Issue No. 21, October 2018
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 Macrophage Activation in Hepatocellular Carcinogenesis

  • Chronic inflammation is a hallmark of cancer and is central to the onset and progression of hepatocellular carcinoma (HCC). Hepatic macrophages play a critical role in the inflammatory process leading to HCC. The oncogene Astrocyte Elevated Gene-1 (AEG-1) regulates NF-B activation, and germline knockout of AEG-1 in mice (AEG-1-/-) results in resistance to inflammation and experimental HCC. Conditional hepatocyte- and myeloid cell-specific AEG-1-/- mice (AEG-1-/-HEP and AEG-1-/-MAC, respectively) were developed to dissect the role of AEG-1 in these cell types in regulating HCC.

  • HCC was induced by treatment with N-nitrosodiethylamine and phenobarbital. AEG-1-/-HEP mice exhibited a significant reduction in disease severity compared to control littermates, while AEG-1-/-MAC mice were profoundly resistant.

  • AEG-1-/- hepatocytes exhibited increased sensitivity to stress and senescence.

  • AEG-1-/- macrophages were resistant to M1 and M2 differentiation with significant inhibition in migration, endothelial adhesion and efferocytosis activity, indicating that AEG-1 ablation renders macrophages functionally anergic.

  • These results identify a central role for AEG-1 in regulating macrophage activation, and indicate that AEG-1 is required in both tumor cells and the tumor microenvironment to stimulate hepatocarcinogenesis.

Targeted inhibition of AEG-1 in both tumor cells and tumor microenvironment cells is a potential strategy for therapeutic intervention of HCC

The risk factors for HCC include viral hepatitis, alcoholism and non-alcoholic fatty liver disease, all of which lead to chronic inflammation. Liver-resident macrophages (Kupffer cells) constitute ~20% of the total cells in the liver and play a vital role in establishing a pro-inflammatory, pro-tumorigenic environment. During initial tumorigenesis, damaged hepatocytes release cytokines, such as IL-1, which stimulate Kupffer cells to activate NF-B resulting in the release of IL-6 that activates the oncogenic STAT3 signaling in the hepatocytes, thereby promoting proliferation of transformed cells. Concomitantly, tumor-associated macrophages also secrete various cytokines and chemokines, including IL-1, TNF, IL-6, CCL2 and CXCL10, which increase HCC cell proliferation and NF-B-mediated protection from HCC cell apoptosis, as well as angiogenic and growth factors, such as VEGF, PDGF, TGF and FGF, which support HCC growth. Understanding the mechanism of macrophage activation is, therefore, vital to controlling the chronic inflammatory process leading to HCC.

Astrocyte Elevated Gene-1 (AEG-1)/metadherin (MTDH) functions as a major oncogene for HCC and is highly overexpressed in HCC patients of diverse etiologies by multiple mechanisms including genomic amplification. AEG-1 knockout mice (AEG-1-/-) exhibit complete resistance to N-nitrosodiethylamine (DEN) and phenobarbital (PB)-induced HCC. Furthermore, AEG-1 ablation results in markedly reduced inflammation in mice because AEG-1 is fundamentally required for activation of NF-B, a key regulator of inflammatory process. Indeed both AEG-1-/- hepatocytes and macrophages show an inherent inability to activate NF-B upon lipopolysaccharide (LPS) treatment. Conversely, hepatocyte-specific AEG-1 transgenic mice (Alb/AEG-1) develop highly aggressive DEN-induced HCC compared to WT littermates indicating a key regulatory role of AEG-1 in HCC cells. AEG-1 expression in macrophages is markedly higher than in hepatocytes. This finding coupled with the observations that AEG-1 is required for NF-B activation and inflammation suggest that AEG-1 may be important in regulating both tumor cells and tumor-associated macrophages.

In this paper, we demonstrate that AEG-1 -/-HEP mice show partial resistance to development of experimental HCC, while AEG-1 -/-MAC mice show almost complete resistance mimicking the phenotype of AEG-1-/- mice. In vitro studies demonstrated that AEG-1 deletion renders macrophages functionally anergic, establishing a pivotal role for AEG-1 in regulating macrophage function. Thus, targeting AEG-1 in both hepatocytes and macrophages might be an effective way to combat HCC. We recently demonstrated that hepatocyte-targeted nanoparticles delivering AEG-1 siRNA profoundly inhibit growth of orthotopic xenografts of human HCC cells in nude mice. In the case of endogenous HCC, both hepatocyte- and macrophage-targeted nanoparticles delivering AEG-1 siRNA may exert a robust and sustained anti-HCC effect. Studies are in progress to evaluate this hypothesis.

This investigation was a fruitful collaboration between the laboratories of colleagues Devanand Sarkar, MBBS, PhD, Shobha Ghosh, PhD, Zhao Lai, PhD, Mikhail Dozmorov, PhD, Nitai D. Mukhopadhyay, PhD, Mark A. Subler, PhD, Jolene J. Windle, PhD, and Paul B. Fisher, MPh, PhD. Chadia L. Robertson was a postdoctoral fellow in Dr. Sarkar’s laboratory at VCU and is the first author of this paper, and performed much of the biological and animal work. The mouse models were generated in collaboration with the VCU Massey Cancer Center (MCC) Transgenic/Knock-out Mouse Shared Resource directed by Dr. Windle, and Next Generation Sequencing analyses were performed by Dr. Lai at the University of Texas Health Science Center San Antonio and Dr. Dozmorov at VCU. Dr. Mukhopadhyay performed Bayesian analysis of tumor probability. This research was supported by NIH/NIDDK grant R01 DK107451 to Dr. Sarkar, a Virginia Commonwealth University Massey Cancer Center pilot project grant (D. Sarkar and S. Ghosh), and an NCI Cancer Center Support Grant to the VCU MCC (P30 CA016059). The study was accepted in Cancer Research on September 04, 2018*.

Figure legend: A. AEG-1 -/-HEP and AEG-1 -/-MAC mice are resistant to experimental HCC. AEG-1 fl/fl , AEG-1 -/-HEP and AEG-1 -/-MAC mice (7 per group) received an initial DEN injection followed by PB treatment in drinking water. The mice were sacrificed at 32 weeks when all data points were analyzed. Representative photographs of the livers. B. Heat map of differentially expressed genes in AEG-1+/+ and AEG-1-/- bone marrow derived macrophages (BMDM), isolated from AEG-1 fl/fl and AEG-1 -/-MAC mice, respectively. C. Upstream regulators inhibited in AEG-1-/- BMDM. p-value was <10 -7 thus approaching 0 in the graph. Inflammatory regulators are inhibited in AEG-1-/- BMDM. D. Representative images of adherent AEG-1+/+ and AEG-1-/- Kupffer cells to liver sinusoidal endothelial cells (LSEC). Magnification: 400x. Scale bar: 20 m m. (Robertson et al., Cancer Res, 2018).
Publications:
* Robertson CL, Mendoza RG, Jariwala N, Dozmorov M, Mukhopadhyay ND, Subler MA, Windle JJ, Lai Z, Fisher PB, Ghosh S, Sarkar D. Astrocyte elevated gene-1 (AEG-1) regulates macrophage activation in hepatocellular carcinogenesis. Cancer Res. 2018, Sep 4. pii: canres.0659.2018. DOI: 10.1158/0008-5472.CAN-18-0659 .

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, Siddiq A, Gredler R, Shen X-N, Rajasekaran D, Robertson CL, Subler MA, Windle JJ, Dumur CI, Mukhopadhyay ND, Garcia D, Lai Z, Chen Y, Balaji U, Fisher PB, Sarkar D. Astrocyte elevated gene-1 and c-Myc cooperate to promote hepatocarcinogenesis in mice. Hepatology. 2015, 61: 915-929. PMCID: PMC4309751 .

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 a Professor in the Department of Human and Molecular Genetics (HMG), Virginia Commonwealth University (VCU) 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. Shobha Ghosh, PhD, is Professor and Associate Chair for Research in the Department of Internal Medicine, VCU School of Medicine. Zhao Lai, PhD, is the Director of the Genome Sequencing Facility at the Greehey Children’s Cancer Research Institute at the University of Texas Health Science Center San Antonio. 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. 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. Chadia L. Robertson, PhD was a postdoctoral fellow, Nidhi Jariwala, PhD, was a graduate student, and Rachel Mendoza, BS is a laboratory technician in Dr. Sarkar’s laboratory.