Metabolomics by Magnetic Resonance: From Molecules to Man

1. Metabolomics by Magnetic Resonance: From Molecules to Man John Griffiths, Yuen-Li Chung, Helen Troy Cancer Research UK Biomedical Magnetic Resonance Research Group Department of Basic Medical Sciences, St George�s Hospital Medical School, London SW17 ORE, UK

2. Metabolomics by NMR NMR-based methods offer quick, simple ways for studying the metabolome. For instance, 1H NMR can be used ex vivo to obtain metabolic profiles from genetically manipulated cells or biopsies from patients or experimental animals. Soluble metabolites can be extracted with perchloric acid, and lipids with chloroform/methanol. Solid specimens can be studied by MAS-NMR. A unique advantage is that by MRS we can also study some of the same metabolites in vivo.

3. Metabolites detected in cancer by NMR

4. Medical Uses of Metabolomics We use metabolic profiling to study Genetically modified cells, Tissues from genetically modified organisms, Biopsies from patients. Actions of novel drugs Our general strategy is to take a metabolic profile from the abnormal cell or tissue and compare it with the corresponding metabolic profile of �wild type� cells, control organisms or tissues. This method is even more effective if the corresponding transcriptomic or proteomic profiles are also available.

5. Metabolic Profiling of Anticancer Drug Mechanisms We have also used metabolic profiling by NMR methods, both in vivo and ex vivo, to study novel anticancer drugs: Magnetic Resonance Spectroscopic pharmacodynamic markers of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17AAG) in human colon cancer models. Chung et al., J Natl Cancer Inst. 95: 1624-1633 (2003). Non-invasive measurements of capecitabine metabolism in bladder tumors over-expressing thymidine phosphorylase by 19F MRS. Chung et al., Clinical Cancer Research 10: 3863-3870 (2004). Tumor dose response to the vascular disrupting agent, 5,6-dimethylxanthenone-4-acetic acid, using in vivo magnetic resonance spectroscopy. McPhail, et al. Clin Cancer Res 11: 3705-3713 (2005).

6. The Role of HIF in the Tumour Metabolome It has been known since Otto Warburg�s work in the 1920s that cancer cells have an abnormal metabolome. In particular, they rely more on glycolysis for energy metabolism. Glycolytic enzyme and glucose transporter expression are induced by the HIF (Hypoxia Inducible Factor) pathway. HIF-1, the main cellular O2 detector, is upregulated in cancer cells by two mechanisms.

8. HIF-1� Deficiency We set out to study the metabolomes of Hepa c4 cancer cells which are deficient in the HIF-1� subunit (also known as ARNT). We assumed that as they could not form the HIF-1 dimer they would be unable to upregulate their glycolytic pathways. Surprisingly, these HIF-1 � deficient cancer cells had only 20% of normal [ATP]. 1H NMR metabolic profiles of tumour extracts showed significant changes in several other metabolites.

15. Was Our Original Hypothesis Correct? We had originally assumed (based on numerous published studies) that HIF-1� deficient c4 tumours would be unable to upregulate glycolysis. We therefore attributed their deficiency of ATP to lack of glycolytic precursors for purine synthesis (Griffiths et al., Cancer Research, 62: 688-95, 2002). However, cultured c4 cells (which failed to form active HIF complex) produced the same amount of lactate and other glycolytic products as WT cells.

16. Normal Lactate Output from Cultured c4 Cells

18. Early studies on small c4 tumours (Maxwell et al, PNAS 94: 8104, 1997) reported slower growth rates than in WT tumours. The larger tumours that we grew for our 31P MRS studies in vivo showed a more complex pattern. Initially our c4 tumours grew slowly, but after 3 weeks they started secreting VEGF and grew faster than WT tumours. Growth Rate Studies in WT and c4 Tumours

19. Conclusions Despite their defective HIF pathway, cultured c4 cells took up glucose and produced lactate at normal rates, so their glycolytic flux was normal. They could even upregulate glycolysis in hypoxic conditions. Large Hepa-c4 tumours had normal glycolytic enzyme, glucose transporter and VEGF expression. After 3 weeks they grew even faster than normal tumours. Nevertheless their [ATP] was 20% of normal, suggesting a defect in purine synthesis. Perhaps HIF-deficient tumours eventually express another transcription factor that upregulates glycolysis and VEGF secretion, accelerating growth, but which fails to upregulate ATP synthesis.

20. Metabolic Profiling Studies on the Role of Succinate and Fumarate in the HIF Pathway Recently we have provided metabolic profiles for two studies that have shown how succinate dehydrogenase and fumarate hydratase can act as oncogenes because of their effect on the HIF pathway. These papers are discussed in a review by Esteban & Maxwell in Nature Med (News and Views) 11: 1047-1048, 2005 Isaacs et al, Cancer Cell 8: 143-153 (2005) Pollard et al, Human Mol. Genet. 14: 2231-2239 (2005)

22. Fumarate Hydratase as an Oncogene The inherited cancer Hereditary Leiomyomatosis Renal Cell Carcinoma is characterised by germ line mutations in the fumarate hydratase gene. Fumarate hydratase is a well-known �housekeeping� energy metabolism enzyme in the tricarboxylic acid cycle. It catalyses conversion of fumarate to malate. We collaborated in a study (Isaacs et al 2005) which demonstrated that these mutations of fumarate hydratase elevate intracellular fumarate which then upregulates HIF by competitively inhibiting HIF prolyl hydroxylase. NMR metabolic profiling showed that siRNA knockdown of fumarate hydratase in A459 cells elevated fumarate levels and increased glycolysis.

23. siRNA Knockdown of Fumarate Hydratase in Cultured Cells

24. siRNA Knockdown of Fumarate Hydratase in Cultured Cells

25. siRNA Knockdown of Fumarate Hydratase in Cultured Cells

26. Metabolic Profiling of Human Cancers Caused by FH and SDH In another study (Pollard et al, 2005) metabolic profiling was performed on biopsies from patients with: Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome (HLRCC) which is caused by fumarate hydratase mutations. Hereditary Paraganglioma and Phaeochromocytoma Syndrome (HPGL) which is caused by mutations of another TCA cycle enzyme, succinate dehydrogenase.

27. Elevated Succinate in Gangliomas With and Without Germline Succinate Dehydrogenase Mutations

28. Elevated Succinate in Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome

29. Elevated Fumarate in Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome

31. Acknowledgements Oxford University Adrian Harris Hammersmith Hospital Eric Aboagye Molecular & Population Genetics, St George�s, London Patrick Pollard Shirley Hodgson Urologic Oncology, NCI Jennifer Isaacs Len Neckers Institute of Cancer Research Martin Leach Ian Judson Paul Workman

33. Metabolic Profiling by NMR Methods However, NMR metabolic profiles measure only 50 or so metabolites. How can a method that gives such partial information about the metabolome provide useful insights into the phenotypic effects of gene knockouts, anticancer drug actions etc? Kevin Brindle has proposed two explanations: Pathways intersect so densely that any perturbation rapidly affects the metabolites we can measure. Because we quantify all the metabolites simultaneously in the same sample, with minimal processing, their relative concentrations are known with great precision.