Metastasis development in a novel mouse model of advanced liver cancer

Introduction: Liver cancer is one of the leading causes of cancer related mortality worldwide. It is often diagnosed in advanced stages when treatment options remain limited. Novel diagnostic markers and therapies are therefore urgently needed. The absence of reliable mouse models has been a major limitation in the study of advanced, metastatic liver cancer in the past. Here, we present a novel genetically engineered mouse model of primary liver cancer. In this model, activation of oncogenic Kras together with functional inactivation of the tumor suppressor proteins RB and p53 mimics molecular events that occur in the majority of human liver cancer and leads to rapid formation of hepatic tumors with extrahepatic metastases.

Methods: Adult Rblox/lox;p53lox/lox;KrasLSL-G12D/+ mice were injected intravenously with a liver specific AAV-Cre. Upon liver tumor formation individual lesions were microdissected and tissue was analyzed for RNA and protein expression. Microarray analysis was performed on an Affymetrix platform (Mouse Genome 430 2.0 Array) and mRNA expression was validated using qPCR. Protein expression was visualized by immunohistochemistry and SDS page gel electrophoresis.

Results: The RPK mouse model combines liver specific inactivation of RB and p53 with activation of Ras signaling. After gene recombination, RPK mice rapidly develop hepatic tumors within 3 to 4 months. Cancer lesions in this mouse model express hepatocytic (AFP+ and/or ALB+) as well as biliary (CK19+) differentiation markers. Comparison of gene expression microarrays from RPK tumors with data from human liver cancer samples confirmed clustering with human un-differentiated hepatocellular carcinomas and cholangiocarcinoma. Strikingly, selective targeting of hepatocytes in RPK mice showed that liver tumors of mixed or even biliary differentiation arise from mature hepatocytes. Analysis for activation of oncogenic pathways revealed activated ERK and AKT signaling downstream of Kras in RPK liver tumors. Additionally, numerous genes related to metastasis development were highly expressed in RPK cancer lesions. In line with these findings we detected extrahepatic metastases in the majority of mice with advanced tumor lesions.

Summary: The RPK mouse model presents a novel platform to study key molecular mechanisms in liver tumor initiation and development. Importantly, the highly consistent occurrence of metastatic lesion allows for the investigation of molecular drivers of metastasis development in liver cancer. Preliminary data from our mouse model indicate for an important role of Ras signaling in the metastatic process. The underlying molecular mechanisms are currently under investigation. The RPK model will help to answer important question in tumor biology of hepatic malignancies and aims to identify novel therapeutic targets for the treatment of human liver cancer.

Corresponding author: Ehmer, Ursula

E-Mail: ursula.ehmer@tum.de