Malignant melanoma, as all solid tumours, are dependent on angiogenesis for their growth. Vascular Endothelial growth factor, the predominant angiogenic factor is known to be upregulated in melanoma. We recently described a novel isoform of VEGF -VEGF165b (Bates et al. 2002) – that is down-regulated in renal and prostate cancer and inhibits endothelial cell proliferation, migration, vasodilatation and angiogenesis. To determine whether VEGF165b was similarly downregulated in melanoma, we have measured the expression VEGF165b mRNA in aggressively metastatic malignant melanoma.
10-16 serial sections were taken from eight formalin fixed and paraffin embedded primary cutaneous metastatic melanomas (0.88-8.mm thick) from the pathology archives at Frenchay Hospital with local Ethics Committee approval. mRNA was extracted from sections using the technique described by Krafft et al. (1997) and reverse transcribed. The cDNA was then subjected to a PCR reaction using primers designed to amplify the region from exon 7 (primer sequence, GTAAGC TTG TAC AAG ATC CGC AGA CG) to the 3’UTR region (ATG GAT CCG TAT CAG TCT TTC CTG G), or with primers specific for VEGF165b (across the exon 7/9 boundary, TTA AGC TTT CAG TCT TTC CTG GTG AGA CTG CA) or VEGF165 (in exon 8, TCA CCG CCT CGG CTT GTC ACA T). Agarose gel electrophoresis and ethidium bromide staining showed expression of both VEGF165 and VEGF 165b in all eight melanomas. The intensity of the bands produced by gel electrophoresis was measured using NIH Image, compared to a control band and then subjected to statistical analysis using a paired t test. Although both isoforms appear to be equally expressed in normal skin, the intensity of the VEGF165b band was surprisingly significantly greater than VEGF165 (P < 0.001) in these metastatic melanomas. (VEGF165b-35.6 ± 14.9 % of control cDNA. VEGF165 5.9 ± 16.6 %).
These findings with cutaneous melanoma suggest a mechanism of angiogenic factor expression and regulation that is different in metastatic malignant melanoma than it is in prostate and renal cancer.
This work was supported by The Skin Cancer Research Fund, and the Wellcome Trust.