Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS

Abstract

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

Keywords: JNK; Kupffer cell; Tnf; cholastasis; intrahepatic cholangiocarcinoma; mitochondrial dysfunction; pro-inflammatory niche; reactive oxygen species; unfolded protein response.

Figure 1.. Lack of Hspd1 Causes Acute Liver Damage, Hepatic Proliferation, and Cholangiocellular Tumorigenesis

(A) Electron microscopy and 8-OHdG staining of WT and Hspd1^ΔLPC livers. Arrowhead indicates a classic mitophagosome. Scale bars, 0.5 μm (upper), 50 μm (lower). (B) Six- and 8-week-old Hspd1^ΔLPC mice and WT littermates. (C) Survival curve of Hspd1^ΔLPC and WT mice. (D) Eight-week-old Hspd1^ΔLPC and WT littermate livers. Scale bar, 5 mm. (E) Serum from 8-week-old Hspd1^ΔLPC mice and WT littermates. Scale bar, 1 cm. (F) Upper: Ki67 staining of 6-week-old Hspd1^ΔLPC and WT livers. Black arrowheads, Ki67⁺ cholangiocytes; white arrowheads, Ki67⁺ hepatocytes. Lower: double staining of Hspd1 (red) and Ki67 (brown) of 8-week-old Hspd1^ΔLPC and WT livers, indicating foci of hepatocyte (white arrowhead) and cholangiocellular (black arrowhead) proliferation. Scale bar, 50 μm. (G) Quantification of cholangiocellular lesions and Ki67⁺ hepatocytes over time in Hspd1^ΔLPC mice and WT littermates. (H) H&E staining of Hspd1^ΔLPC liver. Arrowheads indicate biliary intraepithelial neoplasia. Scale bar, 100 μm. (I) IHC analysis in 8-week-old WT and Hspd1^ΔLPC livers. Scale bar, 50 μm. (J) Genomic hybridization profiles of CK19⁺ cholangiocellular neoplasia. (K) Macroscopy (left) and CK19 staining (right) of tumor graft from severe combined immunodeficiency Beige liver. Scale bars, 5 mm (left), 100 μm (right). (L) Quantification of cholangiocellular lesions over time in AAV8-Cre Hspd1^loxP/loxP and control mice. (M) H&E and CK19 staining of AAV8-Cre Hspd1^loxP/loxP livers. Scale bar, 100 μm. Data are represented as the mean ± SEM. *p < 0.05, ***p < 0.001. ns, not significant. See also Figures S1 and S2.

Figure 2.. Oxidative and Pro-carcinogenic Liver Microenvironment in Hspd1^ΔLPC Mice

(A) Heatmap of the expression of anti-oxidant genes in Hspd1^ΔLPC livers compared with WT over time. Red, upregulated; green, downregulated. (B) Hspd1 and 8-OHdG staining of 8-week-old Hspd1^ΔLPC and WT livers. Scale bar, 50 μm. (C) Western blot analysis of Hspd1^ΔLPC and WT livers from the indicated ages. (D) Western blot analysis of Hspd1^ΔLPC and WT livers from the indicated ages. (E) qRT-PCR for the top upregulated pro-survival genes in Hspd1^ΔLPC liver compared with 8-week-old WT liver. Data are represented as the mean ± SEM. (F) Hspd1 and c-Myc IHC in consecutive liver sections from 8-week-old WT and Hspd1^ΔLPC mice. The dashed lines indicate the border between the Hspd1⁺ and Hspd1⁻ areas. Scale bar, 20 μm. See also Figure S3.

Figure 3.. Anti-oxidants Attenuate ROS Accumulation and Premalignant Cholangiocellular Lesions in Hspd1^ΔLPC Mice

(A) Survival plots for Hspd1^ΔLPC mice on normal or BHA diet. (B) Macroscopy of the liver of 6- and 8-week-old Hspd1^ΔLPC or 8-week-old WT mice on normal or BHA diet. Scale bar, 1 cm. (C) H&E of the liver of 8-week-old Hspd1^ΔLPC mice on normal versus BHA diet and quantification of necrotic areas. Scale bar, 100 μm. (D) Serum ALT, AST, and bilirubin in 8-week-old Hspd1^ΔLPC mice on normal and BHA diet. (E) A6 IHC in 8-week-old normal versus BHA diet Hspd1^ΔLPC livers and quantification of cholangiocellular overgrowth. Scale bar, 20 μm. (F) qRT-PCR of livers from 8- and 20-week-old normal versus BHA diet Hspd1^ΔLPC mice for CK19 and Hnf4α. (G and H) Western blot of liver lysates from 8-week-old normal versus BHA diet Hspd1^ΔLPC mice (G) and quantifications the p-JNK to total JNK and the p-Erk1/2 to total Erk1/2 ratio (H). (I) IHC for p-JNK and CHOP in consecutive liver sections from normal versus BHA diet 8-week-old Hspd1^ΔLPC mice. Scale bar, 50 μm. (J) qRT-PCR of livers from 8- and 20-week-old normal versus BHA diet Hspd1^ΔLPC mice for genes indicated. (K) A schematic depicting the action of BHA in Hspd1^ΔLPC mice. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. See also Figure S4.

Figure 4.. Kupffer Cells Promote Cholangiocellular Tumorigenesis

(A) Left: schematic of key signals regulating hepato- or cholangiocellular proliferation. Right: qRT-PCR of Hspd1^ΔLPC livers at different ages for the expression of key ligands. (B and C) Tnf ELISA (B) and qRT-PCR (C) of WT and normal versus BHA diet 8-week-old Hspd1^ΔLPC livers. (D) Tnf and F4/80 IHC in consecutive sections of 8-week-old Hspd1^ΔLPC and WT livers. Scale bar, 20 μm. (E) Immunofluorescence (IF) of Tnfr1 and CK19 in 8-week-old Hspd1^ΔLPC and WT livers. Scale bar, 20 μm. (F) CK19 IHC and Tnfr1 in situ hybridization in consecutive sections from 8-week-old Hspd1^ΔLPC and WT livers. The dashed lines indicate the border between bilary cells and hepatocytes. Scale bar, 20 μm. (G) Upper: timeline of clodronate administration on Hspd1^ΔLPC mice. Lower: survival plot for Hspd1^ΔLPC mice treated with mock or clodronate. (H) F4/80 and CK19 IHC in consecutive sections from 8-week-old Hspd1^ΔLPC livers treated with mock or clodronate. Quantification of each staining was shown in the right panels. Scale bar, 50 μm. (I) Tnf ELISA from 8-week-old Hspd1^ΔLPC livers treated with mock or clodronate. (J) Quantification of Ki67⁺ hepatocytes in Hspd1^ΔLPC livers treated with mock or clodronate. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. See also Figure S5.

Figure 5.. Tnfr1 Signaling Promotes Premalignant Cholangiocellular Lesions

(A) Cell proliferation of WT or Tnfr1^−/− cholangiocytes was analyzed by bromodeoxyuridine (BrdU) incorporation. (B) Hepatoblasts were kept in basal medium or basal medium supplemented with DMSO or Tnf. IF of A6 and Hnf4α was performed. Scale bar, 20 μm. (C) Survival analysis for Hspd1^ΔLPC, Hspd1^ΔLPC Tnfr1^+/−, and Hspd1^ΔLPC Tnfr1^−/− mice. (D) Macroscopic appearance of 8-week-old Hspd1^ΔLPC, Hspd1^ΔLPC Tnfr1^+/−, and Hspd1^ΔLPC Tnfr1^−/− livers. Scale bar, 1 cm. (E and F) H&E, A6 and Ki67 IHC, and Tnfr1 in situ hybridization of 8-week-old WT, Hspd1^ΔLPC, and Hspd1^ΔLPC Tnfr1^−/− livers (E), as well as quantification of necrosis and cholangiocellular overgrowth (F). The dashed lines indicate the border between bilary cells and hepatocytes. Scale bar, 50 μm. (G) IHC of 8-week-old Hspd1^ΔLPC and Hspd1^ΔLPC Tnfr1^−/− livers. Scale bar, 20 μm. (H) qRT-PCR of 6- and 8-week-old WT, Hspd1^ΔLPC, and Hspd1^ΔLPC Tnfr1^−/− livers for indicated genes. (I) Percentage of Ki67⁺ hepatocytes in WT, Hspd1^ΔLPC, and Hspd1^ΔLPC Tnfr1^−/− livers. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. See also Figure S5.

Figure 6.. JNK/c-Jun Activation Is Required for Premalignant Cholangiocellular Lesions

(A) p-JNK, p-Stat3, and Rela IHC in 8-week-old WT and Hspd1^ΔLPC livers. The black arrowhead indicates a p-JNK⁺ cholangiocyte, the white arrowhead indicates a p-JNK⁺ hepatocyte. Black arrows indicate positive staining in non-parenchymal cells, while white arrows indicate negative staining in liver parenchymal cells. Scale bar, 20 μm. (B) Rela and c-Jun IF in hepatoblasts cultured in basal medium supplemented with DMSO or Tnf. White arrowheads indicate c-Jun nuclear staining. Scale bar, 20 μm. (C) Timeline of SP600125 administration on Hspd1^ΔLPC mice and survival of Hspd1^ΔLPC mice not treated (mock) or treated with SP600125. (D) CK19 and Ki67 IHC in 8-week-old Hspd1^ΔLPC livers treated with mock or SP600125, and quantification of cholangiolar cancerous lesion areas and Ki67⁺ cholangiocytes relative to total cholangiocytes. Scale bar, 50 μm. (E) c-Jun IF in hepatoblasts cultured in Tnf-containing medium with or without SP600125. White arrowheads indicate the c-Jun nuclear staining. Scale bar, 20 μm. (F and G) Phase contrast and A6 and Hnf4α IF images (F) and qRT-PCR (G) of hepatoblasts in basal or Tnf-containing medium with or without SP600125. Scale bar, 20 μm. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. See also Figure S6.

Figure 7.. Kupffer Cell-Derived Tnf/JNK Axis Is Activated in Different ICC Models

(A) IHC of CK19 and p-JNK in liver sections from four ICC models as indicated. The dashed lines indicate the border between ICC and the surrounding liver tissues. Scale bar, 100 μm. (B) F4/80 and Tnf IHC in consecutive sections from CRISPR/cas9-mediated multiplex mutagenesis livers. Scale bar, 20 μm. (C–F) Survival plot (C and E) and representative macroscopic view of ICC and quantification of tumor incidence (D and F) for JNK1/2^ΔLPC and control C57BL/6 mice in HDTV-Akt/Notch-induced (C and D) or HDTV-p53/Kras-induced (E and F) ICC model. Scale bar, 1 cm. (G) H&E and IHC staining of Ki67 and p-JNK in livers as indicated in (C and E). Scale bar, 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. See also Figure S7.

Figure 8.. Tnf and p-JKN Are Strongly Increased in ICC Patient Specimens

(A) H&E and IHC staining of Tnf, p-JNK, and 8-OHdG in a representative human ICC sample. Scale bar, 50 μm. (B and C) Representative IHC images and quantification of Tnf (B) and p-JNK (C) in human ICC and HCC. Arrowheads indicate positive staining. Scale bars, 100 μm (left), 20 μm (right). (D) Model depicting a non-cell-autonomous mechanism of hepatocyte mitochondrial dysfunction in regulating cholangiocellular tumorigenesis. ***p < 0.001. ns, not significant. See also Figure S8.