oncogenic stress sensed by the

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Cancers arise in a mti-step process that invoves thedysregation of oncogenes, tmor sppressors andpro-apoptotic signas1. Genetic and epigenetic aterationsead to changes in ce groth cyces, ce differentiationprogrammes and ce death pathays1. Stdies of cooncancer have served as a paradigm for considering thestepise deviations from norma processes that cmi-nate in metastatic cancer2(FIG. 1). Initia genetic changesrest in hyperproiferation of previosy norma ces,and sbseqent oncogene mtations and epigeneticchanges ead to the deveopment of increasingy dyspasticpre-canceros adenomas or other pre-canceros esions2.These initia changes activate key tmor sppressors,sch as p53, hich sppress ce cyce progression andmay indce ce senescence or apoptosis2. Mtations ofthese tmor sppressor genes, or other genes in thereevant pathays, remove this defence against tmordeveopment and correate ith the appearance of can-

cer. The accmation of other mtations can infenceangiogenesis, migration and metastasis. Athogh theorder of occrrence of these mtations may vary in dif-ferent types of cancer or even different instances of thesame type of cancer, evidence sggests that homozygosmtations in p53 say become predominant atein tmorigenesis2. This highights the fact that earyevents in tmorigenesis activate p53 and create seectivepressre for oss of this key tmor sppressor.

In addition to these argey ce-intrinsic defencesagainst tmorigenesis, evidence sggests there arece-extrinsic barriers to tmor deveopment, someof hich are mediated by the immne system. Severa

cancers are inked to infectios agents, and in some ofthese cases transformation depends on direct infectionof the pre-maignant ce3. Exampes that are reevant tohman disease incde cervica carcinoma, some ym-phomas and Kaposis sarcoma. In these instances, thetransformed ce may express non-sef antigens encodedby the pathogen that can be targeted by B and T ces, inthe same ay that they might respond dring an infec-tion. Other cancers arise by spontaneos genetic and/orepigenetic changes. In these tmors, sef antigens aresometimes overexpressed and can activate the adaptiveimmne system oing to their nnatray high abn-dance, hich can overcome sef toerance. Hoever, inother cases adaptive immne responses are not readiydetected and may not have a major roe in tmor sp-pression4. In these instances, tmor sppression maybe carried ot by the innate immne system. In gen-era, adaptive immne responses are initiated by signas

that are associated ith infammation that is cased byinnate immne responses4, and this is probaby aso trefor responses to tmors. Therefore, the initia innateimmne response to tmors may be decisive in deter-mining hether immne srveiance is effective. It mstbe emphasized that many immne responses to tmorsare not ony non-protective, bt paradoxicay promotecancer. Important research in this area has been revieedesehere4,5 and i not be addressed in detai here.

Natra kier (NK) ces are an integra componentof the innate immne response to tmors. NK ces areymphocytes that differ from B and T ces in that they senmeros receptors, none of hich is encoded by genes

Department of Molecular and

Cell Biology, Cancer Research

Laboratory, 485 Life Science

Addition, University of

California, Berkeley,

California 94720, USA.

Correspondence to D.H.R.

email: [email protected]

doi:10.1038/nri2604

p53

A tumour suppressor that is

mutated in ~50% or more of

all human cancers. p53 is a

transcription factor that is

activated by DNA damage,anoxia, expression of certain

oncogenes and several other

stress stimuli. Target genes

activated by p53 regulate cell

cycle arrest, apoptosis, cell

senescence and DNA repair.

Kaposis sarcoma

A tumour of endothelial

cell origin that is found

most frequently in

immunosuppressed patients,

particularly individuals with HIV.

Kaposis sarcoma-associated

herpesvirus has been

implicated as a cofactor in

the development of Kaposis

sarcoma.

Oncogenic stress sensed by theimmune system: role of natural killercell receptorsDavid H. Raulet and Nadia Guerra

Abstract | A growing body of research is addressing how pathways that are dysregulated

during tumorigenesis are linked to innate immune responses, which can contribute to

immune surveillance of cancer. Components of the innate immune system that are localizedin tissues are thought to eliminate early neoplastic cells, thereby preventing or delaying the

establishment of advanced tumours. This Review addresses our current understanding of

the mechanisms that detect cellular stresses that are associated with tumorigenesis and

that culminate in the recognition and, in some cases, the elimination of the tumour cells by

natural killer cells and other lymphocytes that express natural killer cell receptors.

R E V I E W S

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Normal tissue cell

Multi-step process of tumorigenesis

Proliferative signals Replication stress,

DNA damage, DNAdamage response,(ATM, ATR, CHK)

p19ARF activation

DNA damage response, activation ofp53 and other tumour suppressors:

Cell-intrinsic barriers: Cell cycle arrest Senescence Apoptosis

Cell-extrinsic barriers: Immune receptor ligands Immune elimination

Mutations that supersedebarriers and optimize fitnessof malignant cells: cancer

Mutations in gatekeeper andother key tumour suppressorsand oncogenes (including RAS, MYC)

Mutations and loss ofheterozygosity in keytumour suppressors (such as p53)that regulate the cell cycle

Mutations inregulators of othercellular processes

Immune receptor

ligand

Malignant cancer

Apoptoticcancer cell

Early cancerPre-cancerous lesion

that ndergo rearrangement. Each NK ce expressessevera stimatory and inhibitory NK ce receptors thatcan fnction ith some independence, enabing NK cesto separatey target ces that increase or decrease theirexpression of varios igands6. Many of the NK ce inhibi-tory receptors are specific for MHC cass I moeces,hich are expressed by norma ces bt are often ost from

infected ces or tmor ces7. ligands for NK ce stim-atory receptors are say poory expressed by heathyces bt are pregated by nheathy ces, sch as trans-formed, infected or stressed ces6,7. NK ce activation iscontroed by the baance of stimatory and inhibitorysignaing incrred hen target ce engagement occrsand the varios receptors engage their igands68. Hence,some norma ces dispay stimatory igands bt fai tobe kied by NK ces becase their MHC cass I moecesengage inhibitory receptors that conteract stimatorysignaing. Increased expression of stimatory igands bya target ce can overcome inhibitory signaing in the NKce, resting in target ce ysis. loss of inhibitory MHC

igands can aso rest in target ce ysis. Both occr in thecontext of disease, sometimes in the same ce8. In additionto their roe in NK ces, some stimatory NK ce recep-tors are aso commony expressed by sbsets of T ces, inhich they are thoght to provide an innate signa thatenhances T ce activation9.

Recent evidence sggests that the host immne

response to tmor ces is in some cases inked to spe-cific events that are associated ith cear transforma-tion and tmorigenesis. In this Revie, e focs on themechanisms that ink oncogenic stress to innate immnestimi, in particar the stimi that act throgh NK cereceptors and the ces that express them.

Immune surveillance of primary tumours

Immne srveiance of cancer is a concept that as dis-credited for some time, bt has received strong experi-menta spport in the past 10 years (TABLE 1). Beforerevieing the evidence, it is sef to smmarize theexperimenta systems sed in sch stdies.

Figure 1 | Stewise cacer rressi ad itrisic ad extrisic barriers t cacer. On the basis of

histopathological, clinical and molecular data generated from the analysis of colon carcinomas, it was proposed that

cancer generally develops in a stepwise manner as depicted in a Vogelgram diagram which is the basis of the figure2,145.

Although the order of certain specific events probably varies in different instances of cancer, certain early events,

including oncogene activation, result in DNA replication stress and DNA damage and therefore activation of the DNA

damage response. Oncogene activation leads to the induction of p19ARF expression by a distinct mechanism.

Independently of each other, the DNA damage response and activated p19ARF activate key tumour suppressors such as

p53. Depending on numerous factors, activated p53 results in cell cycle arrest, cell senescence or apoptosis, all of which

are intrinsic barriers to tumorigenesis. The DNA damage response also induces the expression of ligands for the receptor

natural killer group 2, member D (NKG2D), and probably other immune receptors, which can activate extrinsic antitumour

immune responses. Similarly, cell senescence induced by p53 triggers an immune response that eliminates the senescent

cells, although the specific receptors involved in these mechanisms have not yet been defined. Because these barrierstypically arise downstream of oncogene activation, selection for homozygous p53 mutations is usually delayed relative to

oncogene activation and often correlates with a transition to malignancy. Subsequently, the tumour undergoes additional

evolution that optimizes its fitness and capacity to metastasize. ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3

related; CHK, checkpoint kinases.

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Many past stdies of the immne systems roe incancer have reied on tmor transpant modes, typi-cay sing tmor ce ines that are impanted sbc-taneosy in recipient animas. Athogh this approachis sef, it is faed in that impanted tmors deveopfrom ce ines that ceary escaped immne srvei-ance in the anima from hich they ere derived andcan aso be aberrant oing to proonged ctring.Frthermore, they are say impanted in ectopicsites in arge nmbers and fai to recapitate the eariest

stages of transformation and tmorigenesis. As a rest,the physioogica reevance of observations shoingimmne destrction of impanted tmors is oftenqestionabe. Aternative approaches incde stdiesof fy spontaneos cancer, cancer indced by trans-genic expression of oncogenes and carcinogen-indcedcancer (TABLE 1). These approaches are considered morereiabe for identifying the roe of the immne systembecase the tmors arise in the norma tisse site, typi-cay from a singe ce, and proceed throgh the varios

Table 1 | Ie deficiecies assciated with reater tr icidece r seerit i ice

mse strai Sste t rtetrieesis

Te f tr Defectie iecet

Refs

Spontaneous tumours

129/Sv None Colon and lung RAG2 10

129/Sv None Colon and mammary RAG2 and STAT1 10

C57BL/6 and BALB/c None B cell lymphoma 2-microglobulin andperforin

11,14

C57BL/6 None Lymphoma TRAIL 13

C57BL/6 None Lymphoma Perforin 12

Transgenic and knockout cancer models

129/Sv Tp53/ Lymphoid and other STAT1 22

129/Sv Tp53/ Lymphoid and other IFNR 22

C57BL/6 Tp53+/ Lymphoid and other TRAIL 13

C57BL/6 Tp53+/ Lymphoid Perforin* 12

C57BL/6 Tp53+/ Lymphoid and other TCRJ28 and CD1d 23

C57BL/6 TRAMP Prostate NKG2D 18

C57BL/6 TRAMP Prostate TCR 17

C57BL/6 EMyc B cell lymphoma NKG2D 18

C57BL/6 EMyc B cell lymphoma TRAILR 28

C57BL/6 EMyc B cell lymphoma RAG1 19

Carcinogen-induced tumours

129/Sv MCA Fibrosarcoma RAG2 10

129/Sv MCA Fibrosarcoma IFNR 10,22

129/Sv MCA Fibrosarcoma IFNR 27

129/Sv MCA Fibrosarcoma STAT1 10,22

129/Sv MCA Fibrosarcoma RAG2 and STAT1 10

C57BL/6 MCA Fibrosarcoma IFN 26,31

C57BL/6 MCA Fibrosarcoma Perforin 25,26

C57BL/6 MCA Fibrosarcoma TRAIL 29

C57BL/6 DEN Hepatocarcinoma TRAILR 28

C57BL/6 MCA Fibrosarcoma TCRJ28 26

FVB MCA Fibrosarcoma TCR 30

FVB MCA Fibrosarcoma TCR 30

FVB DMBA and TPA Cutaneous TCR 30,35

C57BL/6 MCA Fibrosarcoma TCR 31

*Contrary results have been observed in a mixed 129/Sv x C57BL/6 genetic background 27. Contrary results have been observed inRAG1-deficient C57BL/6 mice24. Contrary results have been observed24. DEN, diethylnitrosamine; DMBA, dimethylbenz(a)anthracene; IFNR, interferon- receptor; MCA, methylcolanthrene; NKG2D, natural killer group 2, member D; RAG, recombination-activating gene; STAT1, signal transducer and activator of transcription 1; TCR, T cell receptor; TPA, 12- O-tetradecanoyl phorbol13-acetate; TRAILR, TNF-related apoptosis-inducing ligand receptor; TRAMP, transgenic adenocarcinoma of the mouse prostate.

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Recombination-activating

gene 2

(Rag2). A gene encoding a

protein that mediates V(D)J

recombination in preB cells

and thymocytes, which is

necessary for the production

of B and T cell receptors, and

thus for the development of

B and T cells.

Perforin

A component of the cytolytic

granules of cytotoxic T cells

and natural killer cells that

participates in the

permeabilization of plasma

membranes, allowing

granzymes and other cytotoxic

components to enter target

cells.

Large T antigen

A multifunctional protein

product of the simian virus 40

(SV40) early region that is

necessary to establish a

permissive host cell

environment for viral

replication by interactions with

host proteins. Large T antigen

binds and functionally

inactivates the tumour

suppressor proteins

retinoblastoma and p53.

T cell

A T cell that expresses a T cell

receptor consisting of a -chain

and a -chain. T cells are

present in several epithelial

locations as intraepithelial

lymphocytes (IELs) and in

lymphoid organs. Although the

functions of T cells (or IELs)

are still mostly unknown, it has

been suggested that mucosal

T cells mediate innate-type

mucosal immune responses,

and epidermal T cells in

mice have been implicated in

tumour surveillance and wound

repair.

Natural killer T (NKT) cell

A subpopulation of T cells that

expresses both NK cell and

T cell markers. In the C57BL/6

mouse strain, NKT cells

express the NK1.1 (NKRP1C)

molecule and the T cell

receptor (TCR). Some NKT cells

recognize CD1d-associated

lipid antigens and express a

restricted repertoire of TCRs

(invariant NKT cells). After TCR

stimulation of naive cells,

NKT cells rapidly produce

interleukin-4 and interferon-.

stages of tmor deveopment. Nevertheess, they varyin their ease of se and have other shortcomings, assmmarized beo.

Fully spontaneous tumorigenesis. Cancer that deveopsin norma ntreated mice is presmaby the most reiabemose mode of hman cancer and has been stdied insome anayses of immne srveiance. Hoever, thesestdies have imitations becase of the o incidence offy spontaneos cancers in most mose strains and theheterogeneity of cancers that arise.

Despite these difficties, a key report shoedincreased incidence of spontaneos ng and intestinacarcinomas in 129/Sv mice that acked a B and T cesoing to mtations in recombination-activating gene 2(Rag2)10. Interestingy, mammary carcinomas ere notdetected in RAG2-deficient mice, bt arose in micedeficient in both RAG2 and signa transdcer and acti-

vator of transcription 1 (STAT1), a component of theinterferon (IFN) receptor signaing compex, sggest-ing a roe for innate immne responses in the contro

of mammary tmors. A genera and robst roe forperforin in immne srveiance of cancer as shon bythe deetion of the gene encoding perforin in to mosestrains11,12,14. TNF-reated apoptosis-indcing igand(TRAIl; aso knon as TNFRSF10), another mediatorof cytoysis, has aso been impicated in responses againstcancer13. These stdies sggest a roe for both the innateand adaptive immne responses in the contro of fyspontaneos tmors.

Transgenic models of spontaneous cancer. Geneticayengineered mice that overexpress oncogenes or haveatered tmor sppressor fnction deveop sponta-neos cancers that mimic the deveopment of natramaignancies in most respects. The tmors in thesemice arise and progress atochtonosy (at their natrasites), incding dring the eariest stages, and are ikeyto refect the natra interactions beteen the tmorand the immne system in initiay norma tisses.In most of these modes, most or a animas deveoptmors of the same type, and in some cases tmorsdeveop ith predictabe kinetics, aoing systematicanaysis of the varios stages of cancer. A draback ofsome of these modes is their high penetrance, sch thattmors are repeatedy initiated in the same host andcan overhem the immne response. A fe reevantmodes i be smmarized here.

In the transgenic adenocarcinoma of the mose pros-tate (TRAMP) mode, the rat probasin promoter directsthe expression of the eary genes (sma t and large T anti-gens) of simian virs 40 (SV40) by the prostate epithe-im of adt mice. Prostate tmors deveoping in thesemice are infitrated by ekocytes, incding CD8+ T cesthat are specific for tmor-associated antigens15,16. Otherce types that infitrate the tmors incde NK ces, T cells and natural killer T(NKT) cells (P. Savage, per-sona commnication). One stdy recenty shoed that T ces cod sppress high-grade prostate tmors inthe TRAMP mode17, and another recent stdy shoedthat the immnoreceptor NK group 2, member D (NKG2D)

contribted to the contro of the high-grade, aggressiveform of carcinomas that deveop in some of these mice18.As discssed ater, NKG2D is a stimatory receptorexpressed by NK ces and some T ces, sggesting thatone or both of these ce types is invoved in the controof aggressive prostate adenocarcinomas.

The EMyc transgene mode consists of the Myconcogene expressed nder the contro of the E immno-gobin heavy chain enhancer and the Myc promoter.EMyc transgenic mice that aso ack B and T cesoing to a mtation in Rag1 sccmb to pre-B ce ym-phomas a fe eeks earier than their Rag1-sfficientittermates, sggesting that matre B or T ces mayimit the deveopment of these tmors19. Frthermore,ymphomagenesis as aso acceerated in EMyc micethat acked the gene encoding NKG2D, hich sggeststhat NKG2D has a roe in promoting either NK ce-dependent or T ce-dependent eimination of ympho-mas18. A EMyc transgenic mice eventay deveopymphomas, perhaps becase the initiation of netmors finay overhems immne system contro18,20.

Mtations of the gene encoding the tmor sppres-sor p53 (Tp53) occr spontaneosy in most natrayarising cancers. Deficiency in p53 removes an importantbarrier to tmorigenesis and contribtes to genomicinstabiity21. Mice ith homozygos germine mtationsin Tp53 (Tp53/ mice) deveop mainy ymphomas ofthymic origin, hereas Tp53+/ mice deveop both dis-seminated ymphomas and non-ymphoid tmors,mainy sarcomas. Stdies sho that tmor incidenceis higher in p53-deficient mice that are aso deficient inIFN, the IFN receptor and/or STAT1 (REF. 22). IFN-insensitive Tp53/ mice deveoped a broader range oftmors compared ith mice acking p53 aone 22. Inaddition, invariant NKT ces, the cassica sbset ofNKT ces, have been shon to contribte to the controof tmor deveopment in Tp53+/ mice23; both perforin-and TRAIl-mediated apoptotic pathays participate inthis response12,13.

Carcinogen models. Tmors arising as a rest of chemi-ca carcinogenesis deveop atochtonosy and pro-ceed throgh the stages of spontaneos tmorigenesis.Carcinogen-based modes, athogh sef, have beencriticized becase the potent chemicas may indce ocatisse infammation that cod ater experimenta ot-comes24. Hoever, an advantage of these modes com-pared ith some transgenic modes is that the carcinogen

can be titrated so that tmor incidence is imiting.Among the most commony stdied modes of mose

tmorigenesis is the indction of fibrosarcomas in micetreated sbctaneosy or intradermay ith the car-cinogen methychoanthrene (MCA). Genetic stdiesshoed an increased incidence of MCA-indced fibro-sarcomas in mice ith defects in perforin25,26, the IFNsignaing pathay10,22,27 or the TRAIl-mediated cytotox-icity pathay28,29. Both and T ces ere impicatedin the kiing of MCA-indced fibrosarcomas30,31. Amongces ith an T ce receptor, NKT ces32 and possi-by CD4+ T ces10 ere fond to be most important. Inaddition, NK ces have a crcia roe as direct effectors

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NK group 2, member D

(NKG2D). A lectin-type

activating receptor encoded by

killer cell lectin-like receptor

subfamily K, member 1 (Klrk1)

located in the natural killer cell

gene complex. NKG2D

associates with signalling

adaptor molecules, including

DAP10 (in both humans and

mice) and DAP12 (in mice but

not humans). DAP10 activates

phosphoinositide 3-kinase, and

its signalling mechanism

resembles that of

co-stimulatory receptors, such

as CD28. By contrast, DAP12

activates spleen tyrosine

kinase, and its signalling

resembles that of B and T cell

receptors.

of tmor ce ysis and possiby as coaborators ithinvariant NKT ces in the contro of these tmors 33,34.By contrast, CD8-deficient mice exhibited no defect inthe contro of MCA-indced tmors25.

Another idey sed carcinogen mode is the indc-tion of skin cancer invoving the tmor initiator andpromoter combination dimethybenz(a)anthracene(DMBA) and 12-O-tetradecanoy phorbo 13-acetate(TPA). Mice deficient in skin-associated T ces hada higher incidence of DMBATPA-indced tmors30.By contrast, CD8+ T ces cod promote tmorigenesisin this mode nder some conditions35.

Receptors mediating immune surveillance

As revieed esehere36, evidence spports a roe for theadaptive immne system in immne srveiance, indicat-ing a roe for B or T ce receptors. Here e focs on recep-tors sed by NK ces and in some cases shared by T cesthat have defined antigen specificities and have beenimpicated in tmor srveiance. Among these receptorsare NKG2D, NKp30, NKp44, NKp46, NKp80, 2B4 and

DNAX accessory moece 1 (DNAM1; aso knon asCD226). Severa of the receptors seem to mediate indcedsef recognition; that is, the igands are encoded by thehosts genome, are poory expressed by norma ces andare pregated by stressed or diseased ces8. This phe-nomenon is best characterized in the case of the NKG2Dreceptor. For this reason, and becase its roe in tmorsrveiance has been examined extensivey, e focs ordiscssion on NKG2D and its igands, after introdcingsome of the other key NK ce receptors.

NKp30, NKp44, NKp46 and NKp80.These for stim-atory receptors are coectivey caed natra cyto-toxicity receptors (NCRs)3740, athogh NKG2D andother receptors aso have important roes in the recogni-tion and cytotoxicity of tmor ces by NK ces, as dis-cssed beo. The for receptors have been e stdiedin hman NK ces, bt ony one of them, NKp46, hasbeen characterized in mice. In hmans, NKp30, NKp46and NKp80 are expressed by a NK ces, hereas NKp44is ony expressed by activated NK ces41,42. Bocking oneor more of these receptors ith antibodies often inhibitskiing of tmor ce ines by hman NK ces in vitro3739.

Frthermore, mice ith a targeted mtation in the geneencoding NKp46 ere impaired in their abiity to reject atransferred ymphoma ce ine that expressed igands forthat receptor43.

A srprising diversity of nreated igands has beenreported for NCRs, incding vira haemaggtinins(for NKp46 and NKp44)44,45, heparan sphate proteo-gycans (for NKp30 and NKp46)46, the ncear fac-tor HlA-B-associated transcript 3 (for NKp30)47 andactivation-indced C-type ectin (for NKp80)48.Additiona stdies i be needed to determine theimportance of these varios igands for NCRs.

2B4.2B4 is a member of the signaing ymphocyte acti-vation moece (SlAM)-reated famiy of receptors andis expressed by a NK ces, T ces, a sbset of CD8 +T ces and a hman CD14+ monocytes49. The niqeigand for 2B4 is CD48 (aso a SlAM-reated receptor),hich is expressed by a haematopoietic ces. 2B4 canfnction as either a stimatory or inhibitory receptordepending on the spice isoform that is expressed, the

identity of the signaing adaptor moece it associatesith and the extent of cross-inking of the receptor5052.2B4 signaing has been shon to have a roe in rejectingtmors that express CD48 (REF. 53).

DNAM1. DNAM1 is an adhesion moece that isconstittivey expressed by most NK ces, T ces, macro-phages and dendritic ces54,55. ligands for DNAM1incde CD112 (aso knon as nectin 2 and PVRl2) andCD155 (aso knon as PVR)56,57. These igands are oftenexpressed by tmor ces and can activate or enhancetmor ce ysis in vitro58,59. Recent stdies shoedthat DNAM1-deficient mice have redced capacity toreject certain tmor ces and to imit the formation ofcarcinogen-indced tmors in vivo34,60.

NKG2D.NKG2D is a ectin-ike type II transmembranehomodimer that has received considerabe attentionoing to evidence of its roe in immne responses in thecontext of cancer, infection and atoimmnity. NKG2Dis expressed by virtay a NK ces and activated CD8+T ces, and sbsets of T ces and NKT ces61,62. Incertain conditions, NKG2D is aso expressed by hmanCD4+ T ces6367.

Nmeros different NKG2D igands have been iden-tified, a of hich are reated sef proteins that are simi-ar to MHC cass I moeces (BOX 1). Hoever, norma

ces typicay do not express the igands at sbstantiaeves, hereas they are often specificay pregated incanceros or stressed tisses (TABLE 2).

Evidence has accmated shoing that NKG2Dhas an important roe in the immne srveiance oftmors (FIG. 2). NKG2D-dependent eiminationof tmor ces that express NKG2D igands hasbeen e docmented in vitro39,62,68,69 and in vivo intmor transpant experiments70,71. In hmans, specificNKG2D gene poymorphisms have been associated ithssceptibiity to cancer72.

The most direct evidence spporting a roe forNKG2D in tmor srveiance came from anaysis

Box 1 | The iads fr nKg2D

Natural killer group 2, member D (NKG2D) ligands are self proteins that are related toMHC class I molecules, although they differ in that they do not present molecular cargo

and fail to bind 2-microglobulin133. The ligands include MHC class I polypeptide-related

sequence A (MICA) and MICB in humans62, which have no mouse homologues, and the

cytomegalovirus UL16-binding protein (ULBP) or retinoic acid early transcript 1 (RAET1)

ligand families, which exist in both humans and mice134137. Each human or mouse strain

can express approximately 510 different NKG2D ligands. Most normal cells, however,

do not express substantial levels of NKG2D ligands on the cell surface. By contrast,

most tumour cell lines express one or more NKG2D ligand39,62,135,136. Furthermore, many

primary human tumours express NKG2D ligands117,138,139. Similarly, expression of the

NKG2D ligands RAET1 or murine ULBP-like transcript 1 (MULT1) was observed on

primary lymphomas generated in EMycmice18,140 and on primary adenocarcinomas

generated in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice18. Ligand

expression is also induced in cells that are infected with certain pathogens141.

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of tmor incidence in gene-targeted mice that ackNKG2D and carry transgenes that increase the incidenceof specific cancers18. In the TRAMP mose mode, the

incidence of a highy aggressive form of prostate adeno-carcinoma as markedy increased hen the mice ereaso deficient for NKG2D18. Simiary, in mice carryingthe EMyc transgene that cases B ce ymphoma,onset of ymphoma as acceerated by 7 eeks if themice ere aso deficient for NKG2D18. It has not yetbeen estabished hether NKG2D-dependent controof tmors in these modes is mediated by NK ces orone or more type of NKG2D-expressing T ce, or byboth NK ces and T ces.

The invovement of NKG2D in tmor srveiancein the TRAMP mose mode as aso sggested by thefinding that many of the aggressive adenocarcinomasthat arose in NKG2D-deficient mice expressed one ormore of the NKG2D igands, hereas simiar tmorsthat arose in NKG2D-sfficient mice generay ackedexpression of NKG2D igands. A probabe expanationis that aggressive prostate adenocarcinoma tmorsthat arose in NKG2D-sfficient mice ere sbjected toNKG2D-mediated immne srveiance, resting inseection for variant tmor ces that had ost expres-sion of the igands (FIG. 2). In contrast to this pattern inTRAMP mice, the B ce ymphomas that arose in EMycmice commony expressed NKG2D igands regardess ofhether the mice ere NKG2D deficient. These datasggested that some tmors can escape cytotoxicitymediated by NKG2D-expressing ces despite contined

expression of NKG2D igands. This is consistent iththe observation that many primary tmors in normaanimas or hmans express NKG2D igands.

A roe for NKG2D in the srveiance of skin canceras sggested by the increased expression of transcriptsfor NKG2D igands in carcinogen-treated skin sam-pes30. Interestingy, transgenic mice that constittiveyexpressed high eves of NKG2D igands, hich restsin dampened NKG2D fnction, shoed an increasedincidence of carcinogen-indced ctaneos maignan-cies73. Hoever, these data did not definitey impicateNKG2D in ctaneos immne srveiance becasethe NK ces in these transgenic mice ere defective at

eiminating tmor ces that acked NKG2D igands inaddition to those ces that expressed NKG2D igands.

NKG2D deficiency did not rest in the increased

incidence or severity of some types of cancer, incdinga ate arising, ess maignant form of adenocarcinomain TRAMP mice or fibrosarcomas indced by MCA18.The observation that NKG2D deficiency did not restin more MCA-indced fibrosarcomas as srprising inight of the contrasting findings of an earier stdyin hich MCA-treated mice ere repeatedy injected ithan NKG2D-specific antibody to bock the receptor74. It ispossibe that sstained engagement of NKG2D by anti-bodies impairs both NKG2D-dependent and NKG2D-independent NK ce fnctions, as has been reported forsstained engagement of NKG2D by its igands73,7577. Inany case, the observation that the incidence or sever-ity of tmors as naffected by NKG2D deficiency insome modes sggests that these types of tmor readiyevade NKG2D-dependent immne srveiance, fai toexpress NKG2D igands at a sfficienty eary stage or areotherise seqestered or insensitive to immne destrc-tion. Possibe mechanisms of immne evasion incdepregation of inhibitory MHC cass I moeces toconteract the higher eves of stimatory igandsand/or oss of distinct stimatory or adhesion igands bythe tmor ces. By contrast, the receptor DNAM1 hasbeen impicated in immne srveiance of MCA-indcedsarcomas, sggesting that its action is not readiy evadedin this system34.

Considering the capacity for cancers to evove in the

host and the heterogeneity of oncogenic mechanismsthat operate in different cancer modes, it is not srpris-ing that the NKG2D system, or indeed any other system,is ineffective at impeding disease in some cancer modes.A detaied expanation of hy NKG2D is ineffective inthese specific modes has not yet been docmented, btit is notabe that in some systems NKG2D igands areexpressed on the srface of tmor ces bt fai to pro-mote tmor rejection, hereas in other cases NKG2Digands are ost from the tmor ces. In the first caseit is probabe that the response of NKG2D+ ymphocytesis sppressed or avoided, hereas in the second casetmor variants that ack expression of igands may arise

Table 2 | Idcti f nKg2D iads b stress athwas

lee f reati uderi athwa liads reated Reatr cets Refs

mRNA (transcriptionor mRNA stabilization)

Heat shock response MICA and MICB(in humans)

Heat shock transcription factors 96


DNA damageresponse

ULBP, MICA (in humans);RAET1, MULT1 and H60a(in mice)

ATR-, ATM- andCHK-dependent; p53 notrequired

90


Cell senescence MICA, MICB and ULBP2(in humans)

ATR- and ATM-dependent insome cases

57,93


Wounding H60c (in mice) ND 105

Protein stabilization Heat shock response;UV irradiation

MULT1 (in mice) Independent of DNA damageresponse

97

ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3 related; CHK, checkpoint kinase; H60, histocompatibility 60; MICA,MHC class I polypeptide-related sequence A; MULT1, murine ULBP-like transcript 1; ND, not determined; RAET1, retinoic acidearly transcript 1; ULBP, cytomegalovirus UL16-binding protein; UV, ultraviolet.

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Aggressiveprostatetumour cell

Less aggressiveprostate tumour cell

Aggressive tumour

Escape (occurs ina fraction of cases)

Less aggressive, late developing tumours

Normal tissue Normal tissue

Initial expansion; rare loss of NKG2D ligands

Potential escape mechanisms Downregulation of NKG2Dand/or Loss of ligands for activating receptorsother than NKG2D

Upregulation of inhibitory ligands Loss of ligands for adhesion molecules Immunosuppressive environment Resistance to apoptosis

a b

NKG2Dligand

Apoptoticcancer cell

NKG2D-mediated elimination of early tumours

Tumorigenic events, NKG2D ligand induction

Resistance to NKG2D-mediated elimination

readiy. why some tmors may ose igand expressionmore readiy than others is nknon, bt this cod bereated to the fact that the miie of activated oncogenes,mtated tmor sppressors and other mediators variesin tmors of different origins.

Cancer-associated immune activation

Tmorigenesis is a compex process invoving the dysreg-ation of many cear pathays, in many cases rest-ing from mtations in oncogenes and tmor sppressorgenes. Recognition of cancer ces by innate immne cesdepends on their abiity to distingish these dysregatedces from norma ces. Many of the distinctive featres of

cancer ces, sch as proiferation, invasiveness and repres-sion of ce death pathays are featres that can be exhib-ited by norma ces in other contexts. Other featres aremore specific to diseased ces, athogh ntransformedstressed ces may aso be simiary affected. For a hostdefence system to be effective against cancer, the mecha-nism in the cancer ce that aerts the immne system mostprobaby invoves signaing pathays that process severatypes of information that coectivey, bt not individay,identify the ce as a cancer ce.

There are nmeros featres that distingish cancerces from most norma ces. To mention ony a fe,mtations that activate oncogenes probaby occr eary

Figure 2 | mde f nKg2D-ediated tr sreiace f rstate adecarcia. In C57BL/6 transgenic

adenocarcinoma of the mouse prostate (TRAMP) mice, immune responses depending on natural killer group 2,

member D (NKG2D) limit the development of an early highly malignant form of prostate adenocarcinoma, but not

a late developing, less malignant form. a | The highly malignant tumours in NKG2D-deficient TRAMP mice generally

express NKG2D ligands, whereas the rarer tumours of this type in NKG2D-sufficient mice generally lack NKG2D

ligands, suggesting that the immune response modifies these tumours by selecting for variant tumour cells that fail

to express NKG2D ligands. b | By contrast, the less aggressive, late developing tumours express NKG2D ligands (albeit

heterogeneously) regardless of whether NKG2D is expressed, suggesting that these tumours evade NKG2D-dependent

elimination by a distinct mechanism.

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Unfolded protein response

A response that increases the

ability of the endoplasmic

reticulum to fold and

translocate proteins,

decreases the synthesis of

proteins and causes cellcycle arrest and apoptosis.

Ataxia telangiectasia

(also known as LouisBar

syndrome). A familial recessive

disease that is characterized by

progressive cerebellar ataxia,

oculocutaneous telangiectases

and susceptibility to

pulmonary infections. It is

caused by germline mutations

in ataxia telangiectasia

mutated(ATM), which encodes

a sensor that activates the

DNA damage response.

in the deveopment of most tmors and provide per-sistent proiferative signas. Oncogene-indced signasactivate tmor sppressors by at east to mechanisms,one invoving p19ARF (encoded by cycin-dependentkinase inhibitor 2A (CDKN2A)) and the other invov-ing the DNA damage response, hich is activated after

DNA damage is sensed by the protein kinases ataxia te-angiectasia mtated (ATM) and ATM and Rad3 reated(ATR) (BOX 2). Activated p19ARF and the DNA damageresponse can each independenty indce the expressionof p53 and other mediators that arrest the ce cyce andcan ead to ce senescence (BOX 3) or apoptosis henpersistenty activated78,79.

Mtations in tmor sppressor genes, or in compo-nents of the pathays that activate tmor sppressors,enabe contined ce proiferation of nascent tmorces bt dysregate DNA repication and repair ina manner that timatey rests in instabiity of thegenome and the accmation of chromosoma abnor-maities80,81. Hence, at eary stages of tmorigenesis,ces may have eary arning signs sch as activationof p19ARF, the DNA damage response and tmorsppressors, and activation of the gene programmethat is associated ith ce senescence. At ate stagesthe ces have other defects sch as genomic instabiity.Frthermore, certain other stress pathays are com-mony activated in cancer ces, incding the heat shockresponse82 and the unfolded protein response83. The roesof some of these pathays as arning signas that triggeranticancer immne responses are discssed beo.

DNA damage response. The roe of the DNA damageresponse as an important barrier to tmorigenesis as

highighted by stdies of hman tisses shoing thateary pre-neopastic esions in the breasts, ngs, bad-der and coon of patients have chronic activation of theDNA damage response, manifested by the phosphory-ation of ATM, checkpoint kinase 2 homooge (CHK2)and histone -H2AX, another marker of DNA damageresponse activation84,85(FIG. 1). Other stdies shoedthat transgenic expression of the proto-oncogeneMycin mice eads to DNA damage, conseqent activation ofATM and p53 and apoptosis of affected ces in vivo86,87.DNA damage indced by MYC may rest from the pro-dction of increased reactive oxygen species, independ-enty of ce cyce entry, and from the indction of rapid

DNA repication88. ATM activation nder these condi-tions obstrcts tmorigenesis, as deficiency of ATMincreases MYC-indced tmorigenesis in mice86,87.These data are consistent ith the fact that mtationsinATMin hmans, hich case the syndrome ataxiatelangiectasia hen present in the homozygos state,increase the incidence of ymphoma, ekaemia andbreast cancer89.

A ink beteen the DNA damage response andantitmor immne responses as initiay estab-ished in stdies of NKG2D igands 90 (TABLE 2).Genotoxic stress that activates ATM or ATR canindce the expression of NKG2D igands (retinoicacid eary transcript 1 (RAET1), mrine ulBP-iketranscript 1 (MulT1), histocompatibiity 60a (H60a),MHC cass I poypeptide-reated seqence A (MICA)and cytomegaovirs ul16-binding proteins (ulBPs))on the srface of reativey norma ctred ces, incd-ing fibrobast ce ctres8,90. Indction of NKG2D ig-and expression as prevented by inhibiting or knockingdon the expression of ATR or ATM, depending on

the natre of the genotoxic stress8,90. Most estabishedtmor ce ines express NKG2D igands constittivey,and knockdon stdies estabished that constittiveATM or ATR activation had an important roe in main-taining constittive igand expression by those ces8,90.These stdies indicated that the dispay of NKG2D ig-ands on tmor ces is mediated in part by an activatedDNA damage response. This pregation of NKG2Digand expression is accompanied by increased eves ofthe corresponding mRNA transcripts, bt it has not beendetermined hether this is de to increased transcriptionor aterations in mRNA processing.

The DNA damage response aso has a roe in indc-ing the expression of igands for a distinct NK ce recep-tor, as indicated by a stdy shoing that the DNAM1igand CD155 as indced by DNA-damaging drgson the srface of mtipe myeoma ces in an ATM-and ATR-dependent manner57. Finay, the DNA dam-age response directy regates the expression of deathreceptor 5 (DR5), a igand for TRAIl91. Engagement ofDR5 by TRAIl indces apoptosis, and evidence indi-cates that TRAIl is expressed by NK ces and T ces andfnctions as an important effector moece in tmorsrveiance by these ces28,92.

Box 3 | Ce seescece

Cell senescence, in which tumour cells can survive for a

time in an irreversibly senescent state, is associated with

a blockade in cell proliferation and an induction of a

specific programme of gene expression that results in

the secretion of several pro-inflammatory cytokines and

chemokines. Paradoxically, many of these modulators

promote tumour cell growth, and studies suggest that

the senescent state can, in some cases, promote

tumorigenesis143. However, abundant evidence has

accumulated that suggests senescence restricts

tumorigenesis78,79,144 by inhibiting tumour cell growth,

inducing cell death and activating immune responses

that help to eliminate cancer cells.

Box 2 | DnA daae resse

The DNA damage response protects the genome by facilitating the repair of minor

DNA damage in cells and functions as a key barrier to tumorigenesis (see alsoFIG. 1).

Rapid DNA replication in the context of oncogene activation is thought to result in

the disruption of DNA replication forks (replication stress) and accompanying DNA

breaks, both of which can trigger the DNA damage response by activating the

protein kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR),

which are key sensors in the DNA damage response pathway142

. ATM and ATR initiatea cascade that ultimately induces cell cycle arrest and DNA repair functions.

Depending on the cell type and other factors, prolonged or severe activation of the

DNA damage response results in the activation of apoptotic or cell senescence

programmes. The DNA damage response therefore helps to preserve the integrity of

the genome and eliminate severely damaged cells.

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Cell senescence. Ce senescence has been inked toimmne-mediated tmor eimination mechanisms bya stdy of iver tmors generated from transformedces that initiay acked p53 expression. when p53expression as sbseqenty sitched on, the tmorsnderent groth arrest, exhibited featres of cesenescence and ere graday eiminated by NK cesand other infitrating ces78. These stdies sggested thatce senescence associated ith p53 activation in tmorces is in some cases connected ith the activation ofimmne responses that destroy cancer ces or theirpre-maignant conterparts.

The detais of the ay the senescence programmeis inked to anticancer immne responses have not yetbeen determined, bt stdies sggest that nmerosigands that stimate immne responses are pregatedin senescent ces. These incde intercear adhesionmoece 1 (ICAM1), a igand for ymphocyte fnction-associated antigen 1 (lFA1), hich has an important roein NK ce activation, NKG2D igands sch as MICA andulBP2, and CD155 (REFS 78,93)(TABLE 2). As aready noted

above, the expression of some of these igands is indcedby the DNA damage response. So, it remains ncearhether the indction of these immne-stimatingigands is reated to the senescence programme itsef,hich normay takes severa days to estabish, or theDNA damage response, hich may be invoved in estab-ishing the senescence programme. Notaby, the indctionof NKG2D igand expression in ctred ces by DNA-damaging agents did not reqire p53 (REF. 90), hereastmor senescence and NK ce-dependent eiminationof senescent tmor ces as indced by p53 reactiva-tion in vivo78. Therefore, it seems probabe that bothp53-dependent and p53-independent processes inkedto tmorigenesis might regate the sensitivity of tmorces to NK ce-mediated tmor eimination in vivo.

The heat shock response. It has ong been knonthat the heat shock response is activated in many formsof cancer82. Mice deficient in heat shock transcriptionfactor 1 (HSF1), hich fnctions as a key indcer ofthe heat shock response, ere ess ssceptibe to cancerthan id-type mice in experimenta modes, sggestingthat the heat shock response is hijacked by tmors toenhance their srviva94. Monting an immne responseagainst ces expressing heat shock proteins cod thereforetarget a response that is otherise beneficia to tmors,ths imiting tmorigenesis.

Stdies in ctred hman epitheia ces impicatedthe heat shock response in transcriptiona activationof the hmanMICA andMICB genes95(TABLE 2). Thepromoter regions of both genes contain HSF1 bind-ing eements, hich ere necessary for transcriptionaactivation96. Distinct eements in the promoters erereqired to spport transcription in virs-infectedces or proiferating ces, sggesting that there assome independence in the contro ofMICA andMICBtranscription nder different conditions.

So far, heat shock-indced transcriptiona activationhas not been observed in the case of other NKG2D igandfamiies, incding hman ulBP and RAET1 proteins

and mose RAET1, MulT1 and H60 proteins. In thecase of MulT1, hoever, the heat shock response hasan important roe in regating ce srface expressionof the protein at a post-transcriptiona step (TABLE 2). Infibrobasts and other ces, the cytopasmic tai of MulT1is sbject to biqityation, hich targets the protein fordestrction in ysosomes97. Exposre of these ces to heatshock reversed the biqitin-dependent destrction ofMulT1, resting in a marked increase in protein eves atthe ce srface. Interestingy, travioet (uV) irradiationof fibrobasts had the same effect97. Athogh uV irradia-tion indces the DNA damage response, the stabiizationof MulT1 protein by uV irradiation as independent ofthe DNA damage response. uV irradiation aters severaother pathays in ces, and it is possibe that its effectson MulT1 stabiization are exerted throgh a pathaythat overaps ith the heat shock response.

MulT1 expression provides an exampe of combi-natoria regation by distinct cancer-associated stresspathays, the DNA damage response90 and the heatshock response97. Coping MulT1 expression ith

mtipe stress pathays, in this case operating at differ-ent stages of biogenesis of the moece, may representa paradigm for mechanisms that restrict the dispay ofimmne-activating igands to seriosy diseased ces.

Other mechanisms that regulate NKG2D ligands.Evidence sggests there are additiona modes of reg-ation of stimatory igands that activate NK ces. InF9 embryocarcinoma ces, the transcription ofRaet1genes as indced by retinoic acid98, hich as the basisfor the first identification of these genes. Retinoids exertgroth sppressive effects on norma ces and tmorces and have been considered promising agents for can-cer therapy99. Hoever, the roe of retinoids in regatingNKG2D igands remains ncear. The transcription ofRaet1e as inhibited by the transcription factor JuNB ince ines and mose tisses100. This finding is of particarinterest in ight of the evidence that Jn famiy transcrip-tion factors sho compex regation in conditions ofstress and injry101.

NKG2D is expressed by a T ces that reside inthe mose epidermis68. These T ces participate in thedestrction of ctaneos maignancies and in ondheaing102,103. The NKG2D igand H60c is expressedseectivey in the epidermis104,105. Engagement of H60cdispayed on keratinocytes by NKG2D on epiderma T ces is essentia for triggering T ce activation

and ysis of the keratinocytes, sggesting that NKG2Dfnctions as a key co-stimatory receptor in the activa-tion of epiderma T ces105. Interestingy, expressionof H60c is pregated in onded skin, and ctredkeratinocytes pregate H60c bt not other NKG2D ig-ands, sggesting that H60c may be the main NKG2Digand that is invoved in ctaneos immne srveianceof tmors105(TABLE 2). The mechanisms that preg-ate H60c expression in diseased and stressed tisses arenot yet knon. Athogh hman and mose ctaneosT ces differ in many respects, it is interesting that oneof the hman NKG2D igands, ulBP4, is aso seectiveyexpressed in hman skin106.

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Regation of NKG2D igands by microRNAs asreported for MICA and MICB. In hman ce ines,microRNAs that target the 3 ntransated regions of the

MICA andMICB mRNA transcripts inhibited steady stateMICA and MICB expression107. Exposre of the ces tostress ampified MICA and MICB expression to an extentthat cod overcome the inhibition that as imposed bythe microRNAs. whether these microRNAs are them-seves regated in norma tisses by stress remains tobe estabished. Hoever, it as of interest that certaintmors overexpressed the microRNAs, hich may serveas a mechanism to evade immnosrveiance107.

An additiona, important mechanism of regationof NKG2D igands is throgh shedding from the cesrface, hich has been reported for hman MICA,MICB, ulBP2 and ulBP4 (REFS 108111) bt has notbeen docmented for the mose NKG2D igands.Shedding of NKG2D igands is thoght to be mediatedby a disintegrin and metaoproteinase (ADAM) famiymetaoproteinases112114 and is assisted by endopasmicreticm protein 5 (ERP5; aso knon as PDIA6)115. The

presence of free NKG2D igands in the serm of patientsith ate-stage cancer109,110 correated ith redced evesof NKG2D at the srface of NK ces and T ces anddecreased fnction of these ces108,116,117; therefore, thiscod be a mechanism that enabes tmors to escapeimmne srveiance118. Shedding of NKG2D igands isaso seen in cases of atoimmne disease in hich thereis no evasion process66, sggesting that shedding mayoccr spontaneosy in the case of ces that expressthe igands. Hoever, it is aso possibe that the extentof shedding increases as tmors progress, perhapsrefecting seection for immne-evading variants.

Influences of the tumour microenvironment.In thetmor microenvironment, nmeros events are thoghtto have an impact on the expression and fnction ofimmnoreceptors, incding NKG2D and its igands.In principe, these infences may increase or decreaseNKG2D-dependent immne srveiance. For exampe,as noted earier, in some cases infammation associatedith tmorigenesis can increase tmor groth and sp-press protective antitmor immne responses146; it ispossibe that inhibition of NKG2D fnction is one exam-pe of ho sch inhibition occrs. It is aso conceivabethat in some tmor microenvironments NKG2D signa-ing indces a pro-tmor gene activation programme.Athogh the roes of these regatory events in promot-

ing or inhibiting antitmor immne responses remainncertain, they mst be acconted for hen consideringthe roe of NKG2D in tmor immnity.

Cytokines that are present in the tmor micro-environment are an important potentia determinantof NKG2D igand expression. RAET1 expression asdonregated fooing exposre to transforminggroth factor- (TGF)114, hich might therefore be oneof the mechanisms of immnosppression mediated bythis cytokine. Srprisingy, exposre to IFN and IFNdonregated H60a bt not RAET1 or MulT1 expres-sion by sarcoma ce ines119. In addition, the expressionof MICA and, in some cases, ulBP2 by meanoma and

gioma ce ines as donregated by IFN treat-ment120,121. These findings ere srprising becase IFNsgeneray promote antitmor immne responses.

The expression and fnction of NKG2D itsef isaso infenced by the tmor microenvironment. Pro-infammatory cytokines that are invoved in proiferationand srviva of NK ces and T ces, sch as interekin-2(Il-2) and Il-15, stimate NKG2D expression and poten-tiate the cytotoxic and IFN secretory fnction of NKces and T ces122,123. Conversey, the immnosppressivecytokine TGF, hich is secreted by severa tmors andis fond in the serm of cancer patients124, can directyindce NKG2D donregation hen secreted in thetmor microenvironment, as a membrane-bondcytokine on regatory immne ces or hen presentin tmor-derived exosomes125,126. Donregation ofNKG2D expression aso occrs in response to macro-phage migration inhibitory factor, hich is anothercytokine that favors tmor groth127.

In addition to cytokines, it is probabe that NKG2Digands expressed in the tmor microenvironment can

adversey affect NKG2D fnction. Sstained engagementof NKG2D by its igands in vivo compromises the capac-ity of NK ces to attack tmors. Persistent engagementby RAET1 (REF. 73), H60a128 and MICA75, or by sobeforms of MICA and MICB108, is knon to indce NKG2Dinternaization and sbseqent degradation. In somecases, sch persistent NKG2D engagement impairs NKce fnctions more broady, even inhibiting responses toNK ce-sensitive target ces that ack NKG2D igands73,76.These findings raise the possibiity that hen tmorgroth overhems protective responses, NKG2D andother NK ce fnctions are timatey inhibited as a restof persistent stimation.

Concluding remarks

As a rest of the signaing associated ith tmorigen-esis, many deveoping tmor ces dispay ce srfaceigands that engage activating receptors expressed byNK ces and in some cases T ces. Some of the samesignaing pathays that activate this extrinsic responseare responsibe for activating intrinsic tmor sp-pressor mechanisms sch as p53-indced apoptosisand ce senescence. Hoever, there are probaby somedifferences in the specific mediators of the to typesof response, as sggested by the finding that indction ofNKG2D igands by the DNA damage response occrs inces acking p53 (REF. 90).

As exempified by stdies of the NKG2D igandMulT1, antitmor responses that depend on NKce receptors are in some cases regated by coop-eration of distinct stress pathays that act at differenteves of biogenesis of the immne-activating igands.Combinatoria regation by distinct stress pathayspresmaby heps to prevent inappropriate responses tonorma ces. On the donside, hoever, these featresmay provide mtipe targets for mtationa inactivationof immne recognition of tmor ces.

An interesting qestion is hether the igand-indction mechanisms that are inked to tmor sp-pressor pathays evoved specificay for antitmor

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responses or hether they have a more genera roe indisease responses, sch as in responses to infections.In considering this qestion, it is often arged thatbecase cancer is predominanty a disease of oderindividas ho are presmed to be post-reprodctive,natra seection cannot directy seect for anticancermechanisms. It shod be kept in mind, hoever, thatthis argment appies eqay to intrinsic tmor sp-pressor mechanisms and immne-based tmorsppressor mechanisms. In ight of this, some of theassmptions behind this argment may be qestionedas the varios barriers to tmorigenesis presmabyork together to ensre that cancer is say deayedto a ater stage of ife.

Immne-based antitmor mechanisms have thepotentia to compement, rather than simpy sppement,tmor ce-intrinsic barriers in at east to respects.First, the triggers of the immne-based mechanisms mayoperate in cases in hich expression of intrinsic tmorsppressors is ost from tmor ces, sch as in the indc-tion of NKG2D igands by the DNA damage response that

occrs in ces that have ost the expression of the p53tmor sppressor90. Secondy, the innate immne systemresponses that are indced by these mechanisms may insome cases promote strong adaptive immne responsesthat have the capacity for the indction of immnoogicamemory and sstained systemic protection.

Athogh NK ce receptor-dependent responses totmors can be protective to the host, some tmorsmay avoid detection by donregating or sheddingigands, as sggested by stdies of NKG2D igands. Themechanisms by hich igand expression is ost are notcompetey nderstood and mst be addressed in ftrestdies. In some cases, it is probabe that oss of igandexpression rests from mtations in deveoping tmorces that inactivate the pathays hich indce igandexpression, hereas in other cases the ces may acqiremtations in the igand genes themseves. It is asoprobabe that athogh expression of igands for NK cereceptors is often protective against cancer, in some casesthe tmor can evove to expoit igand expression toprevent a protective immne response. Desensitizationof NKG2D as a rest of shedding of NKG2D igands isprobaby one exampe, bt there are ikey to be others.

A thorogh nderstanding of the pathays that areinvoved in the activation of the immne response by

tmors may enabe the design of therapetic drgs.In cases in hich tmors acqire mtations that dis-abe pathays hich indce the expression of igandsfor immne ces, a sef approach cod be to designdrgs that bypass the missing steps or re-indce ig-ands by another mechanism. It is possibe, for exampe,that the efficacy of some chemotherapy drgs sch as5-fororaci and cispatin may be reated to increas-ing NKG2D igand expression by tmor ces throghthe activation of the DNA damage response8,129. In addi-tion, proteasome inhibitors and/or histone deacetyaseinhibitors are promising candidates for enhancing theexpression of NKG2D igands130,131. Given the probabi-ity that igand expression is indced by the synergisticaction of severa signas that are associated ith thecanceros state, sch interventions may be seective intargeting tmor ces and not norma ces. In casesin hich inhibitory cytokines have a roe in sppressingigand expression on tmor ces, bockade of cytokineaction may serve as a sef therapetic intervention. Asan aternative approach, athogh NKG2D igands are

absent from some tmor ces in vivo, vaccines com-prising ces that express tmor antigens and NKG2Digands may nevertheess be effective at indcing effec-tive adaptive immne responses against tmors insome cases70,132.

Hoever, many, if not most, advanced cancers con-tine to express igands for NKG2D and other activat-ing receptors on NK ces ith impnity(FIG. 2). Tmorescape in these instances may be de to impairedfnctioning of NK ce receptors oing to inhibi-tory cytokines in the microenvironment, in hichcase cytokine bockade may be beneficia. Shedding ofigands, sch as NKG2D igands, are thoght to repressprotective immne responses, sggesting that antibod-ies that systemicay bock or remove the shed proteinsmay be of therapetic benefit111. In other cases, persistentstimation of the NK ces throgh NKG2D and possibyother activating receptors may rest in NK ce anergy.A deeper nderstanding of ho sch persistent stim-ation inactivates NK ce receptors and NK ce activitymay provide approaches to reverse the nresponsivestate of NK ces. Fina y, NKG2D igands are an invit-ing target for therapetic antibodies that are designedto eiminate igand-expressing tmor ces in cases inhich they contine to be expressed by tmor ces.

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