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

Hye-Lim Ju,Sang Hoon Ahn,Jinsil Seong,Sook In Chung,Kwang-Hyub Han,Do Young Kim,Sinhwa Baek,Simon Weonsang Ro,William B Coleman

doi:10.1371/journal.pone.0059869

Abstract

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.

Highlights

Liver cancer is a complex multistep process that involves genetic alterations in multiple proto-oncogenes and tumor suppressor genes [1,2,3]
Compared to cells transfected with pT2/enhanced green fluorescent protein (EGFP), confirming the constitutive activation of Ras signaling by HrasG12V (Fig. 2A)
We presented a simplified methodology with which the tumorigenic potential of individual genes and combinations of genes can be tested in the liver in vivo

Summary

Introduction

Liver cancer is a complex multistep process that involves genetic alterations in multiple proto-oncogenes and tumor suppressor genes [1,2,3]. An oncogenic mutation in a single gene, usually fails to induce liver cancer, as shown in transgenic mouse models expressing single oncogenes [5,6]. This implies that oncogenic collaboration among multiple cancer-related genes is required to induce HCC. An activating mutation in ras genes (a mutation at codon 12 resulting in the substitution of valine for glycine, for example) leads to constitutive activation of the Ras signaling pathways. An activating mutation in Smo (such as a mutation at codon 535 leading to substitution of leucine for tryptophan) causes hedgehog signaling to be constitutively active and is found in a variety of tumors [9,10]. Loss of p53 function is frequently found in tumors of diverse cellular origins, including HCC, and is considered a critical step in tumor development [1,11]

Methods

Results

Conclusion