PERK/eIF2α signaling inhibits HIF-induced gene expression during the unfolded protein response via YB1-dependent regulation of HIF1α translation
Nucleic Acids Research
PERK/eIF2 signaling inhibits HIF-induced gene expression during the unfolded protein response via YB1-dependent regulation of HIF1 translation
Iglika G. Ivanova 0
Catherine V. Park 0
Adrian I. Yemm 0
Niall S. Kenneth 0
0 Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University , Newcastle upon Tyne NE2 4HH , UK
HIF1 (hypoxia inducible factor 1 ) is the central regulator of the cellular response to low oxygen and its activity is deregulated in multiple human pathologies. Consequently, given the importance of HIF signaling in disease, there is considerable interest in developing strategies to modulate HIF1 activity and down-stream signaling events. In the present study we find that under hypoxic conditions, activation of the PERK branch of the unfolded protein response (UPR) can suppress the levels and activity of HIF1 by preventing efficient HIF1 translation. Activation of PERK inhibits de novo HIF1 protein synthesis by preventing the RNA-binding protein, YB-1, from interacting with the HIF1 mRNA 5 UTR. Our data indicate that activation of the UPR can sensitise tumor cells to hypoxic stress, indicating that chemical activation of the UPR could be a strategy to target hypoxic malignant cancer cells.
Cellular hypoxia can occur as a consequence of low
atmospheric oxygen or locally in tissues due to
inflammation, ischemia, injury or poor vascularisation (
). At the
cellular level, hypoxia is characterised by a switch in
energy metabolism coupled with a rapid change in the
transcriptional program, primarily mediated by the hypoxia
inducible factor (HIF) family of transcription factors (
Activation of HIF promotes the expression of specific
target genes that play critical roles in the adaptive response to
hypoxia and the restoration of cellular homeostasis (2).
HIF1 is a ubiquitously expressed heterodimeric
transcription factor, composed of an oxygen labile HIF1
subunit and a constitutively expressed HIF1 subunit (
HIF1 and HIF1 are essential for development as both
HIF1 and HIF1 knockout mice die in utero between 9.5
and 10.5 days of gestation, largely due to defects in
embryonic vascularisation (
). HIF1 stability is primarily
regulated through the action of several proline hydroxylases
(PHDs), which act to modify proline residues in the
oxygendependent degradation (ODD) domain of HIF1 (
Hydroxylated HIF1 is recognised by the von-Hippel
Lindau (VHL) E3-ubiquitin ligase, which promotes the
ubuiquitination and subsequent degradation of HIF1 by the
26S proteasome (
). As a consequence, the half-life of the
HIF1 protein is <5 min in normal conditions, resulting
in the HIF1 protein being virtually undetectable in
adequately oxygenated cells and tissues (
). In hypoxic cells
PHD enzymes are inhibited resulting in rapid HIF1
accumulation, this allows HIF1 to dimerise with HIF1 to
promote the expression of HIF target genes (
Although HIF1 levels are primarily regulated by
proteasomal degradation alternative mechanisms exist to
modulate HIF activity such as transcriptional regulation of
HIF genes or post-translational modification of HIF
). Control of HIF1 biogenesis through
regulation of protein translation is also emerging as an
important mechanism for regulating HIF in hypoxic cells. In fact,
HIF1 protein biogenesis is responsible for 40–50% of the
increased levels of HIF1 protein in response to hypoxic
). HIF1 has both 5 and 3 UTRs that can
regulate its translation; with the 5 UTR containing an
internal ribosome entry site that can upregulate HIF1
translation, and the 3 UTR mainly responsible for controlling
mRNA stability (13). 5 -UTR-dependent upregulation of
HIF1 translation is observed in metastatic cell lines,
indicating that this mechanism of HIF1 elevation may be
critical for the malignant phenotype (
In actively growing eukaryotic cells, protein translation
accounts for ∼75% of the total energy expenditure of a cell
). During severe hypoxia/anoxia (<0.2% O2), cellular
energy consumption is limited and global protein synthesis
is inhibited through activation of the unfolded protein
response (UPR) (
). The UPR is a highly conserved pathway
that allows cells to effectively manage cellular stress
triggered by chemical and environmental factors (
to the UPR is the PKR-like ER kinase (PERK)-dependent
phosphorylation of eukaryotic initiation factor 2 (eIF2 )
which represses global translation while promoting the
preferential translation of mRNA that encode stress-responsive
factors to restore cellular homeostasis (
severe hypoxia/anoxia the UPR and hypoxia response
pathways interact to potentiate the expression of HIF target
genes (18). However, inhibition of the PHD enzymes and
stabilisation of HIF1 occurs at relatively moderate levels
of hypoxia (<2%), which is not sufficient to activate the
In this present study, we examined the consequences of
activating the UPR in conditions of moderate hypoxia to
investigate if this could potentiate the HIF-dependent
hypoxic response. Surprisingly, we find that chemical
activation of the UPR during moderate hypoxia impairs HIF1
stabilisation and results in the down regulation of
hypoxiainduced HIF1 activity. Our data indicate that activation of
the UPR in low oxygen severely reduces HIF1 activity by
blocking HIF1 mRNA translation in a PERK-dependent
manner. Activation of the UPR reduces the interaction
between the RNA binding protein, YB-1, and the 5 -UTR of
the HIF1 mRNA, thus preventing its efficient translation.
Chemical inhibition of PERK rescues the HIF1 defect and
the levels of HIF1 activity in hypoxic cells treated with UPR
agonists. Impairment of the HIF pathway by UPR
activation results in a reduction of cell viability in low oxygen,
indicating that targeting the UPR may be a strategy to target
hypoxic malignant cells.
MATERIALS AND METHODS
PC-3 cells were grown in RPMI with 25mM HEPES,
supplemented with 10% FBS and L-glutamine. U2OS, MCF7
and COV-434 cells were gown in DMEM supplemented
with 10% FBS, L-gluatamine, and penicillin streptomycin.
U2OS HRE luciferase and U2OS NF- B luciferase cells
have been previously described (
Cells were incubated at 1% O2 in an in vivo 400 hypoxia
work station (Ruskin, UK). Cells were lysed for protein
extracts, and RNA extraction in the chamber to avoid
Thaspsigargin (Enzo), tunicamycin (Calbiochem),
DMOG (Calbiochem), GSK2606414 (Calbiochem),
MG132 (Sigma), lactacyctin (Calbiochem), ionomycin
(Sigma) were dissolved in DMSO and added to cells at
the concentrations indicated in the figure legends. DTT
(Sigma) and EGTA (Sigma) were prepared in ultrapure
Cell lysis and immunoblotting
Cells were lysed in 8 M urea lysis buffer and
immunoblotted as described (
). Antibodies used were HIF1 (Clone
241809, R&D systems), HIF2 (#7096, Cell Signaling
Technologies), phospho- eIF2 (Ser 51) (#9721, Cell
Signaling Technologies), eIF2 (#5324, Cell Signaling
Technologies), PERK (#3192, Cell Signaling Technologies),
YB-1 (A303–231A, Bethyl), -tubulin (#2146, Cell
Signaling Technologies), VHL (#68547, Cell Signaling
Technologies), -actin (AC-74, Sigma).
Lysates for luciferase assay were prepared in 1× passive
lysis buffer (Promega), 100 l per well of a 24-well plate.
10 l of lysate was incubated with 50 l luciferase reagent
(Promega) and measured for 10 s using (Lumat LB9507,
EG&G Berthold). Graphs are represent raw RLUs readings
from three independent experiments.
PC-3 cells were grown to 80% confluency and incubated in
100 mg/ml cycloheximide for 3min and resuspended in
hypotonic polysome extraction buffer (5 mM Tris [pH 7.5], 2.5
mM MgCl2, 1.5 mM KCl, 1% Triton X-100, 100 mg/ml
cycloheximide, 100 U/ml RNasin). Cell were lysed through
the addition of Triton X-100 (0.5%) and sodium
deoxycholate (0.5%) to solubilise the cytosolic and endoplasmic
reticulum-associated ribosomes. Extracts were normalised
by OD 260 nm and layered onto 10 ml of 10–50% sucrose
steps and centrifuged at 222 228 × g (36 000 rpm) for 2 h at
4◦C using SW41Ti rotor. The sucrose steps were
fractionated into twelve 0.75 ml fractions. Absorbance at OD254 nm
and visualisation by RNA agarose electrophoresis
following Trizol (Invitrogen) purification was used to determine
the monosomal and polysomal fractions.
To probe for direct interactions between YB-1 protein and
HIF1 transcripts PC-3 were resuspended in hypotonic
polysome extraction buffer and lysed through the addition
of Triton X-100 (0.5%) and sodium deoxycholate (0.5%).
Clarified lysates were incubated with 1 ug of YB-1 antibody
and antibody/protein/RNA complexes were isolated by
using 20 l packed volume protein A sepharose beads. Beads
were washed with polysome extraction buffer and RNA
isolated using the PeqGold RNA isolation kit. cDNA was
prepared using Qiagen Quantanova cDNA synthesis kit.
Quantitative reverse transcription-PCR
Quantitative PCR data was generated on a Rotor-Gene
Q (Qiagen) using the following experimental settings:
hold 50◦C for 3 min; hold 95◦C 10 min; cycling (95◦C
for 30 s; 58◦C for 30 s; 72◦C for 30 s with
fluorescence measurement for 45 cycles). All values were
normalised to 18S rRNA or RPL13A levels using the Pfaffl
method as indicated. Primers sequences: HIF1 For- 5
CATAAAGTCTGCAACATGGAAGGT-3 , HIF1 Rev
5 -ATTTGATGGGTGAGGAATGGGTT-3 ; 18S rRNA
For 5 -GTAACCCGTTGAACCCCATT-3 , 18S rRNA
Rev 5 - CCATCCAATCGGTAGTAGCG- 3 ; RPL13A
sense 5 -CCT GGA GGA GAA GAG GAA AGA GA -3 ,
antisense 5 -TTG AGG ACC TCT GTG TAT TTG TCA
A-3 ; BNIP3 sense 5 -GCC CAC CTC GCT CGC AGA
CAC-3 ; GLUT1 sense 5 -CTG GCA TCA ACG CTG TCT
TC-3 , antisense 5 -GCC TAT GAG GTG CAG GGT C-3 ;
PDK1 sense 5 -AGT TCA TGT CAC GCT GGG TA-3 ,
antisense 5 -CAG CTT CAG GTC TCC TTG GA-3 .
Cell viability assays
Cell viability was measured using Prestoblue assay
(Invitrogen) and performed according to the manufacturer’s
protocol. Briefly, PC-3 cells were seeded at a density of 5000
cells/well. Cells were pre-treated with inhibitors for 30 min
before incubation at 1% O2 or 21% O2 for 24 h. The
absorbance was recorded at 570 nm after 30 min incubation
of cells with Presto Blue reagent. The cell viability was
expressed as a percentage relative to untreated controls
ER stress suppresses HIF levels and activity in moderate hypoxia
The endoplasmic reticulum (ER) is responsible for
performing multiple functions essential for cellular
homeostasis, development, and stress responsiveness (
hypoxia/anoxia (<0.2% O2) induces ER stress and results
in potent activation of the UPR (
). However, there are
conflicting reports as to whether activation of the UPR
during moderate hypoxia contributes positively or negatively
to the HIF-dependent transcriptional response (
To examine the effect of activating the UPR on the HIF
response during moderate hypoxic stress (1% O2), PC-3
prostate cancer cells were treated with thapsigargin to
induce ER stress (
). Eukaryotic initiation factor 2 (eIF2 )
is rapidly phosphorylated in cells treated with
thapsigargin, consistent with the UPR being activated (Figure 1A).
Moderate hypoxia (1% O2) was not sufficient to induce the
UPR, as no hypoxia-dependent increase in phospho-eIF2
was observed (Figure 1A). Surprisingly, hypoxia-dependent
HIF1 stabilisation was markedly decreased in hypoxic
PC3 cells pretreated with thapsigargin compared to controls
(Figure 1A). The effect of thapsigargin on HIF1
stabilisation was tested in MCF7 (breast), COV-434 (ovarian)
and U2OS (osteosarcoma) treated with thapsigargin and
exposed to hypoxia. In each of the cell lines tested HIF1
stabilisation was impaired in cells in which the UPR is
activated indicating that this effect is conserved between cell
lines (Figure 1B–D).
HIF activity was then assessed using U2OS cells
containing an integrated luciferase reporter construct
possessing three copies of the hypoxia-responsive element (HRE)
consensus-binding site. A robust activation of luciferase
activity was observed in cells exposed to 1% O2, which was
reduced by treatment with thapsigargin in a dose-dependent
manner (Figure 1E). Thapsigargin treatment did not alter
TNF-induced NF- B activation, indicating the effect on
HIF activity is specific (Figure 1F).
Previous reports have suggested that modulation of
intracellular calcium in hypoxic cells can contribute to
HIFdependent gene expression, both positively and negatively,
by modulating its levels and/or its activity (
thapsigargin activates the UPR by significantly altering
calcium homeostasis we tested if modulation of
intracellular calcium was sufficient to control HIF1 levels. PC-3
cells pre-treated with the calcium ionophore, ionomycin,
had similar levels of hypoxia induced HIF1 stabilisation
as compared to controls (Supplementary Figure S1A and
B). Similarly, chelating excess calcium using EGTA did
not significantly alter the levels of HIF1 stabilised by low
oxygen (Supplementary Figure S1C). Co-treatment of cells
with thapsigargin and EGTA did not alter the
thapsigargindependent inhibition of HIF1 stabilisation, indicating that
ER stress rather than modulation of calcium homeostasis
alters hypoxia-induced HIF1 levels (Supplementary
Activation of the UPR impairs HIF activity
To investigate whether alternative activators of the UPR
interfere with HIF activity PC-3 cells were treated with
diethiothretol (DTT) or tunicamycin. These agents robustly
activate the UPR with modes of action distinct from one
another, and from thapsigargin (
). DTT and tunicamycin
both activate the UPR, as measured by phospho-eIF2 , to
a level similar to that seen following thapsigargin treatment
(Figure 2A–C). Treatment with both DTT and tunicamycin
suppress HIF1 levels following hypoxic stress, consistent
with data from thapsigargin treated cells (Figure 2B and C).
Stabilisation of HIF1 using the PHD inhibitors DMOG or
CoCl2 is also sensitive to activation of the UPR, indicating
that UPR-dependent decrease in HIF activity is oxygen
independent (Figure 2D–F, Supplemental Figure S2). HIF1
levels and activity were also compromised in U2OS
HRELuc cells pre-treated with UPR activators and exposed to
hypoxia or DMOG, consistent with results from PC-3 cells
Activation of the UPR does not alter HIF1 protein stability
HIF1 protein levels are primarily controlled by
ubiquitinmediated proteolysis (
). Previous work has indicated that
inducing ER stress through modulation of calcium levels
can interfere with the ubiquitin-mediated degradation of
). However, treatment with the proteasome
inhibitor, MG132, did not significantly increase HIF1
levels in thapsigargin treated cells indicating thapsigargin is
not acting to modulate HIF1 protein stability
(Supplemental Figure S3A and B). HIF1 could not be stabilised
with MG132, or an alternative proteasome inhibitor,
lactacystin, in the presence of thapsigargin, suggesting that
UPR-dependent modulation of HIF1 is independent of
proteasomal degradation (Supplemental Figure S3C and
D). HIF1 protein levels can also be modulated by
autophagic degradation (
), however lysosome inhibitors
failed to rescue the levels of HIF1 in thapsigargin treated
PC-3 cells, indicating UPR-dependent control of HIF1 is
independent of this signaling pathway (Supplemental
Figure S3E and F). Together these data indicate that UPR
activation does not modulate HIF1 levels by altering HIF1
Activation of the UPR reduces HIF1 translation
Levels and activation of HIF can be altered by
transcriptional control of HIF subunits (
). However, steady state
levels of HIF1 mRNA are unaltered by treatment with
thapsigargin, hypoxia or hypoxia mimetics as indicated by
qRT-PCR analysis of HIF1 mRNA levels (Figure 3A,
B, Supplemental Figure S4A–E). HIF1 mRNA
associated with active ribosomes was measured to determine the
rate of HIF1 mRNA translation. Cellular protein
synthesis, as well as translation rates of individual mRNAs, can
be measured by polysome profiling; a technique in which
free ribosomes can be separated from mRNA bound
ribosomes (polysomes) on a sucrose gradient. Hypoxia results
in a moderate decrease in the translation rates in PC-3 cells
as indicated by the increase in the number of monosomes
detected in cells exposed to 1% O2 (Figure 3C). The
decrease in global translation rates is independent of HIF
activity, as the hypoxia mimetic, DMOG, has no significant
effect on protein synthesis (Figure 3D). Surprisingly,
activation of the UPR using thapsigargin did not significantly
alter the global translation rates as measured by the
number of actively translating ribosomes (polysomes) in either
hypoxic or DMOG treated cells (Figure 3C and D,
Supplemental Figure S5). To investigate the levels of
individual mRNAs associated with actively translating ribosomes,
cDNAs were prepared from fractions containing polysomes
(Fractions 8–11) (Figure 3C, D and Supplemental Figure
S3). Quantitative RT-PCR analysis of polysome-associated
HIF1 mRNA revealed a reduction of actively translating
HIF1 in thapsigargin treated samples in both hypoxic and
DMOG treated cells (Figure 3E and F). These data suggest
mRNA translation is sensitive to activation of
UPR-dependent suppression of HIF activity requires PERK
eIF2 is phosphorylated on S51 by four distinct kinases;
PERK, heme-regulated inhibitor (HRI), protein kinase R
(PKR), and general control non-depressible 2 (GCN2)
which are activated by ER-stress, heme depletion, viral
infection and amino acid starvation, respectively (
Inhibition of PERK using GSK 2606414, a small molecule
inhibitor, prevented thapsigargin-induced eIF2
phosphorylation consistent with the induction of ER stress (Figure
4A). Immunoblot analysis demonstrated that PERK
inhibition reversed thapsigargin-dependent reduction of HIF1
levels (Figure 4A). Inhibition of PERK rescued the HIF1
defect from both hypoxic and DMOG treated cells exposed
to DTT and tunicamycin (Figure 4B–F). PERK
inhibition alone did not increase HIF1 levels in hypoxic cells,
suggesting it is acting to reverse inhibition, rather than
as a direct activator (Supplemental Figure S6). Inhibition
of PERK was also sufficient to restore HIF1 levels in
U2OS HRE-Luc cells exposed to UPR agonists (Figure
5A–F). Treatment with PERK inhibitor alone was not
sufficient to elevate HIF1 levels or activity in the absence
of UPR agonists (Supplemental Figure S6). Importantly,
treatment with UPR agonists, either alone or in
combination with GSK2606414 did not significantly alter
levels of TNF induced NF- B activity; indicating that
UPRdependent changes in HIF activity are specific
(Supplemental Figure S7A–D). Together these results demonstrate that
activation of the UPR inhibits HIF signaling in a
UPR-dependent suppression of HIF-target genes is reversed by PERK inhibition
To examine the role of the UPR on the expression of
hypoxia-responsive genes, we performed quantitative real
time PCR analysis of the expression of HIF1 target genes in
PC-3 and U2OS cells treated with thapsigargin. PC-3 and
U2OS cells were treated with thapsigargin and the PERK
inhibitor and subjected to hypoxia for 7 h (Figure 6A and
B). RNA was isolated from these cells and was used to
quantitate the transcript levels of the canonical HIF1 target
genes; CAIX, GLUT1 and PDK1. As expected, exposure
to low oxygen resulted in an increase in expression of all of
these genes, as compared to control (Figure 6A and B).
Expression of HIF target genes is suppressed in hypoxic cells
treated with thapsigargin and inhibition of PERK was
sufficient to reverse the UPR-dependent suppression of
HIFresponsive genes in both PC-3 and U2OS cells (Figure 6A
PERK Inhibition rescues HIF1 translation in thapsigargin treated cells
Polysomal profiling was performed on PC-3 cells treated
with thapsigargin and GSK2606414 to measure actively
translating ribosomes. Surprisingly, PERK inhibition did
not have an obvious effect on global protein synthesis, as
measured by the number of actively translating ribosomes
in hypoxic or DMOG treated cells (Figure 7A and B).
Treatment with GSK2606414 did however significantly reverse
the thapsigargin-dependent suppression of HIF1 mRNA
translation in both hypoxic and DMOG treated PC-3,
indicating that the UPR-dependent decrease in HIF1
translation is in part dependent on PERK activity (Figure 7C and
HIF1 mRNA/ YB-1 interaction is disrupted in thapsigargin
Maintaining high levels of HIF1 mRNA translation is
critical for full activation of HIF during prolonged hypoxia
). Several regulatory factors have been identified that
enhance or reduce HIF1 translation by directly binding to
HIF1 mRNA. One such factor is the Y-box binding
protein 1 (YB-1) that binds directly to the 5 UTR of HIF1
mRNA to sustain high levels of HIF1 in hypoxia (31).
Previous studies have reported YB-1 activity and
subcellular localisation is sensitive to conditions of ER-stress.
YB-1, normally diffusely present in the cytosol, relocalises
to ribonucleoprotein complexes known as stress granules
in thapsigargin treated cells (
). We therefore examined
whether YB-1 played a role in UPR-dependent reduction
in HIF1 translation. As the total levels of YB-1 are not
altered by either hypoxia or activation of the UPR (Figure
8A), we investigated if the YB-1/ HIF1 mRNA was
disrupted by thapsigargin treatment. YB-1 was efficiently
precipitated from cell extracts using a specific polyclonal
antibody (Supplemental Figure S8A). cDNA prepared from
YB-1 precipitates and analysed by quantitative RT-PCR
revealed an increased association between YB-1 and the
HIF1 transcript, consistent with its role in maintaining
HIF1 translation in low oxygen (Figure 8B). In the
presence of the UPR agonist, thapsigargin, the levels of
YB1 associated with the HIF1 transcript are reduced
(Figure 8C). The reduction in the YB-1 / HIF1 mRNA
interaction is consistent with the reduction in HIF1
translation. YB-1 interaction with HIF1 mRNA was specific
as no significant binding of the IL-8 transcript to YB-1
was observed (Supplemental Figure S8B).
Thapsigargindependent reduction of the YB-1/ HIF1 mRNA
interaction was partially reversed by GSK2606414, consistent with
the increase in HIF1 translation (Figure 8C). Our data
indicate that activation of the UPR reduces the interaction
between the translational activator YB-1 and the HIF1
mRNA in a PERK-dependent manner.
Thapsigargin sensitises cells to hypoxic stress
Activation of the HIF family of transcription factors is a
critical component of the cellular response to low oxygen. In
solid tumor cells elevated levels of HIF1 contribute to the
malignant phenotype by promoting the expression of
proangiogenic and pro-survival gene products. As thapsigargin
decreases HIF1 levels and activity we examined if prostate
cancer cell lines were more sensitive to thapsigargin in
hypoxic cells. PC-3 cells were treated with thapsigargin and
incubated at normoxia and hypoxia for 24 h. The viability
of hypoxic PC-3 cells was significantly reduced by
thapsigargin treatment, consistent with the reduction of HIF1
activity (Figure 9A). Increased sensitivity to thapsigargin in low
oxygen conditions was specific, as normoxic and hypoxic
PC-3 cells were equally sensitive to the antimitotic agent,
docetaxel (Figure 9B). The data suggest that activation of
the UPR reduces the HIF-dependent hypoxic response by
impairing HIF1 mRNA translation.
Clinical and experimental data suggest that oxygen
homeostasis and HIF activity is disrupted in multiple human
pathologies such as heart disease, cancer, cardio vascular
disease and chronic obstructive pulmonary disease (
The majority of studies examining how HIF is deregulated
have focused on protein stability, however control of HIF1
translation remains relatively understudied despite its
major contribution to the HIF-dependent hypoxic response.
The results from the present study indicate that activation of
the UPR during moderate hypoxia can attenuate the
HIFdependent transcriptional program. UPR agonists prevent
the full activation of HIF by inhibiting HIF1 mRNA
translation, resulting in a decrease in HIF1 transcriptional
activity and a suppression of HIF1-target gene expression
in a variety of cell lines.
The UPR is a major determinant of cell survival in
response to various conditions of cellular stress and is
associated with various human pathologies including
inflammatory diseases, diabetes and cancer (
). The UPR
is characterised by the activation of three parallel
signalling pathways: PERK-dependent phosphorylation of
eIF2 , inositol-requiring protein 1 (IRE1 )–X-box
binding protein 1 (XBP1) and activating transcription factor 6
(ATF6 ). Our data indicate that activation of the PERK
pathway suppresses the full activation of HIF target gene
expression. Polysome profiling suggests that HIF1 mRNA
translation is extremely sensitive to UPR activation. These
data were unexpected, as HIF1 is efficiently stabilised in
severely hypoxic cells in which the UPR is activated (
Our results indicate that activation of the UPR does not
promote HIF1 accumulation, but severely suppresses it,
indicating that alternative signaling pathways must be
activated to maintain HIF1 biogenesis in severely hypoxic
cells. Interestingly, our data show that hypoxia-dependent
accumulation of HIF2 is unaffected by UPR activation,
suggesting the translational control of HIF1 mRNA by
YB-1 is specific for the HIF1 subunit (Figure 8A).
Accumulating evidence indicates HIF1 translation is
an important mechanism to control HIF activity in cells
). HIF1 mRNA has non-coding regions at both the
5 and 3 ends of the transcript that can be bound by
regulatory proteins to control rates of HIF1 biogenesis (
However, the signaling pathways that regulate their binding
to the HIF1 transcript remain poorly defined (
YB1 is a multifunctional nucleic acid-binding protein that can
directly bind to and activate translation of HIF1 mRNA
to promote sarcoma cell invasion and enhanced metastatic
capacity in vivo (31). In our present study we find that
activation of the UPR under conditions of hypoxic stress
reduces the interaction of YB1 with HIF1 mRNA, reducing
HIF1 mRNA translation and causes a decrease in HIF1
levels. Total YB-1 is not altered by activation of the UPR
or hypoxic stress, however when cells experience ER stress
YB-1 alters its subcellular distribution to localise to stress
granules; discrete riboprotein complexes in the cytoplasm
of cells (
). This rapid redistribution of YB-1 results in less
YB-1 being associated with HIF1 mRNA, suppressing its
In addition to its role in ER homeostasis, the UPR has
emerged as a key mediator of DNA replication, energy
metabolism and cellular activation of apoptosis (
UPR components are often deregulated in malignant cells
and the activation of the UPR is thought to contribute to
tumor development (37). However, whether activation of
the UPR is positive or negative for tumor progression
remains unclear (
). UPR agonists such as thapsigargin
and tunicamycin have been shown to be effective in
promoting tumor cell death (
). Indeed, analogues of
thapsigargin are currently in clinical trials as prostate cancer
). The effectiveness of these agents in
targeting tumor cells may be due to their ability to target the
HIF pathway. Indeed, histological analysis of implanted
tumors in nude mice treated with tunicamycin revealed
reduced growth, vasculature and VEGF levels, classical signs
of reduced HIF activity (
). Our data suggest that hypoxic
tumor cells display increased sensitivity to UPR agonists,
indicating that activation of the UPR may be a strategy to
target hypoxic, solid tumors.
Collectively our data reveal that ER-stress regulates the
interaction between the YB-1 protein and HIF1 mRNA,
which can alter rates of HIF1 protein synthesis.
UPRdependent suppression of HIF1 may provide a novel
strategy for targeting aberrant HIF activity in human disease.
Supplementary Data are available at NAR Online.
We would like to thank Dr Jill Hunter, Prof. Neil Perkins
and Prof. Sonia Rocha for helpful discussions and reagents.
In addition, we are grateful to Dr Claudia Schneider for her
help in analysing and preparing RNA samples.
Cancer Research UK [C1443/A22095 to I.G.I.];
Biotechnology and Biological Sciences Research Council
Doctoral Training Partnership Studentship (to C.V.P.);
Biotechnology and Biological Sciences Research Council
[BB/M018318/1 to A.I.Y.]; Newcastle University
Independent Researcher Establishment Scheme Award (to N.S.K.).
Funding for open access charge: Newcastle University.
Conflict of interest statement. None declared.
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