炎症介导的肿瘤微环境是诱导肿瘤转移的重要因素。我们前期报道了炎症介质补体C5a通过结合肿瘤表面C5a受体（C5aR）促进了肺癌侵袭与转移,肿瘤表面C5aR活性及表达强弱直接影响了肺癌细胞的侵袭能力。结合多种蛋白可通过偶联C5aR调控其功能，我们前期通过免疫共沉淀结合质谱分析发现C5aR与钙蛋白酶Capn4等多种蛋白形成复合物，干扰Capn4后可抑制C5a/C5aR介导的肺癌侵袭转移。Capn4是一类蛋白水解酶，通过调控底物的分子结构及活性，影响细胞增殖及迁移，其表达与肿瘤的侵袭转移密切相关。此外，钙蛋白酶和补体共同参与了一些炎症性疾病病理进展，因此，基于国内外研究进展及我们的前期工作基础，本项目拟通过研究Capn4对C5a/C5aR系统的影响，进一步明确Capn4对C5a/C5aR补体系统介导的肺癌转移的调控作用及分子机制，将为炎症诱导的肺癌转移的早期诊断及有效治疗提供新的靶点

Inflammatory microenvironment plays decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion and metastasis. We previously reported anaphylatoxin C5a promoted lung cancer invasion and motility via aberrantly C5a-receptor activation，and the expression and activation of C5aR directly influence the invasion and motility of lung cancer cell.It has been reported a wide range of protein can couple with C5aR and control its activity. Therefore, we performed co-immunoprecipitation combined with mass spectrometry analysis confirmed that C5aR coupled Capn4 and other proteins，and the reduction of Capn4 expression could impaired lung cancer cell invasion induced by C5a/C5aR. As one isoform of calpain family,Capn4 proteolyses substrates at one or limited number of sites to transform and modulate their structure and activities, Capn4 is considered as a modulator that governs various cellular functions such as survival and motility, and the expression of Capn4 was reported to be associated with tumor invasion and metastasis. Additionally, studies had revealed a both roles of complement and calpain in inflammatory disease.Taking into account of previous researches and our previous study, here, we try to explore the regulatory role of Capn4 in C5a/C5aR mediated lung cancer invasion which can be clinically used in early diagnosis and potential therapeutic target against lung cancer metastasis.

炎症介导的肿瘤微环境是诱导肿瘤转移的重要因素。我们前期报道了炎症介质补体 C5a 通过结合肿瘤表面 C5a 受体（C5aR）促进了肺癌侵袭与转移,肿瘤表面 C5aR 活性及表达强弱直接影响了肺癌细胞的侵袭能力。进一步发现C5aR表达与E-cadherin表达负相关，C5aR参与了非小细胞肺癌的上皮间质转化。C5aR下游激活AKT及ERK通路，并促进MMP-2及MMP-9的表达。结合多种蛋白可通过偶联 C5aR 调控其功能，我们通过免疫共沉淀发现 C5aR 与钙蛋白酶 Capn4 相互作用。钙蛋白酶(Calpain)是一类钙依赖的高度保守的蛋白水解酶，调控细胞的生长及迁移，Capn4被证实在肝癌、胆管癌中具有促进肿瘤转移的作用。已知C5aR激活后细胞内钙会内流，进而激活钙蛋白酶，那么钙蛋白酶是否参与了其信号，有待进一步研究。通过western blot，我们发现Capn4在非小细胞肺癌内表达升高，干扰其表达后肺癌细胞系的增殖与侵袭能力均受到抑制。免疫组化检测208例非小细胞肺癌内Capn4的表达，发现Capn4与非小细胞肺癌病人肿瘤大小（p=0.044）及淋巴结转移（p=0.025）密切相关，为病人不良预后的独立预测因素。Capn4可以调控C5aR下游MMP-2的表达发挥促癌作用。已知Capn4为钙蛋白酶小亚基，Calpain 2为大亚基，Capn4作为小亚基辅助大亚基功能，因此我们进一步研究了大亚基Calpain 2的表达情况。发现Calpain 2在肺癌中表达水平上调，并促进肺癌细胞的增殖与迁移能力，calpain 2激活EGFR-AKT信号通路发挥促癌作用，并参与了紫杉醇耐药的作用。因此，本课题完整阐述了钙蛋白酶 Capn4在非小细胞肺癌中的生物学功能，并发现其为病人独立不良预后因素，Capn4调控了C5aR下游MMP-2的表达，为临床筛查高复发人群并给予治疗提供新的理论依据及潜在应用价值。同时，Calpain 2作为Capn4对应大亚基，通过激活EGFR-AKT信号通路促进非小细胞肺癌增殖与迁移，提高非小细胞肺癌对紫杉醇类化疗药物耐药性的作用，为提高患者化疗敏感性提供新的方向。