酪氨酸代謝酶HPD低表达激活mTOR通路重编程肝癌干细胞代谢的机制研究

肿瘤干细胞可能是肿瘤复发、转移和耐药的根源。本实验室首先报道了CD133分子是一个重要的肝癌干细胞标志物。我们前期利用RNA-Seq测序分析发现4-羟基苯丙酮酸双加氧酶（HPD）是肝癌CD133+细胞亚群中最具有特异性的酪氨酸代谢基因，通过细胞功能学实验证实HPD敲低能够促进肝癌细胞的生长和、自我更新和代谢重编程。临床样本显示HPD的表达与患者预后密切相关，但它调控肝癌细胞干性特征的分子机制尚不清楚。本项目拟利用多种体内外实验模型和技术，从分子、细胞、动物模型以及临床肝癌标本等不同层次系统，研究HPD对肝癌发生发展的作用；阐明调控分子机制；明确临床意义。本项目的预期研究成果将为揭示HPD在肝癌发生发展中的功能与作用分子机制，为CD133+肝癌干细胞的根源性治疗提供有效策略。

Hepatocellular carcinoma (HCC), the most malignant form of liver cancers, is the 5th most common form of all primary malignancies and is a major health burden as the second leading cause of cancer-related death worldwide. HCC is often diagnosed at advanced stage where treatment regimens are only palliative and the disease progresses very fast, causing the survival rates and the prognosis of HCC patients to remain dismal. Therefore, identification of factors and underlying mechanisms which are critical for the development and progression of HCC remains a high priority for the design of novel therapeutic interventions. Liver is one of the most metabolically active organs in our body with critical roles in regulating different metabolic processes. Metabolic reprogramming, as a major hallmark of cancer, provides a selective advantage to cancer development and progression. Emerging evidence suggests metabolic reprograming of glucose, glutamine and fatty acids to contribute to HCC development and progression. HCC is a particularly heterogenous disease, which contributes to inefficacy of current treatments. Intratumor molecular heterogeneity of HCC is partly attributed to the presence of cancer stem cells (CSCs), which we now know represents a critical root of tumor recurrence and therapy resistance. We and others have previously found CD133 to mark an important functional marker of liver CSCs. However, the role of metabolic reprogramming in regulating self-renewal and tumorigenic abilities of liver CSCs has not been elucidated. Therefore, we performed RNA sequencing on sorted CD133+ and CD133- subpopulations from Huh7 and PLC8024 HCC cells to identify the deregulated metabolic processes. Tyrosine catabolic process was identified as the most significantly deregulated pathway between CD133+ and CD133- subpopulations. We observed a significant down-regulation of the second tyrosine catabolic enzyme HPD in CD133+ subpopulations. Further, downregulation of the second enzyme HPD was detected in HCC samples compared with paired non-tumor liver specimens. HPD downregulation was also significantly correlated with overall survival in HCC patients. Moreover, an upregulation of serum tyrosine levels was detected in HCC patients compared with healthy individuals. By metabolomics profiling, we detected increased levels of TCA cycle intermediates and decreased levels of glycolytic intermediates in HPD suppressed cells. Mechanistically, we observed activation of mTOR signaling and increased expression of its downstream targets SLC1A5 and GLS which are proteins involved in glutamine metabolism, suggesting enhanced TCA cycle and glutamine metabolism for energy production. Taken together, the present data suggests an unreported alteration of tyrosine metabolism in CD133+ CSCs which promotes metabolic reprogramming and drives cancer development in HCC. In this study, we will (1) investigate the metabolic functions of HPD in HCC development by a series of in vitro and in vivo functional assays; (2) delineate the metabolic vulnerability of HCC and its intricate mechanism resulted from tyrosine/HPD deregulation by isotopic flux analysis; and (3) evaluate the clinical value of serum tyrosine and HPD in early stage HCC diagnosis, prognosis and predicting treatment response in targeted therapy.