Melissa M. Keenan

Acidosis induces reprogramming of cellular metabolism to mitigate oxidative stress.

BackgroundA 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.ResultsAcidosis increased both glutaminolysis and fatty acid β-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.ConclusionsAcidosis 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.

Alternative fuels for cancer cells.

AbstractTumor metabolism is significantly altered to support the various metabolic needs of tumor cells. The most prominent change is the increased tumor glycolysis that leads to increased glucose uptake and utilization. However, it has become obvious that many nonglucose nutrients, such as amino acids, lactate, acetate, and macromolecules, can serve as alternative fuels for cancer cells. This knowledge reveals an unexpected flexibility and evolutionarily conserved model in which cancer cells uptake nutrients from their external environment to fulfill their necessary energetic needs. Tumor cells may have evolved the ability to utilize different carbon sources because of the limited supply of nutrients in their microenvironment, which can be driven by oncogenic mutations or tumor microenvironmental stresses. In certain cases, these factors permanently alter the tumor cells’ metabolism, causing certain nutrients to become indispensable and thus creating opportunities for therapeutic intervention to eradicate tumors by their metabolic vulnerabilities.

Comprehensive profiling of amino acid response uncovers unique methionine-deprived response dependent on intact creatine biosynthesis.

Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine/glycine-dependent creatine biosynthesis.

Fish Oil Slows Prostate Cancer Xenograft Growth Relative to Other Dietary Fats and is Associated with Decreased Mitochondrial and Insulin Pathway Gene Expression

BACKGROUND:Previous mouse studies suggest that decreasing dietary fat content can slow prostate cancer (PCa) growth. To our knowledge, no study has yet compared the effect of multiple different fats on PCa progression. We sought to systematically compare the effect of fish oil, olive oil, corn oil and animal fat on PCa progression.METHODS:A total of 96 male severe combined immunodeficient mice were injected with LAPC-4 human PCa cells. Two weeks following injection, mice were randomized to a Western diet based on fish oil, olive oil, corn oil or animal fat (35% kilocalories from fat). Animals were euthanized when tumor volumes reached 1000 mm3. Serum was collected at death and assayed for PSA, insulin, insulin-like growth factor-1 (IGF-1), IGF-1-binding protein-3 and prostaglandin E-2 (PGE-2) levels. Tumors were also assayed for PGE-2 and cyclooxygenase-2 levels, and global gene expression was analyzed using Affymetrix microarrays.RESULTS:Mice weights and tumor volumes were equivalent across groups at randomization. Overall, fish oil consumption was associated with improved survival relative to other dietary groups (P=0.014). On gene expression analyses, the fish oil group had decreased signal in pathways related to mitochondrial physiology and insulin synthesis/secretion.CONCLUSIONS:In this xenograft model, we found that consuming a diet in which fish oil was the only fat source slowed tumor growth and improved survival compared with that in mice consuming diets composed of olive oil, corn oil or animal fat. Although prior studies showed that the amount of fat is important for PCa growth, this study suggests that the type of dietary fat consumed may also be important.

ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate.

In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

Cystine Deprivation Triggers Programmed Necrosis in VHL-Deficient Renal Cell Carcinomas

Oncogenic transformation may reprogram tumor metabolism and render cancer cells addicted to extracellular nutrients. Deprivation of these nutrients may therefore represent a therapeutic opportunity, but predicting which nutrients cancer cells become addicted remains difficult. Here, we performed a nutrigenetic screen to determine the phenotypes of isogenic pairs of clear cell renal cancer cells (ccRCC), with or without VHL, upon the deprivation of individual amino acids. We found that cystine deprivation triggered rapid programmed necrosis in VHL-deficient cell lines and primary ccRCC tumor cells, but not in VHL-restored counterparts. Blocking cystine uptake significantly delayed xenograft growth of ccRCC. Importantly, cystine deprivation triggered similar metabolic changes regardless of VHL status, suggesting that metabolic responses alone are not sufficient to explain the observed distinct fates of VHL-deficient and restored cells. Instead, we found that increased levels of TNFα associated with VHL loss forced VHL-deficient cells to rely on intact RIPK1 to inhibit apoptosis. However, the preexisting elevation in TNFα expression rendered VHL-deficient cells susceptible to necrosis triggered by cystine deprivation. We further determined that reciprocal amplification of the Src-p38 (MAPK14)-Noxa (PMAIP1) signaling and TNFα-RIP1/3 (RIPK1/RIPK3)-MLKL necrosis pathways potentiated cystine-deprived necrosis. Together, our findings reveal that cystine deprivation in VHL-deficient RCCs presents an attractive therapeutic opportunity that may bypass the apoptosis-evading mechanisms characteristic of drug-resistant tumor cells. Cancer Res; 76(7); 1892-903. ©2016 AACR.

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.

Abstract 3004: Contextual RNAi screen identifies ACLY and ACC1 as mediators of hypoxia-induced apoptosis through metabolic and transcriptional mechanisms

To become established as a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. While many stress-signaling mechanisms have been well-studied, much remains unknown about how tumor cells survive these stresses during tumor progression. To identify genes that modulate cellular survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that the genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis through both metabolic and transcriptional mechanisms. First, while depleting α-ketoglutarate in control cells, hypoxia unexpectedly increased levels of α-ketoglutarate in the cells depleted of ACC1 or ACLY. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis in a mitochondria-dependent manner. Second, loss of ACLY or ACC1 reduced protein levels and activity of the oncogenic transcription factor ETV4. Silencing of ETV4 protected cells from hypoxia-induced apoptosis and triggered remarkably similar global transcriptional responses as with silenced ACLY or ACC1. Importantly, within tumor expression datasets, ETV4 transcriptional activity was highly correlated with ACLY or ACC1 gene expression signatures. Therefore, ETV4 acted as a key regulator of the transcriptional output of lipogenic activity via ACLY or ACC1. These results reveal a novel interconnectedness between cellular metabolic and transcriptional responses for life or death decisions under stress. Since lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future. Citation Format: Melissa M. Keenan, Beiyu Liu, Xiaohu Tang, Jianli Wu, Derek Cyr, Robert D. Stevens, Olga Ilkayeva, Joseph Lucas, Deborah M. Muoio, So Young Kim, Jen-Tsan Chi. Contextual RNAi screen identifies ACLY and ACC1 as mediators of hypoxia-induced apoptosis through metabolic and transcriptional mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3004. doi:10.1158/1538-7445.AM2015-3004

Introduction: Molecular Genetics of Acid Sensing and Response

Since most biological reactions in human body occur within narrow ranges around neutral environments, any changes in the pH environment have great impacts on a wide variety of functions, including gene expression, protein folding, enzymatic activities, cell proliferation, and cell death. When the pH homeostasis is disrupted and results in tissue acidity, how various cell types sense and respond in their physiology, metabolism, and gene expression play a dramatic role in modulating disease outcomes. Therefore, understanding how various cells sense and react to pH imbalance have broad impact in a wide variety of human diseases, including cancer, stroke, myocardial infarction, diabetes, and renal and infectious diseases. In this book, several experts in the field have highlighted various aspects of the molecular genetics on how mammalian cells sense and respond to acidosis and their implications in the normal physiological adaptations and pathogenesis. These reviews highlight at least three levels of complexity in the acid sensing and response among different cell types and disease settings.

A Genomic Analysis of Cellular Responses and Adaptions to Extracellular Acidosis

Even though lactic acidosis is a prominent feature of solid tumors, we have limited understanding of the mechanisms by which lactic acidosis influences the genetic, epigenetic, proteomic, and metabolic phenotypes of cancer cells. This chapter aims to (1) briefly outline the tumor microenvironment and how acidity relates to its biology; (2) briefly discuss traditional hypothesis-driven or single-gene studies that have explored cancer cells’ responses to acidosis or lactic acidosis; (3) explain what we have learned from “-omics” approaches that have been applied to studying cellular response to acid and lactic acid; and (4) reflect on the projections of these studies in (2) and (3) to in vivo human tumor biology and how we can use this information to better inform disease treatments.