Mechanisms of hypoxic gene regulation of angiogenesis factor Cyr61 in melanoma cells.

Hypoxia has a profound influence on progression and metastasis of malignant tumors. In the present report, we used the oligonucleotide microarray technique to identify new hypoxia-inducible genes in malignant melanoma with a special emphasis on angiogenesis factors. A commercially available Affymetrix gene chip system was used to analyze five melanoma cell lines of different aggressiveness. A total of 160 hypoxia-inducible genes were identified, clustering in four different functional clusters. In search of putative angiogenesis and tumor progression factors within these clusters, Cyr61, a recently discovered angiogenesis factor, was identified. Cyr61 was hypoxia-inducible in low aggressive melanoma cells; however, it showed constitutive high expression in highly aggressive melanoma cells. Further analyses of transcriptional mechanisms underlying Cyr61 gene expression under hypoxia demonstrated that an AP-1 binding motif within the Cyr61 promoter plays a central role in the hypoxic regulation of Cyr61. It could be shown by use of in vitro luciferase assays, electrophoretic mobility shift assays, and immunoprecipitation that hypoxia-inducible factor-1alpha interacts with c-Jun/AP-1 and may thereby contribute to Cyr61 transcriptional regulation under hypoxia. Taken together, the presented data show that Cyr61 is a hypoxia-inducible angiogenesis factor in malignant melanoma with tumor stage-dependent expression. This may argue for a hypoxia-induced selection process during tumor progression toward melanoma cells with constitutive high Cyr61 expression.