Among the several hallmarks of cancer cells currently gaining attention is the remodeling of cellular metabolism, where glucose and glutamine are imported at high rates and metabolized to provide building blocks for the biosynthesis of lipids, proteins, and DNA required for growth and proliferation. Transformation of normal cells is therefore often accompanied by a dramatic increase in the glycolytic flux although the end product of glycolysis, pyruvate, is diverted from entering the respiratory TCA cycle and instead secreted from the cell as lactate. In order to compensate for the decrease in glucose-derived pyruvate entering the TCA cycle, glutamine is taken up and converted to glutamate by the mitochondrial enzyme glutaminase, where glutamate can then feed an entry point of the TCA cycle upon conversion to ?-ketoglutarate. The increased flux of glutamine and its role in providing biosynthetic precursors for proliferation represents a critical step in the metabolic remodeling of cancer cells. In particular, a specific isoform of glutaminase, glutaminase C (GAC), has been shown to be up regulated and activated in Mycand NF-?B-dependent transformation, and as such may prove to be an important therapeutic target. However, the mechanism of activation of GAC is poorly understood, as well as its role in supporting neoplastic growth. Therefore, the aim of this proposal is to investigate the activation of GAC in vitro using a novel fluorescence resonance energy transfer (FRET) assay, complemented by an in vivo metabolic study of model cell systems engineered to express in a tetracycline-dependent manner, the oncogenesMyc and Dbl, as well as constitutively active GAC mutant constructs. The in vitro FRET assay will provide important information regarding how the ability of GAC to undergo a dimer-to-tetramer transition is coupled to the activation of its enzymatic activity, as well as the mode of inhibition of GAC by small molecule inhibitors. Furthermore, recent X-ray crystallographic structures solved by our laboratory will be used to create constitutively active mutated forms of GAC to complement the in vitro FRET assay. These mutants will be incorporated into the engineered inducible cell system to specifically investigate the impact of hyperactive glutaminase activity on the growth properties of the induced cells. This induced """"""""glutamine addicted"""""""" cell line, as well as the inducible cell lines capable of expressing the oncogenic transcriptional regulator Myc, and the Rho-GTPase guanine nucleotide exchange factor (GEF) Dbl, will ultimately be characterized using state of the art 13C-labeled glucose and glutamine metabolic tracing methods, including mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The expectation is that these studies will ultimately provide a detailed portrait of metabolic transformation and a deeper understanding of the role glutaminase plays in supporting cancer cell growth.
Cancer cells have been historically characterized as having hijacked normal cellular machinery in order to convey a significant growth advantage, thus posing the fundamental challenge in the treatment of cancer. Therefore, a good deal of effort has been devoted toward identifying the unique differences between normal cells and cancerous cells in order to develop novel therapeutics. The motivation for this study is to systematically investigate the newly discovered reprogramming of glutamine metabolism that occurs specifically in cancer cells, with the hope that this will lead to the development of new targets and therapeutic strategies for the treatment of cancer.
|Stalnecker, Clint A; Ulrich, Scott M; Li, Yunxing et al. (2015) Mechanism by which a recently discovered allosteric inhibitor blocks glutamine metabolism in transformed cells. Proc Natl Acad Sci U S A 112:394-9|