BACKGROUND Bag-1 (Bcl-2-associated athanogene) is a multifunctional anti-apoptotic protein frequently overexpressed in cancer. Bag-1 interacts with a variety of cellular targets including Hsp70/Hsc70 chaperones, Bcl-2, nuclear hormone receptors, Akt and Raf kinases. In this study, we investigated in detail the effects of Bag-1 on major cell survival pathways associated with breast cancer. METHODS Using immunoblot analysis, we examined Bag-1 expression profiles in tumor and normal tissues of breast cancer patients with different receptor status. We investigated the effects of Bag-1 on cell proliferation, apoptosis, Akt and Raf kinase pathways, and Bad phosphorylation by implementing ectopic expression or knockdown of Bag-1 in MCF-7, BT-474, MDA-MB-231 and MCF-10A breast cell lines. We also tested these in tumor and normal tissues from breast cancer patients. We investigated the interactions between Bag-1, Akt and Raf kinases in cell lines and tumor tissues by co-immunoprecipitation, and their subcellular localization by immunocytochemistry and immunohistochemistry. RESULTS We observed that Bag-1 is overexpressed in breast tumors in all molecular subtypes, i.e., regardless of their ER, PR and Her2 expression profile. Ectopic expression of Bag-1 in breast cancer cell lines results in the activation of B-Raf, C-Raf and Akt kinases, which are also upregulated in breast tumors. Bag-1 forms complexes with B-Raf, C-Raf and Akt in breast cancer cells, enhancing their phosphorylation and activation, and ultimately leading to phosphorylation of the pro-apoptotic Bad protein at Ser112 and Ser136. This causes Bad's re-localization to the nucleus, and inhibits apoptosis in favor of cell survival. CONCLUSIONS Overall, Bad inhibition by Bag-1 through activation of Raf and Akt kinases is an effective survival and growth strategy exploited by breast cancer cells. Therefore, targeting the molecular interactions between Bag-1 and these kinases might prove an effective anticancer therapy.

中文翻译：

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Bag-1 通过激活 Akt 和 Raf 激酶来刺激 Bad 磷酸化，从而介导乳腺癌细胞的存活。

背景 Bag-1（Bcl-2 相关的 athanogene）是一种多功能抗凋亡蛋白，在癌症中经常过度表达。Bag-1 与多种细胞靶标相互作用，包括 Hsp70/Hsc70 伴侣、Bcl-2、核激素受体、Akt 和 Raf 激酶。在这项研究中，我们详细研究了 Bag-1 对与乳腺癌相关的主要细胞存活途径的影响。方法使用免疫印迹分析，我们检查了具有不同受体状态的乳腺癌患者的肿瘤和正常组织中的 Bag-1 表达谱。我们通过在 MCF-7、BT-474、MDA-MB-231 和 MCF-1 中异位表达或敲低 Bag-1，研究了 Bag-1 对细胞增殖、凋亡、Akt 和 Raf 激酶途径以及 Bad 磷酸化的影响。 10A乳腺细胞系。我们还在乳腺癌患者的肿瘤和正常组织中测试了这些。我们通过免疫共沉淀研究了细胞系和肿瘤组织中 Bag-1、Akt 和 Raf 激酶之间的相互作用，并通过免疫细胞化学和免疫组织化学研究了它们的亚细胞定位。结果我们观察到，Bag-1 在所有分子亚型的乳腺肿瘤中均过表达，即，无论其 ER、PR 和 Her2 表达谱如何。Bag-1 在乳腺癌细胞系中的异位表达会导致 B-Raf、C-Raf 和 A​​kt 激酶的激活，这些激酶在乳腺肿瘤中也会上调。Bag-1 与乳腺癌细胞中的 B-Raf、C-Raf 和 A​​kt 形成复合物，增强其磷酸化和激活，最终导致促凋亡 Bad 蛋白 Ser112 和 Ser136 磷酸化。这导致 Bad 重新定位到细胞核，并抑制细胞凋亡，有利于细胞存活。结论 总体而言，Bag-1 通过激活 Raf 和 A​​kt 激酶进行不良抑制是乳腺癌细胞利用的有效生存和生长策略。因此，针对 Bag-1 和这些激酶之间的分子相互作用可能被证明是一种有效的抗癌疗法。