Zou Man, Li Yanhui, Xia Shu, Chu Qian, Xiao Xiaoguang, Qiu Hong, Chen Yu, Zheng Zu'an, Liu Fei, Zhuang Liang, Yu Shiying
Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Cell Physiol Biochem. 2017;44(2):778-791. doi: 10.1159/000485291. Epub 2017 Nov 23.
BACKGROUND/AIMS: Triple-negative breast cancer (TNBC) is a high-risk breast cancer phenotype without specific targeted therapy options and is significantly associated with increased local recurrence in patients treated with radiotherapy. CAVEOLIN-1 (CAV-1)-mediated epidermal growth factor receptor (EGFR) nuclear translocation following irradiation promotes DNA repair and thus induces radiation resistance. In this study, we aimed to determine whether knockdown of CAV-1 enhances the radiosensitivity of basal-like TNBC cell lines and to explore the possible mechanisms.
Western blotting was used to compare protein expression in a panel of breast cancer cell lines. Nuclear accumulation of EGFR as well as DNA repair and damage at multiple time points following irradiation with or without CAV-1 siRNA pretreatment were investigated using western blotting and confocal microscopy. The radiosensitizing effect of CAV-1 siRNA was evaluated using a clonogenic assay. Flowcytometry was performed to analyse cell apoptosis and cell cycle alteration.
We found that CAV-1 is over-expressed in basal-like TNBC cell lines and barely expressed in HER-2-positive cells; additionally, we observed that HER-2-positive cell lines are more sensitive to irradiation than basal-like TNBC cells. Our findings revealed that radiation-induced EGFR nuclear translocation was impaired by knockdown of CAV-1. In parallel, radiation-induced elevation of DNA repair proteins was also hampered by pretreatment with CAV-1 siRNA before irradiation. Silencing of CAV-1 also promoted DNA damage 24 h after irradiation. Colony formation assays verified that cells could be radiosensitized after knockdown of CAV-1. Furthermore, G2/M cell cycle arrest and apoptosis enhancement may also contribute to the radiosensitizing effect of CAV-1 siRNA.
Our results support the hypothesis that CAV-1 knockdown by siRNA causes increased radiosensitivity in basal-like TNBC cells. The mechanisms associated with this effect are reduced DNA repair through delayed CAV-1-associated EGFR nuclear accumulation and induction of G2/M arrest and apoptosis through the combined effects of CAV-1 siRNA and radiation.
背景/目的：三阴性乳腺癌（TNBC）是一种高危乳腺癌表型，没有特定的靶向治疗方案，并且与接受放射治疗的患者局部复发增加显著相关。照射后，小窝蛋白-1（CAV-1）介导的表皮生长因子受体（EGFR）核转位促进DNA修复，从而诱导放射抗性。在本研究中，我们旨在确定敲低CAV-1是否能增强基底样TNBC细胞系的放射敏感性，并探索其可能的机制。
采用蛋白质免疫印迹法比较一组乳腺癌细胞系中的蛋白质表达。使用蛋白质免疫印迹法和共聚焦显微镜研究在有或没有CAV-1 siRNA预处理的情况下，照射后多个时间点EGFR的核积累以及DNA修复和损伤情况。使用克隆形成试验评估CAV-1 siRNA的放射增敏作用。进行流式细胞术分析细胞凋亡和细胞周期改变。
我们发现CAV-1在基底样TNBC细胞系中过表达，而在HER-2阳性细胞中几乎不表达；此外，我们观察到HER-2阳性细胞系比基底样TNBC细胞对照射更敏感。我们的研究结果表明，敲低CAV-1会损害辐射诱导的EGFR核转位。同时，照射前用CAV-1 siRNA预处理也会阻碍辐射诱导的DNA修复蛋白水平升高。敲低CAV-1还会在照射后24小时促进DNA损伤。集落形成试验证实，敲低CAV-1后细胞对辐射更敏感。此外，G2/M期细胞周期阻滞和凋亡增强也可能有助于CAV-1 siRNA的放射增敏作用。
我们的结果支持以下假设，即通过siRNA敲低CAV-1会导致基底样TNBC细胞放射敏感性增加。与此效应相关的机制是通过延迟与CAV-1相关的EGFR核积累减少DNA修复，并通过CAV-1 siRNA和辐射的联合作用诱导G2/M期阻滞和凋亡。
