Soumen K. Manna

Transcription factor NRF2 regulates miR-1 and miR-206 to drive tumorigenesis

The mechanisms by which deregulated nuclear factor erythroid-2-related factor 2 (NRF2) and kelch-like ECH-associated protein 1 (KEAP1) signaling promote cellular proliferation and tumorigenesis are poorly understood. Using an integrated genomics and ¹³C-based targeted tracer fate association (TTFA) study, we found that NRF2 regulates miR-1 and miR-206 to direct carbon flux toward the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle, reprogramming glucose metabolism. Sustained activation of NRF2 signaling in cancer cells attenuated miR-1 and miR-206 expression, leading to enhanced expression of PPP genes. Conversely, overexpression of miR-1 and miR-206 decreased the expression of metabolic genes and dramatically impaired NADPH production, ribose synthesis, and in vivo tumor growth in mice. Loss of NRF2 decreased the expression of the redox-sensitive histone deacetylase, HDAC4, resulting in increased expression of miR-1 and miR-206, and not only inhibiting PPP expression and activity but functioning as a regulatory feedback loop that repressed HDAC4 expression. In primary tumor samples, the expression of miR-1 and miR-206 was inversely correlated with PPP gene expression, and increased expression of NRF2-dependent genes was associated with poor prognosis. Our results demonstrate that microRNA-dependent (miRNA-dependent) regulation of the PPP via NRF2 and HDAC4 represents a novel link between miRNA regulation, glucose metabolism, and ROS homeostasis in cancer cells.

Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells

AbstractIn this study, a liquid chromatography mass spectrometry (LC/MS)-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the human pancreatic cancer cell line Panc-1. Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in water were compared for sample harvesting. Four different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) with UPLC-QTOF-MS were also evaluated. Global metabolomics profiles were compared; the number of total detected features and the recovery and relative extraction efficiencies of target metabolites were assessed. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen to harvest Panc-1 cells which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was chosen as the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method therefore provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further LC/MS-based global metabolomics study on Panc-1 cell lines and possibly other cancer cell lines with similar chemical and physical properties. FigureOptimized harvesting, extraction and analytical protocols for cell metabolomics analysis.

Noninvasive Urinary Metabolomic Profiling Identifies Diagnostic and Prognostic Markers in Lung Cancer

Lung cancer remains the most common cause of cancer deaths worldwide, yet there is currently a lack of diagnostic noninvasive biomarkers that could guide treatment decisions. Small molecules (<1,500 Da) were measured in urine collected from 469 patients with lung cancer and 536 population controls using unbiased liquid chromatography/mass spectrometry. Clinical putative diagnostic and prognostic biomarkers were validated by quantitation and normalized to creatinine levels at two different time points and further confirmed in an independent sample set, which comprises 80 cases and 78 population controls, with similar demographic and clinical characteristics when compared with the training set. Creatine riboside (IUPAC name: 2-{2-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-oxolan-2-yl]-1-methylcarbamimidamido}acetic acid), a novel molecule identified in this study, and N-acetylneuraminic acid (NANA) were each significantly (P < 0.00001) elevated in non-small cell lung cancer and associated with worse prognosis [HR = 1.81 (P = 0.0002), and 1.54 (P = 0.025), respectively]. Creatine riboside was the strongest classifier of lung cancer status in all and stage I-II cases, important for early detection, and also associated with worse prognosis in stage I-II lung cancer (HR = 1.71, P = 0.048). All measurements were highly reproducible with intraclass correlation coefficients ranging from 0.82 to 0.99. Both metabolites were significantly (P < 0.03) enriched in tumor tissue compared with adjacent nontumor tissue (N = 48), thus revealing their direct association with tumor metabolism. Creatine riboside and NANA may be robust urinary clinical metabolomic markers that are elevated in tumor tissue and associated with early lung cancer diagnosis and worse prognosis.

UPLC-MS-based urine metabolomics reveals indole-3-lactic acid and phenyllactic acid as conserved biomarkers for alcohol-induced liver disease in the Ppara-null mouse model.

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine, were background-dependent. Elevation of ethyl-β-d-glucuronide and N-acetylglycine was found to be a common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Identification of Noninvasive Biomarkers for Alcohol-Induced Liver Disease Using Urinary Metabolomics and the Ppara-null Mouse

Alcohol-induced liver disease (ALD) is a leading cause of nonaccident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively nonspecific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study, the metabolic changes associated with ALD were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-beta-d-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of a metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

Disruption of Endothelial Peroxisome Proliferator-Activated Receptor γ Accelerates Diet-Induced Atherogenesis in LDL Receptor-Null Mice

Objective— Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) is widely expressed in vessel walls, and its activation by agonists showed beneficial effects in cardiovascular diseases. However, the role of endothelial cell (EC) PPAR&ggr; in atherogenesis is not fully understood. Methods and Results— To assess the contribution of endothelial-specific PPAR&ggr; in atherosclerosis, EC-specific PPAR&ggr; disruption and LDL receptor (LDLR) double-knockout (PPAR&ggr;&Dgr;EC/LDLR−/−) mice were developed. When challenged with a high-cholesterol diet for 4 weeks, PPAR&ggr;&Dgr;EC/LDLR−/− mice exhibited severe atherosclerotic lesions compared to either their littermate controls or macrophage-specific PPAR&ggr; disruption and LDLR double knockout (PPAR&ggr;&Dgr;M&PHgr;/LDLR−/−) mice. Metabolic analysis showed severe dyslipidemia and significant increase in systolic blood pressure in the PPAR&ggr;&Dgr;EC/LDLR−/− mice. Histological analysis and real-time quantitative PCR suggested an exacerbated inflammation in PPAR&ggr;&Dgr;EC/LDLR−/− mice, as revealed by the increases of proinflammatory gene expression and macrophage infiltration in vivo and in vitro. Furthermore, in vivo endothelial permeability was also increased by endothelial PPAR&ggr; disruption. Bone-marrow transplantation studies, which reconstituted hematopoietic PPAR&ggr;, demonstrated that the accelerated atherogenesis was due to endothelial PPAR&ggr; deficiency. Conclusion— Endothelial PPAR&ggr; plays an important protective role in atherogenesis.

Biomarkers of Coordinate Metabolic Reprogramming in Colorectal Tumors in Mice and Humans

BACKGROUND & AIMS There are no robust noninvasive methods for colorectal cancer screening and diagnosis. Metabolomic and gene expression analyses of urine and tissue samples from mice and humans were used to identify markers of colorectal carcinogenesis. METHODS Mass spectrometry-based metabolomic analysis of urine and tissues from wild-type C57BL/6J and Apc(Min/+) mice, as well as from mice with azoxymethane-induced tumors, was employed in tandem with gene expression analysis. Metabolic profiling was also performed on colon tumor and adjacent nontumor tissues from 39 patients. The effects of β-catenin activity on metabolic profiles were assessed in mice with colon-specific disruption of Apc. RESULTS Thirteen markers were found in urine associated with development of colorectal tumors in Apc(Min/+) mice. Metabolites related to polyamine metabolism, nucleic acid metabolism, and methylation, identified tumor-bearing mice with 100% accuracy, and also accurately identified mice with polyps. Changes in gene expression in tumor samples from mice revealed that derangement of metabolites were a reflection of coordinate metabolic reprogramming in tumor tissue. Similar changes in urinary metabolites were observed in mice with azoxymethane-induced tumors and in mice with colon-specific activation of β-catenin. The metabolic alterations indicated by markers in urine, therefore, appear to occur during early stages of tumorigenesis, when cancer cells are proliferating. In tissues from patients, tumors had stage-dependent increases in 17 metabolites associated with the same metabolic pathways identified in mice. Ten metabolites that were increased in tumor tissues, compared with nontumor tissues (proline, threonine, glutamic acid, arginine, N1-acetylspermidine, xanthine, uracil, betaine, symmetric dimethylarginine, and asymmetric-dimethylarginine), were also increased in urine from tumor-bearing mice. CONCLUSIONS Gene expression and metabolomic profiles of urine and tissue samples from mice with colorectal tumors and of colorectal tumor samples from patients revealed pathways associated with derangement of specific metabolic pathways that are indicative of early-stage tumor development. These urine and tissue markers might be used in early detection of colorectal cancer.

Global Metabolomics Reveals Urinary Biomarkers of Breast Cancer in a MCF-7 Xenograft Mouse Model

Global metabolomics analysis has the potential to uncover novel metabolic pathways that are differentially regulated during carcinogenesis, aiding in biomarker discovery for early diagnosis and remission monitoring. Metabolomics studies with human samples can be problematic due to high inter-individual variation; however xenografts of human cancers in mice offer a well-controlled model system. Urine was collected from a xenograft mouse model of MCF-7 breast cancer and analyzed by mass spectrometry-based metabolomics to identify metabolites associated with cancer progression. Over 10 weeks, 24 h urine was collected weekly from control mice, mice dosed with estradiol cypionate (1 mg/mL), mice inoculated with MCF-7 cells (1 × 107) and estradiol cypionate (1 mg/mL), and mice dosed with MCF-7 cells (1 × 107) only (n = 10/group). Mice that received both estradiol cypionate and MCF-7 cells developed tumors from four weeks after inoculation. Five urinary metabolites were identified that were associated with breast cancer; enterolactone glucuronide, coumaric acid sulfate, capric acid glucuronide, an unknown metabolite, and a novel mammalian metabolite, “taurosebacic acid”. These metabolites revealed a correlation between tumor growth, fatty acid synthesis, and potential anti-proliferative effects of gut microbiota-metabolized food derivatives. These biomarkers may be of value for early diagnosis of cancer, monitoring of cancer therapeutics, and may also lead to future mechanistic studies.

Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway

The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2–related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD.

Application of Mass Spectrometry-Based Metabolomics in Identification of Early Noninvasive Biomarkers of Alcohol-Induced Liver Disease Using Mouse Model

A rapid, non-invasive urine test for early stage alcohol-induced liver disease (ALD) would permit risk stratification and treatment of high-risk individuals before ALD leads to irreversible liver damage and death. Urinary metabolomic studies were carried out to identify ALD-associated metabolic biomarkers using Ppara-null mouse model that is susceptible to ALD development on chronic alcohol consumption. Two successive studies were conducted to evaluate the applicability of mass spectrometry-based metabolomics in identification of ALD-specific signatures and to examine the robustness of these biomarkers against genetic background. Principal components analysis of ultraperformance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS)-generated urinary metabolic fingerprints showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals. It also showed that a combined endogenous biomarker panel helps to identify subjects with ALD as well as those at risk of developing ALD even without any information on alcohol intake or genetics. Quantitative analysis showed that increased excretion of indole-3-lactic acid and phenyllactic acid was a genetic background-independent signature exclusively associated with ALD pathogenesis in Ppara-null mice that showed liver pathologies similar to those observed in early stages of human ALD. These findings demonstrated that mass spectrometry-based metabolomic analysis could help in the identification of ALD-specific signatures, and that metabolites such as indole-3-lactic acid and phenyllactic acid, may serve as robust noninvasive biomarkers for early stages of ALD.