Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer

  • A Corrigendum to this article was published on 21 January 2015
  • A Corrigendum to this article was published on 18 November 2015

Abstract

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer1,2,3,4,5. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation6,7,8,9,10. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs4,5, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

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Figure 1: Mutant IDH blocks hepatocyte differentiation.
Figure 2: Mutant IDH blocks hepatocyte differentiation by silencing HNF-4α.
Figure 3: Mutant IDH inhibits hepatocyte differentiation and quiescence of liver progenitors.
Figure 4: Mutant IDH cooperates with Kras(G12D) to drive liver progenitor cell expansion and multi-step IHCC pathogenesis.

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Primary accessions

Gene Expression Omnibus

Data deposits

Affymetrix Mouse 420Av2 DNA microarray data have been deposited in the Gene Expression Omnibus under accession number GSE57002.

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Acknowledgements

We thank R. Mostoslavsky, L. Ellisen, A. Kimmelman, and members of the Bardeesy laboratory for valuable input. We also thank S. Thorgeirsson and J. Andersen for sharing unpublished data sets. This work was supported by grants from TargetCancer Foundation and the National Institutes of Health (R01CA136567-02 and P50CA1270003) to N.B. N.B. holds the Gallagher Endowed Chair in Gastrointestinal Cancer Research at Massachusetts General Hospital. S.K.S. is the recipient of a Cholangiocarcinoma Foundation/Conquer Cancer Foundation of ASCO Young Investigator Award, and an American Cancer Society Postdoctoral Fellowship (PF-13-294-01-TBG). C.A.P. is the recipient of a Canadian Institutes of Health Research postdoctoral fellowship. N.B., J.M.L. and D.S are members of the Samuel Waxman Cancer Research Foundation Institute Without Walls. J.M.L. and D.S. are supported by the Asociación Española Contra el Cáncer (AECC).

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Authors

Contributions

S.K.S. and C.A.P. contributed equally to the study. S.K.S., C.A.P., K.S.G., M.S.N. and S.G. carried out the experiments involving hepatoblasts and mouse models. K.N.R. and S.R. performed computational analysis on gene expression data. J.F. performed immunohistochemistry on tissue sections. E.A.A. and K.-K.W. assisted with the generation of the IDH mutant mice. V.D. analysed the histology from the murine liver specimens. C.W. and U.A. carried out the experiments involving the HNF-4α knockout mice. D.S., H.C., O.M. and J.M.L. performed GSEA analysis on human IHCC samples. A.X.Z., A.F.H. and C.R.F. were involved in the study design. K.E.Y., K.S.S., J.T., J.P.-M. and C.G. developed and provided the AGI-5027 compound and measured 2HG in our samples. S.K.S., C.A.P. and N.B. designed the experiments and wrote the paper. N.B. supervised the studies. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Nabeel Bardeesy.

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Competing interests

K.Y., K.S., J.T., J.P.-M. and C.G. are employees of Agios Pharmaceuticals.

Extended data figures and tables

Extended Data Figure 1 Impact of mutant IDH1 and IDH2 on hepatoblast cell differentiation.

a, Primary hepatoblast cells were engineered to express the indicated human IDH alleles or empty vector (EV) under a doxycycline (Dox)-inducible system. Lysates from hepatoblast cells cultured in the presence of increasing doxycycline concentrations were analysed by immunoblot using an antibody that recognizes both murine and human IDH1 (top) or with an antibody specific to human IDH2 (lower panel); actin is the loading control. Note that 25 ng ml−1 doxycycline induces physiological levels of IDH1 expression and was used in the experiments shown in Figs 1 and 2. b, Photomicrographs of cells grown for 2 days on collagen-coated plates in the presence of 25 ng ml−1 doxycycline. WT, wild type. c, Hepatoblast cells cultured in the presence of increasing doxycycline concentrations were analysed by liquid chromatography/mass spectrometry (LC/MS) for levels of intracellular 2HG. d, Growth curve of hepatoblasts cultivated on collagen-coated dishes. e, f, Hepatoblast cells from a were grown on collagen-coated or uncoated plates and analysed for expression of hepatocyte markers by qRT–PCR. g, Enrichment plot showing downregulation of a hepatocyte gene set (Gene Expression Omnibus accession number GSE28892) in R132C-expressing cells. FDR, false discovery rate; NES, normalized enrichment score. h, 2HG levels in hepatoblast cells expressing the indicated IDH alleles or empty vector and treated with AGI-5027 (+) or DMSO vehicle (−). i, j, Wild-type hepatoblasts were treated with 500 μm octyl-(R) or -(S) enantiomers of 2HG or DMSO vehicle, and tested for hepatocyte differentiation upon transfer to uncoated plates by hepatocyte sphere formation (i) or qRT–PCR (j). k, l, Hepatoblast cells expressing the indicated alleles were tested for biliary differentiation upon transfer to matrigel as assessed by qRT–PCR for the induction of biliary makers Bgp and Ggt1. Scale bars, 100 μm. Error bars indicate ± s.d. between technical duplicates. *P < 0.05, Student’s t-test. Data are representative of at least two independent experiments.

Extended Data Figure 2 Mutant IDH represses the HNF-4α-mediated hepatocyte differentiation program.

ac, Gene expression profiling of hepatoblast cells expressing the indicated alleles and grown on collagen. a, b, Clustering analysis (a) and enrichment plot (GSEA) (b) showing downregulation of HNF-4α targets in the IDH-mutant cells relative to controls. c, GSEA plots using the collection of cis regulatory elements from the Molecular Signatures Database (C3 collection) reveals strong downregulation of genes containing consensus HNF-4α- and HNF1α-binding sites in the IDH-mutant hepatoblast cultures. d, qRT–PCR showing expression of Hnf4a and its target genes in hepatoblast cells expressing either empty vector (EV) or IDH2 R172K and grown on collagen-coated plates. e, HNF-4α(7–9) expression (immunoblot) in hepatoblast cells grown on collagen-coated plates (quantification of HNF-4α:actin is indicated). Data are representative of two independent experiments. f, g, Immunofluorescence staining for HNF-4α (using an antibody that detects all isoforms) in hepatoblast cells expressing empty vector or IDH1 R132C and grown on collagen (f). The chart shows quantification of the ratio of the HNF-4α and DAPI signals. The specificity of the antibody was demonstrated by the diminished staining in cells with knockdown of endogenous HNF-4α (g). h, Immunoblot showing that R132C suppresses HNF-4α(1–6) and HNF-4α(7–9) isoforms on uncoated plates. i, j, AGI-5027 restores Hnf4a(1–6) induction in R132C-expressing hepatoblasts as shown by qRT–PCR (i) and immunoblot (j). DMSO = vehicle. k, Wild-type hepatoblasts were treated with 500 μm octyl-(R) or -(S) enantiomers of 2HG, DMSO vehicle, or media alone. Hnf4a(1–6) levels were determined by qRT–PCR. l, m, Cultures of hepatoblast cells expressing the indicated alleles were grown on uncoated plates for 5 days and subjected to chromatin immunoprecipitation (ChIP) for H3K4me3 (l) or H3K27me3 (m). Enrichment for the promoter regions of Hhex (highly expressed gene in hepatoblast cells), Hoxa10 (transcriptionally silent in hepatoblast cells) and P1 Hnf4a was measured by qPCR. n, o, Analysis of wild-type hepatoblasts expressing shRNA control (shCTL) or targeting different HNF-4α sequences (shHnf4a#1, shHnf4a#2): hepatocyte marker expression (qRT–PCR) (n); HNF-4α immunoblot (o). Error bars indicate ± s.e.m. for f and ± s.d. for d, i, kn between technical duplicates. Scale bars, 50 μm. *P < 0.05, Student’s t-test.

Extended Data Figure 3 HNF-4α is dispensable for biliary differentiation.

a, Wild-type hepatoblast cells expressing shRNA control (shCTL) or targeting HNF-4α were tested for ability to undergo biliary differentiation upon transfer to matrigel as assessed by tubule formation 24 h after transfer (left) and qRT–PCR for induction of the biliary markers Bgp and Ggt1 10 days after transfer to matrigel (right). b, Immunoblot showing ectopic expression of HNF-4α(1) in hepatoblast cells. Error bars indicate ± s.d. between technical replicates. Scale bar, 100 μm.

Extended Data Figure 4 Analysis of genetically engineered mouse model with hepatocyte-specific mutant IDH expression.

a, Schematic of doxycycline-inducible IDH2 mutant alleles (see Methods for details). Mice harbouring mutant human IDH2 alleles were crossed with Alb-Cre and Rosa26-LSL-rtTA strains for liver-specific expression. bd, Characterization of expression pattern of mutant IDH2 in Tet-R140Q and Tet-R172K genetically engineered models compared to control wild-type mice, after 4 weeks of doxycycline supplementation, reveals hepatocyte-specific expression, consistent with previous models using this transgenic targeting system36. b, Immunofluorescence analysis of Tet-R140Q and control wild-type livers using an antibody that detects both endogenous and transgenic IDH2 expression. Note that transgenic IDH2(R140Q) is expressed in the hepatocytes that stain for HNF-4α(1–6), but not in the bile ducts that are marked by CK19. c, Immunofluorescence analysis of Tet-R140Q and control wild-type livers using an antibody that is specific to IDH2(R140Q), showing an identical pattern of transgene expression. d, Immunofluorescence analysis of Tet-R172K and control wild-type livers using an antibody that is specific to IDH2(R172K). This allele is also expressed in the hepatocytes, but shows more focal expression compared to R140Q. In b and c an antibody to total HNF-4α was used to label hepatocytes. Scale bars, 50 μm.

Extended Data Figure 5 Characterization of genetically engineered mouse model with hepatocyte-specific mutant IDH expression in the presence or absence of liver injury.

a, Measurement of 2HG levels in liver lysates from IDH2-mutant and control mice treated with doxycycline for 1 month. b, Uninjured wild-type and Tet-R140Q livers exhibit comparable expression of the hepatocyte markers Hnf4a, Adh1, Alb, and Aldob and the biliary markers Sprr1a and Onecut1 by qRT–PCR. c, d, Tet-R140Q mice and littermate controls (WT) receiving doxycycline were fed a DDC-containing diet for 5 days before being switched to normal chow for 1 week (see Fig. 3a for schematic). Mutant IDH2 does not provoke liver injury as reflected by comparable levels of serum AST, Tbili and ALT (c), and absence of cleaved caspase-3 staining in Tet-R140Q compared to wild-type mice (d). Duodenum from a TNF-α-treated mouse was used as a positive control for cleaved caspase-3 staining. Scale bars, 50 μm. NS, not significant. Error bars represent ± s.e.m.

Extended Data Figure 6 Characterization of response to liver injury in genetically engineered mouse model with hepatocyte-specific mutant IDH expression.

af, Tet-R140Q or Tet-R172K mice and littermate controls (WT) receiving doxycycline were fed a DDC-containing diet for 5 days before being switched to normal chow for 7 or 21 days (see Fig. 3a for schematic). a, Representative H&E images of livers from wild-type and Tet-R140Q mice analysed at day 7 and 21. b, Quantification of Ki-67-positive cells for indicated markers as shown in Fig. 3e (N = 3 mice per group, from at least 5 high-powered fields per mouse scored). Error bars show ± s.e.m. Data from Fig. 3f (day 21) is reproduced here for comparison. c, d, Immunofluorescence analysis of IDH2-mutant livers showing that Ki-67 co-localizes with HNF-4α+ (c) and IDH2(R140Q)-expressing cells (d). As shown in Fig. 3e, these cells express lower levels of HNF-4α compared to wild-type hepatocytes. e, f, Tet-R172K mice and littermate controls (WT) receiving doxycycline were fed a DDC-containing diet for 5 days before being switched to normal chow for 1 week. Immunofluorescence analysis of IDH2 mutant livers shows that Ki-67 co-localizes with HNF-4α+ and IDH2(R172K)-expressing cells (e) and greater numbers of Ki-67+, HNF-4α+ cells in Tet-R172K compared to wild-type mice (f). Chart shows quantification. Error bars show ± s.e.m. between 3 mice. Scale bars, 50 μm. *P < 0.05, Student’s t-test.

Extended Data Figure 7 Characterization of IDH2R172K genetically engineered mouse model.

a, Schematic of LSL-IDH2R172K genetically engineered mouse model (see Methods for details). Mice harbouring the LSL-IDH2R172K allele were crossed with the Alb-Cre strain to target expression to the liver. b, Immunofluorescence analysis revealed specific expression of IDH2(R172K) in CK19+ biliary cells, whereas hepatocytes were negative. c, Immunofluorescence analysis showing comparable low levels of proliferation (Ki-67) in IDH2R172K and littermate control livers (WT) at 3 months of age. Scale bars, 50 μm.

Extended Data Figure 8 The impact of HNF-4α ablation on IHCC pathogenesis in vivo.

ac, Alb-CreERT2;Hnf4afl/fl (HNF-4αfl/fl;Cre) mice were treated with DEN on postnatal day 15, and subsequently administered tamoxifen (TAM) or corn oil (CO) control at 8 months of age as described20. DEN is activated to its carcinogenic form by cytochrome P450 (CYP) enzymes, including CYP2E1, specifically in hepatocytes37,38. Livers were harvested 2 months later and analysed by H&E staining, immunofluorescence and immunohistochemistry. a, b, In HNF-4αfl/fl;Cre + TAM livers, HCC arises from HNF-4α+ cells that have escaped HNF-4α ablation (a), while CK19+ IHCC stains negative for HNF-4α (b, right). b, Left, normal liver from control corn-oil-treated livers is shown. c, Representative H&E staining (top) and immunofluorescence images (bottom) show marked expansion of SOX9+ oval cells in TAM-injected mice. Surrounding these oval cells are HNF-4α+ hepatocytes that escaped Hnf4a ablation in TAM-injected mice. d, H&E stain confirming that the liver tumour arising in a KrasG12D (Alb-Cre;LSL-KrasG12D) mouse is an HCC. e, Measurement of 2HG in liver tumours from IDH2R172K;KrasG12D (Alb-Cre;LSL-IDH2R172K;LSL-KrasG12D) mice compared to normal liver from control mice (N = 2). Scale bars, 50 μm. Error bars show ± s.d. *P < 0.05, Student’s t-test.

Extended Data Figure 9 Expression of mutant IDH2 in the IDH2R172K genetically engineered mouse model.

a, b, Immunofluorescence analysis to characterize expression of IDH2 in the liver of IDH2R172K;KrasG12D compound mice. a, Staining with an antibody that recognizes both endogenous wild-type and mutant IDH2 shows that endogenous IDH2 is expressed in the bile duct in non-diseased liver from KrasG12D mice (top). In IDH2R172K;KrasG12D mice, total IDH2 staining is at near endogenous levels in the normal bile ducts (middle) and in the biliary intraepithelial neoplasia lesions (bottom). b, Immunofluorescence using an antibody specific to IDH2(R172K) showing specific expression of the R172K transgene in CK19+ bile ducts (middle) and biliary intraepithelial neoplasias (bottom) in IDH2R172K;KrasG12D animals. Scale bars, 50 μm.

Extended Data Figure 10 Stem cell features in IDH-mutant murine and human IHCC pathogenesis.

a, IDH2(R172K) expression using mutant-specific antibodies in livers from IDH2R172K and IDH2R172K;KrasG12D compound mice showing specific expression of the R172K transgene in SOX9+ oval cells and IHCC. Scale bars, 50 μm. b, Normalized enrichment plot of a cohort of 127 human IHCC samples genotyped for IDH status, showing that the subset of tumours with IDH1/IDH2 mutations exhibits enrichment of a gene signature36 that identifies IHCCs with hepatic stem cell/progenitor features.

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Saha, S., Parachoniak, C., Ghanta, K. et al. Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer. Nature 513, 110–114 (2014). https://doi.org/10.1038/nature13441

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