Compelling evidence identifies a synergism between alcohol and HCV infection for the risk of developing hepatocellular carcinoma (HCC). This application seeks support for research directed to the role of Nanog-positive cancer stem cells in hepatocarcinogenesis induced by HCV and alcohol. A mechanistic link between alcohol and HCV has recently been unveiled by our discovery that HCV NS5A protein induces Toll-like receptor 4 (TLR4) in hepatocytes, which in turn mediates liver tumor development in mice fed alcohol. TLR4 signaling activated by alcohol-induced endotoxemia, induces the progenitor/stem marker Nanog and liver tumors in NS5A transgenic (Tg) mice but not in wild type (wt) or NS5A Tg mice deficient in TLR4. Nanog immunostaining is co-localized with the cancer stem cell marker CD133 and CD49f in liver tumors of these mice. In vitro promoter and mRNA analyses demonstrate TLR4-dependent transcriptional activation of Nanog in NS5A-transfected cells. Transplantation of p53-/- hepatoblasts transduced with TLR4, produces liver tumors in recipient mice after repetitive LPS injection, and this tumor formation is prevented with Nanog shRNA. Transplantation of Nanog+/CD133+/CD49f+ cells but not Nanog-/CD133-/CD49f+ cells isolated from liver tumors of our mouse model, causes tumor growth in nude mice given repetitive LPS injection. Lentiviral cDNA library established from the former cancer stem cells but not the latter cells, transforms the oval cell line in vitro. These findings support the hypothesis that synergistic liver oncogenesis by HCV/NS5A and alcohol is mediated by TLR4-induced oncogenic activity of Nanog+ cancer stem cells. To test this hypothesis and understand the mechanisms of oncogenesis in the model, we will pursue two major aims: 1) to identify and validate oncogenic genes in the Nanog+ cancer stem cells;and 2) to elucidate genetic and epigenetic mechanisms of TLR4-mediated Nanog induction in the cancer stem cells. For the Aim 1, a lentiviral cDNA library established from the cancer stem cells will be used to isolate candidate oncogenic genes;shRNA-based knockdown will be applied to validate these oncogenic genes in vitro and in vivo;and ChIP-seq analysis will be performed for genome-wide analysis for Nanog binding in the cancer stem cells. For the Aim 2, we will elucidate the transcriptional mechanisms by which TLR4 induces Nanog via promoter-reporter assay with deletion constructs and site-directed mutagenesis, and ChIP analysis;and determine epigenetic regulation for sustained Nanog induction via DNA methylation analysis and ChIP analysis for histone modifications. In summary, the proposed R01 application represents novel mechanistic research focused on Nanog+ liver cancer stem cells generated via activated TLR4 signaling by HCV NS5A and alcohol. An ultimate goal of this application is to eventually translate basic knowledge derived from the current study to design novel therapeutic modalities targeted to Nanog+ cancer stem cells for HCV/alcohol-related HCC.

Public Health Relevance

Hepatitis C virus (HCV) is one of the most important causes of liver cancer, which is the third most deadly cancer in the world. The goal of this proposal is to understand how HCV and alcohol induces liver cancer so that better prevention and treatment can be found.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA018857-05
Application #
8516909
Study Section
Special Emphasis Panel (ZAA1-JJ (06))
Program Officer
Wang, Joe
Project Start
2009-09-15
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
5
Fiscal Year
2013
Total Cost
$340,495
Indirect Cost
$130,313
Name
University of Southern California
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Uthaya Kumar, Dinesh Babu; Chen, Chia-Lin; Liu, Jian-Chang et al. (2016) TLR4 Signaling via NANOG Cooperates With STAT3 to Activate Twist1 and Promote Formation of Tumor-Initiating Stem-Like Cells in Livers of Mice. Gastroenterology 150:707-19
Chen, Chia-Lin; Uthaya Kumar, Dinesh Babu; Punj, Vasu et al. (2016) NANOG Metabolically Reprograms Tumor-Initiating Stem-like Cells through Tumorigenic Changes in Oxidative Phosphorylation and Fatty Acid Metabolism. Cell Metab 23:206-19
Chen, Jian; Yao, Zhi-Xing; Chen, Jiun-Sheng et al. (2016) TGF-β/β2-spectrin/CTCF-regulated tumor suppression in human stem cell disorder Beckwith-Wiedemann syndrome. J Clin Invest 126:527-42
Machida, Keigo; Feldman, Douglas E; Tsukamoto, Hidekazu (2015) TLR4-dependent tumor-initiating stem cell-like cells (TICs) in alcohol-associated hepatocellular carcinogenesis. Adv Exp Med Biol 815:131-44
Siddique, Hifzur R; Feldman, Douglas E; Chen, Chia-Lin et al. (2015) NUMB phosphorylation destabilizes p53 and promotes self-renewal of tumor-initiating cells by a NANOG-dependent mechanism in liver cancer. Hepatology 62:1466-79
Tsukamoto, Hidekazu; Mishra, Lopa; Machida, Keigo (2014) Alcohol, TLR4-TGF-β antagonism, and liver cancer. Hepatol Int 8 Suppl 2:408-12
Kondo, Yasuteru; Ninomiya, Masashi; Kimura, Osamu et al. (2014) HCV infection enhances Th17 commitment, which could affect the pathogenesis of autoimmune diseases. PLoS One 9:e98521
Chen, Chia-Lin; Tsukamoto, Hidekazu; Liu, Jian-Chang et al. (2013) Reciprocal regulation by TLR4 and TGF-β in tumor-initiating stem-like cells. J Clin Invest 123:2832-49
Feldman, Douglas E; Chen, Chialin; Punj, Vasu et al. (2013) The TBC1D15 oncoprotein controls stem cell self-renewal through destabilization of the Numb-p53 complex. PLoS One 8:e57312
Tsukamoto, Hidekazu; Zhu, Nian-Ling; Wang, Jiaohong et al. (2012) Morphogens and hepatic stellate cell fate regulation in chronic liver disease. J Gastroenterol Hepatol 27 Suppl 2:94-8

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