Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Steven M Frisch

Committee Co-Chair

Christopher P Cifarelli

Committee Member

Linda V Davis

Committee Member

John Hollander

Committee Member

Elena Pugacheva

Committee Member

Scott A Weed

Abstract

When deprived of connections to their extracellular matrix, normal epithelial cells trigger a process of programmed cell death referred to as anoikis. The transcriptional reprogramming event known as Epithelial-Mesenchymal transition (EMT) confers anoikis resistance and ultimately, increased metastatic potential. Distant metastatic disease contributes to the majority of deaths occurring in many cancer types, especially those originating in breast tissue. The ability to metastasize depends critically upon a cancer cell's ability to evade anoikis. Heterogeneity of tumors containing anoikis, therapy resistant populations likely explains the tendency of disease to reappear in distant locations after a period of apparent remission. The wound healing transcription factor, Grainyhead-like 2 (GRHL2), has been previously demonstrated to play an indispensable role in the maintenance of the epithelial phenotype through both suppression of EMT and promotion of the reciprocal event, Mesenchymal-Epithelial transition (MET), accompanied by suppression of the cancer stem cell (CSC) phenotype and re-sensitization to anoikis. Loss of GRHL2 expression is associated with aggressive, metastatic breast tumor types such as claudin low and basal B subclasses of tumor cell lines. Specifically, GRHL2 expression is lost in the cancer stem cell-like compartment of tumors characterized by their enrichment in the CD44hi/CD24low cell population and an EMT phenotype. Constitutive expression of the epithelial enforcer GRHL2 results in increased anoikis sensitivity as well as reduction in tumor initiating frequency. Here, the effects of GRHL2 upon intracellular metabolism in the context of reversion of the EMT/CSC phenotype, with a view toward understanding how these effects promote anoikis sensitivity were investigated. EMT resulted in enhanced mitochondrial oxidative metabolism. While this was accompanied by higher accumulation of superoxide, the overall level of Reactive Oxygen Species (ROS) declined, due to decreased hydrogen peroxide. Glutamate Dehydrogenase 1 (GLUD1) expression increased in EMT, and this increase, via the product alpha-ketoglutarate (alpha-KG), was important for suppressing hydrogen peroxide and protecting against anoikis. GRHL2 suppressed GLUD1 gene expression, decreased alpha-KG, increased ROS and sensitized cells to anoikis. These results demonstrate a mechanistic role for GRHL2 in promoting anoikis through metabolic alterations.

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