Warburg 效应与HBV感染慢性炎症恶性转化的分子机制

Cancer cells are well documented to rewire their metabolism and energy production networks via aerobic glycolysis, or the Warburg effect, to support and enable rapid proliferation, continuous growth, survival in harsh conditions, invasion and metastasis, and resistance to anti-cancer drugs. During chronic viral infection, replication of the viral genome and packaging and secreting viruses may request the host cells to employ a similar Warburg-like metabolic program and energy production networks operated efficiently in the proliferating cancer cells, in order to provide the high energy needs and building blocks for macromolecular synthesis, such as the synthesis of nucleotides, lipids, and proteins. Although few report documents the involvement of Warburg effect in HBV replication and during the processes of HBV-associated hepatocarcinogenesis, our preliminary studies demonstrate that several proteins that critically control the metabolic flux during glycolysis are deregulated in the replicative-competent HBV stable cells and a hepatocellular cancer (HCC) cell line derived from a HBV-positive patients by an unbiased iTRAQ proteomic analysis. In addition, we showed that the signaling pathway that controls the Warburg glycolytic metabolism of glucose is also involved in HBV infection, viral replication, as well as the clearance of HBV virus by anti-viral drugs. Therefore, it is our central hypothesis that the metabolic adaptation and metabolic reprogramming during chronic inflammation mediated by HBV infection and viral replication is the major mechanism underlying HBV-associated hepatocarcinogenesis and (epi-)genetic alterations that initiate a Warburg-like metabolic reprograming in the HBV-replicating host cells is the critical step toward the malignant transformation. To test this hypothesis, we plan to investigate the cellular intrinsic networks that control the Warburg effect in the replicative HBV-transfected stable cells in the presence or absence of inhibitors that block metabolic flux during glycolysis at various levels; blood and tissue samples obtained from patients with HBV-associated hepatitis, fibrosis, cirrhosis, and HCC; and animal models with chronic HBV-infection in immune competent or immune incompetent mice, and with or without initiation by a chemical carcinogen diethylnitrosamine in a diverse genetic background via the CRISPR-CAS9-mediated genome editing. We expect that data obtained from these experiments, after bio-computational analysis, will help us to unveil how the metabolic program goes awry during the processes from chronic inflammation, initiated by HBV infection, to malignant transformation. We also expect to identify several key players involved in triggering Warburg-like metabolic reprogramming in the host cells with HBV infection during the malignant transition, which may serve as potential biomarkers for early screening of HCC and as interventional targets for prevention of malignant transformation.

在前期研究中，通过对感染HBV病人肝穿样本与正常肝组织的差异蛋白组学检测，发现在HBV感染肝细胞参与调控有氧酵解（即Warburg效应）的代谢酶谱的上调表达变化，并且，通过体内、外的实验，初步验证了HBV的感染、复制与抗病毒治疗反应，也与Warburg效应机制有关联。因此，我们的工作假说是： Warburg效应是细胞为适应和满足HBV病毒复制的特殊代谢需求而启动，在慢性炎症过程中，长期性的HBV病毒复制与宿主细胞对代谢与氧化应急的反复适应的结果，必然引起细胞代谢程序的重组（metabolic reprogramming），而代谢程序的重组则正是“炎－癌”转化的主要诱因和驱动因素。因此， 我们设计了从慢性复制性HBV稳定感染细胞株、“炎－癌”进展不同临床阶段的病人样本、以及不同免疫力与特定遗传修饰背景小鼠HBV感染模型等三个层次的实验，利用现代“组学” 技术，结合生物信息学方法来验证该假说。

全球每年大约有120万人将因慢性HBV感染所引发的肝硬化、肝癌等并发症死亡。在我国现阶段的肝细胞肝癌患者中，HBV 标志物阳性率达90%。因此，如何有效地阻止HBV携带者的慢性炎症向恶性化转变是摆在我们面前的一道难题, 而阐明HBV所导致的慢性非可控性炎症恶性转化的分子机制，正是我们解决这一科学难题的关键。. 在我们的前期研究工作中，通过对慢乙肝病人与正常肝组织的定量差异蛋白组学分析，发现在慢乙肝病人肝组织中与Warburg效应相关的代谢调控酶谱的显著变化。因此，我们从慢性复制性HBV稳定感染细胞株上，系统性地通过对调控Warburg效应与不同代谢分流环节的关键靶点的系列阻断，验证了HBV病毒DNA复制与 Warburg 效应的关联，并寻找可能同时调控病毒复制和影响“炎－癌”转化的早期预警指标与早期干预靶点。并利用PTEN肝脏特异性敲除 （PTEN-KO ）小鼠，建立了慢性HBV感染模型，利用此模型研究病毒感染复制对肝脏病变--从脂肪变、脂肪性肝炎、肝纤维化、肝硬化到肝细胞肝癌过程，尤其是对“炎-癌”转化过程的影响，阐明 Warburg－like 效应与“炎－癌”转化的分子机理和关键调控节点。. 我们发现：1、Warburg效应激酶AKT影响HBV病毒DNA的感染复制； 2、HBV的感染复制依赖丝氨酸的从头合成。特异性敲低丝氨酸从头合成的关键代谢酶或加入其特异性抑制剂可显著抑制HepG2.215细胞的HBV病毒 DNA及模板cccDNA的复制合成，且在细胞培养基中去除丝氨酸或重新添加丝氨酸都可以影响HepG2.215细胞的HBV病毒 DNA及模板cccDNA的复制合成；3、干扰糖代谢的磷酸戊糖旁路（PPP）关键代谢酶G6PD蛋白的表达亦可以影响乙肝病毒DNA及其模板cccDNA的复制合成；4、肝特异性PTEN敲除小鼠模型中，高压注射建立的慢性乙肝感染小鼠模型，可加速肝脏特异性PTEN敲除小鼠的自然成瘤时间和平均成率。. 综上所述，我们在复制性HBV稳定表达细胞系的体外研究与模式动物的体内研究成果，不仅为慢性乙肝感染治疗开辟了新的抗病毒治疗思路与干预途径，而且也可能为“慢性非可控性炎症恶性转变”的防控带来新的范式转变（paradigm-shift），并达到从源头上阻断慢性炎症向恶性化转变的目的。