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  • Accepted Manuscript

    Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

    Saireudee Chaturantabut, Arkadi Shwartz, Kimberley J. Evason, Andrew G. Cox, Kyle Labella, Arnout G. Schepers, Song Yang, Marianna Aravena, Yariv Houvras, Liliana Mancio-Silva, Shannon Romano, Daniel A. Gorelick, David E. Cohen, Leonard I. Zon, Sangeeta N. Bhatia, Trista E. North, Wolfram Goessling

    PII: S0016-5085(19)30031-9 DOI: https://doi.org/10.1053/j.gastro.2019.01.010 Reference: YGAST 62366

    To appear in: Gastroenterology Accepted Date: 7 January 2019

    Please cite this article as: Chaturantabut S, Shwartz A, Evason KJ, Cox AG, Labella K, Schepers AG, Yang S, Aravena M, Houvras Y, Mancio-Silva L, Romano S, Gorelick DA, Cohen DE, Zon LI, Bhatia SN, North TE, Goessling W, Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes, Gastroenterology (2019), doi: https://doi.org/10.1053/j.gastro.2019.01.010.

    This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

    https://doi.org/10.1053/j.gastro.2019.01.010 https://doi.org/10.1053/j.gastro.2019.01.010

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    GPER1 mTOR

    PI3K-AKT

    Growth Regeneration Cancer

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    Title: Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Short Title: GPER1 enhances liver growth Authors: Saireudee Chaturantabut1, Arkadi Shwartz1, Kimberley J. Evason2, Andrew G. Cox1,3, Kyle Labella1, Arnout G. Schepers4, Song Yang5, Marianna Aravena6, Yariv Houvras7, Liliana Mancio-Silva4, Shannon Romano8, Daniel A. Gorelick8, David E. Cohen6, Leonard I. Zon5,9,10,11, Sangeeta N. Bhatia4,12,13, Trista E. North5,10, Wolfram Goessling1,10,11,12,13,14. 1Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. 2Department of Pathology, University of Utah, Salt Lake City, UT 84112, USA 3Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.. 4Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. 5Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA. 6Division of Gastroenterology & Hepatology, Weill Cornell Medical College, New York, NY 10021, USA. 7Departments of Surgery and Medicine, Weill Cornell Medical College, New York, New York 10021, USA. 8Department of Pharmacology & Toxicology, University of Alabama at Birmingham, Birmingham, AL 35293, USA. 9 Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MA 20815, USA. 10Harvard Stem Cell Institute, Cambridge, MA 02138, USA. 11Dana-Farber Cancer Institute, Boston, MA 02215, USA. 12Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, 02139, USA. 13Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 14Divison of Gastroenterology, Massachusetts General Hospital, Boston, MA 02114 Grant Support: This study is supported by NIH NCI 5K08CA172288 (K.J.E.), R24OD017870 (L.I.Z.; W.G), R01DK090311, R01DK105198 (W.G.) and The Claudia Adams Barr Program in Cancer Research (W.G.). W.G. is a Pew Scholar in the Biomedical Sciences. Abbreviations: ANAS, anastrozole; ANT, adjacent normal tissue; Akt, protein kinase B; BPA, bisphenol A; BrdU, bromodeoxyuridine; CC, cholangiocarcinoma; DAPI, 4′,6- diamidino-2-phenylindole; DMBA, dimethylbenzanthracene; DMSO, dimethysulfoxide; EdU, ethynyldeoxyuridine; ERE, estrogen response element; ERK, extracellular signal- regulated kinases; E2, 17β-estradiol; ESR, estrogen receptor; FACS, fluorescence- activated cell sorting; ful, fulvestrant; GO, Gene Ontology; GPER1, G protein-coupled estrogen receptor 1; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; HNF4α, hepatocyte nuclear factor 4α; hpf, hours post fertilization; hpT, hours post treatment; IACUC - Institutional Animal Care and Use Committee; IHC, immunohistochemistry; ISH, in situ hybridization; mTOR, mechanistic target of rapamycin; MO, morpholino antisense oligonucleotide; Mtz, metronidazole; PCNA, proliferating cell nuclear antigen; PI3K, phosphoinositide 3-kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TALEN, transcription activator-like effector

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    nuclease; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; WT, wild-type. Correspondence: Address correspondence to Wolfram Goessling (wolfram_goessling@hms.harvard.edu), Genetics Division, Brigham and Women’s Hospital, NRB458, 77 Ave Louis Pasteur, Boston, MA 02115, U.S.A, phone: 617-525- 7710, fax: 617-525-4751. Disclosure: The authors have no conflict of interest to disclose. Transcript Profiling: Accession number in GEO is GSE92544.

    Author Contributions: S.C. and W.G. conceived the study, designed experiments, and analyzed data. S.C. conducted all zebrafish experiments. A.S. performed confocal microscopy and Western Blots. K.J.E. acquired human tissues and performed pathological analysis of zebrafish and human liver tumors. A.G.C. analyzed metabolomics data. K.L. performed mRNA rescue, xenoestrogens, and long-term anastrozole zebrafish experiments. A.G.S. and L.M-S. performed primary human hepatocyte experiments. S.Y. and Y.H. analyzed RNA sequencing data. M.A. carried out liver cancer cell line experiments. S.R., D.A.G., D.E.C., L.I.Z., S.N.B, and T.E.N. provided overall input. S.C., T.E.N and W.G. wrote the manuscript. All authors reviewed and edited the manuscript. The authors thank Isaac Oderberg from the Goessling lab for creating the visual abstract, John Asara at the Metabolomics Core at the Beth Israel Deaconess Medical Center for assistance with the metabolomics experiments, and staff of Beth Israel Deaconess zebrafish core facility for fish husbandry.

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    Title: Estrogen Activation of G Protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Abstract: Background & Aims: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Methods: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages; liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a co- culture system. We measured levels of G protein-coupled estrogen receptor (GPER1) in HCC and non-tumor liver tissues from 68 patients by immunohistochemistry. Results: Exposure to 17 beta-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the PI3K–Akt–mTOR pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1, compared with non-tumor tissues; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. Conclusions: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. KEY WORDS: sex hormone, signal transduction, transcription regulation, hepatocarcinogenesis

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    Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality

    worldwide, and the fastest-growing cause of cancer deaths in the U.S.1 Clinically relevant

    biomarkers and therapies to detect and prevent HCC do not exist. Interestingly, chronic

    liver disease and liver cancer are much more frequent in males,2 and men with cirrhosis

    and HCC have elevated serum estrogen levels.3, 4 Further, patients und

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