热休克蛋白90调控CD24促进肠癌侵袭和转移的分子机制

D24 is a small glycosyl-phoinositol-anchored protein. It plays an important role in transmembrane signaling pathway associated with tumor invasion and migration including colorectal cancer in vivo and in vitro. But the underlying mechanism is not very clear. Through high throughout mass spectrometry analysis and SDS-PAGE, Hsp90, a molecular chaperone responsible for the proper folding,activity,maturation,metabolism and intracellular trafficking of numerous client protein, was among the most prominent proteins that we identified. Furthermore, our data showed that the positive staining of CD24 significantly correlated to that of Hsp90 in colorectal cancer tissues. In vitro, the expression of CD24 decreased after 17-AAG treatment by Western blotting analysis. CD24-induced colorectal cancer cell migration decreased after 17-AAG treatment by Wound healing assay.In vivo,CD24 promoted hepatic metastasis and 17-AAG reduced the number of hepatic metastasis nodules. Recent studies suggests that extracellular Hsp90 is the most important component involved in tumor migration and invasion.So we hypothesized that CD24 was a new client protein of Hsp90 and extracellular Hsp90 was involved in CD24-induced colorectal cancer invasion and migration. In the present study, Co-IP assay, sucrose density gradient centrifugation and truncation mutants were used to investigate tumor microenvironment, the interaction site and protein metabolism of CD24 and Hsp90. The aim of our present study was to elucidate the role of Hsp90 in CD24-induced colorectal cancer invasion and migration and the underlying mechanism.

CD24是高度糖基化的细胞膜蛋白，通过传递跨膜信号参与调控肿瘤侵袭转移，但如何参与膜信号传导目前仍存在诸多争议。我们通过免疫沉淀及质谱鉴定筛选出CD24潜在相互作用蛋白Hsp90，并进而证实两者在肠癌组织中表达显著相关，在体外实验中，Hsp90特异性抑制剂17-AAG可以减弱肠癌细胞CD24的表达，抑制CD24诱导的肠癌细胞迁移及减少实验动物肝转移模型中肝转移结节数目。那么，Hsp90调控CD24的分子机制是什么？研究证实膜Hsp90是Hsp90调控肿瘤侵袭转移的重要组分，因此，我们首次提出CD24是Hsp90新的"客户蛋白"，以及膜Hsp90通过调控CD24影响肠癌侵袭转移的假设。本项目拟进一步采用CO-IP、脂筏分离、截短突变等技术探讨其作用机制，分析HSP90与CD24相互作用的结构域，以及该位点结合对肠癌侵袭转移的重要作用，旨在阐明肠癌中Hsp90调控CD24的分子机制及生物学意义

CD24参入了人类多种肿瘤的发病和肿瘤转移进程，但是CD24与肿瘤新生血管之间的关系还不清楚。在此项研究中， 我们主要针对CD24调节结肠肿瘤血管生成的机制展开研究。研究结果提示在结肠癌原发灶和转移灶中新生血管密度MVD与CD24的表达密切相关。采用基因敲除技术沉默CD24表达能减弱人脐静脉血内皮细胞迁移和管样结构形成，但是对其增殖影响小。进一步分析其分子机制发现CD24可以通过调节Stat3对VEGF的转录活性，并且确认了HSP90能介导CD24对Stat3的调节作用。在体内我们, 采用HSP90特异性抑制剂17-AAG能明显降低的结肠肿瘤细胞的肝脏转移能力，同时减少了肿瘤血管生成。这些结果综合说明了CD24能通过HSP90介导调节Stat3对VEGF的转录活性。本项目从体内体外提供充足的证据说明了CD24对结肠肿瘤血管生成的重要作用，并且针对CD24的治疗将有易于减少结肠肿瘤转移，提高结肠肿瘤患者的生存率。