Download Acidosis Acts through HSP90 in a PHD/ VHL

Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-15-2630
Cancer
Research
Tumor and Stem Cell Biology
Acidosis Acts through HSP90 in a PHD/
VHL-Independent Manner to Promote HIF
Function and Stem Cell Maintenance in Glioma
€g
u
€ f1, Boyan K. Garvalov1,
€ rcu
€ 1, Sabine Gra
Alina Filatova1, Sascha Seidel1,2, Nuray Bo
1
and Till Acker
Abstract
Hypoxia is a common feature of solid tumors, which controls
multiple aspects of cancer progression. One important function
of hypoxia and the hypoxia-inducible factors (HIF) is the
maintenance of cancer stem-like cells (CSC), a population
of tumor cells that possess stem cell-like properties and drives
tumor growth. Among the changes promoted by hypoxia
is a metabolic shift resulting in acidification of the tumor
microenvironment. Here, we show that glioma hypoxia
and acidosis functionally cooperate in inducing HIF transcription factors and CSC maintenance. We found that these effects
did not involve the classical PHD/VHL pathway for HIF upre-
gulation, but instead involved the stress-induced chaperone
protein HSP90. Genetic or pharmacologic inactivation of
HSP90 inhibited the increase in HIF levels and abolished the
self-renewal and tumorigenic properties of CSCs induced by
acidosis. In clinical specimens of glioma, HSP90 was upregulated in the hypoxic niche and was correlated with a CSC
phenotype. Our findings highlight the role of tumor acidification within the hypoxic niche in the regulation of HIF and CSC
function through HSP90, with implications for therapeutic
strategies to target CSC in gliomas and other hypoxic tumors.
Introduction
tumor environment (7). For example, blockade of carbonic
anhydrase IX (CA IX), a hypoxia upregulated transmembrane
enzyme that converts H2O and CO2 into carbonic acid (Hþ
and HCO3), increased extracellular pH, decreased proliferation, enhanced apoptosis, and markedly suppressed tumor
growth in vivo (5, 8). Acidic extracellular pH has also been
shown to promote tumor cell migration, invasiveness, and
metastasis (8–10). Although tumor hypoxia and acidosis are
linked and both represent crucial aspects of the tumor microenvironment, their interplay in tumor progression remains
poorly understood.
It has become clear that tumor growth and progression is driven
by a subpopulation of cells with stem cell–like properties, termed
cancer stem cells (CSC) or tumor-initiating cells (11). We and
others have previously established that hypoxia plays a key role in
promoting the CSC phenotype through HIFs (12, 13). Acidic
stress has also been shown to promote a cancer stem cell phenotype (14); however, very little is known about the mechanisms
through which microenvironmental pH may impact on hypoxic
signaling and control CSC maintenance within the hypoxic niche.
Interestingly, glioblastoma cells exposed to more acidic conditions show an increased resistance to chemotherapeutics (15),
and elevated expression of VEGF (16), properties characteristic of
CSCs. Thus, the contribution of acidosis to the regulation of CSCs,
particularly within the hypoxic CSC niche, is of high interest and
potential therapeutic relevance.
Here, we show that hypoxia and acidosis synergize to increase
HIF levels and function in glioblastoma, resulting in a marked
potentiation of CSC self-renewal, maintenance, and tumorigenicity. The pH-mediated regulation of HIF, CSC function, and
tumorigenicity is not dependent on PHD/VHL activity, but is
instead controlled by HSP90. Furthermore, increased HSP90
levels in human glioblastomas are associated with a higher
expression of HIF target genes and CSC markers, highlighting
Rapidly growing tumors often outpace their blood supply
generating a hypoxic microenvironment. Tumor hypoxia triggers a set of adaptive responses that ultimately promote a more
aggressive tumor phenotype and are primarily controlled by
the transcription factor system of the hypoxia-inducible factors
(HIF; ref. 1). HIF abundance is tightly regulated by the prolyl
hydroxylase domain (PHD) proteins. PHDs use O2 to hydroxylate HIFs (2, 3), which enables binding of the VHL protein, a
component of an E3 ubiquitin ligase complex that catalyzes
HIF1/2a ubiquitination and degradation. One of the primary
effects of hypoxia is the induction of a metabolic shift from
oxidative phosphorylation to glycolysis, with lactic acid as the
end product (4). This is accompanied by the upregulation of
carbonic anhydrases, transporters for lactate, Hþ, HCO3, and
other ions, leading to a net acidification of the extracellular
tumor environment (5, 6). A decreased pH is a characteristic
feature of many tumor types and a number of studies have
started to delineate critical functions of the acidic extracellular
1
Institute of Neuropathology, University of Giessen, Giessen, Germany.
Institute of Cell Biology and Neuroscience and Buchmann Institute
for Molecular Life Sciences (BMLS), University of Frankfurt, Frankfurt,
Germany.
2
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
€g
u
€rcu
€, B.K. Garvalov, and T. Acker contributed equally
A. Filatova, S. Seidel, N. Bo
to this article.
Corresponding Author: Till Acker, Institute of Neuropathology, University of
Giessen, 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.
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Filatova et al.
the potential importance of HSP90 as a druggable pathway for the
targeting of glioma CSCs.
Materials and Methods
Cell culture
The glioblastoma cell lines G55TL, G121, G141, and G142 were
kindly provided by K. Lamszus and M. Westphal [Department of
Neurosurgery, University Medical Center Hamburg-Eppendorf
(UKE), Hamburg, Germany; ref. 17], the renal carcinoma line
786-O was obtained from ATCC and RCC11 (18) was obtained
from Dr C. Bauer (Institute of Physiology, University of Zurich,
Zurich, Switzerland) between 1998 and 2002. The primary glioblastoma line NCH644 was kindly provided by C. Herold-Mende
(Heidelberg, Germany; ref. 19) in 2010. The primary glioblastoma lines GBM015 and GBM031 were obtained in 2007–2008
from patients undergoing surgery in accordance with a protocol
approved by the institutional review board and cultured in neurosphere medium (13). G55TL, G121, G141, and G142 cells were
cultured in DMEM (Invitrogen) supplemented with 10% FBS
(PAN Systems). NCH644, GBM015, and GBM031 were propagated under neurosphere conditions in neurosphere medium
(DMEM-F12; Invitrogen), 5 mmol/L HEPES, 2% B-27 serum-free
supplement without vitamin A (Invitrogen) supplemented with
20 ng/mL bFGF and 20 ng/mL EGF (PeproTech)]. All cells were
maintained at 37 C in 5% CO2. For experiments with different
pH, G55TL, 786-O, and RCC11 cells were incubated in CO2
-independent medium (#18045-054, Invitrogen) supplemented
with 2 mmol/L L-glutamine and 10% FBS; the primary glioma
cells lines were incubated in CO2-independent medium supplemented with 2% B-27 serum-free supplement without vitamin A,
2 mmol/L L-glutamine, 20 ng/mL bFGF, and 20 ng/mL EGF. The
pH of the medium was adjusted to the values indicated in the
figures with 1 mol/L HCl or 1 mol/L NaOH. For hypoxic treatment
cells were grown at 1% O2 for the indicated periods of time in a
Hypoxic Workstation (Ruskinn Technology). NCH644, GBM031,
and GBM015 were preincubated in medium with pH7.4 at
normoxia and incubated in media with pH7.4 or 6.7 at hypoxia.
For geldanamycin treatment, cells were preincubated with pH 7.4
at 21% O2 for 4 days; in the last two hours of the preincubation 1
mmol/L geldanamycin (Enzo Life Sciences) or control DMSO was
added, followed by incubation at 1% O2 in medium with pH 7.4
or 6.7 supplemented with DMSO control or 1 mmol/L geldanamycin for 6 or 18 hours (for G55 and NCH644 cells, respectively).
Further details on the experimental procedures are included in the
in corresponding figure legends. For stem cell condition, the cells
were incubated in neurosphere medium for the same time. Cells
that showed unusual growth or morphology were tested for
mycoplasma contamination and only noncontaminated cells
were used for experiments. Cell lines obtained from ATCC were
authenticated by the manufacturer. No additional authentication
was performed by the authors for any of the cell lines.
Tumor xenografts
Animal experiments were approved by the veterinary department
of the regional council in Darmstadt, Germany. Xenograft transplantations were performed in athymic 6–8 week old female NMRI
nu/nu mice (Janvier Labs) that were kept in a specific pathogen-free
animal facility according to the institutional guidelines.
For intracranial tumor transplantation, mice were placed
into a stereotactic apparatus, and 1 105 cells in a volume
of 2 mL (NCH644), or 7.5 103 cells in a volume of 1 mL (G55)
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were resuspended in cold, CO2-independent medium and
slowly injected into the left striatum. At the onset of neurologic
symptoms, mice were sacrificed at the same time point (at day
19 after transplantation for the experiments with G55 cells,
and at day 22 posttransplantation for the experiments with
NCH644 cells) through deep anesthesia with ketamine and
xylazine. The chest was opened and the vasculature was perfused with a 0.9% NaCl solution for 2 minutes and fixative
(4% PFA) for 4 minutes. Brains were removed and additionally
fixed overnight in 4% PFA, dehydrated in 30% sucrose for
4 days, and rapidly frozen on dry ice for sectioning with a cryotome. The sections were stained with hematoxylin and eosin.
Tumor volume was determined using stereological quantification 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 injected
with 60 mg/g Hypoxyprobe intraperitoneally 90 minutes prior
to cardiac perfusion with 0.9% NaCl solution. After removal of
the brain and dissection of the tumors, one part of each tumor
was snap-frozen in liquid nitrogen for subsequent protein
isolation. Another part of the tumor was incubated in fixative
overnight and embedded in paraffin for subsequent histologic and immunohistochemical analysis. Tumor volume was
analyzed using the section with the biggest tumor area by
measuring the largest diameter (L) and largest perpendicular
diameter (W), using the formula L W2/2 (G55).
For subcutaneous tumor injections, 7.5 104 G55 cells suspended in 0.1 mL PBS/Matrigel were injected subcutaneously into
the flanks of 6–8-week-old female nude (NMRI nu/nu) mice. The
tumor size was measured at regular intervals using a caliper
according to the formula V ¼ L W2/2. Mice were maintained
until tumors exceeded a volume of 2,000 mm3 or upon the onset
of morbidity symptoms (>20% weight loss, tumor ulceration).
Tumors were excised and snap-frozen in liquid nitrogen for
subsequent protein isolation.
Transfection
G55TL cells were transiently transfected with plasmid DNA
using Lipofectamine 2000 according to the manufacturer's
instructions. siRNAs against HIF1a, HIF2a, or nontargeting control siRNA were obtained as a pool of 4 siRNA oligos (ONTARGETplus SMART pool, Dharmacon). Reverse siRNA transfection of GBM015 cells was performed with 10 pmol siHIF1a and
50 pmol siHIF2a, or 60 pmol nontargeting siRNA, respectively,
with Lipofectamine RNAiMAX (Thermo Scientific) in antibioticfree neurosphere medium according to the manufacturer's
instructions. Twenty-four hours after transfection, the cells were
seeded in CO2-independent neurosphere medium at pH 7.4 and
preincubated under normoxic conditions for 48 hours. Subsequently, the cells were cultured under 1% O2 neurosphere medium at pH 7.4 or 6.7 and harvested after 18 hours (protein and
RNA) or 96 hours (FACS).
Immunoblotting
For immunoblotting, cells were harvested in PBS (4 C), cell
pellets were lysed in 10 mmol/L Tris.HCl (pH 7.5), 2% SDS,
2 mmol/L EGTA, 20 mmol/L NaF, and 15–50 mg of protein lysates
were subjected to SDS-PAGE and Western blot analysis using
antibodies specific for CD133 (Abcam ab19898 and Miltenyi
Biotec 130-092-395), GFAP (Dako Z 0334), HIF1a (Cayman
Chemical 10006421 or BD Transduction Laboratories 610958),
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Acidosis Promotes HIF and Glioma Stem Cells
HIF2a (Novus Biologicals NB 100-122), HSP90 (Enzo/Stressgen
ADI-SPA-830), V5 (Invitrogen R960-25), VHL (Abcam,
ab11189), and Tubulin (Dianova DLN09992) as a loading control. Immunoreactive bands were visualized with the ECL system
(Perkin Elmer or Pierce).
FACS
Spheres were dissociated into single-cell suspension by Accutase (PAA) treatment for 15 minutes at 37 C. After two washing
steps with FACS Staining buffer (PBS, pH 7.2, 0.5% BSA, 2 mmol/
L EDTA), single cells were blocked with 20 mL of normal mouse
IgG (Invitrogen) for 20 minutes at 4 C and stained with 10 mL of
CD133/2 (293C3)-PE–conjugated antibody (Miltenyi Biotec
130-090-853) or 5 mL of CD15-V450–conjugated antibody (BD
Biosciences 642917) for 30 minutes at 4 C. The background
staining was determined using matching isotype control antibodies from the same manufacturers at the same concentration as
the specific antibodies. Five minutes before analysis, 1 mmol/L
SYTOX Blue (Invitrogen; CD133/2-PE) or SYTOX Red (CD15)
nucleic acid stain was added to exclude dead cells. Flow cytometry was performed using BD FACSCanto II (BD Biosciences).
Data were analyzed using FlowJo v7/9 (Tree Star) by gating for
live cells based on SYTOX staining, then for singlets using
forward scatter area versus width and side scatter area versus
width, followed by gating for the CD133 or CD15-positive
populations, respectively.
Sphere forming units and in vitro growth
A total of 1 106 cells were seeded per 10-cm dish and cultured
at pH 7.4 or 6.7 at 1% O2 for 96 hours. For quantification of sphere
forming units (SFU), the cells were then split in 12-well suspension culture plates (n ¼ 12) at a density of 300 cells/well in 1 mL
or 6-well suspension culture plates (n ¼ 6) at a density of
500 cells/well in 2-mL neurosphere medium. After 7 days, the
spheres were counted and the percentage of sphere-forming cells
was calculated. For sphere formation experiments applying
siRNA-mediated knockdowns, cells were transfected as described
above. Twenty-four hours after transfection, medium was changed and the cells cultured at pH 7.4 or 6.7, 1% O2 for 72 hours. For
quantification of SFUs, the cells were then split in 6-well suspension culture plates (n ¼ 6) at a density of 500 cells/well in 2-mL
neurosphere medium. After 7 days, the spheres were counted and
the percentage of sphere-forming cells was calculated.
Real-time quantitative RT-PCR
RNA was extracted with the RNeasy Mini Kit (Qiagen), and
reverse transcribed using standard protocols (RevertAid H Minus
M-MuLV Reverse Transcriptase, Fermentas). cDNA was amplified
using the ABsolute QPCR SYBR Green Mix. Gene-specific PCR
products were measured continuously in a StepOnePlus real-time
PCR system (Applied Biosystems) for up to 45 cycles. The difference in the threshold number of cycles between the gene of
interest and HPRT (hypoxanthine phosphoribosyltransferase 1)
was then normalized relative to the standard chosen for each
experiment and converted into fold difference.
IHC
Immunohistochemical stainings of human biopsies were
approved by the institutional review board and were carried
out using antibodies against HIF1a (Cayman Chemical
10006421), HSP90 (Enzo/Stressgen ADI-SPA-830), and CD133
(Miltenyi Biotec 130-092-395) with an Autostainer (Ventana/
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Roche) according to the manufacturer's instructions. For
Hypoxyprobe detection (Hypoxyprobe-1, NPI Inc.), paraffin
sections were dewaxed in xylene (2 10 minutes) followed
by a series of descending alcohol concentrations (2 5 minutes
100% EtOH, 2 5 minutes 96% EtOH, 2 5 minutes 70%
EtOH) and rehydrated for 5 minutes in H2O and TBS buffer
each. For antigen retrieval, the sections were boiled in preheated
citrate buffer (pH 6) for 10 minutes and subsequently cooled
down at room temperature for 20 minutes, followed by washing
steps in TBS (2 5 minutes). Endogenous peroxidase was
blocked in 0.6% H2O2 for 30 minutes followed by washing
steps with TBS (3 5 minutes). Unspecific antibody binding
was blocked with 10% normal goat serum (NGS) in TBS/0.1%
Tween (TBST) for 1 hour at room temperature, followed by
incubation with FITC-Mab1 antibody (1:100 in 10% NGS/
TBST, NPI Inc.) for 2 hours at room temperature. After three
washing steps in TBST (5-minute each), sections were incubated
with anti-FITC HRP secondary antibody (1:200 in 10% NGS/
TBST, NPI Inc.) for 2 hours at room temperature followed by
three washing steps in TBST. Chromogen reaction was performed using diaminobenzidine (DABþ, Dako) for 3 minutes.
Counterstaining was performed in hematoxylin for 2 minutes.
To achieve color development of the stained nuclei, sections
were rinsed in tab water for 2 minutes followed by a rinse in H2
O. Sections were dehydrated in a series of ascending alcohol
concentrations (2 5 minutes 70% EtOH, 2 5 minutes 96%
EtOH, 2 5 minutes 100% EtOH) with a final incubation in
xylene (2 5 minutes). Sections were permanently mounted
using Cytoseal XYL (Thermo Scientific).
Luciferase reporter assays
A 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 SV40
Renilla luciferase constructs for normalization of transfection
efficiency. To assess the effect of HIFs on VEGF promoter transcription, the cells were cotransfected with 300 ng pcDNA3-HIF1a
or HIF2a overexpression constructs, or pcDNA3 as a control.
The next day, the cells were placed at the indicated concentrations of O2 and CO2 and 24 hours later were lysed for analysis
of luciferase expression downstream of the VEGF promoter, using
a dual luciferase reporter system (Promega), according to the
manufacturer's instructions.
Lentiviral constructs and stable cell lines
pGIPZ lentiviral constructs against HSP90a, HSP90b, and
pGIPZ nonsilencing control vectors were purchased from Open
Biosystems (Thermo Scientific). Lentiviral HA-tagged dominantnegative mutant of HSP90b (HA-HSP90 DN) or a GFP-expressing
control plasmids were constructed as described in the Supplementary Materials and Methods. Lentivirus was packaged by
cotransfection of lentiviral vectors with the packaging plasmids
pCI-VSVG and psPAX into 14-cm plates with HEK293T cells using
Fugene HD (Roche). Medium was changed every 24 hours, the 48and 72-hour supernatants were pooled, filtered through a 0.45mm filter, and ultracentrifuged at 20,000 g, 4 C for 4 hours.
Titers were determined by counting the number of GFP-positive
colonies or crystal violet–stained colonies. The shHSP90 and
control lines were generated by lentiviral transduction and selection with 2 mg/mL puromycin to create the stable shRNA pools.
The HA-HSP90 DN and GFP lines were generated by lentiviral
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transduction and selection with 6 mg/mL blasticidin to create the
stable HA-HSP90 DN or GFP-overexpressing cells.
Bioinformatic analysis
Gene expression data [z-scores (all genes)] for the glioblastoma cohort of The Cancer Genome Atlas (TCGA) Research
Network were downloaded from the cBio portal (http://www.
cbioportal.org/public-portal/index.do). Tumors with z-scores
for HSP90a < 1 (n ¼ 90) were considered as low HSP90expressing, tumors with z-scores of > þ1 (n ¼ 64) were considered as high HSP90 expressing.
Statistical analysis
Results are presented as mean SEM. Sample size was chosen
based on previous empirical experience with the respective
types of assays or animal tumor models. Animals that unexpectedly died before tumors were collected from the remaining
animals were excluded from the analysis. The experiments were
not randomized and the investigators were not blinded to
allocation during experiments and outcome assessment. For
pairwise comparisons, statistical analysis was performed using
the Student t test except for the expression analysis in the TCGA
glioblastoma cohort, where the Mann–Whitney test was used
due to non-normal distribution. For comparisons between
multiple groups, ANOVA with a Bonferroni post test was
performed using GraphPad Prism. Statistical significance was
defined as , P < 0.05; , P < 0.01; , P < 0.001.
Results
Acidosis increases HIF function and the CSC phenotype
We were interested in analyzing to what extent metabolic
parameters of the hypoxic tumor microenvironment other than
pO2 may regulate HIF levels and function in glioblastoma. As
tumor hypoxia is frequently accompanied by acidosis and
hypoglycemia, we cosubjected a panel of glioblastoma cell
lines to different hypercapnic conditions (10% and 20%
CO2), as a physiologic means of lowering the pH, and different
glucose levels (high glucose: 4.5 g/L; low glucose: 1.0 and 0 g/L
glucose; Fig. 1A and B). Under normoxia, only very low-level of
HIF1a and moderate levels of HIF2a were detectable, which
were highly upregulated under hypoxia (Fig. 1A and B) with a
concomitant increase in mRNA expression of the HIF-target
genes VEGF, LDHa, Glut-1, and CAIX (Supplementary Fig. S1).
Glucose deprivation mostly did not alter or even decreased
HIF1a and HIF2a abundance under normoxia and hypoxia. In
contrast, decreasing the pH through hypercapnia moderately
increased HIF1a and HIF2a levels already under normoxia
(Fig. 1A and B) and dramatically elevated the hypoxic upregulation of HIF1a and HIF2a, demonstrating that hypoxia and
acidosis synergistically increase the HIF response (Fig. 1B). In
line 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 regulation
by pH. As CO2 can exert effects on cell physiology that are
independent of acidification of the cellular environment (22),
we next modulated the pH by addition of NaOH and HCl in
CO2-independent medium (Fig. 1D and E). Acidic pH was
sufficient to upregulate HIF1a and HIF2a levels already under
normoxia and even further potentiated the hypoxia-dependent
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increase in HIF1/2a expression (Fig. 1D). Decreasing the pH
down to 6.6–6.8, conditions that are observed in tumors (23),
efficiently induced HIF1a and HIF2a expression under normoxia and hypoxia, whereas further pH reduction decreased
HIF1/2a abundance (Fig. 1E).
Previous work by us and others has demonstrated that hypoxia promotes the CSC phenotype through the induction of
HIFs (12, 13). As expression and activity of HIFs were induced by
low pH, we assessed whether acidosis would also promote the
stem cell properties of glioblastoma cells. We first confirmed the
pH-dependent regulation of HIF and HIF target genes in a panel
of primary glioblastoma lines derived from patient biopsies
and continuously propagated in serum-free sphere (stem cell)
conditions (Fig. 2A and B). Importantly, acidosis concomitantly
increased the expression of a panel of CSC-associated genes
and the fraction of cells positive for the glioma CSC markers
CD133 and CD15 (Fig. 2C and Supplementary Fig. S2A
and S2B), as well as the capacity to form spheres as a measure
of self-renewal (Fig. 2D). Notably, the stimulation of CSC
maintenance by low pH was dependent on HIFs, as HIF1/2a
silencing (Supplementary Fig. S3A and S3B) abrogated not only
the acidosis-induced upregulation of HIF target gene expression
(Fig. 2E), but also the increase in the fraction of cells positive
for the CSC marker CD133 and in self-renewal capacity (Fig. 2F
and G). Collectively, our results identify acidosis as a potent
metabolic factor that regulates HIF function and the CSC phenotype synergistically with hypoxia within the pathophysiologic pH range observed in the tumor microenvironment.
The pH-mediated control of HIF is PHD/VHL independent
We next aimed at identifying the molecular mechanism that
allows acidosis to regulate HIF. It has been previously shown, that
an acidic pH of 5.8–6.2 induces HIF1a by nuclear sequestration of
VHL, resulting in a relative cytoplasmic lack of VHL and inhibiting
VHL-dependent proteasomal degradation (24). To examine
whether an acidic pH in the range of 6.7–6.9, values typically
measured in tumors (14, 23), regulates HIF through VHL we first
analyzed the influence of acidification on HIF levels in the VHLdeficient renal cell carcinoma lines 786-O and RCC11 (which
express 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 overexpression in glioblastoma cells did not affect the acidosisinduced 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 of
HIF proteins by acidosis, nonhydroxylatable mutant proteins of
HIF1a and HIF2a with substitution of both prolyl residues that
are hydroxylated by PHDs (mPPN mutants, ref. 13), were strongly
increased by acidic pH (6.7; Fig. 3D). Taken together, these data
provide compelling evidence that acidosis regulates HIF levels
independently of the PHD/VHL-mediated degradation pathway
in glioma.
Acidosis controls HIF and the CSC phenotype through HSP90
Apart from the oxygen-dependent PHD/VHL pathway, one
of the best characterized mechanisms controlling HIF stability is linked to HSP90. HSP90 interacts with HIF (26, 27)
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Acidosis Promotes HIF and Glioma Stem Cells
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 varying
glucose 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 18
hours. The panel shows nonadjacent lanes from the same exposure of the same Western blot analysis. C, hypercapnic acidosis increases HIF1/2a
transactivation activity. G55 cells were cotransfected with a VEGF promoter luciferase reporter together with empty vector, HIF1a, or HIF2a and exposed to
the 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 or
hypoxia (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.
and competes with the protein RACK1 for association with
HIF, thus protecting HIF from proteasomal degradation (28).
Therefore, we next examined whether the acidosis-dependent
upregulation of HIF levels and the CSC phenotype are coupled
to HSP90. We found that lowered pH induced a marked
upregulation of HSP90 in established and primary glioblastoma cell lines (Fig. 4A and B). Importantly, inhibition of HSP90
function with geldanamycin abolished the acidosis-induced
increase in HIF1/2a and CD133 (Fig. 4C and D). Similarly,
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a dominant-negative HSP90 construct (29) blocked the
increase of HIF levels at acidic pH (Fig. 4E), demonstrating
that HSP90 activity is required to mediate HIF upregulation
under conditions of acidic stress. To corroborate the HSP90
dependent control of HIF we tested whether increasing HSP90
levels would replicate the effects of acidosis. Indeed, overexpression of wild-type HSP90 already at physiologic pH led
to an elevation of HIF1a and HIF2a levels similar to the one
caused by low pH (Fig. 4F).
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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 primary
glioblastoma stem cell lines under hypoxia. Immunoblot (A) and real-time RT-PCR (B) of NCH644, GBM015, and GBM031 primary glioblastoma stem
cell 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 cell
phenotype in primary glioblastoma cells. Quantification of the fraction of CD133þ cells, determined by FACS analysis (n ¼ 3; C) and sphere forming
capacity (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, acidosis
induces 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), and
the sphere-forming capacity (n ¼ 6; G) of GBM015 primary glioblastoma cells transfected with nonsilencing control or HIF1/2a siRNA and incubated
at 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.
We next assessed the function of HSP90 in HIF regulation in
a microenvironmental setting of hypoxia and acidosis in vivo.
Importantly, HSP90 disruption by shRNA-mediated knockdown in glioblastoma cells significantly reduced intracranial
OF6 Cancer Res; 76(19) October 1, 2016
tumor growth (Fig. 5A and B). While both control and shHSP90
glioblastomas displayed typical perinecrotic areas with pronounced hypoxia, as revealed by Hypoxyprobe staining
(Fig. 5C), HIF1/2a levels and the expression of the HIF target
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Acidosis Promotes HIF and Glioma Stem Cells
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 renal
carcinoma 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 does
not 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 6.7
or 7.4 under hypoxia (1% O2), or under normoxia following the inhibition of PHD function by the iron chelator dipyridyl (DP) and the PHD inhibitor
dimethyloxalylglycine (DMOG) for 6 hours. D, PHD-refractory HIF1a and HIF2a mutants remain sensitive to pH regulation. Immunoblot of G55 cells transfected
with 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.
gene VEGF were significantly reduced in shHSP90 tumors
(Fig. 5D–F). A similar decrease of HIF levels and orthotopic
tumor growth was elicited HPS90 inhibition through a dom-
inant-negative HSP90 (Supplementary Fig.S4A–S4D). Moreover, HSP90 silencing increased survival in a subcutaneous
transplantation model, again concomitant with a reduction in
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 the
acidosis-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 the
acidosis-induced HIF upregulation. Immunoblot of G55 cells transfected with control vector (pcDNA3, "") or an HA-tagged dominant negative HSP90
construct 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 transfected
with control vector (pcDNA3, "") or an HA-tagged wild-type HSP90 construct and incubated at pH7.4 or 6.7 and 1% O2.
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Filatova et al.
Figure 5.
HSP90 inactivation suppresses the acidosis-induced HIF increase and tumor growth. A, silencing of HSP90a/b reduces HIF levels in vitro. Immunoblot
of 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 of
HSP90 reduces tumor growth and intratumoral HIF levels and activity. Control and HSP90a/b shRNA G55 tumor cells were orthotopically transplanted
in 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 gene
VEGFA 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 levels
in control and shHSP90 tumors by immunoblot (n ¼ 9). All values are mean þ SEM. , P < 0.05; , P < 0.001. Scale bars, 1 mm (B), 100 mm (D), 20 mm (D, higher
magnifications).
HIF1/2a levels (Supplementary Fig. S5A–S5D). Importantly,
HPS90 disruption reduced the tumor-initiating capacity of the
cells, indicating an involvement of the HSP90-mediated
increase in HIF levels under acidosis in tumor initiation and
CSC maintenance. Indeed, HSP90 inhibition with geldanamycin efficiently blocked the acidosis induced increase in the
CD133-postive tumor cell fraction as well as in self-renewal
(Fig. 6A and B). To further corroborate the role of acidosis and
HSP90 in the promotion of CSC function, we examined the
tumorigenic capacity of primary glioblastoma cells, a defining
OF8 Cancer Res; 76(19) October 1, 2016
property of CSCs. Glioblastoma cells grown under pH 6.7
generated significantly larger intracranial tumors than cells
cultured at physiologic pH (Fig. 6A, C, and D). Crucially,
concomitant treatment of the cells with geldanamycin
completely abolished the increased tumorigenicity under acidosis, but had no effect under physiologic pH (Fig. 6C and D).
These findings demonstrate that acidosis increases HIF levels
and CSC function through HSP90 and that HSP90 inhibition
can efficiently suppress HIF induction, CSC maintenance, and
tumorigenicity.
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Acidosis Promotes HIF and Glioma Stem Cells
Figure 6.
The acidosis-induced increase in CSC
function is dependent on HSP90. A and
B, inhibition of HSP90 abrogates the
acidosis-dependent induction of the
glioma stem cell phenotype. The fraction
of CD133þ cells, determined by FACS
analysis (n ¼ 3; A), and sphere-forming
capacity (n ¼ 12; B) of NCH644 primary
glioblastoma cells treated with
control vehicle (DMSO) or 1 mmol/L
geldanamycin (GA) and cultured
at pH 7.4 or 6.7 and 1% O2. C and D,
acidosis stimulates tumorigenicity in an
HSP90-dependent manner. NCH644
primary glioblastoma cells incubated at
1% O2 in medium with pH 7.4 or 6.7
supplemented with DMSO control or
1 mmol/L geldanamycin for 18 hours were
orthotopically transplanted into
nude mice. Tumor xenografts were
stained with hematoxylin and eosin
and the tumor volume was quantified
(D; n ¼ 7–8). Representative images of
the hematoxylin and eosin–stained
tumor sections are shown in C. All values
are means þ SEM; , P < 0.01;
, P < 0.01; , P < 0.001. Scale bar,
1 mm (C). ns, nonsignificant.
HSP90 is expressed in the hypoxic CSC niche and correlates
with the CSC phenotype
We next wanted to assess the relevance of our findings to
human glioblastomas. Analysis of gene expression in the glioblastoma cohort of TCGA research network (30) revealed that
tumors with high HSP90 expression had increased levels of the
HIF target gene VEGF, as well as of the glioma stem cells markers
CD133 and nestin (Fig. 7A), supporting the notion that HSP90 is
an important factor activated by the CSC microenvironment. To
confirm that HSP90 is specifically upregulated in glioblastoma in
the hypoxic CSC niche, we examined its localization in glioblastoma biopsies. The presence of necrotic areas is a diagnostic
hallmark of glioblastoma and is associated with regions of insufficient blood supply and severe hypoxia. HIF levels were elevated
in the perinecrotic regions (Fig. 7B), indicating that these areas are
hypoxic and thus presumably more acidic as a result of the
hypoxia-induced metabolic response. Importantly, HSP90 levels
in the same region were also increased in 10 of 10 glioblastoma
biopsies examined, with 93.0% 4.0% of perinecrotic/hypoxic
areas showing HIF and HSP90 coexpression. Moreover, CD133
was also prominently enriched in this area (Fig. 7B), supporting
our hypothesis of synergistic stimulation of HIF function by
hypoxia and acidosis in an HSP90-dependent manner, leading
to an expansion of the CSC pool in human glioblastomas.
Discussion
Tumor cells are involved in an intimate crosstalk with their
microenvironment, which critically controls major aspects of
cancer cell biology. In this study, we show that two key characteristics of the tumor microenvironment, hypoxia and acidosis,
synergize to potentiate HIF activity and HIF-dependent functions
through complementary and additive mechanisms. We identify
HSP90 as a key factor responsible for the capacity of the acidic
www.aacrjournals.org
microenvironment to promote the hypoxic response and its
downstream functions, including CSC maintenance and tumorigenicity, pointing to potential strategies for the targeting of this
critical cancer cell population.
Acidosis regulates HIF function through HSP90
Hypoxia and acidosis are characteristic features of many
tumor types. Similarly to hypoxia, the acidic tumor microenvironment regulates key aspects of tumor pathophysiology. For
example acidosis can enhance the resistance of tumor cells to
various chemotherapeutics (15, 31, 32). Furthermore, a number of studies have linked acidosis to an enhanced invasive
and metastatic capacity of tumor cells (9, 33–36). In gliomas,
the acidic microenvironment has been shown to induce VEGF
expression and tumor angiogenesis (16, 37). Importantly, most
of the properties listed above are also regulated by hypoxia and
the HIF pathway. Indeed, our results uncover an interesting
link between acidosis and HIF showing that acidosis increases
HIF levels and function in a synergistic manner with hypoxia.
An increase of both HIF1a and HIF2a levels under acidosis has
been previously reported in primary glioblastoma cells
and other cancer cell types (14, 38). In contrast, other groups
have observed a downregulation of HIF in acidic conditions
(39, 40). By systematically assessing the effects of different
levels of acidosis on HIFs we show that acidosis enhances HIF
levels down to a pH of 6.6–6.8, while lower pH values decrease
HIF expression, pointing to a narrow pH window for acidotic
activation of the HIF pathway. Importantly, these values
correspond to the extracellular pH that has been measured
in the majority of tumors, including glioblastoma (14, 23),
underlining the physiologic relevance of our findings.
The levels and activity of HIFs are regulated by multiple
mechanisms. A central place among those is taken by the
PHD/VHL-dependent HIF hydroxylation, ubiquitination and
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Figure 7.
High HSP90 expression is observed in the hypoxic niche and correlates with hypoxic and stem cell markers in human glioblastomas. A, comparison of
the 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. The
panels on the right show higher magnifications of the staining in the perinecrotic areas. N, necrosis. C, a model of the synergistic regulation of HIF
function and CSC maintenance by the hypoxic/acidotic tumor niche. The hypoxic tumor niche induces the establishment of an acidic environment, which
synergizes with decreased oxygen availability to potentiate the hypoxic response. This represents a positive feedback loop that enhances HIF-dependent
functions 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 hostile
microenvironment. Scale bars, 100 mm (C) and 20 mm (C, inset).
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 to
HIF stabilization (24). However, several lines of evidence that we
present here strongly argue against a role for the VHL/PHD
degradation pathway in mediating the effect of acidosis on HIF
stability in the pH range that is typically encountered in tumors.
For example, renal carcinoma cells lacking VHL still responded to
acidosis with a striking increase in HIF levels. Furthermore, HIF
induction under acidic pH was not affected by inhibition of
PHDs. Instead, we show that acidosis stabilizes HIF1a and HIF2a
through the upregulation of HSP90, independently of PHD and
OF10 Cancer Res; 76(19) October 1, 2016
VHL. Importantly, inhibition of HSP90 function by geldanamycin or a dominant-negative mutant abolished the stabilization of
HIF1/2a at low pH. Among alternative, PHD-VHL–independent
mechanisms that regulate HIF stability HSP90 has been well
characterized (reviewed in ref. 41). The multifunctional scaffold
protein RACK1 binds the PAS-A domain of HIF with subsequent
recruitment of the ubiquitin ligase complex that mediates its
degradation, in a manner analogous to the mechanism activated
by VHL, but independent of O2/PHD-mediated HIF hydroxylation (28). HSP90 competes with RACK1 by binding to the HIF
PAS-A domain (42), thereby stabilizing HIF (28). Taken together,
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Acidosis Promotes HIF and Glioma Stem Cells
our results highlight the central importance of acidosis as a key
microenvironmental factor that regulates the HIF response synergistically with decreased oxygen tension through an HSP90dependent, but PHD/VHL and oxygen-independent mechanism.
Acidosis and HSP90 are important regulators within the
hypoxic niche and a target for antitumor therapy
HSP90 forms a chaperone complex that can stabilize and
activate a number of cellular proteins. In cancer cells HSP90 plays
an additional important role by protecting various mutated or
overexpressed proteins against misfolding and degradation,
thereby facilitating oncogene addiction, counteracting proteotoxic stress and enabling cancer cell survival (43). HSP90 upregulation has been found in several types of cancer and has
been linked to poor prognosis and increased tumor aggressiveness
(44–46). This has led to the development of a number of smallmolecule inhibitors, including geldanamycin and its derivatives
that are currently in clinical trials for various tumor types (47).
Our results highlight a general function of HSP90 in tumor
physiology, revealing a novel role of HSP90 as the key mediator
of the acidosis-dependent potentiation of HIF function. The
clinical relevance of our findings is supported by the fact that
HSP90 is prominently upregulated within the hypoxic niche of
human glioblastomas and a high level of HSP90 is linked to the
upregulation of HIF targets and CSC marker genes. Interestingly, a
higher sensitivity of CSCs to HSP90 inhibitors compared with
neural stem cells or other untransformed cells has recently been
demonstrated (48, 49). Therefore, blockade of HSP90 function in
glioblastoma, where HSP90 inhibitors have not been tested in
clinical trials so far, could represent a viable therapeutic strategy.
An alternative possibility is to aim at neutralizing tumor acidosis
itself, and indeed recent studies have shown that this can be
achieved by simple interventions such as bicarbonate administration, resulting in curtailed tumor growth, invasion, and metastasis (33, 50).
The synergistic increase of HIF levels and function by hypoxia
and acidosis likely affects multiple HIF-dependent processes that
collectively can promote tumor aggressiveness. Among those, the
maintenance and expansion of the CSC pool likely plays an
important role, as it is linked to numerous aspects of tumor
progression that are stimulated by both hypoxia and acidosis.
We and others have previously shown that CSCs are located and
controlled within a hypoxic niche through HIF (12, 13). Our data
support a model in which the hypoxic tumor niche induces an
acidic environment, which synergizes with decreased oxygen
availability to potentiate the hypoxic response and enhance
HIF-dependent downstream functions in a positive feedback loop
(Fig. 7C). Given the crucial role of HIFs in tumor progression, 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 within the hypoxic niche.
Importantly, these two, synergistically acting mechanisms may
allow tumor cells to fine-tune and flexibly induce HIF activity in
response to different microenvironmental parameters (O2 level,
pH) to activate key hallmarks of cancer (Fig. 7C). Thus, by
providing important mechanistic insight into the synergistic
control of HIF function and CSC maintenance through central
physiologic parameters of the tumor microenvironment, our
work uncovers potential avenues for developing novel therapeutic
approaches targeted against hypoxia/acidosis-driven tumor
progression.
Disclosure of Potential Conflicts of interest
T. Acker was a consultant/advisory board member for Merck Serono
(2014/2015; minor relationship). No potential conflicts of interest were
disclosed by the other authors.
Authors' Contributions
Conception and design: T. Acker
€ rc€
Development of methodology: A. Filatova, S. Seidel, N. B€
ogu
u, B.K. Garvalov,
T. Acker
Acquisition of data (provided animals, acquired and managed patients,
€ rc€
provided facilities, etc.): A. Filatova, S. Seidel, N. B€
ogu
u, S. Gr€af,
B.K. Garvalov, T. Acker
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
€ rc€
computational analysis): A. Filatova, S. Seidel, N. B€
ogu
u, B.K. Garvalov,
T. Acker
Writing, review, and/or revision of the manuscript: A. Filatova, S. Seidel,
€ rc€
N. B€
ogu
u, B.K. Garvalov, T. Acker
Administrative, technical, or material support (i.e., reporting or organizing
€ rc€
data, constructing databases): A. Filatova, S. Seidel, N. B€
ogu
u, B.K. Garvalov,
T. Acker
Study supervision: B.K. Garvalov, T. Acker
Acknowledgments
We would like to thank Barbara Lafferton, Gudrun Schmidt, Kerstin Leib,
Carmen Selignow, and Tanja Diem for excellent technical assistance.
Grant Support
This work was supported by grants from the Deutsche Krebshilfe (T. Acker,
B.K. Garvalov), the German Ministry of Education and Research (BMBF) within
the National Genome Network (NGFNplus) and Brain Tumor Network (BTN)
(T. Acker), the DFGKFO210 (T. Acker, B.K. Garvalov), DFG SPP1069, DFG
SPP1190 (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 the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received September 25, 2015; revised June 14, 2016; accepted July 13, 2016;
published OnlineFirst August 3, 2016.
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Cancer Research
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 2016 American Association for Cancer
Research.
Published OnlineFirst August 3, 2016; DOI: 10.1158/0008-5472.CAN-15-2630
Acidosis Acts through HSP90 in a PHD/VHL-Independent
Manner to Promote HIF Function and Stem Cell Maintenance
in Glioma
Alina Filatova, Sascha Seidel, Nuray Bögürcü, et al.
Cancer Res Published OnlineFirst August 3, 2016.
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