Ouyang Wen, Zhang Shimin, Yang Bo, Yang Chunxu, Zhang Junhong, Zhou Fuxiang, Xie Conghua
Department of Radiation and Medical Oncology, Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430071, P.R. China.
Hubei Key Laboratory of Tumor Biological Behaviors, Zhongnan Hospital, Wuhan University, Wuhan, Hubei 430071, P.R. China.
Oncol Rep. 2016 Feb;35(2):717-24. doi: 10.3892/or.2015.4463. Epub 2015 Nov 27.
To investigate the regulatory mechanisms of decoy receptor expression in TRAIL-resistant breast cancer MCF-7 cells, cytotoxicity and apoptosis assays were applied to examine sensitivity to TRAIL in breast cancer cells. Immunofluorescence and immunoprecipitation were used to detect the co-localization and interaction of USP9x and β-catenin. Luciferase assay was used to examine activity of the DcR1/DcR2/OPG reporter. Overexpression/silencing of β-catenin was performed to confirm β-catenin mediated transcription of the decoy receptors. Additionally, silencing of USP9x was performed to prove that USP9X stabilizes β-catenin and mediates TRAIL-resistance. It was found that USP9x interacted with β-catenin and inhibited the degradation of β-catenin through the deubiquitination of β-catenin. Luciferase reporter assays showed induction of DcR1/DcR2/OPG reporter activity observed upon co-transfection of β-catenin and Tcf-4. The overexpression/silencing of β-catenin further confirmed the role of β-catenin in the regulation of transcription of the decoy receptors. Silencing of USP9x directly evidenced that USP9x affected the protein expression level of β-catenin, the transcription level of the decoy receptors, and reversed TRAIL-resistance of MCF-7 cells. In conclusion, USP9x interacted with and stabilized β-catenin through deubiquitination to mediate transcription of the decoy receptors in breast cancer cells. Our results offer new insights into the mechanisms of resistance to TRAIL, and USP9x could potentially be a therapeutic target for TRAIL-resistant breast cancers.
为了研究诱饵受体表达在TRAIL耐药乳腺癌MCF-7细胞中的调控机制，应用细胞毒性和凋亡试验检测乳腺癌细胞对TRAIL的敏感性。采用免疫荧光和免疫沉淀法检测USP9x与β-连环蛋白的共定位和相互作用。用荧光素酶报告基因检测法检测DcR1/DcR2/OPG报告基因的活性。进行β-连环蛋白的过表达/沉默以证实β-连环蛋白介导诱饵受体的转录。此外，进行USP9x的沉默以证明USP9X稳定β-连环蛋白并介导TRAIL耐药。研究发现USP9x与β-连环蛋白相互作用，并通过β-连环蛋白的去泛素化抑制β-连环蛋白的降解。荧光素酶报告基因检测显示，共转染β-连环蛋白和Tcf-4后，DcR1/DcR2/OPG报告基因活性增强。β-连环蛋白的过表达/沉默进一步证实了β-连环蛋白在诱饵受体转录调控中的作用。USP9x的沉默直接证明了USP9x影响β-连环蛋白的蛋白表达水平、诱饵受体的转录水平，并逆转了MCF-7细胞的TRAIL耐药性。总之，USP9x通过去泛素化与β-连环蛋白相互作用并使其稳定，从而介导乳腺癌细胞中诱饵受体的转录。我们的研究结果为TRAIL耐药机制提供了新的见解，USP9x可能是TRAIL耐药乳腺癌的一个潜在治疗靶点。
