Oncogene (2001) 20, 6891 ± 6898 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Metabolic cooperation between dierent oncogenes during cell transformation: interaction between activated ras and HPV-16 E7 Sybille Mazurek*,1,4, Werner Zwerschke2,3,4, Pidder Jansen-DuÈ rr2 and Erich Eigenbrodt1 1Institute for Biochemistry and Endocrinology, Veterinary Faculty, University of Giessen, Frankfurter Strasse 100, 35392 Giessen, Germany; 2Institute for Biomedical Ageing Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria; 3Tyrolean Cancer Research Institute, Innrain 66, A-6020 Innsbruck, Austria The metabolism of tumor cells (tumor metabolome) is linked to the disadvantage that those cells undergo characterized by a high concentration of glycolytic apoptosis under glucose limitation (Carmeliet et al., enzymes including pyruvate kinase isoenzyme type M2 1998; Shim et al., 1998). Glucose limitation is also (M2-PK), a high glutaminolytic capacity, high fructose found in solid tumors. To circumvent this disadvan- 1,6-bisphosphate (FBP) levels and a low (ATP+ tage, tumor cells use the amino acids glutamine and GTP):(CTP+UTP) ratio. The sequence of events serine as alternative sources for energy production. In required for the establishment of the tumor metabolome analogy to glycolysis the conversion of glutamine to is presently unknown. In non-transformed rat kidney lactate has been termed glutaminolysis; the ¯ow of (NRK) cells we observed a high glutaminolytic ¯ux rate serine to lactate has been termed serinolysis (Eigen- and a low (ATP+GTP):(CTP+UTP) ratio, whereas brodt et al., 1998a; Mazurek et al., 2001a,b). FBP levels and M2-PK activity are still extremely low. To coordinate glycolysis, glutaminolysis and serino- After stable expression of oncogenic ras in NRK cells a lysis, proliferating cells express a certain isoenzyme of strong upregulation of FBP levels and of M2-PK activity pyruvate kinase, referred to as pyruvate kinase type was observed. Elevated FBP levels induce a tetrameriza- M2 (M2-PK) (Eigenbrodt et al., 1985). M2-PK occurs tion of M2-PK and its migration into the glycolytic in a tetrameric form with a high anity for its enzyme complex. AMP levels increase whereas UTP and substrate phosphoenolpyruvate (PEP) and in a dimeric CTP levels strongly decrease. Thus, ras expression form with a low PEP anity. The glycolytic completes the glycolytic part of tumor metabolism phosphometabolite fructose 1,6-bisphosphate (FBP) leading to the inhibition of nucleic acid synthesis and induces the reassociation of the dimeric to the cell proliferation. The HPV-16 E7 oncoprotein, which tetrameric form (Eigenbrodt et al., 1992). When cooperates with ras in cell transformation, directly binds M2-PK is dimeric, glucose carbons are channelled to to M2-PK, induces its dimerization and restores nucleic synthetic processes, preferentially catalyzed by trans- acid synthesis as well as cell proliferation. Apparently, ketolase to nucleic acids (Glossmann et al., 1981; the combination of the dierent metabolic eects of ras Eigenbrodt et al., 1992; Boros et al., 1997, 2000; and E7 constructs the perfect tumor metabolome as Zwerschke et al., 1999; Cascante et al., 2000; Mazurek generally found in tumor cells. Oncogene (2001) 20, et al., 2001b) The ¯ow of glutamine to lactate increases 6891 ± 6898. to ensure energy production when M2-PK is inhibited (Mazurek et al., 1999, 2001b). Aside from mitochon- Keywords: metabolome; glycolysis; glutaminolysis; M2- drial glutaminolytic energy production, the glycolytic PK; adenylate kinase; nucleotides pyruvate kinase reaction is the only way to produce energy in tumor cells. Therefore, pyruvate kinase controls the ATP:ADP and GTP:GDP ratio and, in Introduction cooperation with adenylate kinase and 6-phosphofruc- to 1-kinase, the AMP levels (Mazurek et al., 1998; Tumorigenesis is generally characterized by an increase Thomas and Fell, 1998). An increase in AMP levels in the glycolytic enzyme system (Eigenbrodt et al., inhibits DNA synthesis and cell proliferation by a 1985, 1992; Board et al., 1990; Dang and Semenza, reduction of NAD(H) as well as UTP levels and CTP 1999). The increased glycolytic capacity allows tumor synthesis (Henderson and Scott, 1981; Mazurek et al., cells to migrate and survive under hypoxic conditions 1997a,b). By this mechanism M2-PK activity adapts as occur in solid tumors (Eigenbrodt et al., 1985; Kim cell proliferation to the nutrient supply. The metabolic et al., 1997; Reshkin et al., 2000). This advantage is network that is controlled by M2-PK has been termed the metabolome (Mazurek et al., 2001a,b). All tumor cells analysed to date express a high amount of the low PEP ane dimeric form of M2-PK (Eigenbrodt et al., *Correspondence: S Mazurek; 1992). M2-PK was shown to be directly targeted by E-mail: [email protected] 4These authors contributed equally to this paper certain oncoproteins, such as pp60v-src kinase and the Received 23 May 2001; revised 3 July 2001; accepted 5 July 2001 E7 oncoprotein of the human papillomavirus type 16 Interaction between ras and HPV-16 E7 S Mazurek et al 6892 (Presek et al., 1988; Eigenbrodt et al., 1998b; Results Zwerschke et al., 1999). The pp60v-src kinase phosphor- ylates M2-PK in tyrosine; the HPV 16-E7 oncoprotein Increased glutaminolysis and serinolysis in ras-expressing directly binds to M2-PK. Both oncoproteins trigger the rat kidney cells dimerization of M2-PK (Eigenbrodt et al., 1998b; Zwerschke et al., 1999). Due to the central regulatory A cell line (referred to as 14/2 cells) was derived from role of M2-PK, the interaction of the E7 oncoprotein normal rat kidney (NRK) cells by stable transfection with this key glycolytic enzyme dierentially in¯uences with a constitutive expression vector for EJras and a glycolysis, glutaminolysis, serinolysis, phosphometabo- hormone-inducible expression vector for HPV-16 E7 lite levels and nucleic acid metabolism in a way that (Crook et al., 1989). Expression of oncogenic ras leads depends on the individual enzyme features of the host to growth arrest which is overcome by expression of cell line (Mazurek et al., 2001b). E7 (Crook et al., 1989; Serrano et al., 1997). This A detailed metabolome analysis has been performed system allows to compare the phenotype of parental for the investigation of the metabolic eect of E7 rat kidney cells with the properties of ras-expressing transformation on two dierent NIH3T3 cell strains (referred to as ras-expressing RK cells below), and ras/ (high and low glycolytic) (Mazurek et al., 2001b). E7-coexpressing rat kidney cells (referred to as ras/E7- NIH3T3 cells, which are already immortal, are expressing RK cells below), and hence reveals transformed by E7 expression alone whereas transfor- consequences of the sequential expression of both mation of primary normal rat kidney cells (NRK cells) oncogenes. Parental NRK cells were characterized by requires the expression of two cooperating oncopro- low rates of glucose consumption and lactate produc- teins such as ras and E7 (Bedell et al., 1987; Crook et tion. Constitutive ras-expression, as in 14/2 cells, led to al., 1989; Zwerschke and Jansen-DuÈ rr, 2000). Metabo- a drastic increase of both (Table 1). The ratio between lome analysis has not yet been used to investigate the lactate production and glucose consumption decreased metabolic cooperation of two oncoproteins during from 1.9 in NRK cells to 1.4 in ras-expressing RK transformation. To obtain insight in metabolic altera- cells (Table 2). In glycolysis, 1 mole of glucose is tions involved in cell transformation by ras and E7, we converted into 2 moles lactate. A ratio between lactate analysed the metabolome of primary NRK, a ras- production and glucose consumption of nearly two expressing cell line derived from NRK as well as NRK- indicates that all lactate produced must be derived derived cells expressing both ras and E7. In NRK cells, from glucose. Therefore, the decrease in the ratio ras-expression leads to an inhibition of proliferation between glucose consumption and lactate production that is restored by the additional expression of E7. means that in ras-expressing RK cells either more Therefore, oncogene-expressing rat kidney cells are a glucose was channeled to synthetic processes or that good model to investigate links between metabolic more lactate derived from other sources than glucose. control and the regulation of cell proliferation. This question can be clari®ed by the comparison of the Table 1 Nutrient ¯uxes in NRK cells, ras-expressing RK cells as well as ras/E7-expressing RK cells NRK RK+ras RK+ras+E7 Metabolites x+S.E.M. (nmoles/(h×105 cells)) Glucose consumption 43.6+3.7 84.5+3.3*** 120.9+2.5*** Lactate production 83.5+2.0 148.7+2.7*** 218.5+5.7*** Pyruvate consumption 3.8+0.1 9.9+0.5*** 7.3+0.5* Glutamine consumption 11.3+1.5 9.4+3.8n.s. 8.4+3.9n.s. Glutamate production 6.5+0.3 6.5+0.3n.s. 5.7+0.5n.s. Serine consumption 8.5+1.2 30.9+3.7*** 23.2+5.2n.s. Alanine production 3.5+0.3 1.7+0.2*** 1.8+0.2n.s. The ¯ux rates were measured at an average cell density of 256105 cells/dish. NRK cells were compared with ras- expressing RK cells and ras-expressing RK cells were compared with ras/E7-expressing RK cells. *=P50.05; **P50.01; ***P50.001. n.s.=not signi®cant. nNRK=6, nRK+ras=5, nRK+ras+E7=5 Table 2 Correlations between dierent metabolite conversion rates in NRK, ras-expressing RK and ras/E7-expressing RK cells NRK RK+ras RK+ras=E7 Abscissa Ordinate Slope Intercept r Slope Intercept r Slope Intercept r Glucose Lactate 1.9 53 0.974 1.4 558 0.992 1.7 221 0.997 Pyruvate Lactate 23.6 ±294 0.991 10.4 1181 0.973 17.8 1901 0.973 Glutamine Lactate 2.9 1515 0.375 3.3 2622 0.601 5.7 3479 0.678 Alanine Lactate 24.9 130 0.955 28.6 2346 0.960 42.9 3310 0.892 Glucose Pyruvate 0.08 16 0.963 34 ±55 0.961 0.09 ±81 0.980 For these calculations the ®rst metabolite (abscissa (nmoles/(h dish)) was plotted versus the second metabolite (ordinate (nmoles/h dish)).