Microenvironmental and metabolic influences in tumor cell redox homeostasis

Redox reactions dictate protein shape, regulate enzyme activity, and underscore cellular metabolism. When redox poise fails and cells are overcome by oxidation, cellular death soon follows. Redox biology is an especially important aspect of cancer cell biology, because oncogene-induced hyperproliferation results in pro-oxidant tumor cell phenotypes. Thus, tumor cell antioxidant systems are often inherently strained and vulnerable to further oxidative attack. Targeted therapies can be designed to kill tumor cells by pushing them beyond redox thresholds compatible with cellular life without injuring healthy cells that can more effectively dissipate oxidative stress. Radiation therapy and many chemotherapeutic drugs use this mechanism of oxidant-based cell killing to eliminate tumor cell populations. However, in clinical settings, these therapies face tumor cell resistance stemming from multifactorial changes in cellular drug and energy metabolism, environmental selective pressures, and intratumoral heterogeneity. To further advance efficacious and selective anticancer strategies, increased understanding of microenvironmental and metabolic influences in tumor cells redox homeostasis is needed. Poorly arranged and malfunctioning vasculature in the tumor microenvironment causes heterogeneous regions of hypoxia to form throughout the solid mass. Clinically, tumor hypoxia is a negative prognostic indicator for a more aggressive phenotype and is associated with treatment resistance. Metabolically, tumor hypoxia results in hypoxia inducible factor-driven gene network activation and enhanced mitochondrial production of reactive oxygen species. Given this, it is important to match laboratory in vitro studies as closely as possible to the in vivo conditions of the tumor microenvironment. However, even simple in vitro hypoxic studies are complicated by the rigors of hypoxic experimentation and, thus, most studies are conducted at hyperoxic atmospheric levels of oxygen. Engineered microfluidic platforms offer an approach to hypoxic laboratory studies. This report validates an ease-of-use platform suitable to study complex cell behaviors in hypoxia. Herein we present the design and implementation of an open-well microfluidic platform that enables on-chip cell culture, experimentation, and microscopy in a controlled hypoxic microenvironment. This platform has been theoretically and experimentally validated to quickly induce and maintain 0.3 mg/L O2 hypoxia, and it has enabled studies of the stabilization of hypoxia inducible factor in hypoxia and the redox response of the mitochondrial matrix in hypoxia. The glutathione system functions in antioxidant defense and xenobiotic detoxification to preserve subcellular redox poise and maintain enzymes in functional reduced states. Glutathione is produced from its constitutive amino acids in a two-step enzymatic process that occurs in the cytosol. Despite glutathione’s role in cellular antioxidant defense, its production in the pro-oxidant state of hypoxia has been under studied. We investigated whether glutathione production in hypoxia differed by cellular transformation state and malignancy in an isogenic series of breast cancer cells. Unexpectedly, we observed a glutathione biosynthetic gene downregulation and a loss of glutathione content following culture in chronic mild hypoxia. This result did not differ with transformation state and was seen over a range of hypoxic exposures. With further investigation of glutathione synthesis in hypoxia, strategies may be uncovered to specifically promote the loss of glutathione content by cancer cells in the hypoxic tumor microenvironment. Increased glutamine uptake and turnover contributes to the metabolic reprogramming of transformed cells. Oncogenic glutaminolysis results in glutamine utilization that far exceeds the anabolic needs of transformed cells for nitrogen and increases the anaplerotic flux of glutamine through the TCA cycle to support mitochondrial respiration. MYC and RAS-transformed tumor cells are particularly reliant on glutamine metabolism for cellular homeostasis. In these cells, glutaminolysis promotes rapid cellular proliferation by supporting energy generation, cell division, and antioxidant defense. Herein we report an investigation of the metabolic requirements of tumor cells for glutamine to preserve glutathione redox homeostasis. Glutamine is deaminated by glutaminase to glutamate, a TCA cycle carbon that supports mitochondrial energy generation via oxidative phosphorylation and serves as a direct substrate for cytosolic glutathione synthesis. Cytosolic and mitochondrial glutathione pools are distinctly regulated and maintained at independent concentrations. RAS-transformed tumor cells expressing the Glrx-roGFP2 redox biosensor, a GFP-based subcellular probe sensitive to local glutathione redox potentials, were exposed to glutamine deprivation and pharmacologic inhibition of glutaminolytic enzymes. We found that oncogenic RAS increases cellular reliance on glutamine to maintain glutathione redox poise. Furthermore, we report that cytosolic glutathione homeostasis is more independent of glutamine metabolism and far more robust in resisting changes to baseline redox potential than mitochondrial matrix glutathione. The mitochondrial matrix requires transaminase-mediated glutamine metabolism to resist glutathione oxidation. Alpha-ketoglutarate, a TCA cycle carbon and downstream metabolite of glutamate, can prevent mitochondrial glutathione oxidation and aid GSH biosynthesis during glutamine deprivation. However, alpha-ketoglutarate cannot restore proliferation to glutamine-deprived KRAS-transformed tumor cells that require transaminase-mediated glutamine metabolism for rapid proliferation. This study demonstrates that glutaminolysis is needed to maintain glutathione concentrations and mitochondria glutathione redox poise in RAS-transformed cells. Tumor cell reliance on glutamine for mitochondrial redox homeostasis may present targets for the rational design of novel or companion approaches to selectively predispose cancer cells to pathologic oxidation in anticancer therapies. The dissertation research presented herein explores cancer redox biology from a number of vantage points. The Glrx-roGFP2 redox biosensor enabled unique examination of glutathione redox responses to physical, xenobiotic, and metabolic insults to live cancer cells in real time. It is hoped that these studies of the microenvironmental and metabolic influences in tumor cell redox homeostasis contribute to the body of knowledge that is drawn upon to advance cancer treatment.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste