NHC Key Laboratory of Carcinogenesis, Hunan Key Laboratory of Oncotarget Gene, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, 410013, China.
Cancer Research Institute, School of Basic Medical Sciences, Central South University, 110 Xiangya Road, Changsha, 410078, Hunan, China.
Cell Oncol (Dordr). 2024 Oct;47(5):1973-1993. doi: 10.1007/s13402-024-00981-3. Epub 2024 Sep 2.
Bromodomain-containing protein 7 (BRD7) is downregulated and functions as a tumor suppressor in many types of cancers including breast cancer, and the dysregulation of BRD7 expression is closely related to the development and progression of breast cancer. Whereas little attention has been focused on the regulation of BRD7 protein levels in breast cancer, which needs to be further elucidated.
The protein stability of BRD7 in breast cancer cells and BRD7 protein level in breast cancer tissues was examined by Western Blotting. The potential E3 ubiquitin ligase proteins that interact with the BRD7 was screened by coimmunoprecipitation combined with mass spectrometry analysis in MDA-MB-231 cells. We proved the interaction between BRD7 and tripartite motif containing 28 (TRIM28) through Co-Immunoprecipitation (Co-IP) and immunofluorescence assays. Co-IP and ubiquitination assay were used to explore the specific binding domain between BRD7 and TRIM28 and the ubiquitination site of BRD7. The effects of TRIM28 on the BRD7 protein stability and ubiquitination level was investigated by qPCR, Western Blot and Co-IP assay. CCK-8 and clone formation assays were carried out to assess the effect of TRIM28 on proliferation ability of breast cancer ells. Transwell assay and wound healing assay were used to investigate the effect of TRIM28 on breast cancer cell invasion and migration. Flow cytometry was used to detect the effect of TRIM28 on cell cycle and apoptosis of breast cancer cells. In addition, we confirmed effect of TRIM28 on tumor growth and metastasis by xenograft and metastatic mouse models. We designed some recovery assays to explore the role of recovery BRD7 in TRIM28-mediated promotion of malignant progression of breast cancer in vivo and in vitro. Finally, the clinical significance of TRIM28 and BRD7 was proved by immunohistochemistry.
In this study, we demonstrated that BRD7 was an unstable protein and might be regulated by ubiquitination in breast cancer; furthermore, we found that the Coiled-Coil region of TRIM28 could directly bind to N-terminal of BRD7, and TRIM28 mediates BRD7 ubiquitination and degradation dependent on K21 by acting as a potential E3 ubiquitin ligase. Moreover, TRIM28 promoted cell proliferation, migration, invasion, xenograft tumor growth and metastasis, thus playing an oncogenic role in breast cancer. Furthermore, the restoration of BRD7 expression in breast cancer significantly reversed the promotional effects of TRIM28 on malignant progression both in vitro and in vivo. In addition, TRIM28 was highly expressed in the biopsy tissues of breast cancer, and its expression was negatively correlated with BRD7 expression and positively correlated with TNM stage and poor prognosis of BC patients.
Our findings provide a novel mechanism by which TRIM28 significantly facilitates BRD7 ubiquitination and degradation, thus promoting breast cancer malignant progression. Targeting the TRIM28/BRD7 axis might be a novel potential strategy for the clinical diagnosis and treatment of breast cancer.
溴结构域蛋白 7（BRD7）在包括乳腺癌在内的多种癌症中下调并作为肿瘤抑制因子发挥作用，BRD7 表达的失调与乳腺癌的发生和发展密切相关。然而，人们对乳腺癌中 BRD7 蛋白水平的调节关注甚少，这需要进一步阐明。
通过 Western Blotting 检测乳腺癌细胞中 BRD7 的蛋白稳定性和乳腺癌组织中 BRD7 蛋白水平。通过 MDA-MB-231 细胞中的免疫共沉淀结合质谱分析筛选与 BRD7 相互作用的潜在 E3 泛素连接酶蛋白。我们通过共免疫沉淀（Co-IP）和免疫荧光实验证明了 BRD7 与三肽重复含 28 个氨基酸（TRIM28）之间的相互作用。通过 Co-IP 和泛素化实验探索 BRD7 和 TRIM28 之间的特异性结合域和 BRD7 的泛素化位点。通过 qPCR、Western Blot 和 Co-IP 实验研究 TRIM28 对 BRD7 蛋白稳定性和泛素化水平的影响。通过 CCK-8 和克隆形成实验评估 TRIM28 对乳腺癌细胞增殖能力的影响。通过 Transwell 实验和划痕愈合实验研究 TRIM28 对乳腺癌细胞侵袭和迁移的影响。通过流式细胞术检测 TRIM28 对乳腺癌细胞周期和凋亡的影响。此外，我们通过异种移植和转移小鼠模型证实了 TRIM28 对肿瘤生长和转移的影响。我们设计了一些恢复实验来探索在体内和体外恢复 BRD7 在 TRIM28 介导的促进乳腺癌恶性进展中的作用。最后，通过免疫组化证实了 TRIM28 和 BRD7 的临床意义。
在这项研究中，我们证明 BRD7 是一种不稳定的蛋白质，可能在乳腺癌中通过泛素化进行调节；此外，我们发现 TRIM28 的卷曲螺旋区可以直接与 BRD7 的 N 端结合，TRIM28 作为一种潜在的 E3 泛素连接酶，通过介导 BRD7 的泛素化和降解来促进细胞增殖、迁移、侵袭、异种移植肿瘤生长和转移，从而在乳腺癌中发挥致癌作用。此外，在体外和体内，恢复 BRD7 表达显著逆转了 TRIM28 对恶性进展的促进作用。此外，TRIM28 在乳腺癌活检组织中高表达，其表达与 BRD7 表达呈负相关，与 BC 患者的 TNM 分期和不良预后呈正相关。
我们的研究结果提供了一种新的机制，即 TRIM28 显著促进 BRD7 的泛素化和降解，从而促进乳腺癌的恶性进展。靶向 TRIM28/BRD7 轴可能是乳腺癌临床诊断和治疗的一种新的潜在策略。
