DACH1与EGFR-STAT3信号的交互作用调控三阴性乳腺癌恶性生物学行为

Comparing with other types of breast cancer, triple negative breast cancer (TNBC) has a worse prognosis for lack of effective molecular target therapy; therefore, it is vital important to explore the key signaling driving the initiation and progression of TNBC. DACH1 has been shown to regulate breast cancer stemness, EMT and metastasis, and its lower expression is associated with unfavorite prognosis. The expression of DACH1 is barely detected in both TNBC cell lines and tumor tissues; however, its role in TNBC has not been clearly elucidated. Hyper-activation of EGFR-STAT3 signaling is a basic characteristic of TNBC, leading to the stemness, invasion, metastasis and therapeutic resistance to chemotherapy and radiotherapy..Analysis of open gene expression databases and preliminary experiments suggested a significant association between DACH1 and EGFR-STAT3, so far, reciprocal regulation of DACH1 with EGFR-STAT3 has not been reported. Hereby, we proposed that unbalanced function of DACH1 and EGFR-STAT3 attribute to the malignant behaviors of TNBC. By modifying the relative expressions of DACH1 or EGFR-STAT3 through ectopic expression or shRNA-based gene silencing, the proliferation, stemness, EMT, invasion, tumorigenesis and sensitivity to chemotherapy/radiotherapy will be determined; Luciferase-based promoter analysis and chromatin immunoprecipitation will be applied to evaluate the association of DACH1 with EGFR and STAT3; The relationships between DACH1 and EGFR-STAT3 signaling with clinic-pathological parameters, such as tumor size, tissue differentiation, lymph node involvement, TNM stage and prognosis will be systematically analyzed. Altogether, our results will deepen the understanding of cross-talk among various signaling in TNBC, providing a molecular basis for precision target therapy. ...Keywords: Breast cancer, EGFR, STAT3, Stemness, EMT

三阴性乳腺癌(TNBC)缺乏有效的分子靶点，预后差；因此，探索TNBC关键信号通路尤其重要。DACH1与乳腺癌干性、EMT和转移相关，其低表达预示患者预后不良。TNBC细胞系和瘤组织中DACH1的表达基本丢失, 但与TNBC的确切关系仍不明确。TNBC中EGFR-STAT3活化增加干性、侵袭、转移和治疗抗性。大数据分析和前期实验提示DACH1与EGFR-STAT3信号之间负相关, 其相互作用未见研究报告。我们推测两个信号间功能失衡是TNBC恶性生物学的重要因素之一。通过调节DACH1或EGFR-STAT3的相对表达，观察TNBC细胞增殖、干性、EMT、侵袭、致瘤性及治疗敏感性；报告基因和免疫沉淀分析DACH1与STAT3结合的分子基础；评价乳腺癌组织DACH1和EGFR-STAT3信号表达与病理-临床特征的关系。研究结果将丰富TNBC信号网络间交互调节，为乳腺癌靶向治疗提供科学依据。

EGFR、STAT3通路的激活与三阴性乳腺癌（TNBC）的进展和不良预后相关，近来调控器官发育的DACH1-EYA-SIX基因网络与乳腺癌的关系引起关注，本项目探索两种信号之间的交互作用。外源表达DACH1抑制MDA-MB-231细胞的恶性行为，增加对EGFR抑制剂（Erlotinib）和STAT3抑制剂(Statitic)的敏感性；而过表达SIX1促进MDA-MB-231和4T1细胞的增殖，且减低对Erlotinib和Statitic的敏感性。TCGA等数据库分析结果显示EGFR和STAT3表达呈正相关，高表达EGFR和STAT3的TNBC患者无复发生存率（RFS）和总生存期（OS）更短。Erlotinib或Statitic或两药联合治疗乳腺癌细胞，并使用Chou-Talalay方法分析有无协同作用，结果表明双药联合有协同抑癌效果。 Western blot结果显示两药联合可以协同性抑制ERK/AKT信号通路。在乳腺癌小鼠模型中，联合用药组比单药组显示出更好的抗癌效果；IHC染色证实联合用药组对比单药组Ki67的表达更低，而cleaved-caspase 3, caspase 9的表达增加。我们发现EYA2在TNBC组织中高表达，与肿瘤组织的分级成正比，高表达EYA2的患者OS和RFS显著缩短。公共数据库的分析提示EYA2与官腔型标志ER、PR、FOXA1反相关，与基底细胞标志KRT5、EGFR和乳腺癌干性基因YBX-1、KLF5、SOX10成正相关，与DACH1成反比。异位表达EYA2抑制乳腺癌细胞的增殖。蛋白检测提示EYA2增加YBX1、EGFR及PCNA的表达。生信分析显示DACH1与乳腺癌增殖和DNA复制负相关，高表达患者预后好；而EYA2和SIX1与DNA复制、mTOR和Wnt活性成正相关，高表达患者预后差；基于DACH1和SIX1，我们构建乳腺癌预后模型。

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