背景:三阴性乳腺癌(TNBC)的特征在于其高转移潜力,导致患者生存率低下。癌相关成纤维细胞(CAF)在通过诱导线粒体生物发生促进TNBC转移方面至关重要。然而,如何抑制CAF赋予的线粒体生物合成仍需探索。
方法:我们使用伤口愈合和细胞侵袭试验研究了转移,3D文化,anoikis检测,和NOD/SCID小鼠。线粒体生物发生通过MitoTracker绿色FM染色检测,线粒体DNA水平的定量,和蓝色天然聚丙烯酰胺凝胶电泳。表达式,转录,通过蛋白质印迹法检测过氧化物酶体增殖物激活受体共激活因子1α(PGC-1α)的磷酸化,染色质免疫沉淀,双荧光素酶报告分析,定量聚合酶链反应,免疫沉淀,和液相色谱-串联质谱。使用Kaplan-Meier绘图仪数据库和临床乳腺癌组织样本评估了PGC-1α在TNBC中的预后作用。
结果:我们证明PGC-1α提示淋巴结转移,肿瘤血栓形成,TNBC患者的生存率很低,它是由CAFs诱导的,在TNBC中充当线粒体生物发生和转移的诱导剂。紫草素阻碍了CAF诱导的PGC-1α表达,核定位,以及与雌激素相关受体α(ERRα)的相互作用,从而抑制靶向PGC-1α/ERRα的线粒体基因。机械上,PGC-1α的下调是由合酶激酶3β诱导的PGC-1α在Thr295的磷酸化介导的,这与神经前体细胞表达的发育下调的4e1识别以及随后通过泛素蛋白水解的降解有关。PGC-1α在Thr295的突变消除了紫草素对CAF刺激的TNBC线粒体生物发生和转移的体外和体内抑制作用。
结论:我们的研究结果表明,PGC-1α是通过破坏线粒体生物发生来阻断TNBC转移的可行靶标,紫草素具有通过靶向PGC-1α作为线粒体生物发生抑制剂治疗TNBC转移的潜力。
BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore.
METHODS: We investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan-Meier plotter database and clinical breast cancer tissue samples.
RESULTS: We demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3β-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo.
CONCLUSIONS: Our findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.