PDF file - 325K, Nuclear p53 expression correlates positively with loss of wild type allele in serous epithelial ovarian carcinomas in p53m/+ mutant mice

mRNA expression of metabolic genes in RWPE-AKT1 or RWPE-MYC vs. RWPE-EV cells (A), or human prostate tumors relative to a normal prostate tissue pool (B). (C) FASN levels in RWPE-AKT1, RWPE-MYC and control (RWPE-EV) cells, and tumors vs. normal prostate tissues are shown. Association of high (dark grey)/low (white) GLUT-1 with phosphoAKT1 expression (p<0.05) (D) Diagram depicting all analyzed enzyme transcripts within the related metabolic pathways.

Table S1. List of gene expression assays (Applied Biosystems).

Supplementary Data from A Genetically Encoded Multifunctional TRAIL Trimer Facilitates Cell-Specific Targeting and Tumor Cell Killing

PDF file - 267K, Widespread metabolomic alterations consistent with tumor development are found in blood of SEOC mice compared to wild type mice

PDF file - 161K, Analysis of recombination efficiency of targeted alleles in mutant mice

PDF file - 307K, lntrabursal injection of Adeno-Cre activates conditional alleles in OSE cells and induces histopathological changes of murine OSE post induction

PDF file - 253K, Gene expression analysis

PDF file - 53K, Tumor spectrum in Adeno-Cre injected mice

PDF file - 255K, Immunohistochemical staining for CA125, WT1 and calretinin

Real-time dose-response curves for fructose have been non-invasively determined in primary rat hepatocyte alginate spheroids cultured in a NMR-compatible fluidized-bed bioreactor. Using 13C-labeled glucose and glycine culture medium, fructose dose was compared to glucose uptake and glycogen synthesis rate using 13C NMR spectroscopy, and to ATP and fructose-1-phosphate concentration using 31P NMR spectroscopy. A highly efficient multicoaxial perfusion system maintains high density 3-D hepatocyte cultures, permitting 13C and 31P NMR spectral time courses with 1min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16min, glycogen plummeted, lactate became the largest 13C-glucose metabolite via anaerobic glycolysis, while glutathione was the largest 13C-glycine metabolite. ATP depletion and fructose-1-phosphate formation demonstrated a dose response with a 3h EC50 of 19mM±8.9mM and 17.4mM±3.7mM, respectively. Computational modeling of mass transfer corroborated experimental results and helped determine the optimal bioreactor loading densities, oxygen concentration, and perfusion rates to maintain physiologically-relevant nutrient levels. The total bioreactor plus perfusion loop has a dead volume of 2ml, and contains 5 million hepatocytes. Due to the non-invasive measurements, there is a reduction of animal tissue by an order-of-magnitude, depending on the number of time points in an experiment. This dynamic flux approach may have generic utility for dose-response studies monitoring multiple metabolic reactions in other primary mammalian cells, such as human, that have strict oxygen demands.

Metabolomics, the quantification of small biochemicals in plasma and tissues, can provide insight into complex biochemical processes and enable the identification of biomarkers that may serve as therapeutic targets. We hypothesized that the plasma metabolome of asthma would reveal metabolic consequences of the specific immune and inflammatory responses unique to endotypes of asthma. The plasma metabolomic profiles of 20 asthmatic subjects and 10 healthy controls were examined using an untargeted global and focused metabolomic analysis. Individuals were classified based on clinical definitions of asthma severity or by levels of fraction of exhaled NO (FENO), a biomarker of airway inflammation. Of the 293 biochemicals identified in the plasma, 25 were significantly different among asthma and healthy controls (p < 0.05). Plasma levels of taurine, lathosterol, bile acids (taurocholate and glycodeoxycholate), nicotinamide, and adenosine-5-phosphate were significantly higher in asthmatics compared with healthy controls. Severe asthmatics had biochemical changes related to steroid and amino acid/protein metabolism. Asthmatics with high FENO, compared with those with low FENO, had higher levels of plasma branched-chain amino acids and bile acids. Asthmatics have a unique plasma metabolome that distinguishes them from healthy controls and points to activation of inflammatory and immune pathways. The severe asthmatic and high FENO asthmatic have unique endotypes that suggest changes in NO-associated taurine transport and bile acid metabolism.

A systems biology approach was used to comprehensively examine the impact of renal disease and hemodialysis (HD) on patient response during critical illness. To achieve this, we examined the metabolome, proteome, and transcriptome of 150 patients with critical illness, stratified by renal function. Quantification of plasma metabolites indicated greater change as renal function declined, with the greatest derangements in patients receiving chronic HD. Specifically, 6 uremic retention molecules, 17 other protein catabolites, 7 modified nucleosides, and 7 pentose phosphate sugars increased as renal function declined, consistent with decreased excretion or increased catabolism of amino acids and ribonucleotides. Similarly, the proteome showed increased levels of low-molecular-weight proteins and acute-phase reactants. The transcriptome revealed a broad-based decrease in mRNA levels among patients on HD. Systems integration revealed an unrecognized association between plasma RNASE1 and several RNA catabolites and modified nucleosides. Further, allantoin, N1-methyl-4-pyridone-3-carboxamide, and N-acetylaspartate were inversely correlated with the majority of significantly downregulated genes. Thus, renal function broadly affected the plasma metabolome, proteome, and peripheral blood transcriptome during critical illness; changes were not effectively mitigated by hemodialysis. These studies allude to several novel mechanisms whereby renal dysfunction contributes to critical illness.

INTRODUCTION:There is a critical need to develop clinical laboratory assays that provide risk assessment for men at elevated risk for prostate cancer, and once diagnosed, could further identify those men with clinically significant disease.METHODS:Recent advancements in analytical instrumentation have enabled mass spectrometry-based metabolomics methodologies. Further advancements in chromatographic techniques have facilitated high throughput, quantitative assays for a broad spectrum of biochemicals.RESULTS:Screening metabolomics techniques have been applied to biospecimens from large cohorts of men comparing those individuals with prostate cancer to those with no evidence of malignancy. Work beginning in tissues has identified biochemical profiles that correlate with disease and disease severity, including tumor grade and stage. Some of these metabolic abnormalities, such as dramatic elevations in sarcosine, have been found to translate into biological fluids, especially blood and urine, which can be sampled in a minimally invasive manner.DISCUSSION:The differential abundances of these tumor-associated metabolites have been found to improve the performance of clinical prognostic/diagnostic tools.CONCLUSION:The outlook is bright for metabolomic technology to address clinical diagnostic needs for prostate cancer patient management. Early validation of specific clinical tests provides a preview of further successes in this area. Metabolomics has shown its utility to complement and augment traditional clinical approaches as well as emerging genomic, transcriptomic and proteomic methodologies, and is expected to play a key role in the precision medicine-based management of the prostate cancer patient.

Bladder cancer (BCa) is a common malignancy worldwide and has a high probability of recurrence after initial diagnosis and treatment. As a result, recurrent surveillance, primarily involving repeated cystoscopies, is a critical component of post diagnosis patient management. Since cystoscopy is invasive, expensive and a possible deterrent to patient compliance with regular follow-up screening, new non-invasive technologies to aid in the detection of recurrent and/or primary bladder cancer are strongly needed. In this study, mass spectrometry based metabolomics was employed to identify biochemical signatures in human urine that differentiate bladder cancer from non-cancer controls. Over 1000 distinct compounds were measured including 587 named compounds of known chemical identity. Initial biomarker identification was conducted using a 332 subject sample set of retrospective urine samples (cohort 1), which included 66 BCa positive samples. A set of 25 candidate biomarkers was selected based on statistical significance, fold difference and metabolic pathway coverage. The 25 candidate biomarkers were tested against an independent urine sample set (cohort 2) using random forest analysis, with palmitoyl sphingomyelin, lactate, adenosine and succinate providing the strongest predictive power for differentiating cohort 2 cancer from non-cancer urines. Cohort 2 metabolite profiling revealed additional metabolites, including arachidonate, that were higher in cohort 2 cancer vs. non-cancer controls, but were below quantitation limits in the cohort 1 profiling. Metabolites related to lipid metabolism may be especially interesting biomarkers. The results suggest that urine metabolites may provide a much needed non-invasive adjunct diagnostic to cystoscopy for detection of bladder cancer and recurrent disease management.

Mammalian skeletal muscles exhibit age-related adaptive and pathological remodeling. Several muscles in particular undergo progressive atrophy and degeneration beyond median lifespan. To better understand myocellular responses to aging, we used semi-quantitative global metabolomic profiling to characterize trends in metabolic changes between 15-month-old adult and 32-month-old aged Fischer 344 × Brown Norway (FBN) male rats. The FBN rat gastrocnemius muscle exhibits age-dependent atrophy, whereas the soleus muscle, up until 32 months, exhibits markedly fewer signs of atrophy. Both gastrocnemius and soleus muscles were analyzed, as well as plasma and urine. Compared to adult gastrocnemius, aged gastrocnemius showed evidence of reduced glycolytic metabolism, including accumulation of glycolytic, glycogenolytic, and pentose phosphate pathway intermediates. Pyruvate was elevated with age, yet levels of citrate and nicotinamide adenine dinucleotide were reduced, consistent with mitochondrial abnormalities. Indicative of muscle atrophy, 3-methylhistidine and free amino acids were elevated in aged gastrocnemius. The monounsaturated fatty acids oleate, cis-vaccenate, and palmitoleate also increased in aged gastrocnemius, suggesting altered lipid metabolism. Compared to gastrocnemius, aged soleus exhibited far fewer changes in carbohydrate metabolism, but did show reductions in several glycolytic intermediates, fumarate, malate, and flavin adenine dinucleotide. Plasma biochemicals showing the largest age-related increases included glycocholate, heme, 1,5-anhydroglucitol, 1-palmitoleoyl-glycerophosphocholine, palmitoleate, and creatine. These changes suggest reduced insulin sensitivity in aged FBN rats. Altogether, these data highlight skeletal muscle group-specific perturbations of glucose and lipid metabolism consistent with mitochondrial dysfunction in aged FBN rats.

Most of the research on tumor cell metabolism has focused on glucose utilization. However, when glucose is limited, solid tumors are forced to catabolize alternative substrates such as fatty acids and amino acids as an energy source. Measuring these alternations in tumor cell metabolism enables us to track neoplastic changes in the tissue to lead towards a more reliable diagnostic outcome. Although a very small number of elements are used in biochemistry, the metabolome is structurally diverse for the production of simple compounds such as phosphate and amino acids as well as more structurally complex compounds such as nucleotides, oligosaccharides, and complex lipids. Characterization of the metabolome, therefore, requires analytical methods that can handle a wide range of molecular structures and physicochemical properties, including solubility, polarity, and molecular weight. A further factor for consideration in the selection of technology for metabolomics is the wide range of concentrations of biochemical typically present in biological systems. MS has established itself as the high-throughput, information-rich, industrially stable approach to assess both the composition of diverse sample types as well as changes to that composition following perturbation.

BACKGROUND:Current diagnostic techniques have increased the detection of prostate cancer; however, these tools inadequately stratify patients to minimize mortality. Recent studies have identified a biochemical signature of prostate cancer metastasis, including increased sarcosine abundance. This study examined the association of tissue metabolites with other clinically significant findings. METHODS:A state of the art metabolomics platform analyzed prostatectomy tissues (331 prostate tumor, 178 cancer-free prostate tissues) from two independent sites. Biochemicals were analyzed by gas chromatography-mass spectrometry and ultrahigh performance liquid chromatography-tandem mass spectrometry. Statistical analyses identified metabolites associated with cancer aggressiveness: Gleason score, extracapsular extension, and seminal vesicle and lymph node involvement. RESULTS:Prostate tumors had significantly altered metabolite profiles compared to cancer-free prostate tissues, including biochemicals associated with cell growth, energetics, stress, and loss of prostate-specific biochemistry. Many metabolites were further associated with clinical findings of aggressive disease. Aggressiveness-associated metabolites stratified prostate tumor tissues with high abundances of compounds associated with normal prostate function (e.g., citrate and polyamines) from more clinically advanced prostate tumors. These aggressive prostate tumors were further subdivided by abundance profiles of metabolites including NAD+ and kynurenine. When added to multiparametric nomograms, metabolites improved prediction of organ confinement (AUROC from 0.53 to 0.62) and 5-year recurrence (AUROC from 0.53 to 0.64). CONCLUSIONS:These findings support and extend earlier metabolomic studies in prostate cancer and studies where metabolic enzymes have been associated with carcinogenesis and/or outcome. Furthermore, these data suggest that panels of analytes may be valuable to translate metabolomic findings to clinically useful diagnostic tests.

Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. Inhibiting the signaling of the transcription factor nuclear factor kappa B (NF-κB) largely prevents cancer-induced muscle wasting in murine models. We have previously shown the utility of Compound A, a highly selective novel NF-κB inhibitor that targets the IκB kinase complex, to provide clinical benefit in cancer-induced skeletal muscle and cardiac atrophy.