[Show abstract][Hide abstract] ABSTRACT: A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis.
Acidosis increased both glutaminolysis and fatty acid beta-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes.
Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.
[Show abstract][Hide abstract] ABSTRACT: Within solid tumor microenvironments, lactic acidosis, and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here, we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1α protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Furthermore, these data suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Although lactic acidosis is a prominent feature of solid tumors, we still have limited understanding of the mechanisms by which lactic acidosis influences metabolic phenotypes of cancer cells. We compared global transcriptional responses of breast cancer cells in response to three distinct tumor microenvironmental stresses: lactic acidosis, glucose deprivation, and hypoxia. We found that lactic acidosis and glucose deprivation trigger highly similar transcriptional responses, each inducing features of starvation response. In contrast to their comparable effects on gene expression, lactic acidosis and glucose deprivation have opposing effects on glucose uptake. This divergence of metabolic responses in the context of highly similar transcriptional responses allows the identification of a small subset of genes that are regulated in opposite directions by these two conditions. Among these selected genes, TXNIP and its paralogue ARRDC4 are both induced under lactic acidosis and repressed with glucose deprivation. This induction of TXNIP under lactic acidosis is caused by the activation of the glucose-sensing helix-loop-helix transcriptional complex MondoA:Mlx, which is usually triggered upon glucose exposure. Therefore, the upregulation of TXNIP significantly contributes to inhibition of tumor glycolytic phenotypes under lactic acidosis. Expression levels of TXNIP and ARRDC4 in human cancers are also highly correlated with predicted lactic acidosis pathway activities and associated with favorable clinical outcomes. Lactic acidosis triggers features of starvation response while activating the glucose-sensing MondoA-TXNIP pathways and contributing to the "anti-Warburg" metabolic effects and anti-tumor properties of cancer cells. These results stem from integrative analysis of transcriptome and metabolic response data under various tumor microenvironmental stresses and open new paths to explore how these stresses influence phenotypic and metabolic adaptations in human cancers.
[Show abstract][Hide abstract] ABSTRACT: We present an applied study in cancer genomics for integrating data and inferences from laboratory experiments on cancer cell lines with observational data obtained from human breast cancer studies. The biological focus is on improving understanding of transcriptional responses of tumors to changes in the pH level of the cellular microenvironment. The statistical focus is on connecting experimentally defined biomarkers of such responses to clinical outcome in observational studies of breast cancer patients. Our analysis exemplifies a general strategy for accomplishing this kind of integration across contexts. The statistical methodologies employed here draw heavily on Bayesian sparse factor models for identifying, modularizing and correlating with clinical outcome these signatures of aggregate changes in gene expression. By projecting patterns of biological response linked to specific experimental interventions into observational studies where such responses may be evidenced via variation in gene expression across samples, we are able to define biomarkers of clinically relevant physiological states and outcomes that are rooted in the biology of the original experiment. Through this approach we identify microenvironment-related prognostic factors capable of predicting long term survival in two independent breast cancer datasets. These results suggest possible directions for future laboratory studies, as well as indicate the potential for therapeutic advances though targeted disruption of specific pathway components.
Preview · Article · Dec 2009 · The Annals of Applied Statistics
[Show abstract][Hide abstract] ABSTRACT: Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a "common currency" that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies--in cancer and other diseases--have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes.
[Show abstract][Hide abstract] ABSTRACT: The tumor microenvironment has a significant impact on tumor development. Two important determinants in this environment are hypoxia and lactic acidosis. Although lactic acidosis has long been recognized as an important factor in cancer, relatively little is known about how cells respond to lactic acidosis and how that response relates to cancer phenotypes. We develop genome-scale gene expression studies to dissect transcriptional responses of primary human mammary epithelial cells to lactic acidosis and hypoxia in vitro and to explore how they are linked to clinical tumor phenotypes in vivo. The resulting experimental signatures of responses to lactic acidosis and hypoxia are evaluated in a heterogeneous set of breast cancer datasets. A strong lactic acidosis response signature identifies a subgroup of low-risk breast cancer patients having distinct metabolic profiles suggestive of a preference for aerobic respiration. The association of lactic acidosis response with good survival outcomes may relate to the role of lactic acidosis in directing energy generation toward aerobic respiration and utilization of other energy sources via inhibition of glycolysis. This "inhibition of glycolysis" phenotype in tumors is likely caused by the repression of glycolysis gene expression and Akt inhibition. Our study presents a genomic evaluation of the prognostic information of a lactic acidosis response independent of the hypoxic response. Our results identify causal roles of lactic acidosis in metabolic reprogramming, and the direct functional consequence of lactic acidosis pathway activity on cellular responses and tumor development. The study also demonstrates the utility of genomic analysis that maps expression-based findings from in vitro experiments to human samples to assess links to in vivo clinical phenotypes.