Wnt-Notch和Wnt-ERBB信号通路调控NSCLC上皮间质转化和耐药的机制研究

Significant improvements in the outcome of non-small cell lung carcinoma (NSCLC) have been reported in patients treated with the epidermal growth factor receptor (EGFR) inhibitors(EGFR-TKIs). However, EGFR-TKIs) elicit markedly different clinical response rates due to differences in drug bypass signaling as well as genetic variations of drug target and downstream drug-resistant genes. Less than 50% patient EGFR-TKIs response can be accurately predicted by the current developed drug response biomarkers. It is crucial to identify the novel mechanisms involved in the EGFR-TKIs resistance. Cancer cells that change from stationary cells (epithelial) that line organs into mobile mesenchymal cells become more invasive, resistant to treatment and are associated with metastasis and stem cell characteristics. In our previous study, we analyzed the gene expression of 54 non-small cell lung cancer lines and find sets of genes involved in epithelial-to-mesenchymal transition (EMT), Notch and ERBB signaling. Little is known about Notch and ERBB signaling in EMT dependent drug resistant. Based on these observations and established experimental systems, we expect to further evaluate the importance of Notch and ERBB signaling in epithelial and mesenchymal NSCLC models. In the current application ,we extend our hypothesis as follows: NSCLC cells might activate Wnt/β-catenin and potentates its effect by activating Notch2 and amphiregulin(AREG), which may promote the EMT and drug resistant characters of NSCLC cell-lines. Thus, the activated Wnt-Notch and Wnt-ERBB signaling are crucial switches between EGFR-TKIs resistant and sensitive of NSCLC cells. Biochemical and molecular biological functional analyses employing western blotting, immuno-fluorescence assay, gene transfection and RNAi techniques will be used to provide evidences for the possible mechanisms and nature interactions between Wnt-ERBB and Wnt-Notch signaling. This study will not only clarify the underlying novel EGFR-TKIs resistant mechanisms but also facilitate the discovery of novel biomarkers for clinical application of reduces the risks of EGFR-TKIs treatment in NSCLC patients.

分子靶向药物吉非替尼在EGFR激酶受体突变的晚期NSCLC患者中显示了显著的疗效。由于晚期肺癌肿瘤生物学行为的多样性，患者最终都会不可避免地出现药物抗药性，从而导致治疗失败和疾病的恶化，系统研究其分子机制是目前临床亟待解决的问题。我们前期研究表明上皮细胞间质化转化（EMT）是诱导NSCLC肿瘤细胞耐药特性出现的分子基础。Wnt/βcatenin信号可能与Notch和ERBB通路形成复杂的交互网络，调控EMT依赖的原发和继发性耐药。在此基础上，我们拟通过扰动Wnt/β-catenin信号通路及其靶基因Notch2和AREG的表达，观察其对细胞形态和周期、细胞增殖和侵袭能力以及吉非替尼药物敏感性的影响，探索Wnt信号通过TCF4/LEF调控Notch2和AREG基因表达的分子机制，验证Notch2及AREG基因/蛋白表达预测吉非替尼的临床意义和价值，发展早期诊断和预测药物耐药风险的分子标记物。

本课题的研究目的是通过研究Wnt信号转导通路通过与Notch和ERBB等信号通路的交互，诱导细胞依赖EMT过程及耐药发生的机制，从而发现早期诊断和预测药物耐药风险的分子标记。课题主要研究内容包括⑴通过扰动Wnt/β-catenin信号通路，初步探索对Notch2和AREG基因表达的影响，并观察其对细胞形态和周期、细胞增殖和侵袭能力以及吉非替尼药物敏感性的影响；⑵探索Wnt信号通过TCF4/LEF调控Notch2和AREG基因表达的分子机制；⑶验证Notch2、AREG等相关基因/蛋白表达预测吉非替尼耐药的临床意义和价值。目前本课题针对前2项研究内容按计划开展了有序的研究，第3项研究内容由于正在收集临床样本，尚未能按计划开展。通过本课题的支助发表SCI文章2篇，申请软件著作权1项，研究成果分别参加1次国际会议和1次国内会议并做相关报告，拟申请专利1项。.在对Wnt与Notch信号通路交互的研究中，我们筛选EGFR-TKI敏感和EGFR-TKI耐药的NSCLC细胞系，Wnt、Notch以及STAT3等蛋白在NSCLC细胞系中呈现出不同的表达。以HCC827细胞系为研究体系表明Wnt可能通过与Notch信号通路的相互作用影响NSCLC从上皮向间质转化的表型，影响细胞的药物敏感性。Wnt与Notch的交互主要依赖于Notch2配体Jag1的表达。此外，研究首次在NSCLC中发现Wnt2是通过与FZD7而不是其他受体的相互作用诱导细胞迁移和侵袭。在对Wnt与ERBB信号通路交互的研究中，Wnt1/Wnt2通路信号的增强未能显著影响AREG基因的转录和表达，因此Wnt与ERBB信号通路的交互及由此产生的细胞耐药机制尚不明确。在研究中，我们同时在HCC827中发现Wnt诱导STAT3的磷酸化，通过从数十种中药的有效成分中筛选出靶向Wnt\STAT3的中药锦灯笼有效活性成分physalin A能够在15μM的浓度有效抑制H292、H1975和HCC827Wnt1细胞的增殖，并进一步研究了靶向性药物Gefitinib以及中药锦灯笼有效活性成分physalin A在Wnt\STAT3信号通路过度激活细胞系中的抑癌敏感性及作用机制。研究中我们通过文献挖掘的方法建立了蛋白质翻译后修饰数据库SysPTM，并收集非小细胞肺癌针对EGFR激酶抑制剂和HER2激酶抑制剂的耐药机制数据(建立数据库