Author manuscript, published in "Nature 2009;457(7226):200-4"
DOI : 10.1038/nature07475
Frequent in-frame somatic deletions activate gp130 in inflammatory
hepatocellular tumours
Rebouissou Sandra 1 2 , Amessou Mohamed 1 2 , Couchy Gabrielle 1 2 , Poussin Karine 1 2 , Imbeaud Sandrine 3 4 , Pilati Camilla 1 2 ,
Izard Tina 5 , Balabaud Charles 6 7 , Bioulac-Sage Paulette 6 8 , Zucman-Rossi Jessica 1 2 *
1 Genomique Fonctionnelle des Tumeurs Solides INSERM : U674, Université Denis Diderot - Paris VII, IFR105, Hopital Saint-Louis - IFR
105 PARIS VII 27, Rue Juliette Dodu 75010 PARIS ,FR
2 IUH, Institut Universitaire d'Hématologie Université Denis Diderot - Paris VII, Hôpital St Louis,FR
3 Genexpress, Génomique Fonctionnelle et Biologie Systémique pour la Santé CNRS : UMR7091, Université Pierre et Marie Curie - Paris VI
, 7 rue Guy Moquet, BP8, 94801 Villejuif cedex,FR
4 DNA Microarray Platform (GODMAP) Centre de G??n??tique Mol??culaire, UPR 2167 CNRS , Gif-sur-Yvette, F-91198 France;
Universit?? Paris-Sud 11, Orsay, F-91405 France
5 Department of Cancer Biology The Scripps Research Institute, Jupiter, Florida, US
6 Fibrose hépatique et cancer du foie INSERM : U889, Université Victor Segalen - Bordeaux II, IFR66, 146 rue léo saignat, 33076 Bordeaux
Cedex,FR
7 Service de Hépato-gastroentérologie CHU Bordeaux, Hôpital Saint-André, Bordeaux,FR
inserm-00340859, version 1 - 25 May 2009
8 service d'anatomie pathologique CHU Bordeaux, Groupe hospitalier Pellegrin, Bordeaux,FR
* Correspondence should be adressed to: Jessica Zucman-Rossi
Abstract
Inflammatory hepatocellular adenomas (IHCA) are benign liver tumours defined by the presence of inflammatory infiltrates and by
the elevated expression of inflammatory proteins in tumour hepatocytes1,2. Here we show a striking activation of the IL6 signalling
pathway in this tumour type, and sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the
IL6ST gene that encodes the signalling co-receptor gp130. Indeed, 60% of IHCA harbour small in-frame deletions that target the
binding site of gp130 for IL6, and expression of four different gp130 mutants, in hepatocellular cells, activates STAT3 in absence of
ligand. Further, analysis of hepatocellular carcinomas revealed that rare gp130 alterations are always accompanied by β
-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 having STAT3 activation.
MESH Keywords Adenoma, Liver Cell ; genetics ; pathology ; Cell Line, Tumor ; Cytokine Receptor gp130 ; genetics ; metabolism ; Gene Expression Profiling ; Gene
Expression Regulation, Neoplastic ; Humans ; Inflammation ; genetics ; pathology ; Interferons ; metabolism ; Interleukin-6 ; metabolism ; STAT3 Transcription Factor ;
metabolism ; Sequence Deletion ; genetics ; Signal Transduction
Author Keywords hepatocellular adenoma ; heaptocellular carcinoma ; gp130 ; inflammation ; oncogene ; mutation
Several recent studies have shown STAT3 activation in epithelial tumours, underscoring the importance of IL6 signalling and the
inflammatory response in tumourigenesis, which provides an opportunity for therapeutic intervention3. However, the mechanisms that
provoke sustained STAT3 activation in tumours are largely unresolved. To define the interaction between the inflammatory response and
carcinogenesis in liver tumours, we assessed inflammatory hepatocellular adenomas (IHCA), benign tumours predominately found in
women and frequently associated with obesity and alcohol use1,2. Tumour hepatocytes of these adenomas express elevated levels of serum
amyloid A (SAA) and C-reactive (CRP) proteins, two members of the acute-phase inflammatory response, whereas SAA and CRP are not
expressed in inflammatory cells, Kupffer cells, or other sinusoidal cells in IHCA (Fig. 1a, Supplementary Fig. S1, and Ref. 1).
Inflammatory infiltrates were largely localised to arterial vessels, but were also found within the sinusoidal lumens of IHCA. Here, CD45 +
CD3+ T lymphocytes (CD4:CD8, 2:1) were intermingled with less numerous CD20+CD79A+ B cells, some plasma cells and a few
polymorphonuclear cells; no CD30+ lymphocytes, nor CD56+ or CD57+ NK cells were observed. In addition, CD68+ histiocytes were
present in infiltrates, as well as prominent Kupffer cells in sinusoidal lumens (Supplementary Fig. S1). Overall, inflammatory infiltrates
observed in IHCA were highly polymorphous.
To resolve the underlying pathogenesis of these inflammatory lesions, a genome-wide transcriptome analysis of four IHCA was
compared to four normal liver tissue samples. Among the 285 genes significantly overexpressed in IHCA ( Supplementary Table S1), gene
ontology analysis identified a strong enrichment for genes associated with inflammation and the immune response, accounting for 40 % of
the overall terms significantly enriched (Supplementary Table S2). High levels of significance were found for “antigen processing and
presentation of peptide antigen” (P=2.10−11) and “regulation of the JAK/STAT cascade” (P=10−5) (Supplementary Table S2). We
Page 1/9
Nature. Author manuscript
confirmed this inflammatory signature in an additional 14 IHCA with a clear activation of the acute-phase inflammatory response affecting
both type-1 and type-2 acute-phase genes (Fig. 1b and 1c, Supplementary Table S3). Consistent with the known roles of IL6 and
JAK-STAT signalling in the acute-phase response4,5, STAT3 mRNA and protein were significantly elevated in IHCA (Fig. 1b and 1c).
IHCA also overexpressed several effectors of type-1 and type-2 interferon signalling pathways (e.g., JAK2, STAT1 and STAT2) and their
downstream targets (Fig. 1b, 1c). Collectively, these data suggest that IL6 and interferon signalling are the main inflammatory pathways
activated in IHCA (Fig. 1d).
Since IL6 was not overexpressed in IHCA and because the inflammatory response was restricted to tumour hepatocytes ( Fig. 1a), we
reasoned that somatic genetic mutation(s) might account for activation of IL6 receptor signalling in IHCA. We selected IL6ST as a
candidate gene since it encoded the cell surface signalling receptor gp130 shared by at least six different cytokines including IL6, IL11,
LIF, OSM, CTNF and CT-14,6. We sequenced the entire gp130 coding region in 43 IHCA and 33 non-inflammatory hepatocellular
adenomas. Remarkably, 26 mutations in gp130 were identified specifically in 60% (26/43) of IHCA, and these included 16 unique, small
in-frame deletions and one 33 bp in-frame duplication in exon 6 (Fig. 2a and Table 1). Notably, all IL6ST mutations were found in IHCA
and all were of somatic origin, as they were not observed in adjacent normal liver tissues. In all cases, IL6ST mutations were monoallelic,
and IHCA with these mutations expressed both the wild type and mutated alleles at comparable levels, as judged by sequencing RT-PCR
products of IL6ST mRNA (Supplementary Fig. S2).
inserm-00340859, version 1 - 25 May 2009
Binding of IL-6 to its cognate receptor gp80 (encoded by IL6R) induces formation of a high affinity ternary hexameric complex
consisting of two molecules each of IL6, IL6R and gp1307,8. Gp130 engagement then activates JAK/Tyk tyrosine kinases and the STAT
family of transcription factors9–11. The consequences of the IL6ST in-frame deletions observed in IHCA included the removal of 1 to 26
amino acids neighbouring the IL6/IL6R binding site (also known as CHR E-F loop) located in D2 domain of gp130 ( Fig. 2b). We
modelled the different deletions and the duplication in the known crystal structure of the wild type IL6/IL6R/gp130 ternary complex (PDB
1P9M)7. All of these mutations are predicted to disrupt key residues involved in the gp130-IL6 interface. Specifically, the most frequent
alterations target residues 186–191, which direct the gp130-IL6 interaction, whereas the remaining deletions and duplication affect the
other two loops that contribute to gp130-IL6 interactions (Fig. 2b). Therefore, the gp130-IL6 interface is targeted in IHCA.
To investigate possible functional consequences of these gp130 mutations, we tested the effects of enforced expression of two frequent
deletions (S187_Y190del and Y186_Y190del) and two infrequent mutants (V184_Y186del, S187A and K173_D177del) in Hep3B cells, a
hepatocellular carcinoma line that activates the acute inflammatory phase following IL6 treatment 12. In the absence of IL6 ligand and
serum, overexpression of wild type gp130 alone was not sufficient to activate STAT3 and the downstream acute-phase inflammatory genes
(Fig. 3 and Refs. 7,13). In contrast, all gp130 IHCA mutants activated an acute phase inflammatory response and induced typical targets of
this response, including CRP, SAA2, SPINK1 and FBG (Fig. 3a, 3c). Further, as observed in IHCA, all of these gp130 mutants induced
the expression of SOCS3, which normally serves to harness cytokine signalling (Fig. 3a). Mutant gp130 S187_Y190del was constitutively
tyrosine phosphorylated and the activity of STAT3 was clearly increased in gp130 S187_Y190del-expressing Hep3B cells ( Fig. 3b).
Similarly, immunohistochemical analyses of IHCA demonstrated marked increases in nuclear STAT3 phosphorylated at Tyr-705 (
Supplementary Fig. S4e–f). Finally, IL6 augmented the induction of CRP in gp130 S187_Y190del-expressing cells, but mutant receptors
were not hypersensitive to low doses of IL6 (Fig. 3d). Therefore, gp130 mutants are constitutively active, and they activate STAT3 and
inflammatory response genes in the absence of IL6.
A critical step in the activation of intracellular signalling after IL6 binding on gp130 is the formation a hexameric structure that
juxtaposes the membrane proximal domains of two gp130 molecules at the cell surface 7,14,15. Using co-immunoprecipitation, we showed
that the gp130 S187_Y190del IHCA mutant was able to homodimerize or heterodimerize with wild-type gp130 independently of IL6,
whereas wild-type gp130 cannot homodimerize (Fig. 3f). Homodimerization of gp130 in absence of ligand has also been previously
described for two other gp130 mutants, Y190FV to AAA and Y190A 16. Moreover, overexpression of wild-type gp130 impaired the
activity of the mutant gp130 S187_Y190del in a dose-dependent manner (Fig 3e); therefore, mutant gp130 activity appears to be driven by
its homodimerization that can be competed by the wild-type protein. Interestingly, using a reverse genetic approach in mice, Ernst and
collaborators have recently shown that IL11 promotes chronic gastric inflammation and associated tumourigenesis mediated by gp130 and
STAT3 activation17. In IHCA, we also found significant increases (6-fold) in the levels of IL11 mRNA (P 90% confidence). Only genes whose expression significantly differed between inflammatory HCA versus non-tumour
liver tissues (considering absolute fold change > 1.5 and p-values ≤ 0.01) were selected.
Gene ontology (GO) categories that were significantly over-represented in genes significantly up-regulated or suppressed in
inflammatory HCA were determined by the hypergeometric test using the web-based tool GOTree Machine (GOTM) (
http://bioinfo.vanderbilt.edu/gotm/) by comparison with the distribution of the overall genes included in the HG-U133A Affymetrix array.
Quantitative RT-PCR
Quantitative RT-PCR was performed in duplicate as described30. Ribosomal 18S RNA (R18S) was used to normalize expression data
and 2−ΔΔCT method was applied. Final results were expressed as the n-fold differences in target gene expression in tested samples when
compared with the mean expression value of non-tumour tissues (for tumour analysis) or with control cell line.
DNA sequencing
DNA sequencing was performed as described26 using primers provided in Supplementary Table S6. All mutations were validated by
sequencing a second independent PCR product on both strands.
Cell culture
Hep3B cells (ATCC) were grown in DMEM supplemented with 10% fetal calf serum. For transfections, cells were plated 16h prior to
transfection to produce monolayers that were 60% confluent and these were transfected by using Lipofectamine™ LTX according to the
manufacturer’s instructions (Invitrogen). Transfection efficiency was monitored by measuring the level of either wild-type and mutated
gp130 mRNA using quantitative RT-PCR and western blot analysis. The same gp130 expression level was observed in wild-type and
mutant transfected cells. 48h after transfection, cells were maintained in serum-free medium, then cells were either left untreated or were
stimulated with rhIL-6 (100 ng/ml) in a serum-free medium for 3 hr, just before cell harvest and protein and RNA extraction. For
luciferase assay, Hep3B cells were co-transfected with Stat3 luciferase reporter vector (pSTAT3-luc 0.5μg, Panomics) and the expression
plasmid for wild type or S187_Y190del mutant (ΔS) gp130 (1μg). Two days after transfection, the cells were lysed and the luciferase
activity was measured according to the manufacturer’s recommendations (Promega). The activities were normalized to protein in each cell
lysate.
Generation of gp130 mutants
Page 4/9
Nature. Author manuscript
A full-length gp130 open reading frame cloned in pORF9 vector was purchased from Invivogen (pORF9-hIL6ST). Mutagenesis
reactions were performed using the QuickChange site-directed mutgenesis kit (Stratagen). All constructs were verified by sequencing.
Using the same method, we also introduced two different epitope tags (myc and flag) at the C-terminal end in the wild-type and
S187_Y190del constructs.
Western blot analysis and immunoprecipitation
Western blot analyses were performed as described30 using the antibodies specific for STAT3, phospho-STAT3 Tyr705, STAT1,
phospho-JAK2 Tyr1007/1008 (Cell Signaling Technology, diluted 1:500), JAK2 (Santa Cruz Biotechnology, 1:500), CRP (Sigma, 1:500),
gp130 (C-20 Santa Cruz Biotechnology, 1:200), and VEGF (Novus Biologicals, 1:100). The phosphorylated form of gp130 (p-gp130) was
analysed by SDS-PAGE after immunoprecipitation from cell lysates containing 750 μg of proteins using 5μg of the C-20 anti-gp130
antibody. The membrane was then probed with antibodies directed against phosphotyrosine (clone 4G10, Upstate Biotechnology) and
gp130 (C20). For dimerization assays, cell lysates were incubated with Protein G agarose (Pierce) and anti-flag antibody (Cell signaling
technology, 1:50), at 4°C overnight. The immune complexes were sedimented, washed, separated by SDS-PAGE and analysed by Western
blot using anti-flag (1:1000) and anti-myc (1:1000) antibodies. In western blot, red ponceau staining or actin (Sigma, 1:3000) expression
was analysed to appreciate protein loading.
Immunohistochemistry
inserm-00340859, version 1 - 25 May 2009
Immunohistochemistry was performed using a Dako autostainer, on paraffin sections of 10% fixed tumour tissue using 2 monoclonal
antibodies against inflammatory proteins: anti-C-reactive protein (CRP), (Abcam, 1:1500), anti-serum amyloid A (SAA), (Dako, 1:50),
anti-phospho-STAT3 Tyr705 (Cell signaling Technology, 1:50), anti-gp130 (C-20 Santa Cruz Biotechnology, 1:200). For inflammatory
cells immunotyping, the following antibodies (Dako) were used: CD45 (clone EB11+PD7/26, 1:300), CD3 (polyclonal, 1:100), CD4
(clone OPD4, 1:100), CD8 (clone C8/144B, 1:20), CD20 (clone L26, 1:100), CD68 (clone PG-M1, 1:50). For each immunohistochemical
procedure, antigen retrieval was performed in citrate buffer, detection was amplified by the Dako Envision system.
Ackowledgements:
We are indebted to Philippe Bois (The Scripps Research Institute, Jupiter) and Olivier Bernard (Inserm E0210, Paris) for scientific discussion
and critical reading of this manuscript. We warmly thank Cristel Thomas and Gaelle Cubel for their participation to this work that is dedicated
to Jean-Philippe Salier (Inserm U519, Rouen). We also thank Jean Saric, Christophe Laurent, Antonio Sa Cunha, Brigitte Le Bail, Anne
Rullier for contributing to the tissue collection (CHU Bordeaux). This work was supported by Inserm (R éseaux de recherche clinique et ré
seaux de recherche en santé des populations), the Ligue Nationale Contre le Cancer (“Cartes d’identité des tumeurs” program), ARC (grant
5158), and the Fondation de France. S.R. and M.A. are supported by a fellowship from la Ligue Nationale Contre le Cancer and the Inca,
respectively. JZR is supported by an interface contract between Inserm and Bordeaux hospital. TI is supported by the National Institutes of
Health grants GM071596, AI055894, and AI067949.
References:
1. Bioulac-Sage P Hepatocellular adenoma subtype classification using molecular markers and immunohistochemistry. Hepatology. 46: 740- 8 2007;
2. Zucman-Rossi J Genotype-phenotype correlation in hepatocellular adenoma: new classification and relationship with HCC. Hepatology. 43: 515- 24 2006;
3. Grivennikov S , Karin M Autocrine IL-6 signaling: a key event in tumourigenesis?. Cancer Cell. 13: 7- 9 2008;
4. Akira S Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell. 77: 6371 1994;
5. Wegenka UM , Buschmann J , Lutticken C , Heinrich PC , Horn F Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly
activated by interleukin-6 at the posttranslational level. Mol Cell Biol. 13: 276- 88 1993;
6.. Hibi M Molecular cloning and expression of an IL-6 signal transducer, gp130. Cell. 63: 1149- 57 1990;
7. Boulanger MJ , Chow DC , Brevnova EE , Garcia KC Hexameric structure and assembly of the interleukin-6/IL-6 alpha-receptor/gp130 complex. Science. 300: 2101- 4
2003;
8.. Ward LD High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6, interleukin-6 receptor, and gp-130. J Biol Chem.
269: 23286- 9 1994;
9. Lutticken C Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science. 263: 89- 92 1994;
10.. Murakami M IL-6-induced homodimerization of gp130 and associated activation of a tyrosine kinase. Science. 260: 1808- 10 1993;
11. Stahl N Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components. Science. 263: 92- 5 1994;
12. Coulouarn C Genome-wide response of the human Hep3B hepatoma cell to proinflammatory cytokines, from transcription to translation. Hepatology. 42: 946- 55 2005;
13.. Kishimoto T , Akira S , Narazaki M , Taga T Interleukin-6 family of cytokines and gp130. Blood. 86: 1243- 54 1995;
14. Chow D , He X , Snow AL , Rose-John S , Garcia KC Structure of an extracellular gp130 cytokine receptor signaling complex. Science. 291: 2150- 5 2001;
15.. Skiniotis G , Boulanger MJ , Garcia KC , Walz T Signaling conformations of the tall cytokine receptor gp130 when in complex with IL-6 and IL-6 receptor. Nat Struct
Mol Biol. 12: 545- 51 2005;
16. Li H , Nicholas J Identification of amino acid residues of gp130 signal transducer and gp80 alpha receptor subunit that are involved in ligand binding and signaling by
human herpesvirus 8-encoded interleukin-6. J Virol. 76: 5627- 36 2002;
17. Ernst M STAT3 and STAT1 mediate IL-11-dependent and inflammation-associated gastric tumourigenesis in gp130 receptor mutant mice. J Clin Invest. 118: 1727- 38
2008;
18.. Maione D Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver. Embo J. 17: 5588- 97 1998;
19. Judd LM Gastric cancer development in mice lacking the SHP2 binding site on the IL-6 family co-receptor gp130. Gastroenterology. 126: 196- 207 2004;
20. Judd LM STAT3 activation regulates growth, inflammation, and vascularization in a mouse model of gastric tumourigenesis. Gastroenterology. 131: 1073- 85 2006;
Page 5/9
Nature. Author manuscript
21. Tebbutt NC Reciprocal regulation of gastrointestinal homeostasis by SHP2 and STAT-mediated trefoil gene activation in gp130 mutant mice. Nat Med. 8: 1089- 97 2002;
22.. Boulton TG , Stahl N , Yancopoulos GD Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine
phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors. J Biol Chem. 269: 11648- 55 1994;
23. Croker BA SOCS3 negatively regulates IL-6 signaling in vivo. Nat Immunol. 4: 540- 5 2003;
24. Bioulac-Sage P Clinical, morphologic, and molecular features defining so-called telangiectatic focal nodular hyperplasias of the liver. Gastroenterology. 128: 1211- 8 2005
;
25. Schutyser E , Struyf S , Van Damme J The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev. 14: 409- 26 2003;
26. Bluteau O Bi-allelic inactivation of TCF1 in hepatic adenomas. Nat Genet. 32: 312- 5 2002;
27. Chen YW , Jeng YM , Yeh SH , Chen PJ P53 gene and Wnt signaling in benign neoplasms: beta-catenin mutations in hepatic adenoma but not in focal nodular hyperplasia
. Hepatology. 36: 927- 35 2002;
28.. Boyault S Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets. Hepatology. 45: 42- 52 2007;
29. Naugler WE Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science. 317: 121- 4 2007;
30.. Rebouissou S HNF1alpha inactivation promotes lipogenesis in human hepatocellular adenoma independently of SREBP-1 and carbohydrate-response element-binding
protein (ChREBP) activation. J Biol Chem. 282: 14437- 46 2007;
31. Gautier L , Cope L , Bolstad BM , Irizarry RA Affy--analysis of Affymetrix GeneChip data at the probe level. Bioinformatics. 20: 307- 15 2004;
32. Gentleman RC Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5: R80- 2004;
33.. Li C , Hung Wong W Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biol. 2: Research00322001;
34. Irizarry RA Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res. 31: e15- 2003;
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
inserm-00340859, version 1 - 25 May 2009
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 PG, 574_582del
575_583del
577_579del, 580A>T
583_588del
614_646dup
643_645del
Y186_F191del, V192F
S187_F191del
T188_F191del, V192T
Y190_V192del, N193S
F191L, V192_I194del
V192_I194del
N193del, I194F
E195_V196del
K206_P216dup, G205R
D215del
Codons and mutated nucleotides are numbered according to IL6ST cDNA ORF
gp130 mutants analysed in Hep3B cells
**
Page 9/9
Nature. Author manuscript
