The long-term objectives of this application are to identify the cellular origins of hepatocellular carcinoma ('HCC'). Many intrahepatic candidates might serve as HCC-progenitor cells, including mature hepatocytes, tissue-specific bipotential stem cells called 'oval'cells, multipotential liver stem cells, and postulated malignant HCC cells with spheroid-forming and stem cell-like properties ('liver cancer stem cells'[LCSCs]). Thus far, rodent models of chemical hepatocarcinogenesis support HCC-progenitor roles only for hepatocytes, although the validity of their roles to the exclusion of other candidates has not been established rigorously, and appear to depend on the carcinogen and its regimen of administration. That most markers for intrahepatic HCC progenitors are not specific to a single cell type or HCC has confounded simple fate-mapping. Therefore, to define a population for study, a unifying hypothesis is proposed that hepatic oval cells, determined in part here by characteristic tiny size and combination of markers, are, in fact, premalignant and HCC-spheroid, HCC, and LCSC progenitors. Accordingly, {a standard hepatocarcinogenic regimen} known to induce oval cells will be utilized in combination with mice capable of being expression-specifically, heritably targeted. The carcinogen- induced ?1-fetoprotein expressing ('AFP+') oval cells, a minority population in both rodent and human adult liver and a controversial, but longstanding HCC-progenitor candidate, will then be followed with a new two-step fate mapping approach. Experiments will be guided by three specific Aims:
in Aim 1, {ROSA26 ZsGreen1 ('ZSG1') mice will be fed an hepatocarcinogenic diet (choline-deficiency + ethionine)} to monitor the induction of AFP+ oval cells and their putative descendants (HCCs;HCC-derived spheroids;postulated LCSCs), as evaluated by standard biological methods of histology, immunohistochemistry, immunofluorescence, {epifluorescence} and cell culture;
in Aim 2, a control ('Ad-E'), and an experimental adenoviral vector expressing Cre recombinase governed by mouse 5'Afp regulatory elements ('Ad-Afp-Cre'), will be constructed and validated by methods of molecular biology, 1o culture and immunostaining. {Potential vector liver toxicity will also be assessed};
in Aim 3, carcinogen-induced AFP+ oval cells will be 'marked'specifically in ROSA mice after carcinogen feeding, by precisely timed, and transient Ad-Afp-Cre infection, to activate heritable ROSA {ZSG1} reporter genes solely in AFP+ oval cells from this narrow time window, and their subsequent descendants. This approach, plus ROSA and liver cell marker co-staining will be used to follow, and characterize the carcinogen-induced oval cell fates. In vitro growth and in vivo transplantation studies will be performed with ROSA {ZSG1+} HCCs, and HCC- derived spheroids to determine their stem cell-like and tumorigenic properties. {The WHO reports that 696,000 people died from HCC in 2008;these numbers are increasing, and environmental chemicals are among major causes.} It is expected that knowledge of the basic cellular etiologies of hepatocarcinogenesis will lead to targeting strategies for treatment of HCC. This research addresses missions of the NCI, NIDDKD and NIAID.

Public Health Relevance

More than 690,000 people throughout the world die yearly from liver cancer (hepatocellular carcinoma ['HCC']), an insidious, aggressive cancer which is caused by many factors, including environmental exposure to chemical carcinogens. The intrahepatic cells that are attacked by chemical carcinogens, and eventually produce malignant and metastatic HCCs, have not been identified unequivocally, and although hepatocytes are implicated as one such progenitor cell type, controversy, speculation and limited approaches have continued to confound this conclusion, as well as {further studies} and isolation of the other candidates. Investigations in this proposal will be based on a new hypothesis and new fate-mapping approach, broadly applicable to other cell populations and systems, to mark heritably, identify, and follow HCC-progenitor cells (including recently proposed 'liver cancer stem cells'), thus contributing to knowledge of the cellular etiology of HCC, and enabling its potential clinical targeting.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Molecular Oncogenesis Study Section (MONC)
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Johnson, Ronald L
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University of California San Diego
Schools of Medicine
La Jolla
United States
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