A crossroad to tumor metabolome: pyruvate kinase type M2 Sybille
Mazurek 1 Institute of Biochemistry & Endocrinology, Veterinary
Faculty, University of Giessen, 35392 Giessen, Germany 2 ScheBo
Biotech AG, Giessen, Germany Slide 2 This lecture is dedicated to
Prof. Dr. med. vet. Erich Eigenbrodt (1949 - 2004) Head of the
Department Comparable Biochemistry of Animals Institute of
Biochemistry & Endocrinology, Veterinary Faculty, University of
Giessen, Giessen, Germany Slide 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 Slide 4 Slide 5 Slide 6 Slide 7 Slide 8 Tumor M2-PKLung M2-PK
Slide 9 Slide 10 Slide 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 Slide 12 Gelpermation of
purified M2-PK in NIH 3T3 cells control pp60 v-src 30405060 0 20 40
60 80 100 120 140 fractions [mU/ml] Slide 13 Genome of the HPV 16
Slide 14 HPV-16 E7 Tumor M2-PKM2-PK E7 oncoprotein of HPV-16 Slide
15 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 10 fractions Gel permeation of
M2-PK in NRK cells NRK 30204050 [U/ml] NRK + ras NRK + ras + E7
Slide 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 6.7 0 2 4 6 8 10 12 3545
fractions [U/ml] 304550 NRK NRK + ras Slide 17 PEP affinity of the
tetrameric and dimeric M2-PK 0.0 0.2 0.4 0.6 0.8 1.0 012345
phosphoenolpyruvate [mM] v vmax 543210 phosphoenolpyruvate [mM] 0.2
0.4 0.6 0.8 1.0 0.0 Km + FBP FBP v vmax Slide 18 glucose
fructose-1,6 P 2 pyruvate lactate ADP ATP normal proliferating
cells: DNA Energy production M2-PK Slide 19 glucose ADP ATP
pyruvate lactate fructose-1,6 P 2 oncoproteins + nucleic acids
phospholipids amino acids tumor cells: PEP Slide 20 temperature
sensitive RSV transformed chicken embryo cells 0 1 2 3 4 5 35C42C
nmoles/mg *** fructose 1,6-P 2 42C35C P-ribose-PP *** Slide 21
fructose 1,6-bisphosphate 0 10 20 30 40 [nmoles/mg protein] ***
NRKNRK + ras * NRK + ras + E7 x + SD Slide 22 glucose ADP ATP
pyruvate lactate fructose-1,6 P 2 oncoproteins + nucleic acids
phospholipids amino acids tumor cells: PEP GDP GTP (UTP, CTP) Slide
23 NRK cells 0 1 2 3 4 5 6 7 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) Slide 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 Slide 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 Slide 26 glucose ADP
ATP pyruvateglutaminelactate fructose-1,6 P 2 tumor cells: oxygen
starvation oncogenes + DNA glutaminolysis + oxygen starvation Slide
27 lactate production glucose consumption 010203040102030
uptakerelease glutamine [nmoles/g min] 0.5 1.0 1.5 2.0 Correlation
between the lactate production : glucose consumption ratio and
glutamine release in xenotransplanted human cervix carcinomas slope
= 0.02 r = 0.86 Slide 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. Slide 29 Metabolome ProteomeGenomeTumor-
http://www.metabolic-database.com Slide 30 melanoma Ugurel et al.:
Int. J. Cancer (2005), 117: 825-830 pancreatic cancer Ventrucci et
al.: Dig. Dis. Sci. (2004), 49: 1149-1155 cervical carcinoma Kaura
et al.: J. Obstet. Gynaecol. Res. (2004), 30: 193-196 thyroid
carcinoma Bena-Boupda et al.: Anticancer Res. (2003), 23: 5237-5240
gastric cancer Kim et al.: Korean J. Gastroenterol. (2003), 42:
387-393 breast cancer Hoopmann et al.: Cancer Lett. (2002), 187:
223-228 Lftner et al.: Anticancer Res. (2000), 20: 5077-5082 lung
cancer Schneider et al.: Cancer Detec. Prev. (2000), 24: 531-535
bile duct cancer Kim et al.: Korean J. Gastroenterol. (2003), 42:
387-393 oesophageal carcinoma Schulze: Anticancer Res. (2000), 20:
4961-4964 colorectal cancer Zhang et al.: World J. Gastroenterol.
(2004), 10: 1643-1646 Increase of Tumor M2-PK in EDTA-Plasma renal
cell carcinoma Wechsel et al.: Anticancer Res. (1999), 19:
2583-2590 Oremek et al.: Anticancer Res. (1999), 19: 2599-2601
Slide 31 Robson score Tumor M2-PK (U/ml) 0 100 200 300 400
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: 5095-5098. 11 36 55 14 n = Slide 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), 471-475 0 20 40 60
80 100 120 Stage IStage IIStage IIIStage IV n =2365155 Tumor M2-PK
[U/ml] Cut off [15 U/ml] 25 % 50 % 75 % 241189 sensitivity 28 %33
%64 %73 % Slide 33 Tumor M2-PK concentrations in EDTA-Plasma
samples of patients with Colorectal, Gastric,Oesophageal and
Pancreatic Cancer Schulze, Anticancer Res. (2000), 20: 4961-4964 0
20 40 60 80 100 120 140 controls 141 Cut off [U/ml] 50 % 75 %
colorectal cancer gastric cancer oesophageal cancer pancreatic
cancer n =1631378726 25 % Slide 34 N Tumor M2-PK CEA CA 19-9CA 72-4
Best Combination Non- metastasized 117 48% 34% 18% n.d.
Metastasized 46 54% 72% 50% n.d. Colorectal cancer Total 163 50%
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 130
67% 26% 45% 41% Tumor M2-PK + CA 72- 4 82 % 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 19-9 96 % 65 % Sensitivity of
Tumor M2-PK in comparison to CEA, CA 19-9, CA 72-4 Schulze,
Anticancer Res. (2000), 20: 4961-4964 Slide 35 Tumor M2-PK
concentrations in EDTA-plasma samples Follow-up study of a patient
with a RCC with metastases Complete Remission 0 20 40 60 80 100 120
140 0 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), 471-475 Slide 36 x x x x 364612 0 20 40
60 80 100 120 0weeks [ng/ml] Tumor M2-PK [U/ml] CEA CYFRA-21 SCC
NSE tumor resection diagnosisrelapse Schneider et al., Anticancer
Res. (2002), 22: 311-318 Follow-up study of a patient with a
squamous cell carcinoma of the lung Slide 37 973530n =
NORMALCANCERADENOMA 60 50 40 30 20 10 0 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 % Slide 38 0 10 20 30 40 50 60 70 80 90 100 110 120
Controls n =191 1.7 Dukes A n = 23 52.2% Dukes B n = 24 76.0% Dukes
C n = 26 80.8% 12.2 46.3 Dukes D n = 17 82.4% 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: 980-984 Slide 39 Collaborators Robert R.
Friis RSV transformed cells University of Bern, Switzerland Werner
Zwerschke, Pidder Jansen-Drr 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
