Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma

Abstract

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.

Engraftment and Repopulation Potential of Late Gestation Fetal Rat Hepatocytes

“…Principal component analysis (PCA) was performed in R as described. 29 Identification of differentially expressed genes using a false discovery rate (FDR) of <0.05 30 was performed with Partek Genomic Suite version 6.6 (Partek, St. Louis, MO). Volcano plots used the adjusted p value from pair-wise analysis of variance (ANOVA) results and fold-change in gene expression.…”

MicroRNAs in Animal Models of HCC

“…Interestingly, a global proteomic analysis from glutathione S-transferase-P (GST-P) positive laser micro-dissected nodules identified G6PD among protein markers discriminating R-H model-derived focal lesion from normal liver with or without progenitor cell activation, demonstrating LCM coupled with mass-spectrometry-based proteomics as an effective approach to characterize preneoplastic lesions and to identify early diagnostic markers for effective clinical intervention. In addition, mTOR pathway was found activated at early stages of carcinogenesis and, consistently, its transient inhibition by rapamycin treatment impaired the growth of focal lesions and induced a less aggressive phenotype attenuating the loss of differentiating functions, as detected by both transcriptomic and proteomic genome-wide analyses [177,178]. The AKT/mTOR pathway is frequently activated in HCC, characterizing a subgroup of patients with a high proliferation signature and sorafenib resistance [179][180][181].…”

Enrichment of progenitor cells by 2‐acetylaminofluorene accelerates liver carcinogenesis induced by diethylnitrosamine in vivo

“…7 The Solt and Farber HCC model, also referred as the "resistant hepatocyte model," induces the HCC along with the expression of progenitor cell markers in 9 months after a single intraperitoneal (i.p) injection of the carcinogen diethylnitrosamine (DEN), 2-acetylaminofluorene (2AAF) in the diet and finally, a partial hepatectomy (PH) as the proliferative stimulus for liver regeneration. 8 In this protocol, the combination of 2AAF with PH is the key intervention for the HPC activation during liver regeneration in the rat. 9 For this ending, the hepatocytes DNA is preferentially cross-linked by 2AAF to prevent their proliferation, and as a result, HPC niches are expanded by the hepatic regeneration stimulus.…”