Date on Master's Thesis/Doctoral Dissertation

8-2018

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Martin II, Robert C.G.

Committee Co-Chair (if applicable)

Hein, David

Committee Member

Hein, David

Committee Member

Bhatnagar, Aruni

Committee Member

Bodduluri, Haribabu

Committee Member

Li, Yong

Author's Keywords

hepatocellular carcinoma (HCC); cancer stem cells (CSCs); Wnt/β-catenin signaling; epithelial cell adhesion molecule (EpCAM); orthotopic HCC mouse models

Abstract

Hepatocellular carcinoma (HCC) is one of the deadliest cancers, with variable presentation, high chemotherapy resistance and early tumor recurrence. In this dissertation, we have systematically enriched, identified and characterized HCC cancer stem cells (CSCs) from 3 different HCC cell lines (Hepa1-6, HepG2, and Hep3B) using in vitro serum-free culture method. Enriched spheroids acquired CSC properties, primarily self-renewal capability, spheroid formation ability, and drug resistance. Heterogeneous CSCs enriched from Hepa1-6 cells demonstrated higher tumor initiation and proliferation capability in vivo, compared to control HCC cells in orthotopic immunocompetent mouse model. EpCAM+ CSCs were significantly enriched within heterogeneous Hepa1-6 CSC spheroids. Consequently, we studied the fate of EpCAM+ CSCs in orthotopic immunocompetent mouse model with 3 different liver microenvironments i.e. control normal liver, steatosis (fatty liver induced), and nonalcoholic steatohepatitis (NASH). EpCAM+ CSC mediated HCC carcinogenesis was observed in NASH livers, but failed to develop in control and steatosis liver microenvironment. This is the first study evaluating CSCs in immunocompetent mouse model and demonstrated the importance of liver microenvironment for EpCAM+ CSC mediated tumor initiation. We then performed a lineage tracking study by stably incorporating copGFP or mCherry in Hepa1-6 cells by lentivirus transduction, and examined the origin of tumors in NASH liver microenvironment using orthotopic C57L/J immunocompetent mouse model. Our findings suggest that tumor growth was dose dependent and most tumor nodules arose from copGFP expressing EpCAM+ CSCs. The Wnt/β-catenin pathway components were also found to be overexpressed in CSCs when compared to control. Human HCC specimen analyses suggested concomitant changes in β-catenin and EpCAM levels. Gene analyses of step-wise spheroid formation process identified possible dedifferentiation mechanisms regulated by β-catenin in CSC spheroids. Loss of function analysis by siRNA mediated transient knockdown of β-catenin confirmed its role in spheroid enrichment and doxorubicin resistance. Canonical Wnt pathway study using chemical inhibitors identified β-catenin mediated CSC activation was regulated at nuclear level, and not at the cytoplasmic GSK3β destruction complex level. Analysis of human and mouse tumors suggested that constitutive activation of β-catenin transcription failed to respond to NOTUM, a feedback inhibitor of canonical Wnt/β-catenin signaling.

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