Use of nuclear magnetic resonance-based metabolomics in detecting drug resistance in cancer

Abstract

Cancer cells possess a highly unique metabolic phenotype, which is characterized by high glucose uptake, increased glycolytic activity, decreased mitochondrial activity, low bioenergetic and increased phospholipid turnover. These metabolic hallmarks can be readily assessed by metabolic technologies - either in vitro or in vivo - to monitor responsiveness and resistance to novel targeted drugs, where specific inhibition of cell proliferation (cytostatic effect) occurs rather than direct induction of cell death (cytotoxicity). Using modern analytical technologies in combination with statistical approaches, 'metabolomics', a global metabolic profile on patient samples can be established and validated for responders and nonresponders, providing additional metabolic end points. Discovered metabolic end points should be translated into noninvasive metabolic imaging protocols.

Figure 1. Potential applications and benefits of genomics, proteomics and metabolomics technologies in individualized medicine for cancer patients

Conventional genomics (global gene or DNA mapping) and functional genomics (transriptomics or RNA mapping) has been used mostly on biopsy samples for early assessment of tumor predisposition and classification. The routine ana lysis of large endogenous molecules such as proteins and peptides, both in tissues and body fluids, became feasible with the advances in MS and can be used for early diagnosis and patient selection for targeted therapies. NMR spectroscopy and MS are metabolomics techniques that allow for the simultaneous assessment of hundreds of low-molecular weight endogenous metabolites as well as their dynamic fluxes. Identified metabolic markers can then be translated into in vivo metabolic magnetic resonance spectroscopy/MRSI and PET imaging protocols for therapeutic monitoring. IEF: Isoelectric focusing; MRSI: Magnetic resonance spectroscopical imaging; MS: Mass spectroscopy; NMR: Nuclear magnetic resonance; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Figure 2. [1-¹³C]glucose metabolic pathway

In cancer cells, increased uptake of [1-¹³C]glucose through the glucose transporter Glut-1 is detected by analyzing extracellular levels of [1-¹³C]glucose in media. Increased activity of glycolysis is calculated by the amount of intracellular [3-¹³C]lactate in the cellular fraction, as well as the amount of lactate exported into media through the MCT4. Decreased mitochondrial activity is detected by low levels of ¹³C-enrichment into [4-¹³C]glutamate (through pyruvate dehydrogenase) and [2,3-¹³C]glutamate (through pyruvate carboxylase). Finally, exported acetyl-CoA (from the truncated tricarboxylic acid cycle) into the cytosol is used for increased ¹³C-fatty-acid synthesis. ¹³C-fluxes through the pentose phosphate pathway can be detected by alternative methods, such as gas or liquid chromatography. 3P: Tri-phosphate; ACL: ATP citrate lyase; FAS: Fatty acid synthase; LDH: Lactate dehydrogenase; M2-PK: M2 pyruvate kinase; MCT: Monocarboxylate transporter; OXPHOS: Oxidative phosphorylation; PFK: Phosphofructokinase. Adapted from [6].

Figure 3. Metabolic reprogramming by PI3K/AKT/TOR and Ras/Raf/ERK/MAPK signal transduction pathways

Two major oncogenic pathways, the GTPase Ras/Raf/ERK/MAPK and the lipid kinase PI3K/AKT/mTOR pathways, are downstream signaling pathways for various oncogenic surface tyrosine kinases, such as IGF-1R or EGF receptor. The AKT signaling pathway has been shown to upregulate glucose metabolism (increased activity of Glut-1 and glycolytic enzymes, as well as inhibition of pyruvate dehydrogenase by overexpressing of PDK). The Ras pathway is involved in overexpression of Chok in the Kennedy pathway. Cho: Choline; Chok: Choline kinase; IGF-1R: IGR receptor 1; IRS: Insulin receptor substrate; LDH: Lactate dehydrogenase; PDK: Pyruvate dehydrogenase kinase; PFK: Phosphofructokinase.

Figure 4. Biosynthetic (solid lines) and catabolic (dashed lines) pathways of choline phospholipid metabolism (the Kennedy pathway)

Choline is an essential nutrient and is transported into the cell by choline transporters. Choline transporter-like protein is the major choline transporter in human cancer cells. Phosphatidylcholine is the major membrane phospholipid. Choline kinase, which phosphorylates choline into phosphocholine, acts as a regulatory enzyme for phosphatidylcholine synthesis. Overexpression and increased activity of choline transporters and choline kinase has been demonstrated in malignant cells, resulting in increased levels of phosphocholine and phosphatidylcholine. CDP: Cytidine 5-diphosphocholine; CDP-choline: Citicholine; CMP: Cytidine mono-phosphocholine; CTP: Cytidine choline transferase; PPi: Pyrophosphate. Adapted from [17].

Figure 5. PLS-DA ana lysis and marker validation to distinguish metabolic signature of responsiveness and resistance to imatininb in human Bcr-Abl-positive cells from CML patients

The leukemic cells, sensitive and resistant to the Bcr-Abl tyrosine kinase inhibitor imatinib, were treated with 1 µm imatinib for 24 h. Statistical PLS-discriminant ana lysis on high-resolution ¹H-NMR spectra (both extracts and medium spectra sets were used) allows for group clustering: sensitive untreated cells (squares) versus sensitive cells treated with imatinib (circles) versus resistant cells treated with imatinib (triangles). The group clustering was based on changes in glucose, lactate, choline intermediates and glutamine, with minor contribution of creatine and alanine (see 2). For three steps in metabolomics ana lysis (pattern recognition:metabolite identification:biomarker validation) refer to (1). Cho: Choline; CML: Chronic myeloid leukemia; GPC: Glycerophosphocholine; PC: Phosphatidylcholine; PLS-DA: Partial least square discriminant analysis.