Hypoxia promotes ligand-independent EGF receptor
signaling via hypoxia-inducible factor–mediated
upregulation of caveolin-1
Yi Wanga
, Olga Rochea
, Chaoying Xua
, Eduardo H. Moriyamab,c
, Pardeep Heira
, Jacky Chungd
, Frederik C. Roosa,e
,
Yonghong Chenb
, Greg Finakf,g,h
, Michael Milosevicb
, Brian C. Wilsonb,c
, Bin Tean Tehi
, Morag Parkf,g,h
,
Meredith S. Irwind,j
, and Michael Ohha,1
a
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5S 1A8; b
Division of Biophysics and Bioimaging and
c
Department of Medical Biophysics, University Health Network, Princess Margaret Hospital, Toronto, ON Canada M5G 2M9; d
Cell Biology Program, Hospital
for Sick Children Research Institute, Toronto, ON, Canada M5G 1L7; e
Department of Urology, Johannes Gutenberg University, 55101 Mainz, Germany;
Departments of f
Biochemistry, g
Oncology, and h
Medicine, McGill University, Montreal, QC, Canada H3A 1A1; i
Van Andel Research Institute, Grand Rapids,
MI 49503; and j
Department of Paediatrics, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8
Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health
Network, Toronto, ON, Canada, and approved February 7, 2012 (received for review July 28, 2011)
Caveolin-1 (CAV1) is an essential structural constituent of caveolae,
specialized lipid raft microdomains on the cell membrane involved in
endocytosis and signal transduction, which are inexplicably deregulated
and are associated with aggressiveness in numerous cancers.
Here we identify CAV1 as a direct transcriptional target of oxygenlabile
hypoxia-inducible factor 1 and 2 that accentuates the formation
of caveolae, leading to increased dimerization of EGF receptor
within the confined surface area of caveolae and its subsequent
phosphorylation in the absence of ligand. Hypoxia-inducible factor–
dependent up-regulation of CAV1 enhanced the oncogenic potential
of tumor cells by increasing the cell proliferative, migratory, and
invasive capacities. These results support a concept in which a crisis
in oxygen availability or a tumor exhibiting hypoxic signature triggers
caveolae formation that bypasses the requirement for ligand
engagement to initiate receptor activation and the critical downstream
adaptive signaling during a period when ligands required
to activate these receptors are limited or are not yet available.
Cellular adaptation to compromised oxygen availability or hypoxia
is critically dependent on the activity of heterodimeric
hypoxia-inducible factor (HIF) family of transcription factors (1, 2).
The catalytic HIFα subunit is oxygen labile by the virtue of the
oxygen-dependent degradation (ODD) domain that is targeted for
ubiquitin-mediated destruction under normal oxygen tension or
normoxia via the von Hippel–Lindau (VHL) tumor suppressor
protein-containing E3 ubiquitin ligase, elongins/Cul2/VHL (ECV)
(3). Under hypoxia, HIFα escapes the destructive recognition
of VHL, recruits p300/Creb-binding protein, and binds to the
constitutively expressed and stable HIFβ (also known as “aryl hydrocarbon
receptor nuclear translocator,” ARNT) to form an active
transcription complex (3). HIF engages hypoxia-responsive
element (HRE; 5′-RCGTG-3′) within enhancers/promoters to
initiate transcription of numerous hypoxia-inducible genes that regulate
adaptive responses such as angiogenesis, erythropoiesis, and
anaerobic metabolism (2).
Deregulation of HIF has been well documented in common
human pathologic conditions such as heart disease, cerebrovascular
disease, chronic obstructive pulmonary disease, and cancer
(4–6). The extent of HIFα expression, in particular, is correlated
with cancer aggressiveness, resistance to radiation and chemotherapy,
and poor prognosis (3). Perhaps the most heuristic
association is between the loss of VHL and the resulting upregulation
of HIF activity in the development of VHL disease,
characterized by tumors in multiple organs, including retinal and
cerebellar hemangioblastoma, pheochromocytoma, and clearcell
renal cell carcinoma (CCRCC), the most common form of
kidney cancer (3). In addition to causing rare VHL disease-associated
tumors, biallelic inactivation of VHL is associated with
the vast majority of sporadic CCRCC, which typically and expectedly
exhibit strong hypoxic profiles.
Caveolin-1 (CAV1) is the major structural component of caveolae,
which are 50- to 100-nm flask-shaped vesicular invaginations
of the plasma membrane (7). CAV1, through scaffolding domains
(CSD), also has been shown to bind several proteins involved in
signaling (8). Intriguingly, similar to HIFα, elevated CAV1 expression
has been associated with larger tumor size, higher tumor grade
and stage, resistance to conventional therapies, and poor prognosis
in numerous cancer types in several organs, including colon, liver,
stomach, prostate, breast, lung, brain, and kidney (but with the exception
of extrahepatic bile duct carcinoma and mucoepidermoid
carcinoma of the salivary gland, in which increased CAV1 expression
has been correlated with favorable clinical outcome) (9–27).
Although these observations suggest a possible correlation between
HIF and CAV1, the molecular mechanisms regulating CAV1 expression
and CAV1-mediated signaling remain largely unknown.
Results
Hypoxia Promotes CAV1 Expression via HIF. Most of primary CCRCC
tumor extracts showed markedly higher expression of CAV1 (12/
14) and glucose transporter type 1 (GLUT1) (6/6), a hypoxia indicator,
in comparison with matched normal kidney samples (Fig.
1A and Fig. S1A). CAV1 mRNA expression, similar to hypoxiainducible
genes EGLN3, CA9, VEGFA, PDK1, GLUT1, HIG2, and
LOXL2, was significantly up-regulated in primary CCRCC (n = 10)
in comparison with the nondiseased renal cortex (n = 12) (Fig. 1B).
The correlation between CAV1 and the aforementioned HIF target
genes, as determined by Pearson’s correlation coefficient, was
strong (r > 0.80) and significant (P < 0.0001). Moreover, samples of
primary papillary renal cell carcinoma (RCC), the second most
common form of kidney cancer, with a strong hypoxic signature
displayed increased CAV1 mRNA and protein levels (Fig. S1 B and
C). These results suggest that kidney tumors with elevated hypoxic
profiles are associated with increased CAV1 expression.
We next asked whether CAV1 expression is regulated by HIF.
786-MOCK (VHL−/−
HIF1α−/−
) CCRCC cells, which have a high,
stabilized level of HIF2α because of the loss of VHL, exhibited
elevated CAV1 levels in comparison with isogenically matched
786-O cells stably reconstituted with wild-type VHL (786-VHL)
(Fig. 1C). Similar results were obtained using a different CCRCC
cell line, RCC4 (Fig. S2A). Furthermore, 786-VHL cells, as well as
Author contributions: Y.W., E.H.M., M.M., B.C.W., B.T.T., M.P., M.S.I., and M.O. designed
research; Y.W., O.R., C.X., E.H.M., P.H., J.C., F.C.R., Y.C., and G.F. performed research; Y.W.,
O.R., C.X., E.H.M., P.H., J.C., F.C.R., Y.C., G.F., M.M., B.C.W., B.T.T., M.P., M.S.I., and M.O.
analyzed data; and Y.W. and M.O. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: michael.ohh@utoronto.ca.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1112129109/-/DCSupplemental.
4892–4897 | PNAS | March 27, 2012 | vol. 109 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1112129109
other VHL-null CCRCC cell lines stably reconstituted with VHL
(RCC4-VHL and UMRC2-VHL), but not 786-MOCK cells,
maintained under hypoxia showed a time-dependent increase in
CAV1 that correlated positively with the induction of HIF2α (Fig.
1D and Fig. S2 B–D). Similar results were observed in nonCCRCC
cell lines that are not associated with VHL mutations,
including cervical cancer (HeLa), glioma (CNS-1), metastatic
breast cancer (MTC-1), epidermoid carcinoma (A431), and murine
pro-B (Ba/F3) cell lines, and primary wild-type mouse embryonic
fibroblasts (MEFs) (Fig. S2 E–J). Consistent with CAV1
protein level, relative CAV1 mRNA expression was elevated under
hypoxia or in the absence of VHL (Fig. 1E).
We next asked whether the increased expression of CAV1 under
hypoxia or upon the loss of VHL was mediated via HIF. 786-O cells
with stable shRNA-mediated knockdown of HIF2α expressed
CAV1 levels that were markedly lower than the CAV1 levels of
parental 786-O cells expressing nontargeting scrambled shRNA
(Fig. 1F). Moreover, individual attenuation of either HIF1α or
HIF2α in RCC4-MOCK cells down-regulated CAV1 expression,
and simultaneous knockdown of HIF1α and HIF2α most profoundly
down-regulated CAV1 expression (Fig. S3). Conversely, in HEK293
(VHL+/+
) cells the ectopic expression of stable HIF1α(P564A) or
HIF2α(P531A), in which the proline-to-alanine substitution negates
the otherwise prolyl-hydroxylation within ODD that is necessary for
VHL recognition, increased CAV1 levels (Fig. S4). Consistent with
these findings, the depletion of HIF1α and/or HIF2α in MCF-7
breast cancer cells likewise has been associated with decreased
CAV1 mRNA levels (Gene Expression Omnibus accession number
GDS2761) (28). These results demonstrate that hypoxia induces the
expression of CAV1, at least in part, via HIF1 or HIF2.
CAV1 Is a Direct Transcriptional Target of HIF via Conserved HRE. The
aforementioned expression profiling (Fig. 1B and Fig. S1C) and
quantitative real-time PCR analyses (Fig. 1E) strongly suggest
that the HIF-mediated regulation of CAV1 occurs, at least in
part, at the level of transcription. Consistent with this notion, we
identified a conserved HRE within the CAV1 promoter (Fig. 2A)
that was capable of binding in vitro-translated radio-labeled
HIF1 (HIF1α+ARNT) or HIF2 (HIF2α+ARNT) (Fig. 2B). In
addition, the levels of HIF1α, HIF2α, and RNA Polymerase II
(Pol II) at the endogenous CAV1 promoter as determined by
ChIP were markedly lower in RCC4-VHL and 786-VHL cells
than in RCC4-MOCK and 786-MOCK cells (Fig. 2 C and D).
These results demonstrate that HIF1α and HIF2α physically bind
the CAV1 promoter via HRE concomitant with the engagement
of Pol II and the induction of CAV1 transcription.
Loss of VHL Function Is Associated with Increased Caveolae Formation.
A major component of caveolae is CAV1. Thus, we asked whether
the status of HIF affected caveolae formation by taking advantage
of stable 786-O(VHL−/−
; HIF1α−/−
) subclones expressing various
mutant forms of VHL that are either deficient or proficient in
degrading HIF2α. VHL-null 786-O and 786-VHL(C162F) (an α
domain mutant that has lost the ability to degrade HIFα because
Tissue type:
Subject Number:
Anti-β-actin
Anti-CAV1
Anti-p-ERK
786-MOCK
786-VHL(WT)
Anti-CAV1
Anti-tubulin
Anti-VHL
Anti-HIF2α
0 12 24 48Hypoxia (h)
Anti-CAV1
Anti-tubulin
Anti-HIF2α
786-VHL(WT)
1 2 3 4
786-shSCR 786-shHIF2α
1 2 3 1 2 3
Anti-CAV1
Anti-tubulin
Anti-HIF2α
Repeats
1 2 3 4 5 6
0
0.2
0.4
0.6
0.8
1
1.2
1.4
786-MOCK 786-VHL(WT) 786-VHL(WT)
Normoxia Hypoxia
CAV-1relativeexpression
Normoxia
0
0.5
1
1.5
2
2.5
786-MOCK 786-VHL(WT) 786-VHL(WT)
Normoxia Hypoxia
VEGFrelativeexpression
Normoxia
1 2 3 4 5 6 7 8 9 10 11 12
P=0.02
P<0.001
Normal CCRCC
CAV1realtiveexpression
P<0.0001
0
1
2
3
4
1 1.51 3.95 6.16
A
B
C D E
F
1 2
Anti-GLUT1
3085 3086 3097 3099 3100 3101
N TN T N TN TN TN T
CCRCC NormalCAV1mRNAexpression
P<0.0001
CCRCC Normal
CAV1
CA9
EGLN3
VEGFA
GLUT1
PDK1
ACTA1
-5.00
5.00
0
-3.33
3.33
-1.67
1.67
Correlation
with CAV1 P value
0.8261
0.8584
0.8235
0.8567
0.8307
0.0206
P<0.0001
P<0.0001
P<0.0001
P<0.0001
P<0.0001
P=0.9276
-2
0
2
4
HIG2 0.8063
LOXL2 0.9264 P<0.0001
P<0.0001
Fig. 1. Hypoxia induces the expression of CAV1
through HIF. (A) (Left) Cellular extracts generated
from primary CCRCC and matched normal kidney
samples were resolved by SDS/PAGE and immunoblotted
with the indicated antibodies. (Right) CAV1
signals were normalized to β-actin as measured by
densitometry, and CAV1 signal in normal kidney
samples was arbitrarily set at 1. Error bars indicate
SD. N, normal tissue; T, tumor. The P value was
calculated using Student’s t test. (B) (Left) Relative
gene expression levels of CAV1 in CCRCC (n = 10)
and nondiseased kidney (Normal; n = 12) are shown
as a box-whisker plot. Whiskers indicate 0% and
100% quartiles; the boxes indicate 25% and 75%
quartiles; the horizontal lines in the boxes indicate
the 50% quartile. P value was calculated by using
a two-sided Welch’s t test. (Right) Relative gene
expression levels of CAV1, several HIF-target genes,
including EGLN3, CA9, VEGFA, PDK1, GLUT1, HIG2,
and LOXL2, and a negative control ACTA1 (encoding
α-actin) are shown as a heat map. The colorimetric
scale indicates relative mRNA expression
levels. The correlation between CAV1 and the indicated
hypoxia-inducible genes is represented by
Pearson’s correlation coefficients. (C) Total cell lysates
of 786-MOCK and 786-VHL cells were resolved
by SDS/PAGE and immunoblotted with the indicated
antibodies. (D) Equal amounts of total cell
lysates generated from 786-VHL cells maintained in
hypoxia for the indicated times were separated by
SDS/PAGE and immunoblotted with the indicated
antibodies. CAV1 signals were normalized to tubulin
as measured by densitometry, and the CAV1
signal in 786-VHL lysate without hypoxia treatment
(lane 1) was arbitrarily set at 1. (E) mRNA was extracted
from 786-MOCK and 786-VHL cells maintained
in normoxia or hypoxia, and the expression
levels of CAV1 (Lower) and VEGF (Upper) mRNAs
were normalized against U1AsnRNP1 as measured
by real-time PCR. CAV1 and VEGF levels in 786MOCK
cells were set arbitrarily at 1. Error bars indicate SD. P values were calculated using Student’s t test. (F) Equal amounts of the total cell lysates generated
from 786-O cells stably expressing either HIF2α-specific shRNA (786-shHIF2α) or nontargeting scrambled shRNA (786-shSCR) were separated by SDS/PAGE and
immunoblotted with the indicated antibodies.
Wang et al. PNAS | March 27, 2012 | vol. 109 | no. 13 | 4893
CELLBIOLOGY
of a failure to form the E3 ligase ECV) (29) showed markedly
increased CAV1 mRNA and protein levels in comparison with
786-VHL(WT) and 786-VHL(L188V) (a mutant that has retained
E3 ligase function to degrade HIFα) (Fig. 3 A and B) (29). Ultrastructural
analysis using transmission electron microscopy
showed that 786-O and 786-VHL(C162F) cells exhibited greater
numbers of caveolae than did 786-VHL(WT) or 786-VHL
(L188V) cells (Fig. 3C). Importantly, maintenance of 786-VHL
(WT) cells under hypoxia increased the appearance of caveolae
(Fig. 3D). These results indicate that hypoxia promotes the formation
of caveolae via HIF-dependent up-regulation of CAV1.
CAV1 Binds to and Promotes Ligand-Independent Activation of the
EGF Receptor. Loss of VHL is associated with increased EGF receptor
(EGFR)-mediated signaling, which is thought to contribute
to the oncogenic potential of CCRCC (30–32). High concentrations
of surface EGFR also have been shown to activate
EGFR spontaneously without the participation of ligand (33).
Moreover, CAV1 binds to the C terminus of EGFR through the
CSD (8), and the cytoplasmic domain of EGFR hasbeen shown
to be required for ligand-independent dimer formation (34).
Therefore, we asked whether the increased formation of caveolae
under hypoxia promoted ligand-independent EGFR clustering
and activation on a restricted surface area of caveolae.
Stable shRNA-mediated CAV1 knockdown (786-shCAV1) resulted
in reduced levels of caveolae structures (Fig. 4A) concomitant
with attenuated EGFR phosphorylation in the serum-starved
condition (Fig. 4B, compare lanes 1 and 3), suggesting that CAV1
promotes EGFR activation in the absence of ligand. In support of
this notion, CAV1 coprecipitated EGFR preferentially in the absence
of EGF, and the level of EGFR dimerization decreased upon
stable shRNA-mediated CAV1 knockdown in 786-O cells (Fig. 4 C
and D). Stable shRNA-mediated CAV1 knockdown in MTC-1
cells likewise decreased EGFR dimerization and phosphorylation
(Fig. S5 A and B). Furthermore, shRNA-mediated CAV1 knock-218
gaaccttggg gatgtgccta gacccggcgc agcacacgtc cgggccaacc gcgagcagaa
-158 caaacctttg gcgggcggcc aggaggctcc ctcccagcca ccgcccccct ccagcgcctt
-98 tttttccccc catacaatac aagatcttcc ttcctcagtt cccttaaagc acagcccagg
-38 gaaacctcct cacagttttc atccagccac gggccagcat gtctgggggc aaatacgtag
Supershift HIF2α
Supershift HIF1α
HIF1/2α
1 2 3 4 5 6 7 8 9 1011 1312 14
MOCK
MOCK
250XHRE
Y11Ab
Anti-HIF1αAb
HA-HIF2α
+
ARNT
HA-HIF2α
+
ARNT
HA-HIF1α
+
ARNT
HA-HIF1α
+
ARNT
VEGF HRE CAV1 HRE
250xScramble
250XHRE
250xScramble
250XHRE
Y11Ab
Anti-HIF1αAb
250xScramble
250XHRE
250xScramble
A
15161718
B
C
RelativeIPDNA
-5
0
5
10
15
20
25
RCC4-MOCK RCC4-VHL
P=0.0166
RCC4-MOCKRCC4-VHL
Input
Beads
Anti-HIF1α
CAV1
gapdh
D
Anti-HIF2α
Input
Beads
Anti-HIF1α
Anti-HIF2α
1 2 3 4 5 6 7 8
Fig. 2. HIF engagement of HRE on the CAV1 promoter is associated with increased
Pol II recruitment. (A) Human CAV1 promoter sequence showing the
conserved HRE (box) and the translation start site (underlined). (B) EMSA was
performed using 32
P-labeled VEGF and CAV1 HRE probes mixed with the indicated
in vitro-translated gene products. A 250-fold molar excess of unlabeled
wild-type VEGF HRE (lanes 3 and 7) or CAV1 HRE (lanes 12 and 16) was mixed with
the reaction mixtures. HIF1 (HA-HIF1α/ARNT) and HIF2 (HA-HIF2α/ARNT) complexes
bound to HRE were supershifted with anti-HIF1α (lanes 9 and 18) and antiHIF2α
(lanes 5 and 14) antibodies, respectively. (C) Anti-Pol II ChIP analysis was
conducted on the endogenous CAV1 promoter in RCC4-VHL and RCC4-MOCK
cells using anti-RNA Pol II antibody. The relative immunoprecipitated (IP) DNA
was determined as described in Materials and Methods. Error bars represent SDs
of indicated IP DNA performed in triplicate in a representative experiment. The P
value was calculated using Student’s t test. (D) Anti-HIF1α and anti-HIF2α ChIP
analysiswasperformedonRCC4-VHL and RCC4-MOCKcells,andsemiquantitative
PCR was performed using primers specific for the CAV1 and Gapdh promoters.
A B
C
D
Anti-CAV1
Anti-tubulin
Anti-VHL
786-MOCK
786-VHL(WT)
786-VHL(C162F)
786-VHL(L188V)
1 2 3 4
786-O
786-VHL(WT)
786-VHL(C162F)
786-VHL(L188V)
786-O
786-VHL(W
T)
786-VH
L(C
162F)786-VH
L(L188V)
Caveolaenumber/10μm
P=0.001P=0.001
0
1
2
3
4
0
0.2
0.4
0.6
0.8
1
1.2
1.4
CAV1relativeexpression
786-mock
786-VHL(WT
786-VHL(C162
786-VHL(L188V
786-MOCK
786-VHL(WT)
786-VHL(C162F)
786-VHL(L188V)
HypoxiaNormoxia
Hypoxia
N
orm
oxia
Caveolaenumber/10μm
0.0
0.5
1.0
1.5
P=0.04
Anti-HIF2α
1 0.18 0.96 0.22
Fig. 3. HIF promotes the formation of caveolae via positive regulation of
CAV1. (A) Equal amounts of total cell lysates generated from the indicated
isogenically matched subclones were resolved by SDS/PAGE and immunoblotted
with the indicated antibodies. CAV1 signals were normalized to tubulin
as measured by densitometry, and the CAV1 signal in 786-MOCK lysate
(lane 1) was set arbitrarily at 1. (B) mRNA was extracted from the indicated
cells, and the expression level of CAV1 mRNA was normalized against
U1AsnRNP1 as measured by real-time PCR. The CAV1 level in 786-MOCK was
set arbitrarily at 1. Error bars indicate SD. (C) (Left) The indicated cells were
fixed and visualized by Hitachi H-7000 transmission electron microscopy.
(Magnification: 100,000×.) Arrowheads indicate caveolae. (Scale bar.100 nm.)
(Right) The number of caveolae structures on the cell membrane was quantified
by transmission electron microscopy as described in Materials and
Methods. Error bars indicate SD. P values were calculated using Student’s t test.
(D) (Left) 786-VHL cells maintained in normoxia or hypoxia for 48 h were fixed
and visualized by Hitachi H-7000 transmission electron microscopy. (Magnification:100,000×.)
Arrowheads indicate caveolae. (Scale bar: 100 nm.) (Right)
The number of caveolae structures on the cell membrane was quantified by
transmission electron microscopy as described in Materials and Methods. Error
bars indicate SD. P values were calculated using Student’s t test.
4894 | www.pnas.org/cgi/doi/10.1073/pnas.1112129109 Wang et al.
down attenuated the increased EGFR phosphorylation observed in
the serum-starved condition upon hypoxic treatment (HeLa cells)
or inactivation of VHL (786-O cells) (Fig. S6A). Subcellular fractionation
indicated that under the serum-starved condition, CAV1
coexists with EGFR in few common fractions and that a fraction of
EGFR colocalizes with CAV1 as detected by immunofluorescence
microscopy (Fig. S7 A and B). These results suggest that CAV1
binds and promotes EGFR dimerization and activation in the
confined space of caveolae in the absence of ligand.
The downstream Ras–(C)Raf–MEK–ERK signaling and consequential
cell proliferation were decreased significantly upon CAV1
knockdown in 786-O cells maintained in serum-free medium
[Fig. 4 E (compare lanes 1 and 2) and F]; this decrease might be
attributed, at least in part, to reduced EGFR phosphorylation.
In support of this notion, treatment with an EGFR-specific inhibitor,
AG1478, attenuated Ras–(C)Raf–MEK–ERK signaling
and BrdU incorporation in both serum-starved 786-shSCR and
786-shCAV1 cells (Fig. 4 E and F). This association also was observed
in primary CCRCC specimens where high CAV1 expression
generally was associated with increased phosphorylated ERK
levels (Fig. 1A and Fig. S1A). Notably, phosphorylation of A-Raf,
B-Raf, and mTOR remained relatively unaffected by changes
in CAV1 expression level (Fig. 4E). Overexpression (2.2-fold) of
CAV1 in 786-VHL cells increased phosphorylated EGFR and
ERK levels (Fig. 4G). Furthermore, shRNA-mediated CAV1
knockdown attenuated the level of BrdU incorporation observed
Anti-tubulin
Anti-p-ERK1/2
Anti-ERK1/2
Anti-p-MEK
Anti-p-C-Raf
786-shSCR
786-shCAV1
1 2 3 4
786-shSCR
786-shCAV1
786-sh-
CAV1
786-sh-
SCR
EGF – + – +
Anti-EGFR
Anti-p-EGFR
Anti-CAV1
Anti-tubulin
1 2 3 4
Anti-EGFR
Anti-CAV1
dimer
monomer
1 2
786-shSCR
786-shCAV1
Anti-tubulin
P=0.0013
786-shSC
R
786-shC
AV1
EGFRdimer/monomerratio
0.00
0.05
0.10
0.15
0.20
IP CAV1
EGF – +
Anti-EGFR
Anti-CAV1
1 2
Caveolaenumber/10μm
786-shSC
R
786-shC
AV1
P=0.005
0
1
2
3
4
P<0.05
P<0.05
P>0.05
P<0.05
Anti-p-EGFR
Anti-EGFR
Anti-MEK
Anti-C-Raf
Anti-mTOR
Anti-p-mTOR
786-shSCR
786-shCAV1
–AG1478
Anti-CAV1
+
0
10
20
30
40
50
BrdUincoporation(%)
786-shSCR
786-shCAV1
786-shSCR
786-shCAV1
–AG1478 +Anti-p-A-Raf
Anti-p-B-Raf
Anti-tubulin
Anti-p-ERK1/2
Anti-p-EGFR
Anti-EGFR
Anti-CAV1
CAV1-WT +-
1 2
E
BA
DC
F G
Fig. 4. CAV1 binds EGFR and promotes
ligand-independent EGFR
phosphorylation and Ras–Raf–MEK–
ERK activation. (A) (Left) 786-shSCR
and 786-shCAV1 cells were fixed and
visualized by transmission electron
microscopy. (Magnification:100,000×.)
Arrowheads indicate caveolae; arrow
indicates clathrin-coated pit. (Scale
bar: 100 nm.) (Right) The number of
caveolae structures on the cell membrane
was quantified by transmission
electron microscopy. Error bars indicate
SD. The P value was calculated
using Student’s t test. (B) Equal
amounts of total cell lysates generated
from serum-starved 786-shSCR
and 786-shCAV1 cells that were treated
with (+) or without (−) EGF for 5
min were resolved by SDS/PAGE and
immunoblotted with the indicated
antibodies. (C) Serum-starved 786shSCR
cells treated with (+) or without
(−) EGF for 5 min were lysed; comparable
amounts of total cell lysates
immunoprecipitated with anti-CAV1
antibody were resolved by SDS/PAGE
and visualized by the indicated antibodies.
(D) (Left) Equal amounts of
total cell lysates generated from serum-starved
and chemical cross-linked
786-shSCR and 786-shCAV1cells were
resolved by SDS/PAGE and immunoblotted
with the indicated antibodies.
(Right) The signals of dimer were
normalized to that of monomer as
measured by densitometry. Error bars
indicate SD. The P value was calculated
using Student’s t test. (E) 786shSCR
and 786-shCAV1 cells were serum
starved overnight with (+) or
without (−) AG1478 treatment for
16 h. Equal amounts of total cell
lysates separated by SDS/PAGE were
immunoblotted with the indicated
antibodies. (F) BrdU incorporation by
786-shSCR and 786-shCAV1 cells was
measured following serum starvation
with (+) or without (−) AG1478
treatment for 16 h. Values indicate
the mean percentage of BrdU-positive
cells in relation to total DAPIstained
nuclei from three independent
experiments. Error bars indicate
SD. P values were calculated using
one-way ANOVA followed by a Newman–Keuls
post hoc test. (G) 786VHL
cells with (+) or without (−) ectopic CAV1 overexpression were serum starved for 16 h. Equal amounts of total cell lysates separated by SDS/PAGE were
immunoblotted with the indicated antibodies.
Wang et al. PNAS | March 27, 2012 | vol. 109 | no. 13 | 4895
CELLBIOLOGY
in serum-starved 786-O cells maintained under normoxia or
hypoxia in comparison with 786-shSRC cells (Fig. S6B). CAV1
knockdown likewise reduced the rate of VHL-positive HeLa and
MTC cell proliferation under hypoxia (Fig. S8 D and E). Taken
together, these results suggest that, under compromised oxygen
availability, the stabilization of HIFα triggers CAV1-dependent
cell proliferation and activation of caveolae-sequestered EGFR
during limited ligand availability.
CAV1 Overexpression via HIF2 Accentuates Tumor Cell Proliferation.
Several lines of evidence have shown that the accumulation of
HIF2α upon the loss of VHL has a critical oncogenic role in CCRCC
(4, 5, 35, 36). We asked whether CAV1-specific up-regulation via
HIF2α had discernable oncogenic property in vivo. 786-O cells stably
expressing luciferase-tagged shCAV1 (786-GL-shCAV1) or shSCR
(786-GL-shSCR) were injected into the dorsal skin-fold window
chamber in SCID mice. The tumor volume over time determined by
bioluminescence imaging (BLI) indicated that CAV1 promotes tumor
cell proliferation, because the growth rate of 786-GL-shCAV1
xenografts was markedly attenuated in comparison with 786-GLshSCR
xenografts (Fig. S9A). In accord, phosphorylated ERK
staining was much more prominent in the resected 786-GL-shSCR
xenografts than in 786-GL-shCAV1 xenografts (Fig. S9B). These
results suggest a role for CAV1 overexpression via HIF2α in augmenting
tumor cell proliferation in vivo.
Furthermore, CAV1 knockdown in HeLa cells, which express
low EGFR levels in comparison with 786-O cells, resulted in
a marked down-regulation of PDGF receptor (PDGFR) and type I
insulin-like growth factor receptor (IGF-1R) phosphorylation and
downstream ERK phosphorylation under serum-starved conditions
(Fig. S8A). CAV1 knockdown inhibited ligand-independent
cell migration and cell invasion as determined by wound healing
and Matrigel invasion assays, respectively (Fig. S8 B and C). These
results are consistent with the role of PDGFR and IGF-1R in cell
motility (37, 38) and support a role for CAV1 in hypoxia-mediated
activation of potentially multiple receptor tyrosine kinases (RTKs).
Discussion
Hypoxia triggers essential physiologic adaptive responses ranging
from increased production of oxygen-carrying red blood cells to the
formation of new blood vessels and switching cellular energy production
from aerobic respiration to anaerobic glycolytic metabolism
(32). These and other hypoxia-inducible biological processes
are governed principally by HIF transcriptional factor that becomes
active upon stabilization of the otherwise oxygen-labile
HIFα subunit under hypoxia. Recently, we showed that HIF decelerates
clathrin-mediated endocytosis at the early endosome
sorting stage and thereby prolongs RTK-mediated signaling to
promote cell survival under hypoxia (30, 32). Here we show that
CAV1, an integral structural component of caveolae, is a direct
target of HIF1 and HIF2 and under hypoxia binds to and promotes
ligand-independent activation of EGFR within the substantially
smaller surface area of caveolae relative to the plasma membrane.
The observation that CAV1–EGFR interaction is diminished
in the presence of ligand is intriguing (Fig. 4C) and suggests that
CAV1 may not have a significant role in RTK-mediated signaling
when the availability of ligand is no longer limited. Consistent
with this notion, we found robust and comparable EGFR
phosphorylation irrespective of CAV1 expression level in the
presence of exogenous EGF (Fig. 4B). These observations suggest
either that ligand-mediated clustering of EGFR on plasma
membrane may hinder trafficking to caveolae or that ligand
engagement triggers internalization of EGFR via clathrin-mediated
endocytosis and thereby limits the accessibility of EGFR
to caveolae. However, in the absence of ligand, some fraction of
EGFR in the fluid cell membrane milieu localizes to the caveolae,
the formation of which increases markedly under hypoxia.
Thus, CAV1-mediated RTK signaling may represent a unique
adaptive response triggered under a deleterious situation where
the availability of oxygen is compromised and ligands required to
activate certain critical adaptive responses are not yet available.
Cancer cells invariably hijack the otherwise normal physiologic
responses to hypoxia to potentiate their own survival and growth.
HIFα overexpression commonly associated with tumors (by virtue
of the general oxygen-sensing pathway in regions of hypoxia
or cancer-causing mutations in a growing list of tumor-suppressor
genes such as TSC2, PTEN, p53, and VHL that bypass the
necessity of low oxygen tension to initiate a pseudo hypoxic response)
would be predicted to increase CAV1 expression to
promote autoactivation of EGFR and other RTKs bound in
caveolae. Intriguingly, Martinez-Outschoorn et al. (39) reported
that in cancer-associated stromal fibroblasts hypoxia leads to
a loss of CAV1 via autophagy, suggesting an indirect negative
regulation of CAV1 that may be cell-context dependent. Furthermore,
CAV1 was identified originally as an inhibitor of
EGFR signaling through receptor sequestration (40, 41). Recently,
however, CAV1 has been shown to promote EGFR signaling.
For example, under conditions of oxidative stress, CAV1
was shown to transport EGFR to a perinuclear location where
EGFR no longer is degraded and remains active (42). CAV1overexpressing
MCF-7 breast cancer cells (MCF-7/CAV1)
stimulated with EGF displayed higher EGFR signaling, with
enhanced proliferative and motility rates, than seen in MCF-7
parental cells (43). These examples of differential roles of CAV1
in the regulation of EGFR also may suggest the cell contextdependent
nature of CAV1 function.
The direct link between CAV1 and HIF presented herein likely
explains why, in various tumors including CCRCC, CAV1 overexpression,
similar to HIFα, is associated with larger tumor size,
higher tumor grade and stage, resistance to conventional therapies,
and poor prognosis (10–15, 44). Consistent with this notion,
primary CCRCC cells with a strong hypoxic signature exhibited
elevated expression of CAV1 and phosphorylated ERK levels, and
molecular suppression of CAV1 attenuated Ras–(C)Raf–MEK–
ERK signaling, cell proliferation, and migratory capacity in the
absence of ligand. Thus, the present study unveils CAV1 as an
integral direct component of HIF-mediated signaling in response
to tumor hypoxia or pseudohypoxia that transforms cellular architecture
to drive receptor-mediated proliferative signaling in the
absence of appropriate extracellular cues/ligand (Fig. S9C).
Up-regulation of the EGFR signaling pathway has been observed
and reported to promote tumorigenesis in CCRCC (45).
We show here that HIF-dependent overexpression of CAV1
promotes EGFR signaling in CCRCC. Interestingly, gefitinib, an
EGFR-specific inhibitor, inhibited the proliferation of MCF-7/
CAV1 breast cancer cells more efficiently than it prohibited the
proliferation of MCF-7 parental cells (46). This finding suggests
that gefitinib may have beneficial effect on CCRCC displaying
a CAV1 overexpression signature. Moreover, combined treatment
of metastatic RCC with gefitinib and sunitinib, a multitargeted
tyrosine kinase inhibitor of VEGF receptor (VEGFR)
and PDGFR, demonstrated efficacy comparable to sunitinib
monotherapy with an acceptable safety profile in a Phase I/II
clinical trial (47). Notably, sunitinib, a drug approved by the Food
and Drug Administration for the treatment of metastatic RCC,
has shown significant advantage over IFN-α therapy, the previous
first-line therapy for metastatic RCC (48). We show here that
CAV1 promotes the activation of other RTKs, such as PDGFR
and IGF-1R. Thus, the efficacy of sunitinib for the treatment of
metastatic RCC may be caused in part by CAV1 activation of
several RTKs involved in the oncogenic processes of CCRCC.
Materials and Methods
Clinical Samples. Institutional review board approval was obtained from each
participating institution, and informed consent was obtained from all participants.
For preparation of protein extracts, renal tumor tissue was pulverized
in a mortar under liquid nitrogen and was suspended on ice in lysis
buffer [20 mM Hepes (pH 7.7), 0.2 M NaCl, 1.5 mM MgCl2, 0.4 mM EDTA, 1%
Triton X-100, 0.5 mM DTT, 100 μg/mL leupeptin, 100 μg/mL aprotinin, 10 mM
benzamidine, 2 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerophosphate,
and 0.1 mM sodium-orthovanadate].
4896 | www.pnas.org/cgi/doi/10.1073/pnas.1112129109 Wang et al.
Cells. HEK293 embryonic kidney, 786-O (VHL−/−
; HIF1α−/−
) CCRCC, HeLa cervical
cancer, CNS-1 glioma, MTC-1 metastatic breast cancer, and A431 epidermoid
carcinoma cell lines were obtained from the American Type Culture Collection
and maintained in DMEM (Gibco) supplemented with 10% (vol/vol) heatinactivated
FBS (Sigma) at 37 °C in a humidified 5% (vol/vol) CO2 atmosphere.
Mouse pro-B Ba/F3 cells were generously provided by Mignon Loh (University
of California, San Francisco) (49). These cells were maintained in RPMI-1640
with 10% (vol/vol) FCS (HyClone), penicillin, streptomycin, L-glutamine, and 10
μg/mL mouse IL-3 (Peprotech). Primary MEFs were derived from embryonic day
12.5 wild-type C57BL/6 embryos. 786-O subclones ectopically expressing wildtype
hemagglutinin [HA)-VHL(WT)], HA-VHL(C162F), or HA-VHL(L188V) were
described previously (50, 51). RCC4 (VHL−/−
) CCRCC subclones stably expressing
HA-VHL (RCC4-VHL) or empty plasmid (RCC4-MOCK) were described previously
(52). For hypoxia treatment, cells were maintained at 1% O2 for the
indicated times in a humidified 5% (vol/vol) CO2 ThermoForma hypoxia incubator
at 37 °C.
Additional methodology is described online in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank S. Doyle and B. Temkin for assistance with
the transmission electron microscopy. This work was supported by Canadian
Institutes of Health Research (CHIR) Grant MOP77718 and by Canadian Cancer
Society Grant 18460. Y.W. and O.R. are recipients of CIHR postdoctoral
fellowships. M.O. holds a Canada Research Chair in Molecular Oncology.
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