Parinaz Mahdavi and Eirikur Steingrimsson
Introduction: Melanoma, is the deadliest form of skin cancer. Melanoma cells can switch their phenotype from proliferative, non-invasive cells to quiescent, invasive cells and back (phenotype Swithching), thus escaping therapeutic intervention. The Microphthalmia-associated transcription factor (MITF) has been shown to be instrumental in phenotype switching; although the mechanism has not been fully revealed yet. Recent work from our lab has shown that MITF suppresses the expression of FLT1 and VEGFA, a receptor ligand pair that may lead to an autoregulatory loop. Here we have analyzed the relationship between MITF and FLT1/VEGF.
Methods: RNA-seq data was analyzed; qPCR and ELISA used to measure mRNA and protein expression in melanoma cells with and without MITF; ChIP-Seq identified MITF binding sites, and Luciferase reporter assays used to determine effects of the sites. FLT1 knockdown in MITF knockout cells were made to assess effects on proliferation and migration. FLT1 isoforms were overexpressed in melanoma cells and treated with VEGFA to determine effects on proliferation and migration.
Results: Both RNAseq and CCLE data showed a negative correlation between FLT1/VEGFA and MITF expression. qPCR and Luciferase assays demonstrated elevated FLT1/VEGFA expression in MITF knock-out cells, particularly the soluble form of FLT1. FLT1 silencing affected cell migration and proliferation. Overexpression of mFLT1 rescued proliferation and migration in cells treated with VEGFA, while sFLT1-VEGFA decreased cell migration.
Conclusion: Our results show that MITF indirectly inhibits sFLT1 and VEGFA expression. How sFLT1 and VEGFA mediate their effects is not clear. However, it is unlikely to involve a ligand-receptor activity.
