by ecancer reporter Clare Sansom

The process of angiogenesis, or the growth of new blood vessels, is required by all solid tumours if they are to grow larger than, say, 1-2 mm³. It is therefore an important drug target; there are several angiogenesis inhibitors in the clinic, the first being the monoclonal antibody bevacizumab, and many others in development.

Angiogenesis requires the growth factor VEGF (vascular endothelial growth factor), which is the target of bevacizumab and similar drugs. This protein exists in several isoforms that are created by alternative splicing.

There are two main families of VEGF isoforms: those formed by splicing to a proximal splice site in exon 8, including one termed VEGF₁₆₅, promote angiogenesis, whereas those formed by splicing to a distal splice site in the same exon, including VEGF₁₆₅b, are anti-angiogenic. The balance between these isoforms, and thus between pro- and anti-angiogenic factors, is controlled by alterations in the proportion of each splice variant that is generated from the same mRNA transcript, and this process is poorly understood.

However, patients carrying mutations in the Wilms’ tumour suppressor gene WT1, who generally develop kidney tumours during childhood, have been observed to lack the anti-angiogenic isoform. A large, international team of researchers led by Dave Bates from the University of Bristol and Michael Ladomery from the University of the West of England, Bristol, UK, have now investigated the link between WT1 mutations and alternative splicing of VEGF.

The researchers first used an immortalised cell line from a patient bearing a loss-of-function mutation in the WT1 gene to show that cells without functional WT1 produce much less anti-angiogenic VEGF₁₆₅b, but that this can be restored by the addition of wild-type WT1. Transfection of HEK cells with WT1 bearing any one of four mutations also reduced VEGF₁₆₅b expression relative to that of VEGF₁₆₅, whereas transfection of the same cells with wild type WT1 increased the relative expression of VEGF₁₆₅b.

Over-expression of a splicing factor known as arginine/serine-rich 1 (SRSF1) has previously been associated with increased production of VEGF₁₆₅ relative to VEGF₁₆₅b. Bates, Ladomery and their co-workers showed this splicing factor to be localised in the nucleus and cytoplasm of cells with wild type WT1 but in the nucleus only of those bearing WTI splice site mutations.

Next, they showed using immunoblotting that nuclear localisation of SRSF1 was associated with its enhanced phosphorylation. Several kinases, including the splicing-factor kinase SRPK1, have been implicated in SRSF1 phosphorylation, and the researchers were able to prove that SRPK1 was over-expressed in cells lacking functional WT1.

Inhibition of SRPK1 with a small-molecule kinase inhibitor restored both the cytoplasmic location of SRSF1 and expression levels of VEGF₁₆₅b relative to VEGF₁₆₅ in these cells.

The researchers next investigated the mechanism through which WT1 suppresses expression of SRPK1 and, thus, expression of the pro-angiogenic isoform of VEGF.

Using chromatin immunoprecipitation (ChIP), they showed that WT1 bound to a specific consensus binding site on the promoter region of the SRPK1 gene. Although it is likely that other regulators are involved, the researchers were able to implicate lack of WT1 binding to this site in at least some of the over-expression of SRPK1 in WT1 negative cells.

Upregulation of SRPK1 and nuclear localization of SRSF1 were also observed in kidney cells taken from patients carrying Wilms’ tumour mutations.

Finally, the researchers used a mouse model of vascular growth in an eye tissue to show that inhibition of SRPK1 with a small-molecule inhibitor both increased the ratio of VEGF₁₆₅b production relative to VEGF₁₆₅ and decreased endothelial growth and thus angiogenesis.

Tumours in which SRPK1 had been inhibited also grew more slowly. Taken together, these results suggested that inhibition of the kinase SRPK1 could provide a novel mechanism for preventing angiogenesis in solid tumours.

Reference

Amin, E.M., Oltean, S., Hua, J. and 17 others (2011). WT1 Mutants Reveal SRPK1 to Be a Downstream Angiogenesis Target by Altering VEGF Splicing. Cancer Cell 20, 768–780

doi: 350:2335-2342