Galectin-3 Enhances the Malignant Melanoma Phenotype by Regulating Autotaxin

In melanoma patient specimens and cell lines, the over expression of galectin-3 is associated with disease progression and metastatic potential. Herein, we have sought out to determine whether galectin-3 affects the malignant melanoma phenotype by regulating downstream target genes. To that end, galectin-3 was stably silenced by utilizing the lentivirus-incorporated small hairpin RNA in two metastatic melanoma cell lines, WM2664 and A375SM, and subjected to gene expression microarray analysis. We identified and validated the lysophospholipase D enzyme, autotaxin, a promoter of migration, invasion, and tumorigenesis, to be down regulated after silencing galectin-3. Silencing galectin-3 significantly reduced the promoter activity of autotaxin. Interestingly, we also found the transcription factor NFAT1 to have reduced protein expression after silencing galectin-3. Electrophoretic mobility shift assays from previous reports have shown that NFAT1 binds to the autotaxin promoter in two locations. ChIP analysis was performed, and we observed a complete loss of bound NFAT1 to the autotaxin promoter after silencing galectin-3 in melanoma cells. Mutation of the NFAT1 binding sites at either location reduces autotaxin promoter activity. Silencing NFAT1 reduces autotaxin expression while over expressing NFAT1 in NFAT1 negative SB-2 melanoma cells induces autotaxin expression. These data suggest that galectin-3 silencing reduces autotaxin transcription by reducing the amount of NFAT1 protein expression. Rescue of galectin-3 rescues both NFAT1 and autotaxin. We also show that the re-expression of autotaxin in galectin-3 shRNA melanoma cells rescues the angiogenic phenotype in vivo. Furthermore, we identify NFAT1 as a potent inducer of tumor growth and experimental lung metastasis. Our data elucidate a previously unidentified mechanism by which galectin-3 regulates autotaxin and assign a novel role for NFAT1 during melanoma progression.