The VEGF-A exon 8 splicing-sensitive fluorescent reporter mouse is a novel tool to assess the effects of splicing regulatory compounds in vivo.

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.

Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma.

BACKGROUND: Current therapies for metastatic melanoma are targeted either at cancer mutations driving growth (e.g., vemurafenib) or immune-based therapies (e.g., ipilimumab). Tumour progression also requires angiogenesis, which is regulated by VEGF-A, itself alternatively spliced to form two families of isoforms, pro- and anti-angiogenic. Metastatic melanoma is associated with a splicing switch to pro-angiogenic VEGF-A, previously shown to be regulated by SRSF1 phosphorylation by SRPK1. Here, we show a novel approach to preventing angiogenesis-targeting splicing factor kinases that are highly expressed in melanomas. METHODS: We used RT-PCR, western blotting and immunohistochemistry to investigate SRPK1, SRSF1 and VEGF expression in tumour cells, and in vivo xenograft assays to investigate SRPK1 knockdown and inhibition in vivo. RESULTS: In both uveal and cutaneous melanoma cell lines, SRPK1 was highly expressed, and inhibition of SRPK1 by knockdown or with pharmacological inhibitors reduced pro-angiogenic VEGF expression maintaining the production of anti-angiogenic VEGF isoforms. Both pharmacological SRPK1 inhibitors and SRPK1 knockdown reduced growth of human melanomas in vivo, but neither affected cell proliferation in vitro. CONCLUSIONS: These results suggest that selective blocking of pro-angiogenic isoforms by inhibiting splice-site selection with SRPK1 inhibitors reduces melanoma growth. SRPK1 inhibitors may be used as therapeutic agents.

Intraluminal polyethylene glycol stabilizes tight junctions and improves intestinal preservation in the rat.

Rapidly progressing mucosal breakdown limits the intestinal preservation time below 10 h. Recent studies indicate that intraluminal solutions containing polyethylene glycol (PEG) alleviate preservation injury of intestines stored in UW-Viaspan. We investigated whether a low-sodium PEG solution is beneficial for intestines stored in histidine-tryptophane-ketoglutarate (HTK) preservation solution. Rat intestines used as control tissue (group 1) were perfused with HTK, groups 2 and 3 received either a customized PEG-3350 (group 2) or an electrolyte solution (group 3) intraluminally before cold storage. Tissue injury, brush-border maltase activity, zonula occludens-1 (ZO-1) and claudin-3 expression in the tight junctions (TJ) were analyzed after 8, 14 and 20 h. We measured epithelial resistance and permeability (Ussing chamber) after 8 and 14 h. Group 2 had superior morphology while maltase activity was similar in all groups. TJ proteins rapidly decreased and decolocalized in groups 1 3; these negative events were delayed in group 2, where colocalization persisted for about 14 h. Intestines in group 2 had higher epithelial resistance and lower permeability than the other groups. These results suggest that a customized PEG solution intraluminally reduces the intestinal preservation injury by improving several major epithelial characteristics without negatively affecting the brush-border enzymes or promoting edema.

Serine-arginine protein kinase 1 (SRPK1) inhibition as a potential novel targeted therapeutic strategy in prostate cancer.

Angiogenesis is required for tumour growth and is induced principally by vascular endothelial growth factor A (VEGF-A). VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer (PCa). In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by serine-arginine protein kinase 1 (SRPK1). Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared with benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in PCa may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards the anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knockdown (KD). PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1-KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Small-molecule inhibitors of SRPK1 switched splicing towards the anti-angiogenic isoform VEGF165b in PC-3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of PCa. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumour angiogenesis by regulation of VEGF splicing can alter prostate tumour growth and supports further studies for the use of SRPK1 inhibition as a potential anti-angiogenic therapy in PCa.

Hallmarks of alternative splicing in cancer.

The immense majority of genes are alternatively spliced and there are many isoforms specifically associated with cancer progression and metastasis. The splicing pattern of specific isoforms of numerous genes is altered as cells move through the oncogenic process of gaining proliferative capacity, acquiring angiogenic, invasive, antiapoptotic and survival properties, becoming free from growth factor dependence and growth suppression, altering their metabolism to cope with hypoxia, enabling them to acquire mechanisms of immune escape, and as they move through the epithelial-mesenchymal and mesenchymal-epithelial transitions and metastasis. Each of the 'hallmarks of cancer' is associated with a switch in splicing, towards a more aggressive invasive cancer phenotype. The choice of isoforms is regulated by several factors (signaling molecules, kinases, splicing factors) currently being identified systematically by a number of high-throughput, independent and unbiased methodologies. Splicing factors are de-regulated in cancer, and in some cases are themselves oncogenes or pseudo-oncogenes and can contribute to positive feedback loops driving cancer progression. Tumour progression may therefore be associated with a coordinated splicing control, meaning that there is the potential for a relatively small number of splice factors or their regulators to drive multiple oncogenic processes. The understanding of how splicing contributes to the various phenotypic traits acquired by tumours as they progress and metastasise, and in particular how alternative splicing is coordinated, can and is leading to the development of a new class of anticancer therapeutics-the alternative-splicing inhibitors.