Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Keywords: 589 melanoma •
744 tumors •
748 vascular endothelial growth factor