Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a highly heterogeneous disease, and prior attempts to develop genomic-based classification for HCC have yielded highly divergent results, indicating difficulty in identifying unified molecular anatomy. We performed a meta-analysis of gene expression profiles in data sets from eight independent patient cohorts across the world. In addition, aiming to establish the real world applicability of a classification system, we profiled 118 formalin-fixed, paraffin-embedded tissues from an additional patient cohort. A total of 603 patients were analyzed, representing the major etiologies of HCC (hepatitis B and C) collected from Western and Eastern countries. We observed three robust HCC subclasses (termed S1, S2, and S3), each correlated with clinical parameters such as tumor size, extent of cellular differentiation, and serum alpha-fetoprotein levels. An analysis of the components of the signatures indicated that S1 reflected aberrant activation of the WNT signaling pathway, S2 was characterized by proliferation as well as MYC and AKT activation, and S3 was associated with hepatocyte differentiation. Functional studies indicated that the WNT pathway activation signature characteristic of S1 tumors was not simply the result of beta-catenin mutation but rather was the result of transforming growth factor-beta activation, thus representing a new mechanism of WNT pathway activation in HCC. These experiments establish the first consensus classification framework for HCC based on gene expression profiles and highlight the power of integrating multiple data sets to define a robust molecular taxonomy of the disease.

Fig. 1. HCC subclasses predicted in nine independent datasets

Predicted subclasses are shown in red (S1), blue (S2) and yellow (S3) with expression pattern of the HCC subclass signature. The proportion of the cases with confident prediction (FDR < 0.05) in HCC-A, B, C, D, E, F, G, H, and I were 96%, 96%, 90%, 81%, 79%, 87%, 94%, 83%, and 83%, respectively. Red bars attached to HCC-H and HCC-I indicate positive beta-catenin mutations and nuclear staining of p53, respectively. FDR: false discovery rate.

Fig. 2. Molecular pathways associated with HCC subclasses

Immuno-histochemistry analysis of (A) phospho-AKT, (B) p53, and (C) beta-catenin proteins in HCC-I. Left panels show proportions of the cases with positive staining in each HCC subclass. Right panels show representative positive staining (arrow heads, magnification: ×20). (D) Growth inhibition of SNU-387 cells (predicted to be subclass S1) by knocking down beta-catenin protein using two different shRNA constructs.

Fig. 3. Interaction between WNT pathway and TGF-beta

(A) Up-regulation of an experimentally-defined WNT target gene set, “KENNY_WNT_UP” (FDR<0.001), by TGF-beta. Genes were rank-ordered based on differential expression between TGF-beta-treated and untreated Huh-7 cells (predicted to be subclass S2). A database of target gene sets for experimental perturbations (377 gene sets) was assessed by Gene Set Enrichment Analysis (GSEA). (B) Up-regulation of the S1 signature by TGF-beta treatment. Genes were rank-ordered based on differential expression between treated and untreated Huh-7 cells, and induction of the subclass signature was evaluated by GSEA (FDR=0.04). (C) Suppression of alpha-fetoprotein (AFP) protein expression by TGF-beta treatment. Loading control is non-specific for AFP antibody to show that equal amounts of protein were loaded. FDR: false discovery rate.

Fig.4. Activation of WNT pathway by TGF-beta

(A) Huh7 cells were transfected with the indicated reporter constructs and increasing amounts of mutant beta-catenin (2, 5, 10ng of plasmid). (B) TGF-beta pathway activation was confirmed by phosphorylation of SMAD3. Abundance of beta-catenin protein was not changed by TGF-beta treatment (100pM, 48h). Loading control is non-specific for phosphor-SMAD3 antibody to show that equal amounts of protein were loaded. (C) Huh7 cells were stimulated as above and stained for beta-catenin. Cellular distribution of beta-catenin changed from predominantly membranous to cytoplasmic and perinuclear, and clustered cells spread out with more elongated and flattened morphology.

Fig. 5. Schematic summary of the characteristics of HCC subclasses

AFP: alpha-fetoprotein.