Untargeted LC-HRMS-based metabolomics to identify novel biomarkers of metastatic colorectal cancer

Colorectal cancer is one of the main causes of cancer death worldwide, and novel biomarkers are urgently needed for its early diagnosis and treatment. The utilization of metabolomics to identify and quantify metabolites in body fluids may allow the detection of changes in their concentrations that could serve as diagnostic markers for colorectal cancer and may also represent new therapeutic targets. Metabolomics generates a pathophysiological ‘fingerprint’ that is unique to each individual. The purpose of our study was to identify a differential metabolomic signature for metastatic colorectal cancer. Serum samples from 60 healthy controls and 65 patients with metastatic colorectal cancer were studied by liquid chromatography coupled to high-resolution mass spectrometry in an untargeted metabolomic approach. Multivariate analysis revealed a separation between patients with metastatic colorectal cancer and healthy controls, who significantly differed in serum concentrations of one endocannabinoid, two glycerophospholipids, and two sphingolipids. These findings demonstrate that metabolomics using liquid-chromatography coupled to high-resolution mass spectrometry offers a potent diagnostic tool for metastatic colorectal cancer.This study was supported by a grant (n° 15CC056/DTS17/00081- ISCIII-FEDER) from the Fundación para la Investigación Biosanitaria de Andalucía Oriental (FIBAO) and Roche Pharma S.L. Authors from the Fundación MEDINA acknowledge the receipt of financial support from this public-private partnership of Merck Sharp & Dohme de España S.A. with the University of Granada and Andalusian Regional Government (PIN-0474-2016)

17-allyamino-17-demethoxygeldanamycin treatment results in a magnetic resonance spectroscopy-detectable elevation in choline-containing metabolites associated with increased expression of choline transporter SLC44A1 and phospholipase A2

Abstract Introduction 17-allyamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is currently in clinical trials in breast cancer. However, 17-AAG treatment often results in inhibition of tumor growth rather than shrinkage, making detection of response a challenge. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are noninvasive imaging methods than can be used to monitor metabolic biomarkers of drug-target modulation. This study set out to examine the MRS-detectable metabolic consequences of Hsp90 inhibition in a breast cancer model. Methods MCF-7 breast cancer cells were investigated, and MRS studies were performed both on live cells and on cell extracts. 31P and 1H MRS were used to determine total cellular metabolite concentrations and 13C MRS was used to probe the metabolism of [1,2-13C]-choline. To explain the MRS metabolic findings, microarray and RT-PCR were used to analyze gene expression, and in vitro activity assays were performed to determine changes in enzymatic activity following 17-AAG treatment. Results Treatment of MCF-7 cells with 17-AAG for 48 hours caused a significant increase in intracellular levels of choline (to 266 ± 18% of control, P = 0.05) and phosphocholine (PC; to 181 ± 10% of control, P = 0.001) associated with an increase in expression of choline transporter SLC44A1 and an elevation in the de novo synthesis of PC. We also detected an increase in intracellular levels of glycerophosphocholine (GPC; to 176 ± 38% of control, P = 0.03) associated with an increase in PLA2 expression and activity. Conclusions This study determined that in the MCF-7 breast cancer model inhibition of Hsp90 by 17-AAG results in a significant MRS-detectable increase in choline, PC and GPC, which is likely due to an increase in choline transport into the cell and phospholipase activation. 1H MRSI can be used in the clinical setting to detect levels of total choline-containing metabolite (t-Cho, composed of intracellular choline, PC and GPC). As Hsp90 inhibitors enter routine clinical use, t-Cho could thus provide an easily detectable, noninvasive metabolic biomarker of Hsp90 inhibition in breast cancer patients

Metabolic assessment of the action of targeted cancer therapeutics using magnetic resonance spectroscopy

Developing rational targeted cancer drugs requires the implementation of pharmacodynamic (PD), preferably non-invasive, biomarkers to aid response assessment and patient follow-up. Magnetic resonance spectroscopy (MRS) allows the non-invasive study of tumour metabolism. We describe the MRS-detectable PD biomarkers resulting from the action of targeted therapeutics, and discuss their biological significance and future translation into clinical use

Bicarbonate and dichloroacetate: Evaluating pH altering therapies in a mouse model for metastatic breast cancer

BACKGROUND:The glycolytic nature of malignant tumors contributes to high levels of extracellular acidity in the tumor microenvironment. Tumor acidity is a driving force in invasion and metastases. Recently, it has been shown that buffering of extracellular acidity through systemic administration of oral bicarbonate can inhibit the spread of metastases in a mouse model for metastatic breast cancer. While these findings are compelling, recent assessments into the use of oral bicarbonate as a cancer intervention reveal limitations.METHODS:We posited that safety and efficacy of bicarbonate could be enhanced by dichloroacetate (DCA), a drug that selectively targets tumor cells and reduces extracellular acidity through inhibition of glycolysis. Using our mouse model for metastatic breast cancer (MDA-MB-231), we designed an interventional survival study where tumor bearing mice received bicarbonate, DCA, or DCA-bicarbonate (DB) therapies chronically.RESULTS:Dichloroacetate alone or in combination with bicarbonate did not increase systemic alkalosis in mice. Survival was longest in mice administered bicarbonate-based therapies. Primary tumor re-occurrence after surgeries is associated with survival rates. Although DB therapy did not significantly enhance oral bicarbonate, we did observe reduced pulmonary lesion diameters in this cohort. The DCA monotherapy was not effective in reducing tumor size or metastases or improving survival time. We provide in vitro evidence to suggest this outcome may be a function of hypoxia in the tumor microenvironment.CONCLUSIONS:DB combination therapy did not appear to enhance the effect of chronic oral bicarbonate. The anti-tumor effect of DCA may be dependent on the cancer model. Our studies suggest DCA efficacy is unpredictable as a cancer therapy and further studies are necessary to determine the role of this agent in the tumor microenvironment.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Time-dependent effects of imatinib in human leukaemia cells: a kinetic NMR-profiling study

The goal of this study was to evaluate the time course of metabolic changes in leukaemia cells treated with the Bcr-Abl tyrosine kinase inhibitor imatinib. Human Bcr-Abl+ K562 cells were incubated with imatinib in a dose-escalating manner (starting at 0.1 μM with a weekly increase of 0.1 μM imatinib) for up to 5 weeks. Nuclear magnetic resonance spectroscopy and liquid-chromatography mass spectrometry were performed to assess a global metabolic profile, including glucose metabolism, energy state, lipid metabolism and drug uptake, after incubation with imatinib. Initially, imatinib treatment completely inhibited the activity of Bcr-Abl tyrosine kinase, followed by the inhibition of cell glycolytic activity and glucose uptake. This was accompanied by the increased mitochondrial activity and energy production. With escalating imatinib doses, the process of cell death rapidly progressed. Phosphocreatine and NAD+ concentrations began to decrease, and mitochondrial activity, as well as the glycolysis rate, was further reduced. Subsequently, the synthesis of lipids as necessary membrane precursors for apoptotic bodies was accelerated. The concentrations of the Kennedy pathway intermediates, phosphocholine and phosphatidylcholine, were reduced. After 4 weeks of exposure to imatinib, the secondary necrosis associated with decrease in the mitochondrial and glycolytic activity occurred and was followed by a shutdown of energy production and cell death. In conclusion, monitoring of metabolic changes in cells exposed to novel signal transduction modulators supplements molecular findings and provides further mechanistic insights into longitudinal changes of the mitochondrial and glycolytic pathways of oncogenesis

Cellular changes in boric acid-treated DU-145 prostate cancer cells

Epidemiological, animal, and cell culture studies have identified boron as a chemopreventative agent in prostate cancer. The present objective was to identify boron-induced changes in the DU-145 human prostate cancer cell line. We show that prolonged exposure to pharmacologically-relevant levels of boric acid, the naturally occurring form of boron circulating in human plasma, induces the following morphological changes in cells: increases in granularity and intracellular vesicle content, enhanced cell spreading and decreased cell volume. Documented increases in β-galactosidase activity suggest that boric acid induces conversion to a senescent-like cellular phenotype. Boric acid also causes a dose-dependent reduction in cyclins A–E, as well as MAPK proteins, suggesting their contribution to proliferative inhibition. Furthermore, treated cells display reduced adhesion, migration and invasion potential, along with F-actin changes indicative of reduced metastatic potential. Finally, the observation of media acidosis in treated cells correlated with an accumulation of lysosome-associated membrane protein type 2 (LAMP-2)-negative acidic compartments. The challenge of future studies will be to identify the underlying mechanism responsible for the observed cellular responses to this natural blood constituent

Distinct choline metabolic profiles are associated with differences in gene expression for basal-like and luminal-like breast cancer xenograft models

<p>Abstract</p> <p>Background</p> <p>Increased concentrations of choline-containing compounds are frequently observed in breast carcinomas, and may serve as biomarkers for both diagnostic and treatment monitoring purposes. However, underlying mechanisms for the abnormal choline metabolism are poorly understood.</p> <p>Methods</p> <p>The concentrations of choline-derived metabolites were determined in xenografted primary human breast carcinomas, representing basal-like and luminal-like subtypes. Quantification of metabolites in fresh frozen tissue was performed using high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS).</p> <p>The expression of genes involved in phosphatidylcholine (PtdCho) metabolism was retrieved from whole genome expression microarray analyses.</p> <p>The metabolite profiles from xenografts were compared with profiles from human breast cancer, sampled from patients with estrogen/progesterone receptor positive (ER+/PgR+) or triple negative (ER-/PgR-/HER2-) breast cancer.</p> <p>Results</p> <p>In basal-like xenografts, glycerophosphocholine (GPC) concentrations were higher than phosphocholine (PCho) concentrations, whereas this pattern was reversed in luminal-like xenografts. These differences may be explained by lower choline kinase (<it>CHKA</it>, <it>CHKB</it>) expression as well as higher PtdCho degradation mediated by higher expression of phospholipase A2 group 4A (<it>PLA2G4A</it>) and phospholipase B1 (<it>PLB1</it>) in the basal-like model. The glycine concentration was higher in the basal-like model. Although glycine could be derived from energy metabolism pathways, the gene expression data suggested a metabolic shift from PtdCho synthesis to glycine formation in basal-like xenografts. In agreement with results from the xenograft models, tissue samples from triple negative breast carcinomas had higher GPC/PCho ratio than samples from ER+/PgR+ carcinomas, suggesting that the choline metabolism in the experimental models is representative for luminal-like and basal-like human breast cancer.</p> <p>Conclusions</p> <p>The differences in choline metabolite concentrations corresponded well with differences in gene expression, demonstrating distinct metabolic profiles in the xenograft models representing basal-like and luminal-like breast cancer. The same characteristics of choline metabolite profiles were also observed in patient material from ER+/PgR+ and triple-negative breast cancer, suggesting that the xenografts are relevant model systems for studies of choline metabolism in luminal-like and basal-like breast cancer.</p

Differential Role of Human Choline Kinase α and β Enzymes in Lipid Metabolism: Implications in Cancer Onset and Treatment

11 pages, 6 figures, 1 table.Background The Kennedy pathway generates phosphocoline and phosphoethanolamine through its two branches. Choline Kinase (ChoK) is the first enzyme of the Kennedy branch of synthesis of 1phosphocholine, the major component of the plasma membrane. ChoK family of proteins is composed by ChoKα and ChoKβ isoforms, the first one with two different variants of splicing. Recently ChoKα has been implicated in the carcinogenic process, since it is over-expressed in a variety of human cancers. However, no evidence for a role of ChoKβ in carcinogenesis has been reported. Methodology/Principal Findings Here we compare the in vitro and in vivo properties of ChoKα1 and ChoKβ in lipid metabolism, and their potential role in carcinogenesis. Both ChoKα1 and ChoKβ showed choline and ethanolamine kinase activities when assayed in cell extracts, though with different affinity for their substrates. However, they behave differentially when overexpressed in whole cells. Whereas ChoKβ display an ethanolamine kinase role, ChoKα1 present a dual choline/ethanolamine kinase role, suggesting the involvement of each ChoK isoform in distinct biochemical pathways under in vivo conditions. In addition, while overexpression of ChoKα1 is oncogenic when overexpressed in HEK293T or MDCK cells, ChoKβ overexpression is not sufficient to induce in vitro cell transformation nor in vivo tumor growth. Furthermore, a significant upregulation of ChoKα1 mRNA levels in a panel of breast and lung cancer cell lines was found, but no changes in ChoKβ mRNA levels were observed. Finally, MN58b, a previously described potent inhibitor of ChoK with in vivo antitumoral activity, shows more than 20-fold higher efficiency towards ChoKα1 than ChoKβ. Conclusion/Significance This study represents the first evidence of the distinct metabolic role of ChoKα and ChoKβ isoforms, suggesting different physiological roles and implications in human carcinogenesis. These findings constitute a step forward in the design of an antitumoral strategy based on ChoK inhibition.This work has been supported by grants to JCL from Comunidad de Madrid (GR-SAL-0821-2004), Ministerio de Ciencia e Innovación (SAF2008-03750, RD06/0020/0016), Fundación Mutua Madrileña, and by a grant to ARM from Fundación Mutua Madrileña.Peer reviewe