背景:三阴性乳腺癌(TNBC)是一种异质性癌,其特征是所有乳腺癌亚型中最具侵袭性的表型。然而,由于缺乏特异性靶标和有效的靶向治疗,TNBC患者的治疗选择具有有限的临床疗效。
目的:研究一种新的雌激素受体(ER)-α剪接变体ER-α30在乳腺癌细胞中的生物学特性,以及它在calycosin抗癌作用中的可能作用,一种典型的植物雌激素来源于本草植物黄芪,反对TNBC。这也可以更好地理解calycosin对TNBC进展的抑制活性。
方法:收集乳腺癌组织和癌旁组织,并使用免疫组织化学(IHC)分析ER-α30的表达水平,通过Westernblot和qRT-PCR检测其在两种TNBC细胞系(MDA-MB-231和BT-549)中的表达。然后细胞活力的改变,凋亡,迁移,通过CCK-8,Hoechst33258,伤口愈合,在两个TNBC细胞系中进行transwell和western印迹分析。接下来,通过CCK-8、集落形成、流式细胞术,Hoechst33258和蛋白质印迹测定,以及ER-α30在这些效应中的作用以及ER-α30可能的下游靶标。此外,体内实验是使用用calycosin腹膜内处理的MDA-MB-231异种移植模型进行的。测量异种移植瘤的体积和重量以评估calycosin的体内抗癌活性,同时通过IHC检测肿瘤组织中ER-α30表达的相应变化。
结果:证明了新型ER-α剪接变体ER-α30主要分布在TNBC细胞核中。与正常乳腺组织相比,ER-α30在ER-和孕激素受体(PR)阴性亚型乳腺癌组织中的表达水平明显升高,与正常乳腺细胞系MCF10A相比,在TNBC细胞系(MDA-MB-231和BT-549)中也是如此。此外,ER-α30过表达显着增强细胞活力,迁移,侵袭和EMT进程和减少TNBC细胞凋亡,而shRNA介导的ER-α30敲低显示了相反的结果。值得注意的是,calycosin以剂量依赖性方式抑制ER-α30的表达,伴随着TNBC生长和转移的抑制。对于从MDA-MB-231细胞产生的异种移植物观察到类似的发现。用calycosin治疗抑制了肿瘤生长并降低了肿瘤组织中ER-α30的表达。此外,calycosin的这种抑制作用在ER-α30敲低细胞中更为明显。同时,我们发现ER-α30与PI3K和AKT活性呈正相关,它也可以通过calycosin处理灭活。
结论:第一次,已证明,新型雌激素受体-α剪接变体ER-α30可以通过参与细胞增殖而在TNBC的背景下充当促肿瘤发生因子,凋亡,侵袭和转移,因此,它可能成为TNBC治疗的潜在治疗靶点.毛黄蛋白酶可以降低ER-α30介导的PI3K/AKT通路的激活,从而抑制TNBC的发展和进展,提示calycosin可能是TNBC的潜在治疗选择。
BACKGROUND: Triple-negative breast cancer (TNBC) is a heterogeneous carcinoma characterized by the most aggressive phenotype among all breast cancer subtypes. However, therapeutic options for TNBC patients have limited clinical efficacy due to lack of specific target and efficient targeted therapeutics.
OBJECTIVE: To investigate the biological characteristics of a novel estrogen receptor (ER)-α splice variant ER-α30 in breast cancer cells, and its possible role in the anticancer effects of calycosin, a typical phytoestrogen derived from the herbal plant Astragalus membranaceus, against TNBC. This may also provide a better understanding of the inhibitory activity of calycosin on TNBC progression.
METHODS: Breast cancer tissues and para-cancer tissues were collected and analyzed for the expression levels of ER-α30 using immunohistochemistry (IHC), and its expression in two TNBC cell lines (MDA-MB-231 and BT-549) was detected by western blot and qRT-PCR assays. Then the alteration of cell viability, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in response to overexpression or knockdown of ER-α30 was separately determined by CCK-8, Hoechst 33258, wound healing, transwell and western blot assays in two TNBC cell lines. Next, the anticancer effects of calycosin on MDA-MB-231 cells were evaluated through CCK-8, colony formation, flow cytometry, Hoechst 33258 and western blot assays, along with the role of ER-α30 in these effects and the possible downstream targets of ER-α30. In addition, the in vivo experiments were carried out using MDA-MB-231 xenograft model intraperitoneally treated with calycosin. The volume and weight of xenograft tumor were measured to evaluate the in vivo anticancer activities of calycosin, while the corresponding changes of ER-α30 expression in tumor tissues were detected by IHC.
RESULTS: It was demonstrated that the novel ER-α splice variant ER-α30 was primarily distributed in the nucleus of TNBC cells. Compared with normal breast tissues, ER-α30 expression was found in significantly higher levels in breast cancer tissues of ER- and progesterone receptor (PR)-negative subtype, so did in TNBC cell lines (MDA-MB-231 and BT-549) when compared to normal breast cell line MCF10A. Moreover, ER-α30 overexpression strikingly enhanced cell viability, migration, invasion and EMT progression and reduced apoptosis in TNBC cells, whereas shRNA-mediated knockdown of ER-α30 revealed the opposite results. Notably, calycosin suppressed the expression of ER-α30 in a dose-dependent manner, accompanied with the inhibition of TNBC growth and metastasis. A similar finding was observed for the xenografts generated from MDA-MB-231 cells. The treatment with calycosin suppressed the tumor growth and decreased ER-α30 expression in tumor tissues. Furthermore, this inhibition by calycosin was more pronounced in ER-α30 knockdown cells. Meanwhile, we found a positive relationship between ER-α30 and the activity of PI3K and AKT, which could also be inactivated by calycosin treatment.
CONCLUSIONS: For the first time, it is demonstrated that the novel estrogen receptor-α splice variant ER-α30 could function as pro-tumorigenic factor in the context of TNBC by participating in cell proliferation, apoptosis, invasion and metastasis, thus it may serve as a potential therapeutic target for TNBC therapy. Calycosin could reduce the activation of ER-α30-mediated PI3K/AKT pathway, thereby inhibited TNBC development and progression, suggesting that calycosin may be a potential therapeutic option for TNBC.