An expression signature of phenotypic resistance to hepatocellular carcinoma identified by cross-species gene expression analysis

Abstract

Background and aims: Hepatocarcinogenesis is under polygenic control. We analyzed gene expression patterns of dysplastic liver nodules (DNs) and hepatocellular carcinomas (HCCs) chemically-induced in F344 and BN rats, respectively susceptible and resistant to hepatocarcinogenesis.

Methods: Expression profiles were performed by microarray and validated by quantitative RT-PCR and Western blot.

Results: Cluster analysis revealed two distinctive gene expression patterns, the first of which included normal liver of both strains and BN nodules, and the second one F344 nodules and HCC of both strains. We identified a signature predicting DN and HCC progression, characterized by highest expression of oncosuppressors Csmd1, Dmbt1, Dusp1, and Gnmt, in DNs, and Bhmt, Dmbt1, Dusp1, Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13 in HCCs of resistant rats. Integrated gene expression data revealed highest expression of proliferation-related CTGF, c-MYC, and PCNA, and lowest expression of BHMT, DMBT1, DUSP1, GADD45g, and GNMT, in more aggressive rat and human HCC. BHMT, DUSP1, and GADD45g expression predicted patients' survival.

Conclusions: Our results disclose, for the first time, a major role of oncosuppressor genes as effectors of genetic resistance to hepatocarcinogenesis. Comparative functional genomic analysis allowed discovering an evolutionarily conserved gene expression signature discriminating HCC with different propensity to progression in rat and human.

Fig. 1

Unsupervised hierarchical cluster analysis of gene expression patterns of rat liver tissues. Microarray experiments with 4 normale livers, 8 DNs, and 10 HCCs, per each strain, were made with Agilent Rat 4130A or B. Out of 22,575 gene features, 1,362 gene features, showing more than 2-fold difference compared to median expression value in more than 4 arrays, were selected for cluster analysis. Expression values were Log 2 transformed before clustering. Rows represent individual genes and columns represent each tissue,

Fig. 2

(a) qPCR analysis of Anxa5, Myc, Igfbp3, Gnmt, Ctgf, Igfbp1, Bhmt, Dmbt1, and Dusp1 expression in normal liver (C), dysplastic nodules (N) and HCC (H) of F344 and BN rats. Results are means ± SD of 6 normal livers, 15 DNs, and 14 HCCs per each strain of differences between target gene and RNR-18 expression, named Number Target (NT). NT = 2-ΔCt, ΔCt = (CT(target) – CT(RNR-18)). Tukey-Kramer test: N and H vs. C, BN vs. F344, at least P<0.05 for all genes tested. (b) Correlation analysis of c-Myc and Dusp1 expression determined by microarray and quantitative RTPCR, on 4 normal livers, 8 DNs and 10 HCCs per each strain. (c) Left panel: representative Western blot of c-Myc, Ghmt, Bhmt, Dusp1, and Pp2A. Right panel: Optical densities of the peaks normalized to β-actin values and expressed in arbitrary units. Data are means ± SD of 3 normal livers, 5 DNs, and 5 moderately-differentiated HCCs from F344 and BN rats. Tukey-Kramer test: (*) N and H vs. C, at least P<0.05. (†) F344 vs. BN, P<0.001

Fig. 3

(a) Unsupervised hierarchical cluster analysis of integrated 26 human surrounding non-tumorous liver, 25 HCCB, and 35 HCCA, and 36 rat liver lesions. Orthologous genes with an expression ratio differing at least 2 from the reference in one of the data sets were selected for hierarchical analysis (6,132 genes). (b) Labeling index (percentage of 2-bromo-3-deoxyuridine incorporating nuclei; LI) and apoptotic index (percentage of apoptotic bodies; AI) of DN and HCC of F344 and BN rats. 3,000 hepatocytes per lesion were counted, and results are means ± SD of 8 DNs, and 10 HCCs per each strain. (c) Left panels: representative propidium iodide staining of HCC from F344 and BN rats. Arrows indicate nuclear changes representing apoptosis. Right panel: percentage of apoptotic cells in HCC (AI). Data are means ± SD of 5 HCC per each strain. Tukey-Kramer test: (*) BN versus F344, P<0.001 for LI and AI of both hematoxylin/eosin- and propidium iodide stained sections.

Fig. 4

(a) qPCR analysis of BHMT, DMBT1, DUSP1, GADD45, GNMT, CTGF, c-MYC, and PCNA expression in human HCC with poorer (A) and better (B) prognosis. Results are differences between Number Target (NT) of HCC and mean value of 5 normal livers. NT = 2-ΔCt, ΔCt = (CT(target) – CT(RNR-18)). Box plots show the dispersion of the results of 35 HCCA and 25 HCCB, Negative values on y-axes indicate decrease with respect to normal liver. Mann–Whitney U-test: HCCA vs HCCB, P<0.0001 for all genes tested. (b) Kaplan-Meier survival plots of distinct subgroups of human HCC identified by integrated gene expression data. Mantel-Cox statistical analysis: differences between survival plots, P<0.0001.