Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours

Abstract

Inflammatory hepatocellular adenomas are benign liver tumours defined by the presence of inflammatory infiltrates and by the increased expression of inflammatory proteins in tumour hepatocytes. Here we show a marked activation of the interleukin (IL)-6 signalling pathway in this tumour type; sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the IL6ST gene, which encodes the signalling co-receptor gp130. Indeed, 60% of inflammatory hepatocellular adenomas harbour small in-frame deletions that target the binding site of gp130 for IL-6, and expression of four different gp130 mutants in hepatocellular cells activates signal transducer and activator of transcription 3 (STAT3) in the absence of ligand. Furthermore, analysis of hepatocellular carcinomas revealed that rare gp130 alterations are always accompanied by beta-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant conversion of hepatocytes. The recurrent gain-of-function gp130 mutations in these human hepatocellular adenomas fully explains activation of the acute inflammatory phase observed in tumourous hepatocytes, and suggests that similar alterations may occur in other inflammatory epithelial tumours with STAT3 activation.

Figure 1. Activation of the interleukin-6 and interferon pathways in inflammatory HCA

a, Immunohistochemical analysis of CRP expression: high level of expression in tumour hepatocytes (IHCA); adjacent normal non-tumour liver hepatocytes (NTL) and inflammatory cells located in tumour (arrow) are negative. b, qRT-PCR validation of gene array expression data comparing IHCA (n=14, black) to NTL (n=6, white). Graphs plot mean +/− SD. *, **, *** difference between groups at P<0.05; 0.01 and 0.001, respectively (two-tailed Mann-Whitney test). c, Western-blot analysis comparing expression levels of STAT1 (84, 91 kDa), STAT3 (79, 86 kDa), CRP (24 kDa), VEGF (22 kDa), Jak2 and phospho-Jak2 (125 kDa) between IHCA and NTL. d, Schematic representation of the IL6 and interferon pathways showing all genes overexpressed (red) or suppressed (green) in IHCA compared to NTL. Genes validated by qRT-PCR are underlined (IHCA n=14 versus NTL n=6); others were extracted from the microarray analysis (IHCA n=4 versus NTL n=4).

Figure 2. gp130 mutations in inflammatory hepatocellular tumours

a, The spectrum of mutations in gp130 detected in IHCA (S, signal peptide; TM, transmembrane domain). The location of the 16 deletions, the duplication (in blue), and the resulting amino acid substitutions (in yellow) identified in the D2 domain of gp130 are indicated above the wild type sequence (in bold font). The most frequent mutation is indicated in red. b, Residues 186–192 (shown in pink stick presentation) of the D2 domain of gp130 that are deleted in IHCA are intimately involved in interactions with IL6, as shown in the crystal structure of the IL6R:IL6:gp130 complex (PDB 1P9M). IHCA deletions 173–177 and 215 (labelled in red) are also predicted to disrupt the IL6-gp130 interface. The numbering of residues corresponds to the IL6ST cDNA, which has 22 additional N-terminal residues compared to the polypeptide chain due to the nuclear export signal.

Figure 2. gp130 mutations in inflammatory hepatocellular tumours

a, The spectrum of mutations in gp130 detected in IHCA (S, signal peptide; TM, transmembrane domain). The location of the 16 deletions, the duplication (in blue), and the resulting amino acid substitutions (in yellow) identified in the D2 domain of gp130 are indicated above the wild type sequence (in bold font). The most frequent mutation is indicated in red. b, Residues 186–192 (shown in pink stick presentation) of the D2 domain of gp130 that are deleted in IHCA are intimately involved in interactions with IL6, as shown in the crystal structure of the IL6R:IL6:gp130 complex (PDB 1P9M). IHCA deletions 173–177 and 215 (labelled in red) are also predicted to disrupt the IL6-gp130 interface. The numbering of residues corresponds to the IL6ST cDNA, which has 22 additional N-terminal residues compared to the polypeptide chain due to the nuclear export signal.

Figure 3. Gain-of-function mutations of gp130

a, Gp130 mutants (Δ) including S187_Y190del (S), Y186_Y190del (Y), V184_Y186del, S187A (V) and K173_D177del (K) or controls including gp130 wild type (Wt) and empty plasmid (EP) were transfected in Hep3B. Graphs plot qRT-PCR results relative to EP transfected unstimulated cells, mean +/− SD (transfections in triplicate). b, Graph plots luciferase activity (mean) measured in triplicate transfections (+/−SD) with pSTAT3-luc alone or together with Wt or the ΔS gp130. Expression of total and phosphorylated (p-STAT3 and p-gp130) proteins were analysed using western blotting, after immunoprecipitation (IP) for gp130. c, CRP mRNA expression after transfection with increasing amounts of plasmids expressing Wt or ΔS gp130 (baseline, mock-transfected cells). d, Hep3B transfected with EP, Wt or ΔS gp130 and exposed to increasing concentrations of IL6. Graph plots mean CRP expression value relative to cells transfected with EP +/− SD (transfections in triplicate). e, Effect of increasing amounts of Wt gp130 on the ΔS gp130 mutant activity, without IL6. Graph plots mean level of expression of SOCS3 and CRP relative to cells transfected with EP +/− SD in duplicate (left) or in 5 independent transfections (right). f, Flag and Myc-tagged constructs expressing either the wild-type or the mutant ΔS gp130 were co-transfected (1:1) into Hep3B. gp130 dimers formation was detected after immunoprecipitation using the anti-flag antibody, followed by western blot analysis with the anti-myc and anti-flag antibodies. Shown is one representative of three independent experiments. *, **, *** indicate differences between groups at P<0.05; 0.01 and 0.001, respectively (two-tailed t-test).