GFPT1通过AMPK调控肿瘤代谢的分子机制研究

L-Glutamine: D-fructose-6-phosphate (F6P) 1 aminotransferase (GFPT1) is the first and rate limiting enzyme in the hexosamine biosynthetic pathway. GFPT1 is highly expressed in tumor, which is closely associated with clinical poor prognosis, however, the mechanism and function in tumor of GFPT1 is incompletely understood. Our previous study demonstrated that GFPT1 was significantly upregulated in prostate cancer cells. Consistent with this, cell proliferation was markedly postponed in GFPT1-suppressed human prostate cancer (DU145) cells. Moreover, following the treatment with 2-deoxyglucose (2-DG), the AMPK activity was elevated more obviously in GFPT1-Knockdown cells compare with the control cells. Although AMPK is a critical regulator in energy and nutrient metabolism, the mechanism of GFPT1 regulates AMPK phosphorylation has not been described yet. Based on these results, this project will focus on the potential function of GFPT1 in tumor progression and the regulatory mechanism of GFPT1 on AMPK phosphorylation. Furthermore, the novel chromatography–mass spectrometry method will be applied to study the differential metabolites of the cellular metabolism between the control cells and GFPT1 abnormal-expressed cells. And the metabolism and metabolic protein related to the AMPK pathway will be analyzed. In addition, the potential effect of these key metabolic pathways on GFPT1 regulation of tumor malignant behaviors will be investigated. Eventually, we will define the GFPT1 regulation mechanisms in cancer metabolism through regulation AMPK. Furthermore the mechanism will be validated in the nude mice model and clinical data .Collectively, our study will provide a novel insight for the therapy of cancer.

GFPT1是己糖胺合成途径的限速酶。GFPT1在肿瘤中高表达并与临床不良预后密切相关,但关于其在肿瘤中的详细生物学功能及机制研究尚不明确。我们前期研究发现GFPT1在前列腺癌细胞中高表达,抑制GFPT1导致肿瘤细胞增殖能力下降,而且用AMPK激活剂处理的情况下,与对照组细胞相比敲低GFPT1表达肿瘤细胞中AMPK磷酸化水平上调更加明显。AMPK是调控肿瘤代谢关键分子,关于GFPT1调控AMPK磷酸化的机制还未见报道。在此基础上,本项目拟研究GFPT1在肿瘤中的生物学功能,同时探讨GFPT1调控AMPK磷酸化的机制,结合代谢组学分析GFPT1差异表达引起的代谢物变化,筛选出与AMPK相关差异代谢物及关键基因,并验证筛选出的代谢通路对GFPT1调控肿瘤致瘤性的影响,阐明GFPT1通过AMPK调控肿瘤代谢的机制。并通过裸鼠模型及临床数据分析验证该机制,为临床肿瘤治疗提供新的理论依据。

GFPT1在肿瘤细胞中高表达，抑制GFPT1表达可以特异性降低肿瘤细胞的增殖，但对肿瘤细胞的迁移无明显影响。AMPK是调控细胞能量代谢的重要蛋白，在肿瘤的发生发展中发挥重要作用。本项目中我们首次发现GFPT1调控AMPK的活性影响其下游信号通路，尤其是在营养缺陷的条件下。对GFPT1稳定低表达及对照组细胞系进行蛋白质的氧糖基化水平研究，提取细胞代谢物进行CE-MS质谱分析，发现GFPT1的异常表达对细胞中的蛋白质的糖基化水平及影响AMPK活性的关键代谢物AMP、ADP、ATP的水平无明显影响，排除了GFPT1通过影响细胞代谢调控AMPK的机制。而且实验中我们发现GFPT1对肿瘤细胞增殖的调控依赖于调控AMPK的关键激酶LKB1。通过进一步对调控AMPK的上游信号通路的研究发现，GFPT1对AMPK活性的调控依赖于LKB1信号通路但独立于CAMKKII信号通路，在此基础上,我们证实了GFPT1与LKB1-AMPK形成三元复合物，GFPT1影响AMPK-LKB1复合物的稳定性，同时GFPT1的细胞质定位为GFPT1参与细胞的能量调控及AMPK的活化提供了基础。.综上所述，GFPT1与AMPK及其上游激酶LKB1形成三原复合物并通过影响AMPK-LKB1复合物的稳定性，调控AMPK的活性，进而影响AMPK下游的信号通路调控肿瘤细胞的增殖。本项目不但在理论上详细阐述了GFPT1调控AMPK活性影响肿瘤增殖的机制，而且为GFPT1作为肿瘤标志物的临床诊断及以GFPT1为靶点进行药物筛选与肿瘤治疗提供了新的理论依据。