THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 41, Issue of October 11, pp.

38205–38211, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Insulin-like Growth Factor 1 Induces Hypoxia-inducible Factor

1-mediated Vascular Endothelial Growth Factor Expression, Which
is Dependent on MAP Kinase and Phosphatidylinositol 3-Kinase
Signaling in Colon Cancer Cells*
Received for publication, April 18, 2002, and in revised form, July 3, 2002
Published, JBC Papers in Press, July 30, 2002, DOI 10.1074/jbc.M203781200

Ryo Fukuda‡, Kiichi Hirota‡§, Fan Fan¶, Young Do Jung¶, Lee M. Ellis¶, and Gregg L. Semenza‡储
From the ‡McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21287 and ¶University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Stimulation of human colon cancer cells with insulin- vated protein (MAP) kinase and phosphatidylinositol 3-kinase
like growth factor 1 (IGF-1) induces expression of the (PI3-kinase) pathways plays a critical role in transformation
VEGF gene, encoding vascular endothelial growth fac- and tumorigenesis (1). IGF2 gene expression is up-regulated to
tor. In this article we demonstrate that exposure of the greatest extent of any gene in colon cancer cells relative to
HCT116 human colon carcinoma cells to IGF-1 induces normal colonic epithelium (2), resulting in autocrine stimula-
the expression of HIF-1␣, the regulated subunit of hy- tion of cells which express both receptor and ligand. In addition
poxia-inducible factor 1, a known transactivator of the to the effects of IGF-1R on cell transformation and prolifera-
VEGF gene. In contrast to hypoxia, which induces
tion, treatment of colon cancer cells with IGF-1 also induces
HIF-1␣ expression by inhibiting its ubiquitination and
transcription of the VEGF gene encoding vascular endothelial
degradation, IGF-1 did not inhibit these processes, indi-
cating an effect on HIF-1␣ protein synthesis. IGF-1 stim- growth factor, which is essential for tumor angiogenesis (3, 4).
ulation of HIF-1␣ protein and VEGF mRNA expression Treatment of mice with IGF-1 increases colon cancer growth
was inhibited by treating cells with inhibitors of phos- and metastasis as well as tumor VEGF expression and vascu-
phatidylinositol 3-kinase and MAP kinase signaling larization (5). A variety of growth factor-receptor tyrosine ki-
pathways. These inhibitors also blocked the IGF-1- nase signaling pathways induce VEGF expression in cancer
induced phosphorylation of the translational regulatory cells. In the case of oncogenic RAS signaling, VEGF expression
proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus provid- is dependent upon activity of the MAP kinase/extracellular
ing a mechanism for the modulation of HIF-1␣ protein signal-regulated kinase (ERK) kinase 1 (MEK-1) in fibroblasts
synthesis. Forced expression of a constitutively active but is dependent upon PI3-kinase activity in epithelial cells (6).
form of the MAP kinase kinase, MEK2, was sufficient to Cellular signaling pathways modulate gene expression by
induce HIF-1␣ protein and VEGF mRNA expression. In- altering the activity or expression of specific transcription fac-
volvement of the MAP kinase pathway represents a tors. The major physiological stimulus for VEGF expression is
novel mechanism for the induction of HIF-1␣ protein
cellular hypoxia; hypoxia-induced transcription of the VEGF
expression in human cancer cells.
gene is mediated by hypoxia-inducible factor 1 (HIF-1) (7–10).
Recently, the expression of VEGF in response to heregulin-
induced activation of the HER2neu receptor tyrosine kinase in
The insulin-like growth factor-1 (IGF-1)1 receptor tyrosine
kinase (IGF-1R) is activated by binding either of its ligands, breast cancer cells was shown to be mediated by HIF-1 via the
IGF-1 or IGF-2. IGF-1R signaling through the mitogen-acti- PI3-kinase pathway (11), demonstrating that HIF-1 regulates
both hypoxia- and growth factor-induced VEGF expression in
tumor cells. HIF-1 is a heterodimer composed of a constitu-
* This work was supported by Grants R01-DK39869 (to G. L. S.) and tively expressed HIF-1␤ subunit and an inducibly expressed
R01-CA74821 (to L. M. E.) from the National Institutes of Health. The
costs of publication of this article were defrayed in part by the payment HIF-1␣ subunit (12). Under nonhypoxic conditions, HIF-1␣ is
of page charges. This article must therefore be hereby marked “adver- subject to O2-dependent prolyl hydroxylation (13, 14), which is
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate required for binding of the von Hippel-Lindau tumor suppres-
this fact. sor protein (VHL), the recognition component of an E3 ubiq-
§ Current address: Human Stress Signal Research Center, National
Institute of Advanced Industrial Science and Technology, 1-8-31 Mi- uitin-protein ligase, which targets HIF-1␣ for proteasomal deg-
dorigaoka, Ikeda, Osaka 563-8577, Japan. radation (15). Under hypoxic conditions, O2 becomes limiting
储 To whom correspondence should be addressed: Johns Hopkins for prolyl hydroxylase activity (16) and ubiquitination of
University School of Medicine, CMSC-1004, 600 North Wolfe St., HIF-1␣ is inhibited (17). As a result, HIF-1␣ accumulates,
Baltimore, MD 21287-3914; Fax: 410-955-0484; E-mail: gsemenza@
jhmi.edu. dimerizes with HIF-1␤, and activates transcription of target
1
The abbreviations used are: IGF, insulin-like growth factor; IGF- genes.
1R, IGF-1 receptor; HIF-1, hypoxia-inducible factor 1; VEGF, vascular Signaling via receptor tyrosine kinases can induce HIF-1␣
endothelial growth factor; MAP, mitogen-activated protein; PI3-kinase,
expression by an independent mechanism. HER2neu activation
phosphatidylinositol 3-kinase; 4E-BP1, eIF-4E-binding protein 1; eIF-
4E, eukaryotic initiation factor 4E; ERK, extracellular signal-regulated in breast cancer cells stimulates increased rates of HIF-1␣
kinase; MEK, MAP kinase/ERK kinase; HER2neu, human epidermal protein synthesis via PI3-kinase and the downstream serine-
growth factor receptor 2; VHL, von Hippel-Lindau tumor suppressor; threonine kinases, AKT (protein kinase B) and FRAP (FKBP/
FRAP, FKBP/rapamycin-associated protein; mTOR, mammalian target
rapamycin-associated protein), which is also known as mTOR
of rapamycin; p70s6k, p70 ribosomal protein S6 kinase; CHX, cyclohex-
imide; FBS, fetal bovine serum; HA, hemagglutinin; GST, glutathione (mammalian target of rapamycin) (11). FRAP/mTOR phospho-
S-transferase; CMV, cytomegalovirus. rylates and activates the translational regulatory proteins eIF-

This paper is available on line at http://www.jbc.org 38205

This is an Open Access article under the CC BY license.
38206 IGF-1 Induces HIF-1 and VEGF via MAP Kinase and PI3-kinase Signaling

FIG. 2. Effect of IGF-1, CoCl2, and 1% O2 on HIF-1␣ expression

and stability. A, analysis of HIF-1␣ stability. HCT116 cells were
exposed to 100 ␮M CoCl2 (top panel) or 100 ng/ml IGF-1 (bottom panel)
for 4 h, cycloheximide (CHX) was added to a final concentration of 100
␮M, the cells were incubated for 0 – 60 min, and whole cell lysates were
subject to immunoblot assay using an anti-HIF-1␣ monoclonal anti-
body. The proportion of HIF-1␣ remaining at each time point relative to
time 0 is indicated. B, induction of HIF-1␣ protein and VEGF mRNA
expression by CoCl2 or 1% O2 in the presence or absence of IGF-1.
Serum-starved HCT116 cells were exposed to 100 ␮M CoCl2 (lanes 3 and
4), 1% O2 (lanes 5 and 6), or neither (lanes 1–2) in the presence (lanes 2,
4, and 6) or absence (lanes 1, 3, and 5) of 100 ng/ml IGF-1 for 4 or 24 h
prior to analysis of HIF-1␣ protein or VEGF mRNA expression,
respectively.

4E-binding protein 1 (4E-BP1) and p70 S6 kinase (p70S6K)

(18 –20). Phosphorylation of 4E-BP1 disrupts its inhibitory in-
teraction with eukaryotic initiation factor 4E (eIF-4E), whereas
activated p70S6K phosphorylates the 40 S ribosomal protein S6.
The effect of HERneu signaling on the translation of HIF-1␣
protein is dependent upon the presence of the 5⬘-untranslated
region of HIF-1␣ mRNA (11). These pathways thus provide a

HIF-1␣ protein (top) or total cellular RNA was isolated and analyzed by
blot hybridization using a HIF-1␣ cDNA probe (middle) following RNA
transfer from an ethidium bromide (EtBr)-stained gel (bottom; migra-
tion of 28 S and 18 S rRNA indicated). B, kinetics of HIF-1␣ induction.
Serum-starved cells were exposed to vehicle (lane 1) or 100 ng/ml IGF-1
for 2–24 h (lanes 2– 8) prior to immunoblot analysis of whole cell lysates
using monoclonal antibodies specific for HIF-1␣ (top panel) or HIF-1␤
(bottom panel). C, analysis of VEGF mRNA expression. Serum-starved
cells were exposed to vehicle (lane 1), 10 –100 ng/ml IGF-1 (lanes 2–3),
or 1% O2 (lane 4) for 24 h; total cellular RNA was isolated and analyzed
by blot hybridization using a VEGF cDNA probe (top) following transfer
of RNA from an EtBr-stained gel (bottom). D, effect of IGF-1R inhibitor.
FIG. 1. Effect of IGF-1 treatment on HIF-1␣ and VEGF expres- Cells were pretreated with vehicle (lanes 1 and 2) or 1–100 ␮M H-1356
sion in HCT116 cells. A, analysis of HIF-1␣ expression as a function (lanes 3– 6), exposed to IGF-1 (lanes 2–5) or 1% O2 (lane 6), and har-
of IGF-1 concentration. Duplicate plates of HCT116 cells were cultured vested after 6 h for analysis of HIF-1␣ protein expression by immuno-
in the absence of serum for 24 h, exposed to vehicle (lane 1), 1–1000 blot assay (top) or at 24 h for analysis of VEGF mRNA expression by
ng/ml IGF-1 (lanes 2–5), or 100 ␮M CoCl2 (lane 6) for 6 h. Then either blot hybridization (middle) following RNA transfer from an EtBr-
whole cell lysates were subject to immunoblot assay for expression of stained gel (bottom).
 IGF-1 Induces HIF-1 and VEGF via MAP Kinase and PI3-kinase Signaling 38207

FIG. 3. Analysis of HIF-1␣ ubiquiti-

nation and interaction with VHL. A,
in vitro ubiquitination assay. Lysates pre-
pared from cells exposed to vehicle (⫺) or
IGF-1 (⫹) were incubated with in vitro
translated and 35S-labeled HIF-1␣ in the
presence of ubiquitin and ATP for 0, 30, or
150 min. Polyubiquitinated forms of
HIF-1␣ (Ubi-HIF-1␣) were identified by
their reduced mobility after PAGE. B,
VHL interaction assay. A GST-HIF-1␣ fu-
sion protein was incubated with in vitro
translated and 35S-labeled VHL in the
presence of phosphate-buffered saline
(PBS) (lane 1) or lysates prepared from
cells that were untreated (lane 2) or ex-
posed to 100 ng/ml IGF-1 (lane 3) or 100
␮M CoCl2 (lane 4). Glutathione-Sepharose
beads were used to capture GST-HIF-1␣,
and the presence of associated VHL in the
samples was determined by PAGE and
autoradiography. One-fifth of the input
VHL protein was also analyzed (lane 5).

molecular basis for stimulation of HIF-1␣ protein synthesis in total p70S6K, phosphorylated (Ser-209) or total elF-4E, and phosphoryl-
response to HER2neu activation. ated (Ser-65) or total 4E-BP1 antibodies (purchased from Cell Signaling
Technology and Santa Cruz Biotechnology). Anti-hemagglutinin (HA)
Treatment of cultured cells with IGF-1 or IGF-2 also induces
antibody was from Santa Cruz. Horseradish peroxidase-conjugated
HIF-1␣ protein expression, HIF-1 DNA binding activity, and mouse monoclonal antibodies for mouse and rabbit IgG (1:2500 dilu-
transactivation of target genes (21, 22). The demonstration tion) and ECL reagents were from Amersham Biosciences.
that IGF2 is a HIF-1 target gene (22), that HIF-1␣ is overex- RNA Blot Hybridization—Total RNA was extracted from HCT116
pressed in human colon cancers (23), and that forced overex- cells using TRIzol reagent (Invitrogen) 6 –24 h after IGF-1 stimulation
pression of HIF-1␣ in HCT116 colon carcinoma cells increases and 48 h after plasmid transfection. 10-␮g aliquots of RNA were frac-
tionated by electrophoresis in 1.5% agarose, 2.2 M formaldehyde gels,
tumor growth and vascularization in vivo (24) suggest that
transferred to Hybond N⫹ membranes (Amersham Biosciences), and
HIF-1 may play an important role in autocrine IGF-1R signal- hybridized with a 32P-labeled human HIF-1␣ or VEGF cDNA probe as
ing and angiogenesis in colon cancer. We therefore investigated described previously (11).
the mechanisms by which IGF-1 stimulation increases the ex- In Vitro Ubiquitination Assay—HCT116 cells were serum-starved,
pression of HIF-1 and VEGF. treated with IGF-1 for 0, 30 or 150 min, washed twice with cold hypo-
tonic extraction buffer (20 mM Tris (pH 7.5), 5 mM KCl, 1.5 mM MgCl2,
EXPERIMENTAL PROCEDURES 1 mM dithiothreitol), and lysed in a Dounce homogenizer. The cell
Tissue Culture and Reagents—HCT116 cells were cultured in Mc- extract was centrifuged at 10,000 ⫻ g for 10 min at 4 °C, and the
Coy’s 5A medium with 10% FBS, 100 units/ml penicillin, and 100 ␮g/ml supernatant was stored in aliquots at ⫺70 °C. Ubiquitination assays
streptomycin (Invitrogen). Unless otherwise stated, cells were main- were performed as described previously (26) at 30 °C in a total volume
tained at 37 °C in a humidified 5% CO2, 95% air incubator. IGF-1, of 40 ␮l containing 27 ␮l (50 ␮g) of cell extract, 4 ␮l of 10 ⫻ ATP-
PD98059, wortmannin, rapamycin, cycloheximide (CHX), and cobalt regenerating system (20 mM Tris (pH 7.5), 10 mM ATP, 10 mM magne-
chloride (CoCl2) were purchased from Sigma. H-1356 (JB1) was pur- sium acetate, 300 mM creatine phosphate, 0.5 mg/ml creatine phos-
chased from Bachem Biochemica GmbH. CHX was dissolved in ethanol phokinase), 4 ␮l of 5 mg/ml ubiquitin (Sigma), 0.83 ␮l of 150 ␮M
at 100 mM. PD98059, wortmannin, and rapamycin were dissolved in ubiquitin aldehyde (Sigma), and 2 ␮l of HA-HIF-1␣ that was in vitro
Me2SO at 50 mM, 200 ␮M, and 100 ␮M, respectively. For hypoxic expo- translated (TNT Quick Coupled Transcription/Translation System, Pro-
sures, cells were placed in a modulator incubator chamber (Billups- mega) in the presence of [35S]methionine. HA-HIF-1␣ was recovered
Rothenberg) that was flushed with a gas mixture consisting of 1% O2, using anti-HA-agarose beads, which were then mixed with SDS sample
5% CO2, with balance N2, sealed, and incubated at 37 °C. buffer and boiled for 5 min; the eluates were then analyzed by SDS-
IGF-1 and Inhibitor Treatments—HCT116 cells were plated at a PAGE and autoradiography.
density of 2.4 ⫻ 106/10-cm dish or 8.6 ⫻ 105/6-cm dish. Subconfluent In Vitro HIF-1␣-VHL Interaction Assay—[35S]Methionine-labeled
cells were serum-starved (0.1% FBS in all experiments except Fig. 1A in VHL protein was synthesized in vitro and glutathione S-transferase
which FBS was completely eliminated) for 24 h before IGF-1 was added. (GST)-HIF-1␣-(429 – 608) fusion protein was expressed in E. coli as
The IGF-1R antagonist H-1356 and the kinase inhibitors PD98059, described previously (27). HCT116 cells were serum-starved and
wortmannin, and rapamycin were added 1 h before exposure to IGF-1, treated with IGF-1 or CoCl2 for 4 h prior to lysate preparation. GST-
1% O2, or 100 ␮M CoCl2. CHX was added to the medium of HCT116 cells HIF-1␣-(429 – 608) was preincubated with 10 ␮l of the HCT116 lysate
that had been serum-starved and treated with CoCl2 or IGF-1 for 4 h, for 30 min at 30 °C. Five-␮l aliquots of the GST-HIF-1␣-(429 – 608)
and whole cell extracts were prepared at 15, 30, and 60 min. preincubation and VHL in vitro translation reactions were mixed in 150
Immunoblot Assays—Whole cell extracts were prepared using radio- ␮l of NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris-HCl (pH
immune precipitation buffer, fractionated by SDS-PAGE, transferred to 8.0), 0.5% (v/v) Nonidet P-40). After 90 min at 4 °C, 20 ␮l of glutathione-
a nitrocellulose filter, and subjected to immunoblot assays. For HIF-1␣ Sepharose-4B (Amersham Biosciences) was added. After 30 min of
and HIF-1␤, 150-␮g aliquots of protein were analyzed using a mono- mixing on a rotator, beads were washed three times with NETN buffer.
clonal antibody against HIF-1␣ (H1␣67) or HIF-1␤ (H1␤234) (Novus Proteins were eluted in 2⫻ SDS sample buffer, fractionated by SDS-
Biologicals) at 1:1000 dilution as described previously (23, 25). 50-␮g PAGE, and detected by autoradiography.
aliquots were analyzed using antibodies (1:1000 dilution) specific for Transient Transfection—8.6 ⫻ 105 HCT116 cells were plated/6-cm
phosphorylated (Thr-202/Tyr-204) or total p44/p42 MAP kinase, phos- dish, cultured overnight, and transfected with 1.25 ␮g of pCMV-HA-
phorylated (Ser-473) or total AKT, phosphorylated (Thr-421/Ser-424) or MEK-2DD (kind gift of S. Meloche, Institut de Recherches Cliniques de
38208 IGF-1 Induces HIF-1 and VEGF via MAP Kinase and PI3-kinase Signaling

FIG. 5. MAP kinase and PI3-kinase pathway signaling in IGF-

1-treated cells. HCT116 cells were pretreated for 1 h with 50 ␮M
PD98059, 200 nM wortmannin, or 100 nM rapamycin and then exposed
to 100 ng/ml IGF-1 as indicated. Whole cell extracts were prepared after
15 min (left) or 6 h (right) of IGF-1 stimulation and subject to immu-
noblot assays using antibodies specific for phosphorylated (Thr-202/
Tyr-204) or total p42/p44 MAP kinase and phosphorylated (Ser-473) or
total AKT.

Montreal) or empty pCMV (Stratagene) in the presence of Fugene-6

(Roche Molecular Biochemicals). After 24 h, cells were cultured in 0.1%
FBS for an additional 24 h. Whole cell extracts and total RNA were
prepared for immunoblot and blot hybridization assays, respectively.
For transfected cells exposed to PD98059, the drug was added at the
time of serum starvation.

RESULTS
Exposure of serum-starved HCT116 human colon carcinoma
cells to IGF-1 for 6 h resulted in a concentration-dependent
induction of HIF-1␣ protein expression with a maximal effect
observed in the presence of 100 ng/ml of IGF-1 (Fig. 1A, top).
Similar results were obtained with IGF-2 (data not shown).
HIF-1␣ expression was also induced by exposure of cells to
CoCl2 (Fig. 1A, lane 6), which blocks HIF-1␣ degradation. In
contrast, neither IGF-1 nor CoCl2 induced HIF-1␣ mRNA ex-
pression (Fig. 1A, middle), demonstrating the specific effects of
these agents on HIF-1␣ protein expression. In the presence of
IGF-1, HIF-1␣ protein levels peaked at 8 h and declined there-
after (Fig. 1B, top). HIF-1␤ levels were unaffected by IGF-1
treatment (Fig. 1B, bottom). IGF-1 treatment also induced
VEGF mRNA expression in a concentration-dependent manner
(Fig. 1C, lanes 2 and 3). H-1356, a selective inhibitor of IGF-1R
tyrosine kinase activity, inhibited the induction of HIF-1␣ pro-
tein and VEGF mRNA expression in IGF-1-treated cells in a
dose-dependent manner (Fig. 1D, lanes 3–5), thus demonstrat-
ing a requirement for signal transduction via the IGF-1R. In
contrast, hypoxic cells expressed HIF-1␣ protein and VEGF
mRNA expression at high levels even in the presence of H-1356
(Fig. 1D, lane 6). Under all conditions, there was a strong
correlation between the levels of HIF-1␣ protein and VEGF
mRNA (Fig. 1D, compare top and middle panels).
To determine whether IGF-1 treatment affected HIF-1␣ pro-
tein half-life, HCT116 cells were treated with CoCl2 or IGF-1
FIG. 4. Effect of kinase inhibitors on the induction of HIF-1␣ for 4 h to induce HIF-1␣ expression, and then CHX was added
and VEGF. A, serum-starved HCT116 cells were exposed to vehicle to block ongoing protein synthesis. In the presence of CHX, the
(lane 1) or 100 ng/ml IGF-1 in the presence of no kinase inhibitor (lane
2) or a 1-h pretreatment with 50 ␮M PD98059 (lane 3), 200 nM wort- half-life of HIF-1␣ was ⬎60 min in CoCl2-treated cells but ⬍30
mannin (lane 4), or 100 nM rapamycin (lane 5). Cells were harvested min in IGF-1-treated cells (Fig. 2A). These results indicate that
after 6 h for analysis of HIF-1␣ protein and mRNA or after 24 h for HIF-1␣ expression in IGF-1-treated cells is dependent upon
analysis of VEGF mRNA. B, HCT116 cells were exposed to vehicle (lane ongoing protein synthesis. If IGF-1 induces HIF-1␣ expression
1) or 100 ng/ml IGF-1 in the presence of 0 –50 ␮M PD98059 (lanes 2–5)
for 6 h, and HIF-1␣ protein expression was determined by immunoblot
by stimulating synthesis of the protein, then it would be ex-
assay. C, cells were exposed to vehicle (lane 1) or 100 ng/ml IGF-1 in the pected to have an additive effect with that of CoCl2 or hypoxia,
presence of 0 –200 nM wortmannin (lanes 2– 6). D, cells were exposed to which act by increasing the stability of the protein. Exposure of
IGF-1 after pretreatment with the indicated concentrations of PD98059 HCT116 cells to the combination of IGF-1 and either CoCl2 or
and wortmannin. E, cells were exposed to 100 ␮M CoCl2 (lanes 1–3) or
hypoxia resulted in a greater induction of HIF-1␣ protein (Fig.
100 ng/ml IGF-1 (lanes 4 – 6) in the presence of no kinase inhibitor
(lanes 1 and 4), 50 ␮M PD98059 (lanes 2 and 5), or 200 nM wortmannin 2B, top) and VEGF mRNA (Fig. 2B, middle) expression than
(lanes 3 and 6). exposure of cells to IGF-1, CoCl2, or hypoxia alone.
 IGF-1 Induces HIF-1 and VEGF via MAP Kinase and PI3-kinase Signaling 38209

FIG. 6. Phosphorylation of the

translational regulators 4E-BP1,
p70S6K, and eIF-4E in IGF-1-treated
cells. Serum-starved HCT116 cells were
pretreated with inhibitors for 1 h prior to
IGF-1 treatment as indicated. Whole cell
extracts were prepared after 15 min (left)
or 6 h (right) of IGF-1 stimulation and
subjected to immunoblot assays using an-
tibodies specific for phosphorylated (Ser-
65) or total 4E-BP, phosphorylated (Thr-
421/Ser-424) or total p70S6K, and phos-
phorylated (Ser-209) or total eIF-4E.

Ubiquitination of HIF-1␣ is inhibited under hypoxic condi- 4E), providing further evidence that IGF-1 and CoCl2 act by
tions (13–17). To determine whether IGF-1 treatment affects distinct molecular mechanisms.
ubiquitination, an in vitro assay was performed using lysates To determine whether the MAP kinase and PI3-kinase path-
prepared from control and IGF-1-treated cells. The lysates ways were activated serially or independently in IGF-1-treated
were incubated with 35S-labeled in vitro translated HIF-1␣ in cells, the phosphorylation of p42ERK/p44ERK and AKT were
the presence of ubiquitin and ATP for 0, 30, or 150 min followed analyzed. The increased phosphorylation of p42ERK/p44ERK
by SDS-PAGE to resolve non-ubiquitinated and ubiquitinated that was induced by IGF-1 treatment was blocked by PD98059
forms of HIF-1␣. Prior to incubation (time 0), no ubiquitinated but not by wortmannin or rapamycin (Fig. 5). The increased
HIF-1␣ was detected, whereas the ratio of ubiquitinated to phosphorylation of AKT that was induced by IGF-1 treatment
non-ubiquitinated forms of HIF-1␣ increased over time with no was blocked by wortmannin, but neither PD98059 nor rapamy-
difference observed between IGF-1-treated and untreated ly- cin affected the ratio of phosphorylated to total AKT. Thus,
sates (Fig. 3A). Incubation of a GST-HIF-1␣ fusion protein with whereas both MAP kinase and PI3-kinase activities are re-
control lysate from untreated cells resulted in prolyl hydroxy- quired for induction of HIF-1␣ protein expression, IGF-1 in-
lation of HIF-1␣, which is required for its interaction with VHL duces the activity of each pathway independently.
(Fig. 3B, lane 2). Lysate from CoCl2-treated cells did not pro- The signal transduction pathway involving PI3-kinase, AKT,
mote the interaction of GST-HIF-1␣ with VHL (Fig. 3B, lane 4). and FRAP has been shown to regulate protein translation via
In contrast, lysates from IGF-1-treated cells (Fig. 3B, lane 3) phosphorylation of 4E-BP1 and p70s6k (18 –20). In HCT116
promoted the interaction of GST-HIF-1␣ with VHL as effi- cells, the phosphorylation of both 4E-BP1 and p70s6k, which
ciently as control lysates, providing further evidence that was induced by IGF-1 stimulation, could be blocked by wort-
IGF-1 treatment does not induce HIF-1␣ expression by inhib- mannin or rapamycin (Fig. 6) as expected. PD98059 also
iting VHL-mediated ubiquitination. blocked the phosphorylation 4E-BP1 and p70s6k, an effect con-
To determine the signal transduction pathways mediating sistent with its inhibition of IGF-1-induced HIF-1␣ protein and
the effects of IGF-1 on HIF-1␣ protein and VEGF mRNA ex- VEGF mRNA expression. The mRNA cap-binding protein, eIF-
pression, HCT116 cells were pretreated with PD98059, wort- 4E, was also transiently phosphorylated by IGF-1 treatment of
mannin, or rapamycin, which are selective pharmacologic in- HCT116 cells, and this process was inhibited by PD98059. This
hibitors of MEK, PI3-kinase, and FRAP/mTOR kinase activity, result is consistent with studies indicating that ERK activates
respectively. All three agents inhibited the induction of HIF-1␣ the MAP kinase signal integrating kinases, MNK1 and MNK2,
protein expression in IGF-1-treated cells (Fig. 4A). At the con- which in turn phosphorylate eIF-4E (28, 29).
centrations used, the rank inhibitory effect of these agents was Involvement of MEK and ERK in the induction of HIF-1␣
PD98059 ⬎ wortmannin ⬎ rapamycin. None of the inhibitors expression in IGF-1-treated colon cancer cells represents a novel
had any effect on the expression of HIF-1␣ mRNA. However, signaling pathway. We investigated whether activation of this
the induction of VEGF mRNA expression was inhibited by pathway was sufficient to induce HIF-1␣ and VEGF expression.
these agents with the same rank potency as seen for the inhi- Transient transfection of HCT116 cells with a plasmid encoding
bition of HIF-1␣ protein expression. The induction of HIF-1␣ by a constitutively active form of MEK-2 (MEK-2DD) resulted in
IGF-1 was inhibited in a dose-dependent manner by PD98059 increased levels of phosphorylated p42ERK/p44ERK MAP kinases
(Fig. 4B) or wortmannin (Fig. 4C). The effects of these inhibi- and increased expression of HIF-1␣ protein and VEGF mRNA
tors were synergistic: 10 ␮M PD98059 or 25 nM wortmannin (Fig. 7A). PD98059 has previously been shown to block the phos-
had little effect alone but in combination markedly inhibited phorylation of ERK1 and ERK2 by constitutively active forms of
IGF-1-induced HIF-1␣ expression (Fig. 4D). In contrast to their MEK (30, 31). The activation of p42ERK/p44ERK and the induction
effects on the expression of HIF-1␣ induced by IGF-1 treat- of HIF-1␣ protein expression in MEK-2DD-transfected cells were
ment, PD98059 or wortmannin had little inhibitory effect on inhibited by PD98059 in a dose-dependent manner (Fig. 7B).
the expression of HIF-1␣ in CoCl2-treated HCT116 cells (Fig. These results indicate that constitutive MAP kinase kinase ac-
38210 IGF-1 Induces HIF-1 and VEGF via MAP Kinase and PI3-kinase Signaling

FIG. 8. Molecular mechanisms of HIF-1-mediated VEGF ex-

pression in IGF-1-treated HCT116 cells. PD, PD98059; PI3K, PI3-
kinase; RAP, rapamycin; WM, wortmannin. The arrow and blocked
arrow (no arrowhead) indicate stimulation and inhibition, respectively.

iments. In the present study of colon cancer cells, we have

confirmed that IGF-1 treatment had no effect on HIF-1␣ pro-
tein stability in IGF-1-treated HCT116 cells and also demon-
strated that IGF-1 did not inhibit the interaction of HIF-1␣
with VHL or its subsequent ubiquitination. Thus, as in the case
of heregulin-treated cells, the increased expression of HIF-1␣
FIG. 7. Effect of constitutively active MEK on HIF-1␣ and protein in IGF-1-treated cells is due to stimulation of its syn-
VEGF expression. A, HCT116 cells were transiently transfected with
thesis. However, in contrast to heregulin-stimulated breast
an empty vector (EV) or an expression vector encoding HA-tagged
MEK2DD, a constitutively active form of MEK-2. 24 h after transfection cancer cells, this effect is dependent upon activity of both the
the cells were serum-starved for 24 h and analyzed for the expression of PI3-kinase and MAP kinase pathways in IGF-1-stimulated
HIF-1␣, HA-MEK2DD, and phospho-p42/p44 proteins and for the ex- colon cancer cells (Fig. 8). Whereas signaling from constitu-
pression of VEGF mRNA. B, cells were transfected with empty vector tively active forms of a G protein-coupled receptor, RAF-1, or
(lane 1) or MEK2DD expression vector (lanes 2– 6) and exposed to 0 –50
␮M PD98059 for 24 h. Aliquots of cells lysates were subjected to immu- RAS to MEK and MAP kinases has been shown to stimulate
noblot assay using antibodies specific for HIF-1␣ (top), phosphorylated HIF-1␣ transactivation domain function (35–37), the data re-
p42ERK/p44 ERK (middle), and total p42ERK/p44 ERK (bottom). ported here represent the first demonstration that the MAP
kinase pathway can also stimulate HIF-1␣ expression.
tivity is sufficient to induce increased HIF-1␣ protein and VEGF Dependence on MEK activity for phosphorylation of 4E-BP1
mRNA expression in colon cancer cells. and p70s6K has been demonstrated in other cellular contexts
(38 – 40). In the case of interleukin 6-stimulated myeloma cells,
DISCUSSION both MEK and PI3-kinase are required for activation of p70s6K,
Recent studies have demonstrated that in addition to medi- with MEK inhibitors preventing the phosphorylation of Thr-
ating proliferative and anti-apoptotic signals, receptor tyrosine 421/Ser-424 in the autoinhibitory domain, which is required for
kinases also promote tumor angiogenesis and that the thera- subsequent phosphorylation at Thr-389 by FRAP/mTOR (38).
peutic efficacy of receptor tyrosine kinase inhibitors may derive ERK has been shown to phosphorylate 4E-BP1 in vitro (38).
in part from their anti-angiogenic effects (32, 33). A principal Our data demonstrate a striking correlation between the inhi-
mediator of tumor angiogenesis is VEGF, and a major tran- bition of IGF-1-induced HIF-1␣ protein and VEGF mRNA ex-
scriptional activator of the VEGF gene is HIF-1 (34). We pre- pression and the inhibition of 4E-BP1 and p70s6K phosphoryl-
viously demonstrated that whereas hypoxia decreases HIF-1␣ ation by wortmannin, rapamycin, and PD98059 in HCT116
protein degradation, heregulin stimulation of breast cancer cells. The IGF-1 3 MEK 3 ERK pathway also stimulated the
cells increases HIF-1␣ synthesis, an effect that is dependent on phosphorylation of eIF-4E, which is required for its mRNA cap
HER2neu, PI3-kinase, AKT, and FRAP/mTOR (but not MEK-1) binding activity. Thus, IGF-1 signaling both de-represses (via
activity and the 5⬘-untranslated region of HIF-1␣ mRNA (11). phosphorylation of 4E-BP1) and activates (via phosphorylation of
The studies reported above demonstrate that IGF-1 stimu- eIF-4E and p70s6K) protein synthesis in HCT116 cells (Fig. 8).
lation of human colon cancer cells also increases HIF-1␣ pro- In experimental tumors, increased eIF-4E activity stimu-
tein and VEGF mRNA expression via effects on the transla- lates tumor growth, invasion, and metastasis (41). Although it
tional machinery (Fig. 8). In the previous study of MCF-7 increases global protein synthesis, elevated eIF-4E activity
breast cancer cells, the effect on protein synthesis was docu- disproportionately stimulates the translation of specific pro-
mented by cycloheximide inhibition and by pulse-chase exper- teins with important roles in tumor progression, including
 IGF-1 Induces HIF-1 and VEGF via MAP Kinase and PI3-kinase Signaling 38211
VEGF (41). FRAP/mTOR also has a disproportionate effect on Kriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell,
P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Science 292, 468 – 472
the translation of specific proteins (42). In heregulin-treated 15. Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C., Vaux, E. C.,
MCF-7 cells, increased translation of luciferase mRNA was Cockman, M. E., Wykoff, C. C., Pugh, C. W., Maher, E. R., and Ratcliffe,
dependent upon the presence of HIF-1␣ 5⬘-untranslated se- P. J. (1999) Nature 399, 271–275
16. Epstein, A. C. R., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O’Rourke, J.,
quences, demonstrating that the stimulation of translation was Mole, D. R., Mukherji, M., Metzen, E., Wilson, M. I., Dhanda, A., Tian,
mRNA-specific (11). Y.-M., Masson, N., Hamilton, D. L., Jaakkola, P., Barstead, R., Hodgkin, J.,
Maxwell, P. H., Pugh, C. W., Schofield, C. J., and Ratcliffe, P. J. (2001) Cell
Taken together, these results provide evidence that activa- 107, 43–54
tion of different receptor tyrosine kinases (HER2neu, IGF-1R) in 17. Sutter, C. H., Laughner, E., and Semenza, G. L. (2000) Proc. Natl. Acad. Sci.
different human cancers (breast, colon) have in common the U. S. A. 97, 4748 – 4753
18. Hara, K., Yonezawa, K., Kozlowski, M. T., Sugimoto, T., Andrabi, K., Weng,
stimulation of HIF-1␣ protein synthesis and increased expres- Q.-P., Kasuga, M., Nishimoto, I., and Avruch, J. (1997) J. Biol. Chem. 272,
sion of the downstream target VEGF. The effects of receptor 26457–26463
tyrosine kinase activation on HIF-1␣ expression are additive to 19. Gingras, A.-C., Gygi, S. P., Raught, B., Polakiewicz, R. D., Abraham, R. T.,
Hoekstra, M. F., Aebersold, R., and Sonenberg, N. (1999) Genes Dev. 13,
the effects of hypoxia, emphasizing the importance of two par- 1422–1437
allel pathways for induction of HIF-1 in human cancer, one 20. Peterson, R. T., Desai, B. N., Hardwick, J. S., and Schreiber, S. L. (1999) Proc.
Natl. Acad. Sci. U. S. A. 96, 4438 – 4442
based on physiologic stimulation and the other on genetic al- 21. Zelzer, E., Levy, Y., Kahana, C., Shilo, B. Z., Rubinstein, M., and Cohen, B.
terations. HIF-1␣ overexpression is associated with tumor an- (1998) EMBO J. 17, 5085–5094
giogenesis and increased mortality in cancers of the breast, 22. Feldser, D., Agani, F., Iyer, N. V., Pak, B., Ferreira, G., and Semenza, G. L.
(1999) Cancer Res. 59, 3915–3918
central nervous system, oropharynx, ovary, and uterine cervix 23. Zhong, H., De Marzo, A. M., Laughner, E., Lim, M., Hilton, D. A., Zagzag, D.,
(34). HIF-1␣ overexpression is observed in colon cancer (23), Buechler, P., Isaacs, W. B., Semenza, G. L., and Simons, J. W. (1999) Cancer
and the results presented in this study suggest that HIF-1␣ Res. 59, 5830 –5835
24. Ravi, R., Mookerjee, B., Bhujwalla, Z. M., Sutter, C. H., Artemov, D., Zeng, Q.,
overexpression may contribute significantly to angiogenesis Dillehay, L. E., Madan, A., Semenza, G. L., and Bedi, A. (2000) Genes Dev.
and other important aspects of colon cancer progression. 14, 34 – 44
25. Zagzag, D., Zhong, H., Scalzitti, J. M., Laughner, E., Simons, J. W., and
Acknowledgment—We thank Dr. Sylvain Meloche for generously pro- Semenza, G. L. (2000) Cancer 88, 2606 –2618
viding pCMV-MEK2DD. 26. Cockman, M. E., Masson, N., Mole, D. R., Jaakkola, P., Chang, G. W., Clifford,
S. C., Maher, E. R., Pugh, C. W., Ratcliffe, P. J., and Maxwell, P. H. (2000)
REFERENCES J. Biol. Chem. 275, 25733–25741
27. Mahon, P. C., Hirota, K., and Semenza, G. L. (2001) Genes Dev. 15, 2675–2686
1. Valentinis, B., and Baserga, R. (2001) Mol. Pathol. 54, 133–137 28. Waskiewicz, A. J., Flynn, A., Proud, C. G., and Cooper, J. A. (1997) EMBO J.
2. Zhang, L., Zhou, W., Velculescu, V., Kern, S. E., Hruban, R. H., Hamilton, 16, 1909 –1920
S. R., Vogelstein, B., and Kinzler, K. W. (1997) Science 276, 1268 –1272 29. Waskiewicz, A. J., Johnson, J. C., Penn, B., Mahalingam, M., Kimball, S. R.,
3. Warren, R. S., Yuan, H., Matli, M. R., Ferrara, N., and Donner, D. B. (1996) and Cooper, J. A. (1999) Mol. Cell. Biol. 19, 1871–1880
J. Biol. Chem. 271, 29483–29488
30. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995)
4. Akagi, Y., Liu, W., Zebrowski, B., Xie, K., and Ellis, L. M. (1998) Cancer Res.
Proc. Natl. Acad. Sci. U. S. A. 92, 7686 –7689
58, 4008 – 4014
31. Schramek, H., Feifel, E., Healy, E., and Pollack, V. (1997) J. Biol. Chem. 272,
5. Wu, Y., Yakar, S., Zhao, L., Hennighausen, L., and LeRoith, D. (2002) Cancer
11426 –11433
Res. 62, 1030 –1035
32. Kerbel, R. S., Viloria-Petit, A., Klement, G., and Rak, J. (2000) Eur. J. Cancer
6. Rak, J., Mitsuhashi, Y., Sheehan, C., Tamir, A., Viloria-Petit, A., Filmus, J.,
Mansour, S. J., Ahn, N. G., and Kerbel, R. S. (2000) Cancer Res. 60, 36, 1248 –1257
490 – 498 33. Viloria-Petit, A., Crombet, T., Jothy, S., Hicklin, D., Bohlen, P., Schlaeppi,
7. Forsythe, J. A., Jiang, B.-H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., J. M., Rak, J., and Kerbel, R. S. (2001) Cancer Res. 61, 5090 –5101
and Semenza, G. L. (1996) Mol. Cell. Biol. 16, 4604 – 4613 34. Semenza, G. L. (2002) Trends Mol. Med. 8, S62-S67
8. Carmeliet, P., Dor, Y., Herbert, J.-M., Fukumura, D., Brusselmans, K., 35. Richard, D. E., Berra, E., Gothie, E., Roux, D., and Pouyssegur, J. (1999)
Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, J. Biol. Chem. 274, 32631–32637
C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. (1998) 36. Sodhi, A., Montaner, S., Patel, V., Zohar, M., Bais, C., Mesri, E. A., and
Nature 394, 485– 490 Gutkind, J. S. (2000) Cancer Res. 60, 4873– 4880
9. Iyer, N. V., Kotch, L. E., Agani, F., Leung, S. W., Laughner, E., Wenger, R. H., 37. Sodhi, A., Montaner, S., Miyazaki, H., and Gutkind, J. S. (2001) Biochem.
Gassmann, M., Gearhart, J. D., Lawler, A. M., Yu, A. Y., and Semenza, G. L. Biophys. Res. Commun. 287, 292–300
(1998) Genes Dev. 12, 149 –162 38. Haystead, T. A., Haystead, C. M., Hu, C., Lin, T. A., and Lawrence, J. C., Jr.
10. Ryan, H. E., Lo, J., and Johnson, R. S. (1998) EMBO J. 17, 3005–3015 (1994) J. Biol. Chem. 269, 23185–23191
11. Laughner, E., Taghavi, P., Chiles, K., Mahon, P. C., and Semenza, G. L. (2001) 39. Herbert, T. P., Kilhams, G. R., Batty, I. H., and Proud, C. G. (2000) J. Biol.
Mol. Cell. Biol. 21, 3995– 4004 Chem. 275, 11249 –11256
12. Wang, G. L., Jiang, B.-H., Rue, E. A., and Semenza, G. L. (1995) Proc. Natl. 40. Shi, Y., Hsu, J.-h., Hu, L., Gera, J., and Lichtenstein, A. (2002) J. Biol. Chem.
Acad. Sci. U. S. A. 92, 5510 –5514 277, 15712–15720
13. Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara, 41. Zimmer, S. G., Debenedetti, A., and Graff, J. R. (2000) Anticancer Res. 20,
J. M., Lane, W. S., and Kaelin, W. G., Jr. (2001) Science 292, 464 – 468 1343–1352
14. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., 42. Gingras, A.-C., Raught, B., and Sonenberg, N. (2001) Genes Dev. 15, 807– 826