Evolution of genomic instability in diethylnitrosamine-induced hepatocarcinogenesis in mice.

Diethylnitrosamine (DEN) is a hepatic procarcinogen which is frequently used as an inducer of hepatocellular carcinoma (HCC) in mice. Although mice after DEN exposure are among the most widely used models for liver tumorigenesis, a detailed, mechanistic characterization of the longitudinal changes in the respective tumor genomes has never been performed. Here we established the chronological order of genetic alterations during DEN carcinogenesis by examining mice at different points in time. Tumor samples were isolated by laser microdissection and subjected to array-comparative genomic hybridization (array-CGH) and sequencing analysis. Chromosomal gains and losses were observed in tumors by week 32 and increased significantly by week 56. Loss of distal chromosome 4q, including the tumor suppressors Runx3 and Nr0b2/Shp, was a frequent early event and persisted during all tumor stages. Surprisingly, sequencing revealed that β-catenin mutations occurred late and were clearly preceded by chromosomal instability. Thus, contrary to common belief, β-catenin mutations and activation of the Wnt/β-catenin pathway are not involved in tumor initiation in this model of chemical hepatocarcinogenesis. Our study suggests that the majority of the current knowledge about genomic changes in HCC is based on advanced tumor lesions and that systematic analyses of the chronologic order including early lesions may reveal new, unexpected findings.