Divergent androgen regulation of unfolded protein response pathways drives prostate cancer

The unfolded protein response (UPR) is a homeostatic mechanism to maintain endoplasmic reticulum (ER) function. The UPR is activated by various physiological conditions as well as in disease states, such as cancer. As androgens regulate secretion and development of the normal prostate and drive prostate cancer (PCa) growth, they may affect UPR pathways. Here, we show that the canonical UPR pathways are directly and divergently regulated by androgens in PCa cells, through the androgen receptor (AR), which is critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1α branch, but simultaneously inhibited PERK signaling. Inhibition of the IRE1α arm profoundly reduced PCa cell growth in vitro as well as tumor formation in preclinical models of PCa in vivo. Consistently, AR and UPR gene expression were correlated in human PCa, and spliced XBP-1 expression was significantly upregulated in cancer compared with normal prostate. These data establish a genetic switch orchestrated by AR that divergently regulates the UPR pathways and suggest that targeting IRE1α signaling may have therapeutic utility in PCa.

Meta-analysis of prostate cancer gene expression data identifies a novel discriminatory signature enriched for glycosylating enzymes

BACKGROUND: Tumorigenesis is characterised by changes in transcriptional control. Extensive transcript expression data have been acquired over the last decade and used to classify prostate cancers. Prostate cancer is, however, a heterogeneous multifocal cancer and this poses challenges in identifying robust transcript biomarkers.

METHODS: In this study, we have undertaken a meta-analysis of publicly available transcriptomic data spanning datasets and technologies from the last decade and encompassing laser capture microdissected and macrodissected sample sets.

RESULTS: We identified a 33 gene signature that can discriminate between benign tissue controls and localised prostate cancers irrespective of detection platform or dissection status. These genes were significantly overexpressed in localised prostate cancer versus benign tissue in at least three datasets within the Oncomine Compendium of Expression Array Data. In addition, they were also overexpressed in a recent exon-array dataset as well a prostate cancer RNA-seq dataset generated as part of the The Cancer Genomics Atlas (TCGA) initiative. Biologically, glycosylation was the single enriched process associated with this 33 gene signature, encompassing four glycosylating enzymes. We went on to evaluate the performance of this signature against three individual markers of prostate cancer, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) expression, prostate specific antigen (PSA) expression and androgen receptor (AR) expression in an additional independent dataset. Our signature had greater discriminatory power than these markers both for localised cancer and metastatic disease relative to benign tissue, or in the case of metastasis, also localised prostate cancer.

CONCLUSION: In conclusion, robust transcript biomarkers are present within datasets assembled over many years and cohorts and our study provides both examples and a strategy for refining and comparing datasets to obtain additional markers as more data are generated.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Salt-inducible kinase 2 regulates mitotic progression and transcription in prostate cancer

UNLABELLED: Salt-inducible kinase 2 (SIK2) is a multifunctional kinase of the AMPK family that plays a role in CREB1-mediated gene transcription and was recently reported to have therapeutic potential in ovarian cancer. The expression of this kinase was investigated in prostate cancer clinical specimens. Interestingly, auto-antibodies against SIK2 were increased in the plasma of patients with aggressive disease. Examination of SIK2 in prostate cancer cells found that it functions both as a positive regulator of cell-cycle progression and a negative regulator of CREB1 activity. Knockdown of SIK2 inhibited cell growth, delayed cell-cycle progression, induced cell death, and enhanced CREB1 activity. Expression of a kinase-dead mutant of SIK2 also inhibited cell growth, induced cell death, and enhanced CREB1 activity. Treatment with a small-molecule SIK2 inhibitor (ARN-3236), currently in preclinical development, also led to enhanced CREB1 activity in a dose- and time-dependent manner. Because CREB1 is a transcription factor and proto-oncogene, it was posited that the effects of SIK2 on cell proliferation and viability might be mediated by changes in gene expression. To test this, gene expression array profiling was performed and while SIK2 knockdown or overexpression of the kinase-dead mutant affected established CREB1 target genes; the overlap with transcripts regulated by forskolin (FSK), the adenylate cyclase/CREB1 pathway activator, was incomplete.

IMPLICATIONS: This study demonstrates that targeting SIK2 genetically or therapeutically will have pleiotropic effects on cell-cycle progression and transcription factor activation, which should be accounted for when characterizing SIK2 inhibitors.

UAP1 is overexpressed in prostate cancer and is protective against inhibitors of N-linked glycosylation

Prostate cancer is the second most common cause of cancer-associated deaths in men, and signaling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Consequently, AR target genes are prominent candidates to be specific for prostate cancer and also important for the survival of the cancer cells. Here we assess the levels of all hexosamine biosynthetic pathway (HBP) enzymes in 15 separate clinical gene expression data sets and identify the last enzyme in the pathway, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), to be highly overexpressed in prostate cancer. We analyzed 3261 prostate cancers on a tissue microarray and found that UAP1 staining correlates negatively with Gleason score (P=0.0039) and positively with high AR expression (P<0.0001). Cells with high UAP1 expression have 10-fold increased levels of the HBP end-product, UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is essential for N-linked glycosylation occurring in the endoplasmic reticulum (ER) and high UAP1 expression associates with resistance against inhibitors of N-linked glycosylation (tunicamycin and 2-deoxyglucose) but not with a general ER stress-inducing agent, the calcium ionophore A23187. Knockdown of UAP1 expression re-sensitized cells towards inhibitors of N-linked glycosylation, as measured by proliferation and activation of ER stress markers. Taken together, we have identified an enzyme, UAP1, which is highly overexpressed in prostate cancer and protects cancer cells from ER stress conferring a growth advantage.

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.

Shared common variants in prostate cancer and blood lipids

BACKGROUND: Epidemiological and clinical studies suggest comorbidity between prostate cancer (PCA) and cardiovascular disease (CVD) risk factors. However, the relationship between these two phenotypes is still not well understood. Here we sought to identify shared genetic loci between PCA and CVD risk factors.

METHODS: We applied a genetic epidemiology method based on conjunction false discovery rate (FDR) that combines summary statistics from different genome-wide association studies (GWAS), and allows identification of genetic overlap between two phenotypes. We evaluated summary statistics from large, multi-centre GWA studies of PCA (n=50 000) and CVD risk factors (n=200 000) [triglycerides (TG), low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol, systolic blood pressure, body mass index, waist-hip ratio and type 2 diabetes (T2D)]. Enrichment of single nucleotide polymorphisms (SNPs) associated with PCA and CVD risk factors was assessed with conditional quantile-quantile plots and the Anderson-Darling test. Moreover, we pinpointed shared loci using conjunction FDR.

RESULTS: We found the strongest enrichment of P-values in PCA was conditional on LDL and conditional on TG. In contrast, we found only weak enrichment conditional on HDL or conditional on the other traits investigated. Conjunction FDR identified altogether 17 loci; 10 loci were associated with PCA and LDL, 3 loci were associated with PCA and TG and additionally 4 loci were associated with PCA, LDL and TG jointly (conjunction FDR <0.01). For T2D, we detected one locus adjacent to HNF1B.

CONCLUSIONS: We found polygenic overlap between PCA predisposition and blood lipids, in particular LDL and TG, and identified 17 pleiotropic gene loci between PCA and LDL, and PCA and TG, respectively. These findings provide novel pathobiological insights and may have implications for trials using targeting lipid-lowering agents in a prevention or cancer setting.

HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.

Maintaining and reprogramming genomic androgen receptor activity in prostate cancer

Prostate cancer treatment is dominated by strategies to control androgen receptor (AR) activity. AR has an impact on prostate cancer development through the regulation of not only transcription networks but also genomic stability and DNA repair, as manifest in the emergence of gene fusions. Whole-genome maps of AR binding sites and transcript profiling have shown changes in the recruitment and regulatory effect of AR on transcription as prostate cancer progresses. Defining other factors that are involved in this reprogramming of AR function gives various opportunities for cancer detection and therapeutic intervention.

Androgen-regulated metabolism and biosynthesis in prostate cancer

Metabolic changes are a well-described hallmark of cancer and are responses to changes in the activity of diverse oncogenes and tumour suppressors. For example, steroid hormone biosynthesis is intimately associated with changes in lipid metabolism and represents a therapeutic intervention point in the treatment of prostate cancer (PCa). Both prostate gland development and tumorigenesis rely on the activity of a steroid hormone receptor family member, the androgen receptor (AR). Recent studies have sought to define the biological effect of the AR on PCa by defining the whole-genome binding sites and gene networks that are regulated by the AR. These studies have provided the first systematic evidence that the AR influences metabolism and biosynthesis at key regulatory steps within pathways that have also been defined as points of influence for other oncogenes, including c-Myc, p53 and hypoxia-inducible factor 1α, in other cancers. The success of interfering with these pathways in a therapeutic setting will, however, hinge on our ability to manage the concomitant stress and survival responses induced by such treatments and to define appropriate therapeutic windows.

HOXB13, RFX6 and prostate cancer risk

A new study shows that HOXB13 is preferentially recruited to the risk allele of a prostate cancer-associated SNP, enhancing the expression of RFX6, a driver of prostate cancer cell migration and predictor of disease progression. The work illustrates how a single risk locus contributes both to prostate cancer incidence and, through functional follow-up, to disease progression.

O-GlcNAc transferase integrates metabolic pathways to regulate the stability of c-MYC in human prostate cancer cells

Metabolic disruptions that occur widely in cancers offer an attractive focus for generalized treatment strategies. The hexosamine biosynthetic pathway (HBP) senses metabolic status and produces an essential substrate for O-linked β-N-acetylglucosamine transferase (OGT), which glycosylates and thereby modulates the function of its target proteins. Here, we report that the HBP is activated in prostate cancer cells and that OGT is a central regulator of c-Myc stability in this setting. HBP genes were overexpressed in human prostate cancers and androgen regulated in cultured human cancer cell lines. Immunohistochemical analysis of human specimens (n = 1987) established that OGT is upregulated at the protein level and that its expression correlates with high Gleason score, pT and pN stages, and biochemical recurrence. RNA interference-mediated siliencing or pharmacologic inhibition of OGT was sufficient to decrease prostate cancer cell growth. Microarray profiling showed that the principal effects of OGT inhibition in prostate cancer cells were related to cell-cycle progression and DNA replication. In particular, c-MYC was identified as a candidate upstream regulator of OGT target genes and OGT inhibition elicited a dose-dependent decrease in the levels of c-MYC protein but not c-MYC mRNA in cell lines. Supporting this relationship, expression of c-MYC and OGT was tightly correlated in human prostate cancer samples (n = 1306). Our findings identify HBP as a modulator of prostate cancer growth and c-MYC as a key target of OGT function in prostate cancer cells.

Disseminated tumor cells and their prognostic significance in nonmetastatic prostate cancer patients

Detection of pretreatment disseminated cells (pre-DTC) reflecting its homing to bone marrow (BM) in prostate cancer (PCa) might improve the current model to predict recurrence or survival in men with nonmetastatic disease despite of primary treatment. Thereby, pre-DTC may serve as an early prognostic biomarker. Post-treatment DTCs (post-DTC) finding may supply the clinician with additional predictive information about the possible course of PCa. To assess the prognostic impact of DTCs in BM aspirates sampled before initiation of primary therapy (pre-DTC) and at least 2 years after (post-DTC) to established prognostic factors and survival in patients with PCa. Available BM of 129 long-term follow-up patients with T1-3N0M0 PCa was assessed in addition to 100 BM of those in whom a pretreatment BM was sampled. Patients received either combined therapy [n = 81 (63%)], radiotherapy (RT) with different duration of hormone treatment (HT) or monotherapy with RT or HT alone [n = 48 (37%)] adapted to the criteria of the SPCG-7 trial. Mononuclear cells were deposited on slides according to the cytospin methodology and DTCs were identified by immunocytochemistry using the pancytokeratin antibodies AE1/AE3. The median age of men at diagnosis was 64.5 years (range 49.5-73.4 years). The median long-term follow-up from first BM sampling to last observation was 11 years. Categorized clinically relevant factors in PCa showed only pre-DTC status as the statistically independent parameter for survival in the multivariate analysis. Pre-DTCs homing to BM are significantly associated with clinically relevant outcome independent to the patient's treatment at diagnosis with nonmetastatic PCa.

The mitochondrial and autosomal mutation landscapes of prostate cancer

BACKGROUND: Prostate cancer (PCa) is the most common cancer in men. PCa is strongly age associated; low death rates in surveillance cohorts call into question the widespread use of surgery, which leads to overtreatment and a reduction in quality of life. There is a great need to increase the understanding of tumor characteristics in the context of disease progression.

OBJECTIVE: To perform the first multigenome investigation of PCa through analysis of both autosomal and mitochondrial DNA, and to integrate exome sequencing data, and RNA sequencing and copy-number alteration (CNA) data to investigate how various different tumor characteristics, commonly analyzed separately, are interconnected.

DESIGN, SETTING, AND PARTICIPANTS: Exome sequencing was applied to 64 tumor samples from 55 PCa patients with varying stage and grade. Integrated analysis was performed on a core set of 50 tumors from which exome sequencing, CNA, and RNA sequencing data were available.

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Genes, mutated at a significantly higher rate relative to a genomic background, were identified. In addition, mitochondrial and autosomal mutation rates were correlated to CNAs and proliferation, assessed as a cell cycle gene expression signature.

RESULTS AND LIMITATIONS: Genes not previously reported to be significantly mutated in PCa, such as cell division cycle 27 homolog (Saccharomyces cerevisiae) (CDC27), myeloid/lymphoid or mixed-lineage leukemia 3 (MLL3), lysine (K)-specific demethylase 6A (KDM6A), and kinesin family member 5A (KIF5A) were identified. The mutation rate in the mitochondrial genome was 55 times higher than that of the autosomes. Multilevel analysis demonstrated a tight correlation between high reactive-oxygen exposure, chromosomal damage, high proliferation, and in parallel, a transition from multiclonal indolent primary PCa to monoclonal aggressive disease. As we only performed targeted sequence analysis; copy-number neutral rearrangements recently described for PCa were not accounted for.

CONCLUSIONS: The mitochondrial genome displays an elevated mutation rate compared to the autosomal chromosomes. By integrated analysis, we demonstrated that different tumor characteristics are interconnected, providing an increased understanding of PCa etiology.

The androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man

The androgen receptor (AR) regulates prostate cell growth in man, and prostate cancer is the commonest cancer in men in the UK. We present a comprehensive analysis of AR binding sites in human prostate cancer tissues, including castrate-resistant prostate cancer (CRPC). We identified thousands of AR binding sites in CRPC tissue, most of which were not identified in PC cell lines. Many adjacent genes showed AR regulation in xenografts but not in cultured LNCaPs, demonstrating an in-vivo-restricted set of AR-regulated genes. Functional studies support a model of altered signaling in vivo that directs AR binding. We identified a 16 gene signature that outperformed a larger in-vitro-derived signature in clinical data sets, showing the importance of persistent AR signaling in CRPC.