TSTA3基因通过异常岩藻糖基化影响食管癌侵袭迁移的作用机制研究

Abnormal fucosylation is one of the prominent features of malignant tumor. TSTA3 encoded protein is rate-limiting enzyme of de novo synthesis of guanosine diphosphate (GDP)-fucose, a substrate for fucosylglycans. TSTA3 is a novel esophageal squamous cell carcinoma (ESCC) associated gene which was identified via whole genome sequencing from a large sample of ESCC cases. We observed not only recurrently and obviously copy number amplification of TSTA3 gene in ESCC, but also significantly enrichment of fructose/mannose metabolism in stage I tumors. Our previous study showed that TSTA3 was highly expressed in ESCC tissues compared to that of paired normal tissues, and significantly correlated with poor survival. The overexpression of TSTA3 promoted cell migration and invasion and resulted in fucose level changes on the cell surface. However, targeted fucosylated protein and the underlying melocular mechanism of tumor promotion of TSTA3 in esophageal cancer remains unrevealing. This proposal aims to validate tumor metastasis promotion of TSTA3 in animal model and identify targeted fucose glycoprotein by lectins imprinting experiment combined with mass spectrometry. To elucidate the melocular mechanism of TSTA3, we will perform transcriptome sequencing and RNAi. Meanwhile, we aim to depict the whole picture of glycosylation in serum of ESCC patients by use of glycoproteomics assay. The results will elucidate the relationship between TSTA3 associated abnormal fucosylation and ESCC and might provide a new marker and therapeutic target for ESCC.

岩藻糖基化修饰异常是恶性肿瘤的显著特征之一，TSTA3基因编码GDP-L-岩藻糖从头合成途径的限速酶，为岩藻糖基化提供供体。该基因是申请者前期利用大样本全基因组测序技术鉴定的新食管癌相关基因，其不仅在食管癌中存在显著而重复的拷贝数扩增，所在的岩藻糖代谢通路异常亦在早期食管癌中显著富集。前期研究表明食管癌组织中TSTA3表达量显著增高，是预测ESCC预后的独立因素，其过表达可促进癌细胞的侵袭迁移，引起表面岩藻糖水平的变化，但对其引起哪些蛋白岩藻糖基的变化及具体作用机制尚不清楚。本项目拟采用动物模型进一步验证TSTA3基因对肿瘤转移的影响；利用凝集素印迹转移结合质谱分析鉴定靶向岩藻糖糖蛋白；采用转录组测序、RNA干扰等方法阐明其具体作用机制；利用糖蛋白质组学揭示患者血清中的岩藻糖基化全貌；从而探讨TSTA3相关的岩藻糖水平的变化对食管鳞癌早诊的警示作用，以及作为食管癌治疗靶点的可能性。

岩藻糖基化修饰异常是恶性肿瘤的显著特征之一，TSTA3基因编码GDP-L-岩藻糖从头合成途径的限速酶，为岩藻糖基化提供供体。该基因是申请者前期利用大样本全基因组测序技术鉴定的新ESCC相关基因，其不仅在ESCC中存在显著而重复的拷贝数扩增，所在的岩藻糖代谢通路异常亦在早期ESCC中显著富集。前期研究表明ESCC组织中TSTA3表达量显著增高，是预测ESCC预后的独立因素，其过表达可促进癌细胞的侵袭迁移，引起表面岩藻糖水平的变化，但对其引起哪些蛋白岩藻糖基的变化及具体作用机制尚不清楚。本项目在国家自然科学基金面上项目的资助下，采用凝集素印迹转移结合质谱鉴定、转录组测序、RNA干扰等方法阐明TSTA3基因影响ESCC侵袭迁移的作用机制。发现TSTA3基因在ESCC中存在显著的拷贝数扩增，其扩增引起ESCC中TSTA3的mRNA及蛋白表达的显著增高；TSTA3的扩增及高表达与ESCC患者的淋巴结转移及不良预后显著相关。TSTA3的下调引起岩藻糖基水平的降低，并显著抑制ESCC细胞的侵袭迁移；相反，外源性过表达TSTA3通过增加岩藻糖基化水平导致体内外ESCC侵袭迁移能力的增加。TSTA3可能通过调控LAMP2核心岩藻糖基化和ERBB2终末岩藻糖基化在ESCC中发挥促侵袭迁移的作用；作为岩藻糖基化抑制剂的岩藻糖类似物2-F-Fuc的处理，在降低LAMP2核心岩藻糖基化和ERBB2终末岩藻糖基化水平的同时，可以显著抑制食管癌的侵袭迁移；2-F-Fuc在一定程度上通过负反馈降低了TSTA3 mRNA及蛋白的表达水平；岩藻糖基转移酶活性的改变可能参与了TSTA3介导的ESCC促转移作用。基于以上结果鉴别出ESCC治疗的新靶点，岩藻糖基化抑制剂可能为ESCC靶向治疗提供新的途径。