Helen Troy

Proteomic and Metabolomic Analyses of Atherosclerotic Vessels From Apolipoprotein E-Deficient Mice Reveal Alterations in Inflammation, Oxidative Stress, and Energy Metabolism

Objective—Proteomics and metabolomics are emerging technologies to study molecular mechanisms of diseases. We applied these techniques to identify protein and metabolite changes in vessels of apolipoprotein E−/− mice on normal chow diet. Methods and Results—Using 2-dimensional gel electrophoresis and mass spectrometry, we identified 79 protein species that were altered during various stages of atherogenesis. Immunoglobulin deposition, redox imbalance, and impaired energy metabolism preceded lesion formation in apolipoprotein E−/− mice. Oxidative stress in the vasculature was reflected by the oxidation status of 1-Cys peroxiredoxin and correlated to the extent of lesion formation in 12-month-old apolipoprotein E−/− mice. Nuclear magnetic resonance spectroscopy revealed a decline in alanine and a depletion of the adenosine nucleotide pool in vessels of 10-week-old apolipoprotein E−/− mice. Attenuation of lesion formation was associated with alterations of NADPH generating malic enzyme, which provides reducing equivalents for lipid synthesis and glutathione recycling, and successful replenishment of the vascular energy pool. Conclusion—Our study provides the most comprehensive dataset of protein and metabolite changes during atherogenesis published so far and highlights potential associations of immune-inflammatory responses, oxidative stress, and energy metabolism.

Noninvasive magnetic resonance spectroscopic pharmacodynamic markers of the choline kinase inhibitor MN58b in human carcinoma models

MN58b is a novel anticancer drug that inhibits choline kinase, resulting in inhibition of phosphocholine synthesis. The aim of this work was to develop a noninvasive and robust pharmacodynamic biomarker for target inhibition and, potentially, tumor response following MN58b treatment. Human HT29 (colon) and MDA-MB-231 (breast) carcinoma cells were examined by proton (1H) and phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment with MN58b both in culture and in xenografts. An in vitro time course study of MN58b treatment was also carried out in MDA-MB-231 cells. In addition, enzymatic assays of choline kinase activity in cells were done. A decrease in phosphocholine and total choline levels (P < 0.05) was observed in vitro in both cell lines after MN58b treatment, whereas the inactive analogue ACG20b had no effect. In MDA-MB-231 cells, phosphocholine fell significantly as early as 4 hours following MN58b treatment, whereas a drop in cell number was observed at 48 hours. Significant correlation was also found between phosphocholine levels (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008) following MN58b treatment. Phosphomonoesters also decreased significantly (P < 0.05) in both HT29 and MDA-MB-231 xenografts with no significant changes in controls. 31P-MRS and 1H-MRS of tumor extracts showed a significant decrease in phosphocholine (P < or = 0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors.

Glut-1 as a therapeutic target: increased chemoresistance and HIF-1-independent link with cell turnover is revealed through COMPARE analysis and metabolomic studies

The facilitative glucose transporter Glut-1 is overexpressed and confers poor prognosis in a wide range of solid tumours. The peri-necrotic pattern of expression often seen in human tumour samples is linked with its transcriptional control in hypoxic conditions by hypoxia-inducible factor HIF-1 or through a reduced rate of oxidative phosphorylation. Hypoxia-regulated genes offer promise as novel therapeutic targets as a means of preventing the proliferation and eventual metastatic spread of tissue originating from residual chemically and radio resistant hypoxic cells that have survived treatment. Inhibiting the expression or functionality of Glut-1 may be a way of specifically targeting hypoxic cells within the tumour that depend upon a high rate of glucose uptake for anaerobic glycolysis. We used an array of formalin-fixed, paraffin-embedded samples of the NCI-60 panel of cell lines to carry out immunohistochemical detection of Glut-1 and to select possible candidate lead compounds by COMPARE analysis with agents from the NCI diversity screen, which may work via inhibition of Glut-1 or Glut-1-dependent processes. “Positive” COMPARE hits were mostly conjugated Pseudomonas toxins binding the epidermal growth factor receptor (EGFR). However, correlations with standard anticancer agents were virtually all negative, indicating a link between Glut-1 and chemoresistance. MTT proliferation assays carried out using stable, Glut-1 overexpressing cell lines generated from the bladder EJ138, human fibrosarcoma HT 1080 and the hepatoma wild type Hepa and HIF-1B-deficient c4 tumour cell lines revealed a cell line-dependent increase in chemoresistance to dacarbazine, vincristine and the bioreductive agent EO9 in Glut-1 overexpressing EJ138 relative to WT and empty vector controls. Metabolomic analysis (31P-MRS and 1H MRS) carried out using cell lysates and xenografts generated from Glut-1 overexpressing Hepa and c4 cell lines showed higher glucose levels in Glut-1 overxpressing c4 relative to parental tumour extracts occurred in the absence of an increase in lactate levels, which were in turn significantly higher in the Glut-1 overexpressing Hepa xenografts. This implies that Glut-1 over-expression without a co-ordinate increase in HIF-1-regulated glycolytic enzymes increases glucose uptake but not the rate of glycolysis. Glut-1 overexpressing xenografts also showed higher levels of phosphodiester (PDE), which relates to the metabolite turnover of phospholipids and is involved in membrane lipid degradation, indicating a mechanism by which Glut-1 may increase cell turnover.

Effects of HIF-1alpha and HIF2alpha on Growth and Metabolism of Clear-Cell Renal Cell Carcinoma 786-0 Xenografts.

In cultured clear-cell renal carcinoma (CCRCC) 786-0 cells transfected with HIF1α (HIF-1+), HIF-2α (HIF-2+), or empty vector (EV), no significant differences were observed in the growth rates in vitro, but when grown in vivo as xenografts HIF-2α significantly increased, and HIF-1α significantly decreased growth rates, compared to EV tumors. Factors associated with proliferation were increased and factors associated with cell death were decreased in HIF-2+ tumors. Metabolite profiles showed higher glucose and lower lactate and alanine levels in the HIF-2+ tumors whilst immunostaining demonstrated higher pyruvate dehydrogenase and lower pyruvate dehydrogenase kinase 1, compared to control tumors. Taken together, these results suggest that overexpression of HIF-2α in CCRCC 786-0 tumors regulated growth both by maintaining a low level of glycolysis and by allowing more mitochondrial metabolism and tolerance to ROS induced DNA damage. The growth profiles observed may be mediated by adaptive changes to a more oxidative phenotype.

Increased tumour extracellular pH induced by Bafilomycin A1 inhibits tumour growth and mitosis in vivo and alters 5-fluorouracil pharmacokinetics

The aim was to determine if a specific inhibitor of vacuolar H(+)-ATPases (V-ATPases), Bafilomycin A1 (BFM), could increase the low extracellular pH (pHe) typical of solid tumours and thus inhibit their growth in vivo. BFM inhibited the proliferation of various human cells and rat pituitary GH3 tumour cells in vitro (IC50: 2.5-19.2 nM), and flow cytometry on GH3 cells showed a marked increase in S and G2M phases after 16-48 h, but no evidence of increased apoptosis. BFM caused significant inhibition of GH3 xenograft growth, and histomorphometry showed a 30% decrease in mitosis but no change in apoptosis. 31P-magnetic resonance spectroscopy (MRS) in vivo of GH3 xenografts showed that BFM increased pHe, but did not affect pHi, resulting in a decrease in the negative pH gradient (-delta pH). BFM decreased lactate formation suggesting a reduction in glycolysis. We suggest that BFM reduces extracellular H(+)-transport by inhibition of V-ATPases leading to an increase in pHe and decreased glycolysis, and thus reduced tumour cell proliferation. 19F-MRS in vivo showed that a smaller -delta pH was associated with decreased retention of 5-fluorouracil (5FU) which was consistent with our previous data in vivo implying the -delta pH controls tumour retention of 5 FU.

Histone deacetylase inhibition increases levels of choline kinase α and phosphocholine facilitating noninvasive imaging in human cancers.

Histone deacetylase (HDAC) inhibitors are currently approved for cutaneous T-cell lymphoma and are in mid-late stage trials for other cancers. The HDAC inhibitors LAQ824 and SAHA increase phosphocholine (PC) levels in human colon cancer cells and tumor xenografts as observed by magnetic resonance spectroscopy (MRS). In this study, we show that belinostat, an HDAC inhibitor with an alternative chemical scaffold, also caused a rise in cellular PC content that was detectable by (1)H and (31)P MRS in prostate and colon carcinoma cells. In addition, (1)H MRS showed an increase in branched chain amino acid and alanine concentrations. (13)C-choline labeling indicated that the rise in PC resulted from increased de novo synthesis and correlated with an induction of choline kinase α expression. Furthermore, metabolic labeling experiments with (13)C-glucose showed that differential glucose routing favored alanine formation at the expense of lactate production. Additional analysis revealed increases in the choline/water and phosphomonoester (including PC)/total phosphate ratios in vivo. Together, our findings provide mechanistic insights into the impact of HDAC inhibition on cancer cell metabolism and highlight PC as a candidate noninvasive imaging biomarker for monitoring the action of HDAC inhibitors.

Adaptation to HIF-1 deficiency by upregulation of the AMP/ATP ratio and phosphofructokinase activation in hepatomas

BackgroundHIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1β-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target.MethodsHepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity.ResultsHIF-1β deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1β deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours.ConclusionsDespite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes.

Noninvasive measurements of capecitabine metabolism in bladder tumors overexpressing thymidine phosphorylase by fluorine-19 magnetic resonance spectroscopy

Purpose: Previous studies have shown that tumor response to capecitabine strongly correlates with tumor thymidine phosphorylase (TP). The aims of our study were to (a) investigate the pharmacological role of TP by measuring the pharmacokinetics (PK) of capecitabine in a human bladder tumor model that was characterized by the overexpression of TP and (b) develop the use of PK measurements for capecitabine by fluorine-19 magnetic resonance spectroscopy as a noninvasive surrogate marker for determining TP levels in tumors and for predicting tumor response to capecitabine in patients. Experimental Design: TP overexpressing (2T10) and control tumors were grown s.c. in nude mice. Mice were given a dose of capecitabine or 5′-deoxy-5-fluorouridine (5′DFUR). 19F tumor spectra were acquired for determination of rate constants of capecitabine breakdown and buildup and subsequent breakdown of intermediates, 5′-deoxy-5-fluorocytidine (5′DFCR) and 5′DFUR. The rate constant of 5′DFUR breakdown was also evaluated. Results: The rate constant of breakdown of intermediates was significantly faster in 2T10 tumors than controls (P < 0.003). No significant differences in the rate of capecitabine breakdown or intermediate buildup were observed. The rate constant of 5′DFUR breakdown in the 2T10 tumors was doubled compared with controls (P < 0.001). Conclusions: This study confirmed the expected pathway of capecitabine metabolism and showed that the level of TP was related to the rate of 5′DFUR conversion. Using in vivo fluorine-19 magnetic resonance spectroscopy to mea-sure the PK of capecitabine and its intermediate metabolites in tumors may provide a noninvasive surrogate method for determining TP levels in tumors and for predicting tumor response to capecitabine in patients.

Metabolic biomarkers of response to the AKT inhibitor MK-2206 in pre-clinical models of human colorectal and prostate carcinoma

BackgroundAKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition.MethodsCellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS.ResultsIn vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model.ConclusionThis MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.

Abstract B56: Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in cancer

Abstracts: AACR Special Conference: Metabolism and Cancer; June 7-10, 2015; Bellevue, WA Background: Autophagy is a cellular catabolic degradation response to starvation or stress and the rerouting of cellular metabolism by autophagic cancer cells, to sustain cellular homeostasis during starvation or treatment-induced stress, remains poorly understood. We investigated the hypotheses that treatment-induced autophagy could prolong cancer cell survival, supported by adapted metabolism reversibly maintaining cellular homeostasis. Methods: We induced autophagy in human prostate (PC3) and colorectal (HT29, HCT116 WT (wild-type) and HCT116 Bax-ko (Bax-knock out)) cancer cells by 6 or 24 hours of amino-acid and serum deprivation (in Hanks balanced salt solution), or by 24 hours of treatment with PI103 (a phosphatidyl inositol-3 kinase inhibitor) or dichloroacetate (DCA, a pyruvate dehydrogenase kinase inhibitor). Cellular metabolism during starvation- or treatment-induced autophagy and subsequent recovery were examined by 1H- and 13C-magnetic resonance spectroscopy metabolomic studies. 13C-labeled glucose was used to assess metabolic flux. In vitro findings were verified in two corresponding colorectal xenograft models treated with PI103 or DCA. Results and Discussion: Increased expression of LC3II by western blots and the increased level of autophagosomes visualized by electron microscopy confirmed the induction of autophagy with minimal apoptosis and necrosis in HCT116 Bax-ko cells following starvation, DCA or PI103 treatment and in HCT116 WT, PC3 and HT29 cells following DCA treatment. PI103-induced autophagy prolonged cell survival, whereas starvation-induced autophagic cells eventually died. In PI103- or DCA-induced autophagy, metabolism was re-routed by i) reduced aerobic glycolysis with unchanged glucose uptake, increased cellular glucose levels (P<0.003) and reduced lactate excretion (P<0.0001); ii) increased uptake of branched-chain amino acids and glutamine (P<0.005), with a net accumulation of many intracellular amino acids and succinate (P<0.003), a TCA cycle intermediate. These metabolic alterations can prolong cancer cell survival during stress in a well-nourished environment by providing energy from amino-acid catabolism. Metabolic changes were reversed on recovery from treatment-induced autophagy. Increased levels of glutamine (P<0.01) and TCA-cycle intermediates (P<0.04) were also observed in DCA- and PI103-treated HT29 and HCT116-Bax-ko tumor xenografts, providing potential non-invasive biomarkers for treatment-induced autophagy. This work is supported by the CR-UK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) grants C1060/A10334 and C1060/A16464, NHS funding to the NIHR Biomedical Research Centre. Chang Gung Medical Foundation (Taiwan) grant CMRPG370441 and MRC-funded studentship (MRC119X). MOL is an NIHR Senior Investigator. We thank Alice Warley at the Kings College London Centre for Ultrastructural Imaging (CUI) for providing facilities for electron microscopy. Citation Format: Gigin Lin, Helen Troy, Gabriela Andrejeva, Anne-Christine LF Wong Te Fong, Dow-Mu Koh, Simon P. Robinson, Ian R. Judson, John R. Griffiths, Martin O. Leach, Yuen-Li Chung. Treatment-induced autophagy increases amino acid uptake and switches glucose addiction to amino acid catabolism in 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 B56.