TRIM28 promotes tumor growth and metastasis in breast cancer by targeting the BRD7 protein for ubiquitination and degradation.

IF 4.9 2区 医学 Q2 CELL BIOLOGY Cellular Oncology Pub Date : 2024-10-01 Epub Date: 2024-09-02 DOI:10.1007/s13402-024-00981-3
Changning Xue, Hanbing Meng, Weihong Niu, Mengna Li, Jianxia Wei, Shipeng Chen, Lemei Zheng, Yumei Duan, Hongyu Deng, Faqing Tang, Songqing Fan, Ming Tan, Wei Xiong, Ming Zhou
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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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TRIM28 通过靶向 BRD7 蛋白进行泛素化和降解,促进乳腺癌的肿瘤生长和转移。
目的:含溴结构域蛋白7(Bromodomain-containing protein 7,BRD7)在包括乳腺癌在内的多种癌症中下调并发挥肿瘤抑制因子的功能,BRD7表达失调与乳腺癌的发生和发展密切相关。然而,人们很少关注乳腺癌中BRD7蛋白水平的调控,这需要进一步阐明:方法:采用Western印迹法检测乳腺癌细胞中BRD7蛋白的稳定性和乳腺癌组织中BRD7蛋白的水平。在MDA-MB-231细胞中,通过免疫共沉淀结合质谱分析,筛选出可能与BRD7相互作用的E3泛素连接酶蛋白。我们通过共免疫沉淀(Co-IP)和免疫荧光试验证明了BRD7与含三方基序28(TRIM28)之间的相互作用。Co-IP和泛素化实验用于探索BRD7和TRIM28之间的特异性结合域以及BRD7的泛素化位点。通过qPCR、Western Blot和Co-IP实验研究了TRIM28对BRD7蛋白稳定性和泛素化水平的影响。CCK-8和克隆形成试验评估了TRIM28对乳腺癌细胞增殖能力的影响。Transwell试验和伤口愈合试验用于研究TRIM28对乳腺癌细胞侵袭和迁移的影响。流式细胞术检测了TRIM28对乳腺癌细胞周期和凋亡的影响。此外,我们还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。我们设计了一些复原实验来探讨复原 BRD7 在 TRIM28 介导的乳腺癌体内和体外恶性进展中的作用。最后,通过免疫组化证明了TRIM28和BRD7的临床意义:结果:本研究表明,BRD7是一种不稳定蛋白,在乳腺癌中可能受到泛素化的调控;此外,我们还发现TRIM28的Coil-Coil区域可直接与BRD7的N-端结合,TRIM28通过作为潜在的E3泛素连接酶,介导BRD7泛素化和降解,降解过程依赖于K21。此外,TRIM28还能促进细胞增殖、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,恢复乳腺癌中BRD7的表达可显著逆转TRIM28在体外和体内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能显著促进BRD7的泛素化和降解,从而促进乳腺癌的恶性进展。靶向TRIM28/BRD7轴可能是临床诊断和治疗乳腺癌的一种新的潜在策略。
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来源期刊
Cellular Oncology
Cellular Oncology ONCOLOGY-CELL BIOLOGY
CiteScore
10.30
自引率
1.50%
发文量
86
审稿时长
12 months
期刊介绍: The Official Journal of the International Society for Cellular Oncology Focuses on translational research Addresses the conversion of cell biology to clinical applications Cellular Oncology publishes scientific contributions from various biomedical and clinical disciplines involved in basic and translational cancer research on the cell and tissue level, technical and bioinformatics developments in this area, and clinical applications. This includes a variety of fields like genome technology, micro-arrays and other high-throughput techniques, genomic instability, SNP, DNA methylation, signaling pathways, DNA organization, (sub)microscopic imaging, proteomics, bioinformatics, functional effects of genomics, drug design and development, molecular diagnostics and targeted cancer therapies, genotype-phenotype interactions. A major goal is to translate the latest developments in these fields from the research laboratory into routine patient management. To this end Cellular Oncology forms a platform of scientific information exchange between molecular biologists and geneticists, technical developers, pathologists, (medical) oncologists and other clinicians involved in the management of cancer patients. In vitro studies are preferentially supported by validations in tumor tissue with clinicopathological associations.
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