Differential Proteomic Analysis of Gender-dependent Hepatic Tumorigenesis in Hras12V Transgenic Mice

Zhuona Rong,Tingting Fan,Huiling Li,Juan Li,Kangwei Wang,Xinxin Wang,Jianyi Dong,Jun Chen,Fujin Wang,Jingyu Wang,Aiguo Wang

doi:10.1074/mcp.m116.065474

Abstract

Male prevalence is an outstanding characteristic of hepatocellular carcinoma (HCC), and the underlying mechanisms for this have remained largely unknown. In the present study, Hras12V transgenic mice, in which hepatocyte-specific expression of the ras oncogene induces male-biased hepatic tumorigenesis, were studied, and altered proteins were detected by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Protein samples from hepatic tumor tissues (T) and peritumor tissues (P) of transgenic males and females and the corresponding normal liver tissues (Wt) of nontransgenic males and females were subjected to pairwise comparisons based on proteomic analysis. Among 2381 autodetected protein spots, more than 1600 were differentially expressed based on a pairwise comparison (|ratio| > = 1.5, p < = 0.05). Of these, 180 spots were randomly selected for matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) identification; finally, 89 distinct proteins were obtained. Among these 89 proteins, 7 and 50 proteins were further validated by Western blotting and literature investigation, respectively. Intriguingly, compared with Wt, the altered proteins were relatively concentrated in T in transgenic females but in P in transgenic males. Consistently, the levels of p-ERK and p-mTOR were significantly higher in the T of females compared with that of males. The pathway enrichment assay showed that 5 pathways in males but only 1 in females were significantly altered in terms of the upregulated proteins in T compared with Wt. These data indicate that female hepatocytes are disturbed by oncogenes with great difficulty, whereas male hepatocytes readily do so. In addition, 33 proteins were gender-dependently altered in hepatic tumorigenesis. Moreover, 4% DNA packaging and 4% homeostasis-related functional proteins were found in females but not in males, and more nucleus proteins were found in females (8%) than in males (3%). In conclusion, the proteomic data and comparative analysis presented here offer crucial clues for elucidating the mechanisms that underlie the male prevalence in HCC.

Highlights

Gender-dependent hepatic tumorigenesis and activation of the MEK/ERK and PI3K/AKT/mTOR pathways in Hras12V transgenic mice—Because of the complex etiological factors, multiple-stage progression, and limitations regarding the availability of clinical samples, it is difficult to elucidate the complex mechanisms underlying hepatocellular carcinogenesis and the characteristic male prevalence
Further incidence analysis of the dependence of hepatic alteration on diameter (according to the histopathological diagnosis criteria described by Frith and Ward [29]) showed that the incidence of nodules (Ͻ 2 mm) did not significantly differ between transgenic 15-F and 10-month-old males (10-M), significant lower incidences of adenoma (2–5 mm), carcinoma (Ͼ 5 mm), and total hepatic alterations were found in 15-F compared with 10-M transgenic mice
Most of the proteins that were expressed differentially and were classified into the ras oncogene-positive or -negative related categories were biased toward males. These findings indicate that the ras oncogene more induces the molecular changes contributing to hepatic tumorigenesis in the hepatocytes of males than in those of females, and this finding may play a crucial role in understanding the mechanism underlying the male prevalence of Hepatocellular carcinoma (HCC)

Summary

Introduction

We previously reported Hras12V transgenic mouse lineages generated using an Hras12V construct with a mouse albumin enhancer/promoter, which induced hepatocyte-specific expression of the ras oncogene resulting in male-biased hepatic tumorigenesis at the appropriate time and with a high level of reproducibility [25]. This transgenic mouse represents a suitable model for investigating the underlying mechanisms of hepatocellular carcinogenesis, in relation to the ras oncogene and in relation to male prevalence

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Conclusion