Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma
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Adeola O. Adebayo Michael1,7,*, Nagib Ahsan2,3,*, Valerie Zabala1, Heather Francois-Vaughan1, Stephanie Post4, Kate E. Brilliant3, Arthur R. Salomon3,5, Jennifer A. Sanders1,6, Philip A. Gruppuso1,5
1Department of Pediatrics, Rhode Island Hospital and Brown University, Providence, RI, USA
2Division of Biology and Medicine, Brown University, Providence, RI, USA
3Center for Cancer Research Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI, USA
4Department of Environmental and Evolutionary Biology, Brown University, Providence, RI, USA
5Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA
6Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA
7Current address: Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
*These authors contributed equally to this work
Philip A. Gruppuso, email: Philip_Gruppuso@brown.edu
Keywords: hepatocellular carcinoma, liver, microdissection, proteomics, pre-neoplasia
Received: December 22, 2016 Accepted: January 27, 2017 Published: February 09, 2017
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.
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