Fig 1: ICCs originate from Traf3/Pten-deficient hepatocytes. Liver phenotypes were examined in WT mice, hepatocyte-specific Pten and Traf3 DKO (HDKO) mice, cholangiocyte-specific Pten and Traf3 DKO (CDKO) mice, and mice with hepatocyte-specific Pten and Traf3 knockout following the administration of AAV8-thyroxine binding globulin Cre (AAVDKO) or pEF-iCre through HTVi (HTViDKO). (A) Hematoxylin and eosin and KRT19 staining of HDKO or CDKO and WT mice at 12 weeks of age. Scale bars, 50 µm. (B) Gene expression levels in HDKO and WT mice at 12 weeks of age. n = 3 per group, *p < 0.05. (C) Macro images of HDKO and CDKO mice at 24 weeks of age. (D) Tumor penetrance (>10 mm) of HDKO mice and WT mice at 24 weeks of age. (E) Hematoxylin and eosin staining and KRT19 staining of HDKO mice at 24 weeks of age. Scale bars, 50 µm. (F) Gene expression levels in tumors from HDKO mice and livers of WT mice. n = 3 per group, *p < 0.05. (G) X-gal and KRT19 staining of HDKO and WT mice at 12 weeks of age. Scale bars, 50 µm. (H) Macro image of WT and AAV DKO mice. (I) Tumor penetrance (>10 mm) of AAV DKO mice and WT mice. (J) Hematoxylin and eosin and KRT19 staining of AAV DKO mice. Scale bars, 50 µm. (K) Macro image of WT and HTVi DKO mice. (L) Tumor penetrance (>10 mm) of HTVi DKO mice. (M) Hematoxylin and eosin and KRT19 staining of HTVi DKO mice. Scale bars, 50 µm. (N,O) Gene expression levels in AAV DKO tumors and WT mice (N) and HTVi DKO tumors and WT mice (O), n = 3 per group, *p < 0.05. HE, hematoxylin and eosin
Fig 2: Dysregulation of the TRAF3–NIK axis is associated with advanced stage and poor prognosis of human ICC. (A) Representative images of ICCs with low TRAF3 expression and high NIK expression (upper panels) and ICCs with high TRAF3 expression and low NIK expression (lower panels). (B) Correlation between TRAF3 and NIK staining intensity in tumor tissue of patients with ICC. Scale bars, 50 µm. DFS (C) and OS (D) based on TRAF3 staining intensity in patients with ICC. DFS (E) and OS (F) based on NIK staining intensity in patients with ICC. HE, hematoxylin and eosin; NT, nontumor; T, tumor
Fig 3: Single-cell RNA sequencing reveals the transdifferentiation of Traf3/Pten-deficient hepatocytes into cholangiocytes. (A–C) t-SNE plot of hepatocyte-specific Pten and Traf3 DKO mice and WT mice. (A) Each cluster of the t-SNE plot. (B) WT-derived cells (blue dots) and DKO-derived cells (red dots). (C) General markers based on principal component analysis clustering. Low expression intensity in yellow and high in red. (D) t-SNE plots and monocle pseudotime analysis. Dots are pseudotime, with blue indicating less time elapsed and yellow indicating more time elapsed. Apoa1, apolipoprotein A1; Ass1, argininosuccinate synthase 1; Spp1, secreted phosphoprotein 1
Fig 4: Inactivation of TRAF3/PTEN induces the transdifferentiation of hepatocytes into proliferative cholangiocytes through NIK activation. Liver phenotypes were examined in WT mice, HDKO mice, mice with liver-specific Pten and Traf3 double knockouts (DKO), and mice with liver-specific Pten and Traf3 double knockout following NIKSMI treatment twice a day at a dose of 200 mg/kg. Cell phenotypes were examined in hepatocytes derived from GFP-expressing lentivirus-infected organoids (GFP Hep); CRE-expressing lentivirus-infected organoids (CRE Hep); HepG2 cells or PHHs with knockdown of PTEN and TRAF3 or of PTEN, TRAF3, and MAP3K14; or control siRNA, HepG2 cells, or PHHs with knockdown of PTEN and TRAF3 treated with NIKSMI at a concentration of 9 µM. (A,B) Gene expression levels in HDKO mice at 2 weeks (n = 3) (A) and at 12 weeks (n = 4) (B) after TAM administration, *p < 0.05. (C) Gene expression levels in GFP Hep and CRE Hep. n = 3 per group, *p < 0.05. (D) Gene expression levels in HepG2 (left) or PHHs (right) with control or with knockdown of PTEN and TRAF3. n = 4 per group, *p < 0.05. (E) Western blot in HepG2 cells or PHHs with control or with knockdown of PTEN and TRAF3. (F) Gene expression levels in HepG2 (left) or PHHs (right) with control, with knockdown of PTEN and TRAF3, and with knockdown of PTEN and TRAF3 treated with NIKSMI at a concentration of 9 µM. n = 4 per group, *p < 0.05. (G) Western blot in HepG2 cells (left) or PHHs (right) with control, with knockdown of PTEN and TRAF3, and with knockdown of PTEN and TRAF3 treated with NIKSMI at a concentration of 9 µM. (H) Gene expression levels in HepG2 (left) or PHHs (right) with control or with knockdown of PTEN and TRAF3 or of PTEN, TRAF3, and MAP3K14. n = 4 per group, *p < 0.05. (I) Western blot in HepG2 (left) or PHHs (right) with control or with knockdown of PTEN and TRAF3 or of PTEN, TRAF3, and MAP3K14. (J,K) Cell growth curve of HepG2 with control or with knockdown of PTEN and TRAF3 or of PTEN, TRAF3, and MAP3K14 (J) or with knockdown of PTEN and TRAF3 or of PTEN, TRAF3, and MAP3K14 (K) analyzed by IncuCyte, n = 8 per group, *p < 0.05. (L) Hematoxylin and eosin and KRT19 staining of DKO mice and DKO NIKSMI mice. Scale bars, 50 µm. (M) Gene expression levels in DKO mice and DKO NIKSMI mice. n = 5 per group, *p < 0.05. ACTB, actin beta; HE, hematoxylin and eosin; NC, negative control; PTKD#1 and PTKD#2, phenotypes examined in HepG2 or PHHs with knockdown of PTEN and TRAF3, respectively
Fig 5: Liver-specific inactivation of Traf3 induces cholangiocyte proliferation and promotes ICC development. (A) Hematoxylin and eosin and KRT19 staining of the liver of liver-SB/Pten mice. Scale bars, 50 µm. (B) Trunk driver genes identified in ICC and cHCC-ICC of liver-SB/Pten mice. Transposon insertion sites with read counts =50 were selected, and 10 trunk drivers were identified. (C) Gene map showing the location of transposon insertions in the Traf3 locus. Each arrowhead indicates a single transposon insertion event, and the direction of the arrowhead denotes sense or antisense orientation. (D–I) Liver phenotypes were examined in WT mice, liver-specific Pten knockout mice, liver-specific Traf3 knockout mice, and liver-specific Pten and Traf3 DKO mice. (D) Macro images of the liver at 6 weeks of age. (E) Hematoxylin and eosin staining and KRT19 staining of the liver at 6 weeks of age. Scale bars, 50 µm. (F) KRT19 liver expression levels at 6 weeks of age, n = 4–8 per group, *p < 0.05. (G) Macro images of the liver at 24 weeks of age. (H) Tumor penetrance (>10 mm) at 24 weeks of age. (I) Hematoxylin and eosin and KRT19 staining of the liver at 24 weeks of age. Scale bars, 50 µm. (J–M) Liver phenotypes were examined in WT mice, liver-specific Kras-mutant (KRAS) mice, and Kras-mutant and Traf3 knockout (KRASTRAF3) mice. (J) Hematoxylin and eosin staining and KRT19 staining of the liver at 6 weeks of age. Scale bars, 50 µm. (K) KRT19 liver expression levels at 6 weeks of age, n = 3–4 per group, *p < 0.05. (L) Tumor penetrance (>10 mm) at 24 weeks of age. (M) Hematoxylin and eosin and KRT19 staining of the liver at 24 weeks of age. Scale bars, 50 µm. HE, hematoxylin and eosin; KO, knockout
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