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1 Cancer Biology Program, Hematology and Oncology Division, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; 2 Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York; 3 Department of Medical Biochemistry, Ehime University School of Medicine, Ehime, Japan; 4 Department of Biochemistry, Osaka University Medical School, Osaka, Japan; and 5 Telik, Inc., Palo Alto, California
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3ß1 (10)
and transduces biological signals to neighboring cells in a nondiffusible manner (7)
. The transmembrane form of HB-EGF is a juxtacrine growth factor, which is immobilized on the surface of the cell and interacts with neighboring cells (1)
. It has also been shown that the transmembrane HB-EGF synthesized by one type of cell can stimulate tyrosine phosphorylation of the EGF receptor in another type of cell in coculture (11)
. A recent study demonstrated that HB-EGF can bind to a novel 140-kDa receptor identified as N-arginine dibasic convertase, a metalloendopeptidase of the M16 family, and that binding to N-arginine dibasic convertase is highly specific for HB-EGF among EGF family members (12)
. Its specific binding modulates HB-EGF-induced cell migration via EGF receptor (12)
. Several laboratories have described HB-EGF as being up-regulated in response to oncogenes and in oncogene-transformed cells (13 , 14) . In chicken embryo fibroblasts transfected with regulatable v-Jun, the expression of HB-EGF is greatly induced as v-Jun levels are increased (15) , suggesting that HB-EGF plays an important part in mediating Jun-induced cell transformation. Furthermore, HB-EGF has been identified as an immediate-early response gene that can be activated by the Ras/Raf signaling pathway that mediates the autocrine activation of the c-Jun kinase in NIH3T3 cells (15) . Phosphorylation of the transcription factor Ets-2 by activation of the Raf1/MAPK cascade regulates the induction of HB-EGF transcription in mouse fibroblasts (16) . Additionally, in nontransformed human mammary epithelial cells, HB-EGF expression is induced by EGF and Ha-Ras overexpression (17) , strongly implying that HB-EGF is a direct target of mitogen-activated protein kinase (MAPK). Our previous observations demonstrated that HB-EGF is induced in response to tumor suppressor p53, as well as DNA damage, and HB-EGF induction antagonizes apoptosis mediated by genotoxic stress through the activation of the Ras/Raf/MAPK cascade and the AKT pathway (18 , 19) , strongly suggesting a survival factor function for HB-EGF.
HB-EGF expression has been implicated in tumor progression because of its overexpression in many tumors, including hepatocarcinoma, colon, melanoma, myeloma, breast, prostate, and bladder tumors. It also has been implicated in increased proliferation and metastasis (20, 21, 22, 23, 24, 25, 26, 27) . Many tumor cells with HB-EGF overexpression are diphtheria toxin sensitive, suggesting that these cells accumulate the transmembrane form of HB-EGF. There is growing evidence of increased HB-EGF expression in tumors compared with normal tissue, e.g., pancreatic (28) , liver (23 , 29) , esophageal (30) , melanoma (20) , bladder (31) , and gastric tumors (32) . Although HB-EGF is widely expressed in tumors and may be enhanced compared with normal tissue, its contribution to tumorigenicity is unknown. Moreover, it is not known whether the membrane-bound or the soluble form of HB-EGF contributes to tumorigenic processes.
In this study, we examined the biological effects of the two forms of HB-EGF on tumor growth and angiogenesis, using a tetracycline (tet)-regulated expression system in human EJ bladder carcinoma cells, which have a low basal level of HB-EGF. EJ cells expressing soluble form HB-EGF (s-HB-EGF) or wild-type HB-EGF (wt-HB-EGF) resulted in an increased growth rate, activation of the cyclin D promoter, colony-forming ability, and tumor growth in athymic nude mice. We also found that s-HB-EGF induced vascular endothelial growth factor (VEGF) expression, implicating an autocrine loop that may play a role in regulating these growth factors. Additionally, in response to s-HB-EGF, cells induced the expression and activities of the metalloproteases MMP-9 and MMP-3, leading to enhanced cell migration. In this study, we provide evidence of the biological activity of HB-EGF in tumorigenesis.
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MATERIALS AND METHODS |
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Northern Blot and Western Blot Analyses.
Total RNA was extracted, denatured, and electrophoresed through a 1% agarose-formaldehyde gel as described previously (19)
. Total protein extracts were immunoblotted with the respective antibodies. Tumors were dissected from the mice and snap frozen by liquid nitrogen. The tumors were then homogenized in lysis buffer containing 1% Triton X-100, 10 mM HEPES (pH 7.5), 130 mM NaCl, 5 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, and 50 µg/ml aprotinin. Twenty µg of proteins were loaded into each well and separated by a 4–12% NuPAGE gel (Invitrogen).
Transwell Migration Assay.
The cells were plated at a cell density of 2 x 105 cells in a 100-mm diameter dish. Each form of HB-EGF was induced by the removal of tet for 24 h, followed by serum starvation for another 24 h. Control cells were plated in similar conditions as HB-EGF-expressing cells, except that tet was added to suppress HB-EGF expression. Migration assays were performed using a chemotaxis chamber (Becton Dickinson and Co., Franklin Lakes, NJ) and transwell tissue culture plates (6.5 mm and 8-µm pore size). The bottom of the chamber was coated with either 10 µg/ml fibronectin, collagen I, or Matrigel (Sigma, St. Louis, MO). The uncoated sites were blocked with 10% BSA. One-hundred µl of 1 x 105 cells/ml were introduced into each well and were allowed to migrate for 6 h. Cells were then fixed with methanol and stained with crystal violet. The migrated cells were quantified by counting the number of cells in five random x200 fields. Each experiment was done in triplicates, and the experiment was repeated twice.
Wound-Healing Assay.
HB-EGF was expressed for 24 h in the absence of tet, and control cells were seeded in the same fashion, except in the presence of tet. Cells were then seeded in a 6-well plate at a density of 1 x 106 cells in the presence or absence of tet for 24 h. A wound was made using the tip of a pipette. After 32 h, the cells were fixed with 3.7% formaldehyde in PBS for 20 min, and pictures of the wound were taken at the different field.
Soft Agar Colonogenic Assay.
Soft agar assay was performed by seeding the cells at a density of 1 x 104 in a 60-mm diameter tissue culture dish containing 0.33% top low-melt agarose-0.6% bottom low-melt agarose. Cells were fed every 3 days. Colonies were counted and measured after 2 weeks.
Gene Reporter Assays.
A total of 2 x 105 cells was seeded in each well of a 6-well dish and transfected with desired plasmids using Lipofectamine 2000 reagent (Invitrogen). The assays were performed with the dual luciferase reporter assay system from Promega. The internal controls used were pRL-TK and pRL-CMV to track transfection efficiency. The cells were harvested after 48 h. Each data point obtained is the mean of three independent experiments.
Xenograft Studies.
EJ-HB-EGF and EJ-CAT cells were cultured in media with and without tet for 24 h. Cells were washed twice with DMEM without antibiotics and serum and finally resuspended at a density of 2 x 106 cells in 0.25 ml. The cell suspension was injected s.c. (bilaterally, 0.25 ml/site) into 6-week-old nude athymic mice [Taconic; Cr:(NCr)-nu f BR]. HB-EGF expression was suppressed by feeding mice with water containing 500 µg of doxycycline (dox)/ml in 1% sucrose. Mice were killed after 3 weeks. Tumors were excised and weighed, fixed for 1 h in 10% formalin, washing with PBS, followed by embedding in ornithine carbamyl transferase, and frozen in liquid nitrogen. Six mice were used for each sample.
Computer-Assisted Morphometric Analysis of Intratumoral Vasculature.
Representative tumor sections were stained with anti-CD31 mouse monoclonal antibodies to visualize the blood vessels. Six tumors were evaluated/each sample, and five areas were evaluated at x6 magnification. Images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI), and blood vessels were quantified using IPLab software (Scanalytics, Billerica, MA). The two-sided unpaired t test was used to analyze differences in microvessel density and vascular size and area.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Using this system, we first examined the role of HB-EGF in cell proliferation. The growth rate of EJ-HB-EGF cells was measured in the presence or absence of tet at 1 x 104 cells in 6-well plates for up to 8 days. Induction of wt- or s-HB-EGF by removal of tet resulted in a substantial growth rate increase, as seen by the 75–100% increase, when compared with HB-EGF-repressed cells (tet+; Fig. 1B
). However, pro-HB-EGF induction in EJ cells resulted in a slight increase of growth rate by 
25%. To examine the effect of HB-EGF on cell colony formation ability, EJ-HB-EGF cells expressing three different types of HB-EGF were seeded at 200 cells/100-mm diameter dish. Cells expressing s- or wt-HB-EGF produced approximately two to three times more colonies than that of pro-HB-EGF-induced cells (Fig. 1C)
. We next evaluated the effect of each HB-EGF on colony formation in soft agarose as an additional measure of tumorigenicity in vitro using tet-regulated EJ-HB-EGF cells. The results of agar colony tests are shown in Fig. 1D
. Soluble- or wt-HB-EGF (tet-)-inducing cells significantly stimulated anchorage-independent growth, but anchorage-independent growth was not seen in pro-HB-EGF-induced cells. A colony formation assay in soft agarose was also used to examine whether HB-EGF could promote a transformed phenotype in a different cell line using HeLa cells. Transfection of HeLa cells with wt- or s-HB-EGF following a selection under G418 significantly increased sizes of colonies in soft agarose as compared with those in cells transfected with vector alone or a plasmid expressing pro-HB-EGF (Fig. 1E)
. Taken together, these results suggest that s-HB-EGF expression causes an enhanced transformed phenotype in vitro but pro-HB-EGF overexpression does not.
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3-fold increase in cyclin D1 protein levels, whereas pro-HB-EGF did not result in an increase in cyclin D expression. (Fig. 2A)
. To determine whether HB-EGF regulates the transcription of cyclin D1, we also measured the effects of HB-EGFs on the cyclin D1 promoter using a cyclin D1-luciferase reporter construct (–1745CD1; Ref. 35
). The reporter containing the cyclin D promoter was strongly activated in response to the expression of s- or wt-HB-EGF in EJ cells and 293T cells (Fig. 2B)
, whereas pro-HB-EGF did not show any significant effect on cyclin D promoter activity, regardless of serum presence. These results indicate that the activation of the cyclin D promoter and the –1745CD1 promoter fragment retains complete responsiveness to s-HB-EGF.
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, EJ cells expressing s- or wt-HB-EGF showed an increased overall tumor size when injected into nude athymic mice (EJ-HB-EGF tumors in –tet condition), compared with EJ cells expressing a control CAT protein (EJ-CAT tumors in –tet and +tet) and EJ-HB-EGF cells in tet+ conditions. The mean size of EJ-HB-EGF tumors maintained in the absence of dox was 1.5 g, significantly larger than tumors from control mice, 0.1 g (Fig. 3C)
. However, tumors expressing pro-HB-EGF were similar to those of control mice. Data from the in vitro tumorigenicity assays and the xenograft assays support the conclusion that s-HB-EGF exerts a potent oncogenic potential but pro-HB-EGF does not.
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, but no increase of VEGF mRNA by pro-HB-EGF induction in EJ-pro-HB-EGF cells was seen. These results implicate the up-regulation of HB-EGF as an angiogenesis-related change, occurring through an autocrine response, which is closely associated with tumorigenesis of epithelial cells.
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were first homogenized and examined for HB-EGF expression levels by Western blotting. Fig. 4B
shows that mice maintained in the absence of dox-expressed HB-EGF at a higher level as compared with dox-fed mice. Cryostat sections of five different tumors expressing HB-EGF for each experimental condition include the following: +dox of EJ-HBEGF (no HB-EGF expression); –dox of EJ-HBEGF (HB-EGF expressed); +dox and –dox of EJ-CAT cells were processed and stained for CD31 (PECAM-1), an endothelial junction molecule (40)
. As shown in Fig. 4C
, these sections demonstrated a dramatic increase of microvessels in s- or wt-HB-EGF expressing tumors (+wt-HB-EGF and +s-HB-EGF, -dox) when compared with the different control tumors (+dox, +CAT and –CAT, and +pro-HB-EGF). To achieve a more detailed quantification of the effects of HB-EGF on tumor angiogenesis, average vessel density, vessel size, and percentage of tissue area covered by vessels were determined by computer-assisted image analysis of representative digital images. The relative tumor area occupied by vessels increased 2–3-folds in s- or wt-HB-EGF-expressing tumors (Fig. 4C
, right panel).
Up-Regulation of the MMP by HB-EGF.
It is well documented that the up-regulation of MMP activities results in the proteolytic degradation of the extracellular matrix and the basement membrane, which promotes tumor growth and metastasis (41)
. To better evaluate the participation of HB-EGF in tumor migration and progression, we tested the effects of HB-EGF on MMP-9 activation. As shown in Fig. 5
, HB-EGF induction after tet removal activated the 92-kDa pro-MMP-9 zymogen to the 67-kDa active form. The processing/activation was analyzed by zymography analysis. MMP-9 expression level was also determined by Northern and Western blotting, which showed that MMP-9 levels correlated with an increase of HB-EGF levels (Fig. 5)
. MMP-3, also known as a stromelysin-1, is the most efficient activator of MMP-9 in vitro and in vivo. Therefore, we evaluated the effect of HB-EGF on MMP-3 activity. Fig. 5
also shows the slightly increased activation and expression of MMP-3 in response to HB-EGF induction. However, HB-EGF-mediated activation of MMP-9 was not observed in response to pro-HB-EGF overexpression (data not shown). These results demonstrate that the expression level and activity of MMP-9 and MMP-3 can be enhanced in response to s-HB-EGF in EJ cells.
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, HB-EGF expression in EJ cells after the removal of tet showed a strong increase in migration on all three substrates but not in cells grown in the presence of tet. Although s-HB-EGF and wt-HB-EGF were more effective, pro-HB-EGF expression also increased cell migration on all three matrixes.
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. Soluble- or wt-HB-EGF expression (–tet condition) increased the effectiveness of wound healing, but no significant effect was seen in pro-HB-EGF-expressing cells. |
DISCUSSION |
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HB-EGF is known as a potent mitogen for keratinocytes, hepatocytes, smooth muscle cells, and fibroblasts (2
, 29
, 44)
. HB-EGF expression is elevated in human cancers, including hepatocellular and gastric carcinoma, breast carcinoma, melanoma, colon cancer, pancreatic cancer, glioma and glioblastoma (23
, 24)
. Furthermore, HB-EGF is induced in NIH3T3 cells transformed by v-Ras or v-Raf (34)
. HB-EGF was identified as a target of v-Jun, a potent oncogene, and HB-EGF plays a role in v-Jun-mediated oncogenic transformation (15)
. The significance of HB-EGF overexpression in tumorigenesis is also supported by our findings that HB-EGF activates the cyclin D promoter, implying that v-Jun could stimulate cyclin D transcription through HB-EGF induction. We have previously reported the identification of HB-EGF as a p53 downstream target gene, and p53 induction of HB-EGF could activate cell survival signaling, including AKT and MAPK cascades (19)
. A recent article has demonstrated that inducible expression of oncogenic Raf in normal epithelial cells strongly induced autocrine expression of HB-EGF, transforming growth factor , and amphiregulin, which were directly implicated in the ability of sustained Raf/MAPK pathway stimulation to protect cells from apoptosis (47)
.
It is now well established that VEGF is an endothelial cell-specific, multifunctional growth factor that plays a major role in the initiation of angiogenesis by acting directly as a mitogenic and chemotactic factor (48) . Although HB-EGF is known to have no effect on the proliferation of endothelial cells, some studies demonstrated that VEGF induces expression of angiogenic growth factors, including HB-EGF, in vascular endothelial cells and suggested that HB-EGF induction in response to VEGF provides a critical endothelial cell-derived signal, perhaps the activation of the MAPK and AKT cascade, for the process of new blood vessel formation and maturation (39) . In addition, the reviews have proposed that HB-EGF acts as one of the recruiting signals for mesenchymal cells during the late phase of angiogenesis (49) , implying that HB-EGF plays a critical role in angiogenesis as well as tumor development. In this study, we also demonstrate that s- or wt-HB-EGF induces VEGF expression, whereas it inhibits thrombospondin-1 expression (data not shown), implying the presence of an autocrine growth factor stimulation. Moreover, HB-EGF promotes tumor angiogenesis in vivo and exerts the migration-stimulating and wound-healing potential for cancer cells.
It is now clear that the up-regulation of MMP activities results in the proteolytic degradation of the extracellular matrix and the basement membrane, which promotes tumor growth, angiogenesis, and metastasis (41) . It is possible that the effect of HB-EGF on the functional interaction between VEGF and MMP-9 is a key mechanism for initiation and maintenance of angiogenesis. In our cell system, we found a significant difference in the levels of active MMP-9 and MMP-3 in s-HB-EGF-induced cells. Moreover, recent studies clearly demonstrated that MMP-9 mediates the release and accumulation of VEGF from the cell matrix and that MMP-9 triggers the angiogenic switch (50) . The generation of HB-EGF overexpressing mice or a targeted deletion of HB-EGF should provide additional understanding of mechanisms underlying HB-EGF-associated functions in angiogenesis.
In summary, our findings provide evidence that HB-EGF enhances transformed phenotypes and is associated with the stimulation of MMP-9, MMP-3, and cyclin D activation, which promotes tumorigenesis and angiogenesis. Given its elevated expression in human cancers along with our findings of HB-EGF contribution to enhanced transformed phenotypes, we hypothesize that HB-EGF may behave as an oncogene and, as such, could have importance as a therapeutic target.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Requests for reprints: Sam W. Lee, Harvard Institutes of Medicine, 4 Blackfan Circle, Room 921, Boston, MA 02115. Phone: (617) 667-8563; Fax: (617) 667-0980; E-mail: slee2{at}bidmc.harvard.edu
Received 3/17/04. Revised 5/27/04. Accepted 6/ 3/04.
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