乙肝病毒复制和变异通过S期激酶相关蛋白2途径与AKT/Ras的串话在肝癌发生发展的作用研究

Global multicenter clinical studies have shown that higher level of HBV replication and virus mutations are independent risk factors for HCC development, respectively. Mounting evidence indicates that activation of AKT and Ras pathways is a key oncogenic event in human hepatocarcinogenesis and tumor progression. Deregulation of S phase kinase associated protein 2 (SKP2) has been found in many human tumors. However, mechanisms by which higer HBV DNA level and mutations inducing hepatocarcinogenesis are not clear. Moreover, treatment options for HCC are limited and generally ineffective. Sorafenib which is the only available chemotherapeutic drug for unresectable HCC has limited efficacy in improving survival of HCC patients. It is most likely due to the activation of unknown or alternative pathways promoting the survival of tumor cells. Therefore, investigations of the molecular pathogenesis of HBV associated HCC is necessary for the development of new targeted therapies against this life-threatening liver disease. Our previous in vitro study showed HBV core promoter mutations found in HCC patients upregulated SKP2 expression, in turn accelerating p21 protein degradation, cellular proliferation, and transformation. Here, we hypothesize that the corsstalk effect between HBV factors (viral replication and mutations) and common oncogenic signals (AKT and Ras) promotes SKP2 activity, and subsequently activates a HBV associated hepatocarcinogenic pathway, which is that "SKP2 overexpression → decreased cell cycle inhibitors and increased cell cycle promoters and accelerated degradation of tumor suppressors → cell cycle progression, cellular proliferation, angiogenesis and decreased apoptosis of tumor cells → HCC development and tumor progression". We will first define the correlation between HBV factors and SKP2 expression as well as its downstream signalings in human HCC samples. Both distribution and protein levels of SKP2 and its substrates will be determined. The proliferation and angiogenesis of tumor cells in HCC tissues will be also detected. Second, we will elucidate the molecular mechanisms by which HBV factors lead to increased expression of SKP2 in human hepatic cells. SKP2 transcription level, messager RNA, and protein stability will be tested and clarified the possible mechanism for HBV modulating SKP2. Third, we will characterize the oncogenic potential of HBV factors in mice with overexpression of AKT and/or Ras. Levels of AKT downstream, tumor formation and its possible pathways will be assessed. Supposely, our findings can imply that SKP2 dependent or independent mechanism may be responsible for HBV combined AKT and/or Ras-driven hepatocarcinogenesis. Elucidating these pathways may lead to the development of novel therapeutic strategies targeted SKP2 to prevent or against HBV-related HCC.

乙肝病毒(HBV)感染是肝癌发生的高危因素，AKT和Ras是肝脏癌变的关键通路，但至今没有关于两者串说促进肝癌发生与进展的深入研究，鉴于目前肝癌的药物疗效欠佳，提示存在未知信号基因有助于癌细胞存活；我们前期体外细胞研究发现，HBV X基因变异上调S期激酶相关蛋白2(SKP2)，而SKP2是AKT的效应器，故本研究致力于探索乙肝相关性肝癌发生的独立危险因素(HBV复制和变异)与AKT和Ras共同促进癌变的机制，提出"HBV串联AKT或/和Ras→ SKP2活化→细胞周期因子失调和抑癌基因降解→细胞周期进程、增殖、转化和血管异生→癌肿形成和进展"假说，将利用人肝癌组织标本评价病毒与SKP2及下游基因关系；细胞模型阐明HBV调控SKP2的机制；动物模型探索HBV与AKT/Ras串说的致癌变特点。本研究将证实病毒与癌基因作用的中心环节；全新角度阐明抗病毒的重要性；促进靶向SKP2的抗肝癌新药开发。

乙肝病毒的核心启动子区（CP区）突变和肝癌的发生发展具有相关性，与CP区重叠的X基因能上调AKT的表达，促进肝癌细胞的进展。然而CP区突变与AKT一起协同作用于肝癌的机制现在还不是很明确。本研究发现CP区的A1762T/G1764A (TA) 双突变联合其他突变（TACO突变)和p-AKT的高表达预示病人不良的预后，同时具备TACO突变和p-AKT高表达的病人预后更差。TACO突变或p-AKT的高表达与细胞增殖能力、肿瘤血管生成能力呈正相关；与细胞的凋亡呈负相关；当两个条件叠加在一起时，上述表型更明显。TACO突变和p-AKT能通过上调SKP2的表达水平，致使下游靶基因p21和p27的表达量下降。TACO突变会促进E2F1和SKP2的蛋白水平和mRNA水平上调，然而p-AKT的高表达会使SKP2的负性抑制因子4EBP1/2下调，SKP2的蛋白水平也会下调，但对SKP2的mRNA没有影响。因此，TACO突变和p-AKT的高表达是对术后肝癌患者预后的独立预测因子。它们的共同靶标基因SKP2，很可能成为促进肝癌的诊断和治疗效果的重要分子。