CD59异构体的鉴定及其促进肿瘤侵袭转移的作用机制

CD59 tipically functions for restricting the assembly of membrane attack complex at the late stage of complement activation. It has been demonstrated to be up-regulated in cancer and highly correlates with resistance to antibody-based cancer therapy. Further, numerous evidence demonstrates the alternative roles of CD59 including involvement in tumor metastasis, angiogenesis and anti-apoptosis, as well as participation in reproduction. The alternative splicing of pre-mRNA is an important determinant for protein diversity, and we previously found each human CD59 or mouse Cd59 express four isoforms, in which hCD59-IV and mCd59b-III most likely contribute to the alternative roles of CD59. However, the underlying mechanisms for their functions and molecular mechanisms require future investigation. In this proposal, we will prepare the specific monoclonal antibodies, in combined with quantitative RT-PCR, to detect the expression levels of the two CD59 isoforms in normal and tumor tissues. In addition, we will alter their expression levels by transducing shRNA and exogenous coding DNA of hCD59-IV in cancer cells, or by knocking-out and exogenously expressing mCd59b-III in mice, by which we will reveal the isoforms’ functions and mechanisms for regulating cell proliferation and mobility, particularly for hCD59-IV in tumor metastasis. We expect that our findings may help to investigate the potential of hCD59-IV as diagnostic biomarker and/or therapeutic target for cancer metastasis.

CD59的经典功能为在补体级联反应末期抑制膜攻击复合物的形成，它在肿瘤细胞中高表达并与肿瘤抗体治疗耐药密切相关。大量研究表明CD59尚具有其它难以解释的功能，包括参与肿瘤侵袭转移、血管生成、抑制凋亡以及参与生殖发育等。前体mRNA的可变剪接是产生蛋白质多样性的重要原因，我们前期发现人类hCD59和小鼠mCd59各存在四种异构体，且很可能异构体hCD59-IV和mCd59b-III与上述非经典功能有关，但机制尚有待于揭示。本申请将制备特异性单抗，与定量PCR方法结合，观察这两种异构体在正常组织与肿瘤组织中的表达水平，进一步在细胞与动物水平改变这两个CD59异构体的表达水平，通过多种手段在体内外观察对细胞增殖与迁移能力的影响并揭示其分子机制，在分子、细胞及整体水平重点阐述hCD59-IV促进肿瘤侵袭转移的功能与机制，探讨其作为诊断标记物和治疗靶点的潜在性，为肿瘤侵袭转移的诊治提供新思路新方法。

CD59是重要的补体膜调控蛋白，但既往研究表明它也存在补体非依赖功能。本课题通过研究CD59的异构体入手，探讨了CD59在肿瘤侵袭转移过程中的补体非依赖作用与机制。另外，我们还研究了CD59在肿瘤放疗抵抗和肿瘤免疫中的作用与机制。结果表明CD59-I是四种CD59异构体的主要表达形式，丰度最高，因此，后续主要研究了CD59-I，也直接称为CD59。CD59与乳腺癌患者预后呈显著负相关，敲低CD59表达水平可显著抑制乳腺癌的增殖与侵袭转移，参与的信号通路主要有PI3K/AKT和ERK分子。另外，我们还发现FOXM1通过对PI3K/AKT的调控而影响CD59的表达水平，FOXM1还可通过调控IRF1的表达而促进食道癌的侵袭转移。进一步，FOXM1还发挥转录非依赖的功能，在胞内与MTA1互作，通过促进MTA2的酶切降解，而影响乳腺癌的侵袭转移。我们还发现CD59与食管癌放疗抵抗呈显著负相关关系，敲低CD59表达水平显著提高放疗的肿瘤杀伤效果，分子机制研究发现Src信号可能参与放疗后DNA损伤秀谷。最后，我们利用小鼠Cd59基因敲除小鼠，发现敲除Cd59后几乎完全抑制肿瘤的体内增殖与转移，机制研究发现与T细胞的MAPK信号通路激活有关。总之，肿瘤细胞高表达CD59可促进肿瘤的增殖与侵袭转移，而T淋巴细胞高表达CD59则可抑制其功能。因此，CD59是一个潜在的预测标志物和抗肿瘤治疗靶点，具有较大的转化应用价值。