YKL-40在胆囊癌病变中的分子作用机制

YKL-40, a secreted heparin-binding glycoprotein, is dramatically elevated in the blood of patients with multiple advanced cancers. The results suggest that YKL-40 serves as a cancer biomarker. However, little is known regarding YKL-40 levels in gallbladder cancer (GBC) and pathological mechanism that mediates the cancer malignancy. The applicant has embarked for many years on the research field related to molecular mechanisms of YKL-40-mediated carcinopathogenesis and completed a number of large research projects supported by USA federal funding agencies, thus gaining remarkable achievements . The current project is to investigate the new pathological role of YKL-40 in GBC development. The preliminary data showed that a large population of GBCs express a high level of YKL-40 which is associated with epithelial-mesenchymal transition (EMT). In addition, the C-terminus of YKL-40 contains multiple basic arginine (R) and lysine (K) residues (RK-rich domain) which can bind heparin and induce cell activation. In this project, we will measure blood and tumor expression levels of YKL-40 in GBC and analyze the relationship of these levels with cancer metastasis and patient survival. We will also utilize YKL-40 wild-type and R or K-point mutant proteins to evaluate their effects on the binding to heparin and tumor cell EMT. It is anticipated that serum and tumor expression levels of YKL-40 are positively correlated with cancer development and metastasis. RK-rich domain is YKL-40's heparin-binding site. Upon binding to heparin, YKL-40 triggers tumor cell to undergo EMT and induces tumor metastasis. This study will discover the key molecular mechanisms by which YKL-40 prompts GBC metastasis. Therefore, the findings will provide immense value for the establishment of a new biomarker and therapeutic target in clinical diagnosis and therapy of patients with GBC.

YKL-40是肝素结合的分泌型糖蛋白，其高血浓度发现于多种上皮性晚期癌症，提示YKL-40是癌症的标志物。但YKL-40在胆囊癌（GBC）中的表达及病理作用机制尚不清楚。申请人近年来一直致力于YKL-40肿瘤病理机制的研究，主持过多项美国重大科研项目，成绩显著。本课题是探索YKL-40在GBC病变时的分子调节机制。预实验显示胆囊癌高表达的YKL-40与肿瘤上皮-间质样细胞转化（EMT）有关，YKL-40多元精氨酸（R）和赖氨酸（K）（RK-rich)碱性基团结合肝素产生活性。本课题将分析YKL-40血浓度与肿瘤表达、肿瘤病变的相关性，检测其野生和R/K突变体的肝素亲和力及对细胞EMT作用。预期胆囊癌YKL-40浓度与肿瘤恶变呈正相关，与硫酸乙酰肝素链（HS）结合诱导上皮性肿瘤细胞进入EMT，导致癌转移。此研究将揭示YKL-40在胆囊癌中的分子机制，为新建胆囊癌标志物及治疗靶点提供理论依据。

本项目是研究分泌型糖蛋白YKL-40对胆囊癌（Gallbladder cancer，GBC）的病理调节机制。GBC是一种由胆管上皮细胞发展成癌细胞的恶性肿瘤，其平均生存率为6-12个月。其主要原因是胆囊癌缺乏特异性症状和体征，早期诊断困难，一经确诊往往处于晚期，多伴有癌转移。.上皮样-间质样细胞的转化过程(epithelial-mesenchymal transition, EMT）已视为上皮性肿瘤转变为恶性肿瘤的一个重要病理过程。这些细胞通常由良性上皮样肿瘤细胞转变成强侵袭力的间质样恶性肿瘤细胞，期间细胞失去E-cad蛋白，而表达间质细胞的特征蛋白如波形蛋白(Vim)；细胞形态也由立方上皮细胞型变成索状间质细胞型，细胞迁移和侵袭能力随之显著升高。.YKL-40是肝素结合的蛋白，其高血浓度发现于多种上皮性晚期癌症，提示YKL-40是晚期癌症的标志物。但YKL-40是否通过调节GBC中的EMT病理机制而发挥作用，还尚不清楚。因此，本课题提出了一个中心假设，即GBC分泌的YKL-40通过其功能结构域与肿瘤细胞膜上硫酸乙酰肝素链（HS）结合而刺激上皮性肿瘤细胞进入EMT，导致肿瘤转移、恶变。课题从三个研究方向来论证此假设：1）解析GBC的YKL-40表达与肿瘤恶性病变的相关性；2）鉴定YKL-40调节GBC肿瘤细胞的EMT作用；3）确立YKL-40的功能性结构域。.本课题我们收集86例GBC病人的样本，结果发现YKL-40肿瘤表达量与肿瘤的发展、EMT转化、病人预后有密切的相关性。在体外实验中，高YKL-40表达的GBC细胞呈EMT的表现型，表达EMT标记蛋白，有强细胞迁移和侵袭功能。并且，我们证明YKL-40的氨基酸序列K337, K342和 R344是肝素的结合位点；此含多元精氨酸、赖氨酸（RK-rich）片段是YKL-40的功能结构域，发挥着肿瘤细胞EMT作用。.本课题首次探索和揭示YKL-40在GBC中表达和血液的浓度，以及它们与肿瘤恶性病变的相关性，从而为胆囊癌诊断提供新的标志物及新靶点；并阐明YKL-40对肿瘤细胞的关键分子调节机制；同时，YKL-40功能性结构域的确立为研发抗YKL-40抗体和寻找针对其蛋白结构的小分子药物提供重要的理论依据。