Hepatocellular carcinoma (HCC) is a heterogeneous disease in which tumor subtypes can be identified based on the presence of adult liver progenitor cells. Having 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. Immunohistochemical staining for phosphorylated ribosomal protein S6 indicated robust mTOR complex 1 (mTORC1) activity in early preneoplastic lesions that peaked during the first week and waned over the subsequent 10 days. Continuous administration of rapamycin by subcutaneous pellet for 70 days markedly reduced the development of focal lesions, but resulted in activation of the PI3K signaling pathway. To test the hypothesis that early mTORC1 activation was critical to the development and progression of preneoplastic foci, we limited rapamycin administration to the 3-week period at the start of the protocol. Focal lesion burden was reduced to a degree indistinguishable from that seen with continuous administration. Short-term rapamycin did not result in the activation of PI3K or mTORC2 pathways. Microarray analysis revealed a persistent effect of short-term mTORC1 inhibition on gene expression that resulted in a genetic signature reminiscent of normal liver. We conclude that mTORC1 activation during the early stages of hepatic carcinogenesis may be critical due to the development of preneoplastic focal lesions in progenitor marker-positive HCC. mTORC1 inhibition may represent an effective chemopreventive strategy for this form of liver cancer.

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to unmanipulated control animals and to animals that underwent an incomplete Solt-Farber protocol to activate hepatic progenitor cells. Regions of formalin-fixed, paraffin-embedded tissue were obtained by laser capture microdissection (LCM). Proteomic analysis yielded 11,070 unique peptides representing 2,227 proteins. Quantitation of the peptides showed increased abundance of known HCC markers (e.g., glutathione S-transferase-P, epoxide hydrolase, 6 others) and potential markers (e.g., aflatoxin aldehyde reductase, glucose 6-phosphate dehydrogenase, 10 others) in foci from placebo-treated and rapamycin-treated rats. Peptides derived from cytochrome P450 enzymes were generally reduced. Comparisons of the rapamycin samples to normal liver and to the progenitor cell model indicated that rapamycin attenuated a loss of differentiation relative to placebo. We conclude that early administration of rapamycin in the Solt-Farber model not only inhibits the growth of pre-neoplastic foci but also attenuates the loss of differentiated function. In addition, we have demonstrated that the combination of LCM and mass spectrometry-based proteomics is an effective approach to characterize focal liver lesions.

Background The limited availability of donor organs has led to a search for alternatives to liver transplantation to restore liver function and bridge patients to transplantation. We have shown that the proliferation of late gestation (embryonic day 19; ED19) fetal rat hepatocytes is mitogen-independent, and that mechanisms regulating of mRNA translation, cell cycle progression and gene expression differ from those of adult rat hepatocytes. In the present study, we investigated whether E19 fetal hepatocytes can engraft and repopulate an injured adult liver. Methods Fetal hepatocytes were isolated using a monoclonal antibody against a hepatic surface protein, leucine amino peptidase (LAP). LAP+ and LAP− fractions were analyzed by immunofluorescence and microarray. Immunopurified E19 liver cells from DPPIV+ rats were transplanted via splenic injection into partial hepatectomized DPPIV- rats that had been pretreated with mitomycin C. Results More than a third of LAP+ fetal hepatocytes expressed ductal markers. Transcriptomic analysis revealed that these dual expressing cells represent a population of less well differentiated hepatocytes. Upon transplantation, LAP+ late gestation fetal hepatocytes formed hepatic, endothelial and ductal colonies within 1 month. By 10 months, colonies derived from LAP+ cells increased so that up to 35% of the liver was repopulated by donor-derived cells. Conclusions Late gestation fetal hepatocytes, despite being far along in the differentiation process, possess the capacity for extensive liver repopulation. This is likely related to the unexpected presence of a significant proportion of hepatocyte marker-positive cells maintaining a less well differentiated phenotype.