Cathepsin D 新的糖基化异构体在肺癌发生发展中的作用及分子机制

Many studies have indicated that Cathepsin D (CD) and/or pro-cathepsin D (pCD) are associated with invasion and metastasis of cancer of beast and ovarian et al. The mutated CD devoid catalytic activity still remained mitogenic for cancer,endothelial and fibroblastic cells, and anti-M6PR antibody and pepstatin A, a natural inhibitor of aspartic proteases can not inhibit above bio-effects.These data suggested that pCD/CD possibly functions through an extract-cellular mode of action of CD involving a triggering of an as yet unidentified cell surface receptor(s), either interacts with other proteins, but nothing is so far known about its precise molecular mechanisms. Recently, during exploring variation of serum-proteomics before and after lung cancer, we discovered that CD was one of important proteins associated with carcinogenesis and progression of cancer, and different expression mode of CD isoform during lung cancer progression. We first observed novel glycosylation isoform of Cathepsin D(66kDa), CD interaction proteins and critical genes (for example, Cdc42) regulated by CD. The purpose of this project is to further investigate diagnosis implication of this novel glycosylation isoform to lung cancer,the roles and molecular mechanisms of this isoform in the initiation and progression of lung cancer. On the basis of our previous study, we speculate that it is possible to find unknown receptor of CD, early diagnose biomarker of lung cancer and antitumor drug target in the future research, which maybe have important theoretic implication and application value.

Cathepsin D(CD)和/或其前体pro-cathepsin D(pCD)与乳腺癌、卵巢癌等肿瘤的侵袭和转移有关，且无酶活性CD突变体同样对肿瘤细胞、内皮细胞和成纤维细胞有促有丝分裂作用，抗CD已知受体M6PR的抗体和天冬氨酰蛋白酶抑制剂pepstatin A也不能抑制上述生物效应，提示pCD/CD可能通过靶细胞上的未知受体或与其他分子相互作用而发挥作用，但机制不明。我们在研究肺癌发病前后血清蛋白质组变化过程中，发现CD是肺癌发生发展的重要蛋白之一，其不同异构体在肺癌发病前后呈现不同的表达模式，并首次发现CD新的糖基化异构体（66kDa）、CD相互作用蛋白和受CD调控的重要基因（如Cdc42）。本课题将进一步研究CD新的糖基化异构体对肺癌诊断的意义；探索该异构体在肺癌发生发展中的作用及分子机制。有望发现CD新的受体和肺癌早期诊断标志物及药物开发新靶点，具有重要的理论意义和应用价值

本项目达到预期研究目标，取得较好的结果。主要成果如下：临床上就诊的肺癌病人多为中晚期，大部分失去手术治疗的机会，故欲获得系列的肺癌组织标本开展相关的研究工作较困难。我们探索出Urethane(氨基甲酸乙酯)腹腔注射诱导法，成功稳定建立处于不同病理时期的小鼠肺腺癌模型，为研究肺腺癌发生、发展和转移的病理生理变化特点及相关机制提供实验素材；使用激光捕获显微切除技术，从早期肺癌组织中获得特异的病理组织或肿瘤细胞，通过iTRAQ定量差异蛋白组学分析，发现192个差异表达蛋白，经免疫组化和Western blotting等进一步验证，发现Cathepsin D、Destrin等多个与肺腺癌发生、发展和转移有关的重要功能基因；通过实验动物模型和临床肺癌病理标本分析，首次较系统的发现Cathepsin D及其糖基化修饰与肺癌发生、发展和转移密切相关；发现66kDa异构体是Cathepsin D新的糖基化异构体，进一步生存分析发现该异构体是影响肺癌患者预期寿命的重要危险因素，可望成为肺癌预后评价的重要指标之一；发现肌动蛋白解聚因子Destrin表达与Cathepsin D表达呈正相关关系，实验动物模型和临床肺腺癌组织芯片分析，首次发现Destrin是影响肺腺癌发展和转移的重要功能基因，其高表达是影响肺癌患者预后生存期的重要危险因素之一；体外实验首次发现，Cathepsin D表达上调和下调，可致Destrin在蛋白及mRNA水平表达上调与下调；Destrin沉默可导致β-catenin在A549细胞的异常膜定位分布，使细胞增殖、迁移和侵袭能力皆降低，推测它可能是Cathepsin D异常表达影响肿瘤发生和转移的重要靶分子之一；发现降低Destrin表达后，可显著提高A549细胞对抗肿瘤药物（顺铂）的致凋亡敏感性，裸鼠荷瘤实验不能形成肿瘤等。本项目取得的研究结果将为肺腺癌早期诊断、预后评价、基因治疗和新的抗肿瘤药物设计提供新的标靶，同时提示肺腺癌患者高表达Cathepsin D和Destrin等可能是肺腺癌易发生侵袭、转移及耐药的重要机制之一。