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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
Institute for Biomedical Technologies, CNR, via Fratelli Cervi 93, 20090 Segrate (Mi), Italy
Universit Telematica San Raffaele Roma, Via Val Cannuta 247, 00166 Rome, Italy
Department of Pharmacology, Universit degli Studi di Milano, Via Vanvitelli 32, 20129 Milan, Italy
a r t i c l e
i n f o
a b s t r a c t
Cancer has been proposed as an example of systems biology disease or network disease. Accordingly, tumor
cells differ from their normal counterparts more in terms of intracellular network dynamics than single
markers. Here we shall focus on a recently recognized hallmark of cancer, the deregulation of cellular
energetics. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been
conrmed as an essential step toward cell transformation. We will consider how the effects of Akt activation
are connected with cell metabolism; more precisely, we will review existing metabolic models and discuss the
current knowledge available to construct a kinetic model of the most relevant metabolic processes regulated
by the PI3K/Akt pathway. The model will enable a systems biology approach to predict the metabolic targets
that may inhibit cell growth under hyper activation of Akt.
2011 Elsevier Inc. All rights reserved.
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Glycolysis and Warburg effect . . . . . . . . . . . . . . . . . . . . . . . .
1.2.
Relevance of Akt in cancer . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.
Role of Akt in cancer cell metabolism . . . . . . . . . . . . . . . . . . . .
1.4.
Integrative systems biology approach to identify molecular targets in anticancer
Kinetic models of glucose/energy metabolism . . . . . . . . . . . . . . . . . . . .
A kinetic model to study the metabolic effects mediated by the PI3K/Akt pathway . .
3.1.
Integration of different kinetic models . . . . . . . . . . . . . . . . . . . .
3.2.
Modeling of the Akt-mediated metabolic effects . . . . . . . . . . . . . . .
Strategies for the analysis of the integrated model . . . . . . . . . . . . . . . . .
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Abbreviations: ACC, acetyl-CoA carboxylase; ACCOA, acetyl-CoA; ACD, acyl-CoA dehydrogenase; ACL, ATP citrate lyase; ACS, acyl-CoA synthetase; ADP, adenosine diphosphate;
ALD, fructose 1,6 bisphosphate aldolase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; BPG, 1,3-bisphosphoglycerate; CAC, carnitine acyl-carnitine carrier; CIT,
citrate; COA, coenzyme A; CPT1A, carnitine palmitoyltransferase 1A; CPT II, carnitine palmitoyltransferase II; DE, differential equation; DHAP, dihydroxyacetone phosphate; ECH,
enoyl-CoA hydratase; EG, extended glycolysis, ENO, enolase; EP, phosphoribulose epimerase; E4P, erythrose-4-phosphate; FA, fatty acid; FASN, fatty acid synthase; F16BP, fructose1,6-bisphosphate; F26BP, fructose 2,6-bisphosphate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; GMM, glutamine mithocondrial metabolism; G6PD, glucose-6phosphate dehydrogenase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLC, glucose; GLUT, glucose transporter; GDH, glutamate dehydrogenase; GLS, glutaminase; G6P,
glucose-6-phosphate; HCD, 3-Hydroxyacyl CoA Dehydrogenases; HK, hexokinase; HPI, hexose-6-phosphate isomerase; KI, Ribose phosphate isomerase; LAC, lactic acid; LDH, lactate
dehydrogenase; MALCOA, malonyl-CoA; MCA, metabolic control analysis; MPM, mitochondrial pyruvate metabolism; NAD, nicotinamide adenine dinucleotides; NADH,
nicotinamide adenine dinucleotides; OCT, 3-oxoacyl-CoA thiolase; PDC, pyruvate dehydrogenase complex; PEP, phosphoenolpyruvate; PFK-1, phosphofructokinase type 1; PFK-2,
phosphofructokinase type 2; 6PG, 6-phosphogluconate; 2PGA, 2-phosphoglycerate; 3PGA, 3-phosphoglycerate; PGAM, 3-phosphoglycerate mutase; 6PGD, phosphogluconate
dehydrogenase; PGK, phosphoglycerate kinase; Pi, inorganic phosphate; PI3K, phosphatidylinositol 3-kinase; PYC, pyruvate carrier; PYK, pyruvate kinase; PYR, pyruvate; PYRt,
pyruvate diffusion between cytosol and mitochondrial intermembrane space; PRPP, phosphoribosyl-pyrophosphate; PRPPS, phosphoribosyl-pyrophosphate synthetase; R5P,
ribose-5-phosphate; Ru5P, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; SA, sensitivity analysis; TA, transaldolase; TCA, tricarboxylic acid; TK1, transketolase 1; TK2,
transketolase 2; TPI, triosephosphate isomerase; X5P, xylulose-5-phosphate.
Corresponding authors.
E-mail addresses: ettore.mosca@itb.cnr.it (E. Mosca), matteo.barcella@itb.cnr.it (M. Barcella), roberta.aleri@itb.cnr.it (R. Aleri), annamaria.bevilacqua@unisanraffaele.gov.it
(A. Bevilacqua), gianfranco.canti@unimi.it (G. Canti), luciano.milanesi@itb.cnr.it (L. Milanesi).
0734-9750/$ see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.08.004
132
5.
Conclusions . . . . . .
Acknowledgments . . . . . .
Appendix A.
Supplementary
References . . . . . . . . .
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data
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1. Introduction
The intrinsic differences between cancer and normal cells are key
when trying to identify new targets for anticancer drugs and to
overcome chemo-resistance to anticancer therapy. Like drivers on busy
roads of big cities begin to turn around to reach their destination,
intracellular networks allow cancer cells to bypass the effect of a drug
using alternative pathways to exploit a critical function for their
survival: it is thus increasingly believed that a systems biology approach,
focused on the analysis of the structure and dynamics of these networks,
can lead to a better comprehension of cancer disease and could aid the
design of safer drugs and therapies (Wang, 2010). To summarize these
concepts, cancer has been designated as a systems biology disease
(Hornberg et al., 2006; Laubenbacher et al., 2009).
However, nowadays the development of systems biology in cancer
research is still limited, especially when more specic applications are
concerned. In this review, we will focus on a particular phenomenon
that has been recently recognized as a hallmark of cancer, the
deregulation of cellular energetics (Hanahan and Weinberg, 2011).
Quite a few tumor cells produce most of their ATP through the glycolytic
pathway, thereby producing more lactate than their untransformed
counterparts (Pedersen, 2007), and increase biosynthetic activities
required to create daughter cells (DeBerardinis et al., 2008). The
constitutive activation of the PI3K/Akt pathway has been conrmed as
an essential step toward cell transformation and contributes to both
metabolic changes in cell energetics and metabolic requirements to
support cell proliferation (Young and Anderson, 2008).
We will review existing models of central metabolism and we will
discuss the current knowledge available to construct a detailed kinetic
model of the most relevant metabolic effects of the PI3K/Akt pathway.
Such a kinetic model enables a systems biology approach to identify
potential metabolic targets that exploit the addiction of tumor cells to
increased glucose uptake and glycolysis.
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139
140
140
140
133
Fig. 1. Map of the glycolysis and pentose phosphate pathway. Detailed map of glycolysis and pentose phosphate shunt with the regulation exerted by the PI3K/Akt signaling pathway
(green dotted arrows where + indicates positive regulation). Black: metabolites; blue: proteins; red: co-factors.
134
Fig. 2. Map of FAs synthesis and -oxidation. Both the FAs synthesis and -oxidation are regulated by the PI3K/Akt signaling pathway (green dotted arrows where + and
indicate positive and negative regulation respectively). The processes of this module span the cytosol, the intermembrane space (IMS) and the mitochondrial matrix (MAT). The
Acyl-CoA is reported between parentheses to indicate that it is not always produced by OCT, as this depends on the acyl group of OCT substrate. Black: metabolites; blue: proteins;
red: co-factors.
135
136
Fig. 3. Summary map of the metabolic network regulated by the PI3K/Akt signaling pathway. The metabolic network regulated by the PI3K/Akt pathway encompasses the
extracellular space, the cytosol and the mitochondrion. The biochemical processes composing the network can be divided into six modules: the glycolysis and the pentose phosphate
pathway, the tricarboxylic acid cycle, the respiratory chain, the fatty acids synthesis and -oxidation, the glutamine mitochondrial metabolism and the metabolite transporters. The
proteins positively and negatively regulated by the PI3K/Akt pathway are reported in green and red color respectively. The map is intended to provide an overall picture of the
metabolic network and, as a consequence, does not report all the molecular species and biochemical processes included in the network.
List of processes
Flux expressions
Glucose transport
Marn-Hernndez et
(2011)
Marn-Hernndez et
(2011)
Marn-Hernndez et
(2011)
Marn-Hernndez et
(2011)
Holzhtter (2004)
Glycolysis pathway
Glycogen production
LDH reaction
Respiratory chain
Fatty acid synthesis
and -oxidation
Pentose phosphate
pathway
PDH reaction
Tricarboxylic acid cycle
Pyruvate carboxylase
Respiratory chain
reactions
ACL reaction
ACC reaction
FASN reaction
ACS reaction
FA transport in
mitochondria
-Oxidation
Lipid metabolism
Glutamine mitochondrial Glutamine mitochondrial
metabolism
transport
GLS reaction
GDH reaction
Metabolite transporters
Malateaspartate
shuttle
Acetyl groups transporter
shuttle
Other transporters
al.
al.
al.
al.
Wu et al. (2007)
Wu et al. (2007)
Yugi and Tomita (2004)
Yugi and Tomita (2004)
Plowman and Cleland
(1967)
Kaushik et al. (2009)
Cox and Hammes
(1983)
Hall et al. (2003)
Yugi and Tomita (2004)
Yugi and Tomita (2004)
Mass-action irreversible
Steib et al. (1986)
Haser et al. (1985)
Bruggeman et al. (2005)
Yugi and Tomita. (2004);
Wu et al. (2007)
Yugi and Tomita (2004);
Swierczyski (1980)
Yugi and Tomita (2004);
Wu et al. (2007)
the FAs synthesis increasing the activity of ACL and inhibits the oxidation decreasing the concentration of CPT1A. FAs synthesis can
be simulated with four enzymatic reactions, respectively catalyzed
by ACL, ACC, FASN and an irreversible reaction representing the ux
toward lipid metabolism. The ACL, ACC and FASN kinetic laws
provided with experimentally determined kinetic parameters can be
found in different studies (respectively Plowman and Cleland, 1967;
Kaushik et al., 2009; Cox and Hammes, 1983), while a mass action
kinetics can be used for the irreversible reaction representing the ux
toward lipids, a boundary of the system. The reactions for FAs entry
into mitochondria and FAs -oxidation are also available in the model
by Yugi and Tomita (2004). This model lacks the pre-step of oxidation, catalyzed by the ACS; however, the kinetic law and related
parameters experimentally determined for puried murine ACS are
available (Hall et al., 2003).
Glutamine, which is highly transported into proliferating cells (Wise
et al., 2008), is a major source for energy and nitrogen for biosynthesis,
and a carbon substrate for anabolic processes in cancer cells (DeBerardinis
and Cheng, 2010). In cancer, glutamine can serve as an alternative
substrate for the TCA cycle in order to produce ATP and can become
critical for biosynthesis and survival. Hence, it can be used as an energy
substrate when glucose supply is limited (Yuneva, 2008), the so-called
glutamine addiction phenomenon (Wise and Thompson, 2010). Thus, it
is important to take into account a series of reactions needed for the
(v) glutamine metabolization in the TCA cycle. One solution consists in
representing this pathway with three biochemical processes. The rst one
is the transport of glutamine from the cytosol into mitochondria, for
which the kinetic law provided by Steib et al. (1986), with kinetic
parameters experimentally determined in rat brain, is suitable. The
second process is the conversion of glutamine into glutamate catalyzed by
137
the mitochondrial GLS; here the kinetic law and parameters experimentally determined in rat kidney (Haser et al., 1985) can be used. The ux
expression for the conversion of glutamate into oxoglutarate, operated by
the GDH, is available in the model of ammonium assimilation in E. coli
(Bruggeman et al., 2005).
Lastly, the (vi) metabolite transporters module, which includes the
malateaspartate shuttle, the acetyl groups transporter shuttle, and
transporters not included in the other modules, is required to
reproduce the metabolites trafc (or translocation) between cytosol
and mitochondria. These carriers can be found in the two models of
Wu et al. (2007) and Yugi and Tomita (2004) with the exception
of the malic enzyme (considered as a part of the acetyl groups
shuttle system), for which specic kinetic law and kinetic parameters
experimentally determined in rat skeletal muscle are described by
Swierczyski (1980).
3.1. Integration of different kinetic models
The linking of different kinetic models consists in the denition of a
unique model that combines the mathematics and possibly contains
some expressions not available in the starting models, but required for a
correct integration. Mathematically, these models are a set of
differential algebraic equations, in which each differential equation
(DE) describes the temporal variation of a metabolite concentration xi as
dxi
1 m
= si;k Jk x; p; t
Vi k = 1
dt
where the stoichiometric coefcient si,k species the amount of xi
produced (si,k 0) or consumed (si,k 0) by a reaction k, Jk is the ux of
reaction k, Vi is the volume of the compartment in which xi is
observed, and m is the total number of reactions. The uxes Jk are
functions of metabolite concentrations x and kinetic parameters p,
and are dened depending on the specic kinetic mechanism (e.g.
MichaelisMenten, Random Bi Bi or Multisite Ping Pong) of the
reaction that they describe. Algebraic equations are used to dene
conservation relations.
Since, as previously mentioned, it is common that the starting
models have been developed in different experimental conditions, a
preliminary step of model tting may be useful to obtain models that
are more compatible according to the known behaviors of the real
system that one wants to represent. For the modeling of the metabolic
network regulated by the PI3K/Akt pathway, the starting models may
Table 2
Preliminary tting of a glycolysis model. Comparison of steady state uxes ratios
computed using the glycolysis model by Marn-Hernndez et al. (2011) before and after
tting to uxes measured in HEK 293 cells by Henry et al. (2011) (Supplementary Fig. 1).
The expression J(X Y) indicates the ux from metabolite X to metabolite Y. The model
has been implemented for MATLAB (2007a, The MathWorks, Massachusetts, U.S.A.) and
simulated using parameters and initial conditions reported, respectively, in Supplementary Table 1 and Supplementary Table 2. Steady states were calculated using MATLAB
function fsolve. PYR_x indicates the mitochondrial concentration of PYR.
Fluxes ratios
J G6PF6P
J GLCG6P
J F6PG3P
J G6PF6P
J G3PPYR
JJ F6PG3P
J PYRPYR x
J PYRLAC
J G3PPYR
J PYRPYR x
Before preliminary
tting
After preliminary
tting
0.850
0.932 0.096
0.883 0.038
1.088
1.006 0.075
1.066 0.020
2.042
2.054 0.006
2.064 0.011
0.296
0.016 0.944
0.135 0.546
4.207
61.263 13.563
5.774 0.373
138
139
Fig. 5. Simulation of the metabolic effects of Akt activation. Simulation of a metabolic network resulting from the integration of the glycolysis model by Marn-Hernndez et al.
(2011) and mitochondrial model by Wu et al. (2007) (see Supplementary materials). Time evolution of a) GLC, b) LAC and c) cytosolic ACCOA computed with four different model
parameterizations that simulate the increasing strength of Akt regulation; x represents the varied parameters, i.e. Vmf, Vm, [F26BP] and Vm of respectively GLUT, HK, PFK1 and ACL ux
expressions (see Supplementary material). The initial conditions used are reported in Supplementary Table 3, while the ux expressions and the kinetic parameters are listed in
Supplementary Table 4. The simulations of the integrated model have been performed using the software MATLAB (2007a, The MathWorks, Massachusetts, U.S.A.): differential
equations were solved with the function ode15s.
140
At the same time, it is becoming more and more evident that targeting
single gene products or pathways yields low rates of response and
should not be expected to cure cancer (Hayden, 2008). The PI3K/Akt
pathway regulates the activity of several proteins that, in turn, control
mass and energy ux distribution through a tightly interconnected
metabolic network. As a consequence, the altered metabolic network
shows different dynamics compared to the normal one. In this
context, an effective therapeutic approach should attack the altered
system in order to switch it off without affecting the normal system,
and having a model allows using SA methods to select robust and
sensitive parts of the system. With the aim of enabling a systems
biology approach for the metabolic network regulated by the PI3K/Akt
pathway, we reviewed and discussed the framework of knowledge
available for the modeling of the considered network, emphasizing
the use of enzyme-specic kinetic laws with experimentally derived
kinetic parameters.
The analysis of the current knowledge suggests that a series of detailed
rate equations are available for the most important biochemical processes
that link together in an integrated metabolic network all the processes
regulated by the PI3K/Akt pathway. The differences existing among the
experimental conditions used for the characterization of enzyme and
transporters kinetics demand a computational phase of parameter
estimation to obtain a coherent model which t the experimental
observations, before it can be exploited to gain in silico predictions.
Nevertheless, new data sets are continuously made available and
measuring everything is not mandatory (Alberghina and Westerhoff,
2005). The systems biology approach discussed here indeed allows
devising testable hypothesis based on a quantitative model that might
ultimately aid in understanding the complex metabolic network
mediated by the PI3K/Akt pathway.
Acknowledgments
This work has been supported by the MIUR FIRB ITALBIONET
(RBPR05ZK2Z), BIOPOPGEN and the Flagship INTEROMICS projects.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.
1016/j.biotechadv.2011.08.004.
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