食管癌中LCN2与LOXL2功能复合物系统鉴定及其分子作用机制研究

In our previous studies, it was found that LCN2 as an important functional protein was significantly and abnormally over-expressed in esophageal cancer. Furthermore, over-expression of LNC2 was correlated with cancer cell invasion, metastasis, lymph node metastasis and poor differentiation, indicating LCN2 as a new molecular marker for predicting prognosis in esophageal cancer. However, the molecular mechanism of LCN2 remains unclear. In our recent experiments, it was found that LCN2 protein interacted with the enzyme protein LOXL2 in an complex, and promoted esophageal cancer development in highly consistence with LOXL2. Based on these evidences, we propose the scientific hypothesis that LCN2 and LOXL2 synergistically promote the malignant development of esophageal carcinoma. At present, multiple profound scientific issues remained to be solved, such as what other functional proteins are contained, the fine interaction patterns between each pair of the protein components, and the molecular mechanisms of LCN2-LOXL2 complex in the malignant progression of esophageal cancer. Therefore, this project will jointly apply the co-immunoprecipitation, protein mass spectrometry, key amino acid point mutation, confocal, gene over-expression or knock-down and other methods in both of cells and animal models, to focus on investigating the above scientific issues and to expound the scientific hypothesis, aiming to profoundly reveal the molecular mechanisms of LCN2 in an unique point view of the LCN2-LOXL2 complex in esophageal carcinoma.

本项目组以往系列研究发现，LCN2在食管癌中显著异常过表达，是食管癌重要功能蛋白，与癌细胞侵袭移动、淋巴结转移、不良分化密切相关，是食管癌预后预警新型分子标志物，但分子作用机制不明确。最近我们在实验中发现，食管癌中LCN2与酶蛋白LOXL2互作，二者居于同一复合物，且在促癌功能方面表现显著一致性。基于此，我们提出LCN2与LOXL2协同促进食管癌恶性演进发展科学假说。目前，有关该复合物中究竟还包含哪些其他功能蛋白、各蛋白成分间的相互作用方式、该功能复合物在食管癌恶性演进中的分子作用机制等更深层次科学问题尚有待研究。为此，本项目拟通过联合运用免疫共沉淀、蛋白质谱、蛋白关键氨基酸位点突变、激光共聚焦、基因高表达或敲降表达等技术方法，同时结合细胞与整体实验动物水平系列实验，着力研究上述科学问题，重点阐明前述科学假说，从与LOXL2形成复合物这一独特角度，深刻揭示LCN2在食管癌中的分子作用机制。

我们通过联合运用基因转染、蛋白质免疫共沉淀、蛋白免疫印迹、划痕愈合、Transwell/Invasion能力检测、流式细胞术、明胶酶谱、荧光基质胶降解实验、3D细胞共培养、细胞免疫荧光等实验技术方法，探讨食管癌中LCN2/LOXL2/MMP9三元复合物的结构形式及其蛋白质相互作用的关键结构域，并在此基础上系统验证三元复合物在食管癌的恶性进展中所发挥的生物学功能及其分子作用机制。研究发现：.1、蛋白互作情况为：1）LCN2与LOXL2在胞内和胞外，细胞饥饿或正常培养均相互作用；2）LCN2与LOXL2在胞内的互作依赖于LOXL2的酶催化活性中心；3）LCN2与MMP9在胞内和胞外上清中均存在蛋白相互作用；4）LOXL2与MMP9的互作仅发生在在胞内。.2、对LCN2+LOXL2、LCN2+MMP9、LOXL2+MMP9三种组合进行过表达，1）这三种蛋白互作均促进肿瘤细胞的移动、侵袭；2）在KYSE410中，三种蛋白互作促进肿瘤细胞的单细胞增殖能力，在KYSE150中，仅LCN2/LOXL2蛋白互作促进肿瘤细胞的单细胞增殖能力；3）三种蛋白互作对肿瘤细胞的克隆形成能力没有影响，且不影响细胞周期的变化。.3、胞外基质分析结果，1）过表达LCN2或者LOXL2增强MMP2和MMP9酶活性；2）三种蛋白相互作用增强MMP2和MMP9酶活性；3）三种蛋白相互作用促进细胞外基质的降解；.4、三种蛋白相互作用促进细胞微丝骨架重排和增强肌动蛋白聚合速率。.5、信号通路分析结果表明，1）过表达LCN2、LOXL2或者MMP9，能激活ERK和AKT信号通路；2）三种蛋白互作异常激活AKT信号通路。.6、LCN2/LOXL2/MMP9 蛋白相互作用促进体内肿瘤生长和转移。.通过上述的实验结果，本项目认为，在食管癌中LCN2/LOXL2/MMP9三元复合物不仅在降解基质方面协同作用，同时还影响细胞微丝骨架系统的重排，提高食管癌细胞的侵袭能力和移动能力，从而促进食管癌的恶性进展。