Issue No. 8, August 2017
VCU Institute of Molecular Medicine (VIMM) NEWS & VIEWS
The VIMM, established in 2008 by Paul B. Fisher, M.Ph., Ph.D., the Founding Director, is comprised of outstanding scientists/clinicians from VCU School of Medicine focusing on important medical-related research in cancer, neurodegeneration and infectious diseases. The purpose of this NEWS & VIEWS is to highlight the exciting research being performed by the VIMM members.          

Astrocyte elevated gene-1 (AEG-1) Regulates Nonalcoholic Steatohepatitis (NASH)

  • Obesity is a health problem of epidemic proportion. One consequence of obesity is fatty liver disease (NASH), which functions as a risk factor for primary liver cancer (hepatocellular carcinoma, HCC). With the increase in obesity, HCC incidence is increasing at an alarming rate in the USA. Identifying key molecules regulating NASH will facilitate development of targeted intervention strategies for NASH as well as NASH-HCC.
  • A transgenic mouse with hepatocyte-specific overexpression of AEG-1 (Alb/AEG-1) developed spontaneous NASH.
  • A hepatocyte-specific conditional AEG-1 knockout mouse was protected from high fat diet (HFD)-induced NASH.
  • In-depth molecular analyses revealed that inhibition of PPAR-alpha activity results in decreased fatty acid beta-oxidation, augmentation of translation of fatty acid synthase resulting in de novo lipogenesis, and increased NF-kappaB-mediated inflammation, which act in concert to mediate AEG-1-induced NASH.
  • A hepatocyte-specific nanoparticle delivering AEG-1 siRNA (PAMAM-AEG-1si) inhibited AEG-1 and protected wild-type mice from HFD-induced NASH indicating that AEG-1 inhibitory strategies might be developed as a potential therapeutic intervention in NASH patients.

Targeted inhibition of AEG-1 is a potential strategy for therapeutic intervention of NASH

Non-alcoholic fatty liver disease (NAFLD), the most common cause of chronic liver disease in the Western world leading to cirrhosis and hepatocellular carcinoma (HCC), is characterized by initial accumulation of triglycerides (TG) in hepatocytes (hepatic steatosis) with subsequent non-alcoholic steatohepatitis (NASH). The pathogenesis of NASH includes an initial metabolic disturbance that increases the influx of free fatty acids (FFA) and de novo lipogenesis, followed by inflammation, oxidative stress and lipid peroxidation. The molecular players regulating these events are gradually being identified leading to evaluation of multiple strategies in clinical trials. However, an optimum therapy is yet to be established, indicating that a more comprehensive understanding of the molecular mechanism of NASH is urgently needed to develop effective therapeutic strategies.

The oncogene Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and LYRIC, is a pleiotropic protein with a diverse array of functions. AEG-1 is fundamentally required for the activation of the NF-kappaB pathway, a pivotal determinant of inflammation, and AEG-1 regulates NF-kappaB at multiple levels. Lipopolysaccharide (LPS)-induced NF-kappaB activation is markedly abrogated in AEG-1 knockout (AEG-1KO) hepatocytes and macrophages and AEG-1KO mice show resistance to aging-associated inflammation. An essential component of AEG-1 function is its ability to interact with and inhibit the function of retinoid X receptor (RXR). RXR heterodimerizes with nuclear receptors, including Liver X Receptor (LXR), Peroxisome Proliferator Activated Receptor (PPAR) and Farnesoid X Receptor (FXR), and regulates corresponding ligand-dependent gene transcription. Cholesterol metabolites, fatty acid derivatives and bile acids serve as endogenous ligands for LXR, PPAR and FXR, respectively, which play an important role in regulating lipid metabolism, hence NASH.

In this paper, we demonstrate that transgenic mice with hepatocyte-specific overexpression of AEG-1 (Alb/AEG-1) develop spontaneous NASH and hepatocyte-specific conditional AEG-1 knockout mice are resistant to high fat diet-induced NASH, and that human NASH patients show overexpression of AEG-1. We demonstrate that AEG-1 promotes NASH by regulating the function of nuclear receptors, mainly PPAR-alpha, promoting translation of lipogenic enzymes and activating inflammation. We also document inhibition of AEG-1 as a potential strategy to protect from NASH. These findings identify AEG-1 as a novel regulator of NASH onset and progression and suggest that AEG-1 inhibition in the liver might serve as an effective interventional therapeutic to halt NASH progression.

This investigation was a fruitful collaboration between the laboratories and colleagues of Devanand Sarkar, MBBS, PhD, Aliasger K. Salem, PhD, Yidong Chen, PhD, Mikhail Dozmorov, PhD, Paul B. Fisher, MPh, PhD, Jolene J. Windle, PhD, and Arun J. Sanyal, MD. Jyoti Srivastava and Chadia L. Robertson, postdoctoral fellows in Dr. Sarkar’s laboratory at VCU are co-first authors of this paper, and performed much of the biological and animal work. The nanoparticles were developed in the laboratory of Dr. Salem at Holden Comprehensive Cancer Center, University of Iowa, the mouse models were generated in collaboration with VCU transgenic/knockout mouse core directed by Dr. Windle, and Next Generation Sequencing analyses were performed by the laboratories of Dr. Chen in the University of Texas Health Science Center San Antonio and Dr. Dozmorov at VCU. This research was supported by NIH/NCI Grant R01 CA138540 and R21 CA183954 and NIH/NIDDK grant R01 DK107451 to Dr. Sarkar, an NCI Cancer Center Support Grant to the VCU Massey Cancer Center (MCC) P30 CA016059, and an NIH/NIDDK fellowship to Dr. Robertson (T32 DK007150). The study was accepted in Hepatology on April 24*.
Figure legend: A. Alb/AEG-1 mice develop NASH. H&E, IHC staining with the indicated antibodies and picrosirius red staining in FFPE sections of livers of 6 months old male littermates. B. Hepatocyte-specific conditional AEG-1 knockout mice are resistant to HFD-induced NASH. H&E, IHC staining with the indicated antibodies and picrosirius red staining in FFPE sections of livers of AEG-1fl/fl and AEG-1 knockout littermates fed HFD for 22 wks. C. PAMAM-AEG-1si protects from HFD-induced steatosis. H & E and oil red O staining and IHC for AEG-1, F4/80 and Collagen I of liver sections at the end of the study. D. Cartoon depicting the molecular mechanism by which AEG-1 is induced in NASH and AEG-1 promotes NASH. (Srivastava and Robertson et al., Hepatology. 2017 Aug;66(2):466-480. PMCID: PMC5519412).

*Srivastava J, Robertson CL, Ebeid K, Dozmorov M, Rajasekaran D, Mendoza R, Siddiq A, Akiel MA, Jariwala N, Shen XN, Windle JJ, Subler MA, Mukhopadhyay ND, Giashuddin S, Ghosh S, Lai Z, Chen Y, Fisher PB, Salem AK, Sanyal AJ, Sarkar D. A novel role of astrocyte elevated gene-1 (AEG-1) in regulating nonalcoholic steatohepatitis (NASH). Hepatology. 2017 Aug;66(2):466-480. PMCID: PMC5519412

Robertson CL, Srivastava J, Siddiq A, Gredler R, Emdad L, Rajasekaran D, Akiel M, Shen XN, Corwin F, Sundaresan G, Zweit J, Croniger C, Gao X, Ghosh S, Hylemon PB, Subler MA, Windle JJ, Fisher PB, Sarkar D. Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis. J Biol Chem. 2015, 290:18227-36. PMCID: PMC4505065

Rajasekaran D, Srivastava J, Ebeid K, Gredler R, Akiel MA, Jariwala N, Robertson CL, Shen XN, Siddiq A, Fisher PB, Salem AK, Sarkar D. Combination of nanoparticle-delivered siRNA for Astrocyte elevated gene-1 (AEG-1) and all-trans retinoic acid (ATRA): an effective therapeutic strategy for hepatocellular carcinoma (HCC). Bioconjug. Chem. 2015, 26:1651-1661. PMCID: PMC4783168

Robertson CL, Srivastava J, Siddiq A, Gredler R, Emdad L, Rajasekaran D, Akiel M, Shen XN, Guo C, Giashuddin S, Wang XY, Ghosh S, Subler MA, Windle JJ, Fisher PB, Sarkar D. Genetic deletion of AEG-1 prevents hepatocarcinogenesis. Cancer Res. 2014, 74:6184-93. PMCID: PMC4216744

Srivastava J, Robertson CL, Rajasekaran D, Gredler R, Siddiq A, Emdad L, Mukhopadhyay ND, Ghosh S, Hylemon PB, Gil G, Shah K, Bhere D, Subler MA, Windle JJ, Fisher PB, Sarkar D. AEG-1 regulates retinoid X receptor and inhibits retinoid signaling. Cancer Res. 2014, 74:4364-77. PMCID: PMC4135401

Srivastava J, Siddiq A, Emdad L, Santhekadur PK, Chen D, Gredler R, Shen XN, Robertson CL, Dumur CI, Hylemon PB, Mukhopadhyay ND, Bhere D, Shah K, Ahmad R, Giashuddin S, Stafflinger J, Subler MA, Windle JJ, Fisher PB, Sarkar D. Astrocyte elevated gene-1 promotes hepatocarcinogenesis: novel insights from a mouse model. Hepatology. 2012, 56:1782-91. PMCID: PMC3449036
About the Investigators

Devanand Sarkar, MBBS, PhD, is an Associate Professor in Human and Molecular Genetics (HMG), Virginia Commonwealth University, School of Medicine, Richmond, VA, Associate Scientific Director of Therapeutics in the VIMM, Associate Director of Education and Training in the VCU Massey Cancer Center (MCC), and a Harrison Foundation Distinguished Professor in Cancer Research in the VCU MCC. Aliasger K. Salem, PhD, is the Bighley Chair of Pharmaceutical Sciences, Head of Division of Pharmaceutics and Translational Therapeutics, and Professor of Pharmaceutics, Chemical and Biochemical Engineering, Biomedical Engineering and Dentistry at the University of Iowa. Yidong Chen, PhD, is Professor of Epidemiology and Biostatistics at the University of Texas Health Science Center San Antonio. Zhao Lai, PhD, is the Director of Genome Sequencing Facility in the Greehey Children’s Cancer Research Institute at the University of Texas Health Science Center San Antonio. Shah Giashuddin, MD, is the Chief of Anatomic Pathology at the New York Presbyterian Brooklyn Methodist Hospital. Shobha Ghosh, PhD, is Professor of Internal Medicine and Associate Chair for Research, VCU School of Medicine. Jolene J. Windle, PhD, is the Irene Shaw Grigg Professor of Genetics, HMG, VCU and Director, VCU Transgenic/Knockout Mouse Core. Mikhail Dozmorov, PhD, is an Assistant Professor and Nitai D. Mukhopadhyay is an Associate Professor in the Department of Biostatistics, VCU. Mark A. Subler is an Assistant Professor in HMG, VCU. Arun J. Sanyal, MD, is a Professor in the Division of Gastroenterology, Department of Internal Medicine, VCU School of Medicine. Paul B. Fisher, MPh, PhD, is Professor and Chair of HMG, VCU, Director of the VIMM and Thelma Newmeyer Corman Chair in Cancer Research in the VCU MCC. Jyoti Srivastava, PhD, Chadia L. Robertson, PhD, and Devaraja Rajasekaran, PhD, were postdoctoral fellows in Dr. Sarkar’s laboraotory. Maaged A. Akiel, PhD, was a graduate student and Nidhi Jariwala, MS, is a current graduate student in Dr. Sarkar’s laboratory. Kareem Ebeid, MS, is a graduate student in Dr. Salem’s laboratory. Rachel Mendoza, BS, Ayesha Siddiq, PhD, and Xue-Ning Shen, MD, are members of the laboratories of Drs. Sarkar and Fisher performing animal studies.