Abstract A18: Changes in cholesterol metabolism support high-density cell growth and therapeutic resistance in glioblastoma multiforme

Abstract

Abstract Glioblastoma Multiforme (GBM) is a highly aggressive, therapeutically resistant brain tumor that arises from cells of astrocytic lineage. The five year survival rate is just below 10% and this statistic has changed very little in the past decade. Amplification and activating mutations of receptor tyrosine kinases (RTKs) are common in GBM; however despite their oncogenic importance, RTK inhibition with tyrosine kinase inhibitors (TKIs) has been clinically ineffective for GBM. The gap between promising laboratory results and clinical success is a common obstacle in cancer research in part because cell culture models do not accurately mimic complex in vivo conditions and cell behavior, such as therapeutic resistance. Many cell culture studies and high throughput screens of pharmacological agents are performed in cells plated at sub-confluence, or low density. We have found that GBM tumor initiating cells (GBM-TICs) are sensitive to TKIs at low density; however as culture density increases they become resistant, despite equal inhibition of the target RTK and downstream signals. Thus, culturing cells at low and high density provides a novel, isogenic system for studying the cellular mechanisms of therapeutic response. While density does not affect the cell cycle profile of GBM-TICs, we have found that high density cells have lower levels of reactive oxygen species and ATP than low density cells and reduced mitochondrial membrane potential. This suggests a switch to aerobic glycolysis (the Warburg Effect); however lactate levels, which should increase under conditions of aerobic glycolysis, are unaffected by cell density. Additionally, assessment of extracellular metabolic flux does not support an increase in aerobic glycolysis in dense GBM-TICs. Extracellular acidification rate (ECAR), which should increase with aerobic glycolysis, does not show a tumor-specific effect. Oxygen consumption rate (OCR), which should decrease during aerobic glycolysis, increases significantly with density in GBM-TICs but shows no density-dependent change in healthy astrocytes. These data indicate a tumor-specific, non-Warburg metabolic shift in dense GBM-TICs that is not present in sparse GBM-TICs or normal human astrocytes at any density. Further analysis has implicated changes in cholesterol metabolism and transport in density-dependent metabolic change. Specifically, densely cultured GBM-TICs have higher levels of cholesterol and elevated expression of cholesterol transport proteins such as ABCA1 and ABCG1 than cells cultured sparsely. Pharmacologic inhibition of cholesterol biosynthesis results in reduced OCR and increased ATP/cell in dense but not sparse GBM-TICs, which corroborates the importance of cholesterol synthesis and metabolism in dense GBM-TICs. We are currently investigating the connection between cholesterol metabolism and therapeutic resistance in GBM in the hope of identifying novel therapeutic targets to improve clinical outcomes for GBM patients. Citation Format: Diane M. Kambach, Tamalee Kramp, Jie Liu, Michael L. Oshinsky, Federico Bernal, Jayne M. Stommel. Changes in cholesterol metabolism support high-density cell growth and therapeutic resistance in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A18.