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.

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

Background: Androgen steroid hormones are key drivers of prostate cancer. Previous work has shown that androgens can drive the expression of alternative mRNA isoforms as well as transcriptional changes in prostate cancer cells. Yet to what extent androgens control alternative mRNA isoforms and how these are expressed and differentially regulated in prostate tumours is unknown. Methods: Here we have used RNA-Seq data to globally identify alternative mRNA isoform expression under androgen control in prostate cancer cells, and profiled the expression of these mRNA isoforms in clinical tissue. Results: Our data indicate androgens primarily switch mRNA isoforms through alternative promoter selection. We detected 73 androgen regulated alternative transcription events, including utilisation of 56 androgen-dependent alternative promoters, 13 androgen-regulated alternative splicing events, and selection of 4 androgen-regulated alternative 3' mRNA ends. 64 of these events are novel to this study, and 26 involve previously unannotated isoforms. We validated androgen dependent regulation of 17 alternative isoforms by quantitative PCR in an independent sample set. Some of the identified mRNA isoforms are in genes already implicated in prostate cancer (including LIG4, FDFT1 and RELAXIN), or in genes important in other cancers (e.g. NUP93 and MAT2A). Importantly, analysis of transcriptome data from 497 tumour samples in the TGCA prostate adenocarcinoma (PRAD) cohort identified 13 mRNA isoforms (including TPD52, TACC2 and NDUFV3) that are differentially regulated in localised prostate cancer relative to normal tissue, and 3 ( OSBPL1A, CLK3 and TSC22D3) which change significantly with Gleason grade and  tumour stage. Conclusions: Our findings dramatically increase the number of known androgen regulated isoforms in prostate cancer, and indicate a highly complex response to androgens in prostate cancer cells that could be clinically important.

Androgen receptor (AR) signalling and the PI3K pathway mediate survival signals in prostate cancer, and have been shown to regulate each other by reciprocal negative feedback, such that inhibition of one activates the other. Understanding the reciprocal regulation of these pathways is important for disease management as tumour cells can adapt and survive when either single pathway is inhibited pharmacologically. We recently carried out genome-wide exon-specific profiling of prostate cancer cells to identify novel androgen-regulated transcriptional events. Here we interrogated this dataset for novel androgen-regulated genes associated with the PI3K pathway. We find that the PI3K regulatory subunits PIK3R1 (p85α) and PIK3R3 (p55γ) are direct targets of the AR which are rapidly repressed by androgens in LNCaP cells. Further characterisation revealed that the PIK3CA p110α catalytic subunit is also indirectly regulated by androgens at the protein level. We show that PIK3R1 mRNA is significantly under-expressed in prostate cancer (PCa) tissue, and provide data to suggest a context-dependent regulatory mechanism whereby repression of the p85α protein by the AR results in destabilisation of the PI3K p110α catalytic subunit and downstream PI3K pathway inhibition that functionally affects the properties of prostate cancer cells.

Prostate is the most frequent cancer in men. Prostate cancer progression is driven by androgen steroid hormones, and delayed by androgen deprivation therapy (ADT). Androgens control transcription by stimulating androgen receptor (AR) activity, yet also control pre-mRNA splicing through less clear mechanisms. Here we find androgens regulate splicing through AR-mediated transcriptional control of the epithelial-specific splicing regulator ESRP2. Both ESRP2 and its close paralog ESRP1 are highly expressed in primary prostate cancer. Androgen stimulation induces splicing switches in many endogenous ESRP2-controlled mRNA isoforms, including splicing switches correlating with disease progression. ESRP2 expression in clinical prostate cancer is repressed by ADT, which may thus inadvertently dampen epithelial splice programmes. Supporting this, treatment with the AR antagonist bicalutamide (Casodex) induced mesenchymal splicing patterns of genes including FLNB and CTNND1. Our data reveals a new mechanism of splicing control in prostate cancer with important implications for disease progression.

Cell migration drives cell invasion and metastatic progression in prostate cancer and is a major cause of mortality and morbidity. However the mechanisms driving cell migration in prostate cancer patients are not fully understood. We previously identified the cancer-associated cell migration protein Tetraspanin 1 (TSPAN1) as a clinically relevant androgen regulated target in prostate cancer. Here we find that TSPAN1 is acutely induced by androgens, and is significantly upregulated in prostate cancer relative to both normal prostate tissue and benign prostate hyperplasia (BPH). We also show for the first time, that TSPAN1 expression in prostate cancer cells controls the expression of key proteins involved in cell migration. Stable upregulation of TSPAN1 in both DU145 and PC3 cells significantly increased cell migration and induced the expression of the mesenchymal markers SLUG and ARF6. Our data suggest TSPAN1 is an androgen-driven contributor to cell survival and motility in prostate cancer.