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Ovarian cancer has few known risk factors, hampering identification of high-risk women. We assessed the association of prediagnostic plasma metabolites (N = 420) with risk of epithelial ovarian cancer, including both borderline and invasive tumors. A total of 252 cases and 252 matched controls from the Nurses' Health Studies were included. Multivariable logistic regression was used to estimate ORs and 95% confidence intervals (CI), comparing the 90th–10th percentile in metabolite levels, using the permutation-based Westfall and Young approach to account for testing multiple correlated hypotheses. Weighted gene coexpression network analysis (WGCNA; n = 10 metabolite modules) and metabolite set enrichment analysis (n = 23 metabolite classes) were also evaluated. An increase in pseudouridine levels from the 10th to the 90th percentile was associated with a 2.5-fold increased risk of overall ovarian cancer (OR = 2.56; 95% CI, 1.48–4.45; P = 0.001/adjusted P = 0.15); a similar risk estimate was observed for serous/poorly differentiated tumors (n = 176 cases; comparable OR = 2.38; 95% CI, 1.33–4.32; P = 0.004/adjusted P = 0.55). For nonserous tumors (n = 34 cases), pseudouridine and C36:2 phosphatidylcholine plasmalogen had the strongest statistical associations (OR = 9.84; 95% CI, 2.89–37.82; P < 0.001/adjusted P = 0.07; and OR = 0.11; 95% CI, 0.03–0.35; P < 0.001/adjusted P = 0.06, respectively). Five WGCNA modules and 9 classes were associated with risk overall at FDR ≤ 0.20. Triacylglycerols (TAG) showed heterogeneity by tumor aggressiveness (case-only heterogeneity P < 0.0001). The TAG association with risk overall and serous tumors differed by acyl carbon content and saturation. In summary, this study suggests that pseudouridine may be a novel risk factor for ovarian cancer and that TAGs may also be important, particularly for rapidly fatal tumors, with associations differing by structural features.Significance:Pseudouridine represents a potential novel risk factor for ovarian cancer and triglycerides may be important particularly in rapidly fatal ovarian tumors. more...

Supplementary Tables 1-6

Supplementary data on Study population, Metabolite profiling, and Statistical methods

Supplementary Figures 1-8

Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify non-genetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.SignificancePancreatic ductal adenocarcinoma (PDA) is a highly lethal malignancy with no effective therapies. PDA aggressiveness partly stems from its ability to grow within a uniquely dense stroma restricting nutrient access. This study demonstrates that PDA clones that survive chronic nutrient deprivation acquire reversible non-genetic adaptations allowing them to switch between metabolic states optimal for growth under nutrient-replete or nutrient-deprived conditions. One contributing factor to this adaptation mTORC1 activation, which stabilizes glutamine synthetase (GS) necessary for glutamine generation in nutrient-deprived cancer cells. Our findings imply that although total GS levels may not be a prognostic marker for aggressive disease, GS inhibition is of high therapeutic value, as it targets specific cell clusters adapted to nutrient starvation, thus mitigating tumor growth. more...

Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Despite recent identification of metabolic alterations in this lethal malignancy, the metabolic dependencies of obesity-associated PDA remain unknown. Here we show that obesity-driven PDA exhibits accelerated growth and a striking transcriptional enrichment for pathways regulating nitrogen metabolism. We find that the mitochondrial form of arginase (ARG2), which hydrolyzes arginine into ornithine and urea, is induced upon obesity, and silencing or loss of ARG2 markedly suppresses PDA. In vivo infusion of 15N-glutamine in obese mouse models of PDA demonstrates enhanced nitrogen flux into the urea cycle and infusion of 15N-arginine shows that Arg2 loss causes significant ammonia accumulation that results from the shunting of arginine catabolism into alternative nitrogen repositories. Furthermore, analysis of PDA patient tumors indicates that ARG2 levels correlate with body mass index (BMI). The specific dependency of PDA on ARG2 rather than the principal hepatic enzyme ARG1 opens a therapeutic window for obesity-associated pancreatic cancer., Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Here the authors show that obesity induces the expression of the mitochondrial form of arginase ARG2 in PDA and that ARG2 silencing or loss results in ammonia accumulation and suppression of obesity-driven PDA tumor growth. more...

Ovarian cancer has few known risk factors, hampering identification of high-risk women. We assessed the association of prediagnostic plasma metabolites (N = 420) with risk of epithelial ovarian cancer, including both borderline and invasive tumors. A total of 252 cases and 252 matched controls from the Nurses' Health Studies were included. Multivariable logistic regression was used to estimate ORs and 95% confidence intervals (CI), comparing the 90th–10th percentile in metabolite levels, using the permutation-based Westfall and Young approach to account for testing multiple correlated hypotheses. Weighted gene coexpression network analysis (WGCNA; n = 10 metabolite modules) and metabolite set enrichment analysis (n = 23 metabolite classes) were also evaluated. An increase in pseudouridine levels from the 10th to the 90th percentile was associated with a 2.5-fold increased risk of overall ovarian cancer (OR = 2.56; 95% CI, 1.48–4.45; P = 0.001/adjusted P = 0.15); a similar risk estimate was observed for serous/poorly differentiated tumors (n = 176 cases; comparable OR = 2.38; 95% CI, 1.33–4.32; P = 0.004/adjusted P = 0.55). For nonserous tumors (n = 34 cases), pseudouridine and C36:2 phosphatidylcholine plasmalogen had the strongest statistical associations (OR = 9.84; 95% CI, 2.89–37.82; P < 0.001/adjusted P = 0.07; and OR = 0.11; 95% CI, 0.03–0.35; P < 0.001/adjusted P = 0.06, respectively). Five WGCNA modules and 9 classes were associated with risk overall at FDR ≤ 0.20. Triacylglycerols (TAG) showed heterogeneity by tumor aggressiveness (case-only heterogeneity P < 0.0001). The TAG association with risk overall and serous tumors differed by acyl carbon content and saturation. In summary, this study suggests that pseudouridine may be a novel risk factor for ovarian cancer and that TAGs may also be important, particularly for rapidly fatal tumors, with associations differing by structural features. Significance: Pseudouridine represents a potential novel risk factor for ovarian cancer and triglycerides may be important particularly in rapidly fatal ovarian tumors. more...

BackgroundOvarian cancer has few known risk factors, hampering identification of high-risk women. Thus, we assessed the association of pre-diagnostic plasma metabolites (N=420) with risk.nnMethodsWe included 252 cases (176 serous/poorly differentiated; 34 non-serous [endometrioid/clear cell]) diagnosed 3-23 years after blood collection and 252 matched controls from the Nurses Health Studies. Multivariable logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) comparing the 90th-10thpercentile in metabolite levels, using permutation tests to account for testing multiple correlated hypotheses. Weighted gene co-expression network analysis (WGCNA) modules (n=10) summarized by the first principal component and metabolite set enrichment analysis (MSEA; n=23) were also evaluated.nnResultsPseudouridine had the strongest statistical association with ovarian cancer risk overall (OR=2.56, 95%CI=1.48-4.45; p=0.001/adjusted-p=0.15). C36:2 phosphatidylcholine (PC) plasmalogen had the strongest statistical association with lower risk (OR=0.11, 95%CI=0.03-0.35; p more...

Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize β4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell β4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition., Inhibition of PI3K/mTOR, which mimics nutrient starvation, causes death of detached but not matrix-attached cancer cells. Here the authors show that nutrient restriction of epithelial cells causes uptake of the matrix protein laminin, which results in increased intracellular amino acids and enhanced mTORC1 signalling. more...

SummaryAdvances in metabolomics technologies have enabled comprehensive analyses of associations between metabolites and human disease and have provided a means to study biochemical pathways and processes in detail using model systems. Liquid chromatography tandem mass spectrometry (LC-MS) is an analytical technique commonly used by metabolomics labs to measure hundreds of metabolites of known identity and thousands of “peaks” from yet to be identified compounds that are tracked by their measured masses and chromatographic retention times. netome is a computational framework that provides tools for analyzing processed LC-MS data. In this framework, we develop and provide various computational resources including individual software modules to inspect and adjust trends in raw data, align unknown peaks between separately acquired data sets, and to remove redundancies in nontargeted LC-MS data arising from multiple ionization products of a single metabolite. These tools are deployed through computing resources such as web servers and virtual machines with detailed documentation in order to support researchers.Availability and implementationnetome is publicly available with extensive documentation and support via issue tracker at https://broadinstitute.github.io/netome under the MIT license. netome includes a set of computational methods that have been designed to execute quality control and post-raw data processing tasks for metabolomics data (e.g. scaling and clustering metabolite abundances), as well as statistical association testing in a network manner (e.g., testing relationship between metabolites and microbes). Each individual tool is available with source code, workshop-oriented documentation which includes instructions for installation and using tools with demonstration examples, and a web server with all services. We also provide a complete image of the netome package with all pre-installed dependencies and support for Google Compute Engine and Amazon EC2. All tools and related services are maintained, and upon new developments, new modules will be added to the environment.Contactrah@broadinstitute.org, clary@broadinstitute.orgSupplementary informationSupplementary data are available at Bioinformatics online. more...

Non–small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC. more...

Summary Sphingolipids are structural membrane components and important eukaryotic signaling molecules. Sphingolipids regulate inflammation and immunity and were recently identified as the most differentially abundant metabolite in stool from inflammatory bowel disease (IBD) patients. Commensal bacteria from the Bacteroidetes phylum also produce sphingolipids, but the impact of these metabolites on host pathways is largely uncharacterized. To determine whether bacterial sphingolipids modulate intestinal health, we colonized germ-free mice with a sphingolipid-deficient Bacteroides thetaiotaomicron strain. A lack of Bacteroides-derived sphingolipids resulted in intestinal inflammation and altered host ceramide pools in mice. Using lipidomic analysis, we described a sphingolipid biosynthesis pathway and revealed a variety of Bacteroides-derived sphingolipids including ceramide phosphoinositol and deoxy-sphingolipids. Annotating Bacteroides sphingolipids in an IBD metabolomic dataset revealed lower abundances in IBD and negative correlations with inflammation and host sphingolipid production. These data highlight the role of bacterial sphingolipids in maintaining homeostasis and symbiosis in the gut. more...

Obesity is a rising pandemic that is increasingly being recognized for its striking correlation with the incidence of/mortality from cancers of different tissues, particularly that of the pancreas. Ranking as the fourth leading cause of cancer death in the United States, pancreatic ductal adenocarcinoma (PDAC) is a highly lethal, insidious malignancy whose poor prognosis is reflected in a 5-year survival rate of only 5%. In an obese state characterized by elevated circulating levels of insulin and insulin-like growth factor-1 (IGF-1), metabolic signaling pathways, including PI3K/Akt might be aberrantly regulated, altering tumor cell metabolism and driving the maintenance of PDAC. Recognized as a hallmark of cancer formation, altered cellular metabolism is evident in Kras-driven PDAC tumors. However, the specific metabolic impact of obesity and increased insulin and IGF-1 levels on PDAC tumors has not been thoroughly investigated. We performed a large orthotopic xenograft experiment involving the injection of human pancreatic cancer cells modified to either express constitutively active, myristoylated Akt, or a control fluorescent protein (CFP) into the pancreata of lean or diet-induced obese immunodeficient mice. We find that control CFP-tumors grown in obese mice and all Akt-tumors, independent of obesity, are significantly larger than CFP-tumors grown in lean mice. Gene expression and metabolomics analyses demonstrate a striking representation of distinct metabolic pathways, including the nitrogen/arginine or urea cycle pathway in pancreatic tumors grown in obese versus lean mice. This pathway enrichment partially overlaps with that observed in the Akt-driven tumors, independent of obesity. Here we describe the validation of these results by altering urea cycle gene expression in the human PDAC cells and assessing their effect on PDAC cell proliferation, survival, metabolism, and response to arginine deprivation both in tissue culture in vitro and as orthotopic xenografts in the pancreata of lean or diet-induced obese mice in vivo. These studies will help identify metabolic dependencies in Akt-driven or non-Akt-driven pancreatic tumors grown under either obese/hyperinsulinemic or lean states, that could be targeted therapeutically in PDAC patients. Citation Format: Tamara Zaytouni, Cory Dubois, Daniel Hitchcock, Nicholas Stylopoulos, Clary Clish, Nada Kalaany. Identifying metabolic liabilities in obesity-associated pancreatic cancer. [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 IA26. more...