First, riluzole does not induce C-terminal Smad phosphorylation, suggesting that the TGFb/receptor complexes are not engagedTable 1. Genes whose expression was regulated by riluzole in the WM793 cell line using the Human TGFb/BMP Signaling Pathway RT2 ProfilerTM PCR Array.The fold increase or decrease (comparing to control) was above 2 for these four genes. BMP3: bone morphogenetic protein 3; GDF5: growth differentiation factor 5; INHBB: inhibin, beta B; PLAU: plasminogen activator, urokinase.upon riluzole treatment. In contrast, the initial step after TGFb activation is the C-terminal phosphorylation of Smad2 and Smad3, and this step is required for the TGFb-induced Smad3 phosphorylation [20]. Second, riluzole does not affect the expression of TGFb1, TGFb2 or TGFb3 as shown by real time RT-PCR (data not shown). Therefore, this does not support the possible hypothesis that riluzole increases Smad linker phosphorylation by inducing TGFb production. Finally, in contrast to TGFb-induced Smad linker phosphorylation, the TbRI inhibitor, SB431542, did not inhibit the riluzole-induced Smad linker phosphorylation. The effect of riluzole on the linker phosphorylation of Smad2 and Smad3, downstream effectors of TGFb, will likely modulate TGFb signaling and the expression of TGFb target genes. Our previous report suggested that Smad3 linker phosphorylation might contribute to the resistance to TGFb-mediated cell growth inhibition in melanoma, by inhibiting the expression of p15 and p21. However, Smad3 linker phosphorylation did not inhibit the expression of PAI-1, involved in TGFb pro-oncogenic effects. Therefore, Smad3 activity would be inhibited on promoters involved in cell growth inhibition, such as p15 and p21, but fully competent for regulating some of the genes involved in TGFb prooncogenic effects [10]. This model is in accordance with the well-documented fact that not all Smad transcriptional activities have been disrupted in melanoma cells [1]. It is now clear that Smad transcriptional activities are modulated by phosphorylation at their linker domain, but the nature of this modulation will likely depend on the promoter of each TGFb target gene, of the other transcription factors (repressors, activators), binding this promoter and the consequences of these phosphorylation events on the interaction between linker phosphorylated Smad and these other transcription factors. In addition, the identity of the sites phosphorylated in Smad2 and Smad3 (Threonine 220, Serines 245,250 and 255 for Smad2; Thr179 and Serines 204, 208 and 213 for Smad3) will play a role in the modulation of the TGFb target genes. We have shown that riluzole induces the phosphorylation of the cluster of serines (245/250/255) in Smad2 and serine 204 in Smad3 via GSK3, in the majority of the melanoma cell lines analyzed. The exact consequences of these phosphorylation events on Smad2 and Smad3 transcriptional activities will be promoter dependent, as mentioned previously. We initiated the characterization of genes associated with the TGFb signaling pathway and whose expression was modulated by riluzole. Our goal was to define possible mediators of riluzole action downstream of the TGFb signaling pathway. Since TGFb exerts pro-oncogenic activities at late stages of melanoma development, it is important to determine whether riluzole can have a negative effect on the expression of genes involved in TGFb pro-oncogenic activities. This could explain the inhibition of melanoma cell growth in mice and the involution of some of the tumors in patients treated with riluzole. Alternatively, a positive regulation of genes involved in TGFb pro-oncogenic activities by riluzole could explain the mixed responses to riluzole and the failure of some patients to respond to riluzole in clinical trials [26,27]. We characterized two genes whose expression was upregulated by riluzole. The first one codes for inhibin beta B. Two inhibin beta B subunits form a homodimer called activin B, which is a member of the TGFb superfamily [47]. The fact that riluzole upregulates the expression of activin B could potentially have a negative impact on riluzole response since activin is suggested to play an active role in several carcinomas and glioma migration, invasion and progression [53,54,55,56,57]. In addition, one study suggested that melanoma cells might be resistant to the growth inhibitory and pro-apoptotic effects of activin [58].

Resembling the dual function of TGFb in melanoma, melanoma cells would be resistant to activin-mediated tumor suppression, but would utilize activin to promote their migration and metastasis. The second gene whose expression was upregulated by riluzole was PLAU coding for the urokinase plasminogen activator, described as a TGFb target gene [48,49]. It was shown that tumor growth was retarded in uPA-deficient mice [59,60]. uPA belongs to the uPAS system, which plays multiple roles in the neoplastic evolution, including angiogenesis, tumor cell proliferation, adhesion, migration, intravasation and growth at the metastatic site [61]. As suggested earlier regarding the upregulation of activin expression by riluzole, riluzole positive regulation of PLAU might contribute to the mixed responses to riluzole and the failure of some patients to respond to riluzole in clinical trials [26,27]. At this point of the study, we cannot directly link the riluzoleinduced linker phosphorylation of Smad2 and Smad3 to the upregulation of INHBB and PLAU expression. This would require an extensive analysis using stable transfectants expressing wildtype and Smad mutant forms unable to be phosphorylated in the specific GSK3 sites. However, our study revealed an important cross-talk between three melanoma signaling pathways, the glutamate signaling, the PI3K/AKT pathway and the TGFb signaling pathway. We then focused on the TGFb signaling pathway as a possible mediator of riluzole actions. We then identified two genes whose upregulation by riluzole might be detrimental to a more complete response to this agent in clinical trials. The identification of cross-talks such as those described in this study could be instrumental in predicting responses to riluzolebased therapy.

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