Zulato Et Al. - 2012 - Metabolic Effects of Anti-Angiogenic Therapy in Tumors

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Review

Metabolic effects of anti-angiogenic therapy in tumors

Elisabetta Zulato 1 Matteo Curtarello 1 Giorgia Nardo Stefano Indraccolo

Istituto Oncologico Veneto - IRCCS via Gattamelata 64 - 35128 Padova Italy

a r t i c l e i n f o

Article history

Received 29 July 2011

Accepted 3 January 2012

Available online 11 January 2012

Keywords

Glycolysis

Angiogenesis

Anti-angiogenic therapy

LKB1-AMPK pathway

a b s t r a c t

Anti-angiogenic therapy has recently been added to the panel of cancer therapeutics but predictive

biomarkers of response are still not available In animal models anti-angiogenic therapy causes tumor

starvation by increasing hypoxia and impairing nutrients supply It is thus conceivable that angiogenesisinhibition causes remarkable metabolic perturbations in tumors although they remain largely

uncharted We review here recent acquisitions about metabolic effects of angiogenesis blockade in

tumors and discuss the possibility that some metabolic features of tumor cells - ie their dependency

from glucose as primary energy substrate - might affect tumor responses to anti-VEGF treatment

2012 Elsevier Masson SAS All rights reserved

1 Introduction

The concept of targeting the vasculature of tumors has been

validated clinically as therapeutic strategy with the approval of

several drugs that block the vascular endothelial growth factor(VEGF)-VEGF receptor pathway [1] In spite of much efforts

however the 1047297ne mechanisms of tumor regression caused by

angiogenesis inhibition remain substantially unknown In experi-

mental tumors anti-VEGF drugs prune the newly formed vascula-

ture thus reducing microvessel density blood 1047298ow and perfusion

and eventually increasing the hypoxic tumor fraction [2e4] In

patients mechanisms might be more complex especially consid-

ering that VEGF neutralization has therapeutic ef 1047297cacy mainly if

combined with conventional chemotherapy [5] It has been

proposed that anti-VEGF therapy could transiently normalize the

tumor vasculature thus improving delivery of drugs and oxygen to

tumor cells [6] Following this initial time window of ldquovascular

normalizationrdquo sustained VEGF blockade hypothetically leads to

regression of the tumor vasculature followed by increased hypoxia[6] Although it is assumed that in addition to oxygen anti-

angiogenic drugs cut nutrients supply the exact identity of the

metabolites involved and their quantitative variations have not

been accurately reported so far

Here we present an overview of the predictive markers for anti-

angiogenic therapy and of the key metabolic alterations occurring

in cancer Moreover we review the emerging studies connecting

anti-angiogenic therapy to metabolism

2 Predictive markers for anti-angiogenic therapy

Validated biomarkers to predict the optimal biological dose of

anti-angiogenic agents and aiding selection of those patients who

are most likely to bene1047297t from anti-angiogenic treatment are

currently not available although a number of potential circulating

tissue and imaging biomarkers have emerged from recently

completed clinical trials [7]

Since most approved anti-angiogenic drugs target the VEGF

pathway VEGF itself has been the most extensively explored

biomarker In a study with bevacizumab plus chemotherapy for

metastatic breast cancer lower circulating VEGF levels were asso-

ciated with longer Time to Progression (TTP) [8] Likewise in lung

cancer patients elevated baseline VEGF serum levels had a negative

prognostic impact on survival [9] In contrast other studies have

not found any correlation between VEGF blood concentration andthe outcome of anti-angiogenic therapy In a randomized phase II

III trial Non-Small Cell Lung Cancer (NSCLC) patients with high

levels of baseline plasma VEGF had an increased response to bev-

acizumab but VEGF levels were not predictive of survival bene1047297t

[10] In three randomized phase III studies in metastatic colorectal

cancer lung cancer and renal cell cancer investigators assessed the

value of circulating VEGF level as a prognostic and predictive

biomarker for outcome of anti-angiogenic therapy Baseline circu-

lating VEGF levels were useful as a prognostic biomarker but

not as a predictive biomarker for bevacizumab-based treatment

bene1047297t [11]

Corresponding author Tel thorn39 049 8215875 fax thorn39 049 8072854

E-mail address stefanoindraccolounipdit (S Indraccolo)1 These Authors equally contributed to this work

Contents lists available at SciVerse ScienceDirect

Biochimie

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e b i o c h i

0300-9084$ e see front matter 2012 Elsevier Masson SAS All rights reserved

doi101016jbiochi201201001

Biochimie 94 (2012) 925e931



Some studies tested the hypothesis that an association between

VEGF polymorphisms and response to anti-VEGF therapy could

exist In advanced breast cancer the VEGF-2578AA and VEGF-

1154AA genotypes predicted a favorable Overall Survival (OS) for

patients in the paclitaxel plus bevacizumab arm but did not predict

a better Progression Free Survival (PFS) interval [12] In ovarian

cancer VEGF SNPs did not correlate with PFS [13]

One of the common side-effects of anti-angiogenic drugs is

hypertension [14] Different retrospective studies involving NSCLC

colorectal and renal carcinoma patients reported a signi1047297cant

improve in OS or PFS among patients with bevacizumab-induced

hypertension [15e17] One limitation of these studies however is

the fact that consensus criteria to measure bevacizumab-induced

hypertension are not yet established [11]

Anti-angiogenic drugs are often cytostatic in action and tumor

shrinkage or regression may not be a realistic estimate of ef 1047297cacy

To overcome the lack of correspondence between the Response

Evaluation Criteria in Solid Tumors (RECIST) and survival in patients

treated with anti-VEGF therapy new radiological methods are

emerging as surrogate biomarkers One potential tool for biomarker

development is dynamic contrast-enhanced magnetic resonance

imaging (DCE-MRI) which could provide information about tumor

blood vessel structure and functions [18] The volume transferconstant of contrast agent (ktrans) is a measure of tumor perfusion

and permeability in DCE-MRI In one randomized trial of sorafenib

in renal cell carcinoma high baseline DCE-MRI parameters

including ktrans and V p (blood plasma volume fraction) correlated

with PFS whereas changes of DCE-MRI parameters after the start of

therapy did not predict PFS [19] In recurrent gliobastoma a marked

reduction in ktrans after one dose of cediranib was seen in patients

with increased PFS [20]

The [18F]1047298uorothymidine Positron Emission Tomography

(FLT-PET) is an imaging technique for measuring in vivo cellular

proliferation in malignant tumor and organ tissue and is used to

monitor tumor responses to cytostatic therapies [21] A prospective

study in patients with recurrent malignant gliomas suggests that

FLT-PET can predict responses to bevacizumab as early as 1e

2weeks after treatment [22]

Finally certain metabolism-associated biomarkers could be

helpful in selecting patients to bene1047297t from anti-angiogenic

therapy In the CONFIRM trials LDH-A GLUT-1 and VEGFR1

mRNA levels predicted responses of colorectal cancer patients to

chemotherapy plus vatalanib [23] In the same trials high tissue

LDH5 correlated with poor PFS in the placebo subgroup whereas

it correlated with improved PFS in the vatalanib subgroup [24]

Moreover although vatalanib did not improve either PFS or OS

compared with placebo when authors strati1047297ed patients by

serum LDH level before random assignment they observed that

patients with high serum LDH had longer median PFS when

treated with vatalanib than with placebo In conclusion the

authors proposed that high serum LDH levels may identify tumorsthat are more dependent on abnormal angiogenesis and may

be more susceptible to VEGF inhibition as also suggested by

others [25]

3 Warburg effect and other metabolic alterations in cancer

In the 1920s Otto Warburg af 1047297rmed the role of metabolism in

carcinogenesis by demonstrating that cancer cells - at variance

with normal cells - rely on glycolysis instead of mitochondrial

oxidative phosphorylation (OXPHOS) to produce ATP even under

aerobic conditions [26] Warburg originally hypothesized that the

glycolytic switch in cancer cells was a consequence of defects in

mitochondria which impair aerobic respiration Currently

however it is known that mitochondria are not damaged in most

cancer cells suggesting that aerobic glycolysis essentially repre-

sents an adaptive choice of tumors [27]

Glucose is a critical nutrient for proliferating cells and it is used

as primary substrate to generate ATP as well as to synthesize amino

acids nucleotides and fatty acid and to regulate the redox

potential so as to minimize the effects of reactive oxygen species

(ROS) that damage cellular membranes and proteins [2829]

Enhanced glucose uptake - visualized in the clinic by [18F]1047298uo-

rodeoxyglucose (FDG)-PET - correlates with poor prognosis in

certain tumor types suggesting that enhanced glycolysis confers

a substantial growth advantage [30]

In recent years there has been a number of studies indicating

that aerobic glycolysis is constitutively up-regulated in tumor cells

through genetic or epigenetic changes The 1047297rst documented

mechanistic link between an activated oncogene and altered

glucose metabolism was the transcriptional activation of lactate

dehydrogenase A (LDH-A) by the MYC oncogene [31] LDH-A

contributes a crucial component of the Warburg effect the

conversion of pyruvate e the end-point of glycolysis e to lactate

that is secreted by monocarboxylate carriers (MCTs) eliminating it

from the pool and keeping glycolysis active [32] The secreted

lactate lowers the extracellular pH which may in1047298uence remod-

eling of the matrix and facilitate invasion Furthermore acidosisallows for the selection of motile cells that can eventually break

through the basement membrane and metastasize [29] Indeed

high levels of lactate have been proposed as prognostic factor in

certain malignancies [33] MYC was also found to regulate other

glycolysis-associated genes such as hexokinase II (HK II ) as well as

glucose transporters [34]

The AKT signaling pathway links growth control to glucose

metabolism and several studies correlated its activity with high

glycolytic rates in cancer cells AKT regulates expression of glucose

transporters and HKII enhancing both glucose uptake and its

retention in the cell [35] Moreover AKT can also increase

activity of hypoxia-inducible factor (HIF) thus further enhancing

glycolysis [36]

HIF-1 - which can accumulate due to hypoxia as well as alter-ations of various signaling pathways in tumors - up-regulates

expression of genes involved in aerobic glycolysis including

glucose transporters glycolytic enzymes and LDH-A [37] In addi-

tion HIF-1 deviates pyruvate away from mitochondria by up-

regulating pyruvate dehydrogenase the rate-limiting enzyme for

pyruvate to acetyl-CoA conversion thus alleviating oxidative stress

derived from mitochondrial metabolism [36]

Activated RAS oncogene was initially linked to increase cellular

glucose uptake but recent studies suggest that metabolic effects of

RAS activation could be mediated by MYC and HIF although the

precise mechanism is not yet established [36]

Although the p53 tumor suppressor has been viewed as the

ldquoguardian of the genomerdquo recently it has been implicated in

metabolism control [36] The p53 protein represses transcription of GLUT-1 and GLUT-4 transporters and can in1047298uence the metabolic

balance between glycolysis and OXPHOS through the transcrip-

tional regulation of the 26 efructose bisphosphatase TP53-induce

glycolysis regulator (TIGAR) and synthesis of cytocrome c oxidase

(SCO2) subunit of complex IV of the electron transport chain

[3839]

Besides glucose glutamine could represent an important energy

substrate in cancer cells DeBerardinis et al recently proposed that

in transformed cells glucose accounts mainly for lipid and nucleo-

tide synthesis whereas glutamine is responsible for re-feeding of

the TCA cycle for amino acid synthesis and for nitrogen incorpo-

ration into purine and pyrimidine for nucleotide synthesis [40]

MYC-addicted tumor cells are particularly sensitive to glutamine

withdrawal [41] andgenes involvedin mitocondrial biogenesis and

E Zulato et al Biochimie 94 (2012) 925e931926



glutamine metabolism specially glutamine transporter and GLS

genes appear to be under both the direct and indirect transcrip-

tional control of MYC [42] The ability of MYC to induceboth aerobic

glycolysis and glutamine oxidation provides cancer cells with the

ability to accumulate biomass [36]

Many human tumor types show altered metabolism of certain

amino acids showing increased uptake and high levels of amino

acid transporter expression

Enhanced expression of L -type amino acid transporter (LAT-1)

which shows high af 1047297nity for several essential amino acids

including leucine tryptophan and methionine (MET) has been

reported in astrocytomas and correlated with poor prognosis [43]

Patients with glioblastoma or other extracranial malignant tumors

such as lung cancer head and neck cancers breast cancer sarcomas

and lymphomas showed increased uptake of MET [44] a feature

which is exploited to make diagnosis and assess therapeutic ef 1047297-

cacy by 18F-MET PET [45]

In addition to increased glycolytic and amino acid activities

recurrent alterations of lipid metabolism are found in cancer cells

conceivably due to their requirement of de novo synthesis of lipids

for membrane assembly Overexpression of fatty acid synthase

(FASN) that catalyzes the de novo synthesis of fatty acids has been

observed in many human cancers including breast prostate lungand colorectal cancers and high levels of FASN were associated

with poor prognosis [46]

Alterations in choline-metabolites (tCho) are also quite

common in cancer cells Tumor cell lines are characterized by an

increased content of phosphocholine (PCho) as compared with

normal epithelial cells [47] The alpha-isoform of Choline Kinase

(ChoK) is often over-expressed in cancer and it is required to

sustain the PCho pool in tumor cells [48] Choline phosphorilation

by ChoK represents the 1047297rst step of choline metabolism in which

choline is 1047297nally converted to phosphatidylcholine a major

constituent of the mammalian cell membrane Choline-

metabolites are of particular interest because they can be moni-

tored in patients by magnetic resonance spectral (MRS) which

detects endogenous PCho or PET which detects altered kinetics of labeled Cho

An interesting area for future studies is to investigate the

predictive and prognostic value of these metabolic features of

cancer cells and to clarify whether they are modulated by anti-

angiogenic therapy

4 Metabolic perturbations after anti-angiogenic therapy

Responses to anti-angiogenic drugs such as sunitinib or bev-

acizumab have been quite heterogeneous in cancer patients In

some cases tumors respond by decreasing tumor volume by more

than 33 qualifying it for a partial response according to RECIST

criteria In other patients however signi1047297cant changes in tumor

density with no decrease in tumor dimensions are observed [49]

This is often associated with central tumor cavitation and necrosis

an observation which suggests that VEGF blockade may perturb the

energy balance in cancer cells

In a recent study [50] we investigated how metabolic param-

eters contribute to determine the pathologic response to VEGF

blockade in tumor xenografts A landmark observation of our study

was that the level of ldquoglucose addictionrdquo of tumor cells dictates the

amount of necrosis caused by angiogenesis inhibition This was

explained by the fact that VEGF blockade acutely perturbs glucose

and ATP levels in tumor xenografts Measurements by biolumi-nescence metabolic imaging indicated that after anti-VEGF therapy

glucose and ATP concentrations in tumors were 130 mmolg and

110 mmolg respectively Values in control tumors were 330 mmol

g (glucose) and 150 mmolg (ATP) Notably glucose uptake

was maintained following anti-angiogenic therapy as shown by

FDG-PET imaging indicating that delivery of glucose through the

vasculature was not compromised despite a substantial decrease in

microvessel density [50] similarly to what has been observed in

patients after bevacizumab monotherapy [51] So it appears that

glucose steady-state levels are very low after anti-angiogenic

therapy whereas glucose uptake is high likely due to HIF-1a

accumulation in treated tumors Intriguingly a preliminary report

showed that a subset of breast cancer patients treated with short-

term 1047297rst-line bevacizumab strongly up-regulated the hypoxiametagene [52] lending support to our observations

Fig 1 Anti-VEGF therapy increases AMPK activation in tumors Representative pictures of pAMPK and pACC staining of ovarian cancer IGROV-1 xenografts following 1 week of

treatment with the anti-VEGF monoclonal antibody A461

E Zulato et al Biochimie 94 (2012) 925e931 927



A related study investigated metabolic changes in glioblastoma

following anti-VEGF treatment and observed a tendency toward

accumulation of lactate alanine choline myo-inositol creatine

taurine and mobile lipids together with induction of HIF-1a and

activation of the phosphatidyl-inositol-3-kinase pathway [53] This

combination of metabolic changes has previously been associated

with increased hypoxia in human brain tumor spectra [54] and

partially overlaps with our 1047297ndings in ovarian cancer xenografts

[50] In future studies global metabolic changes identi1047297ed by mass

spectrometry analysis (including LC-MSMS and GCeMS) will be

helpful to characterize more extensively metabolic changes

induced by anti-angiogenic therapy in tumors There is already

evidence that this technology enables to pick up speci1047297c oncome-

tabolites in prostate cancer [55] and in gliomas [56]

Fig 2 Parameters contributing to determine tumor responses to VEGF neutralization (A) Anti-angiogenic therapy has been demonstrated to perturb glucose levels in tumors

xenografts Levels of ldquoglucose addictionrdquo of tumor cells in1047298uence the amount of necrosis caused by VEGF blockade Highly glycolytic tumors show a signi1047297cant reduction in their sizeand develop large necrotic areas following short-term anti-angiogenic therapy In contrast poorly glycolytic tumors are only marginally affected in size and do not markedly

increase necrosis after anti-angiogenic therapy [based on [50]] (B) AMPK activation as consequence of perturbations of ATP levels in tumors and its effects on the outcome of VEGF

blockade AMPK activation following short-term anti-angiogenic therapy reduces anabolic processes and cell proliferation being associated with minimal necrosis areas In contrast

tumors that fail to activate this pathway (ie due to LKB1 loss or mutations) maintain a high metabolic demand and are committed to develop large necrotic areas (C) Possible

outlook following tumor necrosis Large necrotic areas may induce recruitment of pro-angiogenic bone marrow-derived cells followed by rapid tumor regrowth Alternatively

killing of the majority of tumor cells by necrosis may turn into a therapeutic effect if relapse mechanisms are not engaged




Finally a novel role of the polyamine system in the hypoxic

response of cancer cells has recently been demonstrated The

polyamine system is up-regulated by hypoxia in a variety of cancer

cell lines and in hypoxic tumor regions and inhibition of polyamine

biosynthesis sensitizes cancer cells to hypoxia-induced apoptosis

in vitro [57] These 1047297ndings are relevant in this context if one

considers that the anti-tumor effect of bevacizumab was signi1047297-

cantly enhanced in mice receiving concomitant treatment with the

polyamine biosynthesis inhibitor DFMO which irreversibly inacti-

vates the key enzyme ornithine decarboxylase (ODC) [57] Thus

increased ODC expression and increased intracellular polyamine

levels may occur in tumors treated with anti-angiogenic drugs

likely contributing to protect tumor cells from hypoxia-induced

apoptosis Drugs which block polyamine synthesis could hypo-

thetically be used to increase the pro-apoptotic effects of anti-

vascular therapy

5 Anti-angiogenic therapy AMPK activation and Warburg

effect

AMP-activated protein kinase (AMPK) is a central metabolic

sensor found in all eukaryote systems that governs glucose and

lipid metabolism in response to alterations in nutrients supply andintracellular energy levels as well as cell polarity cell proliferation

and gene expression regulation [5859] In most species AMPK is

a heterotrimer that consists of a catalytic subunit (a) and two

regulatory subunits (b and g) In mammals there are two genes

encoding the AMPKa catalytic subunit two b genes and three g

subunit genes which differ in their tissue speci1047297city and subcel-

lular localization This serineethreonine kinase is mainly activated

in response to an increase in the AMPATP ratio within the cell and

it is phosphorylated at Thr-172 in the catalytic subunit by upstream

kinases including Liver Kinase B1 (LKB1) or calmodulin-dependent

protein kinase kinase beta (CAMKKb) [6061] In addition AMPK

can also be activated by a variety of pharmacological agents

including metformin which is used in the treatment of metabolic

disorders such as type 2 diabetes and obesity [62] AMPK activationreprograms cellular metabolism and enforces metabolic check-

points by acting on mTOR complex 1 (mTORC1) p53 and other

molecules [63] In particular AMPK acts to restore cellular energy

balance by promoting ATP generating processes such as fatty acid

beta oxidation and simultaneously by inhibiting ATP consuming

processes such as fatty acid synthesis gluconeogenesis and protein

synthesis This is initially achieved by direct phosphorylation of

some key metabolic enzymes (such as Acetil-CoA carboxylase ACC)

and subsequently by modulation of gene expression [64] Decoding

substrates of AMPK that have roles in the various cellular processes

controlled by this kinase is a hot area of investigation in the 1047297eld

With respect to cancer several recent studies in cell culture

models and in vivo have shown that growth of tumor cell lines can

be inhibited by AMPK activation highlighting as this kinase mightbe a cancer relevant ldquodruggablerdquo target In particular combination

of chemotherapy with metformin is more effective than chemo-

therapy to suppress tumor growth and to inhibit metastasis in

xenografts of breast lung and ovarian cancer [6566]

Since AMPK is activated when intracellular levels of ATP decline

and intracellular levels of AMP increase as often happens during

nutrient starvation and hypoxia a certain level of AMPK activation

is commonly seen in solid tumors [67] and we also observed AMPK

activation in the peri-necrotic areas of control xenografts [50]

Moreover we found that anti-angiogenic therapy increased AMPK

activation levels in tumors probably as a consequence of the

dramatic glucose depletion and ATP level exhaustion as demon-

strated by immunohistochemistry analysis of pAMPK and pACC

levels in tumor xenografts treated with anti-VEGF (Fig 1 and [50])

Our results are in agreement with a clinical study that showed that

bevacizumab increased total AMPK and pAMPK levels in renal cell

carcinoma patients [68] Moreover in that study AMPK activation

correlated with longer OS and PFS of treated patients

Tumor cells bearing AMPK de1047297ciency are hypersensitive to

energy stress-inducing agents [6970] It is thus possible that

defects of AMPK activation may limit survival of tumor cells under

glucose starvation andor hypoxia in vitro or anti-angiogenic

therapy in vivo Indeed in preclinical models highly glycolytic

cells that failed to activate AMPK developed large necrotic areas

after short-term anti-VEGF therapy Moreover attenuation of

AMPKa2 in poorly glycolytic cells compromised their survival

under glucose deprivation in vitro and increased necrosis following

anti-angiogenic therapy of tumor xenografts [50]

With regard to cell metabolism AMPK activation may decrease

the glycolytic1047298ux for example by inducing the expression of TIGAR

through p53 phosphorylation and activation [38] In support of this

possibility we observed that AMPKa2 silencing increased glucose

consumption and lactate production in ovarian cancer cells [50] On

the other hand it was previously known that HIF-1a and its target

genes including genes encoding for several glycolytic enzymes are

up-regulated in LKB1- AMPK- and TSC-de1047297cient 1047297broblasts indi-

cating that loss of any of these genes is suf 1047297cient to alter cellmetabolism and to switch over a highly glycolytic phenotype

[7172] We indeed observed a slight increase (2-fold) in HIF-1a

activity following AMPKa2 silencing in tumor cells suggesting that

HIF-1a could in part account for modulation of cell metabolism in

this system

Finally given the mechanistic connections between cell prolif-

eration and glycolysis [27] it is possible that AMPK-mediated

inhibition of cellular growth under conditions where nutrients

are scarce could indirectly contribute to down-modulate glycolysis

in tumor cells

In conclusion the integrityof signaling pathwaysinvolved in the

control of cell metabolism and quiescence - such as AMPK - could

be important to sense changes in the tumor microenvironment

caused by angiogenesis inhibition and to instruct tumor cells toadapt

6 Conclusions

Based on the results reviewed here multiple factors appear to

orchestrate the pathologic responses of tumors to VEGF neutrali-

zation In tumor xenografts the level of ldquoglucose addictionrdquo is

certainly important to determine whether or not tumors will suffer

from shortage of this energy substrate caused by VEGF blockade

(Fig 2A) Whether or not this will be con1047297rmed in patients relies in

part on the feasibility to characterize the glycolytic phenotype of

tumors In patients some imaging techniques - including FDG-PET

and MRS - could be exploited to measure levels of glucose uptake

and lactate production respectively Moreover expression levels of glycolysis-associated transporters such as GLUT-1 or MCT-1 could

be used as surrogate IHC markers in retrospective studies

A second important parameter is the integrity of the LKB1-

AMPK pathway (Fig 2B) Genetic events leading to inactivation of

LKB1 are quite common in certain sporadic tumors such as lung

adenocarcinoma [73] Since these malignancies are also treated

with bevacizumab in combination with chemotherapy [74] there is

an opportunity to investigate whether loss of LKB1 is associated

with increased necrosis following anti-angiogenic therapy More-

over the observation that anti-angiogenic therapy activates AMPK

raises the question whether it might be appropriate to combine

anti-angiogenic therapy with other AMPK-activating drugs The

biguanide metformin which has shown ef 1047297cacy in preclinical

models of breast colon and prostate cancer [75] could reinforce




AMPK activation caused by bevacizumab perhaps leading to more

ef 1047297cient control of tumor growth compared with either drug alone

Alternatively since metformin acts as mild inhibitor of complex I of

the respiratory chain [64] forcing the cells to exploit glycolysis to

produce ATP it could transiently make tumors ldquoglucose addictedrdquo

thus increasing treatment-induced tumor necrosis following anti-

angiogenic therapy Future experimental work is needed to clarify

these issues and design appropriate drug combinations

Finally a third critical parameter intimately linked to the energy

balance in tumor is necrosis It is well established treatment with

vascular disrupting agents (VDA) causes massive tumor necrosis

and this has been associated with rapid regrowth promoted by

recruitment of myeloid cells and increased expression of pro-

angiogenic factors (Fig 2C) [7677] On the other hand ablation of

the large majority of tumor cells by necrosis could also be consid-

ered an evidence of therapeutic response and it may not neces-

sarily be followed by tumor relapse (Fig 2C) Necrosis - sometimes

with central cavitation - is also observed in a subset of patients

treated with sunitinib [49] or bevacizumab [52] Therefore an

important goal of future studies is to clarify the prognostic value of

treatment-induced tumor necrosis by utilizing new protocols

which have been optimized to estimate necrosis in CT scans [78]

In conclusion identi1047297cation of clinically feasible methods todetermine the glycolytic phenotype of tumors interrogate the

status of the LKB1AMPK pathway and accurately measure the

extent of necrosis remain a priority in order to test the predictive

value of these upcoming markers in patients treated with anti-

angiogenic drugs Moreover it is certainly possible that a broader

view of the metabolic changes induced by anti-angiogenic drugs in

tumors by using mass spectrometry or other emerging technolo-

gies will uncover additional molecular sensors engaged by meta-

bolic stress and offer an opportunity to understand how

metabolism-based approaches could improve cancer therapy

Acknowledgment

This work was supported in part by grants from Progetto

Oncologico di Medicina Molecolare i tumori femminili Universitagrave

di Padova - Progetto drsquoAteneo 2010 EZ and GN are recipient of

AIRC fellowships

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[13] KD Steffensen M Waldstrom I Brandslund A Jakobsen The relationship of VEGF polymorphisms with serum VEGF levels and progression-free survival inpatients with epithelial ovarian cancer Gynecol Oncol 117 (2010) 109e116

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[15] P Bono H Elfving T Utriainen P Osterlund T Saarto T Alanko H JoensuuHypertension and clinical bene1047297t of bevacizumab in the treatment of advanced renal cell carcinoma Ann Oncol 20 (2009) 393e394

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[22] W Chen S Delaloye DH Silverman C Geist J Czernin J SayreN Satyamurthy W Pope A Lai ME Phelps T Cloughesy Predicting treat-ment response of malignant gliomas to bevacizumab and irinotecan byimaging proliferation with [18F] 1047298uorothymidine positron emission tomog-raphy a pilot study J Clin Oncol 25 (2007) 4714e4721

[23] PM Wilson D Yang MM Shi W Zhang C Jacques JC Barret K Danene-berg T Trarbach G Folprecht G Meinhardt HJ Lenz Use of intratumoralmRNA expression of genes involved in angiogenesis and HIF1 pathway topredict outcome to VEGFR tyrosine Kinase inhibitor (TKI) in patients enrolledin CONFIRM1 and CONFIRM2 ASCO Annu Meet (2008)

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[28] V Fritz L Fajas Metabolism and proliferation share common regulatorypathways in cancer cells Oncogene 29 (2010) 4369e4377

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[32] DA Tennant RV Duran E Gottlieb Targeting metabolic transformation forcancer therapy Nat Rev Cancer 10 (2010) 267e277

[33] S Walenta M Wetterling M Lehrke G Schwickert K Sundfor EK RofstadW Mueller-Klieser High lactate levels predict likelihood of metastases tumorrecurrence and restricted patient survival in human cervical cancers CancerRes 60 (2000) 916e921

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[37] GL Semenza Targeting HIF-1 for cancer therapy Nat Rev Cancer 3 (2003)721e732

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[39] S Matoba JG Kang WD Patino A Wragg M Boehm O GavrilovaPJ Hurley F Bunz PM Hwang p53 regulates mitochondrial respirationScience 312 (2006) 1650e1653

[40] RJ DeBerardinis A Mancuso E Daikhin I Nissim M Yudkoff S WehrliCB Thompson Beyond aerobic glycolysis transformed cells can engage inglutamine metabolism that exceeds the requirement for protein and nucle-otide synthesis Proc Natl Acad Sci U S A 104 (2007) 19345e19350

[41] M Yuneva N Zamboni P Oefner R Sachidanandam Y Lazebnik De1047297ciencyin glutamine but not glucose induces MYC-dependent apoptosis in humancells J Cell Biol 178 (2007) 93e105

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[46] JA Menendez R Lupu Fatty acid synthase and the lipogenic phenotype incancer pathogenesis Nat Rev Cancer 7 (2007) 763e777

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[49] S Faivre G Demetri W Sargent E Raymond Molecular basis for sunitinibef 1047297cacy and future clinical development Nat Rev Drug Discov 6 (2007)734e745

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[55] A Sreekumar LM Poisson TM Rajendiran AP Khan Q Cao J YuB Laxman R Mehra RJ Lonigro Y Li MK Nyati A Ahsan S Kalyana-

Sundaram B Han X Cao J Byun GS Omenn D Ghosh S PennathurDC Alexander A Berger JR Shuster JT Wei S Varambally C BeecherAM Chinnaiyan Metabolomic pro1047297les delineate potential role for sarcosinein prostate cancer progression Nature 457 (2009) 910e914

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of the polyamine system provides opportunities for tumor growth inhibitionby combined targeting of vascular endothelial growth factor and ornithinedecarboxylase Cancer Res 68 (2008) 9291e9301

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[61] A Woods SR Johnstone K Dickerson FC Leiper LG Fryer D NeumannU Schlattner T Wallimann M Carlson D Carling LKB1 is the upstreamkinase in the AMP-activated protein kinase cascade Curr Biol 13 (2003)2004e2008

[62] BB Zhang G Zhou C Li AMPK an emerging drug target for diabetes and themetabolic syndrome Cell Metab 9 (2009) 407e416

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[66] GZ Rocha MM Dias ER Ropelle F Osorio-Costa FA Rossato AE VercesiMJ Saad JB Carvalheira Metformin Ampli1047297es chemotherapy-induced AMPKactivation and Antitumoral growth Clin Cancer Res 17 (2011) 3993e4005

[67] KR Laderoute K Amin JM Calaoagan M Knapp T Le J Orduna M ForetzB Viollet 5rsquo-AMP-activated protein kinase (AMPK) is induced by low-oxygenand glucose deprivation conditions found in solid-tumor microenvironmentsMol Cell Biol 26 (2006) 5336e5347

[68] D Tsavachidou-Fenner N Tannir P Tamboli W Liu D Petillo B TehGB Mills E Jonasch Gene and protein expression markers of response tocombined antiangiogenic and epidermal growth factor targeted therapy inrenal cell carcinoma Ann Oncol 21 (2010) 1599e1606

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AMPK activity signalling through mTOR and survival in response to energeticstress in LKB1-de1047297cient lung cancer Oncogene 26 (2007) 1616e1625[70] RJ Shaw M Kosmatka N Bardeesy RL Hurley LA Witters RA DePinho

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[72] DB Shackelford DS Vasquez J Corbeil S Wu M Leblanc CL Wu DR VeraRJ Shaw mTOR and HIF-1alpha-mediated tumor metabolism in an LKB1mouse model of Peutz-Jeghers syndrome Proc Natl Acad Sci U S A 106(2009) 11137e11142

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E Ancona S Indraccolo A Amadori Vandetanib improves anti-tumor effectsof L19mTNFalpha in xenograft models of esophageal cancer Clin Cancer Res17 (2011) 447e458

[77] Y Shaked A Ciarrocchi M Franco CR Lee S Man AM Cheung DJ HicklinD Chaplin FS Foster R Benezra RS Kerbel Therapy-induced acuterecruitment of circulating endothelial progenitor cells to tumors Science 313(2006) 1785e1787

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Some studies tested the hypothesis that an association between

VEGF polymorphisms and response to anti-VEGF therapy could

exist In advanced breast cancer the VEGF-2578AA and VEGF-

1154AA genotypes predicted a favorable Overall Survival (OS) for

patients in the paclitaxel plus bevacizumab arm but did not predict

a better Progression Free Survival (PFS) interval [12] In ovarian

cancer VEGF SNPs did not correlate with PFS [13]

One of the common side-effects of anti-angiogenic drugs is

hypertension [14] Different retrospective studies involving NSCLC

colorectal and renal carcinoma patients reported a signi1047297cant

improve in OS or PFS among patients with bevacizumab-induced

hypertension [15e17] One limitation of these studies however is

the fact that consensus criteria to measure bevacizumab-induced

hypertension are not yet established [11]

Anti-angiogenic drugs are often cytostatic in action and tumor

shrinkage or regression may not be a realistic estimate of ef 1047297cacy

To overcome the lack of correspondence between the Response

Evaluation Criteria in Solid Tumors (RECIST) and survival in patients

treated with anti-VEGF therapy new radiological methods are

emerging as surrogate biomarkers One potential tool for biomarker

development is dynamic contrast-enhanced magnetic resonance

imaging (DCE-MRI) which could provide information about tumor

blood vessel structure and functions [18] The volume transferconstant of contrast agent (ktrans) is a measure of tumor perfusion

and permeability in DCE-MRI In one randomized trial of sorafenib

in renal cell carcinoma high baseline DCE-MRI parameters

including ktrans and V p (blood plasma volume fraction) correlated

with PFS whereas changes of DCE-MRI parameters after the start of

therapy did not predict PFS [19] In recurrent gliobastoma a marked

reduction in ktrans after one dose of cediranib was seen in patients

with increased PFS [20]

The [18F]1047298uorothymidine Positron Emission Tomography

(FLT-PET) is an imaging technique for measuring in vivo cellular

proliferation in malignant tumor and organ tissue and is used to

monitor tumor responses to cytostatic therapies [21] A prospective

study in patients with recurrent malignant gliomas suggests that

FLT-PET can predict responses to bevacizumab as early as 1e

2weeks after treatment [22]

Finally certain metabolism-associated biomarkers could be

helpful in selecting patients to bene1047297t from anti-angiogenic

therapy In the CONFIRM trials LDH-A GLUT-1 and VEGFR1

mRNA levels predicted responses of colorectal cancer patients to

chemotherapy plus vatalanib [23] In the same trials high tissue

LDH5 correlated with poor PFS in the placebo subgroup whereas

it correlated with improved PFS in the vatalanib subgroup [24]

Moreover although vatalanib did not improve either PFS or OS

compared with placebo when authors strati1047297ed patients by

serum LDH level before random assignment they observed that

patients with high serum LDH had longer median PFS when

treated with vatalanib than with placebo In conclusion the

authors proposed that high serum LDH levels may identify tumorsthat are more dependent on abnormal angiogenesis and may

be more susceptible to VEGF inhibition as also suggested by

others [25]

3 Warburg effect and other metabolic alterations in cancer

In the 1920s Otto Warburg af 1047297rmed the role of metabolism in

carcinogenesis by demonstrating that cancer cells - at variance

with normal cells - rely on glycolysis instead of mitochondrial

oxidative phosphorylation (OXPHOS) to produce ATP even under

aerobic conditions [26] Warburg originally hypothesized that the

glycolytic switch in cancer cells was a consequence of defects in

mitochondria which impair aerobic respiration Currently

however it is known that mitochondria are not damaged in most

cancer cells suggesting that aerobic glycolysis essentially repre-

sents an adaptive choice of tumors [27]

Glucose is a critical nutrient for proliferating cells and it is used

as primary substrate to generate ATP as well as to synthesize amino

acids nucleotides and fatty acid and to regulate the redox

potential so as to minimize the effects of reactive oxygen species

(ROS) that damage cellular membranes and proteins [2829]

Enhanced glucose uptake - visualized in the clinic by [18F]1047298uo-

rodeoxyglucose (FDG)-PET - correlates with poor prognosis in

certain tumor types suggesting that enhanced glycolysis confers

a substantial growth advantage [30]

In recent years there has been a number of studies indicating

that aerobic glycolysis is constitutively up-regulated in tumor cells

through genetic or epigenetic changes The 1047297rst documented

mechanistic link between an activated oncogene and altered

glucose metabolism was the transcriptional activation of lactate

dehydrogenase A (LDH-A) by the MYC oncogene [31] LDH-A

contributes a crucial component of the Warburg effect the

conversion of pyruvate e the end-point of glycolysis e to lactate

that is secreted by monocarboxylate carriers (MCTs) eliminating it

from the pool and keeping glycolysis active [32] The secreted

lactate lowers the extracellular pH which may in1047298uence remod-

eling of the matrix and facilitate invasion Furthermore acidosisallows for the selection of motile cells that can eventually break

through the basement membrane and metastasize [29] Indeed

high levels of lactate have been proposed as prognostic factor in

certain malignancies [33] MYC was also found to regulate other

glycolysis-associated genes such as hexokinase II (HK II ) as well as

glucose transporters [34]

The AKT signaling pathway links growth control to glucose

metabolism and several studies correlated its activity with high

glycolytic rates in cancer cells AKT regulates expression of glucose

transporters and HKII enhancing both glucose uptake and its

retention in the cell [35] Moreover AKT can also increase

activity of hypoxia-inducible factor (HIF) thus further enhancing

glycolysis [36]

HIF-1 - which can accumulate due to hypoxia as well as alter-ations of various signaling pathways in tumors - up-regulates

expression of genes involved in aerobic glycolysis including

glucose transporters glycolytic enzymes and LDH-A [37] In addi-

tion HIF-1 deviates pyruvate away from mitochondria by up-

regulating pyruvate dehydrogenase the rate-limiting enzyme for

pyruvate to acetyl-CoA conversion thus alleviating oxidative stress

derived from mitochondrial metabolism [36]

Activated RAS oncogene was initially linked to increase cellular

glucose uptake but recent studies suggest that metabolic effects of

RAS activation could be mediated by MYC and HIF although the

precise mechanism is not yet established [36]

Although the p53 tumor suppressor has been viewed as the

ldquoguardian of the genomerdquo recently it has been implicated in

metabolism control [36] The p53 protein represses transcription of GLUT-1 and GLUT-4 transporters and can in1047298uence the metabolic

balance between glycolysis and OXPHOS through the transcrip-

tional regulation of the 26 efructose bisphosphatase TP53-induce

glycolysis regulator (TIGAR) and synthesis of cytocrome c oxidase

(SCO2) subunit of complex IV of the electron transport chain

[3839]

Besides glucose glutamine could represent an important energy

substrate in cancer cells DeBerardinis et al recently proposed that

in transformed cells glucose accounts mainly for lipid and nucleo-

tide synthesis whereas glutamine is responsible for re-feeding of

the TCA cycle for amino acid synthesis and for nitrogen incorpo-

ration into purine and pyrimidine for nucleotide synthesis [40]

MYC-addicted tumor cells are particularly sensitive to glutamine

withdrawal [41] andgenes involvedin mitocondrial biogenesis and











































angiogenic therapy











































































vascular therapy


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vascular therapy


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Acknowledgment




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References











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vascular therapy


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this system





in tumor cells





6 Conclusions



























































Acknowledgment




AIRC fellowships

References











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Acknowledgment




AIRC fellowships

References











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Zulato Et Al. - 2012 - Metabolic Effects of Anti-Angiogenic Therapy in Tumors

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