molecular pathways: hypoxia response in immune cells ......molecular pathways molecular pathways:...

Molecular Pathways

Molecular Pathways: Hypoxia Response in Immune CellsFighting or Promoting Cancer

Asis Palaz�on1,2, Juli�an Aragon�es3, Aizea Morales-Kastresana1,2, Manuel Ortiz de Land�azuri3, andIgnacio Melero1,2

AbstractBoth malignant and stromal components in tumors are influenced by the physiologic conditions of the

microenvironment. Hypoxia is a prominent feature of solid tumors as a result of defective vascularization

and intense metabolic activity. The gene-expression control mechanisms that adapt tissues to hypoxia are

exploited by tumors to promote angiogenesis and vasculogenesis. The functions of infiltrating immune cells

(macrophages and lymphocytes) and other stromal components are also influenced by a limitedO2 supply.

Hypoxia-inducible factors (HIF) are themainmolecular transcriptional mediators in the hypoxia response.

The degradation and activity ofHIF-1a andHIF-2a are tightly controlled by the fine-tuned action of oxygen-

sensing prolyl and asparaginyl hydroxylase enzymes. Recent evidence indicates that hypoxia can modulate

the differentiation and function of T lymphocytes and myeloid cells, skewing their cytokine-production

profiles and modifying the expression of costimulatory receptors. This conceivably includes tumor-

infiltrating lymphocytes. Hypoxia not only directly affects tumor-infiltrating leukocytes but also exerts

effects on tumor cells and vascular cells that indirectly cause selective chemokine-mediated recruitment of

suppressive and proangiogenic T-cell subsets. This review focuses on changes induced by hypoxia in

immune cells infiltrating solidmalignancies. Such changesmay either promote or fight cancer, and thus are

important for immunotherapy. Clin Cancer Res; 18(5); 1207–13. �2011 AACR.

BackgroundO2 supplied by hemoglobin from the blood stream is

intracellularly present innormal tissues at concentrations of�10% and even less in metabolically active tissues (1).Under these normoxic conditions, there is sufficient O2

to ensure the correct function of ATP synthesis in mito-chondria. In tissue culture experiments, immune cells areplaced under normoxic conditions, ignoring the fact thatimmune cellsmust work under hypoxia (0.1–3%O2)whenthey perform their functions in infected tissues, tissuesundergoing autoimmunity, or solid tumors (2). Even insecondary lymphoid organs, such as the spleen, the O2

concentration is <5% (3).Hypoxia-inducible factor 1a (HIF-1a) is the main regu-

lator of the adaptation to a shortage of O2 (4). It wasoriginally discovered in cells regulating erythropoietin pro-duction (5). As a way to preserve cell metabolism, mostgenes under the control of HIF-1a andHIF-2a are related to

glucose transport and metabolism, diverting aerobic toanaerobic ATP production. In addition, the HIF-1aresponse induces soluble enhancers of vascularization suchas VEGF (6) and adrenomedullin (7).

Figure 1 shows that O2 concentration-sensitive hydrox-ylase enzymes govern the molecular machinery that regu-lates the function of HIF-1a (8). Under normoxic condi-tions, prolyl-4-hydroxylase isoforms (PHD1, PHD2, andPHD3) act on prolyl residues of HIF-1a (Pro-402 and Pro-564), thereby defining 2 oxygen-dependent degradationdomains in a reaction that is cocatalyzed by iron (Fe2þ)and 2-oxoglutarate [2-OG (ref. 9)]. Once HIF-1a is prolyl-hydroxylated, a protein complex that contains Von Hippel-Lindau tumor-suppressor protein (pVHL), which acts as anE3 ubiquitin ligase, can build up ubiquitin chains linked toHIF-1a (Fig. 1). Lys-48 polyubiquitination in HIF factorsleads to rapid proteolytic degradation by the proteasome(9). If the O2 is below a certain threshold (�3%, but theexact figure is probably dependent on cell type and species),the PHDs become nonfunctional (Fig. 1). TheHIFs are thennot efficiently degraded and translocate to the nucleus todimerize with HIF-b. HIF-1a and HIF-2a form dimers withHIF-b and find short nucleotide sequences (referred to ashypoxia-responsive elements) on which these proteins actas transcription-promoting factors (10). The crucial func-tion of HIF transcription factors is illustrated by the lethalphenotypes of HIF knockout mice, which die as embryosfrom vascularization failures (11). Experimentation to

Authors' Affiliations: 1Center for Applied Medical Research, University ofNavarra, and 2Servicio de Oncolog��a, Clinica Universidad de Navarra,Pamplona, Spain; 3Servicio de Inmunologia, Hospital Universitario de laPrincesa, Universidad Autonoma de Madrid, Madrid, Spain

Corresponding Author: Ignacio Melero, CIMA, Avenida de Pio XII, 55,31008 Pamplona, Spain. Phone: 0034948194700; Fax: 011-0034-948194717; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-1591

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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Published OnlineFirst December 28, 2011; DOI: 10.1158/1078-0432.CCR-11-1591

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clarify the role of the different molecular elements in thepathway has required tissue lineage–restricted conditionalknockout mice of HIF and the 3 PHDs (12).

Hydroxylation on asparaginyl residues (e.g., Asn-804)located on the C-terminal transactivation domain of HIF-1a

by factor-inhibiting HIF (FIH) constitutes another level ofregulation (13). FIH activity is also dependent on O2, Fe

2þ,and 2-oxoglutarate.Whenoxygen is available, hydroxylationonAsn-804blocks the interactionofHIF-1awith the nucleartranscriptional coactivator protein p300 (Fig. 1; ref. 14). The

Figure 1. O2 sensing and molecular response to hypoxia in cells of the immune system. Schematic representation of the O2 control of HIF stabilityand direct and indirect effects of HIF to control the expression of other gene/proteins. Hypoxia exerts effects mainly by controlling the functions of HIF-1abut also by enhancing the concentration of oxygen free radicals and adenosine. HIF-1a levels are controlled mainly by proteasomal degradation and toa lesser extent by transcriptional induction. Degradation ofHIF-1adependsonLys-48polyubiquitinationby theVHLE3-ubiquitin ligase complex.Degradationonly takes place under normoxic conditions because HIF-1a is targeted to ubiquitination by PHDs that are only operational in the presence of O2. Underhypoxic conditions, stabilized HIF-1a relocates to the nucleus and exerts direct and indirect transcription-promoting activities by partnering with theconstitutively expressed HIF-1b protein. Recent evidence also shows a role for HIF-1a in targeting the FOXP-3 protein for ubiquitination and proteasomaldegradation. This is extraordinarily important because FOXP-3 is critical for the differentiation of regulatory T cells. cAMP, cyclic AMP; IL, interleukin; ROR-gt,RAR-related orphan receptor gt; TLR, toll-like receptor.

Palaz�on et al.

Clin Cancer Res; 18(5) March 1, 2012 Clinical Cancer Research1208




nonoverlapping functions of these mechanisms are sup-ported by recent studies that relied on the use of gene-modified animals selectively deficient for PHD1, PHD2,PHD3, or FIH (12).HIF-2a has important functions mainly in erythropoiesis

and vascularization (15, 16), as well as inmacrophages (17),because expression of HIF-2amRNA is cell-type–dependent(18). Because the focus of this review is immune cellsundergoing hypoxia, wewill concentrate on responsesmedi-ated by HIF-1a and refer to HIF-2a only in relation tomacrophages.HIF-1a does more than control gene transcription direct-

ly, and a number of alternative functions and mechanismsof this protein are being revealed. These include (i) protein-to-protein association with other transcription factors, suchas RAR-related orphan receptor-gt [RORgt (19)] and acti-vator protein-1 [AP-1 (Fig. 1; ref. 20)]; (ii) induction ofmicroRNAs (miRNA), such as miR-210 (21); and (iii)induction of the expression of transcription factors that inturn control other genes and/or epigenetic mechanisms,such as histone acetylation involving p300/CBP (22).HIF-1a can also decrease the levels of gene transcription

by different mechanisms (4). Surprisingly, HIF-1a canalso induce the degradation of proteins by targeting themto polyubiquitination, as was recently observed with theFOXP-3 transcription factor in CD4þ T lymphocytes(Fig. 1; refs. 19). The PHDs that hydroxylate HIF have alsobeen discovered to prolyl hydroxylate members ofthe canonical NF-kB pathway (23) that are critically in-volved in inflammation.

Innate immune system and inflammation underhypoxic conditionsTumor stroma contains vascular cells, myeloid cells [e.g.,

tumor-associated macrophages (TAM)], tumor-infiltratinglymphocytes (TIL), and activated fibroblasts associatedwiththe tumor. Vascular cells, including endothelium and peri-cytes, undergo functional changes due to the hypoxicmicro-environment and have been extensively studied in tumors(24). Moreover, components of tumor stroma have beenshown to outperform malignant cells in the production ofVEGF, and thus play a key role in angiogenesis and aberrantvascularization of tumors (Fig. 2; refs. 25, 26).Myeloid cells are highly represented in intratumoral

environments, and macrophages are frequently the mostabundant of these cells that usually invade the intratu-moral hypoxic environment. Indeed, hypoxic tumor areasinduce tumor-homing of bone marrow–derived CD45þ

myeloid cells (27). These effects have been reported to bepartly dependent on the production by hypoxic tumorcells of stromal cell–derived factor 1a (SDF1a), whichbinds to CXCR4. These cells are also sensitive to inter-leukin (IL)-8 (CXCL8)–guided chemotaxis (28), and IL-8is abundantly produced in the tumor at least partly inresponse to hypoxia.TAMs normally contribute to tumor progression (Fig. 2;

ref. 29). Although the cytotoxicity of macrophages in theearly immune response contributes to the death of tumor

cells, the presence of macrophages has also been correlatedwith a poor prognosis for patients. Macrophages that havebeen recruited to inflammatory sites undergo distinct formsof activation depending on the cytokine signals encoun-tered in the surrounding milieu. For example, cytokines orbacterial compounds that usually accumulate during bac-terial infection, such as lipopolysaccharide and helper type-1 (Th1) cytokines (e.g., IFN-g), induce classic macrophagepolarization [M1 (30)]. This M1 polarization is chieflycharacterized by enhanced intracellular bacterial killing,production of nitric oxide (NO), and tissue destruction,but also with resistance to tumor progression. By contrast,helper type-2 (Th2) cytokines such as IL-4, as well as othersignals, induce alternative macrophage polarization (M2)oriented mainly toward immunoregulation, tissue remo-deling, and tumor promotion (30–32).

Recent evidence suggests that HIF-1a and HIF-2a mayplay contrasting roles in macrophage M1 and M2 polari-zation. In this regard,HIF-1a is activated in response stimulithat induce M1 polarization but is not altered under con-ditions in which macrophages undergo M2 polarization inresponse to IL-4 (33, 34). Such HIF-1a upregulation seemsto be dependent on a specific increase in HIF-1a mRNAlevels, which involve the NF-kB transcription factor(17, 35, 36). In addition, HIF-1a induces markers of M1polarization such as inducible NO synthase. Moreover, lossof HIF-1a induces M2 markers such as macrophage scav-enger receptor 1 (37, 38).

In contrast, a recent study indicated that HIF-2a isinduced specifically during M2 polarization, and showedthat HIF-2a is a critical regulator of arginase 1 gene expres-sion, a molecular marker of M2 polarization that reducesthe intracellular L-arginine pool required to produce NO(33, 39). This could suggest opposite roles of the HIF1 andHIF2 systems in NO generation and potentially in M1/M2polarization. However, controversy surrounds the role ofHIF-2a in M2 polarization and arginase expression, prob-ably reflecting the different conditions of the in vitromodelsemployed (40).

A recent study in an adenocarcinomamodel showed thathypoxic TAMs show signs of M2 polarization. Of interest,the majority of TAMs that resemble an M1 phenotype arelocated in normoxic areas, whereas M2 is found in hypoxicregions. This suggests that the proposed hypoxia-HIF2a-M2polarization axis could dominate in the intratumoral hyp-oxic environment (32). In this regard, immunohistologicstudies have found strong HIF-2a expression in TAMs (41).Moreover, genetic studies in myeloid HIF-2–deficient miceshowed altered migratory properties of HIF-2–deficientmacrophages that were associated with reduced tumorprogression (40). It is also important to consider that theHIF activity of TAMs not only alters intratumoral endothe-lial cells but also regulates other cell types that act asimmunosuppressors of T cells, such as myeloid-derivedsuppressor cells (Fig. 2; refs. 42, 43).

Of importance, metastatic niche formation was recentlyreported to depend on HIF-1a, which induces multiplemembers of the lysyl oxidase family that facilitates bone

Tumor-Infiltrating Leukocytes under Hypoxia

www.aacrjournals.org Clin Cancer Res; 18(5) March 1, 2012 1209




marrow–derived cell recruitment prior to metastatic colo-nization by malignant cells (Fig. 2; ref. 44).

The effects exerted by hypoxia on dendritic cells remainunknown (Fig. 2); however, hypoxia has been found tofavor the immune and inflammatory response, therebypresumably enhancing their ability to prime and activateT cells (45). Moreover, dendritic cells cultured with hypoxicglioblastoma tumor lysates are more efficient at priming

effector T cells compared with antigen uptake from nor-moxic tumor-cell lysates (46).

TILs under hypoxiaT lymphocytes in tumors come in different flavors (Fig.

2). Some are potentially endowed with tumoricidal prop-erties that are restrained, whereas others can suppress thefunctions of effector T cells and natural killer cells. Such

© 2011 American Association for Cancer Research

INN

ATE

IMM

UNIT

Y

ADAPTIVE IMM

UN

ITY

CANCER CELLS

↑ Immune response (45)

↑ Inflammatory response (45)

↑ Th17 differentiation (19)

↑ IL-17, ↑ RORγt (19)

↓ TCR and CD28 mediated activation (49–51, 58)

↑ CD137 (A. Palazón et al.; unpublished data)

↑ Protection against activation-induced death (57)

↑ CD73, ↑ Adenosine (57)

Differentiation to TAMs

(43)

(42)Monocyteprecursor

Treg

Th17

Immunesuppression

CD4

CD8DCs

MØs

↑ FOXP-3 degradation (19)

↑ Metastatic niche formation (44)

↑ Metastasis (4)

↑ Angiogenesis (VEGF, Adrenomedullin) (6, 26)

↑ Vascular tone (26)

↑ Adhesion molecules (ICAM-1, VCAM)

↑ Costimulatory molecules (CD137) (62)

↑ Adrenomedullin (7)

↑ Chemokines (CCL28) (61)

Metabolic shift, ↓ pH, ↑ lactic acid (4)

↓ Susceptibility to CTLs (54)

↑ VEGF-A, ↑ Inflammation

↑ Phagocytosis, ↓ Apoptosis

Tumor progression (29–34)

- Hif-1α: M1-like (NOs)

- Hif-2α: M2-like (Arg1?)

TUMOR ENDOTHELIAL CELLS

HYPOXIA

MDSC

Figure 2. Direct and indirect effects of hypoxia on stromal and malignant cells. Schematic representation of the effects of hypoxia on the different cellsubpopulations found in the tumor microenvironment. Literature references pertaining to the indicated phenomena are provided in the scheme. ICAM,intercellular adhesion molecule; TCR, T-cell receptor; VCAM, vascular cell adhesion molecule.

Palaz�on et al.





immunosuppressor lymphocytes are called regulatory Tcells [Treg (47)]. Among effector T lymphocytes, CD8 andCD4 can be found in the tumor stroma. TILs enter tumorsfrom blood vessels. A variety of factors in the tumor represstheir functional activity, although there is evidence fortumor antigen recognition by TILs. The balance betweenregulatory and effector T cells in the stroma correlates withthe prognosis of patients and the response to immunother-apeutic interventions (48).When T cells enter tumors, they reach progressively more

hypoxic regions as they penetrate deeper into the tumor.Nonetheless, T cells infiltrating tumors tend to remainassociated with vessels in subregions that ought to be lesshypoxic. It is well known that areas of solid tumors canbecome completely anoxic andundergonecrosis. Very fewTcells are found around these necrotic areas.Unfortunately, little research has been carried out regard-

ing the activation of T cells under hypoxia and the differ-ences with activation under normoxic conditions. Somepioneer studies seem to suggest a less efficient activation byT-cell receptor (TCR)-derived signals and costimulation(49–51). Moreover, it has been reported that HIF-1a–defi-cient T cells respond with higher intensity to stimulation ofthe antigen receptor. In addition, a study of bacterial sepsisshowed a functional advantage for T cells upon conditionaldeletion of the HIF-1a gene in T cells in the clearance ofdisseminated bacteria inmice (52). The CD4 and CD8HIF-1-deficient T cells of these mice also showed a more intenseability to proliferate and to produce IFN-g (53). There-fore, it was concluded that HIF-1a is a repressor of T cells,and hypoxia should be an intrinsic immunosuppressivecondition for T cells, including the ability to kill TCR-recognized targets (53, 54). In contrast, a recent studysuggested that HIF-1a deficiency in T cells does not affectproliferation and Th-1/Th-2 polarization (19). It was alsoshown that hypoxia leads to less efficient target cell killingbyCTLs (54).Overall, the T-cell HIF1a-response to hypoxiacan be considered a factor that contributes to immunosup-pression in the tumor microenvironment. At present, thereis a conspicuous lack of information about how hypoxiaaffects Treg functions in the tumor microenvironment oreven in ex vivo culture studies.Themechanisms involved in themodulation of effector T

cells by hypoxia include the release to the extracellularmilieu of adenosine, which acts onA2R inhibitory receptors(55), production of oxygen free radicals, and HIF-1acontrol of genes that downmodulate the immune response(Fig. 1).Dying cells release ATP that is metabolized to adenosine

by the coordinated extracellular enzymatic functions ofCD73 and CD39, which are also upregulated by hypoxia(Fig. 1; ref. 56). Soluble adenosine in turn acts on sur-face receptors of T cells (A2Rs) to augment intracellularcAMP, a second messenger known to repress T-cell func-tions (Fig. 1; ref. 57).Under hypoxic conditions, the respiratory chain in the

mitochondria becomes less efficient, to the point of releas-ing reactive oxygen species that are produced both by tumor

and stromal cells. There is evidence that reactive oxygenspecies can inhibit NF-kBnuclear translocation and therebyswitch off cytokine production. In addition, this oxidativestress has been shown to promote the selective degradationof components of the TCR-CD3 complex, such as CD3z(58).

Very recently it was discovered that HIF-1a favors thedifferentiation of Th17 cells via direct transcriptionalinduction of RAR-related RORgt and indirectly IL-17(19). For these functions, HIF-1a partners with phosphor-ylated STAT-3, which enhances the transcription of HIF-1aand cooperates withHIF-1a at the RORgt promoter (Fig. 1).Moreover, CD4 T cells that are starting to differentiatetoward FOXP-3þ Treg cells experience another surprisingeffect of HIF-1a that involves degradation of the forkheadbox P3 (FOXP-3) protein (Fig. 1). Amazingly, HIF1-a bindsprotein-to-protein to FOXP-3, and as a result recruits theVHL E3 ubiquitination machinery to mark FOXP-3 fordegradation (19).

We have observed that HIF-1a can modulate the expres-sion of costimulatory receptors such as CD137 (A. Palaz�onet al.; unpublished data). Indeed, T-cell activation underhypoxia leads to amuchmore ready upregulation of surfaceCD137 upon antigen stimulation through a mechanismthat is dependent on HIF-1a. This explains why TILs intumors are brightly CD137þ, including effectors and Tregs.Of importance, CD137 is a receptor that upon targeting byimmunostimulatorymonoclonal antibodies leads to a ther-apeutic enhancement of antitumor immunity (59). In theabsence of any agonist ligand expressed by neighboringcells, CD137 is useless for the antigen-primed T cells.However, if immunotherapeutic agonist antibodies arereceived, a strengthened function of the TILs ensues andtumor eradication becomes a frequent outcome. Of note,recent findings indicate that CD137 expression on TILsinside human lymphomas has an important impact as aprognostic factor (60).

Indirect mechanisms of hypoxia on T lymphocytesIn addition to affecting T cells directly, hypoxia can

modify other components of the tumor tissue that in turndictate the behavior of T cells. Hypoxic tumor cellsproduce high levels of CC-chemokine ligand 28 (CCL28).This chemokine has been shown to selectively attractTregs that express CXCR10, which also induces antigentolerance and promotes angiogenesis (61). Hypoxia act-ing on vascular endothelium in tumors gives rise tosurface CD137 expression (62). If this molecule is acti-vated, proinflammatory changes take place that increaseT-cell infiltration (62).

It has also been observed that tumor cells under the effectof hypoxia activate autophagia, and that autophagia pro-tects malignant cells from being killed by cytotoxic T cells(63).

The overall picture is that TILs are restrained in tumorsdue to a partial deprivation of oxygen, which leads to HIF-1a stabilization and extracellular adenosine accumulation(55).






Clinical–Translational AdvancesWe must rethink tumor immunotherapy by taking local

tumor hypoxia into consideration. Innate and adaptiveimmune cells are profoundly modified under hypoxia(Fig. 2). The response to hypoxia as mediated by HIF-1ain T cells and macrophages is entangled with key transcrip-tion factors that modify the intensity and quality of theimmune/inflammatory response, including STAT-3,NF-kB,and RORgt (64).

Two immunotherapy strategies, adoptive T-cell transfer(65) and immunostimulatorymonoclonal antibodies (66),currently stand out because of their potential benefit in theclinical arena. The impact of local tumor hypoxia on thesetherapies should be reexamined. In the most clinicallyeffective form of adoptive T-cell therapy, TILs are takenfrom tumors, reinvigorated, and expanded to be given backto a host who has been preconditioned by chemotherapyand total-body irradiation (65). The TILs need to find theirway back to the tumor and destroy it. The exact state ofhypoxia in the tumor lesions, which modulates all of thesephenomena (i.e., T-cell activation, memory differentiation,migration, and extravasation), needs to be experimentallyaddressed.

In the case of immunostimulatory monoclonal antibo-dies (66), we know virtually nothing about the function ofcostimulatory receptors of the TNF receptor and immuno-

globulin superfamilies when the concentration ofO2 drops.As in the case of CD137, even the expression of the targetsfor the immunotherapeutic antibodies can be modified bythe hypoxia response.

If hypoxia is found to be a guilty accomplice of perni-cious immunoregulatory networks in the tumor micro-environment, interventions with antioxidants, pharma-cologic adenosine inhibitors, and drugs acting on the HIFresponse pathway, including proteasome inhibitors suchas bortezomib, may be considered as interesting adju-vants for a variety of immunotherapies. In any case, thebottom line is that immunomodulatory mechanisms thatare controlled directly or indirectly by the hypoxiaresponse certainly do exist, and such mechanisms offerreal opportunities for intervention.

Disclosure of Potential Conflicts of InterestI. Melero has an ownership interest in Bristol-Myers Squibb and has

served as a consultant to Bristol-Myers Squibb, Pfizer Inc., Merk-Serono,Digna Biotech, andMiltenyi Biotec. The other authors disclosed no potentialconflicts of interest.

AcknowledgmentsWe thank Drs. Ana Rouzaut, Sandra Hervas-Stubbs, and Iv�an Martinez-

Forero for helpful discussions, and Dr. Paul Miller for help with the Englishusage.

ReceivedOctober 28, 2011; revisedDecember 1, 2011; acceptedDecember7, 2011; published OnlineFirst December 28, 2011.

References1. Ward JP. Oxygen sensors in context. Biochim Biophys Acta

2008;1777:1–14.2. Vaupel P. Tumor microenvironmental physiology and its implications

for radiation oncology. Semin Radiat Oncol 2004;14:198–206.3. Caldwell CC, Kojima H, Lukashev D, Armstrong J, Farber M, Apasov

SG, et al. Differential effects of physiologically relevant hypoxic con-ditions on T lymphocyte development and effector functions. J Immu-nol 2001;167:6140–9.

4. Semenza GL. Oxygen sensing, homeostasis, and disease. N Engl JMed 2011;365:537–47.

5. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via denovo protein synthesis binds to the human erythropoietin geneenhancer at a site required for transcriptional activation. Mol Cell Biol1992;12:5447–54.

6. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factorinduced by hypoxia may mediate hypoxia-initiated angiogenesis.Nature 1992;359:843–5.

7. Garayoa M, Mart��nez A, Lee S, P��o R, An WG, Neckers L, et al.Hypoxia-inducible factor-1 (HIF-1) up-regulates adrenomedullinexpression in human tumor cell lines during oxygen deprivation:a possible promotion mechanism of carcinogenesis. Mol Endocrinol2000;14:848–62.

8. Majmundar AJ, Wong WJ, Simon MC. Hypoxia-inducible factors andthe response to hypoxic stress. Mol Cell 2010;40:294–309.

9. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ,et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylationcomplex by O2-regulated prolyl hydroxylation. Science 2001;292:468–72.

10. WengerRH,Stiehl DP,CamenischG. Integrationof oxygensignalingatthe consensus HRE. Sci STKE 2005;2005:re12.

11. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumorformation and embryonic vascularization. EMBO J 1998;17:3005–15.

12. Aragon�es J, Fraisl P, Baes M, Carmeliet P. Oxygen sensors at thecrossroad of metabolism. Cell Metab 2009;9:11–22.

13. LandoD,PeetDJ,Gorman JJ,WhelanDA,WhitelawML,BruickRK. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptio-nal activity of hypoxia-inducible factor. Genes Dev 2002;16:1466–71.

14. Lisy K, Peet DJ. Turnme on: regulating HIF transcriptional activity. CellDeath Differ 2008;15:642–9.

15. Rankin EB, Biju MP, Liu Q, Unger TL, Rha J, Johnson RS, et al.Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin invivo. J Clin Invest 2007;117:1068–77.

16. EmaM, TayaS, Yokotani N, SogawaK,MatsudaY, Fujii-KuriyamaY. Anovel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is poten-tially involved in lung and vascular development. Proc Natl Acad SciU S A 1997;94:4273–8.

17. Fang HY, Hughes R, Murdoch C, Coffelt SB, Biswas SK, Harris AL,et al. Hypoxia-inducible factors 1 and 2 are important transcriptionaleffectors in primary macrophages experiencing hypoxia. Blood2009;114:844–59.

18. Tian H, McKnight SL, Russell DW. Endothelial PAS domain protein 1(EPAS1), a transcription factor selectively expressed in endothelialcells. Genes Dev 1997;11:72–82.

19. Dang EV, Barbi J, YangHY, JinasenaD, YuH, ZhengY, et al. Control ofT(H)17/T(reg) balance by hypoxia-inducible factor 1. Cell 2011;146:772–84.

20. Laderoute KR. The interaction between HIF-1 and AP-1 transcriptionfactors in response to lowoxygen.SeminCell DevBiol 2005;16:502–13.

21. Camps C, Buffa FM, Colella S, Moore J, Sotiriou C, Sheldon H, et al.hsa-miR-210 Is induced by hypoxia and is an independent prognosticfactor in breast cancer. Clin Cancer Res 2008;14:1340–8.

22. Arany Z, Huang LE, Eckner R, Bhattacharya S, Jiang C, Goldberg MA,et al. An essential role for p300/CBP in the cellular response to hypoxia.Proc Natl Acad Sci U S A 1996;93:12969–73.

23. Takeda Y, Costa S, Delamarre E, Roncal C, de Oliveira RL, SquadritoML, et al. Macrophage skewing by Phd2 haplodeficiency preventsischaemia by inducing arteriogenesis. Nature 2011;479:122–6.

Palaz�on et al.





24. Hamzah J, Jugold M, Kiessling F, Rigby P, Manzur M, Marti HH, et al.Vascular normalization in Rgs5-deficient tumours promotes immunedestruction. Nature 2008;453:410–4.

25. Crawford Y, Ferrara N. Tumor and stromal pathways mediating refrac-toriness/resistance to anti-angiogenic therapies. Trends PharmacolSci 2009;30:624–30.

26. Carmeliet P, Jain RK. Molecular mechanisms and clinical applicationsof angiogenesis. Nature 2011;473:298–307.

27. Du R, Lu KV, Petritsch C, Liu P, Ganss R, Passegué E, et al. HIF1alphainduces the recruitment of bone marrow-derived vascular modulatorycells to regulate tumor angiogenesis and invasion. Cancer Cell2008;13:206–20.

28. Alfaro C, Su�arez N,Mart��nez-Forero I, Palaz�on A, Rouzaut A, Solano S,et al. Carcinoma-derived interleukin-8 disorients dendritic cell migra-tion without impairing T-cell stimulation. PLoS ONE 2011;6:e17922.

29. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progres-sion and metastasis. Cell 2010;141:39–51.

30. Sica A, Larghi P, Mancino A, Rubino L, Porta C, Totaro MG, et al.Macrophage polarization in tumour progression. Semin Cancer Biol2008;18:349–55.

31. Martinez FO, Sica A, Mantovani A, Locati M. Macrophage activationand polarization. Front Biosci 2008;13:453–61.

32. Movahedi K, Laoui D, Gysemans C, Baeten M, Stang�e G, Van denBossche J, et al. Different tumor microenvironments contain function-ally distinct subsets of macrophages derived from Ly6C(high) mono-cytes. Cancer Res 2010;70:5728–39.

33. Takeda N, O'Dea EL, Doedens A, Kim JW, Weidemann A, StockmannC, et al. Differential activation and antagonistic function of HIF-alphaisoforms in macrophages are essential for NO homeostasis. GenesDev 2010;24:491–501.

34. Rodr��guez-Prados JC, Trav�es PG, Cuenca J, Rico D, Aragon�es J,Mart��n-Sanz P, et al. Substrate fate in activated macrophages: acomparison between innate, classic, and alternative activation.J Immunol 2010;185:605–14.

35. Peyssonnaux C, Datta V, Cramer T, Doedens A, Theodorakis EA, GalloRL, et al. HIF-1alpha expression regulates the bactericidal capacity ofphagocytes. J Clin Invest 2005;115:1806–15.

36. Blouin CC, Pag�e EL, Soucy GM, Richard DE. Hypoxic gene activationby lipopolysaccharide in macrophages: implication of hypoxia-induc-ible factor 1alpha. Blood 2004;103:1124–30.

37. Shirato K, Kizaki T, Sakurai T, Ogasawara JE, Ishibashi Y, Iijima T, et al.Hypoxia-inducible factor-1alpha suppresses the expression of mac-rophage scavenger receptor 1. Pflugers Arch 2009;459:93–103.

38. Werno C, Menrad H, Weigert A, Dehne N, Goerdt S, Schledzewski K,et al. Knockout of HIF-1a in tumor-associatedmacrophages enhancesM2 polarization and attenuates their pro-angiogenic responses.Carcinogenesis 2010;31:1863–72.

39. Bansal V, Ochoa JB. Arginine availability, arginase, and the immuneresponse. Curr Opin Clin Nutr Metab Care 2003;6:223–8.

40. Imtiyaz HZ, Williams EP, Hickey MM, Patel SA, Durham AC, Yuan LJ,et al. Hypoxia-inducible factor 2alpha regulates macrophage functionin mouse models of acute and tumor inflammation. J Clin Invest2010;120:2699–714.

41. Talks KL, TurleyH,Gatter KC,Maxwell PH, PughCW,Ratcliffe PJ, et al.The expression and distribution of the hypoxia-inducible factorsHIF-1alpha and HIF-2alpha in normal human tissues, cancers, andtumor-associated macrophages. Am J Pathol 2000;157:411–21.

42. Doedens AL, Stockmann C, RubinsteinMP, Liao D, ZhangN, DeNardoDG, et al. Macrophage expression of hypoxia-inducible factor-1 alphasuppresses T-cell function and promotes tumor progression. CancerRes 2010;70:7465–75.

43. CorzoCA,Condamine T, LuL,CotterMJ, Youn JI, ChengP, et al. HIF-1aregulates function and differentiation of myeloid-derived suppressorcells in the tumor microenvironment. J Exp Med 2010;207:2439–53.

44. Wong CC, Gilkes DM, Zhang H, Chen J, Wei H, Chaturvedi P, et al.Hypoxia-inducible factor 1 is a master regulator of breast cancer met-astatic niche formation. Proc Natl Acad Sci U S A 2011;108:16369–74.

45. Bosco MC, Pierobon D, Blengio F, Raggi F, Vanni C, Gattorno M, et al.Hypoxia modulates the gene expression profile of immunoregulatoryreceptors in humanmature dendritic cells: identification of TREM-1 as anovel hypoxic marker in vitro and in vivo. Blood 2011;117:2625–39.

46. Olin MR, Andersen BM, Litterman AJ, Grogan PT, Sarver AL,Robertson PT, et al. Oxygen is a master regulator of the immu-nogenicity of primary human glioma cells. Cancer Res 2011;71:6583–9.

47. Zou W. Immunosuppressive networks in the tumour environment andtheir therapeutic relevance. Nat Rev Cancer 2005;5:263–74.

48. Mlecnik B, TosoliniM,KirilovskyA, Berger A, BindeaG,Meatchi T, et al.Histopathologic-based prognostic factors of colorectal cancers areassociated with the state of the local immune reaction. J Clin Oncol2011;29:610–8.

49. Atkuri KR, Herzenberg LA, Herzenberg LA. Culturing at atmosphericoxygen levels impacts lymphocyte function. Proc Natl Acad Sci U S A2005;102:3756–9.

50. Atkuri KR, Herzenberg LA, Niemi AK, Cowan T, Herzenberg LA.Importance of culturing primary lymphocytes at physiological oxygenlevels. Proc Natl Acad Sci U S A 2007;104:4547–52.

51. Neumann AK, Yang J, Biju MP, Joseph SK, Johnson RS, Haase VH,et al. Hypoxia inducible factor 1 alpha regulates T cell receptor signaltransduction. Proc Natl Acad Sci U S A 2005;102:17071–6.

52. Thiel M, Caldwell CC, Kreth S, Kuboki S, Chen P, Smith P, et al.Targeted deletion of HIF-1alpha gene in T cells prevents their inhibitionin hypoxic inflamed tissues and improves septic mice survival. PLoSONE 2007;2:e853.

53. Lukashev D, Klebanov B, Kojima H, Grinberg A, Ohta A, Berenfeld L,et al. Cutting edge: hypoxia-inducible factor 1alpha and its activation-inducible short isoform I.1 negatively regulate functions of CD4þ andCD8þ T lymphocytes. J Immunol 2006;177:4962–5.

54. HasmimM, NomanMZ, Lauriol J, BenlalamH,Mallavialle A, Rosselli F,et al. Hypoxia-dependent inhibition of tumor cell susceptibility to CTL-mediated lysis involves NANOG induction in target cells. J Immunol2011;187:4031–9.

55. OhtaA,Gorelik E, PrasadSJ, RoncheseF, LukashevD,WongMK, et al.A2A adenosine receptor protects tumors from antitumor T cells. ProcNatl Acad Sci U S A 2006;103:13132–7.

56. Zhang B. CD73: a novel target for cancer immunotherapy. Cancer Res2010;70:6407–11.

57. Sitkovsky M, Lukashev D. Regulation of immune cells by local-tissueoxygen tension: HIF1 alpha and adenosine receptors. Nat Rev Immu-nol 2005;5:712–21.

58. Kono K, Salazar-Onfray F, Petersson M, Hansson J, Masucci G,Wasserman K, et al. Hydrogen peroxide secreted by tumor-derivedmacrophages down-modulates signal-transducing zeta moleculesand inhibits tumor-specific T cell-and natural killer cell-mediated cyto-toxicity. Eur J Immunol 1996;26:1308–13.

59. Melero I, Murillo O, Dubrot J, Herv�as-Stubbs S, Perez-Gracia JL.Multi-layered action mechanisms of CD137 (4-1BB)-targeted immunothera-pies. Trends Pharmacol Sci 2008;29:383–90.

60. Alizadeh AA, Gentles AJ, Alencar AJ, Liu CL, Kohrt HE, Houot R, et al.Prediction of survival in diffuse large B-cell lymphoma based on theexpression of 2 genes reflecting tumor and microenvironment. Blood2011;118:1350–8.

61. Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang LP,et al. Tumour hypoxia promotes tolerance andangiogenesis viaCCL28and T(reg) cells. Nature 2011;475:226–30.

62. Palaz�on A, Teijeira A, Mart��nez-Forero I, Herv�as-Stubbs S, Roncal C,Pe~nuelas I, et al. Agonist anti-CD137 mAb act on tumor endothelialcells to enhance recruitment of activated T lymphocytes. Cancer Res2011;71:801–11.

63. Noman MZ, Janji B, Kaminska B, Van Moer K, Pierson S, Prza-nowski P, et al. Blocking hypoxia-induced autophagy in tumorsrestores cytotoxic T-cell activity and promotes regression. CancerRes 2011;71:5976–86.

64. Kuraishy A, Karin M, Grivennikov SI. Tumor promotion via injury- anddeath-induced inflammation. Immunity 2011;35:467–77.

65. Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, PhanGQ, et al. Durable complete responses in heavily pretreated patientswith metastatic melanoma using T-cell transfer immunotherapy. ClinCancer Res 2011;17:4550–7.

66. Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen L. Immu-nostimulatory monoclonal antibodies for cancer therapy. Nat RevCancer 2007;7:95–106.






2012;18:1207-1213. Published OnlineFirst December 28, 2011.Clin Cancer Res Asis Palazón, Julián Aragonés, Aizea Morales-Kastresana, et al. or Promoting CancerMolecular Pathways: Hypoxia Response in Immune Cells Fighting

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