Cancer Treatment Reviews 39 (2013) 171–179

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Cancer Treatment Reviews

journal homepage: www.elsevierheal th.com/ journals /c t rv

Laboratory-Clinic Interface

New strategies for targeting the hypoxic tumour microenvironment in breast cancer

Carol Ward a,1, Simon P. Langdon a,1, Peter Mullen a,3, Adrian L. Harris b,2, David J. Harrison c,3,Claudiu T. Supuran d,4, Ian H. Kunkler e,⇑a Breakthrough Breast Unit and Division of Pathology, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road,Edinburgh EH4 2XU, UKb Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UKc Pathology, Medical and Biological Sciences Building, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9TF, UKd Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Italye Edinburgh Cancer Research Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK

a r t i c l e i n f o

Article history:Received 13 August 2012Accepted 27 August 2012

Keywords:Breast cancerTumour microenvironmentMetabolismWarburg effectHypoxia

0305-7372/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.ctrv.2012.08.004

⇑ Corresponding author. Tel.: +44 0131 537 2213; fE-mail addresses: cward@staffmail.ed.ac.uk (C. Wa

(S.P. Langdon), pm72@st-andrews.ac.uk (P. Mullen)ac.uk (A.L. Harris), david.harrison@st-andrews.ac.uk (an@unifi.it (C.T. Supuran), I.Kunkler@ed.ac.uk, iKunkler).

1 Tel.: +44 0131 537 1763; fax: +44 0131 537 3159.2 Tel.: +44 01865 222457.3 Tel.: +44 01334 464826; fax: +44 01334 467470.4 Tel.: +39 055 457 3251; fax: +39 055 457 3036.

s u m m a r y

Radiation and drug resistance remain major challenges and causes of mortality in the treatment of locallyadvanced, recurrent and metastatic breast cancer. Metabolic reprogramming is a recently recognisedhallmark of cancer with the hypoxic acidic extracellular environment as a major driver of invasion andmetastases. Nearly 40% of all breast cancers and 50% of locally advanced breast cancers are hypoxicand their altered metabolism is strongly linked to resistance to radiotherapy and systemic therapy.The dependence of metabolically adapted breast cancer cells on alterations in cell function presents awide range of new therapeutic targets such as carbonic anhydrase IX (CAIX). This review highlights pre-clinical approaches to evaluating an array of targets against tumour metabolism in breast cancer andearly phase clinical studies on efficacy.

� 2012 Elsevier Ltd. All rights reserved.

Introduction utilise glycolysis for energy production, even in oxygenated condi-

Breast cancer poses a major challenge with women in westerncountries having a lifetime risk of 1 in 8 for developing the disease.Surgery, radiotherapy and systemic treatments remain the corner-stone of primary therapy with loco-regional radiotherapy and sys-temic treatments contributing to reduced mortality. Althoughtargeted therapies such as Trastuzumab have improved outcomesin primary and recurrent disease, resistance to radiotherapy, endo-crine therapy and chemotherapy in locally advanced and meta-static disease remain major therapeutic challenges and causes ofmortality. Metabolic reprogramming is a recently recognised hall-mark of cancer.1 The fundamentally different bioenergetic metab-olism of cancer cells (which depend mainly on aerobic glycolysis),compared to normal cells (which depend predominantly on mito-chondrial oxidative phosphorylation), provides an array of meta-bolic targets that may be exploited therapeutically. Cancer cells

ll rights reserved.

ax: +44 0131 537 1470.rd), Simon.Langdon@ed.ac.uk, adrian.harris@oncology.ox.-D.J. Harrison), claudiu.supur-ankunkler@yahoo.com (I.H.

tions this is known as the ‘Warburg effect’. The molecular mecha-nisms underpinning the Warburg effect are poorly understood.However current evidence points to enhanced glycolysis playingan important part in the metabolic machinery of the malignantphenotype.2 There is mounting evidence that cancer is not only agenetic disorder but also a disease of dysregulated bioenergeticmetabolism.2 Energy metabolism might yet prove to be the Achil-les’ heel of cancer.3 Metabolic targets have been classified as eitherdirect targets such as the metabolic enzymes themselves or indi-rect targets such as signalling pathways switched on or off result-ing in disordered metabolism.4

One third of breast tumours have hypoxic regions with O2 con-centrations less than 0.3%, compared to normal tissue concentra-tions of approximately 9%.5 It is estimated that 40% of all breastcancers and 50% of locally advanced breast cancers have hypoxicregions where chemotherapy and radiation are less effective,5 pro-viding the rationale for targeting altered metabolism in breast can-cer. This disease comprises different molecular subtypes withdiverse biologies which need specific and appropriate treatmentstrategies such as PARP inhibitors in BRCA mutation carriers oranti-HER2 therapy. A ‘one size fits all’ approach is not thereforeappropriate. Identifying which molecular subtypes might differen-tially benefit from this approach will be important. In this review,we put into perspective emerging preclinical and early phase stud-ies targeting tumour metabolism.

172 C. Ward et al. / Cancer Treatment Reviews 39 (2013) 171–179

The tumour microenvironment

Two main forces drive cancer cells to evolve a more resistantand invasive phenotype, hypoxia (low O2 concentration) and aci-dosis.6 Within normal tissues, O2 supply matches metabolic needs,but in tumours, demand commonly outstrips supply. The diffusionlimit for O2 is approximately 100–200 lm from a blood capillary.Consequently, as tumour growth reaches 1–2 mm3, diffusion limitsand malformation and leakiness of blood vessels cause hypoxicareas to develop.6 As O2 and nutrients become limited, metabolites,such as lactate accumulate. Cells in the tumour core become necro-tic, surrounded by viable cells in the peri-necrotic hypoxic region,where microenvironmental changes are most severe.

Activation of Hypoxia Inducible Factor-1 (HIF) and its role inbreast cancer

Tumour cells must adapt to exist in this microenvironment.Most pro-survival mechanisms are orchestrated by the transcrip-tion factor, Hypoxia Inducible Factor-1 alpha (HIF-1a). Other iso-forms of HIF exist, but are not well enough characterised. Thisreview therefore considers only the role of HIF-1a. Between 1%and 5% of human genes are modulated by HIF-1; including genesinvolved in erythropoiesis, angiogenesis, glycolysis, cell cycle con-trol, proliferation, and metastasis.7 Mechanisms involved in HIF-1activation are illustrated in Fig. 1.

Inappropriate HIF-1 activity is avoided by Factor-inhibiting HIF-1 (FIH) which binds HIF-1a impairing HIF/CREB/p300 activation oftarget genes.7 Chromosome 10q24, containing the FIH-1 gene, isfrequently lost in breast cancer.8 COMM domain-containing 1(COMMD1) protein prevents dimerisation of HIF-1a and HIF-1bin normoxia; however levels fall during progression of breast and

Fig. 1. HIF expression and activation in hypoxic and normoxic conditions. HIF-1 is a hetnuclear translocator (ARNT). (A) In normoxic conditions (above 5% O2), HIF-1a is degradHIF-1a on proline residues. Hydroxylation enables HIF-1a to associate with von Hippel–ubiquitin binding and subsequent degradation of HIF-1a by the 26S proteasome. (B) Hypof HIF-1a, causing increased expression of HIF-1a. HIF-1a translocates to the nucleusassociates with the transcriptional co-activator CREB-binding protein, and binds to hypoxincreased due to activation of the mTOR, PI3K and Ras/MAPK signalling pathways byexpression in normoxic cells.

other cancers, allowing HIF-1 activation to occur independentlyof oxidative status.9 The ‘Warburg effect’ is partially controlledby pyruvate kinase M2 (PKM2), which catalyses a rate-limitingstep in glycolysis, and is a HIF-1 target gene. Conversely, PKM2 isalso a co-activator of HIF-1.10,11 This positive feedback loop mayreprogramme glucose metabolism in cancer cells, and suggeststhat HIF-1 activation can occur in such cells before they encounterhypoxia. PKM2 exists as a high activity tetramer allowing high ATPyields from glycolysis or as a low activity dimer that shuttles gly-colytic intermediates to the biosynthetic pentose phosphate path-way, controlling energy production and cell proliferation.10

Hypoxia and HIF-1a overexpression correlate closely with poorprognosis in breast cancer,8 while HIF-1 inhibition causes tumourgrowth suppression.7 A retrospective study of an unselected groupof 745 breast cancer patients linked high HIF-1a levels to early re-lapse and metastatic disease,12 demonstrating an important rolefor HIF-1 in breast cancer induction, maintenance and progression.

Hypoxia-independent HIF activation in cancer cells

Loss of the tumour suppressor genes, phosphate and tensinhomolog deleted on chromosome 10 (PTEN), or VHL can amplifyHIF-1a expression by O2-independent mechanisms. Insulin-likeor epidermal growth factors or constitutive activation of the Ras-MAPK, Src, or PI3K/mTOR pathways can elevate HIF-1a expression(Fig. 1).7,13 Loss of p53 increases HIF-1a expression, because p53promotes MDM2-mediated ubiquitination of HIF-1-a increasingapoptotic resistance and metastasis.14 Approximately 50% of breastcancers have mutated p53, which is associated with cancer pro-gression and metastasis.14 However the interactions between thep53 and HIF-1 pathways are complex, and appear to be cell typeand context specific.15

erodimer consisting of two subunits, HIF-1a and HIF-1b/aryl hydrocarbon receptored rapidly, by O2-dependent activation of prolyl hydroxylase 2, which hydroxylatesLindau tumour suppressor (VHL) protein, an E3 ubiquitin ligase complex, allowing

oxia prevents activation of prolyl hydroxylase 2, and ubiquitination and degradationand dimerises with HIF-1b, to form the transcription factor HIF-1. Nuclear HIF-1ia response elements (HRE) of hypoxia responsive genes.7 HIF-1a expression can bedisruption of tumour suppressor genes or by growth factors7,13 allowing HIF-1a

C. Ward et al. / Cancer Treatment Reviews 39 (2013) 171–179 173

Hypoxic induction of acidosis in the tumour microenvironmentin breast cancer

Activation of HIF-1 amplifies glycolysis by upregulating expres-sion of glucose transporters and glycolytic enzymes such as lactatedehydrogenase which converts pyruvate to lactate.13 In vivo stud-ies using FdG PET show the transition from DCIS to invasive breastcancer is associated with marked increases in tumour glucoseuptake.16 A symbiotic relationship develops between tumour cells.Lactate produced by hypoxic cancer cells is used to regeneratepyruvate for oxidative phosphorylation by cells in aerobic condi-tions, ensuring efficient glucose use by the tumour. Lactate is re-moved from hypoxic cells via the HIF-inducible plasmamembrane monocarboxylate transporter 4 (MCT4) symportedwith hydrogen ions.17 Without this removal, lactate would lowerintracellular pH leading to cell death.

In the tumour, pH drops as distance from blood vessels in-creases. Approximately 300 lm from blood vessels, extracellularpH can decrease from 7.4 to 6.0.6,18 However, the intracellularpH (pHi) of tumour cells remains between 7.0 and 7.4, becausethe pH gradient is preserved by enzymes, ion pumps, and trans-porters.19 All cells express carbonic anhydrases (CAs) which cata-lyse reversible conversion of CO2 and H2O to HCO3

� and H+ asshown in Fig. 2. HIF-1 activation increases expression of the trans-membrane CAs, CAIX and CAXII.3 While H+ acidifies the extracellu-lar environment; HCO3

� can be recycled and used to increaseintracellular pH. CAIX expression is associated with poor prognosisin breast cancer, being rare in normal breast tissue or benignlesions, but increased in pre-invasive DCIS of the breast and occur-ring only in malignant epithelium.20 However, a more recent studyfound that CAIX was an independent prognostic marker for sur-vival in a subset of breast cancer patients who were postmeno-pausal, hormone receptor positive, with one to three positivelymph nodes.21

Other ion exchangers also regulate pHi. The Na+/H+ exchanger 1(NHE1) extrudes H+ for Na+ (Fig. 2) and is activated by hypoxia, lowpHi and oncogenic transformation (Fig. 2).13 In breast cancer cells,activation of NHE1 increases, acidifying the microenvironment,

Fig. 2. Membrane modulators of glycolytic symbiosis and H+ transport and their inhibitomonocarboxylate transporters MCT 1 and 4, NHE1 and CA IX. Glucose is transported intsymported with hydrogen ions.17 This lactate is used by cells in aerobic conditions trandecreasing pHe. CA IX generates HCO3

� and H+ from CO2 and H2O which also acidifies thtrials or experimental models of breast and other cancers.

enhancing motility and invasive potential.22 HIF activation com-pensates for lactate and CO2 production using mechanisms thatlink intracellular alkalinization to extracellular acidification(Fig. 2).13 Other ion exchangers and bicarbonate transporters areimportant in pHi maintenance, but their roles in hypoxic adapta-tion need further investigation.

Hypoxia and acidosis – mechanisms of resistance andprogression in breast cancer

The hypoxic and acidic tumour environment actively selects amore aggressive cancer cell phenotype, since the mechanismslinked with survival in these conditions are associated with inva-sion, metastasis and resistance to radiation and chemotherapy.19,23

These are outlined in Table 1. Radiotherapy requires O2 to generatecytotoxic free radicals which damage DNA, and to stabilise DNAdamage. Radiation resistance in breast cancer occurs because O2

concentrations in 30–40% of tumours fall below 1%; in 0.1% O2:they can be 2–3 times more resistant to a given radiation dose, be-cause fewer double strand breaks are produced.6 Radiation incursmost damage in rapidly dividing cells, but slower proliferativerates in hypoxic conditions prevent such damage.6

Radiation and cytotoxic drug resistance can occur through ge-netic instability.24 DNA damage response (DDR) pathways detectand repair DNA lesions, but hypoxia inhibits proteins involved inthe homologous recombination (HR), non-homologous end-joining(NHEJ) and also the mismatch repair (MMR) pathways.24 Chronichypoxia prevents activation of the G1/S cell cycle checkpoint,allowing DNA errors to accumulate.24 Low O2 concentrations in-crease the frequency of point mutations, inversions, and deletions,and amplify the multidrug-resistant gene ABCB1, through theinduction of chromosomal fragile sites.24 Defects in DDR proteinscan cause telomere shortening or dysfunction triggering chromo-some fusions and chromosomal instability.25 HIF-1 activation en-hances the expression of the DNA polymerase iota, which has anexceptionally high replication error rate. Expression of this poly-merase is increased in breast cancer cells.26 Alkaline pHi, inhibitsmitotic arrest, caused by activated DNA damage checkpoints.19

rs. Hypoxia increases the expression or activity of the glucose transporter GLUT1, theo the cell via GLUT1 and lactate produced by glycolysis is removed through MCT 4sported by MCT 1. NHE1 exchanges one H+ for one Na+ keeping pHi alkaline ande external microenvironment. Shown are inhibitors that have been used in clinical

Table 1Microenvironmental mechanisms driving the development of resistant and aggressive breast cancer.

Mechanism Effect

Vascular and lymphatic abnormalities Increased interstitial pressure causing inhibition of drug delivery6

HypoxiaSlows cell cycling Resistance to drugs that target proliferation and radiation24

Decreases pro-apoptotic Bcl-2 family members BID, BAD and BAX Decreased apoptosis93

Induces IAP-2 expression Decreased apoptosis and increased resistance to treatment92

Activates PI3K/Akt pathway Decreased apoptosis93

Decreased O2 Resistance to radiotherapy24

Inhibits DNA repair pathways Increased genetic instability24

Generates reactive oxygen species DNA damage24

Activation of lysyl oxidase-Snail pathway Loss of E-cadherin and increased invasive potential23

Increased expression of chemokine receptors Increased invasive and metastatic potential23,29

Disturbs immune cell function Loss of anti-tumour defences, accumulation of cytokines and growth factors33

pH changesAlkaline pHiPreservation of ATP Inhibits apoptosis5,19

Bypass of cell cycle checkpoints Genetic instability19

Increased glycolysis Metabolic adaptation19

Acidic pHeAltered drug structure and uptake Resistance to chemotherapy19

Increased proteinase expression ECM damage, increased invasion and metastasis19

Increased expression of IL-8 and VEGF Increases angiogenesis94

Decreased activation of effector immune cells Loss of anti-tumour activities33

174 C. Ward et al. / Cancer Treatment Reviews 39 (2013) 171–179

Such mechanisms increase mutation rates in hypoxic breast cancercells, which are selected on the basis of enhanced survival poten-tial. HIF-1 and hypoxia can trigger apoptosis in breast cancer cellsby inducing transactivation of pro-apoptotic genes, such as BNIP3and NIX,7 but the effect may depend on the presence of wild typep53 (p53wt).15 Apoptosis of p53wt cells permits further expansionof cells with mutant p53, (p53mt) increasing tumour resistance tocell death.24

Loss of E-cadherin is a crucial feature of epithelial to mesenchy-mal transition (EMT) which allows cancer cells to become migra-tory and invasive. Hypoxia deregulates E-cadherin expression byactivating the lysyl oxidase (LOX)-Snail pathway.24 High LOXexpression correlates with poor prognosis in ER negative breastcancer; inhibition of LOX reduces motility and invasiveness of hyp-oxic cancer cells and reduces metastasis in vivo.27 Increasedexpression of proteinases damages the ECM and basement mem-brane, facilitating invasion and metastasis of breast cancer cells.The hepatocyte growth factor receptor c-MET is also associatedwith invasion and metastasis in breast cancer; its expression andactivation correlate with tumour hypoxia and HIF-1 activation.28

The HIF-regulated chemokine receptors CXCR4 and CXCR6 arehighly expressed on breast cancer cells and also involved in migra-tion and metastasis.29

Hypoxia and acidosis disturb immune cell function, compromis-ing natural anti-tumoural defences, increasing cytokine andgrowth factor expression and permitting further growth. Tu-mour-associated macrophages migrate toward and accumulate inhypoxic areas.30 This cell influx increases the release of factorssuch as TGF-b, IL-8, VEGF, Ang2, and HGF, while low pHe increasesIL-8 and VEGF expression. Hypoxia inhibits the ability of macro-phages to phagocytose dead or dying cells, allowing areas of necro-sis and inflammation to form.31 It prevents the presentation ofantigens to T cells, and inhibits anti-tumour effects of macro-phages.32 HIF-1 protects T-cells from activation-induced celldeath,33 while acidosis causes immune cell dysfunction, interferingwith activation of cytotoxic T cells and natural killer cells.33 VEGFimpairs dendritic cell function and differentiation.34 Thus themicroenvironment protects cancer cells from the effector cells ofthe immune system, allowing unopposed tumour growth andspread.

Hypoxia and the unfolded protein response (UPR)

Severe hypoxia (<0.01% O2), reduces the protein folding capacityof the endoplasmic reticulum and induces activation of the UPRand the eukaryotic translation initiation factor 2-a kinase3(PERK).35 The consequent phosphorylation of eIF2a causes expres-sion of ATF4 which regulates genes involved in stress, metastasisand drug resistance amongst others.35 Moderate hypoxia (1% O2),which strongly activates HIF-1, activates PERK only after extendedculture periods. In breast cancer cells simultaneously exposed toboth moderate hypoxia and pathophysiological lactate acidosis,translational inhibition of HIF-1a occurred, but the UPR was syner-gistically activated.36 ATF4 is essential for cell survival through thepost stress recovery phase, suggesting that diverse stresses com-mon to the tumour microenvironment can combine, enhancingsurvival pathways.36 ATF4 is implicated in drug resistance to cis-platin, doxorubicin, etoposide, SN-38 and vincristine, via transcrip-tional upregulation of the ATP-binding cassette (ABC) membranetransporters ABCC2 and ABCG2 and increased glutathione biosyn-thesis.37 ATF4 DNA copy number amplifications have been recentlyreported in a small subset of breast cancer cell lines and tumours.36

The effect of these amplifications is unclear but they may cause afaster upregulation of ATF4 in response to stress or higher expres-sion of ATF4 in unstressed conditions, resulting in constitutivelyhigher levels of amino acids and VEGFA expression. Interestingly,in a xenograft model of triple negative breast cancer, circulatingtumour cells were found to have upregulation of HIF and AT4 path-ways, in comparison to parental cell lines, suggesting this as a clin-ically relevant biomarker for targeted therapy patient selection.38

Some gene targets of the UPR, such as the metastasis-associatedgene LAMP3, although associated with hypoxia, are controlledindependently of HIF-1.39

Strategies for treatment

Inflammatory breast cancer cells, which experience hypoxia asmetastasising tumour cell emboli, become constitutively adaptedto low O2 concentrations, incurring changes that remain evenwhen oxygenation improves.40 Because most breast cancer deaths

C. Ward et al. / Cancer Treatment Reviews 39 (2013) 171–179 175

occur due to metastatic disease, and hypoxic adaptations arestrongly linked to the metastatic phenotype, it is anticipated thattreatment strategies directed at adaptive targets, will damage ordestroy resistant breast cancer cells in the tumour microenviron-ment, and could prove a useful adjunct for treatment of moreaggressive, metastatic disease, particularly if adaptive changesprove to be permanently maintained in secondary growths(Table 2).

Studies using gene transcription/protein expression to measureactivation of the hypoxia pathway using tumour miR-210, GLUT1,or CAIX expression suggest that the hypoxic response is stronger intriple negative breast cancer (TNBC)41,42; these are frequently ofhigher histological grade and exhibit more aggressive clinicalbehaviour than hormone receptor positive breast cancers.43 TNBCsdisplay high rates of proliferation, metastasis and focal areas ofnecrosis, suggesting a hypoxic core.44 A key regulator of HIF-1inTNBC is SHARP1 and this opposes HIF-1 dependent migrationand invasion.44 Effective targeted therapies have been developedfor hormone receptor or HER-2 positive disease, but these willnot benefit patients with TNBC; effective, specific treatment islacking.43 Therefore novel prognostic and predictive factors foranti-tumour agents are urgently required for TNBC.40

HIF-1 inhibitors

Given its role in adaptation of cells to the microenvironment,HIF-1 is an obvious therapeutic target. HIF-1 blockade compro-mises anaerobic glycolysis, decreases proliferation of hypoxic cellsand promotes necrosis/apoptosis while reducing radio- andchemotherapy resistance.45 HIF-1a inhibitors induced radiosensiti-sation of human breast cancer cells, in vitro and in vivo, andprevented metastatic tumour spread of prostate cancer cells tothe lung in a murine model.46 Strategies to inhibit HIF-1 includeinterference with HIF-1a mRNA expression, protein translationand DNA binding, and those which affect HIF-1a degradation andtranscriptional activity.47 Many such approaches are not HIFspecific, e.g. HSP90 inhibitors and microtubule targeting agentssuch as 17 B 2-methoxyestradiol, which induce HIF-1a degrada-tion, doxorubicin and daunorubicin which block HIF-1 binding tothe HRE, and bortezomib, which inhibits HIF-1 transcriptionalactivity.7,47 PX-478, which selectively targets HIF-1a translation,demonstrated activity in several xenograft models, includingbreast.48 Recently two mechanistically and chemically differentHIF-1 inhibitors, Acriflavine and Digoxin were shown to inhibithypoxia induced expression of LOX and LOXL proteins which areinvolved in metastatic niche formation.

Oncogenic mutations causing constitutive activation of the Ras-MAPK, Src, or PI3K/mTOR pathways increase HIF-1 expression innormoxic and hypoxic conditions (Fig. 1).7 HER-2 upregulationand PI3K/AKT activation increase HIF-1 stability through mTOR,particularly in breast cancer.7 Blocking the PI3K/Akt/mTOR path-way enhances the radiation response in vitro in breast cancer mod-els,49 and the mTOR inhibitor CCI-779 has been shown to be aneffective treatment for breast cancer in Phase II clinical trials.50

Several novel PI3K/Akt inhibitors have been developed, such asSF1126, PI-103, and P529 which increase the effects of radiationand chemotherapeutic agents. These appear to act by normalisa-tion of the tumour vasculature, increasing blood flow and decreas-ing hypoxia.51 In the Phase 3 BOLERO-2 clinical trial, the mTORinhibitor everolimus combined with endocrine therapy improvedprogression free survival in hormone-receptor positive advancedbreast cancer.52

Growth factor receptor activation increases HIF-1 expression(Fig. 1).7 The EGFR inhibitor gefitinib decreases hypoxic HIF-1ainduction in some cancers,53 and improves blood flow and oxygen-ation in breast tumours, via a mechanism that appears to decrease

VEGF release from the tumour.53 Further, gefitinib treatment de-creased the proportion of viable hypoxic cells in murine xenograftmodels of breast cancer.53 In breast cancer cells, elevated Srccauses degradation of VHL increasing HIF-1a levels in normoxicconditions,54 and inducing expression of CAIX.55 The Src inhibitor,dasatinib inhibits cell proliferation in TNBC, inhibits breast cancermigration and invasion in in vitro studies and prevents the forma-tion of bone metastases in a mouse model. Combinations of dasat-inib with other cytotoxics show increased effects on proliferation,viability, migration and invasion in vitro, in comparison with eithertreatment alone. The case for dasatinib as a breast cancer treat-ment and the results of several clinical trials have been recentlyreviewed.56,57 Preliminary data suggests breast cancers exhibitingraf mutations may be sensitive to the Ras/MAPK inhibitor, Selu-metinib.58 The above studies suggest that upregulation of HIF-1by O2-independent means may represent a valid treatment targetin normoxic breast tumours.

PARP inhibition and synthetic lethality

PARP inhibitors represent a novel therapeutic strategy in BRCA1or BRAC2 mutated breast cancers.4 These mutations cause faults inthe HR DNA repair pathways that are synthetically lethal when theDNA repair protein, PARP1 is inhibited.4 A clinical trial using thePARP inhibitor olaparib demonstrated anti-tumour activity in bothBRCA1 and 2-deficient breast and ovarian cancers.59 Because acuteand chronic hypoxia inhibit the HR repair pathway, PARP inhibitorsmay effectively treat hypoxic breast tumours regardless of BRCAstatus, since hypoxia represses translation of the Brca1 gene.24 Invitro and xenograft models of breast cancer demonstrate sensiti-sation to PARP inhibition in hypoxia and further, xenografts treatedwith a PARP inhibitor showed increased responses to radiation.60

Several clinical trials are presently examining PARP inhibitors astherapy for breast and other cancers.61 Cancers cells may well har-bour other mutations that combined with PARP inhibition willcause synthetic lethality.

Hypoxia also suppresses DNA repair pathways such as NHEJ,and MMR. Therefore other synthetically lethal targets in hypoxiccells should exist.24 For example, deficiencies in the MMR proteinsMSH2 and MLH1, which are repressed by hypoxia, are syntheti-cally lethal in combination with disruption of the DNA polymer-ases POLB and POLG, respectively.62 Hypoxic cells depend onChk1, (an effector of the G2 DNA damage checkpoint), to preventDNA breaks and maintain replication forks. In human BRCA posi-tive breast cancer cells, inhibition of Chk1 caused responsivenessto PARP inhibitors.63 The Chk1 inhibitor AZD772 acts as a radiosen-sitiser both in vitro and in vivo.64 Of note it seems to be effective inTNBC, in breast xenograft models.

Deacetylation inhibitors

Histone deacetylase (HDAC) inhibitors are implicated in HIF-1regulation by several mechanisms including HIF-1a protein degra-dation and regulation of HIF-1 transcriptional activity. HDACexpression increases in response to hypoxia, and inhibition ofHDACs cause cell death and reduced tumour growth.65 Suberoylan-ilide hydroxamic acid (SAHA) is the first clinical anti-tumour drugof this class of inhibitors, which appear to be useful against breastcancer. The inhibitor AN-7 decreased HIF-1a levels and reducedlung lesions in a breast model,66 while N-hydroxy-4-(3-phenyl-propanamido)benzamide 5j, was particularly effective againstbreast cancer cells, inducing cell cycle arrest at G(2)M, and apopto-sis by modulating p21, caspase-3, and Bcl-xL.67 This drug inhibitedinvasion and significantly delayed growth of breast cancer xeno-grafts in mice. AN-7 has the advantage preclinically of being syner-gistic with doxorubicin while reducing cardiotoxicity.

176 C. Ward et al. / Cancer Treatment Reviews 39 (2013) 171–179

Another HDAC inhibitor, N-hydroxy7-(2-naphthylthio) heptan-omide (HNHA), demonstrated in vitro and in vivo activity againstbreast cancer, arresting the cell cycle at the G1/S phase via p21induction and inhibiting cell proliferation.68 It decreased VEGFand HIF-1a expression in hypoxic conditions, and inhibited tumourneovascularisation in breast cancer xenografts.68 Activated HIF-1interacts with the first cysteine–histidine (CH1) domain of CBP/p300 which is bound by CBP/p300-interacting transactivator withED-rich tail 4 (CITED4) protein. This inhibits HIF-1a transactivationthrough direct competition for p300 binding. CITED4 expression isreduced in breast cancer, but is re-expressed by HDAC inhibition,69

suggesting that HDAC inhibitors could be useful in the treatment ofhypoxic breast tumours.

P53

The tumour suppressor p53 and HIF-1 interact to determine thefate of tumour cells. p53 can bind directly to the ODD domain ofHIF-1a, blocking HIF-1 transcriptional activity by competing withHIF-1a for p300.15 Activation of p53 inhibits HIF-1a protein accu-mulation by increasing proteosomal degradation, while loss of p53correlates with increased HIF-1a protein level and increased HIF-1activity.15 Loss of p53 selects for apoptotic resistance in hypoxia. Inbreast cancer, p53 is often inactivated because of overexpression ofthe repressors, MDM2 and MDMX.14 Further, almost 50% of breastcancers produce a dysfunctional mutant p53 protein (mtp53)caused by point mutations in the p53 gene which prevent DNAbinding, DNA damage-induced cell death and cell cycle regula-tion.14 Reactivation of p53 through targeting mtp53, MDM2 andMDMX is therefore a plausible strategy in breast cancer treatment,allowing hypoxic cells to be targeted. The pharmacological p53reactivators, nutlin-3, p53 reactivation and induction of massiveapoptosis (PRIMA-1), and reactivation of p53 and induction of tu-mour cell apoptosis (RITA) are currently being explored in cancertreatment.

RITA, a small molecule activator of p53, inhibits tumour growthand induces p53 dependent apoptosis in vivo.70 It blocks HIF-1aactivation and inhibits production of HIF-1a target proteins suchas VEGF, potentially decreasing aberrant angiogenesis.70 PRIMA-1activates mtp53 and inhibits growth of human breast cancer cellsby reactivating the functionality of mtp53, both in vitro andin vivo.71 Nutlin-3 antagonises MDM2, the E3 ubiquitin ligasewhich causes degradation of wild type p53, restoring the p53 sup-pressor pathway. XI-011, an inhibitor of MDMX expression, in-duces apoptosis in several human breast cancer cell lines, actingadditively with nutlin-3.72 MDM2 and MDMX are over-expressedin over 25% of breast cancers.5 These small molecule p53 reactiva-tors could influence treatment of hypoxic breast tumours, and in-crease the response of any therapy reliant on p53-inducedapoptosis. Further, the emergence of p53 as a key regulator inthe control of many aspects of cellular metabolism,73 suggests thatreactivators of p53 may normalise many of the metabolic irregu-larities found in tumours.

Glucose/lactate regulation

Hypoxic tumour cells depend on glucose for energy productionand HIF-1 activation increases the expression of glucose transport-ers. Lactate produced by oxidative phosphorylation in hypoxic tu-mour cells is exported from the cells using MCT4, and importedinto normoxic tumour cells via MCT1.17 HIF activation repressesMCT1 expression and increases MCT4. This symbiotic relationshipallows effective use of glucose by cells in the tumour microenvi-ronment.17 Inhibition of MCT1, 4 or glucose transporters (seeFig. 2), prevents lactate usage by normoxic cells which must utiliseglucose, depriving hypoxic cells of this fuel source. This strategy

induced cell death by glucose starvation in hypoxic tumour areasin in vivo models of lung and colorectal cancers and enhanced sen-sitivity of remaining tumour cells to radiation.74 MCT4 is an impor-tant therapeutic target in glycolytic cancer cells. It can negate theeffects of MCT1 inhibition, particularly in hypoxic conditions inboth in vitro and in vivo models.74 The MCT1/4 accessory moleculeCD147, can be targeted to inhibit expression of both MCTs, reduc-ing in vivo tumour growth.74 MCT1 and MCT4 levels are increasedin breast carcinomas, and overexpression of CD147 increasesgrowth and metastasis of breast cancer cells.75 CD147 expressionis higher in metastatic breast cancer cells, and knock down ofCD147 caused decreases in lactate efflux, MCT4 expression, andmigration of invasive breast cancer cells.75

GLUT1 is the dominant glucose transporter in many cancerswith higher expression found in breast cancer and DCIS than innormal tissue.5 It is significantly increased in hypoxic areas ofbreast tumours, and in more proliferative, higher grade breast can-cers and correlates with reduced survival.5 GLUT1 blocking anti-body induced growth arrest, and apoptosis in breast cancer cellsand increased responsiveness to cisplatin, paclitaxel and gefiti-nib.76 The glucose transport inhibitors, WZB27 and WZB115, inhi-bit breast cancer cell proliferation and induce apoptosis (Fig. 2).77

GLUT1 overexpression enhanced breast tumour cell growth, whilereduced levels impaired growth in a murine model.78 Interferencewith glucose/lactate utilisation is a novel therapeutic research areain breast and other hypoxic tumours. Many inhibitors are underdevelopment.10

pH modulation – ion exchange inhibition

Because survival in the tumour microenvironment depends oncontrol of intracellular pH, interference with pHi regulating sys-tems is a novel relevant therapeutic goal (Fig. 2). Tumour cells thatcannot regulate pHi in acidic conditions, show inhibited prolifera-tion and tumour formation.5 In breast cancer, recent studies sug-gest that CAIX and NHE1 in particular, are pertinent targets.5,20,22

CAIX is important for hypoxic tumour cell survival. It potenti-ates acidification of the external microenvironment, allowingECM degradation and cell invasion. Tumour specific expression ofCAIX is a poor prognostic factor in breast cancer79 and increasedexpression of CAIX in basal-like breast tumours is associated withchemotherapy resistance.42 Some studies show CAIX is an adverseprognostic marker and it is an independent biomarker for distantmetastases in a series of over 3600 breast cancers.79 However in945 high risk pre and postmenopausal patients in the Danish post-mastectomy radiotherapy trials, CAIX was only an independentprognostic factor in subgroup analysis in postmenopausal womenwith 1–3 positive nodes and hormone receptor positive patients.21

In vivo preclinical models testing the efficacy of several novel smallmolecule CAIX inhibitors show CAIX is required for growth andmetastasis of human breast cancer cells.80 Studies suggest de-creased CAIX levels are compensated for by increases in CAXIIexpression in some cancers, but not in these breast models; there-fore breast cancer may be particularly sensitive to CAIX inhibi-tion.80 Several CAIX specific small molecule inhibitors are inadvanced preclinical development.81

Models suggest that NHE1 is important in breast cancer sur-vival, invasion and metastasis, and is therefore another valid ther-apeutic target.22 Inhibition of NHE1 sensitises breast cancer cells toapoptosis when exposed to a variety of chemotherapeutic agents.82

Several NHE1 inhibitors have completed Phase II/Phase III clinicaltrials but toxicity issues were reported.83 For example, the NHE1inhibitor cariporide, which inhibited growth of breast cancer cellsin vitro, showed an unacceptable level of stroke during a largePhase III trial, leading to cessation of further clinical develop-ment.83 Possibly, more specific agents that preferentially target

C. Ward et al. / Cancer Treatment Reviews 39 (2013) 171–179 177

tumour cell NHE1 will be developed or co-treatment with otherpHi inhibitors such as CAIX will allow less toxic drug concentra-tions to be utilised: this may be a more effective strategy thantreating with a single agent. Because the acidic microenvironmentcan hinder drug availability, using these inhibitors may improveuptake and efficacy of some chemotherapeutics. The role of micro-environmental pH modulators and some inhibitors is illustrated inFig. 2.

Angiogenesis

Angiogenesis is a therapeutic target for hypoxic tumoursalthough this at first seems counter-intuitive. Activation of HIF-1induces synthesis of the angiogenic factor VEGF, causing vasculardevelopment that can further tumour growth, although oxygena-tion may suffer if these vessels are malformed. In this case, VEGFinhibition causes normalisation of vessels and increased oxygena-tion, allowing access of chemotherapeutic drugs, and increasedresponsiveness to radiation. Bevacizumab, (a monoclonal antibodytargeting VEGF), increases response rates to chemotherapy andtime to progression, but does not increase overall survival rates.43

This is possibly because the reported long-term effects of Bev-acizumab treatment suggest an induction of tumour hypoxia,which may lead to resistance as described above. However, thisrepresents an opportunity for synthetic lethality, since many ofthose induced changes will be much more necessary in hypoxicconditions. A recent example is the effect of combined blockadeof CA9 with Bevacizumab on tumour growth.84 The induction ofhypoxia by anti-angiogenic agents also increases numbers ofbreast cancer stem cells.85 This could be overcome by co-treatmentwith Notch inhibitors, to reduce this population.86,87

The main approaches used to target vessel growth, are vasculardisruptive agents (VDAs), targeting existing blood vessels, andanti-angiogenic agents, inhibiting the actions of angiogenic factors.VDAs increase responsiveness to radiation in large tumours, allow-ing a reduction in dose needed to control growth. VEGF is inducedin tumours by radiation. Treatment of xenografts with a neutralis-ing VEGF antibody in combination with radiation synergisticallyinhibited tumour regrowth.88 Blocking VEGF signalling in murinexenografts using the VEGF-receptor-2 antibody DC101, normalisedvessels and caused development of a hydrostatic pressure gradientacross the vascular wall, increasing drug infiltration intotumours.89 Anti-angiogenic treatments should improve radiationand cytotoxic treatments of hypoxic tumours including those ofthe breast, either through vascular normalisation, increasingoxygenation and drug penetration or interference with newvascular growth after treatment.

Table 2Strategies aimed at microenvironmentally induced therapeutic targets.

Target Strategy Clinicalphase

HIF-1 Inhibitors of HSP90, microtubuleinhibitors, PX-4787,46–49

III

Inhibition of mTOR, AKT50–52 IIIEGFR inhibitors53 IIHistone deacetylase inhibitors65–68 II

DNA repairpathways

PARP inhibitors,60,61 POLB and POLGdisruption62,

III

Chk1 inhibition.63,64 Ip53 Reactivation using RITA, PRIMA-1, nutlin-3

and XI-1170–72I

Glucose/lactateregulation

Inhibition of GLUT1, MCTs, CD14774–78

Ion exchangeinhibition

Interference with NHE1 and CA IX79–81,83,84 III

Angiogenesis Vascular disruptive agents88,89 III

Opportunities for clinical translation in breast cancer

The preoperative setting of operable breast cancer provides atestbed for assessing novel agents targeting the hypoxic microen-vironment combined with conventional adjuvant systemic therapyand irradiations.90 Pathological complete response to neoadjuvantsystemic therapy is accepted as a useful predictor of long term sur-vival. In most cases, the standard approach is to evaluate novelcompounds as a single agent and then to consider combining thenew agent with existing licensed cytotoxics. However, this strategyof drug development may miss the opportunity to assess synergis-tic effects of targeted drugs with radiation in clinical trials.91 Suit-able serial imaging of tumours will be essential to integrate thesemodalities. For example, measuring effects on hypoxia and prolif-eration within in 2 weeks of an anti-angiogenic drug, would allowfurther drug additions based on the response. Similarly effects ofAkt inhibitors could be analysed by FDG uptake prior to radiother-apy. The preclinical efficacy of novel inhibitors of CAIX and NHE1 incombination with RT and/or chemotherapy in breast cancer celllines and xenografts is currently under investigation in METOXIA,an FP & funded European consortium (www.metoxia.uio.no/eu-fp7) on targeting the hypoxic microenvironment.

Conclusion

The important role of the microenvironment in progression ofbreast and other solid cancers is being increasingly recognised.The adaptations to ensure cell survival in the hypoxic/acidic condi-tions of solid tumours allow the development of more aggressiveand invasive cancer cell populations. This knowledge is also open-ing up new therapeutic frontiers for treating breast cancer. Novelstrategies targeting pHi regulation and glycolysis in hypoxic tu-mours are likely to be useful adjuncts in combination with sys-temic and/or radiation therapy.

Conflict of Interest Statement

The authors declare no conflict of interest in the contents of thisreview.

Acknowledgements

C. Ward is funded by METOXIA. S. Langdon is funded by theScottish Funding Council, Charon Fund and METOXIA. P. Mullenis funded by Breakthrough Breast Cancer. C.T. Supuran is fundedby METOXIA. A. Harris is funded by METOXIA, Oxford NIHR Com-prehensive Biomedical Centre, Breast Cancer Research Foundation,Cancer Research UK Imaging Centre. I. Kunkler is funded byMETOXIA.

These funding bodies had no involvement in the preparation ofthis manuscript.

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