EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation

Abstract

Hepatocellular carcinoma (HCC) is a frequent cancer with limited treatment options and poor prognosis. Tumorigenesis has been linked with macrophage-mediated chronic inflammation and diverse signalling pathways, including the epidermal growth factor receptor (EGFR) pathway. The precise role of EGFR in HCC is unknown, and EGFR inhibitors have shown disappointing clinical results. Here we discover that EGFR is expressed in liver macrophages in both human HCC and in a mouse HCC model. Mice lacking EGFR in macrophages show impaired hepatocarcinogenesis, whereas mice lacking EGFR in hepatocytes unexpectedly develop more HCC owing to increased hepatocyte damage and compensatory proliferation. Mechanistically, following interleukin-1 stimulation, EGFR is required in liver macrophages to transcriptionally induce interleukin-6, which triggers hepatocyte proliferation and HCC. Importantly, the presence of EGFR-positive liver macrophages in HCC patients is associated with poor survival. This study demonstrates a tumour-promoting mechanism for EGFR in non-tumour cells, which could lead to more effective precision medicine strategies.

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Figure 1: HCC formation in mice lacking EGFR in hepatocytes or all liver cells.
Figure 2: EGFR expression in Kupffer cells/liver macrophages promotes HCC development.
Figure 3: EGFR expression is induced in activated Kupffer cells/liver macrophages under pathological conditions.
Figure 4: EGFR expression in Kupffer cells of HCC patients correlates with poor prognosis.
Figure 5: EGFR-dependent IL-6 production and release.

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Acknowledgements

We are grateful to L. Bakiri, D. P. Barlow, R. Eferl, M. Oft and E. F. Wagner for critical reading of the manuscript. We thank T. Baykuscheva-Gentscheva and S. Bardakji for genotyping and F. Hucke and G. Heinze for statistical support. This work was supported by the EC programme LSHC-CT-2006-037731 (Growthstop), the Austrian Science Fund (FWF) grants SFB F3518-B20 (to M.S.), F3517 (to M.T.), FWF-DK W1212 and P25925 and the Austrian Federal Government’s GEN-AU program ‘Austromouse’ (GZ 200.147/1-VI/1a/2006 and 820966). H.W. acknowledges funding by the National Natural Science Foundation of China, 30921006.

Author information

A.N. designed, carried out and analysed in vivo tumour experiments with the EGFRΔhep, EGFRΔMx and EGFRΔMx mice. H.L. designed, carried out and analysed in vitro experiments and some western blot analysis and carried out in vivo tumour experiments with EGFRΔhep/Δmac and EGFRΔmac mice. K.K. carried out in vivo analyses with EGFRΔhep/Δmac and EGFRΔmac mice and in vitro analyses with Kupffer cells including western blot analysis. N.A. helped with histology, immunohistochemistry and immunofluorescence. S.K.W. helped with qRT-PCRs, M.H. helped with histology, mouse colony and animal experiments. L.L. and L.C. carried out stainings on all human samples (Chinese and European cohorts) and analysed the Chinese cohort with the supervision of H.W. W.S. analysed the European cohort together with M.T. and M.P-R. R.Z. and M.S. wrote the manuscript with input from H.L., A.N., N.A., W.S., M.T., M.P-R. and H.W. and with major contributions during the revision phase from K.K. M.S. conceived and supervised the whole project.

Correspondence to Maria Sibilia.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 HCC induction and liver damage in mice and hepatocytes.

(a) Tumour development was initiated in male mice of the indicated genotypes by DEN injection at 4 weeks of age (black arrow). At 8 weeks of age tumours were promoted by a diet complemented with phenobarbital (PB) until mice were sacrificed. Top: EGFRΔhep mice (EGFRf/f; Alfp-Cre). Middle: EGFRΔMx mice (EGFRf/f; Mx-Cre) that received 3 pIpC injections with 2 days intervals at 7 weeks of age to delete EGFR in the liver (grey arrow). Bottom: EGFRΔMx mice (EGFRf/f; Mx-Cre), that received pIpC injections on day 9, 11, and 13 (grey arrow) after birth. (b) Southern Blot analysis showing EGFR deletion in EGFRΔMx (left) and EGFRΔhep (right, non-recombined EGFR allele from non-parenchymal cells) livers (f: floxed EGFR allele (6 kb), Δ: Cre-deleted EGFR allele (3.9 kb). (c) Ki67-positive (left, EGFRf/f, n = 45 (six mice); EGFRΔMx, n = 48 (7 mice)) and TUNEL-positive cells (right, EGFRf/f, n = 56 (six mice); EGFRΔMx: n = 55 (six mice)) in adjacent non-tumour tissue. n = HPF. (d) Representative H&E staining of livers 0, 36, 48, and 72 h after DEN intoxication in vivo. Black arrows indicate necrotic areas. Scale bar, 500 μm. (e,f) Alanine transaminase (ALT, 0 h and 24 h: n = 5 mice per genotype, 48 h: EGFRf/f and EGFRΔMx: n = 4, EGFRΔhepn = 5 mice) and Aspartate transaminase (AST, 0 h: EGFRf/f: n = 3, EGFRΔhep: n = 4, EGFRΔMx: n = 4, 24 h: EGFRf/f: n = 3, EGFRΔhep, n = 3, EGFRΔMx: n = 4, 48 h: EGFRf/f: n = 4, EGFRΔhep, n = 4, EGFRΔMx: n = 3 mice) measured in serum 0, 24, and 48 h after DEN intoxication. (g) Representative active caspase-3 staining (Alexa 488, green) and nuclei (DAPI, blue) of EGFRf/f and EGFRΔhep liver sections 24 and 96 h after DEN intoxication, showing increased apoptosis in EGFR-deficient livers after 96 h. Scale bar indicates 500 μm. (h) HMGB1 staining of cultured EGFRf/f and EGFRΔMx hepatocytes 12 h after DEN treatment in vitro. Nuclei (DAPI, blue), actin (Phalloidin, red), HMGB1 (Alexa 488, green). Scale bar indicates 100 μm. (i) Active caspase-3 staining of cultured hepatocytes of EGFRf/f and EGFRΔMx mice incubated with TNFα/CHX for 12 h. Nuclei (DAPI, blue), actin (Phalloidin, red), active caspase-3 (Alexa 488, green). Scale bar indicates 50 μm. (c) Data represent mean ± s.e.m. (e,f) Data represent mean ± s.d. Student’s t-test for independent samples and unequal variances was used to assess statistical significance (P < 0.05,P < 0.01,P < 0.001). Original data are provided in Supplementary Table 1.

Supplementary Figure 2 EGFR deletion in EGFRΔMx mice and cytokine production on DEN injection.

(a) ELISA showing IL-1β in the supernatant of primary hepatocyte cultures 4 h after incubation with increasing amounts of DEN in vitro. (Primary hepatocyte isolates of EGFRf/f (n = 3) and EGFRΔhep (n = 3)). (b) Release of IL-1β and IL-1α to the supernatant of cultured primary hepatocytes of EGFRf/f, EGFRΔhep, and EGFRΔMx mice after incubation with TNFα quantified by ELISA. n.d. = not detectable. Result of two pooled independent experiments is shown. For each experiment hepatocytes isolated from 2 livers per genotype were pooled and analysed as 4 technical replicates (primary hepatocytes of n = 4 mice were analysed in total for each genotype). (c) Western Blot analysis of EGFR in livers of EGFRf/f and EGFRΔMx mice. (d) Representative livers of EGFRf/f and EGFRΔMx mice 46 weeks after tumour initiation. Scale bar indicates 1 cm. (e) Tumour mass (left, EGFRf/f (n = 10) and EGFRΔMx (n = 9)), area (middle, EGFRf/f (n = 7) and EGFRΔMx (n = 9)), and number (right, EGFRf/f (n = 7) and EGFRΔMx (n = 9)). Two pooled independent experiments. Data (a,b) represent mean ± s.d. Data (e) represent mean ± s.e.m. Student’s t-test for independent samples and unequal variances was used to assess statistical significance (P < 0.05, P < 0.01, P < 0.001). Original data are provided in Supplementary Table 1.

Supplementary Figure 3 EGFR expression in tumour cells and Kupffer cells of human HCCs.

(a) Representative Immunohistochemistry showing EGFR expression in tumour cells/hepatocytes of HCC. Scoring (0, +, ++, +++) was performed according to the scale described below resulting in the generation of Table 1a. Scale bar indicates 50 μm. (be) OS (b,d) and DFS (c,e) of HCC patients of the Chinese (b,c: 129 patients (n = 70 negative for EGFR; n = 59 positive for EGFR)) and European cohort (d,e: 108 patients (n = 69 negative for EGFR; n = 39 positive for EGFR)) with or without EGFR expression in hepatocytes. (f) Representative immunohistochemistry showing EGFR and CD68 staining (0, +, ++) in liver macrophages of human HCC. Scoring (0, +, ++, +++) was performed according to the scale described below resulting in the generation of Table 1b. Scale bar indicates 50 μm. (g) Representative immunofluorescent EGFR and CD68 co-staining in fresh frozen human HCC tissue (n = 12). Nuclei (DAPI, blue), CD68 (Alexa 488, green) and EGFR (Alexa 594, red), merged (bottom right). White arrows indicate double positive cells. Scale bar indicates 50 μm. Scoring system: 0 = negative staining (0%–10% positive), 1 =weak signal (10%–20% positive), 2 = intermediate signal (20%–50% positive) and 3 = strong signal (>50% positive) as previously described30. Log-rank test was used to assess statistical significance.

Supplementary Figure 4 IL-6 production by Kupffer cells after various stimuli and inhibitor treatments.

(a,b) ELISA quantifying IL-1β-induced IL-6 secretion by isolated Kupffer cells after preincubation with increasing amounts of the EGFR inhibitors Cetuximab (a) or BIBW2992 (Afatinib) (b). (n = 2 primary Kupffer cell isolates). (c) IL-6 secretion by isolated Kupffer cells following stimulation with polyIC (20 μg ml−1), imiquimod (12 μg ml−1) and LPS (10 ng ml−1). - = unstimulated. (n = 2 primary Kupffer cell isolates). (d) ELISA quantifying IL-1β- or EGF-induced IL-17A, IL-22 and IL-23 secretion by isolated Kupffer cells (n = 2 primary Kupffer cell isolates). (e,f) Representative Western Blot showing activation of the indicated proteins after 15 min stimulation with IL-1β (e) or EGF (f) in the presence of the respective inhibitors. Note: Each lane contains proteins isolated from pooling Kupffer cells of 3 different livers. Because the amount of proteins obtained from Kupffer cells from 3 pooled livers was not sufficient to perform Western blot analysis for all indicated proteins and treatments, 2 different isolates and Western blots for each series of treatment (EGF+ inhibitors and IL-1β+ inhibitors) had to be performed. (e) Blot 1: IL-1β stimulated EGFRf/f and EGFRΔMx+ inhibitors and expression of EGFR and JNK. Blot 2: IL-1β stimulated EGFRf/f and EGFRΔMx+ inhibitors and expression of p38, IKK, NF-kB, Stat3. (f) Blot 1: EGF stimulated EGFRf/f and EGFRΔMx+ inhibitors and expression of EGFR and JNK. Blot 2: EGF stimulated EGFRf/f and EGFRΔMx+ inhibitors and expression of p38, IKK, NF-kB, Stat3. The results were confirmed in a second set of isolates and Westerns. Original data are provided in Supplementary Table 1.

Supplementary Figure 5 Model of EGFR signalling in hepatocytes and Kupffer cells during HCC formation.

EGFR signalling is hepatoprotective during DEN-induced liver damage as in the absence of EGFR, hepatocytes undergo more necrosis and apoptosis thus leading to increased IL-1β production and release. IL-1β stimulation of Kupffer cells in turn leads to release of IL-6, which is required for compensatory proliferation and repair of damaged hepatocytes. IL-1β-induced IL-6 production occurs in a bimodal way involving the activation of the IL-1R/MyD88 pathway to first induce EGFR ligands and ADAM17 expression with subsequent EGFR transactivation required for IL-6 production via JNK, p38 and IKK.

Supplementary Figure 6 Uncropped images of western blots and PCR analysis.

The uncropped films and gel photographs (Fig. 3c) showing the Western blots and PCR analysis (Fig. 3c) displayed in the main figures. Boxed areas indicate the cropped regions displayed in the respective figures.

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Lanaya, H., Natarajan, A., Komposch, K. et al. EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma formation. Nat Cell Biol 16, 972–981 (2014) doi:10.1038/ncb3031

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