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IGF-1 Induces HIF-1-mediated VEGF Expression that is Dependent

on MAP Kinase and PI-3-Kinase Signaling in Colon Cancer Cells

Ryo Fukuda1, Kiichi Hirota1, 3, Fan Fan2, Young Do Jung2, Lee M. Ellis2, and

Gregg L. Semenza1,4

From the 1McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University

School of Medicine, Baltimore, Maryland 21287, and 2University of Texas M. D. Anderson

Cancer Center, Houston, Texas 77030

3Current address: Human Stress Signal Research Center, National Institute of Advanced

Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan

4To whom correspondence should be addressed: Johns Hopkins University School of Medicine,

CMSC-1004, 600 North Wolfe Street, Baltimore, MD 21287-3914; FAX: 410-955-0484; E-mail:

[email protected].

Running Title: IGF-1 induces HIF-1 and VEGF via MAP kinase and PI-3-kinase signaling

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 30, 2002 as Manuscript M203781200 by guest on June 25, 2018

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SUMMARY

Stimulation of human colon cancer cells with insulin-like growth factor 1 (IGF-1)

induces expression of the VEGF gene encoding vascular endothelial growth factor. In this paper

we demonstrate that exposure of HCT116 human colon carcinoma cells to IGF-1 induces the

expression of HIF-1α, the regulated subunit of hypoxia-inducible factor 1, a known

transactivator of the VEGF gene. In contrast to hypoxia, which induces HIF-1α expression by

inhibiting its ubiquitination and degradation, IGF-1 did not inhibit these processes, indicating an

effect on HIF-1α protein synthesis. IGF-1 stimulation of HIF-1α protein and VEGF mRNA

expression was inhibited by treating cells with inhibitors of phosphatidylinositol-3-kinase and

MAP kinase signaling pathways. These inhibitors also blocked the IGF-1-induced

phosphorylation of the translational regulatory proteins 4E-BP1, p70 S6 kinase, and eIF-4E, thus

providing a mechanism for the modulation of HIF-1α protein synthesis. Forced expression of a

constitutively-active form of the MAP kinase kinase MEK2 was sufficient to induce HIF-1α

protein and VEGF mRNA expression. Involvement of the MAP kinase pathway represents a

novel mechanism for the induction of HIF-1α protein expression in human cancer cells.

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The insulin-like growth factor-1 (IGF-1)1 receptor-tyrosine kinase (IGF-1R) is activated by

binding either of its ligands, IGF-1 or IGF-2. IGF-1R signaling through the mitogen-activated

protein (MAP) kinase and phosphatidylinositol-3-kinase (PI-3-kinase) pathways plays a critical

role in transformation and tumorigenesis (1). IGF2 gene expression is upregulated to the

greatest extent of any gene in colon cancer cells relative to normal colonic epithelium (2),

resulting in autocrine stimulation of cells which express both receptor and ligand. In addition to

effects of the IGF-1R on cell transformation and proliferation, treatment of colon cancer cells

with IGF-1 also induces transcription of the VEGF gene encoding vascular endothelial growth

factor which is essential for tumor angiogenesis (3, 4). Treatment of mice with IGF-1 increases

colon cancer growth and metastasis as well as tumor VEGF expression and vascularization (5).

A variety of growth factor-receptor tyrosine kinase signaling pathways induce VEGF expression

in cancer cells. In the case of oncogenic RAS signaling, VEGF expression is dependent upon

activity of the MAP kinase/extracellular signal-regulated kinase (ERK) kinase 1 (MEK-1) in

fibroblasts but is dependent upon PI-3-kinase activity in epithelial cells (6).

Cellular signaling pathways modulate gene expression by altering the activity or

expression of specific transcription factors. The major physiological stimulus for VEGF

expression is cellular hypoxia and hypoxia-induced transcription of the VEGF 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 breast cancer cells was

shown to be mediated by HIF-1 via the PI-3-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 constitutively-expressed HIF-1β subunit and an inducibly-expressed

HIF-1α subunit (12). Under non-hypoxic conditions, HIF-1α is subject to O2-dependent prolyl

hydroxylation (13, 14) that is required for binding of the von Hippel-Lindau tumor suppressor

protein (VHL), the recognition component of an E3 ubiquitin-protein ligase which targets HIF-


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1α for proteasomal degradation (15). Under hypoxic conditions, O2 becomes limiting for prolyl

hydroxylase activity (16) and ubiquitination of HIF-1α is inhibited (17). As a result, HIF-1α

accumulates, dimerizes with HIF-1β, and activates transcription of target genes.

Signaling via receptor tyrosine kinases can induce HIF-1α expression by an independent

mechanism. HER2neu activation in breast cancer cells stimulates increased rates of HIF-1α

protein synthesis via PI-3-kinase and the downstream serine-threonine kinases AKT (protein

kinase B) and FRAP (FKBP/rapamycin-associated protein), which is also known as mTOR

(mammalian target of rapamycin) (11). FRAP/mTOR phosphorylates and activates the

translational regulatory proteins eIF-4E binding protein 1 (4E-BP1) and p70 S6 kinase (p70S6K)

(18-20). Phosphorylation of 4E-BP1 disrupts its inhibitory interaction with eukarytic initiation

factor 4E (eIF-4E), whereas activated p70S6K phosphorylates the 40S ribosomal protein S6. The

effect of HERneu signaling on the translation of HIF-1α protein is dependent upon the presence of

the 5’-untranslated region of HIF-1α mRNA (11). These pathways thus provide a molecular

basis for stimulation of HIF-1α protein synthesis in response to HER2neu activation.

Treatment of cultured cells with IGF-1 or IGF-2 also induces HIF-1α protein expression,

HIF-1 DNA-binding activity, and transactivation of target genes (21, 22). The demonstration

that IGF2 is a HIF-1 target gene (22), that HIF-1α is overexpressed in human colon cancers (23),

and that forced overexpression of HIF-1α in HCT116 colon carcinoma cells increases tumor

growth and vascularization in vivo (24) suggest that HIF-1 may play an important role in

autocrine IGF-1R signaling and angiogenesis in colon cancer. We therefore investigated the

mechanisms by which IGF-1 stimulation increases the expression of HIF-1 and VEGF.


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EXPERIMENTAL PROCEDURES

Tissue Culture and Reagents– HCT116 cells were cultured in McCoy’s 5A medium with

10% FBS, 100 U/mL Penicillin, and 100 µg/mL Streptomycin (Life Technologies). Unless

otherwise stated, cells were maintained at 37oC in a humidified 5% CO2-95% air incubator. IGF-

1, PD98059, wortmannin, rapamycin, cycloheximide (CHX), and cobalt chloride (CoCl2) were

purchased from Sigma. H-1356 (JB1) was purchased from Bachem Biochemica GmbH. CHX

was dissolved in ethanol at 100 mM. PD98059, wortmanin, and rapamycin were dissolved in

DMSO at 50 mM, 200 µM, and 100 µM, respectively. For hypoxic exposures, cells were placed

in a modulator incubator chamber (Billups-Rothenberg) that was flushed with a gas mixture

consisting of 1% O2, 5% CO2, balance N2, sealed, and incubated at 37oC.

IGF-1 and Inhibitor Treatments– HCT116 cells were plated at a density of 2.4 X 106 per

10-cm or 8.6 x 105 per 6-cm dish. Subconfluent cells were serum-starved (0.1% FBS in all

experiments except Fig. 1A in which FBS was completely eliminated) for 24 h before IGF-1 was

added. The IGF-1R antagonist H-1356 and the kinase inhibitors PD98059, wortmannin, and

rapamycin were added 1 h before exposure to IGF-1, 1% O2, or 100 µM CoCl2. CHX was added

to the media of HCT116 cells that had been serum-starved and treated with CoCl2 or IGF-1 for 4

h and whole cell extracts were prepared at 15, 30 and 60 min.

Immunoblot Assays– Whole cell extracts were prepared using RIPA buffer, fractionated

by SDS-PAGE, transferred to a nitrocellulose filter, and subjected to immunoblot assays. For

HIF-1α and HIF-1β, 150-µg aliquots of protein were analyzed using a monoclonal antibody

against HIF-1α (H1α67) or HIF-1β (H1β234) (Novus Biologicals) at 1:1000 dilution as

previously described (23, 25). 50-µg aliquots were analyzed using antibodies (1:1000 dilution)

specific for phosphorylated (Thr202/Tyr204) or total p44/p42 MAP kinase, phosphorylated

(Ser473) or total AKT, phosphorylated (Thr421/Ser424) or total p70S6K, phosphorylated (Ser209)


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or total elF-4E, and phosphorylated (Ser65) or total 4E-BP1 antibodies purchased from Cell

Signaling Technology and Santa Cruz Biotechnology. Anti-hemagglutin (HA) antibody was

from Santa Cruz. HRP-conjugated mouse monoclonal antibodies for mouse and rabbit IgG

(1:2500 dilution) and ECL reagents were from Amersham Pharmacia Biotech.

RNA Blot Hybridization– Total RNA was extracted from HCT116 cells using TRIzol

reagent (Life Technologies) 6-24 h after IGF-1 stimulation and 48 h after plasmid transfection.

10-µg aliquots of RNA were fractionated by electrophoresis in 1.5% agarose/2.2M formaldehyde

gels, transferred to Hybond N+ membranes (Amersham), and hybridized with a [32P]-labeled

human HIF-1α or VEGF cDNA probe as previously described (11).

In Vitro Ubiquitination Assay – HCT116 cells were serum-starved, treated with IGF-1 for

0, 30 or 150 min, washed twice with cold hypotonic extraction buffer (20 mM Tris [pH 7.5], 5

mM KCl, 1.5 mM MgCl2, 1 mM DTT), and lysed in a Dounce homogenizer. The cell extract

was centrifuged at 10,000 g for 10 min at 4oC and the supernatant was stored in aliquots at -70oC.

Ubiquitination assays were performed as previously described (26) at 30oC in a total volume of

40 µl containing 27 µl (50 µg) of cell extract, 4 µl of 10 x ATP-regenerating system (20 mM Tris

[pH 7.5], 10 mM ATP, 10 mM magnesium acetate, 300 mM creatine phosphate, 0.5 mg/ml

creatine phosphokinase), 4 µl of 5 mg/ml ubiquitin (Sigma), 0.83 µl of 150 µM ubiquitin

aldehyde (Sigma), and 2 µl of HA-HIF-1α that was in vitro-translated (TNT Quick Coupled

Transcription/Translation System, Promega) in the presence of [35S]-methionine. HA-HIF-1α

was recovered using anti-HA agarose beads which were then mixed with SDS sample buffer,

boiled for 5 min and the eluates were analyzed by SDS-PAGE and autoradiography.

In Vitro HIF-1α-VHL Interaction Assay– [35S]-methionine-labeled VHL protein was

synthesized in vitro and glutathione-S-transferase (GST)-HIF-1α(429-608) fusion protein was

expressed in E. coli as previously described (27). HCT116 cells were serum-starved and treated

with IGF-1 or CoCl2 for 4 h prior to lysate preparation. GST-HIF-1α(429-608) was pre-


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incubated with 10 µl of the HCT116 lysate for 30 min at 30oC. Five-µl aliquots of the GST-HIF-

1α(429-608) pre-incubation and VHL in vitro-translation reactions were mixed in 150 µl of

NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris-HCl [pH 8.0], 0.5% [v/v] NP-40).

After 90 min at 4oC, 20 µl of glutathione-Sepharose-4B (Amersham) was added. After 30 min

mixing on a rotator, beads were washed three times with NETN buffer. Proteins were eluted in 2

x SDS sample buffer, fractionated by SDS-PAGE, and detected by autoradiography.

Transient Transfection– 8.6 x 105 HCT116 cells were plated per 6-cm dish, cultured

overnight, and transfected with 1.25 µg of pCMV-HA-MEK-2DD (kind gift of S. Meloche,

Institut de Recherches Cliniques de Montreal) or empty pCMV (Stratagene) in the presence of

Fugene-6 (Roche). 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-1α protein expression with a maximal

effect observed in the presence of 100 ng/ml of IGF-1 (Fig. 1A, top panel). Similar results were

obtained with IGF-2 (data not shown). HIF-1α expression was also induced by exposure of cells

to CoCl2 (Fig. 1A, lane 6) which blocks HIF-1α degradation. In contrast, neither IGF-1 nor

CoCl2 induced HIF-1α mRNA expression (Fig. 1A, middle panel), demonstrating specific effects

of these agents on HIF-1α protein expression. In the presence of IGF-1, HIF-1α protein levels

peaked at 8 h and declined thereafter (Fig. 1B, top panel). HIF-1β levels were unaffected by

IGF-1 treatment (Fig. 1B, bottom panel). IGF-1 treatment also induced VEGF mRNA


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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-1α protein and

VEGF mRNA expression in IGF-1-treated cells in a dose-dependent manner (Fig. 1D, lanes 3-5),

thus demonstrating a requirement for signal transduction via the IGF-1R. In contrast, hypoxic

cells expressed HIF-1α 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-1α protein and VEGF mRNA (Fig. 1D, compare top and middle panels).

To determine whether IGF-1 treatment affected HIF-1α protein half-life, HCT116 cells

were treated with CoCl2 or IGF-1 for 4 h to induce HIF-1α expression and then CHX was added

to block ongoing protein synthesis. In the presence of CHX, the half-life of HIF-1α was > 60

min in CoCl2-treated cells but < 30 min in IGF-1-treated cells (Fig. 2A). These results indicate

HIF-1α expression in IGF-1-treated cells is dependent upon ongoing protein synthesis. If IGF-1

induces HIF-1α expression by stimulating synthesis of the protein, then it would be expected to

have an additive effect with that of CoCl2 or hypoxia, which act by increasing the stability of the

protein. Exposure of HCT116 cells to the combination of IGF-1 and either CoCl2 or hypoxia

resulted in a greater induction of HIF-1α protein (Fig. 2B, top panel) and VEGF mRNA (Fig. 2B,

middle panel) expression than exposure of cells to IGF-1, CoCl2, or hypoxia alone.

Ubiquitination of HIF-1α is inhibited under hypoxic conditions (13-17). To determine

whether IGF-1 treatment affects ubiquitination, an in vitro assay was performed using lysates

prepared from control and IGF-1-treated cells. The lysates were incubated with 35S-labeled in

vitro-translated HIF-1α in the presence of ubiquitin and ATP for 0, 30, or 150 min followed by

SDS-PAGE to resolve non-ubiquitinated and ubiquitinated forms of HIF-1α. Prior to incubation

(time 0), no ubiquitinated HIF-1α was detected, whereas the ratio of ubiquitinated to non-

ubiquitinated forms of HIF-1α increased over time with no difference observed between IGF-1-

treated and untreated lysates (Fig. 3A). Incubation of a GST-HIF-1α fusion protein with control


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lysate from untreated cells resulted in prolyl hydroxylation of HIF-1α which is required for its

interaction with VHL (Fig. 3B, lane 2). Lysate from CoCl2-treated cells did not promote the

interaction of GST-HIF-1α with VHL (Fig. 3B, lane 4). In contrast, lysates from IGF-1-treated

cells (Fig. 3B, lane 3) promoted the interaction of GST-HIF-1α with VHL as efficiently as

control lysates, providing further evidence that IGF-1 treatment does not induce HIF-1α

expression by inhibiting VHL-mediated ubiquitination.

To determine the signal transduction pathways mediating the effects of IGF-1 on HIF-1α

protein and VEGF mRNA expression, HCT116 cells were pretreated with PD98059, wortmannin,

or rapamycin which are selective pharmacologic inhibitors of MEK, PI-3-kinase, and

FRAP/mTOR kinase activity, respectively. All three agents inhibited the induction of HIF-1α

protein expression in IGF-1-treated cells (Fig. 4A). At the concentrations used, the rank

inhibitory effects of these agents was PD98059 > wortmannin > rapamycin. None of the

inhibitors had any effect on the expression of HIF-1α mRNA. However, the induction of VEGF

mRNA expression was inhibited by these agents with the same rank potency as seen for the

inhibition of HIF-1α protein expression. The induction of HIF-1α by IGF-1 was inhibited in a

dose-dependent manner by PD98059 (Fig. 4B) or wortmannin (Fig. 4C). The effects of these

inhibitors were synergistic: 10 µM PD98059 or 25 nM wortmannin had little effect alone but in

combination markedly inhibited IGF-1-induced HIF-1α expression (Fig. 4D). In contrast to their

effects on the expression of HIF-1α induced by IGF-1 treatment, PD98059 or wortmannin had

little inhibitory effect on the expression of HIF-1α in CoCl2-treated HCT116 cells (Fig. 4E),

providing further evidence that IGF-1 and CoCl2 act by distinct molecular mechanisms.

To determine whether the MAP kinase and PI-3-kinase pathways were activated serially

or independently in IGF-1-treated cells, the phosphorylation of p42ERK/p44ERK and AKT were

analyzed. The increased phosphorylation of p42ERK/p44ERK that was induced by IGF-1 treatment

was blocked by PD98059 but not by wortmannin or rapamycin (Fig. 5). The increased


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phosphorylation of AKT that was induced by IGF-1 treatment was blocked by wortmannin but

neither PD98059 nor rapamycin affected the ratio of phosphorylated to total AKT. Thus,

whereas both MAP kinase and PI-3-kinase activities are required for induction of HIF-1α protein

expression, IGF-1 induces the activity of each pathway independently.

The signal transduction pathway involving PI-3-kinase, AKT, and FRAP has been shown

to regulate protein translation via phosphorylation of 4E-BP1 and p70s6k (18-20). In HCT116

cells, the phosphorylation of both 4E-BP1 and p70s6k that was induced by IGF-1 stimulation

could be blocked by wortmannin or rapamycin (Fig. 6) as expected. PD98059 also blocked the

phosphorylation 4E-BP1 and p70s6k, an effect consistent with its inhibition of IGF-1-induced

HIF-1α protein and VEGF mRNA expression. The mRNA cap-binding protein eIF-4E, was also

transiently phosphorylated by IGF-1 treatment of HCT116 cells and this process was inhibited

by PD98059. This result is consistent with studies indicating that ERK activates the MAP kinase

signal integrating kinases MNK1 and MNK2, which in turn phosphorylate eIF-4E (28, 29).

Involvement of MEK and ERK in the induction of HIF-1α expression in IGF-1-treated

colon cancer cells represents a novel signaling pathway. We investigated whether activation of

this pathway was sufficient to induce HIF-1α and VEGF expression. Transient transfection of

HCT116 cells with a plasmid encoding a constitutively-active form of MEK-2 (MEK-2DD)

resulted in increased levels of phosphorylated p42ERK/p44ERK MAP kinases and increased

expression of HIF-1α protein and VEGF mRNA (Fig. 7A). PD98059 has previously been

shown to block the phosphorylation of ERK1 and ERK2 by constitutively-active forms of MEK

(30, 31). The activation of p42ERK/p44ERK and the induction of HIF-1α protein expression in

MEK-2DD-transfected cells was inhibited by PD98059 in a dose-dependent manner (Fig. 7B).

These results indicate that constitutive MAP kinase kinase activity is sufficient to induce

increased HIF-1α protein and VEGF mRNA expression in colon cancer cells.


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DISCUSSION

Recent studies have demonstrated that in addition to mediating proliferative and anti-

apoptotic signals, receptor tyrosine kinases also promote tumor angiogenesis and that the

therapeutic efficacy of receptor tyrosine kinase inhibitors may derive in part from their anti-

angiogenic effects (32, 33). A principal mediator of tumor angiogenesis is VEGF and a major

transcriptional activator of the VEGF gene is HIF-1 (34). We previously demonstrated that

whereas hypoxia decreases HIF-1α protein degradation, heregulin stimulation of breast cancer

cells increases HIF-1α synthesis, an effect that is dependent on HER2neu, PI-3-kinase, AKT, and

FRAP/mTOR (but not MEK-1) activity, and the 5’-untranslated region of HIF-1α mRNA (11).

The studies reported above demonstrate that IGF-1 stimulation of human colon cancer

cells also increases HIF-1α protein and VEGF mRNA expression via effects on the translational

machinery (Fig. 8). In the previous study of MCF-7 breast cancer cells, the effect on protein

synthesis was documented by cycloheximide inhibition and by pulse-chase experiments. In the

present study of colon cancer cells, we confirmed that IGF-1 treatment had no effect on HIF-1α

protein stability in IGF-1 treated HCT116 cells and also demonstrated that IGF-1 did not inhibit

the interaction of HIF-1α with VHL or its subsequent ubiquitination. Thus, as in the case of

heregulin-treated cells, the increased expression of HIF-1α protein in IGF-1-treated cells is due

to stimulation of its synthesis. However, in contrast to heregulin-stimulated breast cancer cells,

this effect is dependent upon activity of both the PI-3-kinase and MAP kinase pathways in IGF-

1-stimulated colon cancer cells (Fig. 8). Whereas signaling from constitutively-active forms of a

G-protein coupled receptor, RAF-1, or RAS to MEK and MAP kinases has been shown to

stimulate HIF-1α transactivation domain function (35-37), the data reported here represent the

first demonstration that the MAP kinase pathway can also stimulate HIF-1α expression.


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Dependence on MEK activity for phosphorylation of 4E-BP1 and p70s6K has been

demonstrated in other cellular contexts (38-40). In the case of interleukin 6-stimulated myeloma

cells, both MEK and PI-3-kinase are required for activation of p70s6K, with MEK inhibitors

preventing the phosphorylation of Thr421/Ser424 in the autoinhibitory domain which is required

for subsequent phosphorylation at Thr389 by FRAP/mTOR (38). ERK has been shown to

phosphorylate 4E-BP1 in vitro (38). Our data demonstrate a striking correlation between the

inhibition of IGF-1-induced HIF-1α protein and VEGF mRNA expression and the inhibition of

4E-BP1 and p70s6K phosphorylation by wortmannin, rapamycin, and PD98059 in HCT116 cells.

The IGF-1 � MEK � ERK pathway also stimulated the phosphorylation of eIF-4E, which is

required for its mRNA cap-binding activity. Thus, IGF-1 signaling both de-represses (via

phosphorylation of 4E-BP1) and activates (via phosphorylation of eIF-4E and p70s6K) protein

synthesis in HCT116 cells (Fig. 8).

In experimental tumors, increased eIF-4E activity stimulates tumor growth, invasion, and

metastasis (41). Although increasing global protein synthesis, elevated eIF-4E activity

disproportionately stimulates the translation of specific proteins with important roles in tumor

progression, including VEGF (41). FRAP/mTOR also has a disproportionate effect on the

translation of specific proteins (42). In heregulin-treated MCF-7 cells, increased translation of

luciferase mRNA was dependent upon the presence of HIF-1α 5’-untranslated sequences,

demonstrating that the stimulation of translation was mRNA-specific (11).

Taken together, these results provide evidence that activation of different receptor

tyrosine kinases (HER2neu, IGF-1R) in different human cancers (breast, colon) share in common

the stimulation of HIF-1α protein synthesis and increased expression of the downstream target

VEGF. The effects of receptor tyrosine kinase activation on HIF-1α expression are additive to

the effects of hypoxia, emphasizing the importance of two parallel pathways for induction of

HIF-1 in human cancer, one based on physiologic stimulation and the other on genetic alterations.


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HIF-1α overexpression is associated with tumor angiogenesis and increased mortality in cancers

of the breast, central nervous system, oropharynx, ovary, and uterine cervix (34). HIF-1α

overexpression is observed in colon cancer (23) and the results presented in this study suggest

that HIF-1α overexpression may contribute significantly to angiogenesis and other important

aspects of colon cancer progression.

Acknowledgments– We thank Dr. Sylvain Meloche for generously providing pCMV-

MEK2DD. This work was supported by a grants R01-DK39869 (to G.L.S.) and R01-CA74821

(to L.M.E.) from the National Institutes of Health.


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FIGURE LEGENDS

FIG. 1. Effect of IGF-1 treatment on HIF-1α and VEGF expression in HCT116 cells.

A, analysis of HIF-1α expression as a function of IGF-1 concentration. Duplicate plates of

HCT116 cells were cultured in the absence of serum for 24 h, exposed to vehicle (lane 1), 1-

1000 ng/ml of IGF-1 (lanes 2-5) or 100 µM CoCl2 (lane 6) for 6 h, and either whole cell lysates

were subject to immunoblot assay for expression of HIF-1α protein (top panel) or total cellular

RNA was isolated and analyzed by blot hybridization using a HIF-1α cDNA probe (middle

panel) following RNA transfer from an ethidium bromide (EtBr)-stained gel (bottom panel;

migration of 28S and 18S rRNA indicated). B, kinetics of HIF-1α induction. Serum-starved

cells were exposed to vehicle (lane 1) or 100 ng/ml of IGF-1 for 2-24 h (lanes 2-8) prior to

immunoblot analysis of whole cell lysates using monoclonal antibodies specific for HIF-1α (top

panel) or HIF-1β (bottom panel). C, analysis of VEGF mRNA expression. Serum-starved cells

were exposed to vehicle (lane 1), 10-100 ng/ml of 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 panel) following transfer of RNA from an EtBr-stained gel (bottom panel). D, effect of

IGF-1R inhibitor. Cells were pre-treated with vehicle (lanes 1 and 2) or 1-100 µM H-1356

(lanes 3-6), exposed to IGF-1 (lanes 2-5) or 1% O2 (lane 6), and harvested after 6 h for analysis

of HIF-1α protein expression by immunoblot assay (top panel) or at 24 h for analysis of VEGF

mRNA expression by blot hybridization (middle panel) following RNA transfer from an EtBr-

stained gel (bottom panel).

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

analysis of HIF-1α stability. HCT116 cells were exposed to 100 µM CoCl2 (top panel) or 100

ng/ml IGF-1 (bottom panel) for 4 h, cycloheximide (CHX) was added to a final concentration of


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100 µM, the cells were incubated for 0-60 min, and whole cell lysates were subject to

immunoblot assay using an anti-HIF-1α monoclonal antibody. The proportion of HIF-1α

remaining at each timepoint relative to time 0 is indicated. B, induction of HIF-1α 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 µM CoCl2 (lanes 3-4) or 1% O2 (lanes 5-6) or neither

(lanes 1-2) in the presence (lanes 2, 4, 6) or absence (lanes 1, 3, 5) of 100 ng/ml IGF-1 for 4 h or

24 h prior to analysis of HIF-1α protein or VEGF mRNA expression, respectively.

FIG. 3. Analysis of HIF-1α ubiquitination and interaction with VHL. A, in vitro

ubiquitination assay. Lysates prepared from cells exposed to vehicle (-) or IGF-1 (+) were

incubated with in vitro-translated and 35S-labelled HIF-1α in the presence of ubiquitin and ATP

for 0, 30, or 150 min. Polyubiquitinated forms of HIF-1α (Ubi-HIF-1α) were identified by their

reduced mobility after PAGE. B, VHL interaction assay. A GST-HIF-1α fusion protein was

incubated with in vitro-translated and 35S-labelled VHL in the presence of PBS (lane 1) or lysates

prepared from cells that were untreated (lane 2) or exposed to 100 ng/ml IGF-1 (lane 3) or 100

µM CoCl2 (lane 4). Glutathione-Sepharose beads were used to capture GST-HIF-1α 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).

FIG. 4. Effect of kinase inhibitors on the induction of HIF-1α and VEGF. A, serum-

starved HCT116 cells were exposed to vehicle (lane 1) or 100 ng/ml IGF-1 in the presence of no

kinase inhibitor (lane 2) or 1-h pretreatment with 50 µM PD98059 (lane 3), 200 nM wortmannin

(lane 4), or 100 nM rapamycin (lane 5). Cells were harvested after 6 h for analysis of HIF-1α

protein and mRNA or after 24 h for analysis of VEGF mRNA. B, HCT116 cells were exposed to

vehicle (lane 1) or 100 ng/ml IGF-1 in the presence of 0-50 µM PD98059 (lanes 2-5) for 6 h and


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HIF-1α protein expression was determined by immunoblot assay. C, cells were exposed to

vehicle (lane 1) or 100 ng/ml IGF-1 in the presence of 0-200 nM wortmannin (lanes 2-6). D,

cells were exposed to IGF-1 after pretreatment with the indicatred concentrations of PD98059

and wortmannin. E, cells were exposed to 100 µM CoCl2 (lanes 1-3) or 100 ng/ml IGF-1 (lanes

4-6) in the presence of no kinase inhibitor (lanes 1 and 4), 50 µM PD98059 (lanes 2 and 5) or

200 nM wortmannin (lanes 3 and 6).

FIG. 5. MAP kinase and PI-3-kinase pathway signaling in IGF-1-treated cells.

HCT116 cells were pretreated for 1 h with 50 µM 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 (panels at left) or 6 h (panels at right) of IGF-1 stimulation and subject to

immunoblot assays using antibodies specific for phosphorylated (Thr202/Tyr204) or total

p42/p44 MAP kinase and phosphorylated (Ser473) or total AKT.

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 (panels at

left) or 6 h (panels at right) of IGF-1 stimulation and subject to immunoblot assays using

antibodies specific for phosphorylated (Ser65) or total 4E-BP, phosphorylated (Thr421/Ser424)

or total p70S6K, and phosphorylated (Ser209) or total eIF-4E.

FIG. 7. Effect of constitutively-active MEK on HIF-1α and VEGF expression. A,

HCT116 cells were transiently transfected with an empty vector (EV) or an expression vector

encoding hemagglutinin (HA)-tagged MEK2DD, a constitutively-active form of MEK-2. 24 h

after transfection the cells were serum-starved for 24 h and analyzed for the expression of HIF-


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1α, HA-MEK2DD, and phospho-p42/p44 proteins and for the expression of VEGF mRNA. B,

cells were transfected with EV (lane 1) or MEK2DD expression vector (lanes 2-6) and exposed

to 0-50 µM PD98059 for 24 h. Aliquots of cells lysates were subjected to immunoblot assay

using antibodies specific for HIF-1α (top panel), phosphorylated p42ERK/p44 ERK (middle panel),

and total p42ERK/p44 ERK (bottom panel).

FIG. 8. Molecular mechanisms of HIF-1-mediated VEGF expression in IGF-1-

treated HCT116 cells. PD, PD98059; PI3K, PI-3-kinase; RAP, rapamycin; WM, wortmannin.

Arrow and blocked arrow indicate stimulation and inhibition, respectively.


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1The abbreviations used are: IGF, insulin-like growth factor; HIF-1, hypoxia-inducible

factor 1; VEGF, vascular endothelial growth factor; MAP, mitogen-activated protein; PI,

phosphatidylinositol; 4E-BP1, eIF-4E binding protein 1; eIF-4E, eukaryotic initiation factor 4E;

IGF-1R, IGF-1 receptor; ERK, extracellular signal-regulated kinase; MEK-1, MAP kinase/ERK

kinase 1; HER2neu, human epidermal growth factor receptor 2; VHL, von Hippel-Lindau tumor

suppressor; FRAP, FKBP/rapamycin-associated protein; mTOR, mammalian target of

rapamycin; p70s6k, p70 ribosomal protein S6 kinase; CHX, cycloheximide; DMSO, dimethyl

sulfoxide; FBS, fetal bovine serum; RIPA, radioimmunoprecipitation assay; HA, hemagglutinin;

DTT, dithiothreitol; ATP, adenosine triphosphate; SDS-PAGE, sodium dodecyl sulfate-

polyacrylamide gel electrophoresis; GST, glutathione-S-transferase; CMV, cytomegalovirus;

MNK, MAP kinase signal integrating kinase.


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IGF -1 (ng/ml) 0 1 10 100 1000 0CoCl2 (µM) 0 0 0 0 0 100

HIF-1α protein

HIF-1α mRNA

EtBr

28S

18S

Fukuda et al.Fig. 1A

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IGF- 1 - + + + + + + +

Time (h) 0 2 4 6 8 12 18 24

HIF-1α

HIF-1β

Fukuda et al.Fig. 1B


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IGF-1 (ng/ml) 0 10 100 0

1% O2 - - - +

VEGF mRNA

28S

18S

Fukuda et al. Fig. 1C

EtBr


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IGF-1 - + + + + - 1% O2 - - - - - + H-1356 (µM) 0 0 1 10 100 100

HIF-1α protein

VEGF mRNA

28S

18S

Fukuda et al.Fig. 1D

EtBr


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CHX 0 15 30 60 (min)

CoCl2

IGF-1


1.0 0.85 0.75 0.69

1.0 0.69 0.38 0.24


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IGF-1 - + - + - + CoCl2 - - + + - - 1% O2 - - - - + +

HIF-1α protein

VEGF mRNA

28S

18S


EtBr


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Time (min) 0 30 150

IGF-1 - + - + - +

HIF-1α

Ubi-HIF-1α


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PBS + - - - - Control - + - - - IGF-1 - - + - - CoCl2 - - - + -VHL input - - - - +

VHL


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HIF-1α protein

VEGF mRNA

28S

18S

IGF-1 - + + + + PD98059 - - + - - Wortmannin - - - + - Rapamycin - - - - +


HIF-1α mRNA

EtBr


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PD98059 (µM ) - 0 10 25 50

IGF-1 - + + + +

Fukuda et al.Fig. 4

Wortmannin (nM) 0 0 25 50 100 200

IGF-1 - + + + + +

HIF-1α protein

HIF-1α proteinB

C


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CoCl2 + + + - - - IGF-1 - - - + + + PD98059 - + - - + - Wortmannin - - + - - +

HIF-1α protein

HIF-1α protein

D

E

Fukuda et al.Fig. 4

PD98059 (µM) 10 10 10 0 5 10

Wortmannin (nM) 0 25 50 25 25 25


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Fukuda et al.Fig. 5

IGF-1 - + + + + PD98059 - - + - -Wortmannin - - - + - Rapamycin - - - - +

IGF-1 - + + + + PD98059 - - + - -Wortmannin - - - + - Rapamycin - - - - +

Phospho-p42/p44

Total p42/p44

Phospho-p42/p44

Total p42/p44

Phospho-AKT

Total AKT

Phospho-AKT

Total AKT

15 min 6 h IGF treatment:


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Fukuda et al. Fig. 6

Phospho-4E-BP1

Total 4E-BP1

Phospho-p70S6K

Total p70S6K

Phospho-4E-BP1

Total 4E-BP1

Phospho-p70S6K

Total p70S6K

Phospho-elF-4E

Total elF-4E

Phospho-elF-4E

Total elF-4E

15 min


6 h


IGF-1 treatment:


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EV + -HA-MEK2DD - +

HA-MEK2DD

Phospho-p42/p44

VEGF mRNA

28S

18S

HIF-1α

EtBr


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HIF-1α protein

Phospho-p42/p44

Total p42/p44

EV + - - - - - MEK2DD - + + + + +PD98059 (µM ) 0 0 5 10 25 50


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IGF-1

H-1356

WM PI3K MEK PD

AKT

RAP

ERK

FRAP

p70S6k 4E-BP1 eIF-4E

HIF-1α Protein Synthesis

HIF-1 Transcriptional Activity

MNK

VEGF Expression

IGF-1R

Fukuda et al.Fig. 8


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Ryo Fukuda, Kiichi Hirota, Fan Fan, Young Do Jung, Lee M. Ellis and Gregg L. Semenzaand PI-3-kinase signaling in colon cancer cells

IGF-1 induces HIF-1-mediated VEGF expression that is dependent on MAP kinase

published online July 30, 2002J. Biol. Chem.

10.1074/jbc.M203781200Access the most updated version of this article at doi:

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