Song In-Sung, Jeong Yu Jeong, Jeong Seung Hun, Kim Ji Eun, Han Jin, Kim Tae-Hyun, Jang Sung-Wuk
Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.
National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Cardiovascular and Metabolic Disease Center, Inje University, Busan, Korea.
Cell Physiol Biochem. 2019;52(3):468-485. doi: 10.33594/000000034. Epub 2019 Mar 15.
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.
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.
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.
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.
背景/目的：乳腺癌是一种临床和分子层面上的异质性疾病。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更差。本研究旨在确定雌激素受体（ER）β作为TNBC细胞中的一种新型线粒体靶点，并探究其潜在机制。
通过qRT-PCR、免疫组织化学和免疫印迹法检测ERβ在临床乳腺样本中的表达。通过共聚焦显微镜分析、免疫共沉淀实验以及亚细胞器的有限去污剂提取法来确定ERβ与Grp75的亚细胞分布及结合情况。采用CCK-8法和流式细胞术评估线粒体ERβ（mitoERβ）过表达对细胞增殖和细胞周期分布的影响。使用特异性荧光探针Mito-Sox、TMRE和Rhod-2AM测量线粒体活性氧、膜电位和Ca²⁺水平。通过非锚定依赖性生长试验、球体形成实验和小鼠原位异种移植模型评估mitoERβ过表达的致瘤作用。
乳腺癌患者肿瘤组织中ERβ的表达低于相邻正常组织，线粒体ERβ（mitoERβ）水平较低也与术后肿瘤复发增加有关。mitoERβ过表达抑制了TNBC细胞的增殖以及动物模型中的肿瘤块生长。此外，mitoERβ过表达增加了TNBC细胞和正常乳腺MCF10A细胞中的ATP生成，而MCF10A细胞中mitoERβ敲低则完全逆转了后者的情况。研究发现Grp75通过直接相互作用正向调节ERβ向线粒体的转运。免疫共沉淀和亚细胞分级实验表明ERβ-Grp75复合物在线粒体中是稳定的。
这些结果表明，TNBC细胞中mitoERβ的上调可确保线粒体转录正常，激活氧化磷酸化系统以产生ATP。研究mitoERβ对乳腺癌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、为临床决策提供依据，并确定TNBC治疗中的新药物靶点。
