6磷酸果糖激酶2同工酶3（PFKFB3）介导细胞周期调控机制及其在肿瘤发生中作用的研究

The elevated protein O-GlcNAcylation facilitates pancreatic cancer cell proliferation under hypoxia and glucose deprivation, but the mechanisms need to be further elucidation. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) plays a key role in the activation of glycolysis through its metabolic product fructose-2,6-bisphosphate (F2,6BP). It was reported that PFKFB3 and cell cycle inhibitor p27 could be upregulated during hypoxia. However, the nuclear PFKFB3 activity could promote cell cycle progression via degradation of p27. In our preliminary studies, we found hypoxia induced O-GlcNAcylation of PFKFB3, which is important for nuclear targeting of PFKFB3 and degradation of p27 under hypoxia. During hypoxia, inhibition of O-GlcNAcylation resulted in detention of PFKFB3 in the cytosol might through promoting the interaction between PFKFB3 and G3BP2 mediated by ERK activity. Glucose deprivation inhibits O-GlcNAcylation of PFKFB3 induced by hypoxia in normal cells, but not in pancreatic cancer cells. Collectively, in this project, we expect to prove and demonstrate the underlying mechanism of that, under hypoxia and low glucose, the O-GlcNAcylation of PFKFB3 contribute to pancreatic cancer cell proliferation through inhibition of p27, which drives pancreatic tumorigenesis.

蛋白O-糖基化促胰腺癌细胞在低氧低糖条件下生长增殖，但其机制还有待进一步阐明。6磷酸果糖激酶2同工酶3（PFKFB3）是糖酵解通路重要的激活因子。已报导低氧促进PFKFB3的表达，也可以上调细胞周期抑制因子P27，而PFKFB3在细胞核中的酶活可促P27降解，维持细胞周期进程。我们发现：低氧促PFKFB3蛋白O-糖基化，抑制其O-糖基化会阻止其在低氧条件下细胞核定位及P27降解，阻止细胞周期进程。在O-糖基化受抑条件下，低氧可能通过ERK影响PFKFB3与G3BP2蛋白相互作用来影响PFKFB3的定位。正常细胞低糖会抑制低氧促PFKFB3的O-糖基化，但胰腺肿瘤细胞在低糖低氧条件下仍维持较高水平的PFKFB3的O-糖基化。本项目旨在证明胰腺肿瘤细胞通过PFKFB3的O-糖基化维持其在低糖低氧微环境中的细胞周期进程，并进一步阐明其机制和这一通路在胰腺癌发生中的作用。

O-GlcNAc转移酶(OGT)催化的蛋白质O-GlcNAc修饰受葡萄糖的严格调控，同时蛋白O-GlcNAc修饰在低氧条件下上调。其细胞生物学意义和分子机制尚需阐明。肿瘤细胞中高水平的蛋白质O-GlcNAc修饰促进肿瘤细胞在低氧低糖微环境中增殖生长，我们前期发现代谢酶PFKFB3的O-GlcNAc修饰影响其细胞核定位，而代谢酶PFKFB3作为糖酵解途径激活因子可以在低氧条件下被诱导表达，在多种肿瘤细胞中也异常高表达，而其功能在细胞质中主要促进糖酵解，在细胞核中主要促进细胞周期。本项目通过蛋白O-GlcNAc糖基化质谱组学分析，发现低氧条件促进代谢酶PFKFB3的O-GlcNAc糖基化修饰，并鉴定出修饰位点，研究发现其O-GlcNAc糖基化修饰影响其低氧下细胞亚定位，进而影响细胞周期进程和肿瘤发生。这项工作首次发现肿瘤细胞在低氧微环境中促进代谢酶PFKFB3被OGT糖基化，肿瘤细胞中高水平的O-糖基化可抑制低氧条件下PFKFB3被ERK磷酸化，抑制低氧下PFKFB3与G3BP2结合，维持其入核促P27降解和细胞周期进程，使细胞更为耐受低氧压力微环境。此外，在该项目支持下的相关工作同期揭示了：代谢酶MTHFD2通过一碳代谢和HBP代谢途径调控UDP-GlcNAc水平，进而通过转录调控因子cMyc等的O-GlcNAc修饰促进免疫抑制基因PD-L1等表达，促肿瘤获得性免疫耐受；申请人合作研发的靶向MTHFD2肿瘤治疗药物，已联合申请专利并推进临床转化。代谢酶PHGDH通过丝氨酸和一碳代谢途径调控SAM水平，进而通过表观修饰途径最终抑制固有免疫应答基因表达，促肿瘤免疫耐受等。总体上，在本项目资金的支持下，相关研究成果以本项目为主要标注项目，以负责人为通讯作者发表原创论文在Cell metabolism，Nature Communications，Advanced Science，Oncogenesis等多个主流期刊，作为发明人申请专利2项，同期负责人获得青年拔尖等人才称号。