Hye Lim Ju

Hepatic expression of Sonic Hedgehog induces liver fibrosis and promotes hepatocarcinogenesis in a transgenic mouse model

BACKGROUND & AIMS Liver fibrosis is an increasing health concern worldwide and a major risk factor for hepatocellular carcinoma (HCC). Although the involvement of Hedgehog signaling in hepatic fibrosis has been known for some time, the causative role of activated Hedgehog signaling in liver fibrosis has not been verified in vivo. METHODS Using hydrodynamics-based transfection, a transgenic mouse model has been developed that expresses Sonic Hedgehog (SHH), a ligand for Hedgehog signaling, in the liver. Levels of hepatic fibrosis and fibrosis-related gene expression were assessed in the model. Hepatic expression of SHH was induced in a murine model for hepatocellular adenoma (HCA) and tumor development was subsequently investigated. RESULTS The transgenic mice revealed SHH expression in 2-5% of hepatocytes. Secreted SHH activated Hedgehog signaling in numerous cells of various types in the tissues. Hepatic expression of SHH led to fibrosis, activation of hepatic stellate cells, and an upregulation of various fibrogenic genes. Liver injury and hepatocyte apoptosis were observed in SHH mice. Persistent expression of SHH for up to 13months failed to induce tumors in the liver; however, it promoted liver tumor development induced by other oncogenes. By employing a HCA model induced by P53(R172H) and KRAS(G12D), we found that the SHH expression promoted the transition from HCA to HCC. CONCLUSIONS SHH expression in the liver induces liver fibrosis with concurrent activation of hepatic stellate cells and fibrogenic genes. It can also enhance hepatocarcinogenesis induced by other oncogenes.

Combination of preS Deletions and A1762T/G1764A Mutations in HBV Subgenotype C2 Increases the Risk of Developing HCC

Background: The interactions among hepatitis B virus (HBV) mutations in developing hepatocellular carcinoma (HCC) remain unclear and thus we investigated the risk of HCC related with single or multiple HBV mutations in Korean patients infected with HBV subgenotype C2. Methods: From January 2003 to December 2008, HBV isolates from 135 patients with HCC were compared with those from 135 patients without HCC, matching for age, gender, and HBeAg status. The prevalence of preS deletions and G1896A and A1762T/G1764A mutations was evaluated. Results: The frequency of preS deletions significantly differed between the non-HCC and HCC groups, with 6 (4.4%) versus 25 (18.5%) patients, respectively (p < 0.001). Additionally, the frequency of A1762T/G1764A mutations was higher in the HCC than the non-HCC group [82 (60.7%) versus 30 (22.2%), p < 0.001]. For combined mutations, the odds ratio (OR) was highest in patients with both preS deletions and the A1762T/G1764A mutation, with 1 (0.7%) versus 11 (8.1%) patients (p = 0.005; OR 11.887). Conclusions: HCC was associated with preS deletions and A1762T/G1764A mutations, and the combination of both mutations had a stronger association with HCC in Korean patients infected with HBV subgenotype C2.

Investigation of Oncogenic Cooperation in Simple Liver-Specific Transgenic Mouse Models Using Noninvasive In Vivo Imaging

Liver cancer is a complex multistep process requiring genetic alterations in multiple proto-oncogenes and tumor suppressor genes. Although hundreds of genes are known to play roles in hepatocarcinogenesis, oncogenic collaboration among these genes is still largely unknown. Here, we report a simple methodology by which oncogenic cooperation between cancer-related genes can be efficiently investigated in the liver. We developed various non-germline transgenic mouse models using hydrodynamics-based transfection which express HrasG12V, SmoM2, and a short-hairpin RNA down-regulating p53 (shp53) individually or in combination in the liver. In this transgenic system, firefly luciferase was co-expressed with the oncogenes as a reporter, allowing tumor growth in the liver to be monitored over time without an invasive procedure. Very strong bioluminescence imaging (BLI) signals were observed at 4 weeks post-hydrodynamic injection (PHI) in mice co-expressing HrasG12V and shp53, while only background signals were detected in other double or single transgenic groups until 30 weeks PHI. Consistent with the BLI data, tumors were observed in the HrasG12V plus shp53 group at 4 weeks PHI, while other transgenic groups failed to exhibit a hyperplastic nodule at 30 weeks PHI. In the HrasG12V plus shp53 transgenic group, BLI signals were well-correlated with actual tumor growth in the liver, confirming the versatility of BLI-based monitoring of tumor growth in this organ. The methodology described here is expected to accelerate and facilitate in vivo studies of the hepatocarcinogenic potential of cancer-related genes by means of oncogenic cooperation.

Transgenic mouse model expressing P53 R172H , luciferase, EGFP, and KRAS G12D in a single open reading frame for live imaging of tumor

Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53R172H and KRASG12D, are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53R172H and KRASG12D in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53R172H and KRASG12D.

Analysis of miRNA expression patterns in human and mouse hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC), one of the most common malignancies in adults displays aberrant miRNA expression during its pathogenesis. We assessed expression of miRNA in surgically resected human HCC of an early stage and murine HCC with a high malignancy in order to find miRNA overexpressed in HCC regardless of tumor stage and underlying etiology. Further, the role of the deregulated miRNA in HCC pathogenesis was investigated.

Transgenic mouse models generated by hydrodynamic transfection for genetic studies of liver cancer and preclinical testing of anti-cancer therapy

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide; however, the genetic mechanisms underlying its pathogenesis are incompletely understood. Genetically engineered mouse (GEM) models of HCC have been developed to elucidate the role of individual cancer‐related genes in hepatocarcinogenesis. However, the expensive and time‐consuming processes related to generating a GEM model discourage the development of diverse genotype models. Recently, a simple and inexpensive liver‐specific transgenic approach was developed, in which a hydrodynamics‐based transfection (HT) method was coupled with the Sleeping Beauty transposase system. Various HT models in which different oncogenic pathways are activated and/or tumor‐suppressing pathways inactivated have been developed in recent years. The applicability of HT models in liver cancer research is expected to broaden and ultimately elucidate the cooperation between oncogenic signaling pathways and aid in designing molecular therapy to target altered pathways.