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Updated: May 22, 2025
Studying TGF-β Signaling and TGF-β-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells
Published on: October 27, 2020
OTUD3 inhibits breast cancer cell metastasis by regulating TGF-β pathway through deubiquitinating SMAD7
Chenchen Geng1, Ke Dong2, Junhua An2
1Department of Ultrasound, Qilu Hospital of Shandong University (Qingdao), Qingdao, 266035, China.
Background:
Breast cancer (BRCA) is the most common malignant tumor in women, and distant metastasis is an important cause of death. Epithelial mesenchymal transition (EMT) is an important factor in tumor cell metastasis, in which TGF-β signaling pathway plays an important role. SMAD7 can inhibit TGF-β pathway. Previously, we found that ovarian tumor domain-containing protein 3(OTUD3) could maintain the stability of multiple molecules through deubiquitination. In this study, multiple experiments were conducted to verify whether OTUD3 can inhibit TGF-β pathway by deubiquitinating SMAD7.
Methods:
Firstly, bioinformatics was used to search the expression of OTUD3 in breast cancer and its correlation with SMAD7 in the TCGA database. The correlation between the protein and mRNA expression levels of OTUD3 and SMAD7 in multiple BRCA cell lines was verified. Also, the OTUD3 and SMAD7 expression in human BRCA samples and its influence on prognosis were verified by immunohistochemical experiments. Then, the CO-IP experiment was performed by transfecting OTUD3 and SMAD7 in HEK293T cells to confirm whether OTUD3 could maintain SMAD7 protein stability through deubiquitination. Furthermore, luciferase reporting assay, in vitro protein interaction, and transwell assay were used to verify whether OTUD3 could inhibit TGF-β pathway by deubiquitinating SMAD7 and affect cell invasion. Western blot and RT-qPCR were used to detect the correlation between OTUD3 and molecules regulated by the TGF-β pathway. Finally, the effect of OTUD3 on tumor cells was determined by 3D matrigel cell culture.
Results:
The expression of OTUD3 was low in BRCA and positively correlated with SMAD7. Cytological experiments and immunohistochemistry confirmed that OTUD3 was positively correlated with the expression of SMAD7, and the patients with a low expression of OTUD3 had a short recurrence-free survival (RFS). Cell experiments confirmed that OTUD3 could regulate the TGF-β pathway by deubiquitinating SMAD7, which affected EMT and inhibited cell invasion. OTUD3 was found to inhibit the stemness of tumor cells by 3D matrigel cell culture.
Conclusions:
Our findings indicated OTUD3 inhibited BRCA metastasis associated with TGF-β signaling by deubiquitination to stabilize SMAD7 protein levels.
Insights
Ovarian tumor domain-containing protein 3 (OTUD3) inhibits breast cancer (BRCA) metastasis by stabilizing SMAD7 protein levels, thereby regulating the TGF-β signaling pathway. This finding offers a potential therapeutic target for reducing BRCA cell invasion and stemness.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Breast cancer (BRCA) is a leading cause of cancer death in women, primarily due to distant metastasis.
- Epithelial-mesenchymal transition (EMT), crucial for tumor metastasis, is significantly influenced by the TGF-β signaling pathway.
- SMAD7 acts as an inhibitor of the TGF-β pathway, and ovarian tumor domain-containing protein 3 (OTUD3) is known to stabilize proteins via deubiquitination.
Purpose of the Study:
- To investigate whether OTUD3 inhibits the TGF-β signaling pathway by deubiquitinating and stabilizing SMAD7.
- To explore the correlation between OTUD3, SMAD7, and breast cancer progression and prognosis.
Main Methods:
- Bioinformatic analysis of TCGA database for OTUD3 and SMAD7 expression and correlation in BRCA.
- Immunohistochemistry on human BRCA samples to assess protein/mRNA expression and patient prognosis.
- Co-immunoprecipitation (CO-IP) to confirm OTUD3's role in SMAD7 deubiquitination and stability.
- Luciferase assays, in vitro protein interaction, Transwell, Western blot, and RT-qPCR to analyze TGF-β pathway regulation, EMT, invasion, and stemness.
Main Results:
- Low OTUD3 expression in BRCA was positively correlated with SMAD7 expression and negatively with patient recurrence-free survival (RFS).
- OTUD3 deubiquitinates SMAD7, enhancing its stability and consequently inhibiting TGF-β signaling, EMT, and cancer cell invasion.
- OTUD3 was demonstrated to reduce the stemness of breast cancer cells.
Conclusions:
- OTUD3 inhibits breast cancer metastasis by deubiquitinating and stabilizing SMAD7, thereby suppressing the TGF-β signaling pathway.
- OTUD3 represents a potential therapeutic target for mitigating breast cancer progression and metastasis.
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