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Based on their lobule location, hepatocytes display differential gene expression, including pericentral hepatocytes that surround the central vein, which are marked by Wnt-β-catenin signaling. Activating β-catenin mutations occur in a variety of liver tumors, including hepatocellular carcinoma (HCC), but no specific therapies are available to treat these tumor subsets. Here, we identify a positive relationship between β-catenin activation, its transcriptional target glutamine synthetase (GS), and p-mTOR-S2448, an indicator of mTORC1 activation. In normal livers of mice and humans, pericentral hepatocytes were simultaneously GS and p-mTOR-S2448 positive, as were β-catenin-mutated liver tumors. Genetic disruption of β-catenin signaling or GS prevented p-mTOR-S2448 expression, while its forced expression in β-catenin-deficient livers led to ectopic p-mTOR-S2448 expression. Further, we found notable therapeutic benefit of mTORC1 inhibition in mutant-β-catenin-driven HCC through suppression of cell proliferation and survival. Thus, mTORC1 inhibitors could be highly relevant in the treatment of liver tumors that are β-catenin mutated and GS positive.

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.