METHODS: The interaction between β2M and HFE was confirmed by co-immunoprecipitation, mass spectrometry, yeast two-hybrid screening, and His pull-down. The knockdown and overexpression of β2M or HFE were performed in MDA-MB-453 cells, and ERK signaling pathway was subsequently analyzed via western blotting. Apoptotic cells were detected using flow cytometer. β2M, HFE, and p-ERK1/2 were examined in tumor and paired adjacent tissues via immunohistochemistry.
RESULTS: HFE was found to be an interacting protein of β2M in ER-/HER2+ breast cancer cells MDA-MB-453 by co-immunoprecipitation and mass spectrometry. A yeast two-hybrid system and His-pull down experiments verified that β2M directly interacted with HFE. β2M and HFE as a complex were mainly located in the cytoplasm, with some on the cytomembrane of MDA-MB-453 cells. In addition to breast cancer cells BT474, endogenous β2M directly interacted with HFE in breast cancer cells MDA-MB-453, MDA-MB-231, and MCF-7. β2M activated the ERK signaling pathway by interacting with HFE and induced apoptosis of MDA-MB-453 cells. The expression of HFE and p-ERK1/2 showed significantly high levels in HER2-overexpressing breast cancer tumor tissue compared with adjacent normal tissue, consistent with the results obtained from the cell experiments.
CONCLUSIONS: β2M induced apoptosis of tumor cells via activation of the ERK signal pathway by directly interacting with HFE in HER2-overexpressing breast cancer.
方法:通过免疫共沉淀证实了β2M与HFE之间的相互作用,质谱,酵母双杂交筛选,和他的下拉。在MDA-MB-453细胞中进行β2M或HFE的敲减和过表达,和ERK信号通路随后通过蛋白质印迹分析。使用流式细胞仪检测凋亡细胞。β2M,HFE,通过免疫组织化学检查肿瘤和配对的邻近组织中的p-ERK1/2。
结果:通过免疫共沉淀和质谱,发现HFE是ER-/HER2乳腺癌细胞MDA-MB-453中β2M的相互作用蛋白。酵母双杂交系统和His下拉实验验证了β2M与HFE直接相互作用。β2M和HFE作为复合物主要位于细胞质中,MDA-MB-453细胞的细胞膜上有一些。除乳腺癌细胞BT474外,内源性β2M还与乳腺癌细胞MDA-MB-453,MDA-MB-231和MCF-7中的HFE直接相互作用。β2M通过与HFE相互作用激活ERK信号通路并诱导MDA-MB-453细胞凋亡。HER2过表达的乳腺癌肿瘤组织中HFE和p-ERK1/2的表达明显高于癌旁正常组织,与细胞实验结果一致。
结论:β2M在HER2过表达的乳腺癌中直接与HFE相互作用,通过激活ERK信号通路诱导肿瘤细胞凋亡。