Acidosis Acts through HSP90 in a PHD/ VHL-Independent Manner … · and acidosis functionally cooperate in inducing HIF transcrip-tion factors and CSC maintenance. We found that these

Tumor and Stem Cell Biology

Acidosis Acts through HSP90 in a PHD/VHL-Independent Manner to Promote HIFFunction and Stem Cell Maintenance in GliomaAlina Filatova1, Sascha Seidel1,2, Nuray B€o�g€urc€u1, Sabine Gr€af1, Boyan K. Garvalov1,and Till Acker1

Abstract

Hypoxia is a common feature of solid tumors, which controlsmultiple aspects of cancer progression. One important functionof hypoxia and the hypoxia-inducible factors (HIF) is themaintenance of cancer stem-like cells (CSC), a populationof tumor cells that possess stem cell-like properties and drivestumor growth. Among the changes promoted by hypoxiais a metabolic shift resulting in acidification of the tumormicroenvironment. Here, we show that glioma hypoxiaand acidosis functionally cooperate in inducing HIF transcrip-tion factors and CSC maintenance. We found that these effectsdid not involve the classical PHD/VHL pathway for HIF upre-

gulation, but instead involved the stress-induced chaperoneprotein HSP90. Genetic or pharmacologic inactivation ofHSP90 inhibited the increase in HIF levels and abolished theself-renewal and tumorigenic properties of CSCs induced byacidosis. In clinical specimens of glioma, HSP90 was upregu-lated in the hypoxic niche and was correlated with a CSCphenotype. Our findings highlight the role of tumor acidifica-tion within the hypoxic niche in the regulation of HIF and CSCfunction through HSP90, with implications for therapeuticstrategies to target CSC in gliomas and other hypoxic tumors.Cancer Res; 76(19); 5845–56. �2016 AACR.

IntroductionRapidly growing tumors often outpace their blood supply

generating a hypoxic microenvironment. Tumor hypoxia trig-gers a set of adaptive responses that ultimately promote a moreaggressive tumor phenotype and are primarily controlled bythe transcription factor system of the hypoxia-inducible factors(HIF; ref. 1). HIF abundance is tightly regulated by the prolylhydroxylase domain (PHD) proteins. PHDs use O2 to hydrox-ylate HIFs (2, 3), which enables binding of the VHL protein, acomponent of an E3 ubiquitin ligase complex that catalyzesHIF1/2a ubiquitination and degradation. One of the primaryeffects of hypoxia is the induction of a metabolic shift fromoxidative phosphorylation to glycolysis, with lactic acid as theend product (4). This is accompanied by the upregulation ofcarbonic anhydrases, transporters for lactate, Hþ, HCO3

�, andother ions, leading to a net acidification of the extracellulartumor environment (5, 6). A decreased pH is a characteristicfeature of many tumor types and a number of studies havestarted to delineate critical functions of the acidic extracellular

tumor environment (7). For example, blockade of carbonicanhydrase IX (CA IX), a hypoxia upregulated transmembraneenzyme that converts H2O and CO2 into carbonic acid (Hþ

and HCO3�), increased extracellular pH, decreased prolifera-

tion, enhanced apoptosis, and markedly suppressed tumorgrowth in vivo (5, 8). Acidic extracellular pH has also beenshown to promote tumor cell migration, invasiveness, andmetastasis (8–10). Although tumor hypoxia and acidosis arelinked and both represent crucial aspects of the tumor micro-environment, their interplay in tumor progression remainspoorly understood.

It has become clear that tumor growth andprogression is drivenby a subpopulation of cells with stem cell–like properties, termedcancer stem cells (CSC) or tumor-initiating cells (11). We andothers have previously established that hypoxia plays a key role inpromoting the CSC phenotype through HIFs (12, 13). Acidicstress has also been shown to promote a cancer stem cell phe-notype (14); however, very little is known about the mechanismsthrough which microenvironmental pH may impact on hypoxicsignaling and control CSCmaintenance within the hypoxic niche.Interestingly, glioblastoma cells exposed to more acidic condi-tions show an increased resistance to chemotherapeutics (15),and elevated expression of VEGF (16), properties characteristic ofCSCs. Thus, the contribution of acidosis to the regulation of CSCs,particularly within the hypoxic CSC niche, is of high interest andpotential therapeutic relevance.

Here, we show that hypoxia and acidosis synergize to increaseHIF levels and function in glioblastoma, resulting in a markedpotentiation of CSC self-renewal, maintenance, and tumorige-nicity. The pH-mediated regulation of HIF, CSC function, andtumorigenicity is not dependent on PHD/VHL activity, but isinstead controlled by HSP90. Furthermore, increased HSP90levels in human glioblastomas are associated with a higherexpression of HIF target genes and CSC markers, highlighting

1Institute of Neuropathology, University of Giessen,Giessen,Germany.2Institute of Cell Biology and Neuroscience and Buchmann Institutefor Molecular Life Sciences (BMLS), University of Frankfurt, Frankfurt,Germany.

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

A. Filatova, S. Seidel, N. B€o�g€urc€u, B.K. Garvalov, and T. Acker contributed equallyto this article.

Corresponding Author: Till Acker, Institute of Neuropathology, University ofGiessen, Arndtstr. 16, 35392 Giessen, Germany. Phone: 49-64-1994-1181; Fax:49-64-1994-1189; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2630

�2016 American Association for Cancer Research.

CancerResearch

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the potential importance ofHSP90 as a druggable pathway for thetargeting of glioma CSCs.

Materials and MethodsCell culture

The glioblastoma cell linesG55TL,G121,G141, andG142werekindly provided by K. Lamszus and M. Westphal [Department ofNeurosurgery, University Medical Center Hamburg-Eppendorf(UKE), Hamburg, Germany; ref. 17], the renal carcinoma line786-O was obtained from ATCC and RCC11 (18) was obtainedfrom Dr C. Bauer (Institute of Physiology, University of Zurich,Zurich, Switzerland) between 1998 and 2002. The primary glio-blastoma line NCH644was kindly provided by C. Herold-Mende(Heidelberg, Germany; ref. 19) in 2010. The primary glioblasto-ma lines GBM015 and GBM031 were obtained in 2007–2008from patients undergoing surgery in accordance with a protocolapproved by the institutional review board and cultured in neuro-sphere medium (13). G55TL, G121, G141, and G142 cells werecultured in DMEM (Invitrogen) supplemented with 10% FBS(PAN Systems). NCH644, GBM015, and GBM031 were propa-gated under neurosphere conditions in neurosphere medium(DMEM-F12; Invitrogen), 5 mmol/L HEPES, 2% B-27 serum-freesupplement without vitamin A (Invitrogen) supplemented with20 ng/mL bFGF and 20 ng/mL EGF (PeproTech)]. All cells weremaintained at 37 �C in 5% CO2. For experiments with differentpH, G55TL, 786-O, and RCC11 cells were incubated in CO2

-independent medium (#18045-054, Invitrogen) supplementedwith 2 mmol/L L-glutamine and 10% FBS; the primary gliomacells lines were incubated in CO2-independent medium supple-mented with 2% B-27 serum-free supplement without vitamin A,2 mmol/L L-glutamine, 20 ng/mL bFGF, and 20 ng/mL EGF. ThepH of the medium was adjusted to the values indicated in thefigureswith 1mol/LHClor 1mol/LNaOH. For hypoxic treatmentcells were grown at 1% O2 for the indicated periods of time in aHypoxicWorkstation (Ruskinn Technology). NCH644,GBM031,and GBM015 were preincubated in medium with pH7.4 atnormoxia and incubated in media with pH7.4 or 6.7 at hypoxia.For geldanamycin treatment, cells were preincubated with pH 7.4at 21%O2 for 4 days; in the last two hours of the preincubation 1mmol/L geldanamycin (Enzo Life Sciences) or control DMSOwasadded, followed by incubation at 1%O2 in medium with pH 7.4or 6.7 supplemented with DMSO control or 1 mmol/L geldana-mycin for 6 or 18 hours (for G55 andNCH644 cells, respectively).Further details on the experimental procedures are included in thein corresponding figure legends. For stem cell condition, the cellswere incubated in neurosphere medium for the same time. Cellsthat showed unusual growth or morphology were tested formycoplasma contamination and only noncontaminated cellswere used for experiments. Cell lines obtained from ATCC wereauthenticated by the manufacturer. No additional authenticationwas performed by the authors for any of the cell lines.

Tumor xenograftsAnimal experimentswereapprovedby theveterinarydepartment

of the regional council in Darmstadt, Germany. Xenograft trans-plantationswere performed in athymic 6–8week old femaleNMRInu/numice (Janvier Labs) thatwere kept in a specific pathogen-freeanimal facility according to the institutional guidelines.

For intracranial tumor transplantation, mice were placedinto a stereotactic apparatus, and 1 � 105 cells in a volumeof 2 mL (NCH644), or 7.5� 103 cells in a volume of 1 mL (G55)

were resuspended in cold, CO2-independent medium andslowly injected into the left striatum. At the onset of neurologicsymptoms, mice were sacrificed at the same time point (at day19 after transplantation for the experiments with G55 cells,and at day 22 posttransplantation for the experiments withNCH644 cells) through deep anesthesia with ketamine andxylazine. The chest was opened and the vasculature was per-fused with a 0.9% NaCl solution for 2 minutes and fixative(4% PFA) for 4 minutes. Brains were removed and additionallyfixed overnight in 4% PFA, dehydrated in 30% sucrose for4 days, and rapidly frozen on dry ice for sectioning with a cryo-tome. The sections were stained with hematoxylin and eosin.Tumor volume was determined using stereological quantifica-tion of series of every twelfth 40-mm section (480 mm intervals)throughout the brain using ImageJ, as previously described(NCH644; ref. 20). G55 tumor–bearing mice were injectedwith 60 mg/g Hypoxyprobe intraperitoneally 90 minutes priorto cardiac perfusion with 0.9% NaCl solution. After removal ofthe brain and dissection of the tumors, one part of each tumorwas snap-frozen in liquid nitrogen for subsequent proteinisolation. Another part of the tumor was incubated in fixativeovernight and embedded in paraffin for subsequent histo-logic and immunohistochemical analysis. Tumor volume wasanalyzed using the section with the biggest tumor area bymeasuring the largest diameter (L) and largest perpendiculardiameter (W), using the formula L � W2/2 (G55).

For subcutaneous tumor injections, 7.5 � 104 G55 cells sus-pended in 0.1mLPBS/Matrigel were injected subcutaneously intothe flanks of 6–8-week-old female nude (NMRI nu/nu) mice. Thetumor size was measured at regular intervals using a caliperaccording to the formula V ¼ L � W2/2. Mice were maintaineduntil tumors exceeded a volume of 2,000 mm3 or upon the onsetof morbidity symptoms (>20% weight loss, tumor ulceration).Tumors were excised and snap-frozen in liquid nitrogen forsubsequent protein isolation.

TransfectionG55TL cells were transiently transfected with plasmid DNA

using Lipofectamine 2000 according to the manufacturer'sinstructions. siRNAs against HIF1a, HIF2a, or nontargeting con-trol siRNA were obtained as a pool of 4 siRNA oligos (ON-TARGETplus SMART pool, Dharmacon). Reverse siRNA transfec-tion of GBM015 cells was performed with 10 pmol siHIF1a and50 pmol siHIF2a, or 60 pmol nontargeting siRNA, respectively,with Lipofectamine RNAiMAX (Thermo Scientific) in antibiotic-free neurosphere medium according to the manufacturer'sinstructions. Twenty-four hours after transfection, the cells wereseeded in CO2-independent neurosphere medium at pH 7.4 andpreincubated under normoxic conditions for 48 hours. Subse-quently, the cells were cultured under 1% O2 neurosphere medi-um at pH 7.4 or 6.7 and harvested after 18 hours (protein andRNA) or 96 hours (FACS).

ImmunoblottingFor immunoblotting, cells were harvested in PBS (4�C), cell

pellets were lysed in 10 mmol/L Tris.HCl (pH 7.5), 2% SDS,2mmol/L EGTA, 20mmol/LNaF, and 15–50 mg of protein lysateswere subjected to SDS-PAGE and Western blot analysis usingantibodies specific for CD133 (Abcam ab19898 and MiltenyiBiotec 130-092-395), GFAP (Dako Z 0334), HIF1a (CaymanChemical 10006421 or BD Transduction Laboratories 610958),

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HIF2a (Novus Biologicals NB 100-122), HSP90 (Enzo/StressgenADI-SPA-830), V5 (Invitrogen R960-25), VHL (Abcam,ab11189), and Tubulin (Dianova DLN09992) as a loading con-trol. Immunoreactive bands were visualized with the ECL system(Perkin Elmer or Pierce).

FACSSpheres were dissociated into single-cell suspension by Accu-

tase (PAA) treatment for 15 minutes at 37�C. After two washingsteps with FACS Staining buffer (PBS, pH 7.2, 0.5% BSA, 2mmol/L EDTA), single cells were blocked with 20 mL of normal mouseIgG (Invitrogen) for 20 minutes at 4�C and stained with 10 mL ofCD133/2 (293C3)-PE–conjugated antibody (Miltenyi Biotec130-090-853) or 5 mL of CD15-V450–conjugated antibody (BDBiosciences 642917) for 30 minutes at 4�C. The backgroundstaining was determined using matching isotype control antibo-dies from the same manufacturers at the same concentration asthe specific antibodies. Five minutes before analysis, 1 mmol/LSYTOX Blue (Invitrogen; CD133/2-PE) or SYTOX Red (CD15)nucleic acid stain was added to exclude dead cells. Flow cyto-metry was performed using BD FACSCanto II (BD Biosciences).Data were analyzed using FlowJo v7/9 (Tree Star) by gating forlive cells based on SYTOX staining, then for singlets usingforward scatter area versus width and side scatter area versuswidth, followed by gating for the CD133 or CD15-positivepopulations, respectively.

Sphere forming units and in vitro growthA total of 1� 106 cells were seeded per 10-cmdish and cultured

at pH7.4or 6.7 at 1%O2 for 96hours. For quantificationof sphereforming units (SFU), the cells were then split in 12-well suspen-sion culture plates (n ¼ 12) at a density of 300 cells/well in 1 mLor 6-well suspension culture plates (n ¼ 6) at a density of500 cells/well in 2-mL neurosphere medium. After 7 days, thespheres were counted and the percentage of sphere-forming cellswas calculated. For sphere formation experiments applyingsiRNA-mediated knockdowns, cells were transfected as describedabove. Twenty-four hours after transfection, medium was chan-ged and the cells cultured at pH7.4 or 6.7, 1%O2 for 72 hours. Forquantification of SFUs, the cells were then split in 6-well suspen-sion culture plates (n ¼ 6) at a density of 500 cells/well in 2-mLneurosphere medium. After 7 days, the spheres were counted andthe percentage of sphere-forming cells was calculated.

Real-time quantitative RT-PCRRNA was extracted with the RNeasy Mini Kit (Qiagen), and

reverse transcribed using standard protocols (RevertAid H MinusM-MuLV Reverse Transcriptase, Fermentas). cDNA was amplifiedusing the ABsolute QPCR SYBR Green Mix. Gene-specific PCRproducts weremeasured continuously in a StepOnePlus real-timePCR system (Applied Biosystems) for up to 45 cycles. The differ-ence in the threshold number of cycles between the gene ofinterest and HPRT (hypoxanthine phosphoribosyltransferase 1)was then normalized relative to the standard chosen for eachexperiment and converted into fold difference.

IHCImmunohistochemical stainings of human biopsies were

approved by the institutional review board and were carriedout using antibodies against HIF1a (Cayman Chemical10006421), HSP90 (Enzo/Stressgen ADI-SPA-830), and CD133(Miltenyi Biotec 130-092-395) with an Autostainer (Ventana/

Roche) according to the manufacturer's instructions. ForHypoxyprobe detection (Hypoxyprobe-1, NPI Inc.), paraffinsections were dewaxed in xylene (2 � 10 minutes) followedby a series of descending alcohol concentrations (2� 5 minutes100% EtOH, 2 � 5 minutes 96% EtOH, 2 � 5 minutes 70%EtOH) and rehydrated for 5 minutes in H2O and TBS buffereach. For antigen retrieval, the sections were boiled in preheatedcitrate buffer (pH 6) for 10 minutes and subsequently cooleddown at room temperature for 20minutes, followed by washingsteps in TBS (2 � 5 minutes). Endogenous peroxidase wasblocked in 0.6% H2O2 for 30 minutes followed by washingsteps with TBS (3 � 5 minutes). Unspecific antibody bindingwas blocked with 10% normal goat serum (NGS) in TBS/0.1%Tween (TBST) for 1 hour at room temperature, followed byincubation with FITC-Mab1 antibody (1:100 in 10% NGS/TBST, NPI Inc.) for 2 hours at room temperature. After threewashing steps in TBST (5-minute each), sections were incubatedwith anti-FITC HRP secondary antibody (1:200 in 10% NGS/TBST, NPI Inc.) for 2 hours at room temperature followed bythree washing steps in TBST. Chromogen reaction was per-formed using diaminobenzidine (DABþ, Dako) for 3 minutes.Counterstaining was performed in hematoxylin for 2 minutes.To achieve color development of the stained nuclei, sectionswere rinsed in tab water for 2 minutes followed by a rinse in H2

O. Sections were dehydrated in a series of ascending alcoholconcentrations (2 � 5 minutes 70% EtOH, 2 � 5 minutes 96%EtOH, 2 � 5 minutes 100% EtOH) with a final incubation inxylene (2 � 5 minutes). Sections were permanently mountedusing Cytoseal XYL (Thermo Scientific).

Luciferase reporter assaysA total of 3 � 104 G55 cells were plated in 24-well plates.

Twenty-four later the cells were transfected using SuperFect(Qiagen) with VEGF promoter-firefly luciferase (21) and a SV40Renilla luciferase constructs for normalization of transfectionefficiency. To assess the effect ofHIFs onVEGFpromoter transcrip-tion, the cells were cotransfected with 300 ng pcDNA3-HIF1aor HIF2a overexpression constructs, or pcDNA3 as a control.The next day, the cells were placed at the indicated concentra-tions of O2 and CO2 and 24 hours later were lysed for analysisof luciferase expression downstream of the VEGF promoter, usinga dual luciferase reporter system (Promega), according to themanufacturer's instructions.

Lentiviral constructs and stable cell linespGIPZ lentiviral constructs against HSP90a, HSP90b, and

pGIPZ nonsilencing control vectors were purchased from OpenBiosystems (Thermo Scientific). Lentiviral HA-tagged dominant-negativemutant of HSP90b (HA-HSP90DN) or a GFP-expressingcontrol plasmids were constructed as described in the Supple-mentary Materials and Methods. Lentivirus was packaged bycotransfection of lentiviral vectors with the packaging plasmidspCI-VSVG and psPAX into 14-cmplates withHEK293T cells usingFugeneHD(Roche).Mediumwas changed every 24hours, the 48-and 72-hour supernatants were pooled, filtered through a 0.45-mm filter, and ultracentrifuged at 20,000 � g, 4�C for 4 hours.Titers were determined by counting the number of GFP-positivecolonies or crystal violet–stained colonies. The shHSP90 andcontrol lines were generated by lentiviral transduction and selec-tion with 2 mg/mL puromycin to create the stable shRNA pools.The HA-HSP90 DN and GFP lines were generated by lentiviral

Acidosis Promotes HIF and Glioma Stem Cells

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transduction and selection with 6 mg/mL blasticidin to create thestable HA-HSP90 DN or GFP-overexpressing cells.

Bioinformatic analysisGene expression data [z-scores (all genes)] for the glioblas-

toma cohort of The Cancer Genome Atlas (TCGA) ResearchNetwork were downloaded from the cBio portal (http://www.cbioportal.org/public-portal/index.do). Tumors with z-scoresfor HSP90a < �1 (n ¼ 90) were considered as low HSP90-expressing, tumors with z-scores of > þ1 (n ¼ 64) were con-sidered as high HSP90 expressing.

Statistical analysisResults are presented asmean� SEM. Sample size was chosen

based on previous empirical experience with the respectivetypes of assays or animal tumor models. Animals that unex-pectedly died before tumors were collected from the remaininganimals were excluded from the analysis. The experiments werenot randomized and the investigators were not blinded toallocation during experiments and outcome assessment. Forpairwise comparisons, statistical analysis was performed usingthe Student t test except for the expression analysis in the TCGAglioblastoma cohort, where the Mann–Whitney test was useddue to non-normal distribution. For comparisons betweenmultiple groups, ANOVA with a Bonferroni post test wasperformed using GraphPad Prism. Statistical significance wasdefined as �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

ResultsAcidosis increases HIF function and the CSC phenotype

We were interested in analyzing to what extent metabolicparameters of the hypoxic tumor microenvironment other thanpO2 may regulate HIF levels and function in glioblastoma. Astumor hypoxia is frequently accompanied by acidosis andhypoglycemia, we cosubjected a panel of glioblastoma celllines to different hypercapnic conditions (10% and 20%CO2), as a physiologic means of lowering the pH, and differentglucose levels (high glucose: 4.5 g/L; low glucose: 1.0 and 0 g/Lglucose; Fig. 1A and B). Under normoxia, only very low-level ofHIF1a and moderate levels of HIF2a were detectable, whichwere highly upregulated under hypoxia (Fig. 1A and B) with aconcomitant increase in mRNA expression of the HIF-targetgenes VEGF, LDHa, Glut-1, and CAIX (Supplementary Fig. S1).Glucose deprivation mostly did not alter or even decreasedHIF1a and HIF2a abundance under normoxia and hypoxia. Incontrast, decreasing the pH through hypercapnia moderatelyincreased HIF1a and HIF2a levels already under normoxia(Fig. 1A and B) and dramatically elevated the hypoxic upre-gulation of HIF1a and HIF2a, demonstrating that hypoxia andacidosis synergistically increase the HIF response (Fig. 1B). Inline with the pH-dependent control of the HIF response,hypercapnia efficiently potentiated the hypoxia- and HIF1/2a–driven transcription downstream of the VEGF promoter(Fig. 1C), suggesting a posttranscriptional HIFa regulationby pH. As CO2 can exert effects on cell physiology that areindependent of acidification of the cellular environment (22),we next modulated the pH by addition of NaOH and HCl inCO2-independent medium (Fig. 1D and E). Acidic pH wassufficient to upregulate HIF1a and HIF2a levels already undernormoxia and even further potentiated the hypoxia-dependent

increase in HIF1/2a expression (Fig. 1D). Decreasing the pHdown to 6.6–6.8, conditions that are observed in tumors (23),efficiently induced HIF1a and HIF2a expression under nor-moxia and hypoxia, whereas further pH reduction decreasedHIF1/2a abundance (Fig. 1E).

Previous work by us and others has demonstrated that hyp-oxia promotes the CSC phenotype through the induction ofHIFs (12, 13). As expression and activity of HIFs were induced bylow pH, we assessed whether acidosis would also promote thestem cell properties of glioblastoma cells. We first confirmed thepH-dependent regulation of HIF and HIF target genes in a panelof primary glioblastoma lines derived from patient biopsiesand continuously propagated in serum-free sphere (stem cell)conditions (Fig. 2A and B). Importantly, acidosis concomitantlyincreased the expression of a panel of CSC-associated genesand the fraction of cells positive for the glioma CSC markersCD133 and CD15 (Fig. 2C and Supplementary Fig. S2Aand S2B), as well as the capacity to form spheres as a measureof self-renewal (Fig. 2D). Notably, the stimulation of CSCmaintenance by low pH was dependent on HIFs, as HIF1/2asilencing (Supplementary Fig. S3A and S3B) abrogated not onlythe acidosis-induced upregulation of HIF target gene expression(Fig. 2E), but also the increase in the fraction of cells positivefor the CSC marker CD133 and in self-renewal capacity (Fig. 2Fand G). Collectively, our results identify acidosis as a potentmetabolic factor that regulates HIF function and the CSC phe-notype synergistically with hypoxia within the pathophysio-logic pH range observed in the tumor microenvironment.

The pH-mediated control of HIF is PHD/VHL independentWe next aimed at identifying the molecular mechanism that

allows acidosis to regulate HIF. It has been previously shown, thatan acidic pHof 5.8–6.2 inducesHIF1abynuclear sequestration ofVHL, resulting in a relative cytoplasmic lack of VHL and inhibitingVHL-dependent proteasomal degradation (24). To examinewhether an acidic pH in the range of 6.7–6.9, values typicallymeasured in tumors (14, 23), regulates HIF through VHL we firstanalyzed the influence of acidification on HIF levels in the VHL-deficient renal cell carcinoma lines 786-O and RCC11 (whichexpress high HIF2a, but not HIF1a; refs. 18, 25). Importantly,lowered pH robustly increased HIF levels in these cells (Fig. 3A),demonstrating that the effect of acidosis is not VHL dependent.Consistently, increasing the intracellular levels of VHL by over-expression in glioblastoma cells did not affect the acidosis-induced hypoxic upregulation of HIFs (Fig. 3B). Furthermore,blockade of PHD function using the iron chelator dipyridyl (DP)and the PHD family inhibitor dimethyloxalylglycine (DMOG)did not affect the induction of HIF levels by acidosis (Fig. 3C).Further in line with a PHD/VHL-independent regulation ofHIF proteins by acidosis, nonhydroxylatable mutant proteins ofHIF1a and HIF2a with substitution of both prolyl residues thatare hydroxylated by PHDs (mPPNmutants, ref. 13), were stronglyincreased by acidic pH (6.7; Fig. 3D). Taken together, these dataprovide compelling evidence that acidosis regulates HIF levelsindependently of the PHD/VHL-mediated degradation pathwayin glioma.

Acidosis controls HIF and the CSC phenotype through HSP90Apart from the oxygen-dependent PHD/VHL pathway, one

of the best characterized mechanisms controlling HIF stabi-lity is linked to HSP90. HSP90 interacts with HIF (26, 27)

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and competes with the protein RACK1 for association withHIF, thus protecting HIF from proteasomal degradation (28).Therefore, we next examined whether the acidosis-dependentupregulation of HIF levels and the CSC phenotype are coupledto HSP90. We found that lowered pH induced a markedupregulation of HSP90 in established and primary glioblasto-ma cell lines (Fig. 4A and B). Importantly, inhibition of HSP90function with geldanamycin abolished the acidosis-inducedincrease in HIF1/2a and CD133 (Fig. 4C and D). Similarly,

a dominant-negative HSP90 construct (29) blocked theincrease of HIF levels at acidic pH (Fig. 4E), demonstratingthat HSP90 activity is required to mediate HIF upregulationunder conditions of acidic stress. To corroborate the HSP90dependent control of HIF we tested whether increasing HSP90levels would replicate the effects of acidosis. Indeed, over-expression of wild-type HSP90 already at physiologic pH ledto an elevation of HIF1a and HIF2a levels similar to the onecaused by low pH (Fig. 4F).

Figure 1.

Hypercapnia and acidosis increase HIF levels and activity synergistically with hypoxia. A, hypercapnic acidosis induces HIF1a and HIF2a. Immunoblot of G55,G121, G141 and G142, glioblastoma cell lines exposed to normoxia (21% O2), hypoxia (1% O2), normocapnia (5% CO2), hypercapnia (10% CO2), and varyingglucose levels (high glucose, 4.5 g/L; low glucose, 1.0 and 0 g/L glucose) for 18 hours. B, hypercapnic acidosis synergizes with hypoxia to induce HIF.Immunoblot of G55 cells, exposed to normoxia (21% O2), hypoxia (1% O2), hypocapnia (2.5% CO2), normocapnia (5% CO2), hypercapnia (10%, 20% CO2) for 18hours. The panel shows nonadjacent lanes from the same exposure of the same Western blot analysis. C, hypercapnic acidosis increases HIF1/2atransactivation activity. G55 cells were cotransfected with a VEGF promoter luciferase reporter together with empty vector, HIF1a, or HIF2a and exposed tothe indicated O2 and CO2 concentrations for 18 hours (n ¼ 3). D, acidic pH increases HIF1/2a levels. Immunoblot of G55 cells grown under normoxia orhypoxia (1% O2) in CO2-independent medium with acidic pH (6.7) or physiologic pH (7.4). E, acidosis induces HIF1a and HIF2a within a restricted pH range.Immunoblot of G55 glioblastoma cells cultured in CO2-independent medium with decreasing pH (7.2–6.1) in normoxia and hypoxia (1% O2) for 6 hours.






We next assessed the function of HSP90 in HIF regulation ina microenvironmental setting of hypoxia and acidosis in vivo.Importantly, HSP90 disruption by shRNA-mediated knock-down in glioblastoma cells significantly reduced intracranial

tumor growth (Fig. 5A and B). While both control and shHSP90glioblastomas displayed typical perinecrotic areas with pro-nounced hypoxia, as revealed by Hypoxyprobe staining(Fig. 5C), HIF1/2a levels and the expression of the HIF target

Figure 2.

Acidosis induces HIF function and CSC self-renewal. A and B, acidosis induces expression of HIF1a and HIF2a and their target genes in primaryglioblastoma stem cell lines under hypoxia. Immunoblot (A) and real-time RT-PCR (B) of NCH644, GBM015, and GBM031 primary glioblastoma stemcell lines following exposure to pH7.4 (physiologic pH) or 6.7 (acidic pH) at 1% O2 for 18 hours (n ¼ 3). C and D, acidosis induces a cancer stem cellphenotype in primary glioblastoma cells. Quantification of the fraction of CD133þ cells, determined by FACS analysis (n ¼ 3; C) and sphere formingcapacity (n ¼ 12; D) of NCH644, GBM015, and GBM031 primary glioblastoma cells after exposure to pH 7.4 or 6.7 at 1% O2 for 96 hours. E–G, acidosisinduces the CSC phenotype through HIF1/2a. Quantification of the expression of the HIF target gene CA IX (E), the fraction of CD133þ cells (n ¼ 3; F), andthe sphere-forming capacity (n ¼ 6; G) of GBM015 primary glioblastoma cells transfected with nonsilencing control or HIF1/2a siRNA and incubatedat pH 7.4 or 6.7 at 1% O2 for 96 hours. All values are means þ SEM, � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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gene VEGF were significantly reduced in shHSP90 tumors(Fig. 5D–F). A similar decrease of HIF levels and orthotopictumor growth was elicited HPS90 inhibition through a dom-

inant-negative HSP90 (Supplementary Fig.S4A–S4D). More-over, HSP90 silencing increased survival in a subcutaneoustransplantation model, again concomitant with a reduction in

Figure 3.

HIF regulation by pH is not dependent on the PHD/VHL system. A, the increase of HIF by acidosis is VHL independent. Immunoblot of the VHL-deficient renalcarcinoma lines 786-O and RCC11 (which do not express HIF1a) cultured at pH 6.7 or 7.4 under normoxia for 18 hours. B, increasing intracellular VHL levels doesnot abolish the acidosis-mediated increase in HIF. Immunoblot of G55 cells transfected with empty vector control (pcDNA) or with a VHL expression plasmid,cultured at 1% O2 and pH 7.4 or 6.7 for 6 hours. C, HIF regulation via pH is retained when the PHD/VHL system is inactivated. G55 cells were cultured at pH 6.7or 7.4 under hypoxia (1% O2), or under normoxia following the inhibition of PHD function by the iron chelator dipyridyl (DP) and the PHD inhibitordimethyloxalylglycine (DMOG) for 6 hours. D, PHD-refractory HIF1a and HIF2amutants remain sensitive to pH regulation. Immunoblot of G55 cells transfectedwith nonhydroxylatable/nondegradable V5-tagged mutants of HIF1a (HIF1a mPPN) and HIF2a (HIF2a mPPN) and incubated at pH 7.4 or 6.7 and 1% O2

for 18 hours. The transfected proteins were detected using an antibody against the V5-tag.

Figure 4.

HSP90 mediates the acidosis-induced increase in HIF. A and B, acidosis upregulates HSP90. Immunoblot of G55 cells (A) and NCH644, GBM015,and GBM031 primary glioblastoma cells (B) incubated at pH 6.7 or 7.4 and 1% O2. C and D, inhibition of HSP90 with geldanamycin abrogates theacidosis-dependent induction of HIF1a and HIF2a. Immunoblot of G55 (C) and NCH644 (D) glioblastoma cells treated with control vehicle (DMSO)or 1 mmol/L geldanamycin (GA) and cultured at either physiologic (7.4) or acidic (6.7) pH and 1% O2. E, dominant-negative HSP90 abolishes theacidosis-induced HIF upregulation. Immunoblot of G55 cells transfected with control vector (pcDNA3, "�") or an HA-tagged dominant negative HSP90construct and incubated at pH 7.4 or 6.7 and 1% O2. F, overexpression of HSP90 induces a HIF increase at physiologic pH. Immunoblot of G55 cells transfectedwith control vector (pcDNA3, "�") or an HA-tagged wild-type HSP90 construct and incubated at pH7.4 or 6.7 and 1% O2.






HIF1/2a levels (Supplementary Fig. S5A–S5D). Importantly,HPS90 disruption reduced the tumor-initiating capacity of thecells, indicating an involvement of the HSP90-mediatedincrease in HIF levels under acidosis in tumor initiation andCSC maintenance. Indeed, HSP90 inhibition with geldanamy-cin efficiently blocked the acidosis induced increase in theCD133-postive tumor cell fraction as well as in self-renewal(Fig. 6A and B). To further corroborate the role of acidosis andHSP90 in the promotion of CSC function, we examined thetumorigenic capacity of primary glioblastoma cells, a defining

property of CSCs. Glioblastoma cells grown under pH 6.7generated significantly larger intracranial tumors than cellscultured at physiologic pH (Fig. 6A, C, and D). Crucially,concomitant treatment of the cells with geldanamycincompletely abolished the increased tumorigenicity under aci-dosis, but had no effect under physiologic pH (Fig. 6C and D).These findings demonstrate that acidosis increases HIF levelsand CSC function through HSP90 and that HSP90 inhibitioncan efficiently suppress HIF induction, CSC maintenance, andtumorigenicity.

Figure 5.

HSP90 inactivation suppresses the acidosis-induced HIF increase and tumor growth. A, silencing of HSP90a/b reduces HIF levels in vitro. Immunoblotof G55 cells stably transduced with nonsilencing control or HSP90a/b shRNAs and cultured at pH 7.4 or 6.7 under hypoxia for 18 hours. B–F, loss ofHSP90 reduces tumor growth and intratumoral HIF levels and activity. Control and HSP90a/b shRNA G55 tumor cells were orthotopically transplantedin nude mice. B, tumor xenografts were hematoxylin and eosin–stained and the tumor volume was quantified (n ¼ 9). C, the expression of the HIF target geneVEGFA was analyzed by qPCR in tumor samples (n ¼ 9). D, immunohistochemical staining for Hypoxyprobe and HIF1 in control and shHSP90 tumors. N,necrotic areas. E, immunoblot for HIF1/2a and HSP90 in extracts from representative control and shHSP90 tumors. F, quantification of HIF1a and HIF2a levelsin control and shHSP90 tumors by immunoblot (n¼ 9). All values are meanþ SEM. � , P < 0.05; �� , P < 0.001. Scale bars, 1 mm (B), 100 mm (D), 20 mm (D, highermagnifications).

Filatova et al.





HSP90 is expressed in the hypoxic CSC niche and correlateswith the CSC phenotype

We next wanted to assess the relevance of our findings tohuman glioblastomas. Analysis of gene expression in the glio-blastoma cohort of TCGA research network (30) revealed thattumors with high HSP90 expression had increased levels of theHIF target gene VEGF, as well as of the glioma stem cells markersCD133 and nestin (Fig. 7A), supporting the notion that HSP90 isan important factor activated by the CSC microenvironment. Toconfirm that HSP90 is specifically upregulated in glioblastoma inthe hypoxic CSC niche, we examined its localization in glioblas-toma biopsies. The presence of necrotic areas is a diagnostichallmark of glioblastoma and is associated with regions of insuf-ficient blood supply and severe hypoxia. HIF levels were elevatedin the perinecrotic regions (Fig. 7B), indicating that these areas arehypoxic and thus presumably more acidic as a result of thehypoxia-induced metabolic response. Importantly, HSP90 levelsin the same region were also increased in 10 of 10 glioblastomabiopsies examined, with 93.0% � 4.0% of perinecrotic/hypoxicareas showing HIF and HSP90 coexpression. Moreover, CD133was also prominently enriched in this area (Fig. 7B), supportingour hypothesis of synergistic stimulation of HIF function byhypoxia and acidosis in an HSP90-dependent manner, leadingto an expansion of the CSC pool in human glioblastomas.

DiscussionTumor cells are involved in an intimate crosstalk with their

microenvironment, which critically controls major aspects ofcancer cell biology. In this study, we show that two key character-istics of the tumor microenvironment, hypoxia and acidosis,synergize to potentiate HIF activity and HIF-dependent functionsthrough complementary and additive mechanisms. We identifyHSP90 as a key factor responsible for the capacity of the acidic

microenvironment to promote the hypoxic response and itsdownstream functions, including CSC maintenance and tumor-igenicity, pointing to potential strategies for the targeting of thiscritical cancer cell population.

Acidosis regulates HIF function through HSP90Hypoxia and acidosis are characteristic features of many

tumor types. Similarly to hypoxia, the acidic tumor microen-vironment regulates key aspects of tumor pathophysiology. Forexample acidosis can enhance the resistance of tumor cells tovarious chemotherapeutics (15, 31, 32). Furthermore, a num-ber of studies have linked acidosis to an enhanced invasiveand metastatic capacity of tumor cells (9, 33–36). In gliomas,the acidic microenvironment has been shown to induce VEGFexpression and tumor angiogenesis (16, 37). Importantly, mostof the properties listed above are also regulated by hypoxia andthe HIF pathway. Indeed, our results uncover an interestinglink between acidosis and HIF showing that acidosis increasesHIF levels and function in a synergistic manner with hypoxia.An increase of both HIF1a and HIF2a levels under acidosis hasbeen previously reported in primary glioblastoma cellsand other cancer cell types (14, 38). In contrast, other groupshave observed a downregulation of HIF in acidic conditions(39, 40). By systematically assessing the effects of differentlevels of acidosis on HIFs we show that acidosis enhances HIFlevels down to a pH of 6.6–6.8, while lower pH values decreaseHIF expression, pointing to a narrow pH window for acidoticactivation of the HIF pathway. Importantly, these valuescorrespond to the extracellular pH that has been measuredin the majority of tumors, including glioblastoma (14, 23),underlining the physiologic relevance of our findings.

The levels and activity of HIFs are regulated by multiplemechanisms. A central place among those is taken by thePHD/VHL-dependent HIF hydroxylation, ubiquitination and

Figure 6.

The acidosis-induced increase in CSCfunction is dependent on HSP90. A andB, inhibition of HSP90 abrogates theacidosis-dependent induction of theglioma stemcell phenotype. The fractionof CD133þ cells, determined by FACSanalysis (n ¼ 3; A), and sphere-formingcapacity (n ¼ 12; B) of NCH644 primaryglioblastoma cells treated withcontrol vehicle (DMSO) or 1 mmol/Lgeldanamycin (GA) and culturedat pH 7.4 or 6.7 and 1% O2. C and D,acidosis stimulates tumorigenicity in anHSP90-dependent manner. NCH644primary glioblastoma cells incubated at1% O2 in medium with pH 7.4 or 6.7supplemented with DMSO control or1mmol/L geldanamycin for 18 hourswereorthotopically transplanted intonude mice. Tumor xenografts werestained with hematoxylin and eosinand the tumor volume was quantified(D; n ¼ 7–8). Representative images ofthe hematoxylin and eosin–stainedtumor sections are shown inC. All valuesare means þ SEM; �� , P < 0.01;�� , P < 0.01; �� , P < 0.001. Scale bar,1 mm (C). ns, nonsignificant.






degradation. A previous report has shown that a highly acidic pH(5.8-6.2) can lead to sequestration of VHL in the nucleolus and toHIF stabilization (24). However, several lines of evidence that wepresent here strongly argue against a role for the VHL/PHDdegradation pathway in mediating the effect of acidosis on HIFstability in the pH range that is typically encountered in tumors.For example, renal carcinoma cells lacking VHL still responded toacidosis with a striking increase in HIF levels. Furthermore, HIFinduction under acidic pH was not affected by inhibition ofPHDs. Instead, we show that acidosis stabilizes HIF1a andHIF2athrough the upregulation of HSP90, independently of PHD and

VHL. Importantly, inhibition of HSP90 function by geldanamy-cin or a dominant-negative mutant abolished the stabilization ofHIF1/2a at low pH. Among alternative, PHD-VHL–independentmechanisms that regulate HIF stability HSP90 has been wellcharacterized (reviewed in ref. 41). The multifunctional scaffoldprotein RACK1 binds the PAS-A domain of HIF with subsequentrecruitment of the ubiquitin ligase complex that mediates itsdegradation, in a manner analogous to the mechanism activatedby VHL, but independent of O2/PHD-mediated HIF hydroxyl-ation (28). HSP90 competes with RACK1 by binding to the HIFPAS-A domain (42), thereby stabilizing HIF (28). Taken together,

Figure 7.

High HSP90 expression is observed in the hypoxic niche and correlates with hypoxic and stem cell markers in human glioblastomas. A, comparison ofthe HIF-target VEGF-A and the glioma CSC markers CD133 and nestin in glioblastomas with high and low HSP90 levels in the TCGA cohort (n ¼ 154).B, serial sections of human glioblastoma containing a perinecrotic (hypoxic) region were immunohistochemically stained for HIF1a, HSP90, and CD133. Thepanels on the right show higher magnifications of the staining in the perinecrotic areas. N, necrosis. C, a model of the synergistic regulation of HIFfunction and CSC maintenance by the hypoxic/acidotic tumor niche. The hypoxic tumor niche induces the establishment of an acidic environment, whichsynergizes with decreased oxygen availability to potentiate the hypoxic response. This represents a positive feedback loop that enhances HIF-dependentfunctions and promotes the maintenance of CSC. The additive induction of HIF by hypoxia and acidic pH via PHD- and HSP90-dependent mechanisms,respectively, may provide a means to fully activate HIF signaling and homeostatic responses that support tumor cell growth and survival in a hostilemicroenvironment. Scale bars, 100 mm (C) and 20 mm (C, inset).

Filatova et al.





our results highlight the central importance of acidosis as a keymicroenvironmental factor that regulates the HIF response syn-ergistically with decreased oxygen tension through an HSP90-dependent, but PHD/VHL and oxygen-independent mechanism.

Acidosis and HSP90 are important regulators within thehypoxic niche and a target for antitumor therapy

HSP90 forms a chaperone complex that can stabilize andactivate a number of cellular proteins. In cancer cells HSP90 playsan additional important role by protecting various mutated oroverexpressed proteins against misfolding and degradation,thereby facilitating oncogene addiction, counteracting proteo-toxic stress and enabling cancer cell survival (43). HSP90 upre-gulation has been found in several types of cancer and hasbeen linked topoor prognosis and increased tumor aggressiveness(44–46). This has led to the development of a number of small-molecule inhibitors, including geldanamycin and its derivativesthat are currently in clinical trials for various tumor types (47).Our results highlight a general function of HSP90 in tumorphysiology, revealing a novel role of HSP90 as the key mediatorof the acidosis-dependent potentiation of HIF function. Theclinical relevance of our findings is supported by the fact thatHSP90 is prominently upregulated within the hypoxic niche ofhuman glioblastomas and a high level of HSP90 is linked to theupregulation ofHIF targets andCSCmarker genes. Interestingly, ahigher sensitivity of CSCs to HSP90 inhibitors compared withneural stem cells or other untransformed cells has recently beendemonstrated (48, 49). Therefore, blockade of HSP90 function inglioblastoma, where HSP90 inhibitors have not been tested inclinical trials so far, could represent a viable therapeutic strategy.An alternative possibility is to aim at neutralizing tumor acidosisitself, and indeed recent studies have shown that this can beachieved by simple interventions such as bicarbonate adminis-tration, resulting in curtailed tumor growth, invasion, andmetas-tasis (33, 50).

The synergistic increase of HIF levels and function by hypoxiaand acidosis likely affects multiple HIF-dependent processes thatcollectively can promote tumor aggressiveness. Among those, themaintenance and expansion of the CSC pool likely plays animportant role, as it is linked to numerous aspects of tumorprogression that are stimulated by both hypoxia and acidosis.We and others have previously shown that CSCs are located andcontrolled within a hypoxic niche through HIF (12, 13). Our datasupport a model in which the hypoxic tumor niche induces anacidic environment, which synergizes with decreased oxygenavailability to potentiate the hypoxic response and enhanceHIF-dependent downstream functions in a positive feedback loop(Fig. 7C). Given the crucial role of HIFs in tumor progression, theadditive induction of HIF by hypoxia and acidic pH via PHD- andHSP90-dependent mechanisms, respectively, may provide a

means to fully activate HIF signaling within the hypoxic niche.Importantly, these two, synergistically acting mechanisms mayallow tumor cells to fine-tune and flexibly induce HIF activity inresponse to different microenvironmental parameters (O2 level,pH) to activate key hallmarks of cancer (Fig. 7C). Thus, byproviding important mechanistic insight into the synergisticcontrol of HIF function and CSC maintenance through centralphysiologic parameters of the tumor microenvironment, ourwork uncovers potential avenues for developingnovel therapeuticapproaches targeted against hypoxia/acidosis-driven tumorprogression.

Disclosure of Potential Conflicts of interestT. Acker was a consultant/advisory board member for Merck Serono

(2014/2015; minor relationship). No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: T. AckerDevelopmentofmethodology:A. Filatova, S. Seidel,N. B€o�g€urc€u, B.K.Garvalov,T. AckerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Filatova, S. Seidel, N. B€o�g€urc€u, S. Gr€af,B.K. Garvalov, T. AckerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Filatova, S. Seidel, N. B€o�g€urc€u, B.K. Garvalov,T. AckerWriting, review, and/or revision of the manuscript: A. Filatova, S. Seidel,N. B€o�g€urc€u, B.K. Garvalov, T. AckerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Filatova, S. Seidel, N. B€o�g€urc€u, B.K. Garvalov,T. AckerStudy supervision: B.K. Garvalov, T. Acker

AcknowledgmentsWe would like to thank Barbara Lafferton, Gudrun Schmidt, Kerstin Leib,

Carmen Selignow, and Tanja Diem for excellent technical assistance.

Grant SupportThis work was supported by grants from the Deutsche Krebshilfe (T. Acker,

B.K. Garvalov), the GermanMinistry of Education and Research (BMBF) withinthe National Genome Network (NGFNplus) and Brain Tumor Network (BTN)(T. Acker), the DFGKFO210 (T. Acker, B.K. Garvalov), DFG SPP1069, DFGSPP1190 (T. Acker), the DFG Clusters of Excellence Cardio-Pulmonary System(ECCPS; T. Acker), LOEWE-OSF, UKGM Kooperationsvertrag x2, 3 (T. Acker,B.K. Garvalov), and the von Behring-R€ontgen Foundation (T. Acker,B.K. Garvalov).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 25, 2015; revised June 14, 2016; accepted July 13, 2016;published OnlineFirst August 3, 2016.

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2016;76:5845-5856. Published OnlineFirst August 3, 2016.Cancer Res Alina Filatova, Sascha Seidel, Nuray Bögürcü, et al. Promote HIF Function and Stem Cell Maintenance in GliomaAcidosis Acts through HSP90 in a PHD/VHL-Independent Manner to

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Acidosis Acts through HSP90 in a PHD/ VHL-Independent Manner … · and acidosis functionally cooperate in inducing HIF transcrip-tion factors and CSC maintenance. We found that these