Tonicity-responsive enhancer-binding protein promotes hepatocellular carcinogenesis, recurrence and metastasis

Figures

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  Figure 1. Hepatic TonEBP expression is elevated in HCC and associated with postoperative recurrence and death in patients with HCC. (A–D) Hepatic TonEBP expression in a mouse model of HCC. (A) TonEBP mRNA levels in tumour-free normal tissues from PBS-injected animals (n=8) and non-tumour and tumours from DEN-injected animals (n=18). Mean+SEM, *P<0.05. (B) Immunoblots of non-tumour and tumours from seven animals. (C) Immunohistochemical images of TonEBP (brown) in hepatic tissues from PBS-injected or DEN-injected animals. Nuclei were counterstained with haematoxylin (blue). (D) Tumour number and TonEBP mRNA in non-tumour in individual animals were plotted (n=12). (E–G) TonEBP expression in hepatic tissues of patients with HCC. (E) Representative immunohistochemical images of TonEBP in hepatic biopsies from patients with HCC. (F) TonEBP mRNA in non-tumour and tumour was measured from patients with HBV- (n=23), HCV- (n=7) and alcohol-associated HCC (n=8). Tumour TonEBP expression of each patient was normalised to its non-tumour region TonEBP expression. Mean+SEM, *P<0.05 compared with corresponding non-tumour. (G) Non-tumour TonEBP mRNA was measured from patients who had recurrence within 2…

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  Figure 2. TonEBP haplodeficiency is resistant to DEN-induced hepatocarcinogenesis. TonEBP+/Δ mice and their TonEBP+/+ littermates were treated with PBS or DEN as in figure 1. (A) Non-tumour regions adjacent to tumours were immunoblotted for TonEBP and Hsc70 from DEN-injected animals. (B) Representative liver images from DEN-injected animals. (C) Tumour number, maximal size of tumours and liver weight from PBS-injected (n=8 for each genotype) or DEN-injected mice (n=18 for each genotype). Mean+SEM. #P<0.05 compared with corresponding PBS-injected animals. *P<0.05 compared with DEN-injected TonEBP+/Δ animals. (D) Representative H&E images of hepatic tissues from DEN-injected animals. Magnified images on the right are from small boxes on the left. Boxes are in tumours (top and bottom of (i), top of (ii) and (iii)), dysplastic nodule (bottom of (ii)) or tumour-free area (bottom of (iii)). (E) real time polymerase chain reaction (RT-qPCR) analyses of TonEBP, inflammatory genes (COX-2, TNFα, IL-1β, IL-6, MCP-1), macrophage marker (F4/80), proliferation markers (Ki-67, cyclin D1) and angiogenic factors (HIF-1α, VEGF) in non-tumour regions adjacent to tumours in animals from (C). (F)…

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  Figure 3. Promotion of hepatocarcinogenesis by HFD is tempered by TonEBP haplodeficiency. Animals were injected with DEN as in figure 2, followed by feeding with ND or HFD for 30 weeks (online supplementary figure S7). (A) Non-tumour regions adjacent to tumours were immunoblotted for TonEBP and Hsc70 in TonEBP+/+ animals. (B) Representative liver images. (C) Top: tumour number, maximal tumour size and maximal tumour weight from individual animals fed with ND (n=12 for each genotype) or HFD (n=15 for each genotype). Mean+SEM, *P<0.05 compared with corresponding TonEBP+/+. #P<0.05 compared with corresponding ND. Bottom: tumours larger than 3 mm in diameter from representative individual animals are shown. (D) Representative images of H&E stained liver tissues from animals fed with HFD. (E) RT-qPCR analyses as in figure 2E. (F) Correlation of TonEBP mRNA expression with mRNA expression of COX-2, TNFα and MCP-1 in non-tumour areas of hepatic tissues from TonEBP+/+ animals fed with ND (n=10) and HFD (n=10). COX-2, cyclo-oxygenase-2; DEN, diethylnitrosamine; HFD, high-fat diet; MCP-1, monocyte chemoattractant protein 1; mRNA, messenger RNA; ND, normal diet; TNFα, tumour necrosis factor alpha;…

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  Figure 4. Liver injury and inflammation are tempered by hepatocyte-specific TonEBP deficiency. (A–D) TonEBPfl/fl; Albumin cre+/− mice (HKO) and their TonEBPfl/fl; Albumin cre−/− (WT) littermates were injected with DEN or PBS. Livers and serum samples were analysed 48 hours later. (A) Livers from the WT animals were immunoblotted for TonEBP and Hsc70. (B) Serum ALT levels in PBS-treated (n=5) and DEN-treated animals (n=8). Mean+SEM. *P<0.05 compared with WT. (C) TUNEL signal in hepatic tissues. (D) RT-qPCR analyses of TonEBP, inflammatory genes (COX-2, TNFα, IL-1β, IL-6) and proliferation markers (Ki-67, cyclin D1) in livers of DEN-treated animals. mRNA abundance was expressed relative to WT group. P, PBS; D, DEN. Mean+SEM, #P<0.05 compared with corresponding P. *P<0.05 compared with WT. (E) Immunoblotting of liver samples, three from each genotype, for TonEBP, PCNA, Hsc70, caspase-3, C-Casp-3, COX-2 and iNOS. (F,G) HKO and WT animals were injected with PBS or LPS (5 µg/kg) in combination with D-galactosamine (400 mg/kg) (LPS). The animals were analysed 4 hour later. (F) Serum levels of ALT and AST. (G) RT-qPCR analysis mRNA levels. mRNA abundance was expressed relative to WT, PBS group.…

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  Figure 5. TonEBP-dependent stimulation of COX-2 requires transcription factor YY1. (A) TonEBP versus COX-2 mRNA abundance in tumours from 25 patients with HCC. (B) Immunohistochemical images of COX-2 and TonEBP in consecutive hepatic sections from two representative patients with HCC. (C) Immunoblots of livers from TonEBP+/Δ (+/Δ) and their TonEBP+/+ littermates (+/+) from figure 2A. (D) Immunoblots of HepG2 cells transfected with siRNA followed by treatment with IL-1β. (E) Serum PGE2 levels in PBS-treated (n=5) or DEN-treated mice (n=8 for each genotype) from figure 2. Mean+SEM. *P<0.05 compared with TonEBP +/+. #P<0.05 compared with PBS-injected mice. Serum PGE2 levels in mean+SEM. *P<0.05 compared with DEN-injected TonEBP+/Δ animals. (F) Lysates of HepG2 cells treated with IL-1β or not (con) as indicated were immunoprecipitated (IP) with normal IgG, anti-YY1 IgG (YY1), normal serum (S) or anti-TonEBP serum (T). The immunoprecipitates and cell lysates were immunoblotted as indicated. (G) HepG2 cells were transfected with siRNA indicated followed by transfection with pGL3 (vector), COX-2 (COX-2 promoter reporter in pGL3) or ΔYY1 (COX-2 whose YY1 binding site was disabled by mutagenesis)…

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  Figure 6. TonEBP and YY1 interact via RHD and spacer. (A) Diagrams of TonEBP and YY1 and their deletion constructs. (B–E) Lysates from HEK293 cells transfected with Myc-TonEBP plus Flag-YY1 (B) or various recombinant fragment of Flag-YY1 (C), Myc-Yc1 plus or Flag-YY1 or Flag-YY1-ΔS (D) or Flag-YY1 plus Myc-TonEBP or Myc-TonEBP-ΔRHD (E) were immunoprecipitated with normal IgG, anti-Flag IgG or anti-Myc IgG as indicated. The immunoprecipitates (upper) and lysates (bottom) were immunoblotted as indicated. (F) Model of TonEBP actions in the pathogenesis of HCC. First, TonEBP mediates oxidative stress-induced cell injury and local inflammation. Second, TonEBP expression is enhanced by oxidative stress. On the promoter of the COX-2 gene, the elevated TonEBP recruits the acetyltransferase p300 to YY1 in a manner dependent on IL-1β. TonEBP promotes tumourigenesis, recurrence and metastasis via PGE2 and possibly other pathways (broken line). See text for details. COX-2, cyclo-oxygenase-2; Flag, anti-Flag IgG; GA rich, glycine-alanin rich; GK rich, glycine-lysine rich; HCC, hepatocellular carcinoma; IB, immunoblot; IgG, normal IgG, Myc, anti-Myc IgG; PGE2, prostaglandin E2; IL, interleukin; IP,…