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, being significantly 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.

Vascular endothelial growth factor (VEGF)-A is differentially spliced to give two functionally different isoform families; pro-angiogenic, pro-permeability VEGF-Axxx and anti-angiogenic, anti-permeability VEGF-Axxxb. VEGF-A splicing is dysregulated in several pathologies, including cancer, diabetes, and peripheral arterial disease. The bichromatic VEGF-A splicing-sensitive fluorescent reporter harboured in a transgenic mouse is a novel approach to investigate the splicing patterns of VEGF-A in vivo. We generated a transgenic mouse harbouring a splicing-sensitive fluorescent reporter designed to mimic VEGF-A terminal exon splicing (VEGF8ab) by insertion into the ROSA26 genomic locus. dsRED expression denotes proximal splice site selection (VEGF-Axxx) and eGFP expression denotes distal splice site selection (VEGF-Axxxb). We investigated the tissue-specific expression patterns in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, and determined whether the splicing pattern could be manipulated in the same manner as endogenous VEGF-A by treatment with the SRPK1 inhibitor SPHINX 31. We confirmed expression of both dsRED and eGFP in the eye, skeletal muscle, cardiac muscle, kidney, and pancreas, with the highest expression of both fluorescent proteins observed in the exocrine pancreas. The ratio of dsRED and eGFP matched that of endogenous VEGF-Axxx and VEGF-Axxxb. Treatment of the VEGF8ab mice with SPHINX 31 increased the mRNA and protein eGFP/dsRED ratio in the exocrine pancreas, mimicking endogenous VEGF-A splicing. The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess splicing regulation in the individual cell-types and tissues, which provides a useful screening process for potentially therapeutic splicing regulatory compounds in vivo

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3′ splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A’s terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3′ splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

Two vascular endothelial growth factor (VEGF) protein families are generated via alternative splicing of the VEGF gene terminal exon. Use of the canonical proximal 3' splice site results in pro-angiogenic VEGF isoforms (VEGFxxx). Anti-angiogenic (VEGFxxxb) isoforms are produced through usage of a distal 3' splice site. In a quest to discover novel anti-angiogenic compounds we performed a screen using molecular tools designed to mimic VEGF splicing. Splicing-sensitive fluorescent reporters (SSFRs) can be used to study specific splicing events. A bichromatic SSFR has been engineered to mimic VEGF terminal exon splicing. Endogenous VEGF intron 7 and parts of exon 8 have been inserted into the reporter. These sequences are followed by the coding sequences for two fluorescent proteins. Depending on which alternative 3' splice site is used, a different fluorescent protein is expressed. This splicing reporter allows VEGF alternative splicing to be followed in vitro and in vivo. Reporter validation was achieved by transfection of the reporter in several cell lines with differential endogenous VEGF splicing as well as by treating stable cell lines expressing the reporter with growth factors or small molecule inhibitors known to alter VEGF alternative splicing. Flow cytometric analysis was used to evaluate the alternative splicing pattern. Following successful validation, several small pilot screens looking for novel regulators of the pro/anti-angiogenic splicing switch were undertaken with growth factors and various chemicals. Following validation and pilot experiments, a large screen of 1280 pharmacologically active compounds (LOPAC library) was performed. Changes to reporter splicing were measured using a fluorescent plate reader. Hits from the screen were investigated further using flow cytometry to confirm alteration in splicing pattern. Several compounds of interest were identified and their effect on endogenous pro/anti-angiogenic VEGF being confirmed by RTPCR. Anti-angiogenic activity is being assessed by several in vitro and in vivo angiogenic assays. Citation Format: Eleanor Star, Steven Harper, David Bates, Sebastian Oltean. Use of VEGF-based splicing-sensitive fluorescent reporters to screen for anti-angiogenic molecules. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A14.