ITPR1 Protects Renal Cancer Cells against Natural Killer …cancerres.aacrjournals.org/content/canres/74/23/6820.full.pdf · ITPR1 Protects Renal Cancer Cells against Natural Killer

Microenvironment and Immunology

ITPR1 Protects Renal Cancer Cells against Natural KillerCells by Inducing Autophagy

Yosra Messai1, Muhammad Zaeem Noman1, Meriem Hasmim1, Bassam Janji2, Andr�es Tittarelli1,Marie Boutet1, V�eronique Baud3,4,5, Elodie Viry2, Katy Billot3,4,5, Arash Nanbakhsh1, Thouraya Ben Safta1,Catherine Richon6, Sophie Ferlicot7, Emmanuel Donnadieu8, Sophie Couve1, Betty Gardie1,Florence Orlanducci9, Laurence Albiges10, Jerome Thiery1, Daniel Olive9, Bernard Escudier10, andSalem Chouaib1

AbstractClear cell renal cell carcinomas (RCC) frequently display inactivation of von Hippel-Lindau (VHL) gene leading

to increased level of hypoxia-inducible factors (HIF). In this study, we investigated the potential role of HIF2a inregulating RCC susceptibility to natural killer (NK) cell–mediated killing. We demonstrated that the RCC cell line786-0 withmutated VHLwas resistant to NK-mediated lysis as compared with the VHL-corrected cell line (WT7).This resistance was found to require HIF2a stabilization. On the basis of global gene expression profiling andchromatin immunoprecipitation assay, we found ITPR1 (inositol 1,4,5-trisphosphate receptor, type 1) as a directnovel target of HIF2a and that targeting ITPR1 significantly increased susceptibility of 786-0 cells toNK-mediatedlysis. Mechanistically, HIF2a in 786-0 cells lead to overexpression of ITPR1, which subsequently regulated the NK-mediated killing through the activation of autophagy in target cells by NK-derived signal. Interestingly, bothITPR1 and Beclin-1 silencing in 786-0 cells inhibited NK-induced autophagy and subsequently increasedgranzyme B activity in target cells. Finally, in vivo ITPR1 targeting significantly enhanced the NK-mediatedtumor regression. Our data provide insight into the link between HIF2a, the ITPR1-related pathway, and naturalimmunity and strongly suggest a role for the HIF2a/ITPR1 axis in regulating RCC cell survival. Cancer Res; 74(23);6820–32. �2014 AACR.

IntroductionClear cell renal cell carcinomas (RCC) account for approx-

imately 3% of adult cancers (1). They are characterized by theirhypervascularity and resistance to conventional anticancertreatments. For many decades, immunotherapy based on IL2and IFNa has been the standard first-line treatment, althoughthe response rate was typically less than 20%withmultiple sideeffects (2). Thanks to improved understanding of RCC molec-

ular pathogenesis, targeted therapies blocking angiogenesis orsignal transduction pathways have been developed (3). Whilethese therapies have undoubtedly improved clinical outcome,most patients eventually relapse and develop progressivedisease (4).

Natural killer (NK) cells play an important role as first-linedefenders in the host response to tumors and infections, intransplant rejection, and the development of tolerance (5). Thepresence of intratumoral NK in RCC correlates with improvedpatient survival (6). Recent studies suggest that immunother-apymay be an effective approach for patientswith RCC (7), andemerging strategies are currently under investigation based onadoptive transfer of T or NK cells (8, 9). However, immunetherapies based on adoptive transfer of in vitro activatedautologous NK cells or on T-cell modulating agents such asantibodies against programmed death 1 and cytotoxic Tlymphocyte–associated antigen-4 have not resulted in a sig-nificant clinical response (10). The activation status of NK cellshas been reported as a reason for the lack of clinical responseand evidence clearly indicates that NK cell activation is notsufficient to kill tumor cells due to complex interactions withthe tumor and its inherent features. However, recent data frompreclinical and clinical studies with donor-derived alloreactiveNK cells have sparked interest in the possibility of exploitingthe antitumor effect of NK cells (11). Themajor challenge for aneffective NK-based immunotherapy is to overcome themechanisms of tumor cell resistance toward NK cells. In this

1INSERM U753, Villejuif, France. 2Laboratory of Experimental Hemato-Oncology, Department of Oncology, Public Research Center for Health(CRP-Sant�e), L-1526 Luxembourg City, Luxembourg. 3INSERM, U1016,Paris, France. 4CNRS, UMR8104, Paris, France. 5Universit�e Paris Des-cartes, Sorbonne Paris Cit�e, Paris, France. 6Functional Genomic UnitGustave Roussy Cancer Institute, Villejuif, France. 7Universit�e Paris-Sud,Assistance Publique-Hopitaux de Paris, Service d'Anatomo-Pathologie,Hopital Bicetre, Le Kremlin-Bicetre France. 8Institut Cochin, Universit�eParis Descartes, CNRS (UMR 8104), Paris, France. 9INSERM, U1068,CRCM, Immunity and Cancer, Marseille, France. 10Medical OncologyDepartment, Villejuif, France.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Y. Messai and M.Z. Noman contributed equally to this article.

Corresponding Author: SalemChouaib, U753 INSERM, Gustave Roussy,114 rue Edouard Vaillant, 94805 Villejuif cedex, France. Phone:33142114547; Fax: 33142115288; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-0303

�2014 American Association for Cancer Research.

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regard, the molecular events involved in the susceptibility ofRCC cells to natural cytotoxic effectors should be taken intoconsideration.The majority of renal cancers presents clear cell carcinoma

histology (12), which is usually associated with mutational orfunctional inactivation of the von Hippel-Lindau (VHL) gene(13). The VHL pathway targets the hypoxia-inducible factors(HIF) family of transcription factors, in particular HIF1a andHIF2a, for ubiquitin-mediated degradation via the protea-some (14). Consequently, VHL inactivation leads to theconstitutive stabilization of HIFs and increased expressionof target genes involved in the unfavorable tumor microen-vironment (15). While the genes regulated by HIF1 and HIF2often overlap (16), HIF2 is reported to regulate unique targetgenes and to be the critical factor in RCC development (17,18). Although several reports have established a link betweenhypoxia-induced HIF1a and tumor cell resistance to immuneeffectors (19–21), the specific role of VHL mutations andselective activation of HIF2a in modulating RCC suscepti-bility to cytotoxic immune effector cells remains largelyunknown.Here, using a VHL-mutated RCC cell line that stabilizes only

HIF2a, we demonstrated that HIF2a-dependent inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1) expression regulatesRCC cell susceptibility to NK-mediated cell lysis. BecauseHIF2a induced by VHL mutation or deletion is critical in RCCdevelopment, this study underscores the significance of itstargeting in the future NK cell–based therapeutic strategiesagainst RCC.

Materials and MethodsCell culture and transfection786-0, PRC3, and WT7 were obtained from Dr. William

Kaelin, Jr. (Dana Farber Institute, Harvard Medical School,Boston, MA). WT7 and PRC3 cells were derived from 786-0 bystable transfection with pRC-HAVHL or the empty vectorrespectively (14). NKD cells were purified from healthy donorperipheral bloodmononuclear cells using CD56-positive selec-tion (STEMCELL technologies). NKD, NKL, and NK92 naturalkiller cell lines were maintained in RPMI supplemented with300 U/mL recombinant human IL2 (rhIL2). The Renca murinerenal carcinoma cell line was originally obtained from a tumorthat arose spontaneously in the kidney of Balb/cmice (22). 786-0 cells were transfected with tomato-LC3 encoding vector byLipofectamine 2000 according themanufacturer's instructions(Invitrogen). Autophagy-defective 786-0 cells were generatedby transduction of Beclin-1 shRNA lentivirus particles (SantaCruz Biotechnologies).

VHL-mutant constructionsThe inducible VHL cell lines, 786-0-VHL WT (wild0type

VHL), 786-0-VHL Y98H (VHL c.292T>C mutation associatedwith VHL disease type 2A), 786-0-VHL C162F (VHL c.485G>Tmutation associatedwithVHLdisease type 1), and 786-0 empty(empty vector), were created using the T-Rex lentiviral expres-sion system (Invitrogen) and pLenti4/TO/V5-DEST-VHLaccording to the manufacturer's protocol.

Western blottingWestern blotting was conducted as previously reported (23).

Primary antibodies (Ab) against HIF2a, VHL, ITPR1, andmicrotubule-associated protein 1A/1B-light chain 3 (LC3)werepurchased, respectively, from Novus Biologicals, BD Pharmin-gen, and Cell Signaling Technology.

Cytotoxicity assayFour-hour chromium release assays were performed as de-

scribed previously (19). Briefly, different effector:target (E:T)ratios were used on 1,000 target cells per well in round-bottom96-well plates. After 4 hours of coculture, the supernatantswere transferred to LumaPlate-96 wells (PerkinElmer), drieddown, and counted on a Packard Instrument TopCount NXT.Percentage-specific cytotoxicity was calculated conventionallyas described earlier (19).

Gene silencing by RNAi and shRNA transductionGene silencing was performed using predesigned sequence-

specific siRNA purchased from Sigma (19). Briefly, 8� 106 cellswere electroporated twice over 48 hours in serum-freemediumwith 20 mmol/L siRNA in an EasyJect Plus electroporationsystem (EquiBio; 260 V, 450 mF). siRNA targeting luciferase wasused as a negative control (50-GCAAGCUGACCCUGAAGUU-CAU-30; siRNA-CT). Predesigned lentiviruses (pLKO.1-puro)expressing shRNA against ITPR1, and shRNA control (CT)were purchased from Sigma and transduced according to themanufacturer's instructions. Gene-specific targeting was eval-uated by quantitative real-time PCR (RT-qPCR) and/or West-ern blotting.

SYBR Green RT-qPCRTotal RNA was extracted using TRIzol solution (Invitrogen),

andmRNA levels were quantified by SYBRGreen qPCRmethod(Applied Biosystems; ref. 19). Relative expression was calcu-lated using the comparative Ct method (2�DCt). Primer seq-uences are available upon request.

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) assays were per-

formed as described (24).Primers used were: VEGF-FWD 50-TCAGTTCCCTGGCAAC-

AT-30; VEGF-REV 50-ACCAAGTTTGTGGAGCTGAG-30; ITPR1-FWD 50-TTCACATCTCAGAACTAGCCACA-30; and ITPR1-REV50-GGGTCACTGCCTAACTCATTC-30.

Microarray analysisDNA microarray analysis was performed as previously

reported (19) using Agilent Human Whole Genome Microar-ray: 44,000 spots. Datamining was done using Rosetta Resolversoftware, IPA (Ingenuity Pathways Analysis).

Confocal microscopy and analysis of immunologicsynapse formation

Effector (NKL) and tumor target cellswere allowed to adhereon poly-lysine–coated coverslips (Sigma) at a 2:1 E:T ratio for30 minutes and immunologic synapse formation was analyzedas described earlier (19). Efficiency of conjugate formation

HIF2a/ITPR1 Axis Regulates RCC Natural Killer–Mediated Lysis

www.aacrjournals.org Cancer Res; 74(23) December 1, 2014 6821




between NK and RCC cells was calculated by determining theratio of effector cells forming conjugates with target cells tototal target cells � 100. The formation of autophagosomes intomato-LC3–expressing 786-0 cells was monitored by laserscanning confocal microscopy (LSM)-510-Meta (Carl Zeiss)using 40� oil immersion objective.

Treatment with perforin and granzyme BNative rat perforin was purified from RNK16 cells as

described (25). Animal use was approved by the Animal Careand Use Committee of the Gustave Roussy Institute (Villejuif,France). Human granzyme B was purified from the human NKcell line YT-Indy as described (26). Cells were washed andequilibrated 5minutes in HBSSwith 10mmol/LHEPES, pH 7.5,4 mmol/L CaCl2, 0.4% BSA before adding sublytic perforinand/or granzyme B at the indicated concentration, diluted ingranzyme B buffer (HBSS, 10 mmol/L HEPES, pH 7.5). Sublyticperforin concentration was determined independently foreach experiment as the concentration required to induce5% to 15% propidium iodide (PI) uptake (2 mg/mL; Sigma)measured 20 minutes later by flow cytometry (BD Accuri C6Flow Cytometer).

NK-delivered granzyme B detection in target cellsThe level of granzyme B in target cells was assessed by

coculture of target cells with YT-INDY-NK cells as previouslydescribed (27). The presence of granzyme B in target cells wasevaluated by immunoblot using granzyme B antibody (BDbiosciences). Intracellular active granzyme B in target cellswas measured by using Prizm granzyme B cell–mediatedcytotoxicty assay kit following manufacturer's instructions(Origene).

ImmunohistochemistryImmunohistochemical (IHC) staining for HIF2a and ITPR1

was performed as described earlier (28) using tissue micro-arrays (TMA) from 235 patients with RCC selected during theperiod 1993 to 2004 from the pathology department of HopitalKremlin Bicetre (Bicetre, France). The criterion for immuno-positivity was aminimum of 10% positive cells evaluated usinga binary qualitative score (0, negative; 1, positive).

In vivo tumorigenic assayTo avoid tumor rejection (human 786-0 cells) by the immune

system of mice, we usedmurine tumor cells. Six- to 7-week-oldmice Balb/c mice (Harlan) were used. Mice were housed at theInstitut Gustave Roussy animal facility and treated in accor-dance with institutional animal guidelines. Mice (n ¼ 10 pergroup) were inoculated subcutaneously with 2.5 � 106 Rencacells.

Calcium video imagingMeasurements of the intracellular calcium concentration

were performed at 37�Cwith a diaphot 300microscope (Nikon)and with MetaFluor software. The video acquisition was madefor 10 minutes. Average calcium responses were calculated forall tumor cells withMetaFluor Analyst software, and statisticalanalysis was made with GraphPad Prism software.

ResultsVHL mutation decreased RCC cell susceptibility to NK-mediated lysis independently of the alteration of synapseformation and NK ligand expression

To investigate the influence of VHL mutations on theregulation of RCC susceptibility to NK effector cells, we usedthe VHL-mutated 786-0 and PRC3 cell lines as well as WT7 celllines stably transfected with wild-type VHL (14). As shownin Fig. 1A, transfection of VHL inWT7 cells resulted in the lossof HIF2a expression, whereas parental 786-0 and PRC3 cellsconstitutively expressed HIF2a. Similarly, HIF2a target genes,such as VEGF, carbonic anhydrase-9 (CAIX), and glucosetransporter 1 (SLC2A1), were significantly downregulated inWT7 (Fig. 1B). As shown in Fig. 1C and D, 786-0 and PRC3 celllines were significantlymore resistant to bothNK92- andNKD-mediated lysis than to WT7 at all E:T ratios. We observedsimilar results with another NK cell line (NKL) and 2 differentNKD cells (Supplementary Fig. S1A–S1C). We also performedCr51 cytotoxic assay using A498-pmock RCCVHL-deficient cellline (expressing exclusively HIF2a) and A498 cell line stablytransfected by a vector encoding the wild-type VHL genecocultured with NKD and NKL effector cells. Our data indicatethat reintroduction of a wild-type pVHL in A498 cells resultedin a significant increase inNK-mediated lysis as comparedwithA498-pmock cells (data not shown). These data indicate thatVHLmutations play a critical role in the acquisition of RCC cellresistance to NK-mediated lysis.

We next examined conjugate formation between NKL andthe 3 RCC cell lines by confocal microscopy. 786-0, PRC3, andWT7 were able to form stable conjugates with NKL cells withthe same efficiency (Fig. 1E and F).Moreover, no differencewasobserved in the mean fluorescence intensity of the phospho-tyrosine staining when NK cells were cocultured with 786-0,PRC3, and WT7 cell lines (Fig. 1G). In addition, a panel of NK-activating and -inhibitory receptor ligands was analyzed. Asshown in Supplementary Fig. S3, no significant difference in thesurface expression of NK ligands between 786-0, PRC3, andWT7 cell lines was observed. This indicates that the differentialRCC cell line susceptibility to NK-mediated lysis associatedwith VHL mutations was not due to a differential NK ligandexpression or an alteration of immune synapse formation.

Silencing HIF2a attenuated 786-0 cell resistance to NK-mediated lysis via ITPR1

To determine whether resistance of VHL-mutated 786-0cells to NK-mediated lysis involved the constitutive expressionof HIF2a, we knocked down HIF2a gene expression (Fig. 2A)and its transcriptional activity (Fig. 2B) by 2 different siRNAs.Interestingly, HIF2a silencing resulted in a significant increaseof 786-0 target cell lysis by NK92 (Fig. 2C) and NKD (Fig. 2D).Similar results were also observed with another NKL and 2different NKD (Supplementary Fig. S1D and S1E). In addition,we inhibited HIF2 expression by siRNA in 786-0 cells and thentransfected these cellswith a plasmid encoding pcDNA3-HIF-2.The results showed that overexpression of HIF2 in HIF2knocked down 786-0 cells restored their resistance to NK-mediated lysis, thus emphasizing the critical role of HIF2 in theacquisition of 786-0 cells resistance to NK effectors (data not

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Cancer Res; 74(23) December 1, 2014 Cancer Research6822




shown). Data depicted in Supplementary Fig. S4 demonstratethat knockdown of HIF2a did not affect NK-activating and-inhibitory ligand expression. These data point to an essentialrole of HIF2a in the acquisition of resistance to NK-mediatedlysis in VHL-mutated RCC cells.To gain insight into the mechanism by which HIF2a regu-

lated RCC susceptibility to NK-mediated lysis in VHL-mutated

cells, we performed a global gene expression analysis usingDNA microarray. We used 786-0 cells transfected with HIF2aor control siRNAs. As shown in Fig. 2E and F, 28 genes weredifferentially expressed with more than 2-fold change and anadjusted P value of 0.05. We first focused on 10 downregulatedgeneswithmore than 3-fold change namely: F3, PTHLH, SLC2A,ADM, ITPR1, GFRA2, VEDGFA, EDN1, ANGPTL4, and EPAS1. A

Figure 1. Differential susceptibilityof 786-0, PRC3, and WT7 cell linesto NK-mediated lysis demonstratecomparable immune synapseformation. A, Western blot analysisof VHL and HIF2a proteinexpression levels in 786-0, PRC3,and WT7 cells. B, HIF2a, VEGF,CAIX, and SLC2A1 expression wasevaluated by qRT-PCR in 786-0,PRC3, andWT7cells. C andD,Cr51

cytotoxicity assays wereperformed on 786-0, PRC3, andWT7 cells as targets at different E:Tratios. NK cell line (NK92) and NKDwere used as effectors. Datarepresent three independentexperiments with SD. Statisticallysignificant differences (indicatedby asterisks) are shown (�,P < 0.05;��, P < 0.005; ��, P < 0.0005). E,conjugate formation between NKeffector cells and target tumor cellswas analyzed by confocalmicroscopy. Synapse formation asdefined by phosphotyrosineaccumulation in the cell-to-cellcontact area was assessed usinganti-phosphotyrosine antibody(green fluorescence). Nuclei werestained with TO-PRO-3 iodide(blue fluorescence). Bars, 5 mm.F, scoring of conjugate formationbetween NK and tumor cells byvisual counting in ten differentrandomly selected fields. G,quantification of phosphotyrosinestaining by mean fluorescenceintensity (MFI) was analyzed usingthe region measurement functionof the ImageJ software. Datarepresent three independentexperiments with SD.






strong positive correlationwas found betweenmicroarray dataand qRT-PCR results (Fig. 2G). Three candidate genes(ANGPTL4, ADM, and ITPR1) were then selected on the basisof their fold change and their involvement in cell death andsurvival. While silencing of ANGPTL4 had no effect on 786-0

tumor cell susceptibility to NK-mediated lysis (SupplementaryFig. S2A–S2C), a slight increase was observed following target-ing of ADM (Supplementary Fig. S2D–S2F). Interestingly,siRNA-mediated silencing of ITPR1 (Fig. 2H) resulted in adramatic and significant increase of 786-0 susceptibility to

Figure 2. Targeting HIF2aattenuated 786-0 cell resistance toNK-mediated lysis byamechanisminvolving ITPR1. A, 786-0 tumorcells were transfected with twodifferent sequences of HIF2asiRNA or control siRNA (siRNA-CT). Western blot analysis wasperformed with antibodies asindicated. Actin was used as aloading control. B, inhibition ofHIF2a target genes followingsiRNA-mediated HIF2a targetingwas evaluated by qRT-PCR. C andD, Cr51 cytotoxicity assays wereperformed on 786-0 transfectedwith either siRNA against HIF2a orsiRNA-CT as targets at differentE:T ratios. NK cell line (NK92) andNKD were used as effectors. Datarepresent three independentexperiments with SD. Statisticallysignificant difference (indicated byasterisks) are shown (�, P < 0.05;��, P < 0.005; ��, P < 0.0005). E,volcano plot of gene expression(log2 fold change) and adjusted Pvalues for 786-0 cells transfectedwith siRNA-HIF2a. F, heat map ofHIF2a-regulated genes in 786-0cells. Red, upregulation; green,downregulation. G, validation byqRT-PCR of HIF2a-regulatedgenes with more than 3-foldchange. H, 786-0 tumor cells weretransfected with two differentsiRNAs targeting ITPR1 or controlsiRNA and gene silencing wasconfirmed by qRT-PCR. I and J,Cr51 cytotoxicity assays wereperformed on 786-0 transfectedwith siRNA against ITPR1 orcontrol siRNA as targets atdifferent E:T ratios. NK92 andNKD cells were used as effectors.Data represent three independentexperiments with SD. Statisticallysignificant difference (indicated byasterisks) are shown (�, P < 0.05;��, P < 0.005; ��, P < 0.0005).

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both NK92- and NKD-mediated lysis (Fig. 2I and J). Theseresults clearly demonstrate a significant role of ITPR1 in theresistance of VHL-mutated RCC cells to lysis by NK effectors.

ITPR1 knockdown inhibited NK-mediated autophagyinduction and increased granzyme B activity in 786-0cellsITPR1 has been shown to be involved in the control of

intracellular calcium signaling and the regulation of autophagy(29). To investigate how ITPR1 regulates 786-0 cell resistance toNK-mediated lysis, we first assessed intracellular calciumaccumulation in either 786-0 cells alone or 786-0 cells trans-fected with siRNA-ITPR1 with or without NK stimulation.Surprisingly, no significant difference in intracellular calciumaccumulationwas observed (Supplementary Fig. S3A and S3B).Similarly, we did not observe any difference in autophagyinduction in VHL-mutated (786-0 and PRC3) and VHL-cor-rected RCC (WT7) cells in the absence of NK (SupplementaryFig. S3C). Moreover, 786-0 cells transfected with siRNA-HIF2aor siRNA-ITPR1 neither induced nor inhibited autophagy afterculture under serum starvation conditions or treatment withhydroxychloroquine (Supplementary Fig. S3D and S3E). Final-ly, 786-0 cells were transfectedwith tomato-LC3 vector, and theformation of autophagosomes was assessed by confocalmicroscopy. We did not detect any difference in the level ofautophagosomes in 786-0 cells transfected with siRNA-controlor siRNA-ITPR1 in the absence of NK cells (Fig. 3A).We then examined whether NK cells can induce autophagy

in target cells. For this purpose, 786-0 cells were transfectedwith tomato-LC3 vector and siRNA-CT or siRNA-ITPR1, cocul-tured with NK cells and the formation of autophagosomes intarget cells was assessed by confocalmicroscopy. Interestingly,we show that NK cells were able to induce autophagy processonly in 786-0 cells transfected with siRNA-CT but not in ITPR1knocked down cells (Fig. 3B). The induction of autophagy insiRNA-CT cells seems to be responsible for the resistance of786-0 cells to NK-mediated lysis. Time lapse video microscopyperformed on control and ITPR1-defective cells providedstrong evidence that control cells displaying several autopha-gosomes survived NK-mediated killing; however, ITPR1-defec-tive cells displaying no autophagosomes underwent NK-medi-ated cell killing (Fig. 3C and Supplementary Movies S1 and S2).We further analyzed the LC3-II levels by Western blotting

using NK92 cell line. Figure 3D showed a dramatic increase inLC3-II in 786-0 cells when cocultured with NK cells (þ)compared with cells cultured without NK cells (�). This resultindicates that autophagy in target cells is induced by a signalderived from NK cells. Interestingly, targeting ITPR1 in targetcells blocks the ability of NK cells to activate autophagy intarget cells, indicating that ITPR1 positively controls autop-hagy in target cells.We have recently shown that selective autophagy impairs

innate tumor immune response by degrading NK-derivedgranzyme B (27). We next evaluated whether targeting autop-hagy may affect the level and the activity of NK-derivedgranzyme B in 786-0 cells. Strikingly, our results indicate thattargeting Beclin-1 significantly increased NK-derived gran-zyme B level (Fig. 3E) in 786-0 cells. Furthermore, no NK signal

(NKG2D) was detected in CT or BECN1 siRNA cells aftercoculture, thereby ruling out any contamination by NK cells.Our results (Fig. 3F) demonstrate that 786-0 cells displayed asignificant lower level of granzyme B than WT7 cells.

Results depicted in Fig. 3G further show that the serineprotease activity of granzyme B was significantly increased inITPR1 and Beclin-1 siRNA–treated 786-0 cells after theyencountered NK cells. It is important to note that both ITPR1-and Beclin-1–silenced cells formed the same number ofimmune conjugates as compared with control cells (Fig. 3H).

To validate these results, we measured activation of keysmediators of the granzyme B signaling pathway in 786-0 cellstransfected with either siRNA-control or siRNA-ITPR1 andincubated with sublytic perforin and human granzyme B. Weobserved that Bid (noncleaved form of Bid, 22 kDa), pro-caspase-9, and pro-caspase-3 cleavages (Fig. 3I) were signifi-cantly increased after perforin/granzyme B loading of ITPR1-silenced cells as compared with control cells. Taken together,these results clearly demonstrate that ITPR1 knockdown inhi-bits NK-induced autophagy and subsequently increased NK-delivered granzyme B activity leading to an increased suscep-tibility to granzyme B and NK-mediated cell death.

ITPR1 is a new direct target gene of HIF2a in RCC cellsWe further asked whether ITPR1 could be a direct target of

HIF2a. 786-0 cells transfected with 2 different HIF2a siRNAsresulted in a significant decrease in ITPR1 expression atmRNA(Fig. 4A) and protein levels (Fig. 4B). In addition, varying HIF2astabilization levels in 4 786-0–derived cell lines with differentVHL mutations (inducible wild-type VHL, VHL-Y98H, VHL-C162F, empty vector) revealed that HIF2a expression levelspositively correlated with ITPR1 (Fig. 4C and D). Similarly, inA498 cells transfected with a vector encoding wild-type VHL(A498-pVHL), we also observed a decrease in HIF2 and ITPR1mRNA and protein expression as compared with A498 cellstransfected with a control vector (A498-pmock; data notshown). These results further support our findings using786-0, PRC3, and WT7 cells.

To examine a putative direct transcriptional regulation ofITPR1 by HIF2a, we analyzed the promoter region of ITPR1gene using fuzznuc (EMBOSS) software and found 10 putativehypoxia-response elements (HRE; A/GCGTG), suggesting apossible direct interaction between HIF2a and the ITPR1promoter. Immunoprecipitation of the chromatin complexesformed in 786-0 and PRC3 cells showed significant enrichmentof the ITPR1 promoter fragment containing HRE-7 with thespecific HIF2a antibody as compared with theWT7 cell line orthe IgG antibody. Similar enrichment of VEGF promoterfragment containing HRE, a well-known HIF2a target gene(30), was also observed in PRC3 and 786-0, but not inWT7 cells(Fig. 4E).

IHC staining was finally performed on tissue sections from235 patients diagnosed with RCC to examine the relationshipbetween ITPR1 and HIF2a expression. Of 235 patients, 203were negative for ITPR1 staining and 32 were positivelystained. Among the ITPR1 negatively stained patients (n ¼203), 178 were also negative for HIF2a (87%). For the ITPR1positively stained patients (n¼ 32), 20 were found to be stained






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by HIF2a (62.5%). Statistical analysis showed a significantcorrelation between HIF2a and ITPR1 staining in thesepatients (Table 1).Taken together, these results clearly demonstrate that

ITPR1 is a direct novel target gene of HIF2a in RCC cell linesand point to the existence of a functional link between ITPR1and HIF2a in RCC.

In vivo targeting of ITPR1 in Renca cells promoted tumorregression in miceTo investigate the in vivo relevance of the HIF2a/ITPR1

pathway, we used the Renca murine RCC with constitutivelystabilized HIF2a. When Renca cells were transfected withsiRNA against HIF2a, a significant decrease in ITPR1 wasobserved at both protein (Fig. 5A) and mRNA levels (Fig. 5B).Moreover, treatment of Renca cells with increasing doses ofdigoxin (an inhibitor of HIF1a and HIF2a; ref. 31) resulted ina simultaneous decrease in HIF2a and ITPR1 expression in adose-dependent manner (Fig. 5C and D). Furthermore, usingstably transduced Renca cells with 2 different sequences ofshRNA targeting ITPR1 (Lenti-shRNA–ITPR1-1 and Lenti-shRNA–ITPR1-2) or control shRNA (Fig. 5E), we demonstrat-ed that ITPR1 inhibition resulted in a significant decrease inRenca tumor growth as compared with control tumors.When NK cells were depleted in Balb/c mice using anti-Asialo-GM1 antibodies, a more significant tumor growth wasobserved as compared with untreated mice. Importantly, thecombination of ITPR1 inhibition and NK cell depletionresulted in more robust tumor growth than NK depletionalone (Fig. 5F). These data are consistent with a role of ITPR1in tumor progression, at least in part, by a mechanisminvolving the regulation of tumor cell susceptibility to NK-mediated cell lysis.

DiscussionVHL mutations play a significant role in regulating the

development, invasiveness, and survival characteristics of RCC(32). However, the role of VHL mutations in modulating RCCsusceptibility to cytotoxic immune effector cells remainslargely unknown. In this study, we show that the restoration

of VHL function in 786-0 and A498 cells significantly increasestheir sensitivity to NK-mediated lysis, suggesting a role for VHLmutations in attenuating RCC resistance to lysis. We furthershow that VHL operates by a mechanism independent ofsynapse formation and NK ligand expression, thereby exclud-ing an alteration of target recognition by NK cells. It is wellestablished that VHL mutations in RCC may result in stabi-lization of both HIF1a and HIF2a or in a selective stabilizationof HIF2a (33). Accumulating evidence indicates that HIF2a ismore important for tumor progression, whereas HIF1amostlybehaves as a tumor suppressor (34). These antagonistic effectshave been partially explained by the ability ofHIF2a to regulategenes involved in activation of proliferation (MYC, CyclinD),inhibition of apoptosis (p53), and promotion of metastasis(CXCR4). In contrast, HIF1a regulates preferentially apoptoticgenes (BNIP3, p53) and inhibits proliferation (MYC inhibition,p21, p27; ref. 35). In the RCC cell line (786-0 cells) used in thisstudy, VHL mutation selectively induces HIF2a stabilization,thus representing an ideal model system to specifically exam-ine the effect of HIF2a on RCC susceptibility to NK-mediatedlysis. Using this model, we showed that targeting HIF2a in 786-0 cells attenuates their resistance to NK-mediated lysis, where-as HIF2a overexpression in HIF2 knocked down 786-0 cellsrestored their resistance to NK-mediated lysis, suggesting acritical role of HIF2a in the acquisition of resistance tocytotoxicity associated with VHL mutations. Perier and col-leagues have previously reported that the restoration of VHLfunction in RCC4 and RCC6 cell lines resulted in a decreasedsusceptibility to NK-mediated lysis by a mechanism involvingmodulation of HLA-1 (HLA-A and HLA-G) expression (36).These discrepancies from our findings may be due to the factthat in our model, 786-0 cell line stabilizes only HIF2awhereasthe RCC4 and RCC6 cell lines stabilize both HIF1a and HIF2a.In this regard, it should be noted that hypoxia and HIF (HIF1aandHIF2a) effects onNK ligand expression have been reportedto be cell-type–specific. Very recently, it was shown thathypoxia did not alter the surface expression of NK cell ligandin multiple myeloma (37), whereas Siemens and colleaguesprovided evidence that hypoxic stress promotes the sheddingof MHC I chain–relatedmolecules MICA andMICB in prostate

Figure 3. ITPR1 regulates NK-induced autophagy and NK-derived granzyme B in 786-0 cells. A, the formation of autophagosomes was assessed by confocalmicroscopy in 786-0 cells transfected with siRNA-CT or siRNA-ITPR1 overexpressing tomato-LC3 cultured without NK cells. B, the formation ofautophagosomeswas assessed by confocalmicroscopy in 786-0 cells transfectedwith siRNA-CTor siRNA-ITPR1overexpressing tomato-LC3 culturedwithPKH-67–stained NK92 cells (middle) at 5:1 E:T ratio. The formation of red dot structures corresponding to autophagosomes in target cells was assessed byconfocal microscopy (Top). Bottom corresponds to merge images of top and middle. Scale bar, 10 mm. C, 786-0 cells described in B were recorded by timelapse video microscopy (presented as Supplementary Movies S1 and S2). At the indicated times, representative images were extracted from themovies. After 81minutes of coculture, NKcellswere able to kill ITPR1-silenced cellsmore efficiently. Scale bar, 10 mm.D, analysis of LC3-II level in 786-0 cells.Control (þ) or ITPR1-defective (�) 786-0 cells were cultured alone (�) or in the presence of NK92 cells. After separation ofNK cells from targets by using beadscoated with monoclonal CD56 antibodies, target cell lysates were subjected to Western blotting for LC3-I and -II expression. E, 786-0 cells stablytransfected with shRNA-CT and shRNA-Beclin-1 (BECN1; autophagy-defective cells) and co-presented to NK cells at 5:1 E:T ratio for 30 minutes. Followingseparation fromNKcells, lysates of tumor cellswere subjected to immunoblot analysis to evaluate thegranzymeB (GzmB) intracellular content. NKcells lysatewas used as control for GzmB detection. F, 786-0 cells expressing (þ) or not (�) BECN1 as well as WT7 were cocultured with NK92 cells for 1 hour.NKcells were separated from targets by using beads coatedwithmonoclonal CD56 antibodies. Target cell lysateswere subjected toWestern blot for analysisof GzmB content. G, 786-0 cells were transfected with siRNA-CT or siRNA-ITPR1, incubated with sublytic perforin (PFN) and GzmB, and Westernblotting was performed. Actin was used as loading control. H, 786-0 cells were transfected with siRNA-CTor siRNA-BECN1or siRNA-ITPR1 and coculturedwith NKcells at 3:1 E:T ratio for 60 minutes. Shown is the percentage of target cells with intracellular active GzmB. I, 786-0 cells were transfected withsiRNA-CT or siRNA-BECN1 or siRNA-ITPR1 and cocultured with NK cells at 3:1 E:T ratio for 60 minutes. Shown is the percentage of conjugate formationbetween effector and target cells assessed by flow cytometry. Data represent three independent experiments with SD. Statistically significant differences(indicated by asterisks) are shown (�, P < 0.05; ��, P < 0.005, ��, P < 0.0005).






cancer cells, resulting in a decreased sensitivity to NK-medi-ated lysis (38).

DNA microarray–based global transcriptional profiling inHIF2a–targeted 786-0 cells and RT-qPCR revealed a set ofgenes related to their fold change. Among the differentiallyexpressed genes, we selected 3 genes, ANGPTL4, ADM, andITPR1, on the basis of their fold change and involvement in celldeath and survival. Very interestingly, ITPR1 was found to bethe most prominent gene involved in the regulation of RCCresistance to NK-mediated lysis.

It is well established that ITPR1, a member of IP3 receptorfamily, is a ligand-gated ion channel that mediates calcium

release from the intracellular stores specially the endoplasmicreticulum (ER; refs. 39–41). Under our experimental condi-tions, calcium accumulation was unchanged in cells regardlessof whether they expressed or not ITPR1 in response to NKeffectors, ruling out a role for ITPR1 in global calcium accu-mulation. However, it should be noted that ITPR1 overexpres-sion could lead to a differential distribution of calcium inintracellular compartments (endoplasmic reticulum andmito-chondria; ref. 42), which could modify the cell response toapoptotic stimuli. In addition, compensatory effects by otherITPR family members including ITPR2 and ITPR3 memberscould also occur.

Figure 4. ITPR1 is a direct noveltarget of HIF2a. A, 786-0 tumorcells were transfected with threedifferent sequences of HIF2asiRNA or control siRNA (siRNA-CT). Inhibition of ITPR1 mRNAexpression following siRNA-mediated HIF2a targeting wasevaluatedbyqRT-PCR.B,Westernblot analysis and densitometrywasperformed. Actin was used as aloading control. C and D, 786-0cells were transfected with emptyvector, a vector encodingwild-typeVHL, and two vectors encodingtwo different VHL mutations,C162F and Y98H, respectively,associatedwith high and low levelsof HIF2a. ITPR1 expression wasanalyzed by Western blotting (C)and qRT-PCR (D). SLC2A1expression reflects HIF2atranscriptional activity. E, ChIPwasperformed in 786-0, PRC3, andWT7 cells using anti-HIF2 orcontrol nonspecific antibodies.Samples were analyzed by real-time PCR using HRE-specificprimers. VEGF was used as apositive control. Data representthree independent experimentswith SD. Statistically significantdifference (indicated by asterisks)are shown (�,P <0.05; ��,P<0.005;��, P < 0.0005). F, representativeimage of hematoxylin and eosin(H&E) staining and IHC staining ofHIF2a and ITPR1 expression innormal renal tissue and in tissuefrom patients with RCC. Boxedregions are displayed at highmagnification in insets (�20).

Messai et al.





Because conflicting studies have suggested a role for HIF2aand ITPR1 in the regulation of autophagy (29), we checked inour model whether autophagy was involved in the acquisitionof resistance to NK by the 786-0 cells. We therefore examinedthe effect of autophagy modulators including serum starva-tion and hydroxychloroquine in the 3 RCC cell lines and in

786-0 transfected or not with siRNA-HIF2a and siRNA-ITPR1and no difference in autophagy markers was observed (Sup-plementary Data S4). Menard and colleagues have reportedthat HIF2a was a potent inhibitor of hypoxia-induced autop-hagy (43). Bohensky and colleagues also showed that inmaturing chondrocytes expressing both HIF1a and HIF2a,

Table 1. HIF2a and ITPR1 staining correlates significantly in human patients with RCC

Correlation between HIF2a and ITPR1 staining in RCC patientsHIF2a staining

� % þ % n P ¼ 1.01 � 10�10

ITPR1 staining� 178 87 25 13 203þ 12 37.5 20 62.5 32

NOTE: Association between HIF2a and ITPR1 expression in patients with RCC. Numbers in the "HIF2a staining" and "ITPR1 staining"columns refer to the number of samples meeting each condition. Numbers in the "�" or "þ" columns indicate the total number ofsamples with negative or positive stainings, respectively.

Figure 5. ITPR1 silencing impairsRenca tumor growth in Balb/cmice. A, Renca cells weretransfected with two differentsequences of HIF2a siRNA orcontrol siRNA (siRNA-CT).Westernblot analysis was performed withantibodies as indicated. Actin wasused as a loading control. B,inhibition of HIF2a target genesfollowing siRNA-mediated HIF2atargeting was evaluated by qRT-PCR. C and D, Renca cells weretreated with increasing doses ofdigoxin for 16 hours. C, HIF2a andITPR1 expression was analyzed byWestern blotting. D, digoxininhibition of HIF2 target genesVEGF and SLC2A1 and of ITPR1wasanalyzedby qRT-PCR. E, qRT-PCR validation of ITPR1 silencingefficiency in Renca cellstransduced stably with Lenti-shRNA-ITPR1-1 and Lenti-shRNA-ITPR1-2. F, Renca cells stablyexpressing Lenti-shRNA-ITPR1-1or Lenti-shRNA-CTwere implantedsubcutaneously in Balb/c mice(n ¼ 10). Mice were depleted forNK cells using anti-Asialo-GM1antibody. Tumors were measuredevery 2 days. Data represent threeindependent experiments withSEM. Statistically significantdifferences (indicated by asterisks)are shown (�,P < 0.05; ��,P < 0.005;��, P < 0.0005).






the latter acts as a brake on the autophagy acceleratorfunction of HIF1a (44).

We next evaluated whether NK cells can induce autophagy in786-0 cells. Interestingly, we showed that NK cells were able toinduce autophagy only in 786-0 cells transfected with siRNA-CTbut not in ITPR1 knockeddown cells, suggesting an involvementof ITPR1 in NK-induced autophagy in VHL-mutated tumortarget cells. This result is in agreement with a recent reportindicating that NK cells are able to induce autophagy promotingtumor cell survival and treatment resistance (45). Several studiesdemonstrated that genetic or pharmacologic inhibition of ITPRreceptors can inhibit or stimulate autophagy (42). Inhibition ofITPR1 signaling pathway can suppress the autophagy indirectlyby mechanisms involving endoplasmic reticulum and calciumremodeling (46). ITPR1 has been reported to directly inhibitstarvation-induced autophagy through increased binding toBeclin-1 (47). Recently, Baginska and colleagues demonstratedthat activation of autophagy under hypoxia resulted in thedegradation of NK-derived granzyme B, which compromisesthe ability of NK cells to eliminate tumor cells (27). As ITPR1-regulated autophagywas also involved in the impairment of NK-mediated lysis of 786-0 cells, we asked whether a subsequentdegradation or deactivation of granzyme B occurred in thesecells. Our results strongly support that the increased expressionof ITPR1byHIF2a leads to the activationof autophagy followingcontactwithNK cells, which subsequently decreased the activityof NK-derived granzyme B. Although the precise mechanism bywhich autophagy affects the level or serine protease activity ofgranzyme B is not addressed here, we strongly believe thatITPR1-mediated NK-induced autophagy induction could beassociated with the decrease in NK-derived granzyme B activityin target cellsmaking them less sensitive toNK-mediated killing.

Here, we obtained experimental evidence indicating thatITPR1 is a novel target of HIF2a and that its expression wasregulated by HIF2a at both mRNA and protein levels. This wasfurther confirmed by ChIP assay. Furthermore, using a largegroup (235) of patients with RCC, we demonstrated the exis-tence of a significant correlation between HIF2a and ITPR1expression. Although the existence of a link between ITPR1 andhypoxia in neuronal cells, mouse kidney, and human embry-onic kidney 293 cells has been reported (48), the direct involve-ment of HIFs in the regulation of ITPR1 has not been estab-lished. Nevertheless, as HIF1a and HIF2a are known to havemany common target genes (15, 16), and given that the hypoxiaresponse element (A/G CGTG) is common for HIF1 and HIF2(49), we cannot exclude the possibility that HIF1a could alsoregulate ITPR1 in cells stabilizing HIF1a.

The in vivo data presented here reveal that ITPR1 targetingcombined with NK depletion significantly enhanced tumorgrowth, supporting the involvement of ITPR1 in regulating thein vivo susceptibility of Renca cells to NK activity. This is inagreement with previous reports indicating that overexpres-

sion of ITPR3 in colorectal carcinoma cells reduced apoptosis,whereas ITPR3 inhibition increased cell death (50). However,we cannot exclude the role of other immune cells in regulatingRenca tumor growth. Taken together, these data support a roleof the inositol triphosphate receptor family in tumor progres-sion. Thus, the HIF2a/ITPR1 axis, triggered by VHLmutationsin RCC, may play a critical role in controlling the switch fromantitumor immunity to tumor cell survival and growth.

Because HIF2a has been reported to be a critical factor intumor progression in RCC (18), it is tempting to speculate thatITPR1 could be one of the mechanisms by which HIF2a drivestumor growth in RCC. In this context, future protocols of NKcell–based immunotherapy should integrate the intrinsic fea-tures of tumor cells (i.e., VHL mutations and subsequenthypoxia status) to improve NK cell–mediated antitumor activ-ity and their cross-talk with tumor microenvironment in thecontext of its stressor complexity and heterogeneity.

Further studies are needed to define whether HIF2a orITPR1 may be considered as a potential target in futuretherapeutic protocols that aim to improve NK cell responsesin patients with RCC and other solid malignancies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Messai, M.Z. Noman, B. Janji, V. Baud, D. Olive,S. ChouaibDevelopment of methodology: Y. Messai, M.Z. Noman, M. Hasmim, S. Couve,J. Thiery, D. Olive, B. Escudier, S. ChouaibAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Messai, M.Z. Noman, C. Richon, S. Ferlicot,E. Donnadieu, S. Couve, L. Albiges, B. Escudier, S. ChouaibAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Messai, M.Z. Noman, M. Hasmim, B. Janji,A. Tittarelli, M. Boutet, V. Baud, E. Viry, K. Billot, S. Couve, J. Thiery, D. Olive,B. Escudier, S. ChouaibWriting, review, and/or revision of themanuscript: Y. Messai, M.Z. Noman,M. Hasmim, A. Tittarelli, S. Couve, L. Albiges, B. Escudier, S. ChouaibAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases):Y.Messai,M.Z. Noman, E. Viry, K. Billot,A. Nanbakhsh, T. Ben Safta, B. Gardie, S. ChouaibStudy supervision: Y. Messai, M.Z. Noman, S. ChouaibOther (performed experiments): F. Orlanducci

AcknowledgmentsThe authors thank Dr. William Kaelin, Jr. for providing RCC cell lines used in

this study and for his helpful suggestions; Denis Martinvalet for the humangranzyme B; Didier Bordereaux for technical help in ChIP analysis; and AgnesLaplanche for her assistance in statistical analysis.

Grant SupportThisworkwas supported by theARTuR (Association pour la Recherche sur les

Tumeurs du Rein), ARC (Association pour la Recherche sur le Cancer) 2012-2013:N� SFI20121205624, and a grant from CRP-Sant�e (LHCE-2013 11 05).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 5, 2014; revised August 19, 2014; accepted September 22,2014; published OnlineFirst October 8, 2014.

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2014;74:6820-6832. Published OnlineFirst October 8, 2014.Cancer Res Yosra Messai, Muhammad Zaeem Noman, Meriem Hasmim, et al. Inducing AutophagyITPR1 Protects Renal Cancer Cells against Natural Killer Cells by

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