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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Cancer Biology [N. R., W. L., F. F., Y. D. J., O. S., C. D. B., R. R., L. M. E.] and Surgical Oncology [S. A. A., L. M. E.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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Results: IGF-I increased VEGF expression in parental and vector-transfected cells, whereas IGF-I induction of VEGF mRNA and protein was abrogated in IGF-IR DN cells. The IGF-IR DN cells demonstrated inhibited growth in both monolayer culture and soft agar (P < 0.05). s.c. injections of IGF-IR DN cells in nude mice led to significantly decreased tumor growth (P < 0.05). Immunohistochemical analyses revealed that IGF-I DN tumors demonstrated decreased tumor cell proliferation, VEGF expression, and vessel count and increased tumor cell apoptosis (P < 0.05 for all parameters compared with controls). Furthermore, IGF-IR DN-transfected cells yielded significantly decreased tumorigenicity and growth in the liver.
Conclusions: These studies demonstrate that the IGF ligand-receptor system plays an important role in multiple mechanisms that mediate human colon cancer growth including regulation of VEGF and angiogenesis.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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The role of IGF-I in malignant growth is at times difficult to define because IGFBPs may modulate the biological effects of IGF (reviewed in Ref. 15 ). These factors can bind IGF-I and either enhance or inhibit its ability to bind to its receptor (dependent upon the specific IGFBP), thus affecting its biological activity. However, VEGF induction by IGF-I is most likely not affected by IGFBPs because a truncated IGF-I lacking the binding site for IGFBPs does not alter VEGF induction (9 , 16) .
The IGF-IR, a tyrosine kinase receptor, is the major receptor for both IGF-I and IGF-II and consists of two 
and two ß subunits (4)
. The subunits are entirely extracellular chains, containing a cysteine-rich domain responsible for ligand binding (4
, 17)
. The ß subunits display a highly hydrophobic transmembrane domain, which divides the ß subunit into an extracellular and an intracellular region containing a tyrosine kinase domain (4
, 17)
. Several authors have demonstrated that the IGF-IR is crucial for maintaining normal growth and development (17
, 18)
. Homozygous IGF-IR knockout mice exhibit severe growth deficiency and invariably die at birth (18)
. In human colon cancer, IGF-IR is not only present but is frequently overexpressed (1
, 2)
. However, the biological significance of IGF-IR in colon cancer has not been elucidated.
To further our understanding of the role of the IGF-IR in human colon cancer growth, we stably transfected human colon cancer cells with a truncated IGF-IR (IGF-IR DN) lacking the cytosolic kinase domain. IGF-IR DN forms a nonfunctional heterodimer with endogenous IGF-IR (19 , 20) . Our studies demonstrate that inducible IGF-I signaling was strongly impaired in vitro and that VEGF induction by IGF-I was nearly completely abolished in IGF-IR DN transfectants. Growth of IGF-IR DN-transfected cells was significantly impaired in both monolayer and soft agar cultures. Subcutaneous growth of IGF-IR DN-transfected cells in nude mice was significantly impaired, and this was associated with increased apoptosis and decreased proliferation and blood vessel formation. In addition, IGF-IR DN cells injected directly into the liver formed significantly fewer tumors than controls. These results suggest that the IGF-IR plays a multifunctional role in human colon cancer growth, suggesting that this receptor would be a rational target for antineoplastic therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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(Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-IGF-IRß (Santa Cruz Biotechnology), polyclonal rabbit anti-MAPK (Oncogene Research Products, Cambridge, MA), and HRP-conjugated goat antirabbit IgG (Bio-Rad Laboratories, Hercules, CA).
Cell Lines and Culture Conditions
HT29 human colon cancer cells were purchased from American Type Culture Collection (Manassas, VA) and maintained as described previously (9)
. All in vitro experiments were performed at 50–60% confluence, and cells were used at passages 3–8 after their receipt from the supplier.
Stable Transfections
A mutated IGF-IR truncated at position 952 in the ß subunit transmembrane region (IGF-IR DN) was the generous gift of Dr. D. Prager [Cedars-Sinai Medical Center-University of California Los Angeles School of Medicine, Los Angeles, CA (19
, 20) ]. The IGF-IR DN was subcloned into pcDNA3 vector containing the neomycin (G418; Life Technologies, Inc.) resistance gene. The resulting vector was stably transfected into human HT29 colon cancer cells using FuGENE 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN) following the manufacturer’s protocol. As controls, cells were transfected with the pcDNA3 vector only (mock transfectants). After 48 h, the medium was replaced by medium containing 400 µg/ml G418. Clones were expanded and characterized as described below. To confirm successful transfection, cell extracts of parental and transfected cells were analyzed by nondenaturing Western blot analysis using anti-IGF-IR and anti-IGF-IRß antibodies.
VEGF Induction by IGF-I in Transfected Cells
Parental and transfected cells were grown to 50–60% confluence in standard medium as described above, and medium was changed to 5% FBS-containing medium overnight. Cells were then incubated in the presence or absence of IGF-I (100 ng/ml) in serum-free medium for 24 h. Total RNA was extracted, and VEGF mRNA expression was determined by Northern blot analysis and semiquantitative RT-PCR as described previously (21
, 22)
. The cDNA probes used were as follows: a human VEGF-specific 204-bp cDNA probe (a generous gift of Brygida Berse, Harvard Medical School, Boston, MA; Ref. 23
); and a glyceraldehyde-3-phosphate dehydrogenase probe (purchased from American Type Culture Collection). The VEGF probe identifies all alternatively spliced forms of its mRNA transcripts. In a separate experiment designed to evaluate IGF-I induction of VEGF protein, conditioned medium was harvested after 48 h from cells growing in 1% FBS-containing medium with or without IGF-I (100 ng/ml). The VEGF concentration was determined using an ELISA kit for human VEGF (R&D Systems) as reported previously (Ref. 24
) [the ELISA kit recognizes the VEGF165 and VEGF121 isoforms of the protein because the antibodies used were raised against the NH2-terminal portion of VEGF165 (25)
].
Effect of IGF-IR DN Transfection on IGF-I Induction of Signaling
Previous experiments have shown that VEGF is induced in HT29 colon cancer cells via the Erk1/2 pathway but not through Akt,4
which is consistent with the findings of others (26)
. Parental and transfected cells were grown to 50% confluence. Cells were then incubated in 1% FBS-containing medium overnight, and IGF-I (100 ng/ml) was added for 10 min. Cells were harvested, and protein was extracted. For IRS-1 immunoprecipitation, cell lysates containing 300 µg of protein were incubated with anti-IRS-1 and protein A/G-agarose (Santa Cruz Biotechnology) in rotating tubes overnight at 4°C. Finally, Western blot was performed for phosphorylated tyrosine and IRS-1 as described previously (24)
. For determination of Erk1/2 activity induced by IGF-I, 50–60% confluent cells were incubated overnight with 1% FBS-containing medium. IGF-I (100 ng/ml) was added for 15 min, and cell lysates were harvested. Western blot analysis was performed using antibodies against phosphorylated and total forms of p44/42 (Erk1/2).
Effect of IGF-IR DN Transfection on Monolayer Cell Growth
MTT Assay.
Cells (104) were plated in 96-well plates. After cell attachment, medium was changed to 10% FBS-containing medium for the indicated times. MTT (Sigma, St. Louis, MO) was added to a final concentration of 0.5 mg/ml, and cells were incubated for 90 min. Medium and MTT were removed, DMSO was added for 1 min, and absorption was read at 570 nm.
BrdUrd Labeling.
Cells (2 x 104) were plated on 2-chamber glass slides (Becton Dickinson Labware) and incubated with 10% FBS-containing medium for 36 h. BrdUrd was added to a final concentration of 10 µM, and cells were incubated for 30 min at 37°C. Finally, cells were fixed with acetone and chloroform, and immunohistochemistry was performed as described below.
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Effect of IGF-IR Stop Transfection on Three-dimensional Cell Growth |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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In Vivo Studies |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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Tumor Cell Injection and Therapy.
For in vivo injection, HT29 parental and transfected human colon cancer cells were grown to 50% confluence and incubated in G418-free medium for 24 h to improve cell viability. Cells were harvested from culture flasks after a brief trypsinization and transferred to serum-free HBSS. Single-cell suspensions of >90% viability, as determined by trypan blue dye exclusion (200 µl containing 106 cells), were injected s.c. through a 27-gauge needle in the right flank. All mice were sacrificed when 
3 mice in any group (n = 10 mice/group) had tumors that exceeded 1.2 cm in diameter. In another experiment to determine the role of IGF-IR on growth in the liver, the most common site of colon cancer metastasis, cell suspensions (50 µl containing 106 cells) were prepared as described. Cells were injected beneath the capsule into the left lobe of the liver of nude mice using a 30-gauge needle via a midline incision. Mice were sacrificed when 3 mice became moribund in any group (n = 10 mice/group).
Necropsy Procedures and Histological Studies.
Mice were weighed and sacrificed by cervical dislocation after sedation with methoxyflurane. Tumors were measured, excised, weighed, and processed for immunohistochemical analyses. For evaluation of tumor growth in the liver, livers were excised and weighed, and tumors were measured. If no tumor was visible, the liver was serially sectioned at 1-mm intervals to search for small tumor growth within the parenchyma. A section of each tumor was fixed in formalin and embedded in paraffin, and another section was embedded in OCT compound (Miles Inc, Elkhart, IN), rapidly frozen in liquid nitrogen, and stored at -70°C.
Standard Immunohistochemistry.
Paraffin-embedded tissues were sectioned and stained for H&E, PCNA, IGF-I, IGF-II, and VEGF. Immunohistochemical staining was performed as described previously (27
, 28)
using stable diaminobenzidine (Research Genetics, Huntsville, AL) or AEC (BioGenex Laboratories, San Ramon, CA) as substrates. Sections were counterstained with Gill’s 3 hematoxylin (Sigma) and mounted with Universal Mount (Research Genetics). Negative controls underwent the exact same procedure with omission of the primary antibody. For analysis of VEGF expression, the signal was amplified using the TSA Biotin System (NEN Life Science, Boston, MA), and counterstaining was omitted. Standard immunohistochemistry for CD31 (endothelial cells) was done on frozen tissue sections as described previously (29)
. For BrdUrd labeling, cells were permeabilized with 1% Triton X-100 followed by treatment with 2 N HCl at 37°C before performing standard immunohistochemistry.
Immunofluorescence Staining for Phosphorylated Erk1/2, CD31, and TUNEL.
For immunofluorescence staining, frozen tissue was fixed in cold acetone and chloroform, washed with PBS, and incubated with primary and secondary antibodies as described previously (28, 29, 30)
. The TUNEL assay was performed with a commercial kit (Promega, Madison, WI) according to the manufacturer’s protocol. Immunofluorescence microscopy was done on an epifluorescence microscope equipped with narrow bandpass excitation filters (Chroma Technology Corp., Brattleboro, VT) to individually select for green, red, and blue fluorescence. Images were captured with a C5810 Hamamatsu camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan) mounted on a Zeiss Axioplan microscope (Carl Zeiss Inc.) using Optimas image analysis software (Media Cybernetics, Silver Spring, MD). Images were further processed with Adobe Photoshop software (Adobe Systems, Mountain View, CA). CD31 and TUNEL double-staining was performed where endothelial cells were identified by red fluorescence (CD31 positive), and DNA fragmentation was detected in tumor and endothelial cells by localized green (TUNEL-positive) and yellow (TUNEL- and CD31-positive) fluorescence, respectively. Greater than 95% of cells in these tumor specimens are tumor epithelial cells. Therefore, quantification 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.
Quantification of PCNA, TUNEL, VEGF, Microvessel Density, and BrdUrd Labeling.
For evaluation of immunohistochemical staining in tumors grown in mice, only the tumor edge was examined to avoid geographic bias. Necrotic areas were excluded. The number of PCNA-positive cells was assessed in three areas containing the highest PCNA expression at x100 magnification (0.22 mm2). For determination of TUNEL expression in tissue sections, we counted the number of apoptotic events in five random fields at x100 magnification and divided that number by the total number of cells per field. The intensity of VEGF staining was graded using computer-assisted image analysis with Optimas software. The analysis was based on converting the image into a grayscale image and measuring the absorbance. Four distinct areas with high VEGF expression were evaluated at x100 magnification. To validate the image analysis system, all sections were also semiquantitatively scored subjectively as described previously (27
, 31)
with no significant differences between the objective and subjective techniques. Vessels were counted in four quadrants at x50 magnification according to the method described by Weidner et al. (32)
. For evaluation of BrdUrd staining in vitro, the percentage of BrdUrd-labeled cells was determined in 10 random fields at x200 magnification.
Densitometric Quantification and Statistical Analysis.
Densitometric analysis was performed (Image Quant software, Molecular Dynamics, Sunnyvale, CA) to quantify the results of Northern blot analyses in the linear range of the film. Glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal control for loading of Northern blots. All analyses were done using InStat statistical software (GraphPad Software, San Diego, CA), with P < 0.05 considered to be significant. For comparing tumor-associated parameters, the nonpaired Student’s t test was used unless otherwise specified. If the SDs of two Gaussian distributions were significantly different, the alternate Welch t test was used.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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and ß chain of the IGF-IR, IGF-IR DN cells demonstrated strong additional bands indicating the 300-kDa mutant homodimer and the 360-kDa wild-type/mutant hybrid, respectively (as described in Ref. 19
) (Fig. 1)
. Transfection of the empty vector or IGF-I DN construct had no gross apparent effect on cell morphology. Incubation with IGF-I increased VEGF mRNA expression 8–10-fold in parental and pcDNA3-transfected cells, in contrast to a only slight induction in IGF-IR DN-transfected cells (Fig. 2A)
. Although induction of all isoforms of VEGF mRNA were attenuated in the IGF-IR DN-transfected cells, the band corresponding to the VEGF189 splice variant was decreased to the greatest degree. These results were confirmed by performing quantitative rtPCR (data not shown). The transfected clone (clone 15) showing the least VEGF induction by IGF-I was used for additional experiments. A second clone (clone 9) with a moderate decrease in IGF-IR function was also studied to confirm the results reported below. As determined by ELISA, the baseline and IGF-I-induced VEGF protein in conditioned medium was significantly lower in IGF-IR DN-transfected cells as compared with controls (P < 0.001; Fig. 2B
).
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. We further determined the effect of IGF-IR DN transfection on downstream signaling, specifically activation of Erk1/2. Parental and pcDNA3-transfected HT29 cells showed a strong increase in Erk1/2 phosphorylation after IGF-I treatment. In contrast, IGF-I treatment of IGF-IR DN-transfected cells produced no major induction of Erk1/2 phosphorylation (Fig. 3B)
.
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that were proportional to their decrease in IGF-IR function (clone 9; data not shown). To confirm the effect of IGF-IR on regulating cell proliferation in response to ligand, we also labeled growing HT29 cells by BrdUrd staining. Cells transfected with DN IGF-IR demonstrated significantly fewer BrdUrd positively stained cells when compared with controls (Fig. 4B)
.
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. Tumor size was proportional to the degree of inhibition of IGF-IR activity because the transfected clone with intermediate inhibition of IGF-IR function (clone 9) developed tumors that were smaller than mock transfectants (P < 0.05) but larger than tumors grown from the transfected cell line (clone 15) with the greatest decrease of IGF-IR function (data not shown).
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. There was no significant difference between HT29 parental and pcDNA3-transfected cells (controls) in any of the above parameters. In contrast, IGF-IR DN-transfected tumors demonstrated a decrease in PCNA labeling (proliferation) compared with controls (Fig. 8A)
. In addition, overall apoptosis was enhanced in IGF-IR DN-transfected HT29 cells (Fig. 8B)
. Furthermore, as determined by immunofluorescence staining, the phosphorylation status of Erk1/2 was decreased in IGF-IR DN-transfected cells as compared with controls (data not shown).
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, we performed VEGF staining on the HT29 tumors grown in nude mice using an antibody that recognizes the VEGF165, VEGF189, and VEGF206 splice variants. Using computer-assisted image analysis, tumor cells with impaired IGF-IR function demonstrated significantly less VEGF staining intensity than controls (Fig. 9A)
. Finally, vessels were highlighted by staining sections with an antibody to CD31. IGF-IR DN-transfected tumors had significantly fewer vessels than control groups (Fig. 9B)
. Immunohistochemistry demonstrated the expression of IGF-I and IGF-II in all tumor specimens (data not shown).
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2 analysis). As a gross measure of hepatic tumor burden, the total liver weight was significantly less in mice injected with IGF-IR DN-transfected cells than in controls [IGF-IR DN transfectants, 1.54 ± 0.05 g; mock transfectants, 1.78 ± 0.05 g (mean ± SE); P < 0.05]. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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In addition to the established functions of the IGF-IR listed above, preliminary evidence suggests that it might also be involved in regulating angiogenesis. Our laboratory previously demonstrated that IGF-I increases VEGF expression in human colon carcinoma cells (9) . This observation is of particular interest because the sites of colon cancer growth express IGF-I constitutively. The organ with the highest levels of IGF-I, the liver (15) , is also the primary organ of colon cancer metastasis. In addition to a paracrine effect, IGF-I and IGF-II may also act in an autocrine manner because colon mucosa and colon cancers themselves synthesize significant amounts of IGF-I (1) .
To evaluate the importance of IGF-IR in human colon cancer and its contribution to tumor angiogenesis, we stably transfected HT29 colon cancer cells with a truncated IGF-IR lacking the cytosolic tyrosine kinase domain that formed heterodimers with wild-type IGF-IR, thereby impairing its function (19 , 20) . Incubation with IGF-I led to induction of VEGF mRNA in parental and pcDNA3-empty vector transfected cells, whereas this induction was nearly completely abrogated in IGF-IR DN-transfected cells. Furthermore, constitutive VEGF mRNA expression was partially reduced in IGF-IR DN-transfected cells. VEGF protein secretion was also markedly decreased in IGF-IR DN-transfected cells. The VEGF189 splice variant appeared to be the isoform whose expression was most affected by the decrease in IGF-IR function as determined by Northern blot analysis and RT-PCR. Recently, VEGF189 has been correlated with poor prognosis and increased angiogenesis in several tumors, including colon cancer (42 , 43) .
Binding to the IGF-IR activates several intracellular signal transduction pathways (4 , 17 , 44) . Although the specific signaling cascades activated remain somewhat controversial (44) , key events are the binding of IRS-1, IRS-2, and Shc, which act as docking proteins for various downstream signaling molecules. Several downstream signaling pathways are further activated, including the MAPK pathway that has been shown to be essential for VEGF induction (26) . In contrast, the p85 subunit of the phosphatidylinositol 3'-kinase pathway is activated by IGF-I but does not appear to be involved in VEGF induction in this system (26) .4 In the current study, we demonstrate that IGF-IR DN transfection impairs IGF-IR signaling. Upon IGF-I treatment, IRS-1 is tyrosine-phosphorylated extensively in control cells but only minimally in IGF-IR DN-transfected cells. MAPK activation by IGF-I is also impaired in IGF-IR DN-transfected HT29 cells. These data were confirmed using a second IGF-IR DN-transfected HT29 cell clone with intermediate IGF-IR activity.
The inhibition of IGF-IR function also significantly affected tumor cell proliferation in vitro, possibly also due to impaired MAPK activation. The proliferative rate in IGF-IR DN-transfected cells was lower than that in controls in two separate assays (MTT assay and BrdUrd labeling assay). In soft agar, IGF-IR DN cells formed few colonies that did not respond to IGF-I treatment, whereas there was no significant difference in growth as monolayer or in soft agar between parental and mock-transfected cells. These results are in congruence with a previous study performed by Prager et al. (19
, 20)
, who transfected Rat-1 fibroblasts with the same IGF-IR DN construct used in this study. Although IGF-IR DN-transfected cells also failed to grow in soft agar in their study, Prager et al. did observe a slight increase in the number and size of colonies upon addition of IGF-I (20)
. However, similar to our study, IGF-IR DN transfection did not alter cell morphology in monolayer cultures. Similar results were also reported by Reiss et al. (45)
, who transfected various cancer cell lines with an IGF-IR truncated at residue 486 in the subunit that led to a moderate decrease in monolayer growth but marked inhibition of colony formation in soft agar.
To evaluate the various effects of IGF-IR DN transfection of colon cancer cells on tumor formation in vivo, we injected nude mice s.c. with IGF-IR DN-transfected, pcDNA3-transfected, or parental HT29 cells. IGF-IR DN-transfected cells formed significantly smaller tumors than did controls (weight and volume), findings consistent with those of previous studies in other tumor systems (20 , 45) . However, we observed a decrease of tumor volume dependent on the level of IGF-IR activity, suggesting a dose-dependent effect. To further evaluate the distinct effects of IGF-IR DN transfection on the processes involved in tumor growth, we analyzed the s.c. grown tumors by immunohistochemical techniques. By PCNA labeling, we confirmed the in vivo antiproliferative effect of IGF-IR DN transfection that was demonstrated in our in vitro studies. In addition, cells transfected with IGF-IR DN showed decreased levels of phosphorylated Erk1/2 in vivo, again consistent with our findings in vitro. The antiapoptotic effect of the IGF-IR has been addressed previously (37, 38, 39) and was confirmed in colon cancer growth in vivo because tumors grown from HT29 cells transfected with IGF-IR DN exhibited enhanced apoptosis compared with control tumors.
The studies reported above are novel because they are the first to address the role of IGF-IR on tumor angiogenesis in vivo. In addition to evaluation of the antiproliferative and proapoptotic effects of IGF-IR DN transfection in human HT29 colon cancer cells, we determined the effect of IGF-IR DN on tumor angiogenesis. Because we have previously shown that IGF-I induces VEGF in vitro (9) , we hypothesized that inhibition of the IGF-IR alters human colon cancer angiogenesis in nude mice. IGF-I and IGF-II are expressed by colon cancer cells, including HT29 cells (46) , and therefore may act in an autocrine/paracrine loop in s.c. tumor formation. Because the most active angiogenic site in tumors is the tumor-normal interface (31) , we assessed VEGF expression and microvessel density in this region in s.c. tumors in nude mice. IGF-IR DN-transfected cells expressed significantly less VEGF and produced significantly fewer vessels than control tumors. In addition, expression and activity of other angiogenic factors may also be mediated through the IGF-IR because the vessel count in tumors from IGF-IR DN-transfected cells was decreased to a greater degree than that of VEGF expression in some tumor areas. The contribution of IGF-I-induced angiogenesis may be even more important in organs with constitutive IGF-I expression such as the liver, which is the most frequent distant metastatic site for colon cancer. Therefore, we injected mock and IGF-IR DN-transfected cells directly into the liver of nude mice. Mice injected with IGF-IR DN cells demonstrated significantly less tumorigenicity and decreased liver weights.
Our data demonstrate the critical role of IGF-IR function in colon cancer cells in various processes involved in tumor growth both in vitro and in vivo. Furthermore, this report is the first to demonstrate that inhibition of IGF-IR function may be an indirect means of inhibiting VEGF expression and angiogenesis, similar to principles established in targeting the epidermal growth factor receptor (28) . Due to the multiple functions of the IGF-IR, this receptor appears to be a promising target for therapy of human colon cancer (47 , 48) . Additional studies are clearly needed to further develop a more fundamental understanding of the complex IGF system including the IGF-IR and to evaluate its value and selectivity as a target in anticancer treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Dr. Mildred Scheel Stiftung für Krebsforschung, Deutsche Krebshilfe (N. R.), NIH Grant T-32 09599 (to S. A. A.), NIH Grant CA74821 (to L. M. E.), and NIH Core Grant CCSG CA16672 (to L. M. E.). 
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 444, Houston, TX 77030-4009. Phone: (713) 792-6926; Fax: (713) 792-4689; E-mail: lellis{at}mdanderson.org
3 The abbreviations used are: IGF, insulin-like growth factor; BrdUrd, bromodeoxyuridine; Erk, extracellular signal-regulated kinase; FBS, fetal bovine serum; HRP, horseradish peroxidase; IGFBP, IGF-binding protein; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; VEGF, vascular endothelial growth factor; DN, dominant-negative; rtPCR, reverse transcription-PCR; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
4 Y. D. Jung and L. M. Ellis, unpublished data.
Received 4/ 5/02; revised 6/21/02; accepted 7/ 2/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSEffect of IGF-IR Stop...In Vivo StudiesRESULTSDISCUSSIONREFERENCES |
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B. D. Cohen, D. A. Baker, C. Soderstrom, G. Tkalcevic, A. M. Rossi, P. E. Miller, M. W. Tengowski, F. Wang, A. Gualberto, J. S. Beebe, et al. Combination Therapy Enhances the Inhibition of Tumor Growth with the Fully Human Anti-Type 1 Insulin-Like Growth Factor Receptor Monoclonal Antibody CP-751,871 Clin. Cancer Res., March 1, 2005; 11(5): 2063 - 2073. [Abstract] [Full Text] [PDF]
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D. J. Hicklin and L. M. Ellis Role of the Vascular Endothelial Growth Factor Pathway in Tumor Growth and Angiogenesis J. Clin. Oncol., February 10, 2005; 23(5): 1011 - 1027. [Abstract] [Full Text] [PDF]
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M. Nakamura, S. Miyamoto, H. Maeda, S.-c. Zhang, T. Sangai, G. Ishii, T. Hasebe, Y. Endoh, N. Saito, M. Asaka, et al. Low Levels of Insulin-Like Growth Factor Type 1 Receptor Expression at Cancer Cell Membrane Predict Liver Metastasis in Dukes' C Human Colorectal Cancers Clin. Cancer Res., December 15, 2004; 10(24): 8434 - 8441. [Abstract] [Full Text] [PDF]
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G. Calviello, F. Di Nicuolo, S. Gragnoli, E. Piccioni, S. Serini, N. Maggiano, G. Tringali, P. Navarra, F. O. Ranelletti, and P. Palozza n-3 PUFAs reduce VEGF expression in human colon cancer cells modulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-1{alpha} induction pathway Carcinogenesis, December 1, 2004; 25(12): 2303 - 2310. [Abstract] [Full Text] [PDF]
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Z. Cao, J. Fang, C. Xia, X. Shi, and B.-H. Jiang trans-3,4,5'-Trihydroxystibene Inhibits Hypoxia-Inducible Factor 1{alpha} and Vascular Endothelial Growth Factor Expression in Human Ovarian Cancer Cells Clin. Cancer Res., August 1, 2004; 10(15): 5253 - 5263. [Abstract] [Full Text] [PDF]
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D. Lu, H. Zhang, D. Ludwig, A. Persaud, X. Jimenez, D. Burtrum, P. Balderes, M. Liu, P. Bohlen, L. Witte, et al. Simultaneous Blockade of Both the Epidermal Growth Factor Receptor and the Insulin-like Growth Factor Receptor Signaling Pathways in Cancer Cells with a Fully Human Recombinant Bispecific Antibody J. Biol. Chem., January 23, 2004; 279(4): 2856 - 2865. [Abstract] [Full Text] [PDF]
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 O. Stoeltzing, W. Liu, N. Reinmuth, F. Fan, A. A. Parikh, C. D. Bucana, D. B. Evans, G. L. Semenza, and L. M. Ellis Regulation of Hypoxia-Inducible Factor-1{alpha}, Vascular Endothelial Growth Factor, and Angiogenesis by an Insulin-Like Growth Factor-I Receptor Autocrine Loop in Human Pancreatic Cancer Am. J. Pathol., September 1, 2003; 163(3): 1001 - 1011. [Abstract] [Full Text] [PDF]
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E. K. Maloney, J. L. McLaughlin, N. E. Dagdigian, L. M. Garrett, K. M. Connors, X.-M. Zhou, W. A. Blattler, T. Chittenden, and R. Singh An Anti-Insulin-like Growth Factor I Receptor Antibody That Is a Potent Inhibitor of Cancer Cell Proliferation Cancer Res., August 15, 2003; 63(16): 5073 - 5083. [Abstract] [Full Text] [PDF]
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 G. S. Howarth Insulin-Like Growth Factor-I and the Gastrointestinal System: Therapeutic Indications and Safety Implications J. Nutr., July 1, 2003; 133(7): 2109 - 2112. [Abstract] [Full Text] [PDF]
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O. Stoeltzing, S. A. Ahmad, W. Liu, M. F. McCarty, J. S. Wey, A. A. Parikh, F. Fan, N. Reinmuth, M. Kawaguchi, C. D. Bucana, et al. Angiopoietin-1 Inhibits Vascular Permeability, Angiogenesis, and Growth of Hepatic Colon Cancer Tumors Cancer Res., June 15, 2003; 63(12): 3370 - 3377. [Abstract] [Full Text] [PDF]
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