OTUB2 promotes proliferation and metastasis of triple-negative breast cancer by deubiquitinating TRAF6

Abstract

Objectives: Deubiquitinase OTUB2 plays a critical role in the progression of various tumors. However, its specific role in triple-negative breast cancer (TNBC) remains unclear. This study aims to elucidate the biological function of OTUB2 in TNBC and uncover the underlying mechanisms.

Methods: First, we found that the expression of OTUB2 was upregulated in TNBC by bioinformatics analysis, we then validated its expression in TNBC tissues and cells using immunohistochemistry (IHC) and qPCR and plotted the survival curves by Kaplan-Meier method. Gene set enrichment analysis (GSEA) suggested that OTUB2 may be involved in tumor proliferation and metastasis. Further functional assays, including Cell Counting Kit-8 (CCK-8), colony formation, Transwell, and wound healing assays, were performed to assess the effects of OTUB2 overexpression and knockdown on TNBC cell proliferation and migration. Additionally, UbiBrowser 2.0 was used to identify OTUB2 substrate proteins and western blotting was conducted to clarify the molecular mechanisms involved.

Results: Our results demonstrated that OTUB2 expression was elevated in TNBC and associated with poor prognosis. Overexpression of OTUB2 enhanced the proliferation and migration of TNBC cells, while its knockdown inhibited these processes. Moreover, OTUB2 stabilized tumor necrosis factor receptor-associated factor 6 (TRAF6) by deubiquitinating it, leading to activation of the protein kinase B (AKT) pathway.

Conclusions: OTUB2 exerts its promoting effects on the progression of TNBC by activating the TRAF6/AKT pathway.

Keywords: Deubiquitination; OTUB2; Triple-Negative Breast Cancer (TNBC); Tumor necrosis factor receptor-associated factor 6 (TRAF6).

Figure 1. The expression of OTUB2 was significantly increased in TNBC. (A) Volcano plot showed the differentially expressed genes of deubiquitinases in TNBC based on TCGA datasets; (B) The expression of OTUB2 in pan-cancer based on TCGA cancer and normal data analyzed; (C) The expression of OTUB2 in four subtypes of breast cancer; (D) The prognosis induced by OTUB2 expression was analyzed by Kaplan-Meier in patients with BRCA or TNBC; (E) Immunohistochemistry exhibited OTUB2 expression in tumor tissues was higher than normal; (F) Relative mRNA levels of OTUB2 in TNBC cell lines (MDA-MB-231, BT-549, MDA-MB-157, Hs578T, MDA-MB-468) and breast epithelial cell line MCF10A (*p < 0.05, **p < 0.01).

Figure 2. OTUB2 expression was associated with proliferation and metastasis. (A) In GSEA analysis using the TCGA database, the gene sets associated with proliferation and metastasis were significantly enriched in TNBC samples with high OTUB2 expression; (B, C) qPCR analysis of OTUB2 mRNA expression following the indicated transfection; (D, E) Western Blotting showing the protein expression of OTUB2 in stable MDA-MB-231 and BT-549 cells (**p < 0.01).

Figure 3. OTUB2 promoted TNBC cell proliferation in vitro and tumor growth. (A, B) The effect of OTUB2 on cell proliferation was measured by a CCK8 assay. (C, D) The colony formation assay was used to measure cell proliferation. Representative picture (left) and quantitative statistics (right) of colony numbers. (E–G) Influence of OTUB2 on the growth of MDA-MB-231 cells-derived tumors in mice. Tumor images, growth curves, and tumor weights are shown (**p < 0.01).

Figure 4. OTUB2 promoted migration of TNBC cells. (A, B) The migratory abilities of MDA-MB-231 and BT-549 cells transfected with siOTUB2 determined by Transwell assay; (C, D) Transfected with Flag-OTUB2 plasmid or its empty vector; (E, F) The migratory abilities of MDA-MB-231 and BT-549 cells transfected with siOTUB2 determined by wound healing assay; (G, H) Transfected with Flag-OTUB2 plasmid or its empty vector (*p < 0.05, **p < 0.01).

Figure 5. OTUB2 deubiquitinated TRAF6 and activated AKT. (A) Bioinformatics analysis of interaction molecules of OTUB2; (B) Co-IP with anti-Flag antibody showing interactions between exogenous OTUB2 and TRAF6 in HEK293T cells; (C) qPCR analysis was performed to assess the mRNA levels of TRAF6 in MDA-MB-231 and BT-549 cells following OTUB2 overexpression; (D) OTUB2 promoted TRAF6 protein expression in a dose-dependent manner; (E) HEK293T cells transfected with Flag-TRAF6, HA-Ub and Myc-OTUB2 or the empty plasmids following MG132 treatment (10 μm, 6 h) were subjected to denatured-IP and immunoblotted with the indicated antibodies; (F) UbiBrowser 2.0 was used to predict substrates of E3 ligase; (G) Western blot analysis was performed to assess AKT, p-AKT, and TRAF6 protein expression levels in OTUB2 knockdown MDA-MB-231 and BT-549 cells (ns: p > 0.05).

Figure 6. Proposed working model. The figure was generated using Biorender.com. OTUB2 inhibits the degradation of TRAF6 protein in a ubiquitin-proteasome pathway, promotes TNBC proliferation and migration.