Ryo Fukuda, Kiichi Hirota^§, Fan Fan^¶, Young Do Jung^¶, Lee M. Ellis^¶, and Gregg L. Semenza

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

Received for publication, April 18, 2002, and in revised form, July 3, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 article we demonstrate that exposure of HCT116 human colon carcinoma cells to IGF-1 induces the expression of HIF-1, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. In contrast to hypoxia, which induces HIF-1 expression by inhibiting its ubiquitination and degradation, IGF-1 did not inhibit these processes, indicating an effect on HIF-1 protein synthesis. IGF-1 stimulation of HIF-1 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-1 protein synthesis. Forced expression of a constitutively active form of the MAP kinase kinase, MEK2, was sufficient to induce HIF-1 protein and VEGF mRNA expression. Involvement of the MAP kinase pathway represents a novel mechanism for the induction of HIF-1 protein expression in human cancer cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (PI3-kinase) pathways plays a critical role in transformation and tumorigenesis (1). IGF2 gene expression is up-regulated 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 the effects of 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 PI3-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; 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 HER2^neu receptor tyrosine kinase in breast cancer cells was shown to be mediated by HIF-1 via the 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 constitutively expressed HIF-1 subunit and an inducibly expressed HIF-1 subunit (12). Under nonhypoxic conditions, HIF-1 is subject to O₂-dependent prolyl hydroxylation (13, 14), which 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-1 for proteasomal degradation (15). Under hypoxic conditions, O₂ becomes limiting for prolyl hydroxylase activity (16) and ubiquitination of HIF-1 is inhibited (17). As a result, HIF-1 accumulates, dimerizes with HIF-1, and activates transcription of target genes.

Signaling via receptor tyrosine kinases can induce HIF-1 expression by an independent mechanism. HER2^neu activation in breast cancer cells stimulates increased rates of HIF-1 protein synthesis via PI3-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 (p70^S6K) (18-20). Phosphorylation of 4E-BP1 disrupts its inhibitory interaction with eukaryotic initiation factor 4E (eIF-4E), whereas activated p70^S6K phosphorylates the 40 S ribosomal protein S6. The effect of HER^neu 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 molecular basis for stimulation of HIF-1 protein synthesis in response to HER2^neu activation.

Treatment of cultured cells with IGF-1 or IGF-2 also induces HIF-1 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-1 is overexpressed in human colon cancers (23), and that forced overexpression of HIF-1 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tissue Culture and Reagents– HCT116 cells were cultured in McCoy’s 5A medium with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Unless otherwise stated, cells were maintained at 37 °C 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, wortmannin, and rapamycin were dissolved in Me₂SO 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, with balance N₂, sealed, and incubated at 37 °C.

IGF-1 and Inhibitor Treatments– HCT116 cells were plated at a density of 2.4 × 10⁶/10-cm dish or 8.6 × 10⁵/6-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 medium 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 radioimmune precipitation buffer, fractionated by SDS-PAGE, transferred to a nitrocellulose filter, and subjected to immunoblot assays. For HIF-1 and HIF-1, 150-µg aliquots of protein were analyzed using a monoclonal antibody against HIF-1 (H167) or HIF-1 (H1234) (Novus Biologicals) at 1:1000 dilution as described previously (23, 25). 50-µg aliquots were analyzed using antibodies (1:1000 dilution) specific for phosphorylated (Thr-202/Tyr-204) or total p44/p42 MAP kinase, phosphorylated (Ser-473) or total AKT, phosphorylated (Thr-421/Ser-424) or total p70^S6K, phosphorylated (Ser-209) or total elF-4E, and phosphorylated (Ser-65) or total 4E-BP1 antibodies (purchased from Cell Signaling Technology and Santa Cruz Biotechnology). Anti-hemagglutinin (HA) antibody was from Santa Cruz. Horseradish peroxidase-conjugated mouse monoclonal antibodies for mouse and rabbit IgG (1:2500 dilution) and ECL reagents were from Amersham Biosciences.

RNA Blot Hybridization– Total RNA was extracted from HCT116 cells using TRIzol reagent (Invitrogen) 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, 2.2 M formaldehyde gels, transferred to Hybond N⁺ membranes (Amersham Biosciences), and hybridized with a ³²P-labeled human HIF-1 or VEGF cDNA probe as described previously (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 dithiothreitol), and lysed in a Dounce homogenizer. The cell extract was centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatant was stored in aliquots at 70 °C. Ubiquitination assays were performed as described previously (26) at 30 °C in a total volume of 40 µl containing 27 µl (50 µg) of cell extract, 4 µl of 10 × 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-1 that was in vitro translated (TNT Quick Coupled Transcription/Translation System, Promega) in the presence of [³⁵S]methionine. HA-HIF-1 was recovered using anti-HA-agarose beads, which were then mixed with SDS sample buffer and boiled for 5 min; the eluates were then analyzed by SDS-PAGE and autoradiography.

In Vitro HIF-1-VHL Interaction Assay– [³⁵S]Methionine-labeled VHL protein was synthesized in vitro and glutathione S-transferase (GST)-HIF-1-(429-608) fusion protein was expressed in E. coli as described previously (27). HCT116 cells were serum-starved and treated with IGF-1 or CoCl₂ for 4 h prior to lysate preparation. GST-HIF-1-(429-608) was preincubated with 10 µl of the HCT116 lysate for 30 min at 30 °C. Five-µl aliquots of the GST-HIF-1-(429-608) preincubation 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) Nonidet P-40). After 90 min at 4 °C, 20 µl of glutathione-Sepharose-4B (Amersham Biosciences) was added. After 30 min of mixing on a rotator, beads were washed three times with NETN buffer. Proteins were eluted in 2× SDS sample buffer, fractionated by SDS-PAGE, and detected by autoradiography.

Transient Transfection– 8.6 × 10⁵ HCT116 cells were plated/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 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 expression (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 thereafter (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 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-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).

View larger version (28K): [in this window] [in a new window] Fig. 1. Effect of IGF-1 treatment on HIF-1 and VEGF expression in HCT116 cells. A, analysis of HIF-1 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 IGF-1 (lanes 2-5), or 100 µM CoCl2 (lane 6) for 6 h. Then either whole cell lysates were subject to immunoblot assay for expression of 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; migration 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. Cells were pretreated 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-1 protein expression by immunoblot assay (top) or at 24 h for analysis of VEGF mRNA expression by blot hybridization (middle) following RNA transfer from an EtBr-stained gel (bottom).

To determine whether IGF-1 treatment affected HIF-1 protein half-life, HCT116 cells were treated with CoCl₂ or IGF-1 for 4 h to induce HIF-1 expression, and then CHX was added to block ongoing protein synthesis. In the presence of CHX, the half-life of HIF-1 was >60 min in CoCl₂-treated cells but <30 min in IGF-1-treated cells (Fig. 2A). These results indicate that HIF-1 expression in IGF-1-treated cells is dependent upon ongoing protein synthesis. If IGF-1 induces HIF-1 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-1 protein (Fig. 2B, top) and VEGF mRNA (Fig. 2B, middle) expression than exposure of cells to IGF-1, CoCl₂, or hypoxia alone.

View larger version (55K): [in this window] [in a new window] 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 µM CoCl2 (top panel) or 100 ng/ml IGF-1 (bottom panel) for 4 h, cycloheximide (CHX) was added to a final concentration of 100 µM, the cells were incubated for 0-60 min, and whole cell lysates were subject to immunoblot assay using an anti-HIF-1 monoclonal antibody. 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 µM 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.

Ubiquitination of HIF-1 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-1 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-1. Prior to incubation (time 0), no ubiquitinated HIF-1 was detected, whereas the ratio of ubiquitinated to non-ubiquitinated forms of HIF-1 increased over time with no difference observed between IGF-1-treated and untreated lysates (Fig. 3A). Incubation of a GST-HIF-1 fusion protein with control lysate from untreated cells resulted in prolyl hydroxylation of HIF-1, 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-1 with VHL (Fig. 3B, lane 4). In contrast, lysates from IGF-1-treated cells (Fig. 3B, lane 3) promoted the interaction of GST-HIF-1 with VHL as efficiently as control lysates, providing further evidence that IGF-1 treatment does not induce HIF-1 expression by inhibiting VHL-mediated ubiquitination.

View larger version (35K): [in this window] [in a new window] Fig. 3. Analysis of HIF-1 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-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 fusion 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 exposed to 100 ng/ml IGF-1 (lane 3) or 100 µM 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).

To determine the signal transduction pathways mediating the effects of IGF-1 on HIF-1 protein and VEGF mRNA expression, HCT116 cells were pretreated with PD98059, wortmannin, or rapamycin, which are selective pharmacologic inhibitors of MEK, PI3-kinase, and FRAP/mTOR kinase activity, respectively. All three agents inhibited the induction of HIF-1 protein expression in IGF-1-treated cells (Fig. 4A). At the concentrations used, the rank inhibitory effect of these agents was PD98059 > wortmannin > rapamycin. None of the inhibitors had any effect on the expression of HIF-1 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-1 protein expression. The induction of HIF-1 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-1 expression (Fig. 4D). In contrast to their effects on the expression of HIF-1 induced by IGF-1 treatment, PD98059 or wortmannin had little inhibitory effect on the expression of HIF-1 in CoCl₂-treated HCT116 cells (Fig. 4E), providing further evidence that IGF-1 and CoCl₂ act by distinct molecular mechanisms.

View larger version (26K): [in this window] [in a new window] Fig. 4. Effect of kinase inhibitors on the induction of HIF-1 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 a 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-1 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 HIF-1 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 indicated 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).

To determine whether the MAP kinase and PI3-kinase pathways were activated serially or independently in IGF-1-treated cells, the phosphorylation of p42^ERK/p44^ERK and AKT were analyzed. The increased phosphorylation of p42^ERK/p44^ERK that was induced by IGF-1 treatment was blocked by PD98059 but not by wortmannin or rapamycin (Fig. 5). The increased 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 PI3-kinase activities are required for induction of HIF-1 protein expression, IGF-1 induces the activity of each pathway independently.

View larger version (23K): [in this window] [in a new window] Fig. 5. MAP kinase and PI3-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 (left) or 6 h (right) of IGF-1 stimulation and subject to immunoblot assays using antibodies specific for phosphorylated (Thr-202/Tyr-204) or total p42/p44 MAP kinase and phosphorylated (Ser-473) or total AKT.

The signal transduction pathway involving PI3-kinase, AKT, and FRAP has been shown to regulate protein translation via phosphorylation of 4E-BP1 and p70^s6k (18-20). In HCT116 cells, the phosphorylation of both 4E-BP1 and p70^s6k, which 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 p70^s6k, an effect consistent with its inhibition of IGF-1-induced HIF-1 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).

View larger version (30K): [in this window] [in a new window] 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 antibodies specific for phosphorylated (Ser-65) or total 4E-BP, phosphorylated (Thr-421/Ser-424) or total p70S6K, and phosphorylated (Ser-209) or total eIF-4E.

Involvement of MEK and ERK in the induction of HIF-1 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-1 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 p42^ERK/p44^ERK MAP kinases and increased expression of HIF-1 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 p42^ERK/p44^ERK and the induction of HIF-1 protein expression in MEK-2DD-transfected cells were 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-1 protein and VEGF mRNA expression in colon cancer cells.

View larger version (31K): [in this window] [in a new window] Fig. 7. Effect of constitutively active MEK on HIF-1 and VEGF expression. A, HCT116 cells were transiently transfected with an empty vector (EV) or an expression vector encoding 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-1, HA-MEK2DD, and phospho-p42/p44 proteins and for the expression of VEGF mRNA. B, cells were transfected with empty vector (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-1 (top), phosphorylated p42ERK/p44 ERK (middle), and total p42ERK/p44 ERK (bottom).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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-1 protein degradation, heregulin stimulation of breast cancer cells increases HIF-1 synthesis, an effect that is dependent on HER2^neu, PI3-kinase, AKT, and FRAP/mTOR (but not MEK-1) activity and the 5′-untranslated region of HIF-1 mRNA (11).

The studies reported above demonstrate that IGF-1 stimulation of human colon cancer cells also increases HIF-1 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 have confirmed that IGF-1 treatment had no effect on HIF-1 protein stability in IGF-1-treated HCT116 cells and also demonstrated 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 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 PI3-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-1 transactivation domain function (35-37), the data reported here represent the first demonstration that the MAP kinase pathway can also stimulate HIF-1 expression.

View larger version (15K): [in this window] [in a new window] Fig. 8. Molecular mechanisms of HIF-1-mediated VEGF expression 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.

Dependence on MEK activity for phosphorylation of 4E-BP1 and p70^s6K has been demonstrated in other cellular contexts (38-40). In the case of interleukin 6-stimulated myeloma cells, both MEK and PI3-kinase are required for activation of p70^s6K, with MEK inhibitors preventing the phosphorylation of Thr-421/Ser-424 in the autoinhibitory domain, which is required for subsequent phosphorylation at Thr-389 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-1 protein and VEGF mRNA expression and the inhibition of 4E-BP1 and p70^s6K 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 p70^s6K) protein synthesis in HCT116 cells (Fig. 8).

In experimental tumors, increased eIF-4E activity stimulates tumor growth, invasion, and metastasis (41). Although it increases 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-1 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 (HER2^neu, IGF-1R) in different human cancers (breast, colon) have in common the stimulation of HIF-1 protein synthesis and increased expression of the downstream target VEGF. The effects of receptor tyrosine kinase activation on HIF-1 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. HIF-1 overexpression is associated with tumor angiogenesis and increased mortality in cancers of the breast, central nervous system, oropharynx, ovary, and uterine cervix (34). HIF-1 overexpression is observed in colon cancer (23), and the results presented in this study suggest that HIF-1 overexpression may contribute significantly to angiogenesis and other important aspects of colon cancer progression.

	ACKNOWLEDGEMENT

We thank Dr. Sylvain Meloche for generously providing pCMV-MEK2DD.

	FOOTNOTES

^* This work was supported by Grants R01-DK39869 (to G. L. S.) and 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 of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Current address: Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.

To whom correspondence should be addressed: Johns Hopkins University School of Medicine, CMSC-1004, 600 North Wolfe St., Baltimore, MD 21287-3914; Fax: 410-955-0484; E-mail: gsemenza@jhmi.edu.

Published, JBC Papers in Press, July 30, 2002, DOI 10.1074/jbc.M203781200

	ABBREVIATIONS

The abbreviations used are: IGF, insulin-like growth factor; IGF-1R, IGF-1 receptor; HIF-1, hypoxia-inducible factor 1; VEGF, vascular endothelial growth factor; MAP, mitogen-activated protein; PI3-kinase, phosphatidylinositol 3-kinase; 4E-BP1, eIF-4E-binding protein 1; eIF-4E, eukaryotic initiation factor 4E; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; HER2^neu, human epidermal growth factor receptor 2; VHL, von Hippel-Lindau tumor suppressor; FRAP, FKBP/rapamycin-associated protein; mTOR, mammalian target of rapamycin; p70^s6k, p70 ribosomal protein S6 kinase; CHX, cycloheximide; FBS, fetal bovine serum; HA, hemagglutinin; GST, glutathione S-transferase; CMV, cytomegalovirus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antiangiogenic Therapy Targeting the Tyrosine Kinase Receptor for Vascular Endothelial Growth Factor Receptor Inhibits the Growth of Colon Cancer Liver Metastasis and Induces Tumor and Endothelial Cell Apoptosis¹

Raymond M. Shaheen, Darren W. Davis, Wenbiao Liu, Brian K. Zebrowski, Michael R. Wilson, Corazon D. Bucana, David J. McConkey, Gerald McMahon and Lee M. Ellis²

Departments of Surgical Oncology [R. M. S., B. K. Z., L. M. E.] and Cancer Biology [D. W. D., W. L., M. R. W., C. D. B., D. J. M., L. M. E.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and SUGEN, Inc., South San Francisco, California 94080 [G. M.]

	ABSTRACT

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Increased vascular endothelial growth factor (VEGF) expression is associated with colon cancer metastases. We hypothesized that inhibition of VEGF receptor activity could inhibit colon cancer liver metastases. BALB/c mice underwent splenic injection with CT-26 colon cancer cells to generate metastases. Mice received daily i.p. injections of vehicle, tyrosine kinase inhibitor for Flk-1/KDR (SU5416) or tyrosine kinase inhibitor for VEGF, basic fibroblast growth factor, and platelet-derived growth factor receptors (SU6668). SU5416 and SU6668 respectively inhibited metastases (48.1% and 55.3%), microvessel formation (42.0% and 36.2%), and cell proliferation (24.4% and 27.3%) and increased tumor cell (by 2.6- and 4.3-fold) and endothelial cell (by 18.6- and 81.4-fold) apoptosis (P < 0.001). VEGF receptor inhibitors increased endothelial cell apoptosis, suggesting that VEGF may serve as an endothelial survival factor.

	Introduction

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Angiogenesis is a dynamic and complex process that involves new blood vessel formations from established vasculature (1) . This neovascularization is essential for both primary and metastatic tumor growth; therefore, antiangiogenic therapy may provide a novel addition to current antineoplastic approaches (1) . The development of effective antiangiogenic therapy must first involve identifying biologically relevant molecular targets that are associated with tumor aggressiveness and metastasis formation. Three such angiogenic factors, VEGF³ (2) , bFGF (3) , and PDGF (4) , function by binding to specific high-affinity TK receptors (5) . Antiangiogenic strategies directed toward inhibiting VEGF activity include neutralizing anti-VEGF antibodies, anti-VEGF receptor antibodies, soluble VEGF receptors, antisense VEGF techniques, and VEGF receptor TK inhibitors (6) . VEGF receptor TK inhibitors are small, synthetic, selective molecules that have favorable toxicity profiles, do not induce an immune response, and are not susceptible to enzymatic inactivation (7) . Two novel compounds in this class are SU5416 (8) , a selective inhibitor of only the VEGF receptor, and SU6668, an inhibitor of the receptors for VEGF, bFGF, and PDGF. The purpose of this study was to investigate the effect of these two inhibitors on the angiogenesis and growth of colon cancer liver metastases. A secondary aim was to investigate the effect of these agents on tumor cell proliferation and the induction of tumor and endothelial cell apoptosis.

	Materials and Methods

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Cell Culture.
CT-26 murine colon carcinoma cells were cultured and maintained in MEM supplemented with 5% fetal bovine serum, 2 units/ml penicillin-streptomycin, vitamins, 1 mM sodium pyruvate, 2 mM L-glutamine, and nonessential amino acids at 37°C in 5% CO₂ and 95% air (9) . Cells were harvested from subconfluent cultures with trypsin-EDTA for 1 min, washed in suspended in media, centrifuged at 300 x g for 8 min at room temperature, and then resuspended to a final concentration of 1 x 10⁴ viable cells/50 µl HBSS. Trypan blue exclusion was performed to ensure cell viability. All cell culture reagents were obtained from Life Technologies, Inc. (Grand Island, NY).

Animals and Tumor Cell Inoculation.
Eight-week old male BALB/c mice were obtained from the National Cancer Institute’s Animal Production Area (Fredrick, MD), acclimated for 1 week and caged in groups of five, and fed a diet of animal chow and water ad libitum. Mice were anesthetized in a methoxyflurane (Pitman-Moor, Mundelein, IL) chamber, followed by left upper quadrant laparotomy and splenic exteriorization. Using a 30-gauge needle and a 1-ml syringe, 50 µl of the tumor cell suspension were injected beneath the splenic capsule. The skin and peritoneum were closed in a single layer by using metallic clips (Autoclip; Clay Adams, Parsippany, NJ), which were removed on POD 7. Mice were randomized to one of three groups (15 mice/group), with no statistically significant difference between the mean weights of the three groups. All animal studies were conducted according to a protocol approved by the Animal Care and Use Committee of The University of Texas M. D. Anderson Cancer Center.

Antiangiogenic Therapy.
Beginning on POD 4, therapy was initiated with daily 200-µl i.p. injections of either control vehicle [30% PEG-300 (w/v) in 0.1 M sodium phosphate buffer (pH 8.2)], SU5416 [12 mg/kg in 99% PEG-300 (w/v) with 1% Tween 80 (polyethylene sorbitan monooleate detergent)], or SU6668 (60 mg/kg in control vehicle) using a 30-gauge needle attached to a 1-ml syringe. Animals were sacrificed on POD 22 when the control mice became moribund. Mice were weighed weekly to confirm no drug treatment-associated weight loss. SU5416 and SU6668 were provided by SUGEN, Inc. (South San Francisco, CA); PEG-300 and Tween 80 were obtained from Sigma Chemical Co. (St. Louis, MO), and sodium monophosphate and diphosphate salts were obtained from EM Science (Gibbstown, NJ).

Autopsy and Tissue Preparation.
Mice were sacrificed by cervical dislocation after adequate sedation with methoxyflurane was confirmed by the toe pinch technique. Livers were excised and weighed, and the number of total surface hepatic metastases was counted using a dissection microscope. For IHC staining, a section of the tumor tissue was fixed in Bouin’s solution for 24 h and then fixed in formalin and embedded in paraffin. Another section was embedded in OCT (Miles Inc, Elkhart, IN), frozen in liquid nitrogen, and stored at -70°C.

IHC of Paraffin-embedded and Frozen Tissues.
Paraffin-embedded liver tissues were sliced in 4–6-µm sections, mounted on positively charged Superfrost slides (Fisher Scientific Co., Houston, TX), and allowed to dry overnight at room temperature. Sections were deparaffinized in xylene followed by 100%, 95%, and 80% ethanol and rehydrated in PBS (pH 7.5). These sections were used for H&E staining and detection of PCNA protein expression. Sections analyzed for PCNA were microwaved for 5 min to increase antigen retrieval. Sections analyzed for tumor cell apoptosis by TUNEL were predigested with pepsin (Biomeda, Foster City, CA) for 15 min at 37°C and washed three times for 3 min each time with PBS (Irvine Scientific, Santa Ana, CA).

Liver tissues frozen in OCT were sectioned (8–10 µm), mounted on positively charged slides, and air-dried for 30 min. Frozen tissues were fixed in cold acetone (5 min), 1:1 acetone/chloroform (5 min), and acetone (5 min) and then washed with PBS three times for 3 min each. After these pretreatment procedures, all samples were incubated with 3% H₂O₂ in methanol for 12 min at room temperature to block endogenous peroxidase. Sections were washed three times for 3 min each with PBS (pH 7.5) and then incubated for 20 min at room temperature in a protein-blocking solution consisting of PBS supplemented with 1% normal goat serum and 5% normal horse serum. The primary antibodies directed against CD31 and PCNA were diluted 1:200 and 1:50, respectively, in protein-blocking solution, applied to the sections, and incubated overnight at 4°C. Sections were then rinsed three times for 3 min each in PBS and incubated for 10 min in protein-blocking solution before the addition of peroxidase-conjugated secondary antibody. The secondary antibodies used for CD31 and PCNA staining were diluted 1:200 and 1:100, respectively, in protein-blocking solution. After incubating with the secondary antibody for 1 h at room temperature, the samples were washed and incubated with stable diaminobenzidine (Research Genetics, Huntsville, AL) substrate. Staining was monitored under a bright-field microscope, and the reaction was stopped by washing with distilled water. Sections were counterstained with Gill’s No. 3 hematoxylin (Sigma Chemical Co.) and mounted with Universal Mount (Research Genetics) for 15 s. Control specimens were treated with a similar procedure, except that the primary antibody was omitted.

Immunofluorescence Double Staining and Quantification of Apoptotic Endothelial Cells in Situ.
Frozen tissue sections (8 µm) were fixed with cold acetone for 5 min, acetone plus chloroform (1:1) for 5 min, and acetone for 5 min. Samples were washed three times with PBS and incubated with protein-blocking solution containing 5% normal horse serum and 1% normal goat serum in PBS for 20 min at room temperature. Blocking solution was drained, and the samples were incubated with a 1:400 dilution of rat monoclonal antimouse CD31 antibody (human cross-reactive antibody; PharMingen, San Diego, CA) for 24 h at 4°C. Samples were rinsed with PBS three times for 3 min each and incubated with protein-blocking solution for 10 min at room temperature. Avoiding exposure to light, the blocking solution was drained, and the samples were incubated with a 1:200 dilution of Texas Red-conjugated goat antirat secondary antibody for 1 h at room temperature. Samples were washed two times with PBS containing 0.1% Brij and washed with PBS for 5 min. TUNEL was performed using a commercial kit (Promega, Madison, WI) with the following modifications. Samples were fixed with 4% paraformaldehyde (methanol free) for 10 min at room temperature. The samples were washed with PBS two times for 5 min and then incubated with 0.2% Triton X-100 for 15 min at room temperature. The samples were washed with PBS two times for 5 min and incubated with equilibration buffer (from the kit) for 10 min at room temperature. The equilibration buffer was drained, and reaction buffer containing equilibration buffer, nucleotide mix, and terminal deoxynucleotidyl transferase enzyme was added to the tissue sections and incubated in a humid atmosphere at 37°C for 1 h, avoiding exposure to light. The reaction was terminated by immersing the samples in 2x SSC for 15 min. Samples were washed three times for 5 min to remove unincorporated fluorescein-dUTP. For quantification of endothelial cells, the samples were incubated with 300 mg/ml Hoechst stain for 10 min at room temperature. The samples were then washed with PBS two times for 5 min. Prolong solution (Molecular Probes, Eugene, OR) was used to mount coverslips. Immunofluorescence microscopy was performed using a x40 objective (Zeiss Plan-Neofluar) on an epifluorescence microscope equipped with narrow bandpass excitation filters mounted in a filter wheel (Ludl Electronic Products, Hawthorne, NY) to individually select for green, red, and blue fluorescence. Images were captured using a cooled charge coupled device camera (Photometrics, Tucson, AZ) and SmartCapture software (Digital Scientific, Cambridge, United Kingdom) on a Macintosh computer. Images were further processed using Adobe Photoshop software (Adobe Systems, Mountain View, CA). Endothelial cells were identified by red fluorescence, and DNA fragmentation was detected by localized green and yellow fluorescence within the nucleus (visualized by Hoechst stain) of apoptotic cells. Quantification of apoptotic endothelial cells was expressed as the average of TUNEL positive endothelial cells in five random fields at x40 magnification.

Quantification of Tumor Vessel Counts, PCNA, and TUNEL.
To quantify tumor vessel counts, frozen sections were fixed and stained with primary antibodies to CD31. Five random 0.159-mm² fields at x100 magnification were captured for each tumor by using a Sony three-chip camera (Sony Corporation of America, Montvale, NJ) mounted on a Zeiss universal microscope (Carl Zeiss, Thornwood, NY) and Optimas Image Analysis software (Bioscan, Edmond, WA) installed on a Compaq computer with a Pentium chip, a frame grabber, an optical disc storage system, and a Sony Mavigraph UP-D7000 Digital color printer (Tokyo, Japan). To quantify PCNA expression, the numbers of positive cells were counted in five random 0.159-mm² fields at x100 magnification. To quantify TUNEL positivity in endothelial cells (yellow-stained cells) and tumor cells (green-stained cells) in frozen tissue sections under the Olympus microscope, the numbers of apoptotic events were counted in five random 0.159-mm² fields at x100 per field. More than 95% of cells in these tumor specimens are tumor epithelial cells. Therefore, quantitation of tumor cell apoptosis was made under the assumption that the majority of green-stained cells were tumor cells. This was confirmed by observing the relative amount of apoptotic events in non-CD31 TUNEL-positive cells versus CD31 TUNEL-positive cells when double staining was done in the subsequent study.

Antibodies.
Antibodies for IHC were obtained from the following sources: (a) rat antimouse CD31 antibody, PharMingen; (b) mouse anti-PCNA clone PC 10, DAKO A/S; (c) peroxidase-conjugated goat antirat IgG (H+L) and Texas Red-conjugated goat antirat IgG, Jackson Research Laboratories (West Grove, PA); and (d) peroxidase-conjugated rat antimouse IgG2a, Serotec, Inc. (Raleigh, NC).

Statistical Analysis.
Liver weights; quantification of CD31, PCNA, and TUNEL; and quantitation of apoptotic endothelial cells (by sequential staining for CD31 and TUNEL) were compared by using unpaired Student’s t-tests (InStat for Macintosh; GraphPad Software, San Diego, CA).

	Results

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Tolerance of Therapy and Tumorigenicity.
No significant differences were found in body weight among the three groups at the end of the experiment. Until mice became moribund from tumor burden, no toxic reactions were noted. Autopsy confirmed that 100% of the control mice had surface colon cancer liver metastases.

Effect of SU5416 and SU6668 on Liver Metastases.
Harvested livers were weighed as a gross measure of tumor burden. Relative to control mice, liver weights were decreased in the SU5416 (31.9%; P = 0.002) and SU6668 (35.7%; P < 0.001) groups (Fig. 1A) . Fewer surface liver metastases were present in the SU5416 (48.1%; P < 0.001) and SU6668 (55.3%; P < 0.001) groups than in the control group (Fig. 1B) .

View larger version (28K): [in this window] [in a new window] [Download PPT slide] Fig. 1. Effect of SU5416 and SU6668 on the development of colon cancer liver metastases. Liver weight (a gross measure of tumor burden; A) and the number of surface liver metastases (B) for the three treatment groups are shown. Bars, SE. *, P < 0.001 versus control.

Effect of SU5416 and SU6668 on Tumor Vessel Counts.
Immunohistochemical staining for CD31 to detect vessels in hepatic metastases revealed a significant decrease in tumor vessel counts in the SU5416 (42.0%; P < 0.001) and SU6668 (36.2%; P < 0.001) groups compared with those in the control group (Fig. 2) . Additionally, no significant differences were observed between tumor vessel counts in the SU5416 group and in the SU6668 group.

View larger version (26K): [in this window] [in a new window] [Download PPT slide] Fig. 2. Effect of SU5416 and SU6668 on tumor vessel counts. Immunohistochemical staining for CD31 in tumor-bearing liver sections was used to quantify and compare tumor vessel counts among the three groups. Bars, SE. *, P < 0.001 versus control.

Effect of SU5416 and SU6668 on PCNA Expression and on Endothelial Cell and Tumor Cell Apoptosis.
Immunohistochemical staining for PCNA and immunofluorescent TUNEL staining, with and without concurrent staining for CD31, were performed in tumor-bearing liver sections to evaluate tumor cell proliferation, endothelial cell apoptosis, and tumor cell apoptosis, respectively. SU5416 and SU6668 treatment resulted in a significantly reduced level of tumor cell proliferation (24.4% and 27.3% less than the control group value, respectively; P < 0.001; Figs. 3 and 4 ) and a significantly greater level of apoptosis in endothelial cells (2.6- and 4.3-fold increases, respectively, over that of the control group, P < 0.001) and tumor cells (18.6- and 81.4-fold increases, respectively, over that of the control group, P < 0.001). No differences were found in PCNA expression between the SU5416 and SU6668 groups. However, SU6668 treatment produced 4.4-fold higher endothelial cell apoptosis (P < 0.001) and 1.7-fold higher tumor cell apoptosis (P < 0.048) than SU5416 treatment.

View larger version (20K): [in this window] [in a new window] [Download PPT slide] Fig. 3. Effect of SU5416 and SU6668 on PCNA expression and apoptosis of endothelial and tumor cells. Immunohistochemical staining for PCNA and immunofluorescent staining for TUNEL, with or without staining for CD31, were performed in tumor-bearing liver sections to quantify and compare tumor cell proliferation, endothelial cell apoptosis, and tumor cell apoptosis. Bars, SE. *, P < 0.001 versus control; , P < 0.048 versus SU5416.

View larger version (104K): [in this window] [in a new window] [Download PPT slide] Fig. 4. Immunohistological evaluation of colon cancer liver metastases. Immunohistochemical staining for H&E (row 1; x4), PCNA (row 2; x10), and CD31 (row 3; x10); immunofluorescent double staining for TUNEL and CD31 (row 4; x40); and immunofluorescent staining for TUNEL (row 5; x40) were performed in tumor-bearing liver sections from the three groups. Representative sections demonstrate a significant decrease in tumor cell proliferation (row 2), vascularity (row 3), and induction of apoptosis in endothelial cells (row 4) and tumor cells (row 5) in the SU5416 (column 2) and SU6668 (column 3) groups relative to those in the control group (column 1).

	Discussion

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Tumor growth and metastasis are angiogenesis-dependent processes (10) . Angiogenesis is typically stimulated in response to tumor-secreted angiogenic factors such as VEGF, which bind to high-affinity TK receptors and promote endothelial cell proliferation, invasion, and the formation of new capillaries (11) . We have previously shown that the expression of VEGF and its receptor is associated with tumor vascularity, metastasis, and proliferation of human colon cancer (2) . Based on these fundamental observations regarding the biology of colon cancer, we postulated that inhibition of VEGF action by inhibiting signaling through the VEGF receptor could represent an important antiangiogenic therapeutic modality for inhibiting the growth of colon cancer liver metastases (12 , 13) .

Several small molecule inhibitors that target these growth factor receptors are currently being evaluated in clinical trials (7) . Protein TK inhibitors are promising agents within this class that demonstrate selectivity with minimal toxicity to the host (7 , 8) . Our present results show that treatment of mice with the VEGF receptor TK inhibitors SU5416 and SU668 resulted in marked inhibition of the growth, vascularity, and proliferation of colon cancer liver metastases. We did not observe toxic effects at the doses administered, as evidenced by body weight and grooming habits, which remained similar to those of control mice during the treatment. Our results confirm a recent study that reported decreases in tumor vascularity, growth, and proliferation in multiple tumor types after SU5416 administration (8) . To better understand the mechanism involved in growth inhibition, we also evaluated tumor cell apoptosis. We found that antiangiogenic therapy by TK inhibitors limited tumor growth in association with an increase in tumor cell apoptosis and a decrease in tumor cell proliferation. This finding contrasts with that of a previous study using a Lewis lung carcinoma model in which the inhibition of tumor growth in the presence of angiogenesis suppression was mediated by an induction of apoptosis, without inhibition of tumor cell proliferation (14) . This difference among studies may reflect differences in the model, the agent used, or the duration of the observations.

To determine why antiangiogenic therapy, which specifically targets endothelium, produced an increase in tumor cell apoptosis, we evaluated tumors for endothelial cell apoptosis by combining an immunohistochemical stain for CD31 (vessels) and TUNEL (apoptosis) staining. With this approach, we found a significant induction of endothelial cell apoptosis in the SU5416- and SU6668-treated groups as compared to the control groups. We also observed a more marked increase in the extent of endothelial cell apoptosis relative to tumor cell apoptosis. Because, with rare exceptions, VEGF receptors are expressed exclusively on endothelial cells, it is unlikely that SU5416 directly induces tumor cell apoptosis. Therefore, it is possible that inhibiting the action of VEGF may lead to tumor endothelial apoptosis, which could then lead to a subsequent increase in tumor cell apoptosis. These findings suggest that VEGF may act as a direct survival factor for tumor endothelium and an indirect survival factor for colon carcinoma cells. These findings are supported by other recent reports that have purported VEGF to be crucial to the survival of tumor endothelium (15) . Additional investigations are necessary to confirm whether these causal and temporal relationships exist between VEGF receptor inhibition and endothelial and tumor cell apoptosis.

In addition to targeting the VEGF receptor alone, we also used SU6668 to target three distinct yet homologous, TK receptors that bind to VEGF, bFGF, and PDGF. Our in vivo findings showed significantly greater amounts of tumor and endothelial cell apoptosis in the SU6666-treated group relative to those of the SU5416 group, which suggests that bFGF and PDGF may also have a role as survival factors for tumor endothelium. Therefore, optimal antiangiogenic therapy may require inhibition of the action of several stimulatory angiogenic factors.

In conclusion, we have shown that antiangiogenic therapy targeting the TK receptor for the VEGF receptor inhibits the vascularity, proliferation, and growth of colon cancer liver metastasis and significantly increases endothelial and tumor cell apoptosis. These findings suggest an important role for VEGF as a survival factor for tumor endothelium. Despite the growth inhibition of tumors with no observable toxicity, liver metastases were not eradicated, at least during the brief time that we treated these tumors. Therefore, it remains to be seen whether this growth inhibition could lead to a survival advantage for the treatment groups. Survival studies are currently underway that seek to answer this question. At present, there is a lack of effective systemic therapies that significantly improve survival in patients with metastatic colon cancer. Therefore, our findings suggest that SU5416 and SU6668 are promising antiangiogenic agents that may have clinical utility in the management of colon cancer liver metastases.

	ACKNOWLEDGMENTS

We thank Dr. Michael Andreeff (Department of Molecular Hematology, The University of Texas M. D. Anderson Cancer Center) for the use of the epifluorescent microscope and Christine Wogan for editorial assistance.

	FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Grant T-32 CA 09599 (to R. M. S. and B. K. Z.), the Gillson Longenbaugh Foundation (L. M. E.), the Jon and Suzie Hall Fund for Colon Cancer Research (L. M. E.), and National Institute of Environmental Health Sciences Training Grant T32-ES-07290 (to D. W. D.).

² To whom requests for reprints should be addressed, at Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 106, Houston, TX 77030. Phone: (713) 792-6926; Fax: (713) 792-4689; E-mail: lellis@mdanderson.org.<!–
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³ The abbreviations used are: VEGF, vascular endothelial growth factor; TK, tyrosine kinase; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; IHC, immunohistochemistry; POD, postoperative day; PEG, polyethylene glycol.

Received 7/23/99. Accepted 9/20/99.

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SisCa

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