Riluzole, an inhibitor of
glutamate release, has shown the ability to inhibit
melanoma cell xenograft growth. A phase 0 clinical trial of
riluzole as a single agent in patients with
melanoma resulted in involution of
tumors associated with inhibition of both the
mitogen-activated protein kinase (MAPK) and phophoinositide-3-
kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that
riluzole inhibits AKT-mediated
glycogen synthase kinase 3 (GSK3) phosphorylation in
melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of
transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether
riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that
riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and
serine 204 respectively. Using GSK3 inhibitors and
siRNA knock-down, we demonstrate that the mechanism of
riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The
riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that
riluzole-induced Smad linker phosphorylation is independent of the expression of the
metabotropic glutamate receptor 1 (GRM1), which is one of the
glutamate receptors whose involvement in human
melanoma has been documented. We further show that
riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by
riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage
melanomas.