Modulation of Mitochondrial ERβ Expression Inhibits Triple-Negative Breast Cancer Tumor Progression by Activating Mitochondrial Function.
Modulation of Mitochondrial ERβ Expression Inhibits Triple-Negative Breast Cancer Tumor Progression by Activating Mitochondrial Function.
复制标题
调节线粒体 ERβ 表达可通过激活线粒体功能抑制三阴性乳腺癌肿瘤进展。
DOI：
10.33594/000000034
复制
发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Sung
中科院分区：
文献类型：
--
作者：
I. Song;Y. Jeong;S. Jeong;J. Kim;Jin Han;Tae;Sung
背景/目的
乳腺癌是一种临床和分子异质性疾病。三阴性乳腺癌（TNBC）患者由于缺乏有效的治疗分子靶点，其预后比其他乳腺癌亚型患者差。本研究旨在鉴定雌激素受体（ER）β作为TNBC细胞中的新型线粒体靶点，以及潜在的机制。
方法
采用qRT-PCR、免疫组化和免疫印迹法检测临床乳腺标本中ERβ的表达。通过共聚焦显微镜分析、免疫共沉淀实验和亚细胞器的有限去污剂提取来确定ERβ-Grp 75的亚细胞分布和结合。CCK-8法和流式细胞仪检测线粒体ERβ（mitoERβ）过表达对细胞增殖和细胞周期分布的影响。线粒体活性氧，膜电位，和钙离子水平使用特定的荧光探针Mito-Sox，TMRE，和Rhod-2AM。使用锚定非依赖性生长试验、球体形成和小鼠原位异种移植模型评估mitoERβ过表达的致瘤效应。
结果
乳腺癌患者肿瘤组织中ERβ表达低于癌旁正常组织，低水平的线粒体ERβ（mitoERβ）也与术后肿瘤复发增加相关。在动物模型中，mitoERβ过表达抑制TNBC细胞和肿瘤块的增殖。此外，mitoERβ的过表达增加了TNBC细胞和正常乳腺MCF 10A细胞中的ATP产生，后者在MCF 10A细胞中被mitoERβ敲低完全逆转。Grp 75通过直接相互作用正调控ERβ向线粒体的转运。免疫共沉淀和亚细胞分级分离实验表明ERβ-Grp 75复合物在线粒体中是稳定的。
结论
这些结果表明，TNBC细胞中mitoERβ的上调确保了适当的线粒体转录，激活OXPHOS系统以产生ATP。研究mitoERβ对乳腺癌中线粒体活性和特定线粒体基因表达的影响可能有助于预测肿瘤复发，为临床决策提供信息，并确定治疗TNBC的新药物靶点。
BACKGROUND/AIMS
Breast cancer is a clinically and molecularly heterogeneous disease. Patients with triple-negative breast cancer (TNBC) have poorer outcomes than those with other breast cancer subtypes due to lack of effective molecular targets for therapy. The present study aimed to the identification of estrogen receptor (ER)β as a novel mitochondrial target in TNBC cells, together with underlying mechanisms.
METHODS
Expression of ERβ in clinical breast samples were examined by qRT-PCR, immunohistochemistry and immunoblotting. Subcellular distribution and binding of ERβ-Grp75 was determined by confocal microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. The effect of mitocondrial ERβ(mitoERβ) overexpression on cell proliferation and cell cycle distribution were assessed CCK-8 assays and FACS. Mitochondrial ROS, membrane potential, and Ca²⁺ level were measured using the specific fluorescent probes Mito-Sox, TMRE, and Rhod-2AM. The tumorigenic effect of mitoERβ overexpression was assessed using an anchorage-independent growth assay, sphere formation and a mouse orthotopic xenograft model.
RESULTS
ERβ expression was lower in tumor tissue than in adjacent normal tissue of patients with breast cancer, and low levels of mitochondrial ERβ (mitoERβ) also were associated with increased tumor recurrence after surgery. Overexpression of mitoERβ inhibited the proliferation of TNBC cells and tumor masses in an animal model. Moreover, overexpression of mitoERβ increased ATP production in TNBC cells and normal breast MCF10A cells, with the latter completely reversed by mitoERβ knockdown in MCF10A cells. Grp75 was found to positively regulate ERβ translocation into mitochondria via a direct interaction. Coimmunoprecipitation and subcellular fractionation experiments revealed that ERβ-Grp75 complex is stable in mitochondria.
CONCLUSION
These results suggest that the up-regulation of mitoERβ in TNBC cells ensures proper mitochondrial transcription, activating the OXPHOS system to produce ATP. Studying the effects of mitoERβ on mitochondrial activity and specific mitochondrial gene expression in breast cancer might help predict tumor recurrence, inform clinical decision-making, and identify novel drug targets in the treatment of TNBC.