细胞外铜离子介导CD147自聚合促肝癌细胞恶性表型的结构与功能关系研究

CD147 is a wildely distributed protein and is highly enriched on the surface of malignant tumor cells, which promotes tumor invasion and metastasis. We have identified its ligands CyPA and CD147 isoform binding site on CD147, and clarified the structure-function relationship of the interaction between CD147 and its ligands. Based on these results, we have published several paper on JBC and MCB journals. During the study of CD147 self-association, we found extracellular copper ion can mediate CD147 self-association, and copper ion promoted MMP secretion of hepatoma cell via We have identified CyPA and CD147 isoform binding site on CD147, and clarified the structure-function relationship of the interaction between CD147 and its ligands, which results have been published on JBC and MCB journals. During the study on CD147 self-association mechanism, we found extracellular copper ion can mediate CD147 self-association, and copper ion promoted MMP secretion of hepatoma cell via CD147. In addition, it has been reported that copper level measured by X-ray fluorescence in tumor is higher than normal tissue, and specific copper chelate can inhibit tumor proliferation and angiogenesis, but the mechanism still remains unclear. Based on our previous studies, we will determine the structure of copper mediated CD147 self-association by combined Nuclear Resonance (NMR) and X-Ray crystallography, and then clarify its structure-function relationship and functional targets by using CD147 knock-out cell models and cell biology technology. It will provide a good structure explanation for the copper mediated CD147 aggregation and will further determine the mechanism of extracellular copper in the tumor tissue promotes the malignant properties of hepatoma cells, which is of great interest and pharmaceutical importance for design of clinical treatment.

CD147是高表达于多种癌细胞上的肿瘤标志分子，具有促进癌细胞增殖和转移等功能。申请人前期研究了CD147作为受体与CyPA相互作用以及与其剪切异构体聚合的结构与功能关系，相关文章在JBC等杂志发表。研究中我们发现细胞外铜离子能够介导CD147聚合，并通过膜上CD147促进肝癌细胞分泌基质金属蛋白酶。许多报道显示癌组织中铜离子浓度大幅提高，而铜离子螯合剂能抑制肿瘤增殖和血管生成，但相关机制仍不清楚。本研究计划利用NMR和X射线晶体学等手段深入研究铜离子介导CD147聚合的结构基础，同时利用独有的敲除CD147肝癌细胞模型，结合细胞分子生物学方法，揭示铜离子介导CD147聚合调控肝癌细胞侵袭转移等恶性表型的结构与功能关系及功能靶位，阐明铜离子介导CD147聚合促肝癌细胞恶性表型分子机制。本研究将加深对CD147作用机制的认识，发现铜离子与癌细胞侵袭转移的关系，并为临床治疗提供新思路和新靶位。

大量研究表明，肿瘤微环境中铜离子浓度较正常组织有所升高，其升高的程度与多种癌症的进展呈正相关。已经有研究显示出铜离子能够作为一个促血管生成关键因子，还能促进肿瘤细胞的增殖、促进癌症进展，因此利用铜离子螯合剂进行降铜治疗策略应用于乳腺癌已经进入FDA三期临床阶段，然而其分子机制还不清楚。本项目结合核磁共振研究、细胞功能及生物化学等研究手段，阐明了铜离子能够作为细胞外信号分子，介导肿瘤相关膜抗原CD147发生自聚合，并引起下游PI3K/Akt信号通路开放，从而促进肝癌细胞分泌基质金属蛋白酶2和14，并进一步促进肿瘤的侵袭和转移。我们还明确了相应的功能靶位，为抗转移创新药物的研发提供了理论依据和设计思路。. 在研究中，本项目组确定了肝癌细胞表面的CD147（Basigin）能够作为细胞外二价铜离子的信号受体。细胞外的铜离子能够介导CD147的第二个Ig结构域发生自聚合，从而将信号传递至细胞内，并通过PI3K/Akt信号通路促进肝癌细胞分泌MMP-2和MMP-14。铜离子介导的CD147自聚合还能够增强肝癌细胞刺激周围成纤维细胞分泌MMP2的能力，以及肝癌细胞的侵袭能力，这种促进作用能够被铜离子抑制剂tetrathiomolybdate所抑制。我们通过核磁共振研究确定了铜离子介导CD147自聚合的界面和关键氨基酸残基，通过多种生化和细胞生物学手段筛选得到了一个丧失铜离子结合能力的突变体，该CD147突变体无法响应细胞外二价铜离子的信号。我们的研究揭示了一个全新的细胞外铜离子在促进肿瘤转移方面的信号作用，其相应的靶向铜离子介导的CD147自聚合则成为了一个非常有前景的治疗策略。. 金属离子参与到CD147功能的调控是本研究组首先发现的，这不仅扩充了对CD147调控机理的理解，而且加深了金属离子参与生物学过程的认识。已发表SCI研究论文一篇（Oncotarget. 2016 Feb 23;7(8):9429-47）。另一篇“CD147/Basigin Functions as the Signaling Receptor for Extracellular Divalent Copper in Hepatocellular Carcinoma Cells”投至Oncotarget，已修回（minor revision）。