Author Manuscript Published OnlineFirst on August 5, 2019; DOI: 10.1158/0008-5472.CAN-18-3750 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 SNAI1 promotes the cholangiocellular phenotype, but not 2 epithelial-mesenchymal transition, in a murine hepatocellular carcinoma model 3 4 Meng Xu1,2,3,*, Jingxiao Wang3,4,*, Zhong Xu5, Rong Li6, Pan Wang3,7, Runze Shang3,8, 5 Antonio Cigliano9, Silvia Ribback10, Antonio Solinas11, Giovanni Mario. Pes 12, Katja 6 Evert9, Haichuan Wang3,13, Xinhua Song3, Shu Zhang3,14, Li Che3, Rosa Maria 7 Pascale12, Diego F. Calvisi9,10, Qingguang Liu1, Xin Chen3 8 9 1Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong 10 University, Xi'an Jiaotong University, Xi'an, PR China. 11 2Department of General Surgery, The Second Affiliated Hospital of Xi'an Jiaotong 12 University, Xi'an Jiaotong University, Xi'an, PR China 13 3Department of Bioengineering and Therapeutic Sciences and Liver Center, 14 University of California, San Francisco, CA, USA. 15 4School of Life Sciences, Beijing University of Chinese Medicine, Beijing, PR China 16 5Department of Gastroenterology, Guizhou Provincial People's Hospital, Medical 17 College of Guizhou University, Guiyang, PR China. 18 6Department of Anesthesiology, The Second Affiliated Hospital of Xi'an Jiaotong 19 University, Xi'an, PR China. 20 7Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of 21 Food Science and Nutritional Engineering. 22 8Department of Hepatobiliary Surgery, Xi'jing Hospital, Air Force Military Medical 1 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 5, 2019; DOI: 10.1158/0008-5472.CAN-18-3750 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 23 University, Xi'an, PR China 24 9Institute of Pathology, University Clinic of Regensburg, Regensburg, Germany 25 10Institute of Pathology, University of Greifswald, Greifswald, Germany 26 11Department of Biomedical Sciences, University of Sassari, Sassari, Italy 27 12Department of Medical, Surgical and Experimental Sciences, University of Sassari, 28 Sassari, Italy 29 13Liver Transplantation Division, Department of Liver Surgery, West China Hospital, 30 Sichuan University, Chengdu, PR China. 31 14Department of Radiation Oncology and Department of Head and Neck Oncology, 32 Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan 33 University, Chengdu, Sichuan, PR China. 34 35 *These authors contributed equally to the work 36 37 Running title：SNAI1 promotes cholangiocellular phenotype in vivo 38 39 Corresponding authors: 40 Diego F. Calvisi, M.D.; Institute of Pathology, University Clinic of Regensburg, 41 Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany. E-mail: 42 [email protected] 43 Qingguang Liu, M.D. Ph.D.; Department of Hepatobiliary Surgery, The First Affiliated 44 Hospital of Xi'an Jiaotong University, Xi'an 710061, PR China. Email: 2 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 5, 2019; DOI: 10.1158/0008-5472.CAN-18-3750 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 45 [email protected] 46 47 Conflicts of interest statement 48 The authors declare that they have no conflict of interest. 49 50 Key words: hepatocellular carcinoma; SNAI1; cholangiocellular phenotype; 51 epithelial-mesenchymal transition; mouse model 52 3 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 5, 2019; DOI: 10.1158/0008-5472.CAN-18-3750 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 53 Abstract 54 Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has 55 limited treatment options. Snail family transcriptional repressor 1 (SNAI1) is a master 56 regulator of epithelial-mesenchymal transition (EMT) and has been implicated in HCC 57 initiation and progression. However, the precise role of SNAI1 and the way it 58 contributes to hepatocarcinogenesis have not been investigated in depth, especially 59 in vivo. Here, we analyzed the functional relevance of SNAI1 in promoting 60 hepatocarcinogenesis in the context of the AKT/c-Met driven mouse liver tumor model 61 (AKT/c-Met/SNAI1). Overexpression of SNAI1 did not accelerate AKT/c-Met-induced 62 HCC development or induce metastasis in mice. Elevated SNAI1 expression rather 63 led to the formation of cholangiocellular (CCA) lesions in the mouse liver, a phenotype 64 that was paralleled by increased activation of Yap and Notch. Ablation of Yap strongly 65 inhibited AKT/c-Met/SNAI-induced HCC and CCA development, whereas inhibition of 66 the Notch pathway specifically blocked the CCA-like phenotype in mice. Intriguingly, 67 overexpression of SNAI1 failed to induce EMT, indicated by strong E-cadherin 68 expression and lack of vimentin expression by AKT/c-Met/SNAI tumor cells. SNAI1 69 mRNA levels strongly correlated with the expression of CCA markers including SOX9, 70 CK19 and EPCAM, but not with EMT markers such as E-cadherin and ZO-1, in 71 human HCC samples. Overall, our findings suggest SNAI1 regulates the CCA-like 72 phenotype in hepatocarcinogenesis via regulation of Yap and Notch. 73 74 Significance 4 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 5, 2019; DOI: 10.1158/0008-5472.CAN-18-3750 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 75 Findings report a new function of SNAI1 to promote cholangiocellular 76 transdifferentiation instead of epithelial-mesenchymal transition in hepatocellular 77 carcinoma. 78 79 Introduction 80 Liver cancer is one of the most common tumors and ranks second as a cause of 81 cancer mortality in the world (1, 2). Hepatocellular carcinoma (HCC) and 82 cholangiocarcinoma (CCA) are the two major histotypes of liver cancer. The diagnosis 83 for most HCC and CCA patients is achieved in the advanced stage of these tumors, 84 when only very limited treatment options are available (3, 4). Better understanding of 85 the mechanisms underlying HCC and CCA molecular pathogenesis is obviously of 86 high importance for the development of novel drugs able to treat efficiently these 87 deadly malignancies. 88 Recently, several studies have demonstrated the plasticity of liver cells. 89 Specifically, mounting evidence indicates that mature hepatocytes can transform into 90 cholangiocytes and vice versa (5-7). Besides the stem cell and cholangiocyte 91 compartments, intrahepatic CCA can derive from adult hepatocytes in mice (8, 9). 92 Studies in human HCC also identified a subset of HCC known as cholangiocellular 93 (CC) like HCC, characterized by the expression of biliary markers such as CK19 and 94 EPCAM (10, 11). In addition, mixed/combined HCC and CCA represent a liver cancer 95 entity in humans. Specifically, these mixed tumors have a prognosis similar to CCA 96 and worse than HCC. A recent genomic profile analysis indicated that mixed 5 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 5, 2019; DOI: 10.1158/0008-5472.CAN-18-3750 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 97 HCC/CCA possess molecular features similar to HCC, even in the CCA component 98 (12). The precise mechanisms underlying this conversion remain poorly understood. 99 Recent studies suggest that the Hippo effector Yap and its downstream target Notch 100 are major regulators of cell fate in the liver (13). In mice, overexpression of the Hippo 101 kinase Lats2 prevents CC-like lesions formation in mouse liver tumor models induced 102 by activated AKT and N-Ras oncogenes (14). Furthermore, activated forms of Yap 103 cooperate with AKT to promote CCA development in mice (15-17). Notch signaling 104 has been identified as a major cascade downstream of Yap. Specifically, Yap directly 105 induces the expression of Notch2 and Jag1 genes (13, 18), and ablation of Notch2 106 completely prevents Yap dependent CC formation in vivo (19). Overall, these studies 107 suggest that, during hepatocarcinogenesis, Yap/Notch signaling cascade may 108 promote a CC-like phenotype in the liver. 109 Epithelial to mesenchymal transition (EMT) is a cellular process where epithelial 110 cells lose their cell polarity and transform into mesenchymal-like cells. In tumors, EMT 111 is frequently observed and associated with increased tumor cell proliferation, invasion, 112 and metastasis (20). EMT is recognized by the gain of expression of mesenchymal 113 marker, such as Vimentin and N-cadherin, as well as loss of epithelial markers, 114 including E-Cadherin, ZO-1 (TJP1), and Occludin, in tumor cells. It is well-established 115 that Snail family transcriptional repressor 1 (SNAI1) is a master regulator of EMT 116 during tumor progression (21). It induces EMT via binding to the three E-boxes of the 117 CDH1 (E-cadherin) promoter region, leading to the suppression of CDH1 expression 118