Date of Graduation

12-2015

Document Type

Dissertation (PhD)

Program Affiliation

Experimental Therapeutics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Garth Powis

Committee Member

Michael Davies

Committee Member

Joseph Ludwig

Committee Member

John McMurray

Committee Member

George Calin

Committee Member

Clifford Stephan

Abstract

Normal Glycolytic Enzyme Activity is Critical for Hypoxia Inducible Factor-1α Activity and Provides Novel Targets for Inhibiting Tumor Growth

By Geoffrey Grandjean

Advisory Professor: Garth Powis, D. Phil

Unique to proliferating cancer cells is the observation that their increased need for energy is provided by a high rate of glycolysis followed by lactic acid fermentation in a process known as the Warburg Effect, a process many times less efficient than oxidative phosphorylation employed by normal cells to satisfy a similar energy demand [1]. This high rate of glycolysis occurs regardless of the concentration of oxygen in the cell and is typically at rates significantly higher than those found in normal cells of the same tissue type. Unlike the case of proliferating cancer cells in culture, the internal environment of most solid tumors is hypoxic due to an inadequate or disorganized blood supply leading to even greater increases in glycolytic rates and, therefore, lactic acid production [2]. The regulation of glycolytic enzyme expression by the hypoxia-inducible factor 1 alpha (HIF-1α) transcription factor in states of low oxygen stress is a well described phenomenon [3, 4]. Through the use of a high throughput siRNA screen using an siRNA library targeting all the known open reading frames of the human genome (Dharmacon, Inc.), novel regulators of HIF-1α were identified including many enzymes involved in the glycolytic pathway. Here, I describe a novel relationship between glycolysis and its regulation of HIF-1α activity. Our studies have shown that of the 40 enzymes of glycolysis involving 11 enzymatic steps and various enzyme isoforms, siRNA knockdown of Aldolase A (ALDOA) produces the maximum inhibition of HIF-1α activity in vitro – a finding that I have subsequently confirmed in a murine in vivo model. Further study indicated that this is mediated through a mechanism involving AMPK, a sensor of low ATP levels, and the subsequent dissociation of HIF-1α and p300, a critical co-activator of HIF-1α. ALDOA was chosen as the molecular target for a drug discovery effort because it produced maximum inhibition of cancer growth and would serve as a dual glycolysis and HIF-1α inhibitor, thus blocking two important cancer cell survival mechanisms at the same time.

Tumor hypoxia is a pervasive problem in regards to treatment options as hypoxic tumor cells are resistant to traditional chemo and radiotherapies. Thus, the overreaching goal of this research is to identify viable dual inhibitors of energy production and specific stress survival pathways to be used as a combinatorial therapy for patients in which tumor hypoxia has rendered standard therapeutic tools ineffective.

Keywords

HIF-1a, Glycolysis, Small Molecule, Warburg Effect, Metabolism, Hypoxia