Expression of VEGFxxxb, the inhibitory isoforms of VEGF, in malignant melanoma

Abstract

Malignant melanoma is the most lethal of the skin cancers and the UK incidence is rising faster than that of any other cancer. Angiogenesis -the growth of new vessels from preexisting vasculature -is an absolute requirement for tumour survival and progression beyond a few hundred microns in diameter. We previously described a class of anti-angiogenic isoforms of VEGF, VEGF xxx b, that inhibit tumour growth in animal models, and are downregulated in some cancers, but have not been investigated in melanoma. To determine whether VEGF xxx b expression was altered in melanoma, PCR and immunohistochemistry of archived human tumour samples were used. In normal epidermis and in a proportion of melanoma samples, VEGF xxx b staining was seen. Some melanomas had much weaker staining. Subsequent examination revealed that expression was significantly reduced in primary melanoma samples (both horizontal and vertical growth phases) from patients who subsequently developed tumour metastasis compared with those who did not (analysis of variance (ANOVA) Po0.001 metastatic vs nonmetastatic), irrespective of tumour thickness, while the surrounding epidermis showed no difference in expression. Staining for total VEGF expression showed staining in metastatic and nonmetastatic melanomas, and normal epidermis. An absence of VEGF xxx b expression appears to predict metastatic spread in patients with primary melanoma. These results suggest that there is a switch in splicing as part of the metastatic process, from anti-angiogenic to pro-angiogenic VEGF isoforms. This may form part of a wider metastatic splicing phenotype.

The transcription factor E2F1 and the SR protein SC35 control the ratio of pro-angiogenic versus antiangiogenic isoforms of vascular endothelial growth factor-A to inhibit neovascularization in vivo

“…We observed that expression of the VEGF 165 b transcript increased in response to E2F1 induction, a phenomenon that was not observed with the E2F1 mutant (E132). Similar results were obtained using another set of primers that was complementary to exons 7 and 7/8b, and that allowed the detection of VEGF 165 b mRNA only ( Figure 4b) (Pritchard-Jones et al, 2007). Therefore, although E2F1 decreases VEGF xxx mRNA and protein levels, it seems to affect the expression of VEGF xxx b isoforms in an opposite manner.…”

“…The DNA-binding activity of E2F1 was required in this setting, as a similar decrease was not observed when the E2F1 mutant (E132) was overexpressed. Importantly, we also observed a decrease in both VEGF 165 and VEGF 189 mRNA levels in E2F1-overexpressing cells when we performed RT-PCR analysis using primers targeting exons 7 and 8a of VEGF-A that do not detect VEGF xxx b isoforms (Bates et al, 2006;Pritchard-Jones et al, 2007;Varey et al, 2008) (Figure 1a, lower panel). Therefore, these data indicate that E2F1 downregulates the expression level of pro-angiogenic VEGF xxx transcripts.…”

“…Taken together, these results show that E2F1 can repress VEGF-A promoter activity independently of p53 and under normoxic conditions. (Bates et al, 2002(Bates et al, , 2006Woolard et al, 2004;Pritchard-Jones et al, 2007;Rennel et al, 2008). Using primers that were complementary to exons 4 and 8b, we were able to amplify a fragment with a size that was consistent with the VEGF 165 b transcript ( Figure 4b, right panel).…”

“…For VEGF xxx amplification, the PCR conditions (Cheung et al, 1998) were as follows: 94 1C for 2 min, 29 cycles of 94 1C for 45 s, 60 1C for 1 min and 72 1C for 1 min, and 72 1C for 10 min. The detection of VEGF xxx transcripts using an exon 7 forward primer and a reverse-specific primer for exon 8a that do not detect VEGF xxx b transcripts was performed as described previously (Pritchard-Jones et al, 2007). To detect the antiangiogenic VEGF xxx b splice variants, the PCR conditions were 94 1C for 2 min, 35 cycles of 96 1C for 30 s, 65 1C for 30 s, 72 1C for 1 min, and 72 1C for 10 min for the exon 4/exon 8b primer set.…”

“…VEGF xxx b isoforms are generated by the use of an alternative 3 0 splice acceptor site in the final exon 8 of VEGF-A, leading to the synthesis of proteins of the same length, but with distinct C-terminal six amino acids (Bates et al, 2002). At the biological level, VEGF xxx isoforms are pro-angiogenic and are upregulated in human tumours, whereas VEGF xxx b isoforms are antiangiogenic and are downregulated in some tumours (Bates et al, 2002;Woolard et al, 2004;Pritchard-Jones et al, 2007). It has been shown that VEGF 165 b binds VEGFR-2 and inhibits its downstream classical signalling pathway (Woolard et al, 2004;Kawamura et al, 2008).…”