Persistent Effect of mTOR Inhibition on Preneoplastic Foci Progression and Gene Expression in a Rat Model of Hepatocellular Carcinoma

We previously identified the mTOR pathway as critical to progenitor cell proliferation in a model of liver injury, we investigated the temporal activation of mTOR signaling in a rat model of hepatic carcinogenesis. The model employed chemical carcinogens and partial hepatectomy to induce progenitor marker-positive HCC. Rats were administered the mTOR inhibitor rapamycin for a three week period and liver harvested one month following cessation of rapamycin treatment. Short-term rapamycin treatment resulted in a significant reduction of focal lesion burden. Microarray analysis was performed to characterize the gene expression signature of persistent focal lesions in the rapamcyin and placebo treated animals. This analysis revealed a persistent effect of short-term mTORC1 inhibition on gene expression that resulted in a genetic signature reminiscent of normal liver. Laser capture microdissection was performed to specifically isolate persistent focal lesions from formalin fixed paraffin embedded (FFPE) liver sections derived from triplicate placebo and rapamycin treated rats. In addition, we captured liver tissue from FFPE sections derived from rats that had been administered 2-acetylaminofluorene in combination with 2/3 partial hepatectomy to induce oval cell proliferation and from normal adult rats. RNA was extracted using Qiagen’s FFPE RNeasy Kit and analyzed using Affymetrix Rat Gene ST 1.0 arrays. Unsupervised hierarchical clustering using the 5% of the genes with the highest coefficient of variation across all samples revealed an outlier in the rapamycin treated group. Additional microarray analyses performed on lesions from that animal demonstrated similar results. This animal was not an outlier based on any of the other biochemical or physiologic parameters that were measured, leading us to conclude that the array results reflected the heterogeneity of the genetic signature of the focal lesions and response to rapamycin. The raw expression values from the three arrays on this one animal were averaged for subsequent analyses.