HBP流量调控CD133+肝癌干细胞自我更新能力的代谢机制

Hepatocellular carcinoma (HCC) is the major pathological subtype in primary liver cancer. Liver cancer stem cells (CSCs) were considered to be the root of high proliferation, drug resistance and recurrence in HCC, but the underlying mechanism of self-renewal capacity of liver CSCs is still poorly understood. In our preliminary data, hexosamine biosynthetic pathway (HBP) was identified to be the most significantly upregulated pathway in CD133+ liver CSCs compared with CD133- cancer cells. HBP can rescue the percentage of CD133+ subsets and protein expression level under low glucose conditions, while inhibition of HBP can reduce the percentage of CD133+ subsets and protein expression level in the high glucose condition. We also found the positive correlation between the percentage of CD133+ and the protein expression levels of SOX2 and OCT4. Moreover, because HBP flux drives O-GlcNAcylation via O-GlcNAc donor UDP-GlcNAc, then we hypothesize that HBP flux can regulate the self-renewal capacity of liver CSCs through O-GlcNAcylation of SOX2 and OCT4. To test this hypothesis, firstly, we will establish cell model for quantitative analysis of HBP flux by metabolic flux analysis. Secondly, we will further investigate how HBP flux regulates O-GlcNAcylation of SOX2 and OCT4 and transcriptional activities. Last but not least, we will interrogate whether O-GlcNAcylation of SOX2 and OCT4 modulates their transcriptional activities for the self-renewal capacity of CD133+ liver CSCs. In a word, the successful achievement of this proposed study will highlight the engine role of metabolic flux in liver CSCs and provide evidence for novel metabolic intervention to HCC treatment in clinical researches.

肝癌干细胞被认为是导致肝脏肿瘤快速增殖、耐药及复发的根源，但其自我更新机制尚不清楚。申请人前期工作发现己糖胺合成通路（HBP）在CD133+肝癌干细胞中显著上调，并且CD133的阳性率与SOX2/OCT4的蛋白表达水平高度相关。我们设想在CD133+肝癌干细胞中HBP流量增高导致O-GlcNAc糖基化供体UDP-GlcNAc水平增高，进而通过SOX2/OCT4的O-GlcNAc糖基化修饰调控CD133+肝癌干细胞的自我更新能力。因此，本项目拟1）构建细胞模型并运用代谢流技术定量分析CD133+肝癌干细胞中的HBP流量是否增高；2）阐明SOX2/OCT4的O-GlcNAc糖基化修饰是否调控其转录活性；3）阐明HBP流量是否通过影响SOX2/OCT4的O-GlcNAc糖基化修饰调控肝癌干细胞自我更新能力。本项目的成功实施将确定肝癌干细胞中代谢流的引擎作用，为制定肝癌的代谢干预策略奠定理论基础。

肝癌，特别是肝癌干细胞有别于正常体细胞的代谢模式。除了瓦尔伯格效应，通过代谢组学和稳定同位素标记的代谢流分析技术，有望鉴定到更具特征的代谢亚网络。本项目的研究目标是运用代谢组学和稳定同位素标记的代谢流分析技术，开展肝癌代谢重编程研究，并将相关技术推广到其他肿瘤代谢研究中。在该项目的基金支持下，主要研究内容有：我们考察了转酮醇酶（TKT）在肝癌发生发展中的作用，发现TKT敲除可以阻断非氧化型戊糖磷酸途径，导致5-磷酸核糖不被消耗回到糖酵解通路，从而促进5-磷酸核糖累积，促进核苷酸合成，对基因组稳定性发挥重要作用；这样有助于减少二乙基亚硝胺诱导的急性肝损伤和阻止肝癌的发生发展。我们还运用了体内代谢流分析技术，证实了相关结果。我们也运用多种稳定同位素标记的代谢流分析技术，鉴定受体酪氨酸激酶EGFR和FGFR驱动下的代谢异质性，发现EGFR驱动下倾向于丝氨酸合成通路而FGFR驱动下倾向于乳酸代谢。另外，在另一项肺癌研究中，我们发现谷氨酸转运体SLC1A1促进谷氨酸和胱氨酸的摄取，以及还原型谷胱甘肽的合成，从而促进肺癌发生发展。这些工作都体现了代谢组学和代谢流分析技术的优势，以及在揭示肿瘤代谢重编程中的重要作用。尽管申请书中的肝癌干细胞研究计划不再具有新意，因为CD133作为肝癌干细胞marker不是很充分，且新颖性不太够。我们依然在肝癌、肺癌等肿瘤代谢开展了系列研究，取得了较好成果。目前以共同通讯作者身份发表论文在《Nature Communications》一篇、《Cancer Research》两篇。从体外和体内角度分析肿瘤发生发展过程中的代谢重编程机制，鉴定肿瘤的代谢弱点，有助于制定个体化的治疗方案，找到药物作用靶点，对于解决临床的肿瘤耐药也有启发。在该项目支持下，还在2019年获得了国家自然科学基金糖脂代谢时空调控网络重大研究计划的培育项目，继续开展肝癌代谢组学研究。总之，该项目具有应用前景，未来将开发临床肿瘤代谢组学分析的试剂盒，实现肿瘤的个体化早期诊断。