Graduation Date

Fall 12-15-2017

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Pankaj K. Singh

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal of all cancers with a 5-year survival rate of only 8.2%. This is because PDAC is diagnosed in its advanced stages and is characterized by radio and chemotherapy resistance. Aggressiveness of PDAC tumors is attributed to its high metabolic phenotype, which is characterized by increased glycolysis rate and lactate secretion, while oxidative metabolism is reduced. These metabolic features are required to fulfill the biosynthetic demands of proliferating PDAC cells. However, this increase in metabolic activity results in acidification of the extracellular space because the dense fibrotic stroma of PDAC tumors limits venting of protons into the vasculature thereby creating a chronic low pH microenvironment. Little is known regarding the physiology and metabolism of cancer cells enduring chronic low pH exposure.

To demonstrate effects of low pH, PDAC cells were cultured in low pH 6.9~7.0 to establish chronic low pH as it occurs in tumors. These cells were compared to cells in physiological pH of 7.4, which is also the pH of cell culture, in order to evaluate physiological differences between these pH values. In these experiments, it was observed that cells in low pH have reduced clonogenic capacity and undergo a metabolic shift to oxidative metabolism that is supported by an increase in glutamine uptake. These observations exhibit a robust contrast to PDAC cells in control pH conditions that are highly glycolytic. Furthermore, in low pH there is increased transcription of the GOT1 enzyme, which mediates metabolic flux through the non-canonical glutamine metabolic pathway that allows synthesis of other metabolic substrates from glutamine. Upon shRNA-mediated depletion of GOT1, survival of PDAC cells in low pH was significantly impaired due to increase in ROS to cytotoxic levels. However, supplementing transfected clones with GOT1 metabolic product, oxaloacetate, resulted in growth rescue and reduction in ROS levels. Thus, in chronic low pH stress PDAC cells up-regulate non-canonical glutamine metabolism through increased transcription of GOT1, which allows PDAC cells to generate energy and metabolic co-factors to suppress cytotoxic ROS levels. Low pH is a universal feature of the PDAC tumor microenvironment and further dissection of metabolic adaptations to microenvironment conditions will result in more effective therapy for PDAC.

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