Transcriptomic and lipid profiling analysis reveals a functional interplay between testosterone and growth hormone in hypothyroid liver.

Preclinical and clinical studies suggest that hypothyroidism might cause hepatic endocrine and metabolic disturbances with features that mimic deficiencies of testosterone and/or GH. The absence of physiological interactions between testosterone and GH can be linked to male differentiated liver diseases. Testosterone plays relevant physiological effects on somatotropic-liver axis and liver composition and the liver is a primary organ of interactions between testosterone and GH. However, testosterone exerts many effects on liver through complex and poorly understood mechanisms. Testosterone impacts liver functions by binding to the Androgen Receptor, and, indirectly, through its conversion to estradiol, and cooperation with GH. However, the role of testosterone, and its interaction with GH, in the hypothyroid liver, remains unclear. In the present work, the effects of testosterone, and how they impact on GH-regulated whole transcriptome and lipid composition in the liver, were studied in the context of adult hypothyroid-orchiectomized rats. Testosterone replacement positively modulated somatotropic-liver axis and impacted liver transcriptome involved in lipid and glucose metabolism. In addition, testosterone enhanced the effects of GH on the transcriptome linked to lipid biosynthesis, oxidation-reduction, and metabolism of unsaturated and long-chain fatty acids (FA). However, testosterone decreased the hepatic content of cholesterol esters and triacylglycerols and increased fatty acids whereas GH increased neutral lipids and decreased polar lipids. Biological network analysis of the effects of testosterone on GH-regulated transcriptome confirmed a close connection with crucial proteins involved in steroid and fatty acid metabolism. Taken together, this comprehensive analysis of gene expression and lipid profiling in hypothyroid male liver reveals a functional interplay between testosterone and pulsed GH administration.

Itraconazole Reverts ABCB1-Mediated Docetaxel Resistance in Prostate Cancer.

Docetaxel (DTX) was the first chemotherapeutic agent to demonstrate significant efficacy in the treatment of men with metastatic castration-resistant prostate cancer. However, response to DTX is generally short-lived, and relapse eventually occurs due to emergence of drug-resistance. We previously established two DTX-resistant prostate cancer cell lines, LNCaPR and C4-2BR, derived from the androgen-dependent LNCaP cell line, and from the LNCaP lineage-derived androgen-independent C4-2B sub-line, respectively. Using an unbiased drug screen, we identify itraconazole (ITZ), an oral antifungal drug, as a compound that can efficiently re-sensitize drug-resistant LNCaPR and C4-2BR prostate cancer cells to DTX treatment. ITZ can re-sensitize multiple DTX-resistant cell models, not only in prostate cancer derived cells, such as PC-3 and DU145, but also in docetaxel-resistant breast cancer cells. This effect is dependent on expression of ATP-binding cassette (ABC) transporter protein ABCB1, also known as P-glycoprotein (P-gp). Molecular modeling of ITZ bound to ABCB1, indicates that ITZ binds tightly to the inward-facing form of ABCB1 thereby inhibiting the transport of DTX. Our results suggest that ITZ may provide a feasible approach to re-sensitization of DTX resistant cells, which would add to the life-prolonging effects of DTX in men with metastatic castration-resistant prostate cancer.

Molecular Profiling of Docetaxel-Resistant Prostate Cancer Cells Identifies Multiple Mechanisms of Therapeutic Resistance.

Docetaxel-a taxane-based chemotherapeutic agent-was the first treatment to demonstrate significant improvements in overall survival in men with metastatic castration-resistant prostate cancer (mCRPC). However, the response to docetaxel is generally short-lived, and relapse eventually occurs due to the development of resistance. To explore the mechanisms of acquired docetaxel resistance in prostate cancer (PCa) and set these in the context of androgen deprivation therapy, we established docetaxel-resistant PCa cell lines, derived from the androgen-dependent LNCaP cell line, and from the LNCaP lineage-derived androgen-independent C4-2B sub-line. We generated two docetaxel-resistant LNCaPR and C4-2BR sub-lines, with IC50 values 77- and 50-fold higher than those of the LNCaP and C4-2B parental cells, respectively. We performed gene expression analysis of the matched sub-lines and found several alterations that may confer docetaxel resistance. In addition to increased expression of ABCB1, an ATP-binding cassette (ABC) transporter, and a well-known gene associated with development of docetaxel resistance, we identified genes associated with androgen signaling, cell survival, and overexpression of ncRNAs. In conclusion, we identified multiple mechanisms that may be associated with the development of taxane drug resistance in PCa. Actioning these mechanisms could provide a potential approach to re-sensitization of docetaxel-resistant PCa cells to docetaxel treatment and thereby further add to the life-prolonging effects of this drug in men with mCRPC.

Comprehensive metabolomics analysis of prostate cancer tissue in relation to tumor aggressiveness and TMPRSS2-ERG fusion status.

BACKGROUND: Prostate cancer (PC) can display very heterogeneous phenotypes ranging from indolent asymptomatic to aggressive lethal forms. Understanding how these PC subtypes vary in their striving for energy and anabolic molecules is of fundamental importance for developing more effective therapies and diagnostics. Here, we carried out an extensive analysis of prostate tissue samples to reveal metabolic alterations during PC development and disease progression and furthermore between TMPRSS2-ERG rearrangement-positive and -negative PC subclasses. METHODS: Comprehensive metabolomics analysis of prostate tissue samples was performed by non-destructive high-resolution magic angle spinning nuclear magnetic resonance (1H HR MAS NMR). Subsequently, samples underwent moderate extraction, leaving tissue morphology intact for histopathological characterization. Metabolites in tissue extracts were identified by 1H/31P NMR and liquid chromatography-mass spectrometry (LC-MS). These metabolomics profiles were analyzed by chemometric tools and the outcome was further validated using proteomic data from a separate sample cohort. RESULTS: The obtained metabolite patterns significantly differed between PC and benign tissue and between samples with high and low Gleason score (GS). Five key metabolites (phosphocholine, glutamate, hypoxanthine, arginine and α-glucose) were identified, who were sufficient to differentiate between cancer and benign tissue and between high to low GS. In ERG-positive PC, the analysis revealed several acylcarnitines among the increased metabolites together with decreased levels of proteins involved in ß-oxidation; indicating decreased acyl-CoAs oxidation in ERG-positive tumors. The ERG-positive group also showed increased levels of metabolites and proteins involved in purine catabolism; a potential sign of increased DNA damage and oxidative stress. CONCLUSIONS: Our comprehensive metabolomic analysis strongly indicates that ERG-positive PC and ERG-negative PC should be considered as different subtypes of PC; a fact requiring different, sub-type specific treatment strategies for affected patients.

Gene expression profiles define molecular subtypes of prostate cancer bone metastases with different outcomes and morphology traceable back to the primary tumor.

Bone metastasis is the lethal end-stage of prostate cancer (PC), but the biology of bone metastases is poorly understood. The overall aim of this study was therefore to explore molecular variability in PC bone metastases of potential importance for therapy. Specifically, genome-wide expression profiles of bone metastases from untreated patients (n = 12) and patients treated with androgen-deprivation therapy (ADT, n = 60) were analyzed in relation to patient outcome and to morphological characteristics in metastases and paired primary tumors. Principal component analysis and unsupervised classification were used to identify sample clusters based on mRNA profiles. Clusters were characterized by gene set enrichment analysis and related to histological and clinical parameters using univariate and multivariate statistics. Selected proteins were analyzed by immunohistochemistry in metastases and matched primary tumors (n = 52) and in transurethral resected prostate (TUR-P) tissue of a separate cohort (n = 59). Three molecular subtypes of bone metastases (MetA-C) characterized by differences in gene expression pattern, morphology, and clinical behavior were identified. MetA (71% of the cases) showed increased expression of androgen receptor-regulated genes, including prostate-specific antigen (PSA), and glandular structures indicating a luminal cell phenotype. MetB (17%) showed expression profiles related to cell cycle activity and DNA damage, and a pronounced cellular atypia. MetC (12%) exhibited enriched stroma-epithelial cell interactions. MetB patients had the lowest serum PSA levels and the poorest prognosis after ADT. Combined analysis of PSA and Ki67 immunoreactivity (proliferation) in bone metastases, paired primary tumors, and TUR-P samples was able to differentiate MetA-like (high PSA, low Ki67) from MetB-like (low PSA, high Ki67) tumors and demonstrate their different prognosis. In conclusion, bone metastases from PC patients are separated based on gene expression profiles into molecular subtypes with different morphology, biology, and clinical outcome. These findings deserve further exploration with the purpose of improving treatment of metastatic PC.

Immunoreactivity for prostate specific antigen and Ki67 differentiates subgroups of prostate cancer related to outcome.

Based on gene-expression profiles, prostate tumors can be subdivided into subtypes with different aggressiveness and response to treatment. We investigated if similar clinically relevant subgroups can be identified simply by the combination of two immunohistochemistry markers: one for tumor cell differentiation (prostate specific antigen, PSA) and one for proliferation (Ki67). This was analyzed in men with prostate cancer diagnosed at transurethral resection of the prostate 1975-1991 (n = 331) where the majority was managed by watchful waiting. Ki67 and PSA immunoreactivity was related to outcome and to tumor characteristics previously associated with prognosis. Increased Ki67 and decreased PSA were associated with poor outcome, and they provided independent prognostic information from Gleason score. A combinatory score for PSA and Ki67 immunoreactivity was produced using the median PSA and Ki67 levels as cut-off (for Ki67 the upper quartile was also evaluated) for differentiation into subgroups. Patients with PSA low/Ki67 high tumors showed higher Gleason score, more advanced tumor stage, and higher risk of prostate cancer death compared to other patients. Their tumor epithelial cells were often ERG positive and expressed higher levels of ErbB2, phosphorylated epidermal growth factor receptor (pEGF-R) and protein kinase B (pAkt), and their tumor stroma showed a reactive response with type 2 macrophage infiltration, high density of blood vessels and hyaluronic acid, and with reduced levels of caveolin-1, androgen receptors, and mast cells. In contrast, men with PSA high/Ki67 low tumors were characterized by low Gleason score, and the most favorable outcome amongst PSA/Ki67-defined subgroups. Men with PSA low/Ki67 low tumors showed clinical and tumor characteristics intermediate of the two groups above. A combinatory PSA/Ki67 immunoreactivity score identifies subgroups of prostate cancers with different epithelial and stroma phenotypes and highly different outcome but the clinical usefulness of this approach needs to be validated in other cohorts.

Proteogenomic Characterization of Patient-Derived Xenografts Highlights the Role of REST in Neuroendocrine Differentiation of Castration-Resistant Prostate Cancer.

PURPOSE: An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood.Experimental Design: We applied mass spectrometry-based proteomics to a unique set of 17 prostate cancer patient-derived xenografts (PDX) to characterize the effects of castration in vivo, and the proteome differences between NEPC and prostate adenocarcinomas. Genome-wide profiling of REST-occupied regions in prostate cancer cells was correlated to the expression changes in vivo to investigate the role of the transcriptional repressor REST in castration-induced NEPC differentiation. RESULTS: An average of 4,881 proteins were identified and quantified from each PDX. Proteins related to neurogenesis, cell-cycle regulation, and DNA repair were found upregulated and elevated in NEPC, while the reduced levels of proteins involved in mitochondrial functions suggested a prevalent glycolytic metabolism of NEPC tumors. Integration of the REST chromatin bound regions with expression changes indicated a direct role of REST in regulating neuronal gene expression in prostate cancer cells. Mechanistically, depletion of REST led to cell-cycle arrest in G1, which could be rescued by p53 knockdown. Finally, the expression of the REST-regulated gene secretagogin (SCGN) correlated with an increased risk of suffering disease relapse after radical prostatectomy. CONCLUSIONS: This study presents the first deep characterization of the proteome of NEPC and suggests that concomitant inhibition of REST and the p53 pathway would promote NEPC. We also identify SCGN as a novel prognostic marker in prostate cancer.

Predictive value of combined analysis of pro-NPY and ERG in localized prostate cancer.

This study aimed to investigate if combined analysis of pro-Neuropeptide Y (NPY) and ERG expression in tumor tissue are associated with biochemical failure (BF), castration-based treatment, castration-resistant prostate cancer (CRPC), and prostate cancer (PCa)-specific death for men undergoing radical prostatectomy (RP) for PCa. This study included 315 patients, who underwent RP from 2002 to 2005. Both pro-NPY and ERG expression were analyzed using immunohistochemistry and were scored as low or high and negative or positive, respectively. Risk of BF, castration-based treatment, CRPC, and PCa-specific death were analyzed with multiple cause-specific Cox regression analyses and stratified cumulative incidences using competing risk assessment. Median follow-up was 13.0 years (95% CI: 12.7-13.2). In total, 85.7% were pro-NPY high and 14.3% were pro-NPY low. The combined analyses of pro-NPY and ERG expression was not associated with risk of BF (p = 0.7), castration-based treatment (p = 0.8), CRPC (p = 0.4) or PCa-specific death (p = 0.5). In the multiple cause-specific Cox regression analysis, pro-NPY high and ERG positivity was not associated with BF (HR: 1.02; 95% CI 0.6-1.7; p = 0.94). In conclusion the combination of pro-NPY and ERG expression did not show association with risk of BF, castration-based treatment, CRPC, and PCa-specific death following RP.

SPOP promotes transcriptional expression of DNA repair and replication factors to prevent replication stress and genomic instability.

Mutations in SPOP, the gene most frequently point-mutated in primary prostate cancer, are associated with a high degree of genomic instability and deficiency in homologous recombination repair of DNA but the underlying mechanisms behind this defect are currently unknown. Here we demonstrate that SPOP knockdown leads to spontaneous replication stress and impaired recovery from replication fork stalling. We show that this is associated with reduced expression of several key DNA repair and replication factors including BRCA2, ATR, CHK1 and RAD51. Consequently, SPOP knockdown impairs RAD51 foci formation and activation of CHK1 in response to replication stress and compromises recovery from replication fork stalling. An SPOP interactome analysis shows that wild type (WT) SPOP but not mutant SPOP associates with multiple proteins involved in transcription, mRNA splicing and export. Consistent with the association of SPOP with transcription, splicing and RNA export complexes, the decreased expression of BRCA2, ATR, CHK1 and RAD51 occurs at the level of transcription.

The Proteome of Prostate Cancer Bone Metastasis Reveals Heterogeneity with Prognostic Implications.

Purpose: Bone is the most predominant site of distant metastasis in prostate cancer, and patients have limited therapeutic options at this stage.Experimental Design: We performed a system-wide quantitative proteomic analysis of bone metastatic prostate tumors from 22 patients operated to relieve spinal cord compression. At the time of surgery, most patients had relapsed after androgen-deprivation therapy, while 5 were previously untreated. An extended cohort of prostate cancer bone metastases (n = 65) was used for immunohistochemical validation.Results: On average, 5,067 proteins were identified and quantified per tumor. Compared with primary tumors (n = 26), bone metastases were more heterogeneous and showed increased levels of proteins involved in cell-cycle progression, DNA damage response, RNA processing, and fatty acid ß-oxidation; and reduced levels of proteins were related to cell adhesion and carbohydrate metabolism. Within bone metastases, we identified two phenotypic subgroups: BM1, expressing higher levels of AR canonical targets, and mitochondrial and Golgi apparatus resident proteins; and BM2, with increased expression of proliferation and DNA repair-related proteins. The two subgroups, validated by the inverse correlation between MCM3 and prostate specific antigen immunoreactivity, were related to disease prognosis, suggesting that this molecular heterogeneity should be considered when developing personalized therapies.Conclusions: This work is the first system-wide quantitative characterization of the proteome of prostate cancer bone metastases and a valuable resource for understanding the etiology of prostate cancer progression. Clin Cancer Res; 24(21); 5433-44. ©2018 AACR.

Quantitative Mass Spectrometry-Based Proteomic Profiling for Precision Medicine in Prostate Cancer.

Prostate cancer (PCa) is one of the most frequently diagnosed cancer among men in the western societies. Many PCa patients bear tumors that will not threat their lives if left untreated or if treatment is delayed. Our inability for early identification of these patients has resulted in massive overtreatment. Therefore, there is a great need of finding biomarkers for patient stratification according to prognostic risk; as well as there is a need for novel targets that can allow the development of effective treatments for patients that progress to castration-resistant PCa. Most biomarkers in cancer are proteins, including the widely-used prostate-specific antigen (PSA). Recent developments in mass spectrometry allow the identification and quantification of thousands of proteins and posttranslational modifications from small amounts of biological material, including formalin-fixed paraffin-embedded tissues, and biological fluids. Novel diagnostic and prognostic biomarkers have been identified in tissue, blood, urine, and seminal plasma of PCa patients, and new insights in the ethology and progression of this disease have been achieved using this technology. In this review, we summarize these findings and discuss the potential of this technology to pave the way toward the clinical implementation of precision medicine in PCa.

The ubiquitin ligase Cullin5SOCS2 regulates NDR1/STK38 stability and NF-κB transactivation.

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2-/- mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway.

YAP1 regulates prostate cancer stem cell-like characteristics to promote castration resistant growth.

Castration resistant prostate cancer (CRPC) is a stage of relapse that arises after various forms of androgen ablation therapy (ADT) and causes significant morbidity and mortality. However, the mechanism underlying progression to CRPC remains poorly understood. Here, we report that YAP1, which is negatively regulated by AR, influences prostate cancer (PCa) cell self-renewal and CRPC development. Specifically, we found that AR directly regulates the methylation of YAP1 gene promoter via the formation of a complex with Polycomb group protein EZH2 and DNMT3a. In normal conditions, AR recruits EZH2 and DNMT3a to YAP1 promoter, thereby promoting DNA methylation and the repression of YAP1 gene transcription. Following ADT treatment or when AR activity is antagonized by Bicalutamide or Enzalutamide, YAP1 gene expression is switched on. In turn, YAP1 promotes SOX2 and Nanog expression and the de-differentiation of PCa cells to stem/progenitor-like cells (PCSC), which potentially contribute to disease recurrence. Finally, the knock down of YAP1 expression or the inhibition of YAP1 function by Verteporfin in TRAMP prostate cancer mice significantly suppresses tumor recurrence following castration. In conclusion, our data reveals that AR suppresses YAP1 gene expression through a novel epigenetic mechanism, which is critical for PCa cells self-renewal and the development of CRPC.

The Proteome of Primary Prostate Cancer.

BACKGROUND: Clinical management of the prostate needs improved prognostic tests and treatment strategies. Because proteins are the ultimate effectors of most cellular reactions, are targets for drug actions and constitute potential biomarkers; a quantitative systemic overview of the proteome changes occurring during prostate cancer (PCa) initiation and progression can result in clinically relevant discoveries. OBJECTIVES: To study cellular processes altered in PCa using system-wide quantitative analysis of changes in protein expression in clinical samples and to identify prognostic biomarkers for disease aggressiveness. DESIGN, SETTING, AND PARTICIPANTS: Mass spectrometry was used for genome-scale quantitative proteomic profiling of 28 prostate tumors (Gleason score 6-9) and neighboring nonmalignant tissue in eight cases, obtained from formalin-fixed paraffin-embedded prostatectomy samples. Two independent cohorts of PCa patients (summing 752 cases) managed by expectancy were used for immunohistochemical evaluation of proneuropeptide-Y (pro-NPY) as a prognostic biomarker. RESULTS AND LIMITATIONS: Over 9000 proteins were identified as expressed in the human prostate. Tumor tissue exhibited elevated expression of proteins involved in multiple anabolic processes including fatty acid and protein synthesis, ribosomal biogenesis and protein secretion but no overt evidence of increased proliferation was observed. Tumors also showed increased levels of mitochondrial proteins, which was associated with elevated oxidative phosphorylation capacity measured in situ. Molecular analysis indicated that some of the proteins overexpressed in tumors, such as carnitine palmitoyltransferase 2 (CPT2, fatty acid transporter), coatomer protein complex, subunit alpha (COPA, vesicle secretion), and mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2, protein kinase) regulate the proliferation of PCa cells. Additionally, pro-NPY was found overexpressed in PCa (5-fold, p<0.05), but largely absent in other solid tumor types. Pro-NPY expression, alone or in combination with the ERG status of the tumor, was associated with an increased risk of PCa specific mortality, especially in patients with Gleason score ≤ 7 tumors. CONCLUSIONS: This study represents the first system-wide quantitative analysis of proteome changes associated to localized prostate cancer and as such constitutes a valuable resource for understanding the complex metabolic changes occurring in this disease. We also demonstrated that pro-NPY, a protein that showed differential expression between high and low risk tumors in our proteomic analysis, is also a PCa specific prognostic biomarker associated with increased risk for disease specific death in patients carrying low risk tumors. PATIENT SUMMARY: The identification of proteins whose expression change in prostate cancer provides novel mechanistic information related to the disease etiology. We hope that future studies will prove the value of this proteome dataset for development of novel therapies and biomarkers.

OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

BACKGROUND: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored. METHODS: RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth. RESULTS: Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo. CONCLUSIONS: OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

Lipid profiling and transcriptomic analysis reveals a functional interplay between estradiol and growth hormone in liver.

17ß-estradiol (E2) may interfere with endocrine, metabolic, and gender-differentiated functions in liver in both females and males. Indirect mechanisms play a crucial role because of the E2 influence on the pituitary GH secretion and the GHR-JAK2-STAT5 signaling pathway in the target tissues. E2, through its interaction with the estrogen receptor, exerts direct effects on liver. Hypothyroidism also affects endocrine and metabolic functions of the liver, rendering a metabolic phenotype with features that mimic deficiencies in E2 or GH. In this work, we combined the lipid and transcriptomic analysis to obtain comprehensive information on the molecular mechanisms of E2 effects, alone and in combination with GH, to regulate liver functions in males. We used the adult hypothyroid-orchidectomized rat model to minimize the influence of internal hormones on E2 treatment and to explore its role in male-differentiated functions. E2 influenced genes involved in metabolism of lipids and endo-xenobiotics, and the GH-regulated endocrine, metabolic, immune, and male-specific responses. E2 induced a female-pattern of gene expression and inhibited GH-regulated STAT5b targeted genes. E2 did not prevent the inhibitory effects of GH on urea and amino acid metabolism-related genes. The combination of E2 and GH decreased transcriptional immune responses. E2 decreased the hepatic content of saturated fatty acids and induced a transcriptional program that seems to be mediated by the activation of PPARα. In contrast, GH inhibited fatty acid oxidation. Both E2 and GH replacements reduced hepatic CHO levels and increased the formation of cholesterol esters and triacylglycerols. Notably, the hepatic lipid profiles were endowed with singular fingerprints that may be used to segregate the effects of different hormonal replacements. In summary, we provide in vivo evidence that E2 has a significant impact on lipid content and transcriptome in male liver and that E2 exerts a marked influence on GH physiology, with implications in human therapy.

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer.

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase ß-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

SOCS2 mediates the cross talk between androgen and growth hormone signaling in prostate cancer.

UNLABELLED: Anabolic signals such as androgens and the growth hormone/insulin-like growth factor 1 (GH/IGF-1) axis play an essential role in the normal development of the prostate but also in its malignant transformation. In this study, we investigated the role of suppressor of cytokine signaling 2 (SOCS2) as mediator of the cross talk between androgens and GH signals in the prostate and its potential role as tumor suppressor in prostate cancer (PCa). We observed that SOCS2 protein levels assayed by immunohistochemistry are elevated in hormone therapy-naive localized prostatic adenocarcinoma in comparison with benign tissue. In contrast, however, castration-resistant bone metastases exhibit reduced levels of SOCS2 in comparison with localized or hormone naive, untreated metastatic tumors. In PCa cells, SOCS2 expression is induced by androgens through a mechanism that requires signal transducer and activator of transcription 5 protein (STAT5) and androgen receptor-dependent transcription. Consequentially, SOCS2 inhibits GH activation of Janus kinase 2, Src and STAT5 as well as both cell invasion and cell proliferation in vitro. In vivo, SOCS2 limits proliferation and production of IGF-1 in the prostate in response to GH. Our results suggest that the use of GH-signaling inhibitors could be of value as a complementary treatment for castration-resistant PCa. SUMMARY: Androgen induced SOCS2 ubiquitin ligase expression and inhibited GH signaling as well as cell proliferation and invasion in PCa, whereas reduced SOCS2 was present in castration-resistant cases. GH-signaling inhibitors might be a complementary therapeutic option for advanced PCa.

Androgen-independent effects of Serenoa repens extract (Prostasan®) on prostatic epithelial cell proliferation and inflammation.

Extracts from Serenoa repens are widely used for the treatment of benign prostatic hyperplasia (BPH) and traditionally for prostatitis. In the present study we evaluated the biological effects of Serenoa repens extract (Prostasan®) on prostate cells beyond its known antiandrogenic actions. Prostasan® inhibited epidermal growth factor (EGF) and lipopolysaccharide (LPS) induced proliferation of the prostatic epithelial, androgen independent cell line PC-3. At effective concentrations of 50 µg/mL, Prostasan® partly displaced EGF from EGF receptor (EGFR) but fully blocked EGF-induced cell proliferation of PC-3 cells. Similarly, Prostasan® inhibited LPS-induced proliferation of PC-3 cells without affecting LPS activation of the NFĸB pathway via toll-like receptor-4 (TLR-4). Additionally, Prostasan® reduced the constitutive secretion of monocyte chemotactic protein-1 (MCP-1), the LPS-induced secretion of IL-12 and inhibited MCP-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF) production in the presence of LPS on PC-3 cells. Taken together, our results suggest that S. repens extracts, in addition to other reported effects on BPH development and prostatitis, inhibits EGF-dependent growth and proinflammatory responses of the prostate epithelial cells.