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In most human cancers the Myc proto-oncogene is highly activated. Dysregulation of Myc oncoprotein contributes to tumorigenesis in numerous tissues and organs. Thus, targeting Myc stability could be a crucial step for cancer therapy. Here we report Smad7 as a key molecule regulating Myc stability and activity by recruiting the F-box protein, Skp2. Ectopic expression of Smad7 downregulated the protein level of Myc without affecting the transcription level, and significantly repressed its transcriptional activity, leading to inhibition of cell proliferation and tumorigenic activity. Furthermore, Smad7 enhanced ubiquitylation of Myc through direct interaction with Myc and recruitment of Skp2. Ablation of Smad7 resulted in less sensitivity to the growth inhibitory effect of TGF-b by inducing stable Myc expression. In conclusion, these findings that Smad7 functions in Myc oncoprotein degradation and enhances the cytostatic effect of TGF-b signaling provide a possible new therapeutic approach for cancer treatment.

Myc, a pleiotropic transcription factor, plays a prominent role in human cancer progression. The regulation of Myc stability, therefore, is critical to understand the molecular mechanism of tumorigenesis in various cancers. Myc stability is tightly regulated by the activity of ubiqutin ligases, Skp2 and Fbw7. However, a key mediator of this process has not been addressed yet. Here, we have identified Smad7 as an intermediary regulating Myc stability by recruiting F-box protein, Skp2. Microarray analysis using the cell lines stably expressed Smad7, indicates that Smad7 markedly inhibits transcription of, c-myc-target genes including Id1, Id2, and Id3 without affecting c-myc gene expression. The expression of Myc protein, however, is down-regulated by induction of Smad7 in vitro, and in vivo, Smad7 transgene inducible system in primary epidermal keratinocyte. Conversely, the loss of Smad7 is implicated in the increase of endogenous Myc and Id2 expression, indicating that Smad7 is involved in post-translational regulation of Myc. Indeed, Smad7 ubiquitylates Myc through Smad7 PY motif, showing that mutation or deletion on PY motif diminishes the ubiquitylation of Myc. Treatment of proteasome inhibitor abates Smad7-mediated Myc degradation in HaCaT cell line that conditionally express level of human c-myc. Mapping of Smad7 interactions with Myc and Skp2 indicates that c-terminus, MH2 domain, of Smad7 binds to 3LRR/7LRR domain (aa142-390) of Skp2, and also interacts with Fbw7. Furthermore, intermediate domain (aa 162-236) of Myc has been identified to interact with c-terminus of Smad7. In the presence of Skp2 and Fbw7, Smad7 facilitated the degradation of Myc, thus impairing Myc stability. Smad7 also markedly represses Myc-mediated transcriptional activity and its target gene expression. These findings provide a new insight of Smad7 functions as a key mediator for the proteolysis of Myc proto-oncoprotein. Citation Format: Jin-Muk Kang, Ja-Shil Hyun, Bona Lee, Ja-Kyung Kim, Ji-Hee Lee, Seong-Jin Kim, Tae-Aug Kim. Smad7 is integrated mediator for the proteolysis of Myc. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5178. doi:10.1158/1538-7445.AM2013-5178