Identification of SMURF proteins as negative regulators of the oncogenic Hedgehog/Gli1 pathway

GLI1 is a transcriptional factor known as the main effector of the Hedgehog (Hh) signaling pathway, which is involved in cell proliferation during embryonic development. Although GLI1 has been identified in the context of the Hh pathway, its transcriptional activity can be enhanced through non-canonical mechanisms and its dysregulated activity is strongly associated with tumorigenesis. Since GLI1 is the ultimate effector of the Hedgehog signaling pathway and it can also support and promote cell proliferation when activated by non-canonical mechanisms, it appears to be the most promising therapeutic target, and consequently, understanding its regulatory mechanisms has become increasingly crucial. Among these mechanisms, protein ubiquitination and degradation are already known as central in the GLI1 protein turnover. We focused our attention on the potential role of the two HECT-E3 Ubiquitin Ligases SMURF1 and SMURF2, on GLI1 signaling. We observed that SMURFs overexpression leads to a reduction of GLI1 protein levels and, accordingly, it promotes a downregulation of Hh-GLI1 transcriptional activity. In order to clarify the mechanism used by SMURF proteins to induce GLI1 degradation, we performed co-immunoprecipitation and in vitro-binding assays which confirmed an interaction between SMURFs and GLI1. Moreover, SMURFs overexpression increases GLI1 ubiquitination levels, and this modification leads to GLI1 degradation by proteasome. Finally, we demonstrated that SMURFs overexpression in medulloblastoma cell lines is able to reduce their proliferation rate by inducing GLI1 protein degradation. Taken together, our evidence indicates a previously unknown inhibitory role for SMURFs protein on Gli1 post-translational regulation, suggesting new approaches for medulloblastoma treatments and a SMURFs role as oncosuppressor in this context.