptional activity of full-length and splice variant AR forms at low to sub-micromolar concentrations. The inhibition is lost when residues involved in drug interactions are mutated. Furthermore, the compounds did not impede nuclear localization of the AR and blocked interactions with chromatin, indicating the interference of DNA binding with the nuclear form of the transcription factor. Finally, we demonstrate the inhibition of gene expression and tumor volume in mouse xenografts. Our results indicate that the AR-DBD has a surface site that can be targeted to inhibit all forms of the AR, including enzalutamide-resistant and constitutively active splice variants and thus may serve as a potential avenue for the treatment of recurrent and metastatic prostate cancer.

Anticancer Activity of a Novel Selective CYP17A1 Inhibitor in Preclinical Models of Castrate-Resistant Prostate Cancer

VT-464 is a novel, nonsteroidal, small-molecule CYP17A1 inhibitor with 17,20-lyase selectivity. This study evaluates the anticancer activity of VT-464 compared with abiraterone (ABI) in castrate-resistant prostate cancer cell lines and xenograft models that are enzalutamide (ENZ)-responsive (C4-2) or ENZ-resistant (MR49C, MR49F). In vitro, androgen receptor (AR) transactivation was assessed by probasin luciferase reporter, whereas AR and AR-regulated genes and steroidogenic pathway enzymes were assessed by Western blot and/or qRT-PCR. The MR49F xenograft model was used to compare effects of oral VT-464 treatment to vehicle and abiraterone acetate (AA). Steroid concentrations were measured using LC-MS chromatography. VT-464 demonstrated a greater decrease in AR transactivation compared with ABI in C4-2 and both ENZ-resistant cell lines. At the gene and protein level, VT-464 suppressed the AR axis to a greater extent compared with ABI. Gene transcripts StAR, CYP17A1, HSD17B3, and SRD5A1 increased following treatment with ABI and to a greater extent with VT-464. In vivo, intratumoral androgen levels were significantly lower after VT-464 or AA treatment compared with vehicle, with the greatest decrease seen with VT-464. Similarly, tumor growth inhibition and PSA decrease trends were greater with VT-464 than with AA. Finally, an AR-antagonist effect of VT-464 independent of CYP17A1 inhibition was observed using luciferase reporter assays, and a direct interaction was confirmed using an AR ligand binding domain biolayer interferometry. These preclinical results suggest greater suppression of the AR axis with VT-464 than ABI that is likely due to both superior selective suppression of androgen synthesis and AR antagonism. Mol Cancer Ther; 14(1); 59–69. ©2014 AACR.

Inhibitors of androgen receptor activation function-2 (AF2) site identified through virtual screening.

The androgen receptor (AR) is one of the most studied drug targets for the treatment of prostate cancer. However, all current anti-androgens directly interact with the AR at the androgen binding site, which is prone to resistant mutations, calling for new strategies of the AR inhibition. The current study represents the first attempt to use virtual screening to identify inhibitors of activation function-2 (AF2) of the human AR. By combining large-scale docking with experimental approaches, we were able to identify several small molecules that interact with the AF2 and effectively prevent the transcriptional activation of the AR. The crystallographic structure of one of these inhibitors in complex with the AR provides critical insight into the corresponding protein-ligand interactions and suitable for future hit optimization. Taken together, our results provide a promising ground for development of novel anti-androgens that can help to address the problem of drug resistance in prostate cancer.