Volume 150, Issue 3, Pages 720-733 (March 2016) Epithelial Transforming Growth Factor-β Signaling Does Not Contribute to Liver Fibrosis but Protects Mice From Cholangiocarcinoma  Xueru Mu, Jean-Philippe Pradere, Silvia Affò, Dianne H. Dapito, Richard Friedman, Jay H. Lefkovitch, Robert F. Schwabe  Gastroenterology  Volume 150, Issue 3, Pages 720-733 (March 2016) DOI: 10.1053/j.gastro.2015.11.039 Copyright © 2016 AGA Institute Terms and Conditions

Figure 1 Epithelial TGFβ signaling is activated in murine and human liver disease but does not contribute to toxic and biliary liver fibrosis. (A) Representative images of phospho-Smad2 staining show epithelial TGFβ pathway activation in cirrhotic human liver (n = 5). (B) Phospho-Smad2 staining shows activated TGFβ signaling in epithelial cells in CCl4-induced toxic liver injury as well as BDL- and Mdr2ko-induced biliary liver disease. (C) Dual immunofluorescent staining and subsequent confocal microscopy show phospho-Smad2 in HNF4α-positive hepatocytes and keratin-positive cholangiocytes in CCl4-treated and BDL-treated livers, respectively. (D and E) TBR2lko and TBR2f/f mice were subjected to 8 injections of CCl4 (0.5 μg/mL; TBR2lko, n = 10; TBR2f/f, n = 11) or 15 days of BDL (TBR2lko, n = 16; TBR2f/f, n = 10). Hepatic fibrosis was determined by picrosirius red staining. Hepatic injury was determined by serum alanine aminotransferase (ALT) measurement. (F) Mdr2ko mice carrying floxed TBR2 in the presence (n = 20) or absence (n = 21) of albumin-Cre were killed at age 9 weeks. Liver fibrosis and injury were assessed as described earlier. Scale bars: 100 μm (A, B, D–F), and 50 μm (C). Gastroenterology 2016 150, 720-733DOI: (10.1053/j.gastro.2015.11.039) Copyright © 2016 AGA Institute Terms and Conditions

Figure 2 Epithelial TGFβ signaling does not contribute to genotoxic hepatocarcinogenesis but protects liver from cholangiocarcinoma development in the presence of concomitant PTEN loss. (A) TBR2lko (n = 14) and TBR2f/f mice (n = 12) were injected with DEN (25 mg/kg intraperitoneally) on day 15 postpartum and killed 10 months later. Tumor number, tumor size, and liver–body weight ratio were compared between TBR2lko and TBR2f/f mice (middle graphs). mRNA expression of Afp, Cd133, and mKi67 was determined by qPCR (right graph). (B–E) In contrast to PTENlko mice (n = 9), TBR2 PTENldko mice (n = 12) developed keratin-positive tumors with typical features of cholangiocarcinoma and increased mortality. (F) mRNA expression of markers for cholangiocytes, progenitor cells, biliary injury, and proliferation was determined by qPCR in tumors from PTENlko and TBR2 PTENlko mice. Scale bars: 100 mm (A and B) and 100 μm (C). *P < .05, **P < .01, ***P < .001. Scale bars should be 100mm (A and B) and 100um (C). Gastroenterology 2016 150, 720-733DOI: (10.1053/j.gastro.2015.11.039) Copyright © 2016 AGA Institute Terms and Conditions

Figure 3 Loss of TGFβ signaling promotes cholangiocyte but not hepatocyte proliferation. PTENlko mice and TBR2 PTENldko mice were killed at the age of 2 months. (A) Cholangiocyte expansion in PTENlko and TBR2 PTENldko mice was assessed by keratin immunohistochemistry. (B) Hepatocyte proliferation was compared between PTENlko and TBR2 PTENldko mice by dual immunofluorescent staining for HNF4α and Ki-67 in combination with confocal microscopy. (C) Hepatocyte proliferation was compared between PTENlko and TBR2 PTENldko mice by dual immunofluorescent staining for HNF4α and Ki-67 in combination with confocal microscopy. (D) mRNA expression of progenitor and biliary injury markers was compared between PTENlko and TBR2 PTENldko mice by qPCR. Scale bars: 100 μm (A and C) and 200 μm (B). *P < .05, **P < .01, ***P < .001. Gastroenterology 2016 150, 720-733DOI: (10.1053/j.gastro.2015.11.039) Copyright © 2016 AGA Institute Terms and Conditions

Figure 4 Concomitant loss of TBR2 and PTEN in cholangiocytes promotes cholangiocarcinoma development. (A) Quadruple transgenic mice expressing Prom1-CreERT2 PTENf/f, and mTom/mGFP or Prom1-CreERT2 TBR2f/f, PTENf/f, and mTom/mGFP were treated with DDC diet and tamoxifen as indicated. Two months after treatment initiation, TBR2 PTENΔChol/Prom (n = 4) but not PTENΔChol/Prom (n = 4) mice showed multiple GFP-positive hepatic tumors. (B) Tumors from TBR2 PTENΔChol/Prom mice showed typical features of cholangiocarcinoma as shown by H&E staining and immunohistochemical staining for keratin. (C) Cholangiocyte proliferation was compared between PTENΔChol/Prom and TBR2 PTENΔChol/Prom mice using dual immunofluorescent staining for Ki-67 and GFP, and confocal microscopy. (D) Mice expressing K19-CreERT and PTENf/f (PTENΔChol/K19) (n = 8), or K19-CreERT, TBR2f/f and PTENf/f (PTEN TBR2ΔChol/K19) (n = 9) were treated with DDC diet and tamoxifen as described earlier. Nine weeks after treatment initiation, PTEN TBR2ΔChol/K19 but not PTENΔChol/K19 mice showed hepatic tumors. (E) Tumors from TBR2 PTENΔChol/K19 mice showed typical features of cholangiocarcinoma seen by H&E staining and immunohistochemistry for keratin. (F) Cholangiocyte proliferation was compared between PTENΔChol/K19 and TBR2 PTENΔChol/K19 mice using dual immunofluorescent staining for Ki-67 and keratin 19 and confocal microscopy. Scale bars: 100 mm (A and D) and 100 μm (B, C, E and F). *P < .05. Gastroenterology 2016 150, 720-733DOI: (10.1053/j.gastro.2015.11.039) Copyright © 2016 AGA Institute Terms and Conditions

Figure 5 Concomitant loss of TBR2 and PTEN in hepatocytes promotes cholangiocarcinoma development. (A) PTENf/f (n = 6) and double transgenic mice expressing TBR2f/f and PTENf/f (n = 12) were infected with AAV8-TBG-Cre (1 × 1011 genome copies/mouse, intravenously) for hepatocyte-specific ablation of PTEN (PTENΔHep) or PTEN and TBR2 (TBR2 PTENΔHep), and killed 10 months later. (B) Tumors developed from PTENΔHep and TBR2 PTENΔHep mice, albeit with different macroscopic appearance. (C) H&E and immunohistochemistry for keratin showed cholangiocarcinoma development in TBR2 PTENΔHep but not in PTENΔHep mice. (D) Tumor number was quantified in PTENΔHep and TBR2 PTENΔHep mice. (E) The presence of α-SMA–positive cancer-associated fibroblasts in cholangiocarcinoma from TBR2 PTENΔHep mice was determined by dual immunohistochemistry for K19 (marking tumor cells) and α−SMA (marking cancer-associated fibroblasts). (F) Gene expression of progenitor cell/cholangiocyte (Krt19, Krt7, Cd133, Spp1, and Afp), biliary injury (Muc1, Tff2, and Sprr2a), and cholangiocarcinoma markers (Gprc5a, Ehf, Cldn7, Reg1, Gabrp, Anxa10, Dmbt1, Sema3c) was compared between tumors from PTENΔHep and TBR2 PTENΔHep mice, using qPCR. Scale bars: 100 mm (B), 100 μm (C and E). *P < .05, **P < .01. Gastroenterology 2016 150, 720-733DOI: (10.1053/j.gastro.2015.11.039) Copyright © 2016 AGA Institute Terms and Conditions

Figure 6 Comparison of cholangiocarcinoma developing in mice with hepatocyte-specific deletion of PTEN and TBR2 to human cholangiocarcinoma. (A) Microarray from paired human nontumor liver tissues (n = 23), human intrahepatic cholangiocarcinoma (ICC, n = 23), normal mouse liver (n = 3), liver tumors arising in PTENΔHep mice (n = 4), and liver tumors arising in TBR2 PTENΔHep mice (n = 4). Displayed is a heatmap of all genes with a least a 4-fold and significant (false discovery rate < 0.05) up-regulation in human cholangiocarcinoma vs human normal liver, showing clustering of human ICC and cholangiocarcinoma from TBR2PTENΔHep mice. (B) Comparison of cholangiocarcinoma from TBR2PTENΔHep mice (n = 4) with human ICC (n = 136) showed clustering with the proliferation subclass. Gastroenterology 2016 150, 720-733DOI: (10.1053/j.gastro.2015.11.039) Copyright © 2016 AGA Institute Terms and Conditions