阴虚血瘀证肺腺癌中MACC1蛋白介导非肿瘤干细胞向肿瘤干细胞“逆演进”调控网路障碍和滋阴化瘀法干预机制

It is reported that intervention principle of nourishing yin and removing blood stasis therapy suppressed cell proliferation and induced cell differentiation in lung adenocarcinoma with syndrome of blood stasis due to yin deficiency, however, the mechanism is remain unclear. The dynamic evolution of non-cancer stem cells (non-CSCs) acquire the CSC-like activity under certain conditions is critical for tumor metastatic dissemination in the early stage and recurrence. Recently it has been identified that Metastasis Associated in Colon Cancer 1 (MACC1) is a prognostic biomarker for lung adenocarcinoma metastasis and recurrence. We previously found that MACC1 was correlated with the reverse evolution of non-CSCs to CSCs conversion and cell proliferation in human lung adenocarcinoma significantly in the earlier National Nature Science Foundation Of China (No：81173453). This project aims to establish a dynamic model of non-CSC to CSC conversions in lung adenocarcinoma with syndrome of blood stasis due to yin deficiency, and verify the pivotal role of MACC1 in this conversion. Meanwhile, to explore the dynamic reverse evolution of the MACC1 protein and its downstream factors by analyzing the MACC1 regulated signal pathways, illustrate the mechanism of MACC1 regulatory pathway induces reverse evolution of non-CSC to CSC conversions and the intervention principle of nourishing yin and removing blood stasis therapy in lung adenocarcinoma with syndrome of blood stasis due to yin deficiency. Therefore, this project will provide theoretic base for clinical treatment of nourishing yin and removing blood stasis therapy in lung adenocarcinoma with syndrome of blood stasis due to yin deficiency.

临床研究发现滋阴化瘀法能显著抑制阴虚血瘀证肺腺癌恶性增殖过程并诱导病态细胞趋于正常分化，但其详细机理不明。最新研究显示非肿瘤干细胞获得“干性”逆转为肿瘤干细胞的演变过程是导致肿瘤恶性增殖的关键因素之一。MACC1蛋白是近年发现的介导肺腺癌恶性增殖的重要因子。在我们的前期国家自然科学基金项目（No：81173453）研究中发现MACC1蛋白与肺腺癌非肿瘤干细胞向肿瘤干细胞“逆演进”过程及增殖活性密切相关。本研究拟构建阴虚血虚证肺腺癌非肿瘤干细胞“逆演进”动态转化模型，明确MACC1蛋白在阴虚血瘀证肺腺癌非肿瘤干细胞“逆演进”中的关键作用，并通过体内和体外实验，探索并解析MACC1蛋白信号通路参与调控阴虚血瘀证肺腺癌非肿瘤干细胞增殖过程及滋阴化瘀法干预机制，以期为滋阴化瘀法治疗阴虚血瘀证肺腺癌的分子机制提供理论依据和线索。

阴虚血瘀证肺癌是常见的肺癌证型之一，滋阴化瘀法可有效遏制肺癌的恶性增殖过程，但作用机制不详。肿瘤组织中非肿瘤干细胞通过获得“干性”转变为肿瘤干细胞的动态演变过程是导致肿瘤持续恶性增殖的关键因素之一。本研究通过构建肺癌非肿瘤干细胞向肿瘤干细胞转化的动态演进模型，在体内、体外实验及临床试验中，利用流式细胞分选技术、全基因组测序分析和肿瘤微球形成等技术，探索槐杞黄提取物通过MACC1途径介导非肿瘤干细胞“逆演进”的详细分子机制。研究结果显示：对比GEO数据库中肺癌肿瘤干细胞的数据集，肿瘤干细胞亚群中MACC1基因表达水平显著升高。体外实验中，在肺癌细胞中槐杞黄可下调MACC1蛋白和基因的表达水平，而低表达MACC1能够显著削弱肺癌非肿瘤干细胞向肿瘤干细胞演进过程，同时抑制肺癌细胞的恶性增殖活性，除此敲低MACC1能够增强肺癌干细胞负向调控因子KLF4 mRNA的稳定性。另外我们还发现MACC1能够增强干性因子c-Myc蛋白的稳定性并能够促进癌相关成纤维细胞（CAFs）的侵袭能力，从而干预肺癌非肿瘤干细胞的逆演进进程。体内实验中，槐杞黄通过下调MACC1可有效抑制肺癌细胞的增殖，表现为肿瘤重量和体积的减少。临床试验中，早期阴虚血淤证肺癌患者肿瘤组织中MACC1蛋白表达水平显著高于对应癌旁组织，且MACC1高表达的早期肺癌患者的预后较差。本研究结果表明：阴虚血淤证肺癌发生发展过程中存在非肿瘤干细胞向肿瘤干细胞转变的动态演进过程，而滋阴化淤代表药槐杞黄通过下调MACC1表达水平进而干预MACC1/KLF4与MACC1/c-Myc信号轴，从而达到抑制肺癌非肿瘤干细胞“逆演进”过程，改善肺癌患者预后的目的。本项目为阴虚血瘀证肺癌非肿瘤干细胞“逆演进”为肿瘤干细胞的动态转化提供了实验依据，同时对滋阴化瘀法治疗阴虚血瘀证肺癌，抑制肿瘤恶性增殖提供理论基础和线索。