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A crossroad to tumor metabolome: pyruvate kinase type M2 Sybille Mazurek 1 Institute of Biochemistry & Endocrinology, Veterinary Faculty, University of.

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Presentation on theme: "A crossroad to tumor metabolome: pyruvate kinase type M2 Sybille Mazurek 1 Institute of Biochemistry & Endocrinology, Veterinary Faculty, University of."— Presentation transcript:

1 A crossroad to tumor metabolome: pyruvate kinase type M2 Sybille Mazurek 1 Institute of Biochemistry & Endocrinology, Veterinary Faculty, University of Giessen, Giessen, Germany 2 ScheBo Biotech AG, Giessen, Germany

2 This lecture is dedicated to Prof. Dr. med. vet. Erich Eigenbrodt ( ) Head of the Department Comparable Biochemistry of Animals Institute of Biochemistry & Endocrinology, Veterinary Faculty, University of Giessen, Giessen, Germany

3 glucose PEP lactate pyruvate Pyruvate kinase ADP ATP Glycolysis Nucleogenesis M2-PK lung, proliferating cells: embryonic cells, adult stem cells, tumor cells Gluconeogenesis L-PK liver, kidney Energy production M1-PK muscle, brain

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8 Tumor M2-PKLung M2-PK

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11 kinase 3´ gagpolenvsrc pp60 v-src p19p27p12p15 gp35gp85 core proteinsreverseenvelope tyrosine proteinstranscriptase Genome and gene products of the Rous Sarcoma Virus NH 2 COOH tyr P ATPADP U3U3 R 5´ b (a) pyruvate kinase type M2 enolase lactate dehydrogenase R U 5

12 Gelpermation of purified M2-PK in NIH 3T3 cells control pp60 v-src fractions [mU/ml]

13 Genome of the HPV 16

14 HPV-16 E7 Tumor M2-PKM2-PK E7 oncoprotein of HPV-16

15 fractions Gel permeation of M2-PK in NRK cells NRK [U/ml] NRK + ras NRK + ras + E7

16 Glycolytic enzyme-complex glycolytic enzymes: HK, GAPDH PGK, PGM, PK, LDH other enzymes: NDPK, AK, G6PDH components of the protein kinase cascades: RAF, MEK, ERK AU rich mRNA components: Isoelectric focusing of NRK cells pyruvate kinase activity [U/ml] glycolytic enzyme complex pI 6.0 pI fractions [U/ml] NRK NRK + ras

17 PEP affinity of the tetrameric and dimeric M2-PK phosphoenolpyruvate [mM] v vmax phosphoenolpyruvate [mM] Km + FBP FBP v vmax

18 glucose fructose-1,6 P 2 pyruvate lactate ADP ATP normal proliferating cells: DNA Energy production M2-PK

19 glucose ADP ATP pyruvate lactate fructose-1,6 P 2 oncoproteins + nucleic acids phospholipids amino acids tumor cells: PEP

20 temperature sensitive RSV transformed chicken embryo cells °C42°C nmoles/mg *** fructose 1,6-P 2 42°C35°C P-ribose-PP ***

21 fructose 1,6-bisphosphate [nmoles/mg protein] *** NRKNRK + ras * NRK + ras + E7 x + SD

22 glucose ADP ATP pyruvate lactate fructose-1,6 P 2 oncoproteins + nucleic acids phospholipids amino acids tumor cells: PEP GDP GTP (UTP, CTP)

23 NRK cells parental+ rasras and E7 transformed Correlation between the tetramer : dimer ratio of M2-PK and the (ATP+GTP) : (UTP+CTP) ratio (ATP+GTP) : (UTP+CTP)

24 truncated citrate cycle citrate  -keto- glutarate oxalo- acetate  -keto- glutarate pyruvate fatty acids PEP glucose DNA, amino acids, phospholipids fatty acids M2-PK acetyl-CoA oxalo- acetate  -keto- glutarate pyruvate malate glutaminolysis lactate aspartate glutamine glutamate glutaminase malic enzyme glutamate proline glutamine phospholipids

25 Correlation between glucose consumption and fatty acid release in xenotransplanted human mammary carcinoma glucose consumption [nmole/(g min)] fatty acid release [nmole/(g min)] slope = 58 r = 0.706

26 glucose ADP ATP pyruvateglutaminelactate fructose-1,6 P 2 tumor cells: oxygen starvation oncogenes + DNA glutaminolysis + oxygen starvation

27 lactate production glucose consumption uptakerelease glutamine [nmoles/g min] Correlation between the lactate production : glucose consumption ratio and glutamine release in xenotransplanted human cervix carcinomas slope = 0.02 r = 0.86

28 Metabolic characteristics of tumor cells Expression of the pyruvate kinase isoenzyme type M2 which is mainly in the dimeric form High phosphometabolite pools High channelling of glucose carbons to synthetic processes A low (ATP+GTP) : (UTP+CTP) ratio A high glutaminolytic capacity The pyruvate kinase isoenzyme type M2 which can switch between a highly active tetrameric form and an inactive dimeric form, is an important metabolic sensor to adapt tumor metabolism to varying nutrient and oxygen supply.

29 ® Metabolome ProteomeGenomeTumor-

30 melanoma Ugurel et al.: Int. J. Cancer (2005), 117: pancreatic cancer Ventrucci et al.: Dig. Dis. Sci. (2004), 49: cervical carcinoma Kaura et al.: J. Obstet. Gynaecol. Res. (2004), 30: thyroid carcinoma Bena-Boupda et al.: Anticancer Res. (2003), 23: gastric cancer Kim et al.: Korean J. Gastroenterol. (2003), 42: breast cancer Hoopmann et al.: Cancer Lett. (2002), 187: Lüftner et al.: Anticancer Res. (2000), 20: lung cancer Schneider et al.: Cancer Detec. Prev. (2000), 24: bile duct cancer Kim et al.: Korean J. Gastroenterol. (2003), 42: oesophageal carcinoma Schulze: Anticancer Res. (2000), 20: colorectal cancer Zhang et al.: World J. Gastroenterol. (2004), 10: Increase of Tumor M2-PK in EDTA-Plasma renal cell carcinoma Wechsel et al.: Anticancer Res. (1999), 19: Oremek et al.: Anticancer Res. (1999), 19:

31 Robson score Tumor M2-PK (U/ml) Nephritis I II III IV 42 Cut off Tumor M2-PK concentrations in EDTA plasma samples from patients with renal cell carcinomas Correlation between Tumor M2-PK values and staging Oremek et al., Anticancer Res. (2000), 20: n =

32 Tumor M2-PK concentrations in EDTA plasma samples from patients with lung tumors Correlation between Tumor M2-PK values and staging J. Schneider in: Tumor Markers, AACC Press (2002), Stage IStage IIStage IIIStage IV n = Tumor M2-PK [U/ml] Cut off [15 U/ml] 25 % 50 % 75 % sensitivity 28 %33 %64 %73 %

33 Tumor M2-PK concentrations in EDTA-Plasma samples of patients with Colorectal, Gastric,Oesophageal and Pancreatic Cancer Schulze, Anticancer Res. (2000), 20: controls 141 Cut off [U/ml] 50 % 75 % colorectal cancer gastric cancer oesophageal cancer pancreatic cancer n = %

34 N Tumor M2-PK CEA CA 19-9CA 72-4 Best Combination Non- metastasized % 34% 18% n.d. Metastasized 46 54% 72% 50% n.d. Colorectal cancer Total % 42% 27% n.d. Tumor M2-PK + CEA 67 % Non- metastasized 65 63% 12% 31% 25% Metastasized 72 71% 39% 54% 57% Gastric cancer Total % 26% 45% 41% Tumor M2-PK + CA % Non- metastasized 77 57% 9% 16% 10% Metastasized 10 60% 20% 70% 14% Oesoph. cancer Total 87 59% 15% 43% 12% Tumor M2-PK + CA 19-9 Pancreatic cancer Total 26 73% 42% 85% 43% Tumor M2-PK + CA % 65 % Sensitivity of Tumor M2-PK in comparison to CEA, CA 19-9, CA 72-4 Schulze, Anticancer Res. (2000), 20:

35 Tumor M2-PK concentrations in EDTA-plasma samples Follow-up study of a patient with a RCC with metastases Complete Remission months3 months6 months9 months 1 st cycle 2 nd cycle 3 rd cycle 4 th cycle Cut off Immunochemo [U/ml] W. Wechsel : in: Tumor Markers, AACC Press (2002),

36 x x x x weeks [ng/ml] Tumor M2-PK [U/ml] CEA CYFRA-21 SCC NSE tumor resection diagnosisrelapse Schneider et al., Anticancer Res. (2002), 22: Follow-up study of a patient with a squamous cell carcinoma of the lung

37 973530n = NORMALCANCERADENOMA Tumor M2-PK concentrations in stool samples of patients with colorectal tumors McLoughlin et al. Dublin, Ireland Poster abstract P395, NCRI Cancer Conference, October 2005, Birmingham, UK [U/ml] sensitivity:97 %76 % Cut off 25 % 50 % 75 %

38 Controls n = Dukes A n = % Dukes B n = % Dukes C n = % Dukes D n = % 16.6 Sensitivity: Cut off [U/ml] 5.5 Tumor M2-PK concentrations in stool samples of patients with colorectal tumors 25 % 50 % 75 % Hardt, et al., Brit. J. Cancer (2004), 91:

39 Collaborators Robert R. Friis RSV transformed cells University of Bern, Switzerland Werner Zwerschke, Pidder Jansen-Dürr E7 transformed cells Tyrolean Cancer Institute, Innsbruck Austria Erich Eigenbrodt University of Giessen, Germany Metabolome analysis Peter Vaupel Xenotransplanted human mammary carcinomas University of Mainz, Germany ScheBo Biotech AG Giessen, Germany ELISA Tumor M2-PK


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