Conceptual Framework for Cutting the Pancreatic Cancer Fuel Supply. Anne Le, N.V. Rajeshkumar, Anirban Maitra, and Chi V. Dang. Clin Cancer Res August 15, 2012 18:4285-4290; doi:10.1158/1078-0432.CCR-12-0041

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Conceptual Framework for Cutting the Pancreatic Cancer
Fuel Supply
Anne Le1, N.V. Rajeshkumar2, Anirban Maitra1,2, and Chi V. Dang3
Abstract
Pancreaticductaladenocarcinoma(a.k.a.pancreaticcancer)remainsoneofthemostfearedandclinically
challenging diseases to treat despite continual improvements in therapies. The genetic landscape of
pancreatic cancer shows near ubiquitous activating mutations of KRAS, and recurrent inactivating muta-
tions of CDKN2A, SMAD4, and TP53. To date, attempts to develop agents to target KRAS to specifically kill
cancer cells have been disappointing. In this regard, an understanding of cellular metabolic derangements
in pancreatic cancer could lead to novel therapeutic approaches. Like other cancers, pancreatic cancer cells
rely on fuel sources for homeostasis and proliferation; as such, interrupting the use of two major nutrients,
glucoseandglutamine,mayprovidenewtherapeuticavenues.Inaddition,KRAS-mutantpancreaticcancers
have been documented to depend on autophagy, and the inhibition of autophagy in the preclinical setting
has shown promise. Herein, the conceptual framework for blocking the pancreatic fuel supply is reviewed.
Clin Cancer Res; 18(16); 4285–90. ?2012 AACR.
Introduction
Although inherited and acquired mutations are believed
to cause pancreatic cancer (see review by Iacobuzio-Dona-
hueandcolleaguesinthisissue;ref.1),commonmutations
in canonical oncogenes, such as AKT, MYC, PI3K, and RAS,
andtumorsuppressors,includingTP53andPTEN,alsoalter
cancer metabolism to enable cancer cells to survive and
proliferate in the hypoxic and nutrient-deprived tumor
microenvironment (see review by Feig and colleagues in
this issue; ref. 2). Oncogenes and tumor suppressors alter
metabolism through transcriptional and posttranscription-
al mechanisms. Mutations in genes encoding metabolic
enzymes,suchassuccinatedehydrogenase(SDH),fumarate
hydratase (FH), and isocitrate dehydrogenase (IDH) have
also been linked to tumorigenesis, underscoring the inti-
mateconnectionsbetweenmetabolismandcancer(11,12).
Loss-of-function SDH or FH mutations result in accumu-
lation of precursors such as succinate or fumarate. Mutated
IDH1 or IDH2, on the contrary, are neomorphic enzymes
that reverse the chemical reaction to produce 2-hydroxy-
glutarate (2-HG) from a-ketoglutarate (a-KG; ref. 13).
These accumulated metabolic intermediates all have the
ability to inhibit a-KG–dependent dioxgenases, which are
involved in histone or DNA demethylation and in prolyl
hydroxylases that modulate the hypoxia-inducible factors
(HIF; refs. 14–16). In this regard, mutations in enzymes
causeepigeneticderegulation
tumorigenesis.
To survive and proliferate in an adverse tumor microen-
vironment with limited nutrients and oxygen, cancer cells
must rely on their ability to reprogram canonical biochem-
ical pathways to provide the necessary bioenergetics and
precursors of proteins, nucleic acids, and membrane lipids
(17–19; Fig. 1). In addition to rewiring metabolism, tumor
cells can also activate autophagy, a process that permits
recycling of cellular constituents as internal fuel sources
when external nutrient supplies are limited (20–22).
Although nutrient-rich conditions inhibit autophagy
through activation of mTOR, nutrient-deprived conditions
decrease ATP production, resulting in activated AMPK that
stimulates autophagy (23, 24; Fig. 1). As such, cancer cells
have multiple mechanisms for metabolic adaptation to the
tumor microenvironment.
Proliferatingcancercellstransportglucoseandglutamine
into the cell as major nutrient sources for the production of
ATP and building blocks for macromolecular synthesis (3,
4, 6; Fig. 1). The mitochondrion serves not only as the
cellular powerhouse, producing ATP efficiently from glu-
coseandglutamine,butitisalsoahubfortheproductionof
key intermediates involved in nucleic acid, fatty acid, and
heme synthesis. The proliferating cancer cells, hence, also
produce toxic by-products that must be eliminated or
extruded for cell survival. Reactive oxygen species (ROS)
from the mitochondrion are neutralized; superoxide is
converted by SOD to hydrogen peroxide, which is neutral-
ized by catalase via its conversion to water and oxygen (25–
27). Lactate and carbon dioxide, produced from glucose
and glutamine catabolism, are exported through monocar-
boxylate transporters or neutralized and extruded by
thatcontributesto
Authors' Affiliations: Departments of1Pathology and2Oncology, The Sol
Goldman Pancreatic Cancer Research Center, Johns Hopkins University
School of Medicine, Baltimore, Maryland; and3Abramson Cancer Center,
Perelman School of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania
Corresponding Author: Chi V. Dang, University of Pennsylvania, 1600
PennTower,3400SpruceStreet,Philadelphia,PA19072.Phone:215-662-
3929; Fax: 215-662-4020; E-mail: cvdang@upenn.edu
doi: 10.1158/1078-0432.CCR-12-0041
?2012 American Association for Cancer Research.
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carbonic anhydrase (28). Although this article focuses on
blockingcancerfuelsupplyasapromisingnewapproachto
pancreatic cancer therapy, therapeutic opportunities may
also exist in blocking the exhaust pipes that eliminate
metabolic toxic by-products from cancer cells.
Characteristic Features of Metabolism in
Cancers
Aberrant metabolism is now considered one of the hall-
marks of cancer (29). As a result of genetic alterations and
tumor hypoxia, cancer cells reprogram metabolism to meet
increased energy demand for enhanced anabolism, cell
proliferation, and protection from oxidative damage and
cell death signals. The molecular underpinnings for the
reprogrammedmetabolismofcancercellshavebeenlinked
to the activation of oncogenes orloss of tumor suppressors,
which can function independently of, or through constitu-
tive stabilization of the HIF, HIF-1a. Activated Ras and AKT
oncogenes can increase glycolysis, enhancing the conver-
sion of glucose to lactate (3). The MYC oncogene, which
encodes the transcription factor Myc, increases the expres-
sion of glycolytic genes, thereby enhancing glycolysis and
production of lactate (35).
Glycolysis is a multienzymatic pathway that catabolizes
glucose to pyruvate via more than a dozen different
enzymes with several apparent rate-limiting steps. Phos-
phofructose kinase provides the first rate-limiting step
where fructose-1-phosphate, derived from glucose, is con-
vertedtofructose-1,6-bisphosphate. Theotherrate-limiting
step is the conversion of phosphoenolpyruvate to pyruvate
bypyruvatekinase(muscleform)PKM.Recentstudieshave
raised an intense interest in the embryonic M2 isoform of
pyruvate kinase (PKM2), which is highly expressed in
humantumors.AstudybyGaoandcolleagues(30)showed
that dimeric,nottetrameric PKM2thatlocalizes thecellular
nucleus, activates transcription of MEK5 by phosphorylat-
ing stat3 at Y705 using PEP as a phosphate donor. This
protein kinase activity of PKM2 plays a role in promoting
cell proliferation, revealing an important link between
metabolism alteration and gene expression during tumor
transformation and progression. The xenografted adeno-
carcinoma cells, which carry a mutant form of PKM2 that
preferentially forms dimers, grow more rapidly and seem
more aggressive than cells carrying wild-type (WT) PKM2.
AlthoughthedynamicsofPKM2oligomericstatesandtheir
roles in cancer remain confusing, these studies suggest that
perturbation of glycolysis via manipulation of PKM2 could
have a significant therapeutic effect.
Alteration of glucose metabolism in cancer is known as
aerobic glycolysis or the Warburg Effect (3, 5, 31), which
describes the ability of cancer cells to avidly metabolize
CCR Focus
© 2012 American Association for Cancer Research
Malate
Fumarate
Succinate
Oxaloacetate
Acetyl-CoA
Pyruvate
Citrate
Isocitrate
α-Ketoglutarate
FX11
Glutamine
NADPH
ribose
Glutathione
nucleic
acids
Glutaminolysis
Glutamate
GLS
Glutamine
Glucose
Warburg Effect
LDHA
ATPAMP
BPTES
AMPK
Autophagy
Chloroquine
Lactate
Glycerol
Lipids
Fatty acids
Acetyl-CoA
Figure 1. Diagram depicting the
metabolism of glucose and
glutamine via glycolysis and the
TCA cycle. Both substrates
contribute to the production of
ATP,whichwhendepletedwould
activate AMPK that in turn
triggers autophagy. Autophagy
increases lysosomal recycling of
cellular constituents to recover
ATP. GLS, glutaminase.
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glucose to lactate, even in the presence of oxygen. A
manifestation of the Warburg Effect is the increased
2[18F]fluoro-2-deoxy-D-glucose import by pancreatic can-
cersasdeterminedbypositronemissiontomographic(PET)
scans. However, only about 50% of pancreatic cancers have
positive clinical PET, suggesting additional complexities
and the possibility that pancreatic cancers could use other
fuels or resort to other metabolic survival modes (32, 33).
In addition to glucose, proliferating cancer cells also rely
on glutamine as a major source of energy and building
blocks. In fact, MYC, which is frequently amplified or
overexpressed in pancreatic cancer (34), has been mecha-
nistically linked to the regulation of glutamine metabolism
(35). In this regard, it is reasonable to surmise that PET
negative pancreatic cancers may use glutamine as a major
nutrient source. The Myc transcription factor emerges as a
master regulator of a phlethora of genes involved in cell
growthincludingthoseregulatingribosomeandmitochon-
drial biogenesis and intermediary metabolism. The normal
MYC gene is under the scrutiny of many internal as well as
extracellular cues such as nutrient and oxygen availability,
such that deprivation of these supplies results in the
downregulation of MYC expression. By contrast, many
oncogenic pathways activate MYC or alterations of MYC
itself resulting in a constitutive program of ribosome bio-
genesis and biomass accumulation that renders cancer cells
addicted to nutrients. Indeed withdrawal of either glucose
or glutamine triggers death of cells with MYC overexpres-
sion (35). In this regard, targeting enzymes involved in
glucose or glutamine has also provided proof-of-concept
that metabolic inhibition could provide a beneficial ther-
apeutic effect (36).
Applying metabolomics technologies, with the use of
nuclear magnetic resonance and mass spectrometer–based
stable isotope resolved metabolomics with
glucose and glutamine, a study by Le and colleagues (36)
documentsthatcancercellsuseeitherglucoseorglutamine,
depending on the availability of the nutrients. This flexi-
bility of cancer metabolism enables cancer cells to prolif-
erate and survive even under the hypoxic and nutrient-
deprived conditions, which are often encountered in the
tumor microenvironment. Moreover, in hypoxia, they
observed the enhanced conversion of glutamine to gluta-
thione, an important reducing agent for control of the
accumulation of mitochondrial ROS. Most importantly,
this study uncovered a previously unsuspected glucose-
independent glutamine-driven tricarboxylic acid (TCA)
cycle. Cancer cells subjected to glucose deficiency and/or
hypoxiawouldbenefitfromsuchglucose-independentTCA
cycle activity in the tumor microenvironment. Cell growth
and survival can be sustained by glutamine metabolism
alone. In addition to the links between oncogenes and
tumor suppressors to altered cancer cell metabolism, muta-
tions in specific TCA cycle enzymes contribute to tumori-
genesis of familial or spontaneously acquired cancers. The
study by Mullen and colleagues (37) uncovered other
metabolic reprogrammed pathways in cancer cells, which
have mutations in complex I or complex III of the electron
13C-labeled
transport chain (ETC). These are often found in patient-
derived renal carcinoma cells with mutations in FH, and
also in pharmacologically ETC-inhibited cells with normal
mitochondria. These cancer cells use glutamine-dependent
reductive carboxylation generating acetyl-CoA for lipid
synthesis. These processes use mitochondrial and cytosolic
isoforms of NADPþ/NADPH-dependent IDH. The ETC-
deficient cancer cells, like FH mutations, cannot grow
withoutglutamine.Hence,dependingonthegeneticmake-
up,acancercellcouldbedistinctivelyaddictedtoglucoseor
glutamine.
Given that pancreatic cancers uniquely display increased
levels of fibrotic stroma, the pancreatic cancer cell environ-
ment could be highly nutrient deficient (38, 39). In this
nutrient-deprived state, increased AMPK activity could trig-
ger autophagy (21). The process of self-eating or autophagy
provides starved cells a means to survive by recycling cel-
lular components as bioenergetic substrates for energy
production and building blocks. As such, certain metabolic
hubs should be exploitable for therapy, particularly if a
specificcancertypeis"addicted"tothatpathway.Metabolic
enzymes are readily pharmacologically targetable; in fact,
many historical clinically effective chemotherapeutic drugs
were termed "antimetabolites." Seeking drugs that directly
inhibit these new metabolic targets involved in cancer cell
energy metabolism whilesparingnormalcellsisamongthe
most desirable goal to improve cancer therapy, especially
for the treatment of pancreatic cancer.
Blocking pancreatic cancer fuel supply
Addiction to glucose could be exploited through targeted
inhibition of enzymes involved in glycolysis. One such
target is the glycolytic pathway, in which the most consis-
tent abnormality is its ultimate step of conversion of pyru-
vate to lactic acid by lactate dehydrogenase A (LDHA) to
regenerate NADþthat is required for the further glycolytic
conversion of glucose to pyruvate to generate ATP. Recent
studies (40, 41) have targeted this phenotype of altered
metabolism for therapy. The first drug-like small molecule
(called FX11) that inhibits LDHA was used as proof of
concept for targeting aerobic glycolysis in cancer. This
compound, 7-benzyl-2,3-dihydroxy-6-methyl-4-n-propyl-
1-napthoic acid, has shown an antitumorigenic effect in
mouse models of human lymphoma and pancreatic cancer
through the increased production of ROS and cell death
(40). By blocking LDHA, FX11 diminishes the ability of
malignant cells to metabolize pyruvate to lactate, and halts
the regeneration of NADþfor glycolysis processing. This
study also showed a strong synergy effect in vitro and in vivo
with the use of FX11 in combination with FK866, an
inhibitorof NADþ
biosynthesis, which accentuates
NADþdepletion.
Besides the Warburg Effect, cancer cells also maintain
mitochondrial oxidation of glutamine by glutaminase that
converts glutamine to glutamate,which enters the TCA cycle
as2-oxoglutarate.Tworecentstudiestargetedglutaminolysis
in cancer by a specific glutaminase inhibitor, bis-2-[5-(phe-
nylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide (BPTES;
Blocking Pancreatic Cancer Fuel Supply
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ref. 42). The first study by Seltzer and colleagues (43)
reported a preferable inhibition of mutant IDH1cell growth
byBPTESascomparedwiththeWTenzyme.Mutationatthe
R132 residue of IDH1 creates a novel enzyme function that
produces 2-HG from a-KG, which is from glutamate, a
product of glutamine via glutaminase. Cancer cells with
mutant IDH1 become addicted to glutamine and heavily
depend on glutaminase. The addition of exogenous a-KG
rescued growth suppression ofmutant IDH1cells by BPTES,
which lowered glutamate and a-KG levels, inhibited gluta-
minaseactivity,andincreasedglycolyticintermediates.How-
ever, 2-HG levels were unaffected by BPTES. This presents a
potential therapeutic opportunity. The study by Wang and
colleagues (44) provided another aspect of targeting mito-
chondrial glutaminase activity inhibiting oncogenic trans-
formation. They showed that glutaminase activity, which is
dependent on Rho GTPases and NF-kB activity, increased in
transformed fibroblasts and breast cancer cells. Targeting
glutaminase activity by BPTES to inhibit oncogenic trans-
formation had thus been shown, through a connection
between Rho GTPase activation and cellular metabolism, to
be a promising way forward. The importance of hypoxic
glutaminemetabolisminthestudybyLeandcolleagueswas
alsounderscoredbytheantiproliferativetherapeuticeffectof
BPTES on neoplastic cells in vitro and in a tumor xenograft
model in vivo (36).
Inadditiontotheproof-of-conceptstudiessuggestingthe
feasibility of targeting glucose or glutamine metabolism in
pancreatic cancer, it has been noted that pancreatic cancers
display significant autophagic activities for survival (45–
49).Infact,Ras-transformedcellsdependonautophagyfor
survival (50). Hence, inhibition of autophagy with the
antimalarial agent chloroquine has resulted in significant
preclinical responses of pancreatic cancer xenografts and
allograftsintreatedmiceascomparedwithcontrol(49,51).
Chloroquine also diminishes pancreatic tumorigenesis in a
transgenic model (49). These studies suggest that 2 related
and widely used agents with extremely favorable safety
profiles, chloroquine or hydroxychloroquine, could have
profound clinical effects. Indeed, there are now clinical
trials testing this concept in pancreatic cancer. As for target-
ing glycolysis or glutaminolysis, the field is awaiting phar-
maceutical companies to develop clinically safe, highly
potent drugs to be tested in the clinic. Notwithstanding
the inherent challenges for drug development, the concept
of blocking the pancreatic cancer fuel supply provides a
reasonable framework for the development of what is
hoped to be a new class of anticancer agents.
Future directions
Althoughcancer cells canexhibit unique metabolic path-
ways,theyalsousetheclassicmetabolicpathwaysofnormal
cells. This presents a great challenge to directly target met-
abolic pathways, especially metabolic enzymes as drug
targets. The success of small molecular agents depends on
how much cancer cells are "addicted" to the fuel nutrition
versus normal cells, as shown in the studies mentioned
above. The combination of multiomics technologies will
give a functional perspective of cancer progression, beyond
genes and protein expression profiles. The extensive data
obtained from metabolic flux will be mined to identify
specific pathways active in the tumor compared with
untransformedcells.Thesedataareessentialforunderstand-
ing the metabolic activity in tumor tissue, especially how
cancer cells can respond to different environmental condi-
tions and how they respond to therapy to detect likely
responders and nonresponders to a particular chemothera-
peutic agent. This is highly desirable because it is important
to avoid the use of cytotoxic drugs that have no benefit. The
ultimategoalistocharacterizeandenabletargetedselection
of patients based on predicted metabolic responses. The
metabolic signatures that correlate with sensitivity of pan-
creaticcancerstometabolicinhibitorswillalsobeusedinthe
future to help us combine existing drugs to target multiple
metabolic pathways and attack specific attributes of each
patient’s cancer. In fact, metformin, which inhibits NADH
dehydrogenase and mitochondrial respiration, has preclin-
icalactivityagainstpancreaticcancerxenografts(Kumarand
colleagues, unpublished data) and is used in clinical trials.
However, parameters that predict resistance or response
remain poorly understood. Hence, defining the metabolic
pathways of pancreatic cancer through metabolomics will
pave the way for prediction of response and the identifica-
tionofnewenzymetargetsforpancreaticcancertherapies.A
major technical challenge in this arena is the heterogeneity
of the pancreatic tumor tissue, which is laced with an
extensive fibrotic stroma comprising of host immune cells.
The potential for cancer cells to reprogram their metab-
olism to bypass the targeted enzymatic step may occur and
hence poses a challenge to metabolic therapy. This issue is
especially significant given the interconnectivity of the
cellular metabolic network. In combination with appropri-
ate computational tools (e.g., flux balance analysis), meta-
bolomics offers a powerful way to identify possible "met-
abolic escaperoutes." Withthe insight ofthis guide, we will
eliminatetheseescaperoutesthroughrationalcombination
therapies, for example, in combination with current anti-
metabolites. This will allow for more cost-effective and
personalized cancer treatment.
Further complicating attempts at developing pancreatic
cancer therapies, there is emerging evidence of pancreatic
cancer stem cells in pancreatic cancers and these cells are
responsible for drug resistance to standard chemotherapy,
resulting in relapse and metastasis of pancreatic adenocar-
cinoma (see review by Penchev and colleagues in this issue;
ref. 52). Therefore, recognizing the heterogeneity of multi-
subpopulation ofpancreatictumorsandunderstandingthe
distinguished metabolic modes of each are critical impor-
tant for targeting of these subpopulations.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interests were disclosed.
Authors' Contributions
Conception and design: A. Maitra
Analysis and interpretation of data (e.g., statistical analysis, biosta-
tistics, computational analysis): C.V. Dang
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Writing, review, and/or revision of the manuscript: A. Le, N.V. Rajesh-
kumar, A. Maitra, C.V. Dang
Grant Support
All authors supported by a Stand Up To Cancer Dream Team Transla-
tional Cancer Research Grant, a Program of the Entertainment Industry
Foundation (SU2C-AACR-DT107467). A. Le is supported by the Sol
Goldman Pancreatic Cancer Research fund 80028595 and The Lustgarten
Foundation fund 90049125; C.V. Dang is supported by grants
5R01CA051497-21 and 5R01CA057341-20 and A. Maitra is supported
by R01CA113669, P01CA134292, and R01CA134767.
Received March 23, 2012; revised June 2, 2012; accepted June 27, 2012;
published OnlineFirst August 15, 2012.
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