RESULTS: TCGA datasets were used to evaluate the expression of STAT5A in normal and different cancerous tissues through TIMER2.0, indicating that STAT5A level was decreased in breast cancer tissues compared with normal ones. Gene Set Enrichment Analysis predicted that STAT5A was associated with the activation of immune cells and cell cycle process. We further demonstrated that the infiltration of immune cells was positively associated with STAT5A level. Influorescence staining revealed the expression and distribution of F-actin was regulated by STAT5A, while colony formation assay, wound healing and transwell assays predicted the inhibitory role of STAT5A in the colony formation, migratory and invasive abilities in breast cancer cells. In addition, overexpression of the Notch3 intracellular domain (N3ICD), the active form of Notch3, resulted in the increased expression of STAT5A. Conversely, silencing of Notch3 expression by siNotch3 decreased STAT5A expression, supporting that STAT5A expression is positively associated with Notch3 in human breast cancer cell lines and breast cancer tissues. Mechanistically, chromatin immunoprecipitation showed that Notch3 was directly bound to the STAT5A promoter and induced the expression of STAT5A. Moreover, overexpressing STAT5A partially reversed the enhanced mobility of breast cancer cells following Notch3 silencing. Low expression of Notch3 and STAT5A predicted poorer prognosis of patients with breast cancer.
CONCLUSIONS: The present study demonstrates that Notch3 inhibits metastasis in breast cancer through inducing transcriptionally STAT5A, which was associated with tumor-infiltrating immune cells, providing a novel strategy to treat breast cancer.
结果:TCGA数据集用于通过TIMER2.0评估正常和不同癌组织中STAT5A的表达,表明乳腺癌组织中STAT5A水平较正常组织降低。基因集富集分析预测STAT5A与免疫细胞的激活和细胞周期进程有关。我们进一步证明免疫细胞浸润与STAT5A水平呈正相关。荧光染色显示F-肌动蛋白的表达和分布受STAT5A调控,而集落形成测定,伤口愈合和transwell分析预测了STAT5A在集落形成中的抑制作用,乳腺癌细胞的迁移和侵袭能力。此外,Notch3胞内结构域(N3ICD)的过表达,Notch3的活性形式导致STAT5A的表达增加。相反,通过siNotch3沉默Notch3表达降低STAT5A表达,支持STAT5A在人乳腺癌细胞系和乳腺癌组织中的表达与Notch3呈正相关。机械上,染色质免疫沉淀显示Notch3直接结合STAT5A启动子并诱导STAT5A的表达。此外,过表达STAT5A部分逆转了Notch3沉默后乳腺癌细胞的移动性增强。Notch3和STAT5A的低表达预测乳腺癌患者预后较差。
结论:本研究表明Notch3通过转录诱导STAT5A抑制乳腺癌转移,这与肿瘤浸润的免疫细胞有关,提供一种治疗乳腺癌的新策略。