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Melissa Gammons, Sarah Coupland, Andrew Dick, David Bates; SRPK1 Inhibition, As A Way Of Targeting Pro-Angiogenic VEGF-A Production In Ocular Tumours. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4526.
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Uveal melanoma (UM), although rare, is the most common type of intraocular tumour. Approximately 50% of UM are fatal due to their tendency to metastasise to the liver. Angiogenesis is regulated by vascular endothelial growth factor-A (VEGF-A), a growth factor that is expressed by UM. VEGF-A is alternatively spliced to form two families of isoforms: those that are pro-angiogenic and those that are anti-angiogenic. Splicing of the pro-angiogenic isoforms is mediated by serine-rich protein kinase-1 (SRPK1), a protein kinase that results in the phosphorylation and nuclear localization of serine-rich splicing factor 1 (SRSF1). SRSF1 is a promoter of proximal splice site selection, thus selectively upregulating pro-angiogenic VEGF-A production. Activation of this pathway can be achieved by administration of insulin-like growth factor-1 (IGF-1), a promoter of VEGF mediated tumour growth. We investigated the effect of SRPK1 inhibition on VEGF-A splicing in UM cell lines.
UM cell lines Mel270, Omm2.5 and 92.1 were grown in culture medium. Both before and after 24hours exposure to 1, 5, 10 or 20μM SRPIN340 or combined with 100nM IGF-1 mRNA was extracted, cDNA was synthesised and used in q-PCR and RT-PCR for SRPK1, VEGF165 and VEGF165b expression. Immunofluorescence determined SRSF1 and SRPK1 localisation within the cells, and the protein assessed for VEGF-A expression by Western blot.
SRPK1 was expressed in the UM cytoplasm and translocated to the nucleus during IGF-1 treatment. IGF-1 dose dependently increased pro-angiogenic VEGF-A mRNA expression whereas inhibition of SRPK1 with SRPIN340 reduced pro-angiogenic VEGF-A expression, maintaining the production of anti-angiogenic VEGF-A isoforms.
Targeting SRPK1 reduces the expression of pro-angiogenic VEGF-A in UM cell lines. Selective blocking of pro-angiogenic isoforms may reduce UM metastasis rates whilst maintaining the production of cytoprotective anti-angiogenic isoforms. We wish to validate these results in fresh UM specimens where clinical outcome and genetic status is known.
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