Steroidogenesis in peripheral and transition zones of human prostate cancer tissue

The peripheral zone (PZ) and transition zone (TZ) represent about 70% of the human prostate gland with each zone having differential ability to develop prostate cancer. Androgens and their receptor are the primary driving cause of prostate cancer growth and eventually castration-resistant prostate cancer (CRPC). De novo steroidogenesis has been identified as a key mechanism that develops during CRPC. Currently, there is very limited information available on human prostate tissue steroidogenesis. The purpose of the present study was to investigate steroid metabolism in human prostate cancer tissues with comparison between PZ and TZ. Human prostate cancer tumors were procured from the patients who underwent radical prostatectomy without any neoadjuvant therapy. Human prostate homogenates were used to quantify steroid levels intrinsically present in the tissues as well as formed after incubation with 2 µg/mL of 17-hydroxypregnenolone (17-OH-pregnenolone) or progesterone. A Waters Acquity ultraperformance liquid chromatography coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C18 column was used to measure thirteen steroids from the classical and backdoor steroidogenesis pathways. The intrinsic prostate tissue steroid levels were similar between PZ and TZ with dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), pregnenolone and 17-OH-pregnenolone levels higher than the other steroids measured. Interestingly, 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one, and 5-pregnan-17-ol-3,20-dione formation was significantly higher in both the zones of prostate tissues, whereas, androstenedione, testosterone, DHT, and progesterone levels were significantly lower after 60 min incubation compared to the 0 min control incubations. The incubations with progesterone had a similar outcome with 5-pregnan-3,20-dione and 5-pregnan-3-ol-20-one levels were elevated and the levels of DHT were lower in both PZ and TZ tissues. The net changes in steroid formation after the incubation were more observable with 17-OH-pregnenolone than with progesterone. In our knowledge, this is the first report of comprehensive analyses of intrinsic prostate tissue steroids and precursor-driven steroid metabolism using a sensitive liquid chromatography-mass spectrometry assay. In summary, the PZ and TZ of human prostate exhibited similar steroidogenic ability with distinction in the manner each zone utilizes the steroid precursors to divert the activity towards backdoor pathway through a complex matrix of steroidogenic mechanisms

Clusterin Regulates Drug-Resistance in Melanoma Cells

Clusterin has recently been shown to act as an antiapoptotic protein that confers drug-resistance in models of epithelial tumors. The aim of our work was to provide an insight into a possible role of clusterin in the regulation of drug-resistance in melanoma. In tissue samples, clusterin expression was low in nevi, but high in primary melanoma and melanoma metastases. Clusterin was also strongly expressed in melanoma cell lines, but was barely detectable in cultured melanocytes. To elucidate a possible role of clusterin in drug-resistance of melanoma, clusterin expression was regulated by either plasmid-driven overexpression or by antisense-mediated downregulation. Clusterin overexpression was associated with an increase in drug-resistance, i.e., with an increased survival of melanoma cells in the presence of cytotoxic drugs. In contrast, downregulation of clusterin by 2′-O-(2-methoxy)ethyl (2′MOE)-modified antisense oligonucleotides (AS-ODN) directed against clusterin mRNA significantly reduced drug-resistance, i.e., decreased survival of melanoma cells in the presence of cytotoxic drugs. To evaluate the effects of clusterin-antisense treatment in vivo, we applied an SCID-mouse/human-melanoma xenotransplantation model. Pre-treatment of mice with the 2′MOE-modified clusterin AS-ODN was associated with a significantly improved tumor response to dacarbazine as compared with animals pretreated with a scrambled control oligonucleotide. Taken together, we show that clusterin is strongly expressed in melanoma. Downregulation of clusterin reduces drug-resistance, i.e., reduces melanoma cell survival in response to cytotoxic drugs in vitro and in vivo. Thus, reducing clusterin expression may provide a novel tool to overcome drug-resistance in melanoma

Boolean analysis identifies CD38 as a biomarker of aggressive localized prostate cancer.

The introduction of serum Prostate Specific Antigen (PSA) testing nearly 30 years ago has been associated with a significant shift towards localized disease and decreased deaths due to prostate cancer. Recognition that PSA testing has caused over diagnosis and over treatment of prostate cancer has generated considerable controversy over its value, and has spurred efforts to identify prognostic biomarkers to distinguish patients who need treatment from those that can be observed. Recent studies show that cancer is heterogeneous and forms a hierarchy of tumor cell populations. We developed a method of identifying prostate cancer differentiation states related to androgen signaling using Boolean logic. Using gene expression data, we identified two markers, CD38 and ARG2, that group prostate cancer into three differentiation states. Cancers with CD38-, ARG2- expression patterns, corresponding to an undifferentiated state, had significantly lower 10-year recurrence-free survival compared to the most differentiated group (CD38+ARG2+). We carried out immunohistochemical (IHC) staining for these two markers in a single institution (Stanford; n = 234) and multi-institution (Canary; n = 1326) cohorts. IHC staining for CD38 and ARG2 in the Stanford cohort demonstrated that combined expression of CD38 and ARG2 was prognostic. In the Canary cohort, low CD38 protein expression by IHC was significantly associated with recurrence-free survival (RFS), seminal vesicle invasion (SVI), extra-capsular extension (ECE) in univariable analysis. In multivariable analysis, ARG2 and CD38 IHC staining results were not independently associated with RFS, overall survival, or disease-specific survival after adjusting for other factors including SVI, ECE, Gleason score, pre-operative PSA, and surgical margins

Performance of PCA3 and TMPRSS2:ERG urinary biomarkers in prediction of biopsy outcome in the Canary Prostate Active Surveillance Study (PASS).

BackgroundFor men on active surveillance for prostate cancer, biomarkers may improve prediction of reclassification to higher grade or volume cancer. This study examined the association of urinary PCA3 and TMPRSS2:ERG (T2:ERG) with biopsy-based reclassification.MethodsUrine was collected at baseline, 6, 12, and 24 months in the multi-institutional Canary Prostate Active Surveillance Study (PASS), and PCA3 and T2:ERG levels were quantitated. Reclassification was an increase in Gleason score or ratio of biopsy cores with cancer to ≥34%. The association of biomarker scores, adjusted for common clinical variables, with short- and long-term reclassification was evaluated. Discriminatory capacity of models with clinical variables alone or with biomarkers was assessed using receiver operating characteristic (ROC) curves and decision curve analysis (DCA).ResultsSeven hundred and eighty-two men contributed 2069 urine specimens. After adjusting for PSA, prostate size, and ratio of biopsy cores with cancer, PCA3 but not T2:ERG was associated with short-term reclassification at the first surveillance biopsy (OR = 1.3; 95% CI 1.0-1.7, p = 0.02). The addition of PCA3 to a model with clinical variables improved area under the curve from 0.743 to 0.753 and increased net benefit minimally. After adjusting for clinical variables, neither marker nor marker kinetics was associated with time to reclassification in subsequent biopsies.ConclusionsPCA3 but not T2:ERG was associated with cancer reclassification in the first surveillance biopsy but has negligible improvement over clinical variables alone in ROC or DCA analyses. Neither marker was associated with reclassification in subsequent biopsies

Identification of DEK as a potential therapeutic target for neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer which does not respond to hormone therapy. Research of NEPC has been hampered by a lack of clinically relevant in vivo models. Recently, we developed a first-in-field patient tissue-derived xenograft model of complete neuroendocrine transdifferentiation of prostate adenocarcinoma. By comparing gene expression profiles of a transplantable adenocarcinoma line (LTL331) and its NEPC subline (LTL331R), we identified DEK as a potential biomarker and therapeutic target for NEPC. In the present study, elevated DEK protein expression was observed in all NEPC xenograft models and clinical NEPC cases, as opposed to their benign counterparts (0%), hormonal naïve prostate cancer (2.45%) and castration-resistant prostate cancer (29.55%). Elevated DEK expression was found to be an independent clinical risk factor, associated with shorter disease-free survival of hormonal naïve prostate cancer patients. DEK silencing in PC-3 cells led to a marked reduction in cell proliferation, cell migration and invasion. The results suggest that DEK plays an important role in the progression of prostate cancer, especially to NEPC, and provides a potential biomarker to aid risk stratification of prostate cancer and a novel target for therapy of NEPC

Heterochromatin protein 1α mediates development and aggressiveness of neuroendocrine prostate cancer

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer (PCa) arising mostly from adenocarcinoma via NE transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathological feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor (AR) and RE1 silencing transcription factor (REST) and enriched the repressive trimethylated histone H3 at Lys9 (H3K9me3) mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management

Transcriptional profiling identifies an androgen receptor activity-low, stemness program associated with enzalutamide resistance.

The androgen receptor (AR) antagonist enzalutamide is one of the principal treatments for men with castration-resistant prostate cancer (CRPC). However, not all patients respond, and resistance mechanisms are largely unknown. We hypothesized that genomic and transcriptional features from metastatic CRPC biopsies prior to treatment would be predictive of de novo treatment resistance. To this end, we conducted a phase II trial of enzalutamide treatment (160 mg/d) in 36 men with metastatic CRPC. Thirty-four patients were evaluable for the primary end point of a prostate-specific antigen (PSA)50 response (PSA decline ≥50% at 12 wk vs. baseline). Nine patients were classified as nonresponders (PSA decline <50%), and 25 patients were classified as responders (PSA decline ≥50%). Failure to achieve a PSA50 was associated with shorter progression-free survival, time on treatment, and overall survival, demonstrating PSA50's utility. Targeted DNA-sequencing was performed on 26 of 36 biopsies, and RNA-sequencing was performed on 25 of 36 biopsies that contained sufficient material. Using computational methods, we measured AR transcriptional function and performed gene set enrichment analysis (GSEA) to identify pathways whose activity state correlated with de novo resistance. TP53 gene alterations were more common in nonresponders, although this did not reach statistical significance (P = 0.055). AR gene alterations and AR expression were similar between groups. Importantly, however, transcriptional measurements demonstrated that specific gene sets-including those linked to low AR transcriptional activity and a stemness program-were activated in nonresponders. Our results suggest that patients whose tumors harbor this program should be considered for clinical trials testing rational agents to overcome de novo enzalutamide resistance

Association of TMPRSS2-ERG gene fusion with clinical characteristics and outcomes: results from a population-based study of prostate cancer

Background: The presence of the TMPRSS2-ERG fusion gene in prostate tumors has recently been associated with an aggressive phenotype, as well as recurrence and death from prostate cancer. These associations suggest the hypothesis that the gene fusion may be used as a prognostic indicator for prostate cancer. Methods: In this study, fluorescent in situ hybridization (FISH) assays were used to assess TMPRSS2-ERG fusion status in a group of 214 prostate cancer cases from two population-based studies. The FISH assays were designed to detect both fusion type (deletion vs. translocation) and the number of fusion copies (single vs. multiple). Genotyping of four ERG and one TMPRSS2 SNPs using germline DNA was also performed in a sample of the cases (n = 127). Results: Of the 214 tumors scored for the TMPRSS2-ERG fusion, 64.5% were negative and 35.5% were positive for the fusion. Cases with the TMPRSS2-ERG fusion did not exhibit reduced prostate cancer survival (HR = 0.92, 95% CI = 0.22-3.93), nor was there a significant difference in causespecific survival when stratifying by translocation or deletion (HR = 0.84, 95% CI = 0.23-3.12) or by the number of retained fusion copies (HR = 1.22, 95% CI = .45-3.34). However, evidence for reduced prostate cancer-specific survival was apparent in those cases whose tumor had multiple copies of the fusion. The variant T allele of the TMPRSS2 SNP, rs12329760, was positively associated with TMPRSS2-ERG fusion by translocation (p = 0.05) and with multiple copies of the gene fusion (p = 0.03). Conclusion: If replicated, the results presented here may provide insight into the mechanism by which the TMPRSS2-ERG gene fusion arises and also contribute to diagnostic evaluations for determining the subset of men who will go on to develop metastatic prostate cancer.This work was supported by NIH grants RO1 CA56678, RO1 CA114524, and P50 CA97186; additional support was provided by the Fred Hutchinson Cancer Research Center and the Intramural Program of the National Human Genome Research Institute

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy