Tumor necrosis factor-α (TNF-α) is an immunostimulatory cytokine that is consistently high in the breast tumor microenvironment (TME); however, its differential role in mitochondrial functions and cell survival in ER/PR +ve and ER/PR −ve breast cancer cells is not well understood. In the current study, we investigated TNF-α modulated mitochondrial proteome using high-resolution mass spectrometry and identified the differentially expressed proteins in two different breast cancer cell lines, ER/PR positive cell line; luminal, MCF-7 and ER/PR negative cell line; basal-like, MDA-MB-231 and explored its implication in regulating the tumorigenic potential of breast cancer cells. We also compared the activity of mitochondrial complexes, ATP, and ROS levels between MCF-7 and MDA-MB-231 in the presence of TNF-α. We used Tumor Immune Estimation Resource (TIMER) webserver to analyze the correlation between TNF-α and mitochondrial proteins in basal and luminal breast cancer patients. Kaplan-Meier method was used to analyze the correlation between mitochondrial protein expression and survival of breast cancer patients. The proteome analysis revealed that TNF-α differentially altered the level of critical proteins of mitochondrial respiratory chain complexes both in MCF-7 and MDA-MB-231, which correlated with differential assembly and activity of mitochondrial ETC complexes. The inhibition of the glycolytic pathway in the presence of TNF-α showed that glycolysis is indispensable for the proliferation and clonogenic ability of MDA-MB-231 cells (ER/PR −ve) as compared to MCF-7 cells (ER/PR +ve). The TIMER database showed a negative correlation between the expressions of TNF-α and key regulators of mitochondrial OXPHOS complexes in basal breast vs lobular carcinoma. Conversely, patient survival analysis showed an improved relapse-free survival with increased expression of identified proteins of ETC complexes and survival of the breast cancer patients. The evidence presented in our study convincingly demonstrates that TNF-α regulates the survival and proliferation of aggressive tumor cells by modulating the levels of critical assembly factors and subunits involved in mitochondrial respiratory chain supercomplexes organization and function. This favors the rewiring of mitochondrial metabolism towards anaplerosis to support the survival and proliferation of breast cancer cells. Collectively, the results strongly suggest that TNF-α differentially regulates metabolic adaptation in ER/PR +ve (MCF-7) and ER/PR −ve (MDA-MB-231) cells by modulating the mitochondrial supercomplex assembly and activity.

中文翻译：

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TNF-α 差异调节 ER/PR +ve/−ve 乳腺癌细胞呼吸电子传递复合物的亚基水平，以调节线粒体复合物活性和致瘤潜力


肿瘤坏死因子-α (TNF-α) 是一种免疫刺激细胞因子，在乳腺肿瘤微环境 (TME) 中持续处于高水平；然而，其在 ER/PR +ve 和 ER/PR -ve 乳腺癌细胞中线粒体功能和细胞存活中的不同作用尚不清楚。在本研究中，我们利用高分辨率质谱研究了TNF-α调节的线粒体蛋白质组，并鉴定了两种不同乳腺癌细胞系（ER/PR阳性细胞系）中差异表达的蛋白质； Luminal、MCF-7 和 ER/PR 阴性细胞系；基底样 MDA-MB-231 并探讨了其在调节乳腺癌细胞致瘤潜力中的意义。我们还比较了 TNF-α 存在下 MCF-7 和 MDA-MB-231 之间的线粒体复合物活性、ATP 和 ROS 水平。我们使用肿瘤免疫估计资源 (TIMER) 网络服务器来分析基底癌和管腔乳腺癌患者中 TNF-α 和线粒体蛋白之间的相关性。采用Kaplan-Meier法分析线粒体蛋白表达与乳腺癌患者生存的相关性。蛋白质组分析表明，TNF-α 差异性地改变了 MCF-7 和 MDA-MB-231 中线粒体呼吸链复合物关键蛋白的水平，这与线粒体 ETC 复合物的差异组装和活性相关。 TNF-α 存在时对糖酵解途径的抑制表明，与 MCF-7 细胞 (ER/PR +) 相比，糖酵解对于 MDA-MB-231 细胞 (ER/PR -ve) 的增殖和克隆形成能力是不可或缺的。 ve）。 TIMER 数据库显示基底乳腺与小叶癌中 TNF-α 的表达与线粒体 OXPHOS 复合物的关键调节因子之间呈负相关。 相反，患者生存分析显示，随着 ETC 复合物鉴定蛋白表达的增加，乳腺癌患者的无复发生存得到改善。我们的研究中提供的证据令人信服地证明，TNF-α 通过调节参与线粒体呼吸链超复合物组织和功能的关键组装因子和亚基的水平来调节侵袭性肿瘤细胞的存活和增殖。这有利于线粒体代谢向回补方向重新布线，以支持乳腺癌细胞的存活和增殖。总的来说，结果强烈表明 TNF-α 通过调节线粒体超复合物组装和活性来差异调节 ER/PR +ve (MCF-7) 和 ER/PR -ve (MDA-MB-231) 细胞的代谢适应。