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. 2022 Feb 24;17(1):384-396.
doi: 10.1515/med-2022-0437. eCollection 2022.

Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2

Dongqiang Song  1 , Shuyu Li  2 , Liuxin Ning  2 , Shuncai Zhang  2 , Yu Cai  2
Affiliations

Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2

Dongqiang Song et al. Open Med (Wars). .

Abstract

Purpose: Smurf2, one of C2-WW-HECT domain E3 ubiquitin ligases, is closely related to the development and progression in different cancer types, including hepatocellular carcinoma (HCC). This study aims to illustrate the expression and molecular mechanism of Smurf2 in regulating the progression of HCC.

Methods: The expression of Smurf2 in human HCC and adjacent non-tumor liver specimens was detected using tissue microarray studies from 220 HCC patients who underwent curative resection. The relationships of Smurf2 and HCC progression and survival were analyzed using the chi-square test, Kaplan-Meier analysis, and Cox proportional hazards model. For Smurf2 was low expression in HCC cell lines, Smurf2 overexpression cell lines were established. The effect of Smurf2 on cell proliferation and migration was detected by Cell Counting Kit-8 and colony formation assay, and the epithelial-mesenchymal transition (EMT) markers and its transcription factors were tested by immunoblotting. The interaction and ubiquitination of Smad2 by Smurf2 were detected by co-immunoprecipitation and immunoprecipitation assay. Finally, the effect of Smurf2 on HCC was verified using the mouse lung metastasis model.

Results: Smurf2 was downregulated in HCC tissues compared to that of corresponding non-tumor liver specimens. The low expression of Smurf2 in HCC was significantly associated with macrovascular or microvascular tumor thrombus and the impairment of overall survival and disease-free survival. In vitro and in vivo analysis showed that Smurf2 overexpression decreased the EMT potential of HCC cells by promoting the ubiquitination of Smad2 via the proteasome-dependent degradation pathway.

Conclusion: The expression of Smurf2 was downregulated in HCC specimens and affected the survival of patients. Smurf2 inhibited the EMT of HCC by enhancing Smad2 ubiquitin-dependent proteasome degradation.

Keywords: Smad2; Smurf2; hepatocellular carcinoma; metastasis; ubiquitination.

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Conflict of interest statement

Conflict of interest: The author reports no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Smurf2 expression in HCC tissues and adjacent non-tumor tissues. (a) Representative immunohistochemical staining of smurf2 in tumor tissue and para-tumor tissue. Magnification, ×200. (b) IHC score in HCC tissue and para-tumor tissue (n = 220; P < 0.001). (c and d) OS curve and DFS curve for HCC patients with high vs low expression of Smurf2 IHC score generated with Kaplan–Meier methods. (e) The immunoblotting of Smurf2 protein levels in HCC tissues (T), adjacent non-tumor liver tissues (N), and human HCC cell lines. *P < 0.05.
Figure 2
Figure 2
Smurf2 suppressed the migration of human HCC cell lines. (a and b) Wound healing assay in Hep3B-Smurf2 and Huh7-Smurf2 cells, *P < 0.05; (c and d) transwell migration assay of Hep3B-Smurf2 and Huh7-Smurf2 cells, *P < 0.05; (e) the immunoblotting of E-cadherin, ZO-1, Claudin-1, N-cadherin, and Vimentin protein levels in Huh7-Smurf2 cells; (f) the immunoblotting of EMT transcription factors protein levels in Huh7-Smurf2 cells.
Figure 3
Figure 3
Smurf2 had no significant effect on the proliferation of HCC cell lines. (a) Detection of the proliferation of Hep3B-Smurf2 and Huh7-Smurf2 cells by CCK8 assay; *P < 0.05; (b and c) colony-forming assay of Hep3B-Smurf2 and Huh7-Smurf2 cells; *P < 0.05.
Figure 4
Figure 4
Smurf2 promoting the ubiquitination and degradation of Smad2. (a) The immunoblotting of Smurf2 and Smad2, Smad3, Smad4, and Smad7 protein levels in Huh7-Smurf2 cells. (b) Smurf2 promoting the degradation of Smad2 through proteasome; Huh7-Smurf2 cells treated with the proteasome inhibitor MG132 (2 µM) for 8 h; the protein levels of Smad2, Smad3, Smad4, and Smad7; (c and d) Smurf2 promoting the ubiquitination of Smad2; huh7 and hep3B cells treated with PTCDH-Smurf2, immunoprecipitated with anti-Smad2 antibody and subjected to immunoblotting analysis using indicated antibodies. (e) Co-IP assay to verify the interaction between Smurf2 and Smad2 in L02 cells. (f) Huh7 cells, with or without Smurf2 overexpression, were treated with protein synthesis inhibitor CHX (100 µg/mL) to block protein synthesis. immunoblotting was performed to detect Smad2 levels at different time points. (g) Smurf2 down-regulates the expression of Smad2 and inhibits EMT of HCC cells.
Figure 5
Figure 5
Smurf2 suppressing the HCC tumor metastasis in vivo. (a) Representative images of lungs and PET scan of different groups (white circles represent suspected lesions), and representative images of H&E staining of metastatic lung nodules from different groups. (b) Mouse weight was measured once a week at the indicated time points after injection with Huh7-Smurf2 or control cells. (c) Metastatic nodules in lungs of orthotopic xenograft mice model calculated; Values are presented as mean ± SD; *P < 0.05.
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