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 Review

Proteomics and metabolomics

in cancer drug development
5
Expert Rev. Proteomics 10(5), 000000 (2013)

Angelo DAlessandro In this review article, the main recent advancements in the field of proteomics and
and Lello Zolla* metabolomics and their application in cancer research are described. In the second part of
the review the main metabolic alterations observed in cancer cells are thoroughly dissected, 10
Department of Ecological and Biological
Sciences, University of Tuscia, Largo especially those involving anabolic pathways and NADPH-generating pathways, which
dellUniversita`, snc, 01100 Viterbo, Italy indirectly affect anabolic reactions, other than the maintenance of the redox poise.
*Author for correspondence: Alterations to mitochondrial pathways and thereby deriving oncometabolites are also detailed.
Tel.: +39 0761 357 100
Fax: +39 0761 357 630
The third section of the review is a discussion of how and to what extent (mutations to)
zolla@unitus.it tumor suppressors and oncogenes end up influencing cancer cell metabolism and cell fate, 15
either promoting survival and proliferation or autophagy and apoptosis. In the last section of
the review, an overview is provided of therapeutic strategies that make use of metabolic
reprogramming approaches.

KEYWORDS: cancer drug development mass spectrometry metabolism proteomics

upon mitochondrial oxidative phosphorylation

Metabolomics, the new/old hallmark of
cancer
Human tumor pathogenesis is characterized by
the progressive accumulation of changes to nor-
mal cells, changes that make cells evolve to a
neoplastic state through the gradual acquisition
of a series of hallmark capabilities. This multi-
step process utterly enables normal cells to
become tumorigenic and, ultimately malig-
nant [1]. While metabolic reprogramming has
only recently been included in the list of the so-
called hallmarks of cancer [1], echoes from the
last (at least) 60 years of research already sug-
gested a crucial correlation between chronic and
uncontrolled cell proliferation and deregulated
metabolism [2]. The Warburg effect, named
after Otto Warburg, the first researcher to docu-
ment an exception to the Pasteur effect (inhibi-
tion of glycolysis in presence of oxygen) in
highly proliferating cancer cells, is based upon
the appreciation of an increased glycolytic rate,
at the expenses of mitochondrial metabolism
(preferentially exploited by normal differentiated
cells for energy production purposes), even in
the presence of oxygen [2]. This phenomenon,
often referred to as aerobic glycolysis, was at
first deemed to be counterintuitive, since rapidly
proliferating cells are supposed to have higher
energy requirements, while a strictly glycolytic
metabolism is less efficient than one relying
in terms of ATP production (~18-fold lower
efficiency) [3]. However, since generalization of a 25
Warburg-like metabolism seems to be also appli-
cable to many rapidly dividing embryonic tis-
sues, a tentative explanatory and evidence-based
theory posits that aerobic glycolysis might have
evolved to meet the elevated anabolic demand 30
(for biosynthetic purposes) and favor the uptake
and incorporation of nutrients into biomass by
rapidly dividing cells [3].
Conversely, over generalizations should be
avoided as well, since tumor cells do not 35
always display a Warburg-like metabolism.
Indeed, some tumors are characterized by two
subpopulations of cancer cells, one consisting
of glucose-dependent cells that secrete lactate
(Warburg-wise), while a second subpopulation 40
almost symbiotically relies upon the secreted
lactate to sustain their energy production via
the tricarboxylic acid cycle (TCA cycle, also
known as Krebs cycle) [1].
During the last decade, molecular evidences 45
have underpinned a role for genetic reprog-
ramming in the metabolic regulation observed
in cancer cells, a phenomenon that is often
accompanied by the preferential expression of
cancer-specific isoforms of certain metabolic 50
enzymes, or rather by peculiar and recurrent
cancer-associated mutations, especially in genes
coding for TCA cycle enzymes [4]. In the light

www.expert-reviews.com 10.1586/14789450.2013.840440
2013 Informa UK Ltd ISSN 1478-9450 1

 Review DAlessandro & Zolla
of these observations, Warburgs hypothesis was recently revived
55 and expanded, as it became to be considered one key target for
therapeutic treatments [5]. The implementation of novel mass
spectrometry (MS)-based metabolomics and proteomics
approaches has boosted this area of research, delivering promis-
ing results and suggesting new avenues for further research
60 development in the field of cancer biology, as we will attempt
to review in this paper.
Mass spectrometry-based proteomics & metabolomics
Cancer proteomics still represents a mainstay in cancer research
since the dawn of the post-genomic era [6]. The underlying
65 assumption is that proteins can be regarded as the effectors of
cellular functions and thus, in biological terms, protein profil-
ing might be more informative than mRNA profiling [7].
Nevertheless, despite almost two decades of efforts, the ambi-
tious agenda pursuing the complete annotation of the physio-
70 logical role of all known genes still remains unfulfilled [8].
From a technical standpoint, recent technical advancements
have opened new scenarios in the field of proteomics. While a
decade ago separative and quantitative proteomics approaches
mainly relied upon 2DE-based analyses and the implementa-
75 tion of HPLC-MS-based workflows was only auspicated, cur-
rent proteomics analyses actually take advantage of quantitative
analyses via chromatography-MS approaches. Novel instru-
ments have been indeed implemented (both at the HPLC and
MS levelthe interested reader is referred to reference [7] for
80 further details), as well as bioinformatics suites and tools, which
simplified quantitative analysis by allowing peak alignment,
detection, protein identification and attribution of post-
translational modifications (PTMs).
Quantitative proteomics
85 Among quantitative proteomics approaches that have been
extensively applied in the field of cancer research, three main
strategies have gained momentum: i) in vivo labeling with stable
isotopes; ii) in vitro labeling and iii) label-free approaches [7].
The basic concept behind labeling strategies is that a stable-
90 isotope labeled peptide shares identical chemical features with
its native (unlabeled) counterpart, which results in identical
behavior during chromatographic separation and mass spectro-
metric analysis, though it still allows them to be differentiated
owing to their mass difference. Relative abundances can be
95 then grasped by measuring the ratio of signal intensities for the
labeled and unlabeled peptide pairs under different biological
conditions [7].
Stable isotope labeling of amino acid in culture (SILAC), is
probably the most extensively adopted in vivo labeling
100 approach in cancer research [9]. The SILAC protocol envisages
cell culturing in media containing either normal amino acids
or amino acids labeled with heavy isotopes. The labeled amino
acids are often lysine and arginine (with different combinations
of 13C, 15N, and 3H). The choice to label these basic amino
105 acids stems from the broadly diffused adoption of trypsin as
the protease of choice upstream of HPLC-MS proteomics
2 Expert Rev. Proteomics 10(5), (2013)
analyses. In SILAC, trypsin cleavage thus exposes C-terminally
labeled arginine or lysine, which allows relative quantitation of
each digestion-generated peptide, except for the C-terminus
peptide of the protein [7,9]. While SILAC was at first optimized 110
for unicellular model organisms [7,9], its experimental design
makes it suitable for cell culture experiments and thus amena-
ble for in vivo/ex vivo cancer research investigations. A direct
evolution of SILAC is super-SILAC [10], a method that com-
bines a mixture of multiple SILAC-labeled cell lines. Indeed, 115
SILAC is a particularly accurate quantitative method, although
until recently it was limited to cell lines or animals that could
be metabolically labeled with heavy amino acids. This limita-
tion of SILAC in studying patient tumor samples has been
overcome through the use of a mix of multiple SILAC-labeled 120
cell lines as an internal standard, a technique called super-
SILAC [10]. This mix achieved superior quantification accuracy
compared with a single SILAC-labeled cell line, owing to the
generation of hundreds of thousands of isotopically labeled
peptides in appropriate amounts to serve as internal standards 125
for MS [11].
Isotope-coded affinity tags (ICAT) are one of the most rap-
idly expanding in vitro labeling techniques for protein quantita-
tion. ICAT is based upon specific tagging of cysteine residues
with a reagent containing either eight or no deuterium atoms, 130
along with a biotin group that enables affinity purification strat-
egies to recover and enrich labeled peptides prior to MS analy-
sis [7]. One major limitation of this technique is that it can be
only applied to those proteins that contain cysteine residues.
Isobaric tags for relative and absolute quantification 135
(iTRAQ) is another important in vitro labeling strategy. In
iTRAQ, tagging requires a reporter group, a balance group and
a peptide reactive group. The reactive group binds the N-
terminus and side-chain amines of peptides, while the reporter
(up to eight different labeling patterns are possible) and the 140
balance group are designed as to achieve isobaric balancing in
MS mode, and discriminating fragments in collision induced
dissociation (CID) mode for relative quantitation on the basis
of the relative abundance of different reporter groups [7].
Another labeling strategy for quantitative proteomics implies 145
the use of isotopomer labels, referred to as tandem mass tags
(TMTs) [12]. TMTs are designed to ensure that identical pepti-
des labeled with different isotopomers exactly comigrate in all
chromatographic separations. On the other hand, peptides
from different samples can be identified and relatively quanti- 150
fied using CID-based analysis method. Relative abundance
measurements made in the MS/MS mode using the new tags
are accurate and sensitive [12].
Another strategy aims at quantifying protein abundances by
targeting a so-called proteotypic peptide (defined as an experi- 155
mentally observable peptide that uniquely identifies a specific
protein or protein isoform [13]) through selected reaction moni-
toring (SRM)-MS, which enables isolation and quantitative
assaying of the expected mass to charge ratio (against standards
or in silico predicted values) [14]. Proteotypic standard peptides 160
could be used as external references to determine calibration
 Proteomics, metabolomics & drug development
curves, as resulting from the correlation of MS readings (either
peak intensities or peak areas) in response to a variation in pep-
tide concentrations. However, SRM is not necessarily a synonym
165 for label free quantitation. Early evolutions of the SRM
approach imply the use of isotopically labeled synthetic peptides
(SPIKETIDES [15,16] or AQUA peptides) to enable absolute
quantification of specific proteins in a targeted fashion. The clear
advantage over non-labeled peptides is that isotopically labeled
170 proteotypic peptides can be directly spiked in the sample to be
used as an internal reference, which also helps coping with any
untoward technical bias at the nano HPLC or MS level.
While labeling-based quantitative strategies are rather expen-
sive and often time consuming (especially in vivo, which might
175 require four to six replication cycles of cultured cells to achieve
full labeling), label free approaches are increasingly attracting a
great deal of interest, owing to the reduced costs and ease of
implementation to classic HPLC-MS proteomics workflows.
Recently introduced post hoc algorithms allow calculation of the
180 so-called exponentially-modified protein abundance index
(emPAI), where the number of identified peptides, normalized
against the number of all the possibly identifiable peptides for
a given protein, is used as an indicator of the absolute protein
abundance on a logarithmic basis. Other indicators of absolute
185 protein abundances include spectral counting and peak
intensities [7].
Redox proteomics
Deregulation of metabolism in cancer cells is also intercon-
nected with increased susceptibility to, and exacerbation of, oxi-
190 dative stress (as it will be extensively described in the following
paragraphs). The key approaches to redox proteomics have
been recently reviewed [14,17]. Redox proteomics is a recent
branch of proteomics that is devoted to the determination and,
possibly, quantification of oxidative modifications to proteins
195 (including protein carbonylation, oxidation/nitrosylation of
thiol groups and nitrosylation of tyrosines).
Among all redox modifications, oxidation of thiol groups
might affect the functional activity of several key enzymes (such
as metabolic and redox-homeostasis-related enzymes, including
200 glyceraldehyde 3-phosphate dehydrogenase and peroxiredoxin
2) [14,17]. Owing to the labile nature of thiol groups-targeting
oxidative modifications, an experimental strategy envisages the
temporary quenching of free thiols (by means of trichloroacetic
acid-based acidification or through the use of cell permeable
205 reagents for thiol-groups, such as the alkylating agents iodoace-
tamide or N-ethylmaleimide) and subsequent specific reduction
(also with dithiothreitol, sodium arsenite or dimedone) [14,17].
On the other hand, commercially available antibodies can be
now exploited to determine the extent of S-nitrosylations, via
210 enabling antibody-based enrichment strategies (immunoprecipi-
tation) or direct immunoassay detection (ELISA, western blot).
Other analytical approaches also include biotin labeling for the
enrichment of S-glutathionylated peptides or isotope labeling
(especially ICAT, see above) to enrich, determine and quantify
215 S-oxidative modifications [14,17].
www.expert-reviews.com
Review
Further developments in the field of cancer redox proteomics
are awaited in the next few years.
Phosphoproteomics
Despite the significant body of accumulating knowledge about
genes involved in the development of human cancer (at least 220
300 have been discovered so far), only a limited number of can-
cer genes encode for proteins that are suitable targets for effective
drugs. In this view, protein kinases (such as Abl tyrosine kinase)
are among the best eligible targets for small molecule inhibi-
tors [18,19]. Indeed, sustaining proliferative signaling is a key hall- 225
mark of cancer, which is often achieved through kinase-triggered
phosphorylation cascades [1]. It is thus pivotal to further our
understanding of the biological role of protein phosphorylations
through the introduction of novel analytical strategies to enhance
their detection. Within this context, big strides have been 230
recently made in the field of phosphoproteomics. Phosphoryla-
tion (mostly of S/T, and to a lesser extent to Y amino acid resi-
dues) is a reversible PTM that plays important regulatory
functions in cellular signaling pathways, which can influence cell
growth, differentiation, invasion, metastasis and apoptosis [18,19]. 235
Since protein phosphorylations are often sub-stoichiometric,
enrichment strategies are often necessary to enable determina-
tion of differential phosphorylation events. Enrichment
strategies are either based upon affinity chromatography
(immobilized metal ion affinity chromatography), titanium 240
dioxide, zirconium dioxide, calcium phosphonate precipitation
or strong cation exchange or immunoprecipitation strategies.
Detection via non-MS methods mainly involves antibody-based
approaches, while MS-based approaches rather rely on isotopic
labeling (ICAT, iTRAQ or 32P/33P) and/or alternative (to colli- 245
sion induced dissociationCID) fragmentation strategies, such
as electron transfer dissociation (ETD), which favors generation
of c and z ions upon peptide fragmentation (instead of b and y
ions, which are predominant in CID) [19].
Glycoproteomics 250
Evading growth suppression and activating invasion and meta-
stasis (two key hallmarks of cancer [1]) is mainly achieved by
cancer cells through the modulation of membrane proteins,
which allow cancer cells to bypass contact-triggered growth-
inhibitory signaling. Glycosylation is one of the most common 255
PTMs, estimated to be found in over 50% of human pro-
teins [20] and more than 80% of membrane proteins [21]. Most
membrane biomarkers of cancer cells, which are amenable to
antibody-based therapies, are glycosylated proteins. Other than
extracellular membrane proteins, secreted proteins (which can 260
be thus searched for in the patients body fluids) are often
N-glycosylated in the endoplasmic reticulum or Golgi appara-
tus. It is thus small a wonder that protein glycosylation is
increasingly attracting a great deal of interest in the field of
cancer research. Glycosylations can be further distinguished 265
into: i) N-linked glycosylation, ii) O-linked glycosylation
and iii) C-glycosylations. In like fashion to phosphoproteomics,
glycoproteomics approaches often rely upon preliminary
3
 Review DAlessandro & Zolla
enrichment strategies (lectin affinity or boronic acid chromatog-
270 raphy or immunoprecipitation) to selectively enrich those pro-
teins bearing N-, O- or C-glycosylations. Glycosylated peptides
can then be screened via the use of MS, either MALDI or ESI-
MS, the latter relying both on CID and ETD fragmentation
modes [22].
275 Enriched glycosylated peptides can be thus released via enzy-
matic digestion (with PNGase F for N-glycosylations) and by
chemical methods (for O-glycan release) [20]. Alternative enzy-
matic digestion combinations can help further define the
arrangement of side-chain branches, the most challenging task
280 in glycoproteomics analyses to date [23].
Imaging mass spectrometry
One of the greatest advances over the last decade in the field of
MS-based cancer research has been the introduction of MALDI
imaging approaches. Imaging MS is a molecular analytical tech-
285 nology that enables the simultaneous measurement of multiple
analytes directly from intact tissue sections [24]. Histological fea-
tures of the sample, as gleaned through classic immunohisto-
chemistry staining, can indeed be correlated with molecular
species (proteins, peptides, lipids and metabolites) without the
290 need for target-speci?c reagents such as antibodies.
Imaging MS is based upon matrix spraying on suitably-
treated cryostat sections mounted on conductive indium titai-
num oxide-coated slides. The preliminary treatment depends
on the molecular species under investigation (e.g., organic sol-
295 vents might interfere with on tissue lipid analyses). MALDI
imaging can indeed be applied to determine molecular signa-
tures that are specific of a tumor tissue, while theoretically eas-
ing the individuation of tumor biomarkers and their
discrimination from tumor border biomarkers [25]. The possi-
300 bility to combine it with to routine immunohistochemical
approaches offers the opportunity to validate and complement
the information attainable from a biopsy while obtaining addi-
tional complimentary proteomics/lipidomics/metabolomics-rele-
vant results.
305 Indeed, imaging MS can be applied to obtain tissue profiles
of proteins [26], peptides [27], lipids and metabolites [28] (includ-
ing drug metabolites, thus helping monitoring the efficiency of
a therapeutical treatment). Additionally, protocols have been
developed also to allow imaging analyses of formalin-fixed par-
310 affin-embedded tissue slices [29].
One main limitation of imaging techniques is related to repro-
ducibility (mainly affected by matrix depositing issues), although
recently introduced automatic sprayers dramatically abated tech-
nical variability. Other limitations include the difficulty of moni-
315 toring high molecular weight compounds (especially proteins
above the 5060 kDa threshold) and the constraints related to
the relative abundances of molecular species (e.g., most abundant
compounds are often easily visible, while low abundance ones are
hardly detectable through this approach).
320 However, the flexibility of the method makes it suitable for
targeted quantitative approaches (such as SRM to low molecu-
lar weight compounds, such as drugs [30]) directly on tissue.
4 Expert Rev. Proteomics 10(5), (2013)
From proteomics to metabolomics: MALDI imaging &
MALDI-based metabolomics
Metabolomics is the global quantitative assessment of metabo- 325
lites (low molecular weight compounds below the 1.5 kDa
threshold, including sugars, phosphate compounds, organic
acids, nucleosides, lipids and fatty acids or exogenous com-
pounds) in a biological system [31].
While proteomics investigates the effectors influencing the 330
phenotype, metabolites are the phenotype itself. Indeed, the
historical precursor to metabolomics can be traced back to early
clinical biochemistry approaches [32], while technical advance-
ments in the field NMR and, subsequently, of MS [32,33] have
boosted the refinement of this old/new omics discipline. NMR 335
was at first favored by machine accessibility, established data
handling and the conservative nature of the analysis, which
allows further testing downstream of NMR analyses on the
same samples. Conversely, MS has gradually complemented
and often replaced NMR owing to its higher sensitivity, which 340
results in MS being less demanding in terms of minimum
detectable concentrations of the analyte [33]. Also, MS-based
metabolomics holds the potential to better discriminate metab-
olites, thus improving coverage of the metabolome space, espe-
cially when performing upstream compound-class-specific 345
chromatographic separations [32,33].
In likewise fashion to quantitative proteomics, quantitative
MS-based metabolomics can also rely upon SRM or multiple
reaction monitoring (MRM) approaches [3436], which allow
detection and absolute quantification of a compound and its 350
fragments against a pure standard.
However, most advanced metabolomics studies today rely
upon post hoc alignment, peak detection and metabolite dis-
crimination without any a priori restriction: this approach also
goes by the name of untargeted metabolomics [37] and is 355
already providing decisive insights into cancer biology. In par-
ticular, a rather recent application of MS-based untargeted
metabolomics is based upon carbon flux during catabolism and
anabolism, via supplying 13C-labeled metabolic substrates
(mainly glucose and glutamine) [38]. This approach allows the 360
kinetics of energy fluxes to be monitored during molecular
biology experiments on cell lines (e.g., upon induction or
silencing of an oncogene or tumor suppressor protein), thus
helping further refine the understanding of metabolic networks
in normal and cancer cells. 365
Biomedical application of MALDI MS is technically suited
to monitoring metabolic variations directly on tissue sections
from biopsies, but it also allows the screening of whole body
tissue sections from model organisms (e.g., mice) while looking
for the metabolites derived from catabolism of a specific drug 370
under testing. This is relevant in the context of cancer research
since one of the key steps in drug discovery is the determina-
tion of the areas targeted by a therapeutic (body
accumulation) [39].
MALDI-imaging has recently been applied in cancer metab- 375
olomics research, especially in lipidomics analyses, since lipids
are highly preponderant and easily detectable through imaging
 Proteomics, metabolomics & drug development Review

approaches. One clear advantage with

MALDI imaging approaches is that a
380 molecule can be directly detected on tis- MCT Glut1 GSR GLY SLC38A
GSSG GSH
sue, with a lateral resolution down to GSS
1015 mm with commonly available Glucose GLCY
NADPH NADPH
instruments (while in secondary ion HXK GCLM
imaging MS it can be improved up to G6P GL6P 6PG Ru5P CYST
G6PDH PGLS PGD
385 >50 nm [40]). These advancements have GNPNAT1 GFPT1 GPI
TKT
RPE RPIA GLUT
GlcNAc-6P GlcN-6P F6P X5P R5P GLS
paved the way for a broader application PGM FBPase PFK TALDO1
GLTM
of MALDI-based proteomics and GlcNAc-1P ALDOA
FBP E4P G3P TKT

F6P GLUD1
metabolomics strategies [41]. UAP1
DHAP G3P S7P
UDP-GlcNAc TPI GAPDH
MALDI-based metabolomics, on cell SBP
BPG
390 lysates or tissue homogenates instead of PGK ALDOA NADPH
tissue sections, would have several SER PSER 3PHPYR 3PG E4P DHAP
PSP PSAT 3PGDH PGAM
advantages over routinely used MS- SHMT 2PG ACO ISOCIT IDH
ENO
based metabolomics platforms (such as GLY PEP CITR -KET
PKM
LC-MS), in that it would be amenable Pyruvate
OGDH
ACLY SUCC-CoA
395 to automatization and multiplexing, LDH
PDH
Acetyl-CoA SUCCLG1
especially in combination with robotic Lactate OAA SUCC
MDH SD
auto samplers. MAL FD FUM

Anabolism & the Warburg effect Figure 1. An overview of the main catabolic pathways in normal and proliferat-
The recent introduction of specific ing cells. Glycolysis, Krebs cycle, PPP, serine synthesis, hexosamine synthesis and gluta-
400 metabolomics analytical platforms helped thione homeostasis are included. Enzymes are indicated in light grey, according to their
elucidating metabolic fluxes in highly relative UniProt names.
PPP: Pentose phosphate pathway.
proliferating cells [42,43]. In line with
Kilburns observations [44], dating back to
four decades ago, highly proliferating cells do not have extremely species (ROS) in the form of superoxide anions and hydroxyl
405 higher energy demands (in terms of glucose metabolization) in radicals, which ends up fertilizing the tumor microenviron-
comparison to resting cells. This at least in part justifies the met- ment [46] and promotes the accumulation of further muta-
abolic choice of oxidizing glucose via glycolysis while depressing tions to oncogenes and tumor suppressors [47]. Of note, the 435
the TCA cycle in cancer cells. However, an elevated replication impairment in ROS modulation/production in mitochondria
rate is based upon the accumulation of building blocks to build is often accompanied by mutations to electron transport
410 up mass and cell constituents before replication. In this scenario, chain components [48].
metabolomics analyses contributed significant insights by demon- In parallel, most cancers share distinct features such as
strating how alternative metabolic pathways are indeed activated defects in certain mitochondrial enzymes, including isoci- 440
along with glycolysis, which promote anabolism by constantly trate dehydrogenase (IDH), fumarate dehydrogenase (FD)
providing key reducing coenzymes such as NADPH (that is piv- and succinate dehydrogenase (SD) [49,50]. Alterations to IDH
415 otal, e.g., in lipid synthesis) (FIGURE 1). In parallel to aerobic glycol- or isoform switching (IDH1 vs IDH2) promote the utiliza-
ysis, glucose utilization fuels the main NADPH-generating tion of a-ketoglutarate from glutamine metabolism via
pathway, the (oxidative phase of the) pentose phosphate pathway reductive carboxylation to isocitrate, to fuel acetyl-CoA pro- 445
(PPP). Over-activation of the PPP at the non-oxidative phase duction for fatty acid biosynthetic purposes [5153] or amino
fuels the generation of ribose phosphate substrates for nucleoside acid synthesis via oxaloacetate intermediates (FIGURE 2). Of
420 biosynthesis, another central step toward DNA replication in note, isocitrate to a-ketoglutarate conversion by cytosolic
proliferating cells. It is perhaps worthwhile to recall that IDH is associated with the production of NADPH, analo-
NADPH also plays a fundamental role in the recycling of oxi- gous to the conversion of malate (another TCA cycle inter- 450
dized glutathione (GSSG) back to the reduced form (GSH), mediate) to pyruvate by malic enzyme. Anomalies to
thus contributing substantially to the redox poise (FIGURE 1). IDH1 enzyme (R132H) result in 2-hydroxyglutarate pro-
425 In this complex metabolic scenario, mitochondria are not duction from a-ketoglutarate, which negatively affects a-
just innocent and inactive bystanders in cancer cells [45]. ketoglutarate-dependent dioxygenase enzyme activity and
Mitochondrial metabolism is indeed fueled by glutamine, promotes malignant progression of brain tumors and, in 455
which is one major nitrogen source for biosynthesis reactions particular, gliomas [54].
and carbon source (via glutamate-a-ketoglutarate intermedi- Anomalies to FD and SD results in the accumulation of
430 ate conversion) for the TCA cycle (FIGURE 1). At the same time, fumarate and succinate, respectively. These metabolites (along
mitochondrial activation fuels production of reactive oxygen with the aforementioned 2-hydroxyglutarate) have been recently

www.expert-reviews.com 5
 Review DAlessandro & Zolla

Fatty acid view, fumarate and succinate have been recently referred to as
Synthesis oncometabolites. Also, HIF accumulation diverts IDH-
dependent reductive carboxylation fluxes toward fatty acid 470
Reductive carboxylation
anabolism [56], which further stresses the intertwinement of
ISOCIT IDH
metabolic deregulation with proliferative capacity (FIGURE 3) [57].
CITR -KET GLUT GLTM
Cell proliferation, tumor suppressors, oncogenes &
SUCC-CoA
Acetyl-CoA metabolism
OAA SUCC Metabolic deregulation in cancer cells is partly the cause and 475
mostly the effect of genetic deregulation, at the oncogene and
MAL FUM tumor suppressor level [58]. As described in the previous para-
Amino acid
Synthesis
graph, these metabolic adjustments serve to build up anabolic
products to pursue cell proliferation [59]. At the same time, they
Figure 2. Cancer is often associated with mutations to TCA promote deregulation of oxidative phosphorylation and mito- 480
cycle enzymes, or expression of specific isoforms. In the chondrial events, thus favoring ROS accumulation and altera-
case of IDH, this results in the promotion of reductive carboxyla- tions to the tumor microenvironment. Exacerbation of oxidative
tion from glutamine-derived a-ketoglutarate, instead of regular
stress promotes senescence-like phenomena in cancer cells, while
TCA cycle fluxing towards succinyl Co-A. This utterly results in
acetyl Co-A accumulation, which promotes fatty acid synthesis decreasing glucose uptake, deregulating matrix attachment [59].
and, thus, cell growth and proliferation. At the same time, cancer cells cope with the excess of ROS by 485
IDH: Isocitrate dehydrogenase; TCA: Tricarboxylic acid cycle. activating pro-survival pathways, through the deregulation of
specific oncogenes [60], often complementary to inactivating or
gain of function mutations to tumor suppressor proteins, such
460 found to play a key role as direct inhibitor of dioxygenase and as proteins from the p53 family (p53, p63 and p73), which
AQ2 prolyl hydrolase, enzymes that indirectly catalyze the degrada- normally act as the guardians of the genome stability. One para- 490
tion of the hypoxia-inducible factor (HIF) [5153]. This inhibi- digmatic example is indeed represented by mutations to p53 (e.
tory phenomenon is relevant since accumulation of HIF, which g., R175H and R273H [61]), resulting in cell survival, increased
normally occurs under hypoxia, mediates the activation of pyr- proliferation and promotion of invasiveness.
465 uvate dehydrogenase kinase (PDK1), which in turn inhibits
pyruvate dehydrogenase and thus hinders conversion of pyru- The double role of p53 family members
vate to acetyl-CoA and shunts pyruvate to lactate [55]. In this While early cancer investigation studies indicated that p53 and 495
retinoblastoma protein (RB) mutations (comprehensively
Glucose detected in the great majority of tumors) mainly resulted in the
loss of function of their tumor-suppressor activity [62], recent
study indicate how these proteins (especially p53) might play a
more complex role in modulating the balance of pro-survival 500
ISOCIT and pro-apoptotic signaling, a function that escapes regulation
CITR -KET upon the acquisition of specific mutations to these proteins [63].
Pyruvate
Tp53, for example, is now known to take part in metabolic
SUCC-CoA
Acetyl-CoA modulation at several levels [64]. Analogous roles are increas-
PDH
Lactate OAA SUCC ingly emerging for all the members of the p53 family [65]. For 505
MDH SD
PDK MAL FD FUM example, p53 can inhibit the expression of the glucose trans-
porters GLUT1 and GLUT4 [64], and can increase the expres-
HIF PDH
sion of Tp53-inducible glycolysis and apoptosis regulator
(TIGAR), a fructose-bisphosphatase that inhibits glycolysis by
Figure 3. Cancer is often associated to mutations to Krebs
cycle enzymes, including SD, FD and MDH. These mutations reducing cellular levels of fructose-2,6-bisphosphate and thus 510
end up blocking TCA cycle catabolic fluxes, and promote the promoting a shift backward to the PPP, a process that pro-
accumulation of the respective substrates of these enzymes, suc- motes NADPH accumulation and plays a role in anti-apoptotic
AQ3 cinate, fumarate ad malate. These oncometabolites inhibit prolyl signaling via ROS-damage protection and promotes anabolic
hydrolase activity, thereby indirectly resulting in HIF stabilization. pathways, as summarized in the previous paragraphs [6668]
In turn, this promotes PDK1 activity, an inhibitory enzyme of
pyruvate dehydrogenase. This results in increased lactic acid (FIGURE 4). Besides, p53-responsive elements are present in the 515
fermentation and decreased fluxes from glycolysis to the promoters of PGM and hexokinase II (HK2), which is sugges-
TCA cycle. tive of the fact that p53 can promote at least some steps in gly-
FD: Fumarate dehydrogenase; HIF: Hypoxia-inducible factor; colysis. Of note, under hypoxic conditions, mitochondrial
MDH: Malate dehydrogenase; PDK1: Pyruvate dehydrogenase kin- localization of TIGAR stimulates HK2 (often bound to mito-
ase; SD: Succinate dehydrogenase; TCA: Tricarboxylic acid cycle.
chondrial membrane in tumors) [66]. 520

6 Expert Rev. Proteomics 10(5), (2013)

 Proteomics, metabolomics & drug development
Other than p53, a transactivation-proficient isoform of
p73 has been recently shown to play a role in the promotion
of a metabolic shift toward the PPP, via the upregulation of
enzymes involved in the oxidative phase of the pentose phos-
525 phate shunt such as 6-phosphogluconolactonase [69].
Tp53 also regulates the expression of sestrin proteins, which
activate AMPK to regulate growth and autophagy but also
function as antioxidants, protecting cells from hydrogen
peroxide-induced damage [65]. Furthermore, also in terms of
530 antioxidant defenses, p53 triggers the activation of glutathione
peroxidase, aldehyde dehydrogenase and tumor protein
p53-inducible nuclear protein 1 (TP53INP1), all of them dis-
playing antioxidant functions.
However, it should not be forgotten that many p53-induci-
535 ble proteins participate in apoptotic responses via the promo-
tion of ROS production, including p53-induced gene
3 (PIG3), proline oxidase, BAX, PUMA and p66SHC [65], as
well as of cytochrome C oxidase 2 [70].
Tp53 further influences mitochondrial metabolism by pro-
540 moting the expression of glutaminase 2, glutaminases being a
family of enzymes responsible for glutamine to glutamate con-
version [71]. Analogous effects on glutaminase expression have
been recently reported also for p63 [72]. In turn, glutamate is a
pivotal constituent of the tripeptide GSH, or rather it can be
545 further metabolized to a-ketoglutarate, that can either undergo
reductive carboxylation to isocitrate or further oxidation to
succinyl-CoA via the TCA cycle.
Energy or nutrient deprivation, extreme environmental con-
ditions or Ca2+ release from the lumen of the endoplasmic
550 reticulum (ER) results in the disruption of proper protein-
folding activity in this organelle, a condition that promotes the
so-called ER-stress. ER-stress has been observed to influence
p53 stability by modulating its differential phosphorylation to
serine 315 and serine 376, which promotes p53 localization in
555 the cytoplasm and its degradation [73,74]. Conversely, other
p53 family members, p63 and p73, have been shown to pro-
mote ER stress and scotin (protein shisa-5) [75,76], suggesting an
intricate cross-talk among p53-family members, other than
direct competition for p53 responsive elements or oligomeriza-
560 tion through direct binding. In this view, it is interesting to
note that almost pleiotropic metabolic effects are also expected
for other p53 family members, since, for example,
TAp73 deletion reduces cellular ATP levels, oxygen consump-
tion and mitochondrial complex IV activity, with increased
565 ROS production and oxidative stress sensitivity [77]. This phe-
nomenon involves the mitochondrial complex IV subunit cyto-
chrome C oxidase subunit 4 (Cox4i1), which is a direct
TAp73 [77].
Metabolic starvation experiments
570 On the basis of the evidences for modest energy demands,
albeit extreme substrate uptake, especially of glucose and gluta-
mine (we hereby purposely neglect uptake of lipids from the
medium, which would deserve an entirely dedicated review) for
anabolic and redox homeostasis purposes, new trends in the
www.expert-reviews.com
Review
field of cancer drug research are aimed at evaluating the effects 575
of starvation on cancer cells. One simple approach to promote
cancer cell starvation in vivo would be to prevent angiogenesis,
which would reduce the blood flux and thus oxygen and
nutrient delivery [78]. Anti-angiogenic therapy mainly relies
upon the administration of anti-VEGF-targeting monoclonal 580
antibodies (e.g., bevacizumab).
Within this framework, in vitro metabolomics experiments
have recently provided a clearer understanding of the mecha-
nisms underlying the effects of starvation on cancer cells. Since
the main metabolic substrates for carbon and nitrogen build up 585
in cancer cells are both glucose and glutamine, starvation
experiments have been so far performed through the supple-
mentation of cell media that are depleted of these two com-
pounds [7983]. A complex scenario emerged whereby glucose
might represent the main energy source for certain cell lines 590
(such as head and neck squamous carcinoma cells [79]), while
only glutamine depletion actually triggered apoptosis in other
cell lines [83].
Other than glucose and glutamine, cancer cells necessitate
serine to support anabolism by providing precursors for biosyn- 595
thesis of proteins, nucleotides, creatine, porphyrins, phospholi-
pids and glutathione. Also, up-regulation of the serine synthesis
pathway occurs in some breast cancers [84,85]. It has been
recently demonstrated that p53-mediated cell responses to ser-
ine starvation involve over-activation of the serine synthesis 600
pathway. Besides, it promotes inhibition of glycolysis, since a
rate-limiting enzyme, in particular, the specific and less efficient
cancer isoform, pyruvate kinase M2 (PKM2) [86], is allosteri-
cally activated by serine and thus, serine starvation, ends up
inhibiting glycolysis (FIGURE 4) [87]. This prompts two main 605
effects: increase in TCA cycle fluxes to cope with the decreased
ATP production, and an increase in PPP fluxes, to generate
NADPH and thus cope with oxidative stress arising from the
elevation in TCA cycle-dependent energy production [85]. Of
note, PKM2 expression in cancer cells results in relatively 610
decreased glycolytic rates, which promotes accumulation of
early glycolysis intermediates, including 3-phosphoglycerate, a
precursor to serine de novo synthesis.
Serine metabolism is also at the crossroads between p53 and
starvation-induced autophagic responses [88]. Autophagy is a 615
catabolic mechanism that promotes cell degradation of unneces-
sary or dysfunctional cellular components through the lysoso-
mal machinery to cope with the decrease in energy and
anabolic resources, and it is often regarded as: i) an alternative
option cells might choose to commit suicide, other than apop- 620
tosis, ii) a cells defensive strategy upon cell damage or iii) a
cells major adaptive (survival) strategy to cope with metabolic
stress, such as nutrient deprivation, or starvation in general.
Starvation, the most extensively investigated inducer of auto-
phagic responses, triggers the activation of AMPK [89,90], a kin- 625
ase that is activated by increased AMP/ATP ratios, and is
regulated to some extent by mammalian target of rapamycin
(mTOR), a key molecular sensor for nutrient availability and a
regulator of cell growth and proliferation [9195]. In particular,
7
 Review DAlessandro & Zolla

phosphatidylinositol (4,5)-biphosphate
(PIP2). In this way, PTEN modulates
GLUT1,
GLUT4 PIP3/PIP2 ratios and, indirectly, cell
Anabolism and Antioxidant responses
survival [96].
HXK
Glucose
NADPH NADPH
The list of oncogenes involved in meta- 655
bolic regulation is not only limited to HIF
G6P Oxidative phase PPP
and mTOR, but includes many other play-
F6P ers. One of those is MYC, which encodes
TIGAR Nucleoside a transcription factor c-Myc that links
Non-oxidative phase PPP
biosynthesis
p53 FBP altered cellular metabolism to tumorigene- 660
GLTM sis [97]. Indeed, c-Myc regulates genes
Bax
GLS2 involved in the biogenesis of ribosomes
GLUT and mitochondria, affects glucose and glu-
PUMA 3PG PEP
p66SHC Sestrins tamine metabolism, nucleotide metabolism
PKM2
Glutathione peroxidase
Cytochrome C oxidase 2 Pyruvate Lactate
Aldehyde dehydrogenase
and, along with E2F1, DNA replication 665
SER and miRNA expression [97]. Ectopic c-Myc
TP53INP1
cooperates with HIF to promote the
induction of a transcriptional program for
ROS
hypoxic adaptation, involving up-
Mitochondria regulation of glycolytic genes including lac- 670
Figure 4. Metabolic regulation by p53 at a glance. As a tumor suppressor, p53 is
tate dehydrogenase A, or the repression of
long known to promote apoptosis via enhancing pro-apoptotic factors and ROS- microRNAs (miRNAs) miR-23a/b to
generating mitochondrial metabolism. However, it recently emerged as a role for p53, as increase glutaminase protein expression
a pro-survival mediator under mild stress conditions. This phenomenon appears to involve and glutamine metabolism.
a TIGAR, a fructose-2,6-bisphosphatase that promotes accumulation of early glycolytic Recent flux-balance analyses have indi- 675
precursors thus boosting a diversion towards the PPP. The production of NADPH at the
non-oxidative phase of the PPP sustains anabolic and antioxidant processes. Evidences
cated a key role in glycolytic modulation
also indicate a role for p53 in the induction of anti-oxidant defenses (sestrins, glutathione (toward increase) and glutamine metabo-
peroxidase, aldehyde dehydrogenase, TP53INP1). At the same time, non-oxidative phase lism (towards reductive carboxylation) for
products fuel de novo nucleoside synthesis. Pro-survival effects mediated by p53 under another oncogene, KRAS [98,99]. In partic-
mild stress conditions appear to involve the homeostasis of serine metabolism (SER). ular, KRAS appears to be involved in the 680
PPP: Pentose phosphate pathway; ROS: Reactive oxygen species; TIGAR: Tp53-induced
glycolysis and apoptosis regulator; TP53INP1: Tumor protein p53-inducible nuclear
increase in glycolytic metabolism and
protein 1. channeling of glycolytic intermediates to
non-oxidative phase PPP reactions and
hexosamine biosynthesis (in turn promot-
630 autophagy initiation is associated with downregulation of ing protein glycosylation) [98]. Also, KRAS seems to promote an 685
mTOR complex 1 (mTORC1) activity. mTOR is a well- alternative pathway for glutamine metabolism to glutamate and
conserved serine/threonine kinase that belongs to the phosphoi- alternative downstream pathway. Such alternative route involving
nositide 3-kinase (PI3K)-related kinase family, and it plays an glutamate metabolism appears to be dependent on transami-
important role in the signaling network that controls growth nases, especially aspartate transaminases (GOT), which promote
635 and metabolism in response to environmental cues. The activa- glutamate and oxalacetate production from aspartate and a- 690
tion of mTORC1 (one of the two distinct multi-protein com- ketoglutarate (FIGURE 5). This results in oxaloacetate cytosolic accu-
plexes involving mTOR) requires glutamine and essential mulation (certain GOTs operate only in the cytosol), which
amino acids such as leucine, while it has been recently demon- prompts its conversion to malate (via malate dehydrogenase)
strated to also depend on availability of serine [87]. At the same and, through malic enzyme, to pyruvate, a reaction that concom-
640 time, Akt and AMPK communicate directly with mTORC1, itantly fuels NADPH production [99]. 695
by phosphorylating raptor (an mTORC1 component), leading Analogously, the tumor suppressor promyelocytic leukemia
to 14-3-3 binding and the allosteric inhibition of mTORC1 [94]. (PML) gene acted as both a negative regulator of PPARg coac-
Activation of mTORC1 promotes protein synthesis by phos- tivator 1A (PGC1A) acetylation and a potent activator of
phorylating the kinase S6K and the translation regulator 4E- PPAR signaling and fatty acid oxidation in breast cancer
645 BP1 and lipid biogenesis, via activation of SREBP and PPARg cells [100]. Finally, it is at least worth mentioning the long time 700
transcription factors [94,95]. The phosphatase and tensin homo- established role in metabolic regulation of the insulin-like
log (PTEN) interferes with the whole phosphoinositide 3-kin- growth factors (IGF-I and IGF-II) system [101].
ase (PI3K)/Akt/mTORC1 axis, by dephosphorylating MicroRNAs (miRNAs) are small RNA molecules that regu-
phosphatidylinositol (3,4,5)-trisphosphate (PIP3) at the 3 posi- late gene expression post-transcriptionally [97]. As anticipated in
650 tion of the phosphate of the inositol ring, thus producing the previous paragraphs, miRNA expression can be more or 705

8 Expert Rev. Proteomics 10(5), (2013)

 Proteomics, metabolomics & drug development Review

less directly involved in metabolic mod-

ulation [102105]. Small RNAs may have
an intrinsic function in tumor suppres-
sion, since their levels are globally Cytosol
710 decreased in human cancers cells [88]. In CO2
NADPH NAD+
line with this, the transcription of some ME1 MDH1 GOT1
miRNA genes (such as miR-34) is regu- Lactate PYR MAL OAA ASP GLTM
GLS
lated by p53 [102]. Of note, miR-34a GLUT CYST
appears to be a key regulator of hepatic
715 lipid homeostasis. Together with GLY
miR-34, other miRNAs play a key role GSH
in metabolic modulation, including
miR-33a and miR-33b, which have a ctive
Redu tion
crucial role in controlling cholesterol x yla
carbo
720 and lipid metabolism in concert with
-KET
their host genes, the sterol-regulatory
element-binding protein (SREBP) tran- Acetyl-CoA TCA cycle

TCA
scription factors [104]. Other metabolic OAA
miRNAs, such as miR-103 and
Mitochondrion
725 miR-107, regulate insulin and glucose
homeostasis [104], while Figure 5. Glutamine metabolism can follow different fates in normal and cancer
miR-143 regulates hexokinase-2 expres- cells. Glutamine can be converted to glutamate (via glutaminase enzymesGLS), and thus
sion in cancer cells [105]. AQ4 be metabolized to ketoglutarate, whereby it enters TCA cycle or rather promotes reduc-
tive carboxylation. In parallel, glutamate can represent a building block of the tripeptide
Induction of apoptosis via gene glutathione (GSH). An alternative route involves the malate-aspartate shuttle, a pathway
that involves glutamine-derived aspartate towards the accumulation of oxaloacetate and
730 therapy & metabolic malate intermediates in the cytosol. This pathway is relevant in that NADPH is produced
reprogramming to sustain anabolic and anti-oxidant pathways.
In the previous paragraphs we have sum- GSH: Glutathione; TCA: Tricarboxylic acid cycle.
marized how cancer cells often suffer
from mutations that provide a competi-
735 tive edge over normal cells in terms of biomass accumulation and laboratory testing or already under clinical evaluation (since 760
proliferative capacity. Starting from Warburgs initial hypothesis, they are based on already commercialized drugs).
latest research has revealed that metabolic reprogramming occurs
as a consequence of mutations in cancer genes and alterations in Targeting glycolysis to modulate the Warburg effect
cellular signaling. Thus, we described how these mutations end Substantiation of the Warburg effect derives from the increased
740 up promoting certain pathways (glycolysis, PPP, nucleoside and glucose uptake by cancer cells and increased lactate production
fatty acid biosynthesis, NADPH-generating reactions) at the via glycolysis, even in presence of oxygen. On this ground, 765
expenses of others (above all, oxidative phosphorylation). Appreci- these characteristics have fostered the concept of new classes of
ation of these phenomena was a step forward from the canonic drugs that can be used either to monitor cancer cell metabo-
conception of the Warburg effect and opened new avenues for lism via state of the art diagnostic tools or to make it amenable
745 future developments in the field of cancer treatment [106]. There- to therapeutic interventions [111114].
fore, untuning the metabolic machine [99] might represent the For example, since tumors consume higher levels of glu- 770
new trend in cancer therapies, which might surprisingly be cose, clinicians have long been able to monitor tumor uptake
boosted by decades of research in the field of metabolism-related of a fluorine radioisotope of glucose, 18F-deoxyglucose, by
diseases, including diabetes and drugs for diabetes treatment (such FDG-PET. This technique has proven its usefulness in deter-
750 as metformin) [107]. mining the cancer stage, to identify metastatic sites and mon-
Other metabolic reprogramming strategies envisage the pro- itor treatment effectiveness. In parallel, a correlation has been 775
motion of oxidative stress via mitochondrial uncoupling [108]. observed between the initial degree of FDG-PET positivity
Another approach is related to caloric restriction, ketogenic diets and the overall patient outcome across cancer types and
and modulation of circulating nutrient levels through enzymes subtypes [111].
755 such as asparaginase, these strategies imply that tumor cells are Therapeutic intervention based upon drugs that target glyco-
more demanding in terms of nutrients in comparison to normal lytic enzyme activities are currently under evaluation. Among 780
cells [106110]. the possible targets, hexokinase (HXK), phosphofructokinase
In this section, we will briefly describe the main metabolic (PFK), glyceraldehyde 3-phosphate dehydrogenase (GADPH)
reprogramming strategies [105109] that are currently either under and lactate dehydrogenase (LDH) represent ideal therapeutic

www.expert-reviews.com 9
 Review DAlessandro & Zolla
targets, since chemical inhibitors are already known (though
785 they are often not extremely specific).
Known inhibitors of hexokinase include 2-deoxyglucose,
3-bromopyruvate, 5-thioglucose and mannoheptulose. In par-
ticular, 3-bromopyruvate is a strong alkylating agent toward
the free SH groups of cysteine residues in proteins, which
790 might thus also affect those enzymes with thiol groups in their
active sites (such as GAPDH).
Lonidamine is known to inhibit only the mitochondria-
bound hexokinase, which is a distinctive feature of cancer cells
(please, refer to the previous paragraphs) [111114].
795 Use of a PFK inhibition strategy implies the suppression of
the PFKB3 isozyme, which controls the cellular level of fruc-
tose-2,6-bisphosphate and thus affects the glycolytic flow by
allosterically-modulating PFK activity.
Known inhibitors of GAPDH include a-chlorohydrin,
800 ornidazole and iodoacetate, as well as the pentovalent
arsenate [111].
LDH-A can be knocked down in tumor cells by shRNAs,
thereby stimulating energy fluxes from pyruvate to mitochon-
dria, which in turn promotes mitochondrial uncoupling
805 (ROS production, accumulation of pro-oxidant intermedi-
ates) in those tumors bearing mutation of mitochondrial
enzymes. Targeting LDH might represent a cancer-cell sup-
pressing preferential strategy, while it could be less toxic to
normal cells [111].
810 Other therapeutical interventions might involve the use of
oxythiamine (a thiamine antagonist that inhibits transketolase
and pyruvate dehydrogenase) or glufosfamide. Glufosfamide is
a conjugate of glucose (highly consumed by cancer cells) and
ifosfamide, an alkylating agent with cytotoxic effects. Of note,
815 glufosfamide is uptaken via the SAAT1 glucose transporter,
which is overexpressed in cancer cells [111114].
Owing to its pro-glycolytic potential, HIF-1-targeting drugs
should be included, to a certain extent, in the same category of
glycolysis inhibitors [115]. Several classes of drugs have been
820 designed and tested over the years, targeting HIF transcription,
synthesis, stability, heterodimerization, DNA-binding activity
or targets downstream to HIF-controlled signaling [115].
Targeting NAD-metabolism has recently emerged as a
potential therapeutic approach to tackle cancer cell prolifera-
825 tion, since NAD undergoes crucial changes in cancer cells,
whereby its use in transcription, DNA repair, cell cycle progres-
sion, apoptosis and metabolism processes is deregulated in
comparison to normal cells [116].
Targeting autophagy
830 Although autophagy can result in the suppression of tumor
development, it may also mirror an extreme and desperate
attempt of the cancer cells to survive. Owing to the complexity
of autophagic responses in mediating cancer survival/suppres-
sion, several strategies have been proposed to tackle autophagy
835 over the last few years [117119].
Treatments with rapamycin (targeting mTOR), for example,
induce glucose starvation-like effects in cancer cell [119]. Another
10
recent strategy to promote autophagic responses relies upon the
administration of cannabinoid receptor agonists [120].
Starvation (via nutrient deprivation) of cancer cells has been 840
proposed as a viable strategy to tackle cancer cell prolifera-
tion [121,122], especially when used in combination with
chemotherapy [123,124].
Old drugs, new benefits
Drug discovery is an extremely complicated, expensive and, 845
most of the time a challenging (if not discouraging) area of
research. Patenting, testing and commercializing new effective
drugs is a prohibitive task even for multi-national companies,
which need to invest resources (in terms of funds and trained
personnel) for more than two decades, before (when lucky) 850
receiving the final approval by US FDA. A record of currently
available drugs, the latest version of DrugBank (release 2.0),
includes a list of approximately 4900 drug entries, in which are
enlisted both FDA-approved small molecule and biotech
drugs [125]. Since metabolic diseases have long been investi- 855
gated, especially those involving deregulation of glucose homeo-
stasis, such as diabetes, a long list of currently commercialized
drugs already exists that might be amenable for cancer treat-
ment, in the light of the revisited role of the Warburg effect
and metabolic reprogramming in cancer progression. Bigua- 860
nides (such as metformin) belong to this category of old drugs
with potential new benefits. Biguanides were first isolated in
1920 from the French lilac Galega officinalis, which was known
to contain an agent that reduced the frequent urination associ-
ated with diabetes. Biguanides have step up to the spotlight for 865
their ability to suppress liver gluconeogenesis, which is believed
to occur through activation of hepatic AMP-activated protein
kinase (AMPK) signaling [107]. It was but in recent years that
the antitumor effect of metformin could be observed, an effect
that is mediated by the activation of AMPK and thereby mod- 870
ulating the AMPK/mTOR pathways. At higher doses (than
physiologically achievable in vivo), metformin appears to
directly affect mitochondrial oxidative phosphorylation in
cancer cells.
Targeting fatty acid synthesis & metabolism 875
Owing to their highly proliferating nature, tumor cells need to
build up new membrane to favor replication into daughter
cells. In this view, fatty acid synthesis and uptake from the
medium (tumor microenvironment) are two key pathways that
have attracted a great deal of interest at least during the last 880
10 years [126]. Targeting fatty acid synthesis involves promoting
lipid lowering PPAR-pathways (via fenofibrate, also decreasing
local angiogenesis) [127] or rather by addressing fatty acid oxida-
tion (FAO) [126,128]. While most cancer researchers focused on
glycolysis, glutaminolysis and fatty acid synthesis, the role of 885
fatty acid oxidation in cancer cell metabolic transformation has
not been hitherto carefully examined [129].
FAO is inhibited by oxidative stress and, though indirectly,
it might contribute to counteracting ROS accumulation in can-
cer cells [129]. Indeed, FAO generates one molecule of acetyl 890
Expert Rev. Proteomics 10(5), (2013)
 Proteomics, metabolomics & drug development
CoA in each oxidation cycle and two in the last cycle. Acetyl
CoA enters the Krebs cycle, and combines with oxaloacetate to
give rise to citrate. As described in the previous paragraphs,
IDH-mediated cytosolic conversion of isocitrate to a-
895 ketoglutarate also produces cytosolic NADPH (for anabolic
and antioxidant purposes). This is the same pathway (though
in the opposite direction), according to which fatty acid synthe-
sis under hypoxic conditions (or when mitochondrial respira-
tion is limited) might rely upon glutamine-glutamate-a-
900 ketoglutarate generating reactions, thereby driving reductive
carboxylation toward the accumulation of acetyl CoA, as a
building block for fatty acid synthesis and elongation [130].
It is also worthwhile recalling that glutamine-derived gluta-
mate generates NADPH when converted to a-ketoglutarate by
905 glutamate dehydrogenase (GLUD1). Also, since glutamine-
derived a-ketoglutarate might fuel acetyl CoA accumulation
and thus fatty acid synthesis, glutamine might represent a key
therapeutic target (e.g., at the glutaminase level, through DON
and azaserine) also when attempting to tackle lipid
910 synthesis [131].
Potential pharmacological targets to inhibit FAO are repre-
sented by carnitine palmitoyl transferase (CPT1), the rate-
limiting enzyme in FAO, and 3-ketoacylthiolase (3-KAT),
which catalyzes the final step in FAO [129,132] and ATP-citrate
915 lyase (ACLY), a cytosolic enzyme that catalyzes the generation
of acetyl CoA from citrate [133].
Targeting protein markers of cancer
In the present paper we mainly focused on metabolites or pro-
tein targets mainly related to cancer metabolism. However,
920 there is a long list of emerging molecular markers of cancer
that is continuously expanding [134].
One of the most promising classes of protein biomarkers
that are currently undergoing clinical testing is represented by
molecular chaperones of the heat shock protein (HSP) family.
925 A wide range of human cancers is accompanied by overex-
pression of HSPs, which are implicated in tumor cell prolifera-
tion, differentiation, invasion, metastasis, death and recognition
by the immune system [135]. HSPs are useful biomarkers for
carcinogenesis in some tissues and might be used as a valid
930 indicators of the degree of differentiation and the aggressiveness
of some cancers. Serum levels of HSP and HSP-specific anti-
bodies in cancer patients might help tumor diagnosis and be
related to prognosis of specific cancers. Overexpression of
HSP27, for example, is associated with poor prognosis in gas-
935 tric, liver and prostate carcinoma and osteosarcomas [135]. On
the other hand, HSP70 [136] is correlated with poor prognosis
in breast, endometrial, uterine cervical and bladder carcinomas.
HSPs might interfere with therapetuic treatments and/or
induce resistance to chemotherapy in breast cancer, leukemia
940 patients and osteosarcomas. Two main strategies have been pro-
posed to target HSPs, depending on whether they are related
to an improved or worsened prognosis of a specific cancer.
These strategies include: i) pharmacological modification of
HSP expression or molecular chaperone activity and ii) use of
www.expert-reviews.com
Review
HSPs in anticancer vaccines, exploiting their ability to act as 945
immunological adjuvants [135].
Expert commentary
Big strides have been made over the last decade in the biologi-
cal understanding of the phenomena underpinning cancer
metabolism deregulation. This accumulating body of laboratory 950
science has paved the way for designing new therapeutical strat-
egies and re-discovering of older drugs, with metabolism-
regulatory aptitude.
Mass spectrometry-based proteomics and metabolomics have
been at the core of these basic science advancements, which 955
will undoubtedly translate into actual pharmacological applica-
tions within the next decades. Without the broader distribution
of highly sensitive and accurate MS instruments and new quan-
titative (isotope labeling-based) strategies, cancer research would
have hardly had any chance to set even one single step into the 960
deep forest of metabolic intricacies that we described in the
previous sections. Many molecular biologists have revised their
positions, firmly standing on a reductionistic ground, while
starting to complement classic molecular biology experimental
approaches (to put it cursorily, knock out one gene, induce 965
another gene, knock down another) with emerging
omics disciplines, theoretically encompassing the whole pro-
teome and metabolome (within the current capabilities of the
applied technique).
Metabolism-targeting compounds already include a broad 970
list of patented drugs and food derived nutrient/pharmaceuti-
cal-like molecules, also known as nutraceuticals [137]. Revisiting
the Warburg effect with proteomics and metabolomics tools
has revealed that we might already have an old answer (com-
mercialized drugs) for a new, compelling question (tackling 975
cancer proliferation via metabolic reprogramming).
Five-year view
Further advancements will soon be achieved through the imple-
mentation of in silico prediction tools, based upon machine
learning algorithms, a wind of change that embraces the 980
broader concept of systems biology [138,139]. Based upon bioin-
formatic models, computer predictions will also help predicting
untoward effects (scarce efficiency, scarce specificity) of in vitro
designed drugs [140]. Improvements in drug specificity and
selectivity at the design phase will make it amenable to target 985
cancer specific enzyme isoforms or mutations (PKM2, mito-
chondrial HXK2, monocarboxylate transporters MCT4 for lac-
tate secretion [141]), by exploiting the concept of synthetic
lethality. As summarized by Kaelin [142], two genes are syn-
thetic lethal if mutation of either alone is compatible with via- 990
bility but mutation of both leads to death. Therefore, it could
be possible to target a gene that is synthetic lethal to a cancer-
relevant mutation, as to kill only cancer cells while sparing nor-
mal cells [142].
Finally, as we hope it emerged from this paper, cancer cells 995
display an intricate network of intertwined and mutually-
compensating metabolic pathways. Altering one node of the
11
 Review DAlessandro & Zolla

network might result in the compensation effect promoted by
the so-called plasticity of the metabolic network itself. There-
1000 fore, omics/systems biology-wise efforts should be pursued to
unveil as many pathways as possible, and to gain an improved
understanding of the role of already known, albeit underinvesti-
gated ones, such as the folate and mevalonate pathways (both
producing NADPH at some steps of the cycles). The final
1005 goal will be to determine, case by case, cancer by cancer, the
most suitable targets for multi-targeted pharmaceutical
interventions [143].
Financial & competing interests disclosure
A D Alessandro and L Zolla are supported by funds from the Italian
National Blood Centre (Centro Nazionale SangueCNSIstituto Superiore 1010
SanitaRome, Italy). The authors have no relevant affiliations or finan-
cial involvement with any organization or entity with a financial interest
in or financial conflict with the subject matter or materials discussed in
the manuscript. This includes employment, consultancies, honoraria, stock
ownership or options, expert testimony, grants or patents received or pend- 1015
ing or royalties.
No writing assistance was utilized in the production of this manuscript.

1020 Key issues

The introduction of label-based and label-free quantitative proteomics and metabolomics analyses revived the field of
cancer metabolism.
Eighty years after Warburgs early observations, cancer metabolism is now deemed to be one key hallmark of cancer.
Most of the cancer-specific frequent mutations to tumor suppressors and oncogenes have the potential to affect cancer
1025 cells metabolism.
These changes often promote glycolysis at the expenses of oxidative phosphorylation in terms of energy production, other than anabolic
and NADPH-generating pathways. However, mitochondrial metabolism still plays a key role in producing oncometabolites with regula-
tory effects on key oncogenes, other than providing substrates for anabolic reactions (especially fatty acid de novo synthesis) and contri-
buting to the redox poise (via NADPH generation).
1030 Starvation promotes autophagy, which could provide a therapeutical intervention opportunity for some cancers. Induction of autophagy
might represent a viable alternative to induction of apoptosis.
Small molecule inhibitors of most of the key enzymes involved in the pathways described above are already available and deserve
further testing to understand their specificity and effectiveness. Cancer isoform-targeting (synthetic lethality) specific drugs must also
be designed as well.
1035 Among the thousands of commercially available drugs, those having a validated effect in the treatment of metabolic diseases may also
be suitable for the treatment of some cancers.

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