Synergistic Function Of Kras Mutation And HBx In Initiation And Progression Of Hepatocellular Carcinoma In Mice

Primary liver cancer is one of the most common cancers worldwide. China is acountry with a high incidence of liver cancer, reporting about half of world’s livercancers diagnosed and related deaths. As the major histological subtype,hepatocellular carcinoma (HCC) accounts for70–85%of primary liver cancers. HCCresults from a combination of environmental factors such as hepatitis B virus (HBV)infection and the accumulation of generalized and specific genetic alterations.Although mounting evidence indicates that the dysregulation of some pathways, suchas p53, β-catenin and c-myc, plays an important role in development and progressionof HCC, the molecular pathogenesis of HCC is still under intense investigation.Although KRAS mutations have been detected in only5%of human HCC samples,the activation of RAS signaling pathway has been found in all examined humanHCCs. In vitro, RAS activation leads to fibroblast transformation and hepatocellularproliferation. Hepatocyte specific-activated Kras in zebra fish initiates HCC formation.However, the role of Ras activation in HCC initiation and progression in mice isunderdetermined. HBV infection is epidemiologically associated with thedevelopment of HCC. Our previous studies have demonstrated that as one of theimportant proteins encoded by HBV, X protein (HBx) overexpression leads to HCCintiation in transgenic mice. Whether activation of RAS signaling pathway cansynergistically act with HBx to promote HCC formation and progression remainsundefined.In this study, we generated Kras knock-in mutant mice by breeding a mousestrain carrying the KrasG12Dallele (LoxP-Stop-LoxP-KrasG12D) with a strainexpressing an albumin promoter driven Cre recombinase transgene (Alb-Cre).Hepatocyte-specific Kras activation resulted in liver cancer formation with46%(6/13) penetrance at10month of ages. The liver cancers were identified as HCCs by alphafetal protein (AFP) staining. Pathological analysis of HCCs confirmed that themajority of lesions were well differentiated and accompanied by increased mitosisand anaplastic tumor giant cells. Next, HBx knock-in transgenic mice were bred withKras mutant mice to generate HBx and Kras double transgenic mice. Remarkably,HBx expression significantly promoted the formation and malignant progression ofKras-driven HCC as shown with the accelerated tumor onset, the increased tumorburden and the more poorly differentiated lesions. At the cellular level, concomitantexpression of Kras and HBx resulted in a robust increase in hepatocellularproliferation. At the molecular level, the Akt, MAPK and TGF-β pathways wereupregulated and p53was downregulated in the Kras-driven HCCs, the dysregulationwas more pronounced in the HCCs developed in Kras and HBx double transgenicmice. In addition, the up-regulation of β-catenin, CD44and TBK1and thedown-regulation of E-cadherin were only observed in the Kras and HBx doubletransgenic mice. These results demonstrate that the dysregulation of these signalingpathways and molecules may participate in synergistic function of Kras mutation andHBx in the initiation and progression of hepatocellular carcinoma in mice.In summary, we provided the first in vivo genetic evidence thathepatocyte-specific Kras activation induced HCC and demonstrated that Kras andHBx synergistically promoted the initiation and progression of HCC. The novelgenetic modified mouse models generated in this study closely recapitulated thehistopathologic progression and molecular alterations of human HCC, and maypotentially facilitate the future therapeutic studies.