&get_box_var;ORIGINAL ARTICLE

HOIL-1L Functions as the PKCz Ubiquitin Ligase to Promote LungTumor GrowthMarkus A. Queisser1, Laura A. Dada1, Nimrod Deiss-Yehiely1, Martin Angulo1,2, Guofei Zhou1*, Fotini M. Kouri3,Lawrence M. Knab3, Jing Liu1, Alexander H. Stegh3, Malcolm M. DeCamp3,4, G. R. Scott Budinger1,Navdeep S. Chandel1, Aaron Ciechanover5, Kazuhiro Iwai6, and Jacob I. Sznajder1

1Division of Pulmonary and Critical Care Medicine, 3The Robert H. Lurie Comprehensive Cancer Center, and 4Division of ThoracicSurgery, Northwestern University, Chicago, Illinois; 2Departamento de Fisiopatologıa, Facultad de Medicina, Universidad de la Republica,Montevideo, Uruguay; 5Cancer and Vascular Biology Research Center, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa,Israel; and 6Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Abstract

Rationale: Protein kinase C zeta (PKCz) has been reported to actas a tumor suppressor. Deletion of PKCz in experimental cancermodels has been shown to increase tumor growth. However, themechanisms of PKCz down-regulation in cancerous cells have notbeen previously described.

Objectives: To determine the molecular mechanisms that lead todecreased PKCz expression and thus increased survival in cancercells and tumor growth.

Methods: The levels of expression of heme-oxidized IRP2 ubiquitinligase 1L (HOIL-1L), HOIL-1–interacting protein (HOIP),Shank-associated RH domain-interacting protein (SHARPIN),and PKCz were analyzed by Western blot and/or quantitativereal-time polymerase chain reaction in different cell lines.Coimmunoprecipitation experiments were used to demonstratethe interaction between HOIL-1L and PKCz. Ubiquitination wasmeasured in an in vitroubiquitination assay andbyWestern blotwithspecific antibodies. The role of hypoxia-inducible factor (HIF) wasdetermined by gain/loss-of-function experiments. The effect ofHOIL-1L expression on cell death was investigated using RNAinterference approaches in vitro and on tumor growth in micemodels. Increased HOIL-1L and decreased PKCz expression wasassessed in lung adenocarcinoma and glioblastoma multiforme anddocumented in several other cancer types by oncogenomic analysis.

Measurements andMainResults:Hypoxia is a hallmark of rapidlygrowing solid tumors. We found that during hypoxia, PKCzis ubiquitinated and degraded via the ubiquitin ligase HOIL-1L,a component of the linear ubiquitin chain assembly complex (LUBAC).In vitroubiquitinationassays indicate thatHOIL-1LubiquitinatesPKCzat Lys-48, targeting it for proteasomal degradation. In a xenografttumormodel and lung cancer model, we found that silencing of HOIL-1L increased the abundance of PKCz and decreased the size of tumors,suggesting that lower levels of HOIL-1L promote survival. Indeed,mRNA transcript levels of HOIL-1L were elevated in tumor of patientswith lung adenocarcinoma, and in a lung adenocarcinoma tissuemicroarray the levels of HOIL-1L were associated with high-gradetumors. Moreover, we found that HOIL-1L expression was regulatedby HIFs. Interestingly, the actions of HOIL-1L were independentof LUBAC.

Conclusions: These data provide first evidence of a mechanism ofcancer cell adaptation to hypoxia where HIFs regulate HOIL-1L,which targets PKCz for degradation to promote tumor survival. Weprovided a proof of concept that silencing of HOIL-1L impairs lungtumor growth and that HOIL-1L expression predicts survival rate incancer patients suggesting that HOIL-1L is an attractive target forcancer therapy.

Keywords: hypoxia; hypoxia-inducible factors; tumorigenesis; E3ligase; linear ubiquitin chain assembly complex

(Received in original form March 11, 2014; accepted in final form August 11, 2014 )

*Present address: Department of Pediatrics, University of Illinois at Chicago College of Medicine, Chicago, Illinois.

Supported by National Institute of Health PO1 HL-071643 and HL-48129, the German Research Foundation, the Northwestern University Flow CytometryFacility, Cell Imaging Facility and Mouse Histology and Phenotyping Laboratory, and a Cancer Center Support Grant (NCI CA060553).

Author Contributions: M.A.Q., L.A.D., N.D.-Y., M.A., G.Z., and F.M.K. performed the experiments. M.A.Q., L.A.D., and J.I.S. analyzed the data and conceivedand designed the experiments. J.L., N.S.C., G.R.S.B., L.M.K., A.H.S., M.M.D., A.C., and K.I. provided reagents and guidance. M.A.Q., L.A.D., G.R.S.B.,and J.I.S. wrote the manuscript.

Correspondence and requests for reprints should be addressed to Jacob I. Sznajder, M.D., Division of Pulmonary and Critical Care Medicine, NorthwesternUniversity Feinberg School of Medicine, Chicago, IL 60611. E-mail: j-sznajder@northwestern.edu

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 190, Iss 6, pp 688–698, Sep 15, 2014

Copyright © 2014 by the American Thoracic Society

Originally Published in Press as DOI: 10.1164/rccm.201403-0463OC on August 13, 2014

Internet address: www.atsjournals.org

688 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 6 | September 15 2014

Rapidly proliferating cancers, includinglung adenocarcinoma and glioblastomamultiforme (GBM), must adapt to tissuehypoxia, which develops in the centralregions of tumors (1–3). This is largelyaccomplished by a program activated inresponse to the stabilization of hypoxia-inducible factors (HIFs), which act astranscription factors to induce metabolicchanges and preserve cellular energyhomeostasis (4–6). The importance of HIFsin tumorigenesis is experimentally wellestablished and therapies directed at someHIF target genes are being clinicallyevaluated (3, 7, 8).

The protein kinase C (PKC) family iscomprised of serine-threonine kinases thatregulate cellular adaptation to environmental

stress by interacting with pathways ofsurvival, proliferation, migration, andapoptosis (9–12). It has been recentlydescribed that PKCz acts as a tumorsuppressor because its activity and/orexpression is altered in different types ofhuman cancer including GBM and renalcancer. Also, deletion of PKCz inexperimental cancer models increasestumorigenesis (13–15). These findingsindicate the importance of PKCz asa possible target in anticancer therapies.The mechanism by which PKCz suppressestumor growth has not been completelydescribed but regulation of c-myc,phosphorylation of C/EBPb to inhibit IL-6expression, and inhibition of the serinebiosynthetic cascade by controlling the3-phosphoglycerate dehydrogenase havebeen proposed as downstream targets(13, 14, 16, 17). However, the cellular andmolecular mechanisms involved in down-regulating PKCz in cancer cells have notbeen described and are the main focusof this study.

PKC activity is regulated by itsintracellular localization and by degradation,although the mechanisms controllingPKCz degradation are less well understood.The linear ubiquitin chain assemblycomplex (LUBAC) was reported to bindand ubiquitinate several PKC isoforms (18).LUBAC is composed of two RING-in-between-RING (RBR)–containing proteins:heme-oxidized IRP2 ubiquitin ligase 1L(HOIL-1L), also known as RBCK1, theHOIL-1–interacting protein (HOIP), anda Shank-associated RH domain-interactingprotein (SHARPIN), but only HOIP hasbeen reported to form linear chains(19–22). Recent reports indicate animplication of LUBAC in cancerogenesiswhere excessive LUBAC activation causesabnormal nuclear factor-kB signaling andcancer growth (23–26).

Here, we sought to determine themolecular mechanisms that lead todecreased PKCz expression in cancer cells,which results in increased cancer cellsurvival and tumor growth. Our dataprovide evidence of a novel mechanismwhere HIF promotes cancer cell survivalby orchestrating a pathway of adaptationto hypoxia. We found that the E3 ligaseHOIL-1L is regulated by HIF and targetsPKCz for proteasomal degradation via aLUBAC-independent mechanism. Importantly,patients with lung adenocarcinoma andGBM have high levels of HOIL-1L and low

levels of PKCz, and thus inhibition ofHOIL-1L can be a potential therapyto slow tumor growth.

Methods

PatientsLung tissue was obtained from patientswith lung nodules. Informed consentwas obtained from all participants. Theprotocols were approved by the ScientificReview Committee and the InstitutionalReview Board of Northwestern University.RNA was isolated from human lung cancertumors and adjacent normal tissue stored inRNAlater and used for subsequent analysis.

Cell Culture, Transfections, andHypoxia ExposureIsolation, culture, transfection protocols,and hypoxia exposure of cells weredescribed previously (27, 28). A549, RCC4,RCC41pVHL, and COS-7 cells weregrown in Dulbecco’s modified Eagle mediumas described (28). Hypoxic conditions(1.5% O2, 93.5% N2, and 5% CO2) wereachieved by equilibrating the medium ina humidified workstation (In Vivo 300;Ruskinn Technology, Bridgend, UK)as previously described (27).

Xenograft AssayAnimals were provided with food and waterad libitum, maintained on a 14:10-hourlight-dark cycle, and handled according toNational Institutes of Health guidelines andthe Northwestern University InstitutionalAnimal Care and Use Committee–approvedexperimental protocol. Xenograft TumorAssay was performed as previouslydescribed (29). Briefly, 107 A549 cellsin phosphate-buffered saline were injectedsubcutaneously into the flank of femaleNCr-nu/nu mice (01B74) or 5 3 105

cells for retroorbital injections. The rateof tumor growth was measured by usingcalipers. Tumor samples were homogenizedunder liquid nitrogen with a mortar andpestle and then suspended in lysis-buffer(50 mM N-2-hydroxyethylpiperazine-N-ethane sulfonic acid pH 7.0, 250 mM, NaCl,5 mM ethylenediaminetetraacetic acid, 1mM dithiothreitol, and 0.1% Triton X-100)as previously described (30).

Oncomine DatabaseOncomine 4.4.4.4, a web-based clinicalgenomics database (available at

At a Glance Commentary

Scientific Knowledge on theSubject: Lung and other solid tumorsmust adapt to hypoxic conditions tosurvive and grow. During hypoxia, thehypoxia-inducible factors are stabilizedand promote vascularization andmetabolic pathways, which resultin tumor adaptation and growth.Recently, it has been reported thatprotein kinase C zeta (PKCz) acts asa tumor suppressor factor; silencingPKCz promoted carcinogenesis.However, the mechanisms of howPKCz is down-regulated duringtumorigenesis have not beenelucidated.

What This Study Adds to theField: We report here that PKCz isdegraded in cancer cells by theubiquitin-proteasome system duringhypoxia, which is characteristic ofsolid tumors. The ubiquitin ligaseheme-oxidized IRP2 ubiquitin ligase1L (HOIL-1L) is highly expressed inpatients with lung cancers. Moreover,we identified HOIL-1L as the PKCzubiquitin ligase, which is up-regulatedduring hypoxia via the hypoxia-inducible factor. Silencing of HOIL-1Lincreased cell death during hypoxiaand suppressed tumor growth. Thus,our studies provide in vitro andin vivo evidence for a novel cellularadaptation mechanism to hypoxia,which is of clinical significance.

ORIGINAL ARTICLE

Queisser, Dada, Deiss-Yehiely, et al.: PKCz Ubiquitination and Degradation Promotes Cell Survival during Hypoxia 689

https://www.oncomine.org) was used toanalyze HOIL-1L (rbck1) expression innormal versus cancer expression fromdifferent tissues and differential analysisof highly ranked gene expression fromdifferent cancer types.

StatisticsExperimental conditions were compared byStudent t test for single measurementsand multiple comparisons were performedusing analysis of variance. DCt valuesobtained from quantitative reversetranscriptase polymerase chain reactionwere analyzed for normal distribution usingthe Shapiro-Wilk test. All analyses wereperformed by using GraphPad Prismversion 4.00 (GraphPad Software, La Jolla,CA). Data were expressed as mean 6 SEM(n = 3) unless otherwise indicated.REMBRANDT, a clinical genomicsdatabase, was used to calculate the effectof HOIL-1L (rbck1) expression on overallglioma patient survival. P values werecalculated by the log-rank test. Statisticsfor overall survival were calculated with the

log-rank (Mantel-Cox) test. A P value lessthan or equal to 0.05 was consideredstatistically significant for all tests.

Results

PKCz Is Ubiquitinated by HOIL-1Lduring HypoxiaIn A549 lung adenocarcinoma cells, PKCzprotein abundance was decreased in atime-dependent fashion during exposureto hypoxia (1.5% O2) (Figure 1A; see FigureE1A), without changes in PKCz mRNAlevels or protein synthesis (see Figures E1Band E1C). To identify the mechanism ofPKCz degradation during hypoxia, weexamined the role of the transcriptionfactor HIF. PKCz expression was lower inpVHL-deficient RCC4 cells, which haveconstitutive HIF-1a/2a activation (31),than in RCC4 cells reconstituted withthe HIF E3 ligase pVHL (RCC41VHL),whereas other PKC isoforms were notaffected (Figure 1B). To investigate theeffects of HIF stabilization on PKCz, we

overexpressed a construct in whichtwo prolines in the oxygen-dependentdegradation domain of HIF-1a weremutated to alanine in A549 cells (32). Thestabilization of HIF-1a, which was detectedby a hypoxia response element luciferaseactivity assay (Figure 1C, top), resulted inlower levels of PKCz as compared withnoncoding vector transfected cells ornontransfected cells (Figure 1C, bottom).Moreover, chemical HIF stabilization byCoCl2 also led to decreased PKCz levels(Figure 1D; see Figure E1D). Conversely,silencing of HIF-1a and HIF-2a preventedthe decrease in PKCz during hypoxia(Figure 1E; see Figure E1E). Interestingly,the expression of the classical PKCa andPKCbII were not altered during hypoxia,indicating a specific effect for theexpression of PKCz (see Figure E1F).

On activation, several PKCs aredown-regulated by ubiquitination andproteasomal degradation (33). In A549cells, exposure to hypoxia or CoCl2increased PKCz ubiquitination (Figures 1Fand 1G). Moreover, preincubation with the

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Figure 1. PKCz is degraded during hypoxia. (A) A549 cells were exposed to hypoxia (1.5% O2) and PKCz levels were detected by Western blot (WB).(B) The expression of PKCz was analyzed in RCC4 and RCC41VHL cells by WB. (C) A549 cells were transfected with two different constitutively activeHIF-1a clones (DPA-2 and DPA-5). Hypoxia response element (HRE) luciferase activity and PKCz levels were determined after 24 hours. (D) A549 cellswere treated with CoCl2 (300 mM). PKCz levels were determined by WB. (E) A549 cells were transfected with HIF (HIF1a1HIF2a) siRNA and exposedto hypoxia for 24 hours. PKCz and HIF levels were detected by WB. (F) A549 cells were exposed to normoxia (21% O2) or hypoxia for 6 hours, PKCzwas immunoprecipitated, and ubiquitin-conjugates detected. (G) Cells were exposed to CoCl2 for 6 hours and PKCz was immunoprecipitated and probedwith an ubiquitin antibody. (H) A549 cells were preincubated with UBE1 or lactacystin and exposed to hypoxia for 24 hours. PKCz levels were detectedby WB. Data represent mean 6 SEM of at least three separate experiments. *P < 0.05; **P < 0.01.

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690 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 6 | September 15 2014

E1-inhibitor UBE1–41, or the proteasomeinhibitor lactacystin, prevented PKCzdegradation (Figure 1H; see Figure E2A).We investigated whether the componentsof linear ubiquitination complex, whichhas been described to interact with andubiquitinate several PKC isoforms, wasinvolved in the degradation of PKCzduring hypoxia. Coimmunoprecipitationexperiments showed that during hypoxiaPKCz binds to HOIL-1L but not to HOIPor SHARPIN (Figure 2A). Using an in vitroubiquitination assay, we found that HOIL-1L/HOIP was sufficient to ubiquitinateGST-PKCz (Figure 2B). Characteristic

high-molecular-weight PKCz-ubiquitinconjugates were observed when GST-PKCzwas incubated with wild-type (WT)ubiquitin. The ubiquitin linkage isimportant to determine the fate of thetarget protein. To determine theubiquitination linkages present in PKCz,we performed the in vitro ubiquitinationassay in the presence of Ub K48O, in whichall lysines in ubiquitin were mutated toarginines except Lys-48, or Ub K48RK63Rin which Lys-48 and Lys-63 were mutatedto arginine. We detected mainlyLys-48–linked ubiquitination (Figure 2B,lanes 3 and 4), whereas linear ubiquitination

was ruled out by using N-terminal His-tagged ubiquitin to prevent linear ubiquitinconjugation (34). Ub K48RK63R abolishedall ubiquitination (Figure 2B, lane 3).Consistent with this observation, thedegradation and ubiquitination ofendogenous PKCz during hypoxia wasprevented by silencing HOIL-1L (Figures2C and 2D; see Figure E2B). Takentogether, our results suggest that duringhypoxia HOIL-1L is the E3 ligase for PKCztargeting it for proteasomal degradation.

Because PKCz degradation is HIF-dependent, we investigated whether HIFregulates HOIL-1L expression and found

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Figure 2. HOIL-1L ubiquitinates PKCz and is regulated by HIF. (A) A549 cells were transfected with HA-HOIL-1L and YFP-PKCz and exposed to hypoxiaas indicated. PKCz was immunoprecipitated and proteins analyzed by Western blot (WB). (B) GST-fused PKCz was incubated in the presence ofHOIL-1L1 HOIP for 2 hours at 378C, pulled down with glutathione beads, and analyzed by WB for ubiquitin conjugates. (C) A549 cells were transfected withcontrol or pooled HOIL-1L siRNA and cells were exposed to hypoxia for 24 hours and proteins were detected by WB. (D) A549 cells were transfectedwith control or pooled HOIL-1L siRNA and exposed to hypoxia for 6 hours. PKCz was immunoprecipitated and probed with an ubiquitin antibody.(E) Linear ubiquitin chain assembly complex levels were determined in RCC4 and RCC41VHL cells by WB. (F) A549 cells were exposed to hypoxia for theindicated times. Cell lysates were isolated and HOIL-1L detected by WB. (G) A549 cells were stimulated with CoCl2 (300 mM) for the indicated times. (H)A549 cells were exposed to hypoxia for the indicated times and cell lysates were analyzed by WB. (I) A549 cells were transfected with HIF siRNA, andexposed to hypoxia for 24 hours and the expression of HOIL-1L was analyzed by WB. (J) A549 cells were transfected with HIF siRNA and exposed tohypoxia for 24 hours. RNA was analyzed by quantitative reverse transcriptase polymerase chain reaction. (K) A549 cells were pretreated withcycloheximide (CHX) and exposed to hypoxia for 12 hours. Cell lysates were analyzed by WB. Data represent mean 6 SEM; n > 3; **P , 0.01.

ORIGINAL ARTICLE

Queisser, Dada, Deiss-Yehiely, et al.: PKCz Ubiquitination and Degradation Promotes Cell Survival during Hypoxia 691

that RCC4 cells express higher levels ofHOIL-1L as compared with RCC41 pVHLcells, whereas the levels of HOIP andSHARPIN remained unchanged(Figure 2E). The native heteromericLUBAC exist as a high-molecular-weightcomplex of approximately 600 kD thatforms linear ubiquitin chains. Whenprotein extracts from RCC4 cells wereanalyzed by native gel electrophoresis,HOIL-1L was found in a complex togetherHOIP in a band of approximately 260 kDand also without HOIP in a faster-migrating band of approximately 200 kD.The 200-kD band was not observedafter the separation of proteins fromRCC41pVHL cells, whereas HOIP

migration did not change in eithercondition (see Figure E2C). These resultsare consistent with experiments in UOk111cells, another VHL-deficient cell line (35).Similar results were observed when A549 cellswere exposed to hypoxia (see Figure E2D).Furthermore, HOIL-1L abundanceincreased after 6 hours of hypoxia or CoCl2treatment (Figures 2F and 2G; see FigureE2E) and HOIL-1L mRNA increases beforePKCz degradation occurs (see Figure E2F),whereas HOIP and SHARPIN proteinlevels did not change (Figure 2H). Silencingof HIF-1a and HIF-2a prevented thehypoxia-induced increase in HOIL-1Lprotein and mRNA transcript (Figures 2Iand 2J). In cycloheximide-treated cells, the

up-regulation of HOIL-1L was prevented,and PKCz was not degraded during hypoxia(Figure 2K; see Figure E2G), suggesting thatthe newly synthesized HOIL-1L serves as thePKCz E3 ligase. Taken together, these resultssuggest that hypoxia induced the HIF-mediated up-regulation of HOIL-1L, whichindependently of the other LUBACcomponents conjugates PKCz with Lys-48ubiquitin chains promoting its degradationby the proteasome.

HOIL-1L Ubiquitinates PKCzIndependently of LUBACduring HypoxiaTo elucidate the mechanism by whichHOIL-1L targets PKCz for ubiquitin-mediated degradation, we compared the

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Figure 3. HOIL-1L ubiquitinates PKCz independently of linear ubiquitin chain assembly complex during hypoxia. (A) Schematic representation of theHOIL-1L/HOIP domain structure and its deletions. (B) A549 cells were transfected with wild-type (WT) HA-HOIL-1L, HA-HOIL-1LDUBL, or HA-HOIL-1LDNZF together with YFP-PKCz for 24 hours. YFP-PKCz was immunoprecipitated, lysates and immunoprecipitates were probed for HA and YFP. (C)Lysates and immunoprecipitates from A549 cells overexpressing YFP-PKCz and HA-HOIL-1LWT or HA-HOIL-1LDRING1, HA-HOIL-1LDRING2, and HA-HOIL-1L RING2CS were immunoprecipitated and immunoblotted for HA and YFP. (D) GST-fused PKCz was incubated in the presence of HA-HOIL-1L -WT,-DUBL, -DNZF, -DRING2, or -RING2CS for 2 hours at 378C and then pulled down with glutathione beads and immunoblotted for ubiquitin and ubiquitin-lys48 conjugates. (E) A549 cells were transfected with HA-HOIL-1L or HA-HOIL-1LDUBL, exposed to normoxia or hypoxia for 24 hours, and cell lysatesanalyzed by Western blot (WB). (F) A549 cells were cotransfected with YFP-PKCz, HA-HOIL-1L, and increased amounts of FLAG-HOIP or FLAG-HOIPDUBA for 24 hours as indicated. YFP-PKCz was immunoprecipitated and the interaction with HOIL-1L analyzed by WB. (G) HOIP overexpressionprevents PKCz degradation. A549 cells were transfected with FLAG-HOIP and exposed to normoxia or hypoxia for 24 hours. PKCz degradation was analyzedby WB. Data represented from at least three separate experiments. Asterisks indicate unspecific IgG band. RBR = RING-in-between-RING.

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692 American Journal of Respiratory and Critical Care Medicine Volume 190 Number 6 | September 15 2014

interaction between PKCz and WT HOIL-1L with that of deletion mutants of HOIL-1Land HOIP (Figure 3A). The ubiquitin-like(UBL) domain of HOIL-1L and theubiquitin-associated domain (UBA) ofHOIP are necessary for LUBAC formation,whereas the two RING domains ofHOIP are required for LUBAC’s linearpolyubiquitination activity (35). Furthermore,HOIL-1L Npl4 zinc finger domain (NZF)is required for specific recognition of linearubiquitin chains (36). Interestingly, neitherthe overexpression of HOIL-1LDUBL norHOIL-1LDNZF prevented the interactionbetween PKCz and HOIL-1L (Figure 3B).Apparently, the binding of the mutantHOIL-1L to PKCz is stronger than thatobserved with WT HOIL-1L. RBR-E3ligases transfer ubiquitin via a uniqueRING/HECT-hybrid mechanism in whichthe E2-Ub binds to the RING1 domain ofthe RBR E3 ligase, leading to the ubiquitintransfer from the E2 to the RING2-domain

and subsequently to the substrate. Thedeletion of the RING1 domain in HOIL-1Lhad no effect on the binding to PKCz,whereas overexpression of HOIL-1LDRING2 but not HOIL-1LRING2cs,which cannot form a thioester and iscatalytically inactive as a ligase, preventedthe interaction between PKCz andHOIL-1L (Figure 3C). Furthermore, inan in vitro assay, WT HOIL-1L, HOIL-1LDUBL, and HOIL-1LDNZF inducedthe ubiquitination of PKCz but HOIL-1LDRING2 and HOIL1L-RING2CS failedto do so (Figure 3D). Interestingly,overexpression of HOIL-1LDUBL increasedthe degradation of PKCz during hypoxia(Figure 6E; see Figure E3A). YFP-PKCz andHA-HOIL-1L were coexpressed in A549cells with increasing amounts of FLAG-HOIP to test whether HOIP competeswith the HOIL-1L- PKCz interaction. Asshown in Figure 3F (left) the interactionbetween PKCz and HOIL-1L decreased as

HOIP levels increased. This was confirmedby coexpression of YFP-PKCz and HA-HOIL-1L with increasing amounts ofHOIP-DUBA, which lacks the HOIL-1Lbinding domain and is incapable of forminga functional LUBAC (see Figure E3B).

As expected, HOIP-DUBA did notinterfere with the PKCz-HOIL-1Linteraction (Figure 3F, right). Moreover,HOIP overexpression prevented PKCzdegradation during hypoxia (Figure 3G;see Figure E3C), suggesting that HOIPmight sequester HOIL-1L and competewith binding to PKCz. These findings aresupported by knock-down experimentswhere silencing of HOIP decreases theexpression of PKCz (see Figure E3D).Taken together these results indicate thatduring hypoxia HOIL-1L does not requirethe other components of LUBAC andthat HOIL-1L binds PKCz via its RING2domain, which is also necessary for thecatalytic activity of the E3 ligase.

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Figure 4. PKCz down-regulation by HOIL-1L promotes tumorigenesis. (A) A549 cells were transfected with control, HOIL-1L, HOIP, or SHARPIN siRNA andexposed to hypoxia for 48 hours. Cytotoxicity was measured by lactate dehydrogenase release. (B) Stable A549 shHOIL-1L cells were transfected withcontrol and PKCz siRNA and exposed to hypoxia for 48 hours. (C) A549 cells transfected with shRNA were engrafted into the flanks of nude mice (n = 6).Tumor growth was determined by measuring the tumor size over 5 weeks. (D) Tumor sections were stained with hematoxylin and eosin (H&E; left) and forTUNEL-positive cells (brown; right). Representative pictures are shown. Macroscopic magnification 310. (E) Tumors were removed after 5 weeks andanalyzed by Western blot. HOIL-1L shRNA-derived tumors had higher levels of PKCz in comparison with scrambled shRNA-derived tumors. (F) Quantification ofE. (G) HOIL-1L shRNA-derived tumors had higher levels of PKCz in comparison with HOIL-1L1PKCz shRNA-derived tumors. (H) shHOIL-1L–derivedtumors had decreased weight, which could be rescued with double knock-down of HOIL-1L and PKCz after 5 weeks. Data represent mean6 SEM of n>3. *P < 0.05; **P < 0.01; ***P < 0.001. TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling; VEGF = vascular endothelial growth factor.

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Queisser, Dada, Deiss-Yehiely, et al.: PKCz Ubiquitination and Degradation Promotes Cell Survival during Hypoxia 693

PKCz Down-regulation by HOIL-1LPromotes Tumor GrowthTo evaluate the consequences of PKCzdegradation during hypoxia, the celldeath rate was determined. Duringnormoxia, cell death was approximately8%, which increased to approximately15% after exposing the cells to hypoxiafor 48 hours. Silencing of HOIL-1Lincreased cytotoxicity to approximately30%, whereas knock-down of the otherLUBAC members, such HOIP andSHARPIN, had no effect (Figure 4A).This observation was concordant with thedecrease in cell viability observed duringhypoxia in HOIL-1L–silenced cells (seeFigures E4A and E4B) and the rescue effecton cell death observed after silencing PKCzin these cells (Figure 4B). To confirm thatthe increase in cell death during HOIL-1Ldeficiency was mediated by PKCz, wetransfected A549 cells with an empty vector,WT PKCz, or with a kinase dead (K281R)mutant (PKCz-KD) (37) and exposedthem to hypoxia. Overexpression of WTPKCz during hypoxia dramaticallyincreased cell death, whereas PKCz-KDdid not (see Figure E4C).

To determine whether the degradationof PKCz by HOIL-1L contributes totumorigenesis, we used a murine xenografttumor model. We generated several cell linesin which HOIL-1L was stably silenced (seeFigure E5A); these cells were injectedsubcutaneously into the flanks of nude mice,and tumor progression was followed overa period of 5 weeks (see Figure E5B). StableHOIL-1L–silenced cells responded tohypoxia and stabilized HIF levels in a similarfashion as compared with sh-scr transfectedcells (see Figure E5C, bottom). InjectedHOIL-1L–silenced cells formed smallertumors (Figure 4C; see Figure E5D) andhistologic examination of tumor sections atlow and high magnification revealed thatcontrol sh-scr tumors have necrotic centralareas surrounded by cohesively growing cells(Figure 4D, left). The sh-HOIL-1L tumorsshowed areas of altered architectural featuresindicative of increased cell death, which wasfurther confirmed by increased number ofterminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL)-positive nucleiin the sh-HOIL-1L tumors (Figure 4D,right). In addition, HOIL-1L–silencedengrafted xenografts exhibited higher PKCz

expression, whereas HOIL-1L levels weresignificantly lower (Figures 4E and 4F). Torescue tumor growth in xenografts, doubleknock-down shHOIL-1L1shPKCz cellswere used (see Figure E5E). The decrease ingrowth observed after HOIL-1L silencingwas rescued in the double knock-down cells(Figure 4G; see Figure E5F). Western blotanalysis of the xenograft tissue revealedlow PKCz expression in comparison withshHOIL-1L–derived tumors after 5 weeksand the vascular endothelial growth factorexpression levels were comparable,indicating similar hypoxia (Figure 4H).

HOIL-1L Silencing Decreased LungTumor LoadIn a subsequent series of experiments,we investigated whether HOIL-1Lablation would decrease the growth ofhematogenously delivered lung tumors(Figure 5). We injected A549 cells withsilenced HOIL-1L (shHOIL-1L) or sh-scrA549 cells, described in Figure 4,intravenously and measured lung tumorprogression using micro positron emissiontomography examination. Animals injectedwith sh-HOIL-1L-A549 cells had a smaller

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tumor load as compared with controlsh-scrambled A549 cells (Figure 5A).Hematoxylin and eosin staining revealedcancer cell engraftment in the lungs of allanimals. However, the A549 sh-HOIL-1Lcells inoculated animals had significantlysmaller tumors (Figures 5B and 5C). Also,the sh-HOIL-1L–derived tumors exhibitedmore positive TUNEL staining (Figure 5D;see Figure E6). Taken together these resultssuggest that the absence of HOIL-1L, byincreasing the levels of PKCz, increases cancercell death impairing tumor progression.

HOIL-1L Expression Is Elevatedin AdenocarcinomaWe found higher RNA transcript levelsof HOIL-1L in tumor biopsies from patientswith lung adenocarcinoma compared withnormal adjacent tissue (NAT) (Figure 6A).Consistent with these findings, we foundincreased HOIL-1L expression in lungadenocarcinoma tissue microarray (TMA)by immunohistochemistry (Figure 6B;see Figure E7A). Consistent with the

HOIL-1L expression, Western blots fromadenocarcinoma tissue showed lower PKCzlevels as compared with NAT (Figure 6C;see Figure E7B). Corresponding withelevated HOIL-1L levels, HIF1a expressioncorrelates with the HOIL-1L expressionin adenocarcinoma grade III patients(R2 = 0.4313; P = 0.0203) (Figure 6D).

Tumors from patients with GBM havebeen reported to become severely hypoxic (1,7). In tumor tissue from patients with GBM,we found that HOIL-1L transcript levels weresignificantly increased in comparison withnormal astrocytes (see Figure E8A). Similarly,the relative abundance of HOIL-1L andPKCz protein was increased and decreased,respectively, in the tumors compared with theNAT (see Figures E8B and E8C).Furthermore, we analyzed a microarraydataset from the National Cancer Institutedatabase REMBRANDT, which includes 269glioma patients, and found that high HOIL-1L transcript levels were associated with poorprognosis (P = 0.000132317) (see FigureE8D). To test whether HOIL-1L regulation is

a universal phenomenon in cancer,we analyzed RNA microarray data fromhuman cancer specimens from theOncomine database. HOIL-1L transcriptlevels were up-regulated in 46 different arrays(1.5-fold; P = 0.0001) and HOIL-1L was oneof the top 10% regulated genes with thehighest differential expression in humancolorectal cancer specimens (1–5%) (seeFigure E8E). Taken together, thesedata suggest that tumor hypoxia leads toincreased HOIL-1L expression in differenttypes of cancer cells, which by decreasing thelevels of the tumor suppressor PKCz,facilitates the adaptation to hypoxia.

HOIL-1L Promotes TumorigenesisIndependently of LUBACTo rule out the possibility that HOIL-1Lknock-down was interfering with theLUBAC function, we generated a stable cellline overexpressing HOIL-1LDRING2 (seeFigure E9A). The deletion of RING2domain prevented the degradation of PKCz(see Figure E9B) but did not interfere withthe LUBAC-mediated nuclear factor-kBactivation (see Figure E9C). Comparedwith WT HOIL-1L, HOIL-1LDRING2overexpressing cells produced smallertumors, which was similar to the HOIL-1Lknock-down cells (see Figures E9D andE9E). Increased TUNEL staining was onlyseen in tumors derived from overexpressingHOIL-1LDRING2 cells (see Figure E9F).

Discussion

Cancer cells must adapt to survive hypoxicenvironments, which occur in solid tumors(38–40). We describe a novel mechanismof early response in cancer cell adaptationto hypoxia, where the stabilization of HIFincreases the expression of the E3 ligaseHOIL-1L, which forms Lys48-ubiquitinchains in the tumor suppressor PKCz,targeting it for proteasomal degradation(Figure 7). We found that during prolongedhypoxia PKCz was ubiquitinated anddegraded by the proteasome and haveidentified a novel pathway where HOIL-1Lserves as the essential E3 ligase for PKCzubiquitination. Different E3 ligases, such aspVHL and the HOIL-1L/HOIP complex,have been reported to interact and todegrade PKCs (41–43). We have excludeda role for pVHL in PKCz degradation byshowing that pVHL-deficient RCC4 cellshave lower basal levels of PKCz as compared

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Figure 6. HOIL-1L expression is elevated in adenocarcinoma. (A) HOIL-1L mRNA levels wereanalyzed by quantitative reverse transcriptase polymerase chain reaction in biopsies fromadenocarcinoma patients and compared with normal adjacent tissue (NAT). (B) Adenocarcinoma(grade I-III) tissue microarray was immunostained for HOIL-1L. (C) PKCz and HOIL-1L levels wereanalyzed by Western blot in biopsies from adenocarcinoma patients (grade I-II) and compared withNAT. (D) HOIL-1L expression correlates with HIF-1a expression in adenocarcinoma tissue (grade III).Data represent mean 6 SEM of at least three separate experiments. *P < 0.05; **P < 0.01.

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Queisser, Dada, Deiss-Yehiely, et al.: PKCz Ubiquitination and Degradation Promotes Cell Survival during Hypoxia 695

with RCC41VHL. We then investigated therole of the HOIL-1L/HOIP complex in thehypoxic degradation of PKCz. Nakamuraand coworkers (18) described that HOIL-1Lcoimmunoprecipitated with PKCz and thatthe activation of classical PKC increasesHOIL-1L/HOIP binding. We purified HOIL-1L/HOIP from A549 cells and performed anin vitro ubiquitination assay in which HOIL-1L/HOIP ubiquitinated PKCz forming Lys-48chains. Importantly, in this assay theformation of linear ubiquitin chains wasprevented by the use of recombinant His-tagged ubiquitin, indicating a nontraditionalrole of HOIL-1L in the ubiquitination ofPKCz. We also found that hypoxia increasesthe interaction between PKCz and HOIL-1Lbut not with HOIP or SHARPIN and thatonly the RING2 domain of HOIL-1Linteracted with PKCz and was necessaryfor the catalytic activity. Surprisingly,overexpression of HOIL-1L or HOIL-1LDUBL led to PKCz degradation duringhypoxia and either ubiquitinated PKCzin vitro. These results indicate that HOIL-1L issufficient to degrade PKCz during hypoxia. Inagreement with this, silencing HOIP did notprevent PKCz degradation during hypoxiasuggesting again a HOIP-independent

function of HOIL-1L. In agreement with ourresults, HOIL-1L has been reported to formLys48-polyubiquitin chains in several proteinsincluding iron regulatory protein 2, thetranscription factor Bach, and interferonregulatory factor 3 (44, 45).

The physiologic role of HOIL-1L hasbeen mostly attributed to its functionas a component of LUBAC regulatinginflammation and the immune response(46–48). Patients with HOIL-1L mutationshave inflammatory disorders (49) and HOIL-1L knock-down has been reported to impaircell invasiveness in osteosarcomas (24).Silencing of HOIL-1L inhibited tumor growthand promoted apoptosis in murine modelsof cancer. Lung adenocarcinoma and othernon–small cell lung cancers and GBM areaggressive cancers that grow rapidly resultingin a hypoxic environment (38). Cells overcomehypoxic stress through multiple mechanisms,including the stabilization of HIFs. Bothisoforms, HIF-1a and HIF-2a, areoverexpressed in human non–small cell lungcancer, suggesting that they contribute totumor progression (50). In tumors from lungadenocarcinoma and GBM patients, we foundthat the transcription levels and HOIL-1Lprotein abundance was higher relative to the

NAT and that PKCz levels were reduced.Although tumors develop long-termadaptation mechanisms, an important findingin the current study is that the up-regulationof HOIL-1L via HIF is an early response tofacilitate the fast adaptation of the cancercell to the hypoxic environment. Althougha previous report has shown thatHIF regulatesthe mitochondrial protease LON, which isrequired for COX4–1 degradation andadaption to hypoxia (51), this is the firstreport, to our knowledge, describing a HIF-dependent E3 ligase that promotes theproteasomal degradation of a tumor-suppressor factor. Our data indicate thatHOIL-1L expression is increased in differentcancer types (experimentally andoncogenomic analysis) and in a broad range oftumor types, suggesting a universal response tohypoxia in cancer cells. Taken together, theseresults indicate that HOIL-1L expression incancer is an important mechanism of earlyadaptation and tumorigenesis and may serveas a prognostic biomarker. The clinicalimportance of these findings is supported bydata from human cancer tissues, whereincreased expression of HOIL-1L anddecreased PKCz levels are associated withworse clinical outcomes. Conceivably,the E3-ligase activity of HOIL-1L canpotentially serve as a therapeutic target for thetreatment of solid tumors. n

Author disclosures are available with the textof this article at www.atsjournals.org.

Acknowledgment: The authors thank Dr. OlgaVagin (Department of Physiology, UCLA andVeterans Affairs Greater Los Angeles Health CareSystem) for critical evaluation of the manuscriptand Lucas Sullivan, Mary A. Baker, ErmelindaCeco, Lynn Welch, Kelly Starichia, DaleShumaker, and Alejandro P. Comellas (Divisionof Pulmonary and Critical Care Medicine,Northwestern University) for their technicalassistance. They thank Daniel Procissi from theCenter for Translational Imaging, Departmentof Radiology, Northwestern University forperforming the micro positron emissiontomography imaging and Joseph Phillips whoestablished the lung tumor bank at the LurieCancer Center.

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