Proteomic Analysis of Focal Lesions in a Rat Model of Progenitor Marker-Positive Hepatocellular Carcinoma

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR), markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt‐Farber) model of HCC in the rat. In the present study, we characterized the proteome of persistent, pre‐neoplastic focal lesions in rats subjected to the Solt‐Farber protocol. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and sacrificed 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to animals that underwent a protocol to activate hepatic progenitor cells. Normal rat liver was used as an additional control. Regions of formalin‐fixed, paraffin‐embedded tissue were obtained by laser capture microdissection. Peptides were generated and analyzed by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS), which yielded 11,070 unique peptides representing 2,227 proteins. Peptides that showed increased abundance in placebo foci represented known HCC markers (glutathione S‐transferase‐P, epoxide hydrolase, 6 others) and potential markers (aflatoxin aldehyde reductase, carbonic anhydrase 2, 11 others). Peptides derived from cytochrome P450 enzymes were generally reduced. Neither placebo nor rapamycin samples showed similarity with the progenitor cell samples, indicating that the foci in this model, while considered to be progenitor marker‐positive, do not show evidence of persistent progenitor cells. Pathway analysis, based on non‐parametric identification of affected peptides, was consistent with an effect of rapamycin to reduce dedifferentiation in foci. Finally, we have demonstrated that the combination of LCM and LC‐MS/MS is an effective approach to characterize focal liver lesions. Support or Funding Information NIH R01DK100301 NIH R01HD024455Rhode Island Hospital Department of Pediatrics