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NFκB1 Is a Suppressor of Neutrophil-Driven Hepatocellular Carcinoma

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NFκB1 Is a Suppressor of Neutrophil-Driven Hepatocellular Carcinoma

C L Wilson et al. Nat Commun.

Erratum in

  • Corrigendum: NFκB1 is a suppressor of neutrophil-driven hepatocellular carcinoma.
    Wilson CL, Jurk D, Fullard N, Banks P, Page A, Luli S, Elsharkawy AM, Gieling RG, Bagchi Chakraborty J, Fox C, Richardson C, Callaghan K, Blair GE, Fox N, Lagnado A, Passos JF, Moore AJ, Smith GR, Tiniakos DG, Mann J, Oakley F, Mann DA. Wilson CL, et al. Nat Commun. 2015 Sep 21;6:8411. doi: 10.1038/ncomms9411. Nat Commun. 2015. PMID: 26387912 Free PMC article. No abstract available.

Abstract

Hepatocellular carcinoma (HCC) develops on the background of chronic hepatitis. Leukocytes found within the HCC microenvironment are implicated as regulators of tumour growth. We show that diethylnitrosamine (DEN)-induced murine HCC is attenuated by antibody-mediated depletion of hepatic neutrophils, the latter stimulating hepatocellular ROS and telomere DNA damage. We additionally report a previously unappreciated tumour suppressor function for hepatocellular nfkb1 operating via p50:p50 dimers and the co-repressor HDAC1. These anti-inflammatory proteins combine to transcriptionally repress hepatic expression of a S100A8/9, CXCL1 and CXCL2 neutrophil chemokine network. Loss of nfkb1 promotes ageing-associated chronic liver disease (CLD), characterized by steatosis, neutrophillia, fibrosis, hepatocyte telomere damage and HCC. Nfkb1(S340A/S340A)mice carrying a mutation designed to selectively disrupt p50:p50:HDAC1 complexes are more susceptible to HCC; by contrast, mice lacking S100A9 express reduced neutrophil chemokines and are protected from HCC. Inhibiting neutrophil accumulation in CLD or targeting their tumour-promoting activities may offer therapeutic opportunities in HCC.

Figures

Figure 1
Figure 1. Neutrophils are required for experimentally induced HCC.
(a) Graph shows average total number of hepatic neutrophils ( NIMP-R14 ) in DEN-injured WT or nfkb1−/− mice. Red line denotes basal neutrophil levels in normal WT liver. (b) Average tumour counts and liver/body weight ratio in 30- and 40-week DEN-injured WT and nfkb1−/− mice. Representative pictures of livers from 40-week DEN-injured WT and nfkb1−/− mice. (c) Average total number of PCNA+ hepatocytes in DEN-injured WT and nfkb1−/− mice. (d) Diagram showing the experimental plan to deplete neutrophils (Ly6G antibody) in WT mice from week 32–40 after DEN injury. Average tumour counts, representative pictures of livers and average liver/body weight ratio graph in 40-week DEN-injured WT mice treated with control IgG or Ly6G antibody (Ab) for 8 weeks. n=10 (e) Representative pictures of liver from 20 month (aged under normal conditions) WT and nfkb1−/− mice. Representative photomicrographs of haematoxylin and eosin (H&E)- and Sirius red/fast green-stained liver sections from 20-month nfkb1−/− mice (upper panel) revealed steatohepatitis (fat, blue arrows; inflammation, yellow arrows; ballooned hepatocytes with Mallory–Denk bodies, green arrows) and fibrosis (black arrows). Focal dysplasia (yellow dotted line) with focal inflammation (black arrow) and HCC in H&E-stained nfkb1−/− aged livers (lower panel). Graphs show average liver/body weight ratio and tumour frequency identified histologically using H&E sections from 20-month, aged WT and nfkb1−/− mice. n=7 WT and ten nfkb1−/− mice. (f) Diagram showing the neutrophil depletion experimental protocol in nfkb1−/− mice from week 22–30 after DEN injury. Average tumour counts, representative pictures of livers and graph showing average liver/body weight ratio from 30-week DEN-injured nfkb1−/− mice treated with control IgG or Ly6G for 8 weeks (n=10). All data are means±s.e.m; scale bars, 200 μm. For a and c, n=6, 4, 6, 7, 15 and 9 (nfkb1−/−), and 4, 4, 4, 5, 11 and 14 (WT) for the 5- to 40-week time points). Statistical significance was determined using one-way analysis of variance with Tukey's post-hoc test (a,c) or an unpaired t-test (b,e), *P<0.05, **P<0.01 or ***P<0.001 compared with control.
Figure 2
Figure 2. Dysregulation of hepatic chemokine expression and accelerated HCC in the nfkb1−/− mouse.
(a) Diagram of the experimental plan using whole-body imaging to track WT neutrophils to the liver of acute DEN-injured WT or nfkb1−/− mice. (b) Representative IVIS pictures of mice given NIR815-labelled WT neutrophils intravenously, showing neutrophils tracking to livers of acute DEN-injured WT or nfkb1−/− mice. Graph shows average radians from IVIS-imaged WT or nfkb1−/− mice. (c) Representative ex vivo images of the liver (left), kidney (middle) and the spleen (right), and graph showing average radians from WT or nfkb1−/− livers imaged ex vivo. n=3 (d) Hepatic CXCL1, CXCL2, S100A9 and tumour necrosis factor-α (TNFα) mRNA levels expressed as relative level of transcription difference (RLTD) compared with WT 48 h post acute DEN in WT and nfkb1−/− mice, n=6. (e) Graph shows RLTD of S100A9 in bone marrow macrophages (BMM) compared with hepatocytes isolated from WT and nfkb1−/− mice, n=3. (f) Hepatocyte CXCL1 and CXCL2 mRNA levels expressed as RLTD in nfkb1−/− mice compared with WT, n=3. (g) Graph shows average total number of NIMP-R14 cells in liver sections from WT or nfkb1−/− mice after acute DEN injury-treated±IgG or CXCL1 and two neutralizing antibody, n=5. (h) Average tumour counts, representative pictures of livers and graph showing average liver/body weight ratio from 40-week DEN-injured WT and s100a9−/− mice, n=19. Hepatic CXCL1 and CXCL2 mRNA levels expressed as RLTD in 40-week, chronic, DEN-injured WT and s100a9−/−mice, n=6. Data are means±s.e.m. Statistical significance was determined using an unpaired t-test, *P<0.05, **P<0.01 or ***P<0.001 compared with control.
Figure 3
Figure 3. p50 homodimers complexed with HDAC1 regulate the chemokines CXCL1 and 2.
(a,b) ChIP assay analysis of p50 (a) and HDAC1 (b) recruitment to the CXCL1, CXCL2 and S100A9 promoters in acute DEN-injured WT or nfkb1−/− livers. (c) Model showing repression of the CXCL1, CXCL2 and S100A9 promoters by p50:p50 homodimers complexed with HDAC1. (d) Sequence alignment of nfkb1 gene shows conservation of amino acid serine 340 (in mouse) throughout evolution from purple sea urchin through to human. The conserved serine is shown in bold and red. (e) Sequence alignment of the mouse NF-κB subunits, show conservation of serine 340 (in mouse) throughout all subunits (shown in bold and red) and conservation of serine 337 in NF-κB1, RelA and cRel (shown in bold and blue). (f) X-ray crystal structure of the mouse p50:p50 homodimer (cyan) bound to DNA (deep blue), PDB entry 1NFK. White arrows show serine 340, which is rendered in space-filling representation. (g) Representative western blots of anti-Flag or anti-RelA after Flag IP on lysates from Cos-7 cells transfected with RelA-eGFP±Flag-p50 or Flag-p50 mutant shows that p50 mutants retain interaction with RelA. (h) Western blots for anti-HA or anti-Flag after Flag IP on lysates from Cos-7 cells transfected with Flag-p50 or Flag-p50 mutant±HA-p50 reveals that p50 mutants bind p50, except p50 S342A (human equivalent of mouse 340). Data are means±s.e.m. and representative of five mice per group. Statistical significance was determined using an unpaired t-test, **P<0.01 or ***P<0.001 compared with control.
Figure 4
Figure 4. nfkb1S340A knock-in mice have increased neutrophils and tumour burden in experimentally induced HCC.
(a) Representative photomicrographs of NIMP-R14 staining and graph showing average total number of neutrophils in liver sections from WT or nfkb1S340A mice following acute DEN injury. Black arrows denote NIMP-R14 stained cells, n=5 (b) Hepatic CXCL1, CXCL2 and S100A9 mRNA 48 h post acute DEN in WT and nfkb1S340A mice. n=5 (c) Representative pictures of livers, average tumour counts and liver/body weight ratio in 40-week DEN-injured WT and nfkb1S340A mice. (d) Graphs show average total number of PCNA+ stained cells and representative photomicrographs at × 200 magnification of PCNA staining in WT and nfkb1S340A 40-week DEN-injured livers, black arrows denote PCNA+stained cells, n=6 (e) Graph shows NIMP-R14 cells in liver sections from 40-week DEN-injured WT and nfkb1S340A mice, n=6. Representative photomicrographs at × 200 magnification of NIMP-R14 staining in WT and nfkb1S340A 40-week DEN-injured livers, black arrows denote NIMP-R14 stained cells. Data are means±s.e.m.; all scale bars, 100 μm. Statistical significance was determined using an unpaired t-test, *P<0.05 or ***P<0.001 compared with control.
Figure 5
Figure 5. Neutrophils promote hepatocellular telomere DNA damage.
(a) Representative photomicrographs at × 200 magnification of 8-OHG staining in liver sections from cirrhotic livers and HCC, black arrows denote brown positively stained areas of damage in hepatocytes and red arrows show inflammatory cells. Scale bars, 100 μm . (b) Representative deconvolved maximum intensity projections of telomere FISH and phospho-H2A.X (γH2A.X) staining in alcoholic liver disease (ALD) liver sections (n=2 normal human liver and n=4 ALD). Graph shows per cent TAF+ hepatocytes in ALD liver compared with normal control liver. (b,c) Graphs (i–ii) show the fluorescence intensity and co-localization of telomere FISH and γH2A.X staining of the corresponding single plane images. Scale bars, 3 μm . (c) Representative immuno-FISH images (deconvolved maximum intensity projections) from 30-week DEN-injured nfkb1−/− livers, n=5. Graph per cent TAF TAF+ hepatocytes in WT versus nfkb1−/− 30-week DEN livers. (d) Graph shows per cent TAF+ hepatocytes in 30-week DEN-injured nfkb1−/− mice treated with control IgG or Ly6G antibody for 8 weeks, n=10. All data are means±s.e.m. TAF analysis, a minimum of 50 cells per liver counted for human sections and a minimum of 80 cells counted per mouse section). Statistical significance was determined using an unpaired t-test, **P<0.01 or ***P<0.001 compared with control.
Figure 6
Figure 6. Neutrophil-dependent bystander ROS and hepatocellular cancer is limited by anti-oxidants.
(a,b) Graph shows average total number of HNE- (a) and γH2A.X- (b) positive cells in liver sections from WT or nfkb1−/− mice 5–40 weeks post DEN injury (n=6, 4, 6, 7, 15, 9 (nfkb1−/−) and 4, 4, 4, 5, 11, 14 (WT) for the 5- to 40-week time points, respectively). (a) Representative photomicrographs at × 200 magnification show hepatic 4HNE staining in 40-week DEN-injured WT or nfkb1−/− mice, black arrows denote 4HNE+ hepatocytes; scale bars, 100 μm. (c) Diagram showing either direct (top) or indirect (bottom) transwell co-culture of WT hepatocytes and WT neutrophils. (d) Graphs show mean fluorescence intensity of ROX and dihydroehidium (DHE; ROS production) in WT hepatocytes cultured±WT neutrophils n=3. (e) Representative immunocytochemistry images and cell counts of DAPI/53BP1-stained WT hepatocytes only or WT hepatocytes in either direct or indirect (transwell) co-culture with WT neutrophils n=3; scale bars, 10 μm. (f) Diagram of BHA therapy in 30-week DEN-injured nfkb1−/− mice. Representative liver pictures and average tumour counts in 30-week DEN-injured nfkb1−/− mice±15 weeks dietary supplementation with BHA (n=5 control diet and 8 BHA diet). (g,h) Graphs show average HNE (g) and γH2A.X (h) counts and representative photomicrographs of liver sections from 30-week DEN-injured nfkb1−/− mice±BHA. Black arrows denote 4HNE+ hepatocytes (g) or γH2A.X+ hepatocytes (h); scale bars, 100 μm. All data are means±s.e.m. Statistical significance was determined using an unpaired t-test, *P<0.05, **P<0.01 or ***P<0.001 compared with control.

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