The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Heterogeneity and clinical significance of ETV1 translocations in human prostate cancer

A fluorescence in situ hybridisation (FISH) assay has been used to screen for ETV1 gene rearrangements in a cohort of 429 prostate cancers from patients who had been diagnosed by trans-urethral resection of the prostate. The presence of ETV1 gene alterations (found in 23 cases, 5.4%) was correlated with higher Gleason Score (P=0.001), PSA level at diagnosis (P=<0.0001) and clinical stage (P=0.017) but was not linked to poorer survival. We found that the six previously characterised translocation partners of ETV1 only accounted for 34% of ETV1 re-arrangements (eight out of 23) in this series, with fusion to the androgen-repressed gene C15orf21 representing the commonest event (four out of 23). In 5'-RACE experiments on RNA extracted from formalin-fixed tissue we identified the androgen-upregulated gene ACSL3 as a new 5'-translocation partner of ETV1. These studies report a novel fusion partner for ETV1 and highlight the considerable heterogeneity of ETV1 gene rearrangements in human prostate cancer.

Alterations in beta-catenin expression and localization in prostate cancer

BACKGROUND: Wnt signaling is thought to be important in prostate cancer, in part because proteins such as beta-catenin can also affect androgen receptor signaling. beta-Catenin forms a cell adhesion complex with E-cadherin raising the possibility that loss of expression or a change in beta-catenin distribution in the cell could also alter downstream signaling, decreased inter-cellular adhesion and the promotion of metastasis. A number of studies have reported the altered expression and/or localization of beta-catenin as a biomarker in prostate cancer.

METHODS: Tissue microarrays comprised of BPH and low, moderate and high-grade prostate cancer (n=77) were assessed for beta-catenin expression and distribution using immunohistochemistry. Staining was also performed on a tissue microarray containing tissue from patients before and after hormone manipulation. The effects of fixation and different antibodies was assessed on fixed LNCaP cell pellets and small prostate tissue microarrays.

RESULTS: We have observed increased beta-catenin expression in only high Gleason score (>7) prostate cancer. A nuclear re-distribution of beta-catenin has previously been reported. We noted nuclear beta-catenin in benign prostatic hyperplasia and a gradual loss in nuclear distribution with increasing Gleason grade. We found no evidence for an alteration in beta-catenin expression or re-distribution with hormone ablation. Altered fixation, antibodies and antibody concentration did affect the intensity and specificity of staining.

CONCLUSIONS: A loss of nuclear beta-catenin is the most consistent feature in prostate cancer rather than absolute levels of expression. We also suggest that variation in immunohistochemical protocols may explain variations in the reported literature.

Promoter methylation correlates with reduced Smad4 expression in advanced prostate cancer

BACKGROUND: Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor in a wide range of cell types. A transducer of TGF-beta signaling known as Mothers against decapentaplegic homologue 4 (Smad4) is a known tumor suppressor found on chromosome 18q21.1 and is typically inactivated by deletion or mutation in pancreatic and colorectal cancers. The purpose of the article is to investigate Smad4 expression, gene copy number and methylation status in advanced cases of prostate cancer.

METHODS: We have employed Methylation Specific PCR (MSP) to identify methylation sites within the Smad4 promoter and combined this with quantitative real-time PCR to look for correlates between methylation status and Smad4 expression and to examine androgen receptor (AR) expression. Bacterial artificial chromosome-comparative genomic hybridization (BAC-CGH) has been used to look for genomic amplifications and deletions which may also contribute to expression changes.

RESULTS: We fail to find evidence of genomic deletions or amplifications affecting the Smad4 locus on chromosome 18 but show a correlation between promoter methylation and the loss of Smad4 expression in the same material. We confirm that the AR locus on the X chromosome is amplified in 30% of the advanced clinical samples and that this correlates with increased transcript levels as previously reported by other groups.

CONCLUSION: This indicates that epigenetic changes affect the expression of the Smad4 protein in prostate cancer and points to methylation of the promoter as a novel marker of and contributor to the disease warranting further study.

Pathogenesis of prostate cancer and hormone refractory prostate cancer

Prostate cancer is the second most common malignancy in males and the leading cause of cancer death. Prostate cancer is initially androgen dependent and relies upon the androgen receptor (AR) to mediate the effects of androgens. The AR is also the target for therapy using antiandrogens and LHRH analogues. However, all cancers eventually become androgen independent, often referred to as hormone refractory prostate cancer. The processes involved in this transformation are yet to be fully understood but research in this area has discovered numerous potential mechanisms including AR amplification, over-expression or mutation and alterations in the AR signaling pathway. This review of the recent literature examines the current knowledge and developments in the understanding of the molecular biology of prostate cancer and hormone refractory prostate cancer, summarizing the well characterized pathways involved as well as introducing new concepts that may offer future solutions to this difficult problem.

A role for neurotensin in bicalutamide resistant prostate cancer cells

BACKGROUND: Anti-androgens are administered as a principal treatment for prostate cancer. Aggressive hormone refractory disease is characterized in some cases by the development of a neuroendocrine phenotype. However little attention has been paid to resistance pathways selected for by long-term treatment with non-steroidal anti-androgens.

METHODS: Using a resistant sub-line, LNCaP-Bic, we performed a comparative gene expression profiling using cDNA microarrays and target validation by qRT-PCR. Targets were then explored using cell proliferation, cell cycle analysis and in vitro invasion assays using siRNA technology.

RESULTS: Neurotensin/Neuromedin N (NTS) was upregulated in the LNCaP-Bic line at both the transcript and protein level. The resistant line was found to have an increased proliferation rate, more rapid cell cycle progression and increased invasiveness through Matrigel. Each phenotypic difference could be reduced using siRNA knockdown of NT.

CONCLUSION: Increased expression of NT in bicalutamide resistant prostate cancer cells induces cell proliferation and invasion suggesting that this peptide may contribute to the development of bicalutamide resistant prostate cancer.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, beingsignificantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression.

Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study

BACKGROUND: Understanding the heterogeneous genotypes and phenotypes of prostate cancer is fundamental to improving the way we treat this disease. As yet, there are no validated descriptions of prostate cancer subgroups derived from integrated genomics linked with clinical outcome.

METHODS: In a study of 482 tumour, benign and germline samples from 259 men with primary prostate cancer, we used integrative analysis of copy number alterations (CNA) and array transcriptomics to identify genomic loci that affect expression levels of mRNA in an expression quantitative trait loci (eQTL) approach, to stratify patients into subgroups that we then associated with future clinical behaviour, and compared with either CNA or transcriptomics alone.

FINDINGS: We identified five separate patient subgroups with distinct genomic alterations and expression profiles based on 100 discriminating genes in our separate discovery and validation sets of 125 and 103 men. These subgroups were able to consistently predict biochemical relapse (p = 0.0017 and p = 0.016 respectively) and were further validated in a third cohort with long-term follow-up (p = 0.027). We show the relative contributions of gene expression and copy number data on phenotype, and demonstrate the improved power gained from integrative analyses. We confirm alterations in six genes previously associated with prostate cancer (MAP3K7, MELK, RCBTB2, ELAC2, TPD52, ZBTB4), and also identify 94 genes not previously linked to prostate cancer progression that would not have been detected using either transcript or copy number data alone. We confirm a number of previously published molecular changes associated with high risk disease, including MYC amplification, and NKX3-1, RB1 and PTEN deletions, as well as over-expression of PCA3 and AMACR, and loss of MSMB in tumour tissue. A subset of the 100 genes outperforms established clinical predictors of poor prognosis (PSA, Gleason score), as well as previously published gene signatures (p = 0.0001). We further show how our molecular profiles can be used for the early detection of aggressive cases in a clinical setting, and inform treatment decisions.

INTERPRETATION: For the first time in prostate cancer this study demonstrates the importance of integrated genomic analyses incorporating both benign and tumour tissue data in identifying molecular alterations leading to the generation of robust gene sets that are predictive of clinical outcome in independent patient cohorts.

The Early Effects of Rapid Androgen Deprivation on Human Prostate Cancer

The androgen receptor (AR) is the dominant growth factor in prostate cancer (PCa). Therefore, understanding how ARs regulate the human transcriptome is of paramount importance. The early effects of castration on human PCa have not previously been studied 27 patients medically castrated with degarelix 7 d before radical prostatectomy. We used mass spectrometry, immunohistochemistry, and gene expression array (validated by reverse transcription-polymerase chain reaction) to compare resected tumour with matched, controlled, untreated PCa tissue. All patients had levels of serum androgen, with reduced levels of intraprostatic androgen at prostatectomy. We observed differential expression of known androgen-regulated genes (TMPRSS2, KLK3, CAMKK2, FKBP5). We identified 749 genes downregulated and 908 genes upregulated following castration. AR regulation of α-methylacyl-CoA racemase expression and three other genes (FAM129A, RAB27A, and KIAA0101) was confirmed. Upregulation of oestrogen receptor 1 (ESR1) expression was observed in malignant epithelia and was associated with differential expression of ESR1-regulated genes and correlated with proliferation (Ki-67 expression).

PATIENT SUMMARY: This first-in-man study defines the rapid gene expression changes taking place in prostate cancer (PCa) following castration. Expression levels of the genes that the androgen receptor regulates are predictive of treatment outcome. Upregulation of oestrogen receptor 1 is a mechanism by which PCa cells may survive despite castration.

STAT3 regulated ARF expression suppresses prostate cancer metastasis

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

BACKGROUND: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling.

METHODS: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ(2) tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided.

RESULTS: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts.

CONCLUSIONS: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.