For each cell line, 1×105 of HEK293T or A375 cells were transfected with 22 pmol of siRNA and/or 1 µg of plasmid DNA in 4 µl JetPrime transfectant diluted in 200 µL buffer, according to manufacturer’s instructions (Polyplus Transfection). EDAR-encoding plasmid was complexed with in vivo JetPEI and injected every 2 days into tumors. Once tumors were established (V≈100 mm3), mice were treated by intra-tumoral injection of 5 µg of EDAR (n = 6) or empty pcDNA3 plasmid (n = 6) diluted in jetPEI in vivo transfectant.

Ectodysplasin receptor EDAR is seen as a typical Tumor Necrosis Factor receptor (TNFR) family member known to interact with its ligand Eda-A1, and signaling mainly through the nuclear factor-kappaB (NF-kappaB) and c-jun N-terminal kinases pathways. Mutations in genes that encode proteins involved in EDAR transduction cascade cause anhidrotic ectodermal dysplasia. Here, we report an unexpected pro-apoptotic activity of EDAR when unbound to its ligand Eda-A1, which is independent of NF-kappaB pathway. Contrarily to other death receptors, EDAR does recruit caspase-8 to trigger apoptosis but solely upon ligand withdrawal, thereby behaving as the so-called dependence receptors. We propose that pro-apoptotic activity of unbound EDAR confers it a tumor suppressive activity. Along this line, we identified loss-of-pro-apoptotic function mutations in EDAR gene in human melanoma. Moreover, we show that the invalidation of EDAR in mice promotes melanoma progression in a B-Raf mutant background. Together, these data support the view that EDAR constrains melanoma progression by acting as a dependence receptor.