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Cancer Research 67, 8486, September 15, 2007. doi: 10.1158/0008-5472.CAN-07-0498
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Membrane-Bound Heparin-Binding Epidermal Growth Factor–Like Growth Factor Regulates E-Cadherin Expression in Pancreatic Carcinoma Cells

Fang Wang1, Callum Sloss1, Xiaobo Zhang2, Sam W. Lee3 and James C. Cusack1

1 Division of Surgical Oncology and 2 Gastrointestinal Unit and Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts and 3 Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts

Requests for reprints: James C. Cusack, Division of Surgical Oncology, Massachusetts General Hospital and Harvard Medical School, 7th Floor, Yawkey Building, 55 Fruit Street, Boston, MA 02114. Phone: 617-724-4093; Fax: 617-724-3895; E-mail: jcusack{at}partners.org or Fang Wang, Division of Surgical Oncology, Massachusetts General Hospital and Harvard Medical School, Jackson Building 904, 55 Fruit Street, Boston, MA 02114. Phone: 617-726-5165; Fax: 617-726-8623; E-mail: fwang6{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF growth factor family. Initially synthesized as a membrane-bound precursor (pro-HB-EGF), it is cleaved at the juxtamembrane domain to release the soluble form of HB-EGF (s-HB-EGF) by sheddases, including matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases. This is a process referred to as ectodomain shedding and is implicated in the process of all ligands of the EGF receptor (EGFR) family. The tumorigenic potential of s-HB-EGF has been studied extensively; however, the role of pro-HB-EGF in tumor progression is unknown, despite the fact that a considerable amount of pro-HB-EGF remains on the cell membrane. Our data here clearly indicated the distinct role of pro-HB-EGF in the regulation of E-cadherin expression and the epithelial-mesenchymal transition. We showed here that the expression of pro-HB-EGF was associated with the differentiation status in pancreatic tumors and cell lines. Expression of noncleaved pro-HB-EGF in pancreatic cells resulted in the up-regulation of E-cadherin through suppression of ZEB1, which is a transcriptional repressor of E-cadherin. Inhibition of HB-EGF shedding using a MMP inhibitor, GM6001, also dramatically augmented the E-cadherin expression while suppressing the EGFR activation. Moreover, up-regulation of E-cadherin by pro-HB-EGF not only resulted in cellular morphologic change but also decreased cell motility and enhanced apoptotic sensitivity in response to gemcitabine-erlotinib treatment. Collectively, our data defined a distinct role of pro-HB-EGF in the regulation of E-cadherin, suggesting that inhibition of shedding may be a novel approach to suppress pancreatic metastasis and sensitize cells to cancer therapy. [Cancer Res 2007;67(18):8486–93]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF growth factor family. It is initially synthesized as a membrane-bound precursor (pro-HB-EGF), which is subsequently cleaved at the juxtamembrane domain by matrix metalloproteinases (MMP) and a disintegrin and metalloproteinases, a process often referred as ectodomain shedding (1–3). The soluble form of HB-EGF (s-HB-EGF) is then released from the cell membrane and exerts its mitogenic activity via activating the EGF receptor (EGFR) or other ErbB receptors (4–7). Recent studies have indicated that s-HB-EGF plays a key role in the acquisition of malignancy, such as tumorigenicity (8, 9) and resistance to chemotherapy (10). However, little is known of the pro-HB-EGF and its involvements in tumor development and cancer therapy despite the fact that a considerable amount of pro-HB-EGF remains on the cell membrane uncleaved.

Singh et al. (11) reported recently that pro-HB-EGF modulated hepatocyte growth factor/scatter factor (HGF/SF)–induced cellular responses in Madin-Darby canine kidney cells cells. Expression of mutated noncleavable membrane-bound HB-EGF promoted cell-matrix and cell-cell interaction, decreased cell migration, and HGF/SF–induced cell scattering. By contrast, expression of s-HB-EGF not only increased cell migration but also decreased cell-matrix and cell-cell interactions and promoted the development of long unbranched tubular structures in response to HGF/SF. These findings suggested that pro-HB-EGF and s-HB-EGF had different effects on cell-cell and cell-extracellular matrix interactions. Meanwhile, Iwamoto et al. (12) observed that when interleukin-3–dependent hematopoietic 32D cells were cocultured on a monolayer of Vero-H cells overexpressing pro-HB-EGF, growth inhibition and subsequent apoptosis were induced in the DER cells even in the presence of excess amounts of EGF or s-HB-EGF. They also showed that the inhibitory signal induced by pro-HB-EGF was mediated via EGFR and that the cytoplasmic domain of EGFR was essential for pro-HB-EGF–induced apoptosis. The involvement of pro-HB-EGF in cell-cell contact was also supported by the study showing that coexpression of pro-HB-EGF and CD9 might render the renal epithelial cells more resistant to the disruption of cell-cell and cell-matrix interactions (13).

Our study found that pro-HB-EGF is involved in the epithelial-mesenchymal transition (EMT) via regulating E-cadherin (CDH1) expression in pancreatic carcinoma cells. EMT is a central process implicated in the progression of primary tumors toward metastasis (a switch from the polarized, epithelial phenotype to a highly motile fibroblastoid or mesenchymal phenotype). Associated with these phenotypic changes, loss of E-cadherin is the hallmark of EMT (14). During tumor progression, E-cadherin can be silenced by different mechanisms, such as the promoter hypermethylation (15) or transcriptional repression (16, 17). The primary transcriptional repressors of E-cadherin are zinc finger transcription factors, including Snail, Slug, SIP/ZEB-2, and {delta}EF/ZEB-1 (18–23). These transcription factors down-regulate the expression of E-cadherin via interacting with two 5'-CACCTG (E-box) sequences of the E-cadherin promoter (19, 24).

In this study, we showed a distinct role of pro-HB-EGF in the regulation of E-cadherin. Exogenous pro-HB-EGF was sufficient to augment E-cadherin transcription through the suppression of ZEB1 expression. Furthermore, up-regulation of E-cadherin by pro-HB-EGF not only caused cell morphology change but also resulted in higher sensitivity to chemotherapy treatment. Our data suggest that inhibition of HB-EGF shedding to preserve more pro-HB-EGF may be a novel approach in cancer therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Polyclonal antibody against HB-EGF was purchased from R&D Systems, Inc., which recognizes predominantly the membrane-bound HB-EGF. Polyclonal antibodies against E-cadherin, phospho-EGFR, EGFR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively, were purchased from Cell Signaling. ZEB1 antibody was purchased from Abcam. FITC-labeled E-cadherin antibody was purchased from BD Biosciences. SMARTpool small interfering RNA (siRNA) kits against HB-EGF, ZEB1, and nontargeting scramble siRNA were purchased from Dharmacon, Inc. ZEB1-expressing construct was generously provided by Dr. Michel M. Sanders (Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN). Apoptosis assay kit Annexin V-CY5 was purchased from BD PharMingen (BD Biosciences).

Cell culture and generation of stable clones expressing different forms of HB-EGFs. Human pancreatic carcinoma cell lines, including PANC-1, Capan-1, Capan-2, BxPc-3, and MiaPaCa-2, were purchased from the American Type Culture Collection (ATCC) and cultured according to ATCC recommendation. Generation of stable PANC-1 clones expressing wild-type membrane-bound HB-EGF (wt-pro-HB-EGF) and mutated uncleaved pro-HB-EGF (mut-pro-HB-EGF) was described previously (8, 10).

Wound healing assay. The wound healing assay was done as described (25). Briefly, cells were grown to confluence in six-well plates. Cells were treated with 100 mmol/L hydroxyurea (Sigma-Aldrich) for 24 h in serum-free medium to block cell proliferation. The cross-shaped wound was made on the monolayer using a sterile 200-µL pipette tip. Cells were rinsed with PBS and maintained in serum-free medium for an additional 18 h, and the cross-shaped wounds were photographed.

Immunocytochemistry. Cells were fixed in PBS containing 4% paraformaldehyde for 20 min and washed thrice with PBS followed by permeabilization with 0.1% Triton X-100 for 5 min. Antibodies were diluted in PBS containing 5 mg/mL bovine serum albumin. In membrane localization of E-cadherin, HB-EGF was detected by labeling nonpermeablized cells. Fixed cells were incubated with primary antibodies overnight at 4°C followed by incubation with secondary antibodies for another 60 min. Images were acquired on an inverted immunofluorescence microscope (100x objective, model IX70, Olympus).

Immunohistochemistry. Tissue samples from normal human pancreas (n = 5) and pancreatic ductal adenocarcinoma (PDAC) patients (n = 20) were generously provided by Dr. Sarah Thayer (Division of General Surgery, Massachusetts General Hospital, Boston, MA) or obtained from Massachusetts General Hospital tumor bank. Freshly removed tissue samples were fixed in 10% formaldehyde solution and paraffin embedded for histologic analysis. Following antigen demasking for 20 min in the preheated water bath (95–100°C), sections were subsequently deparaffinized in xylene. Slides were then blocked with normal horse serum (diluted 1:20 in PBS) for 30 min. Antigens were detected using a standard three-step method with primary goat anti–HB-EGF antibody (1:100 working dilution), biotinylated secondary antibody (1:50 working dilution), and streptavidin-horseradish peroxidase (1:300 in PBS). After blocking the endogenous peroxidase activity by 0.3% hydrogen peroxide in methanol and after two washes in PBS, the reaction was visualized with 3,3'-diaminobenzidine (DAKO) according to the manufacturer's instructions. A negative control was set up by replacement of the polyclonal anti–HB-EGF antibody with normal goat IgG. All negative control specimens showed no nonspecific staining.

siRNA-mediated gene silencing of HB-EGF and ZEB1. Cells were grown to 60% to 80% confluence and SMARTpool siRNAs against HB-EGF or ZEB1 were transfected into cells using the TransIT-TKO (Mirus) or DharmaFECT2/4 transfection reagents (Dharmacon) according to the manufacturer's instructions. Nontargeting scramble siRNA was used as a negative control. Briefly, transfection reagent was incubated in serum-free medium for 5 min. Subsequently, the respective siRNA was added. After incubation for 15 min at room temperature, the mixture was diluted with medium and added to each well. The final concentration of each siRNA in transfection was 50 nmol/L. After incubation for 24 h, cells were washed and resuspended in fresh culture medium for an additional 24 to 48 h before harvest.

Detection of gene expression using quantitative reverse transcription-PCR. Expression of HB-EGF, E-cadherin, and ZEB1 genes were measured by quantitative reverse transcription-PCR (q-RT-PCR). Taqman-labeled specific probes of HB-EGF, E-cadherin, and ZEB1 genes were purchased from Applied Biosystems. The reaction of q-RT-PCR was done using Taqman Gene Expression Assays on Applied Biosystems 7300 Real-time PCR Systems (Applied Biosystems). Human endogenous GAPDH gene was used as control group.

Cell motility assay. Motility assay was done in 6.5-mm-diameter Transwell chambers (Costar) with a porous polycarbonate membrane (8.0 micron pore size). In the experiment, after 6 h of serum starvation, the cells (104 each per well) were seeded on the upper side of the filter in serum-free medium and 10% fetal bovine serum (FBS)–containing medium was added to the lower chambers. After 12 h, cells on the upper side of the filters were mechanically removed. Cells migrated to the lower side were fixed with 4% paraformaldehyde and stained with 0.25% Coomassie blue. The filters were photographed and the cells were counted.

Flow cytometry assay. Apoptosis was assessed by fluorescence-activated cell sorting analysis using Annexin V staining (Roche Diagnostics) as described previously (10). Flow cytometric analysis of stained cells was done on the Becton Dickinson FACSCalibur (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of membrane-bound HB-EGF in PDAC tissues and cell lines. Previous clinical studies have indicated that the pro-HB-EGF expression was correlated with small size and early-stage tumor formation and inversely related to lymph node metastasis in colonic neoplasm, hepatocellular carcinoma, and breast cancer (26–29). To investigate the pro-HB-EGF expression in pancreatic cancer, we first analyzed the expression of HB-EGF in PDAC tissue samples using immunohistochemistry. The primary antibody against HB-EGF recognized predominantly the pro-HB-EGF. Stronger HB-EGF immunoreactivity was observed in well-differentiated and moderately differentiated PDAC tissues, whereas faint HB-EGF staining was present in normal pancreatic tissue and poorly differentiated PDACs (Fig. 1A ). Then, the expression of HB-EGF mRNA was analyzed in a panel of pancreatic carcinoma cell lines with different differentiation status (30) compared with normal pancreas using q-RT-PCR. As shown in Fig. 1B, higher expression of HB-EGF mRNA was observed in well-differentiated to moderately differentiated cell lines, including Capan-1, Capan-2, and BxPc-3 cells, whereas in poorly differentiated cell lines, HB-EGF mRNA level was relatively low (in PANC-1 cells) or undetectable (in MiaPaCa-2 cells). Similar results were obtained at the protein level, as we observed that the expression of pro-HB-EGF protein was relatively higher in well-differentiated to moderately differentiated cell lines compared with poorly differentiated cell lines using Western blotting (Fig. 1C). These data indicated that pro-HB-EGF expression in pancreatic carcinoma cells was associated with cell differentiation status.


Figure 1
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  		Figure 1. HB-EGF mRNA and protein expression in human PDAC tissues (A) and in human pancreatic cancer cell lines (B and C). A, HB-EGF immunostaining of specimens from normal pancreas and PDACs. HB-EGF immunoreactivity was faint in normal pancreas and poorly differentiated PDACs, whereas it was abundant in well-differentiated to moderately differentiated PDACs. Original magnification, x20. B, HB-EGF mRNA expression was analyzed by q-RT-PCR in normal pancreas and pancreatic carcinoma cell lines, including Capan-1, Capan-2, and BxPc-3 (well differentiated to moderately differentiated) and PANC-1 and MiaPaCa-2 (poorly differentiated). C, pro-HB-EGF protein was analyzed by Western blotting in normal pancreas and pancreatic carcinoma cell lines. Housekeeping gene GAPDH was used as internal control. Pro-HB-EGF was highly expressed in well-differentiated to moderately differentiated cell lines.

 
HB-EGF expression changes cell morphology via up-regulation of E-cadherin. Because the process of cell differentiation is often related to EMT, we attempted to investigate the potential role of pro-HB-EGF in the regulation of EMT-related gene expression. To differentiate the roles of membrane-bound and s-HB-EGF, we developed stably transfected PANC-1 cells expressing mutated uncleaved pro-HB-EGF (PANC-1/mut-pro-HB-EGF) and vector-transfected control (PANC-1/vector). Meanwhile, Capan-1 cells were also transiently transfected with vector or mut-pro-HB-EGF constructs. We detected stronger expression of E-cadherin in mut-pro-HB-EGF–expressing cells compared with vector control cells by use of Western blotting and q-RT-PCR (Fig. 2A and B ). When cells were grown on collagen-coated two-dimensional surface, PANC-1/mut-pro-HB-EGF cells retained tightly packed cuboidal epithelial-like morphology, whereas PANC-1/vector cells exhibited a fibroblastic spindle-shaped morphology (Fig. 2C). Immunocytochemistry was further done to monitor the subcellular distribution of E-cadherin (Fig. 2D). The induced E-cadherin by mut-pro-HB-EGF expression was observed exclusively in the cellular basolateral membrane, whereas, in the vector control cells, E-cadherin was stained randomly in and around the cytoplasm. Because mut-pro-HB-EGF and E-cadherin were both present at the cell membrane, we attempted to detect whether mut-pro-HB-EGF could physically interact with E-cadherin using coimmunoprecipitation. However, we did not observe any interaction between E-cadherin and mut-pro-HB-EGF or wt-pro-HB-EGF (data not shown).


Figure 2
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  		Figure 2. Pro-HB-EGF expression changes cell morphology via up-regulating E-cadherin expression. A, mut-pro-HB-EGF induces the expression of E-cadherin protein in PANC-1 and Capan-1 cells. B, mut-pro-HB-EGF expression promoted the transcriptional activity of E-cadherin. The mRNA level of E-cadherin in PANC-1 and Capan-1 cells was measured by q-RT-PCR. GAPDH expression was used as endogenous control. C, expression of mut-pro-HB-EGF induces morphologic change in PANC-1 cells. Phase-contrast images of cells (PANC-1/vector and PANC-1/mut-pro-HB-EGF) were cultured for 3 d on collagen I–coated plate. Mut-pro-HB-EGF–expressing cells show highly packed cuboidal epithelial-like morphology, whereas vector cells form a fibroblastic spindle-like morphology. D, immunofluorescent staining of PANC-1/vector– and PANC-1/mut-pro-HB-EGF–expressing cells. Fluorescence signals specific to E-cadherin antibody was visualized as green, and fluorescence signals specific to HB-EGF were visualized as red. DAPI (blue) was used to indicate nuclear. The induced E-cadherin by mut-pro-HB-EGF was expressed exclusively in the cellular basolateral membrane, whereas, in the vector control cells, E-cadherin was stained randomly in and around the cytoplasm.

 
Suppression of endogenous HB-EGF expression using siRNA down-regulates E-cadherin expression. To investigate the role of endogenous HB-EGF in the regulation of E-cadherin expression, we transiently transfected PANC-1, BxPc-3, and Capan-2 cells with siRNA against the HB-EGF gene. Seventy-two hours later, we observed that endogenous HB-EGF and E-cadherin expression were both suppressed as measured by q-RT-PCR and Western blotting (Fig. 3A and B ). These data suggested that HB-EGF expression was necessary to sustain E-cadherin expression.


Figure 3
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  		Figure 3. siRNA-mediated silencing of HB-EGF gene results in the down-regulation of E-cadherin expression. A, PANC-1, BxPc-3, and Capan-2 cells were transiently transfected with SMARTpool siRNA against HB-EGF. Nontargeting scramble siRNA was used as negative control. Seventy-two hours later, the mRNA was extracted and the expression of HB-EGF and E-cadherin mRNA was analyzed by q-RT-PCR. B, the protein level of HB-EGF and E-cadherin was analyzed by Western blotting after transient transfection with siRNA against HB-EGF. Depletion of HB-EGF resulted in the inhibition of E-cadherin expression.

 
Mut-pro-HB-EGF up-regulates E-cadherin due to the suppression of ZEB1 expression. Two major causes of aberrant E-cadherin expression have been established: the promoter hypermethylation (15) and the transcription repression (16, 17). In PANC-1 cells, the promoter of E-cadherin gene remains intact and the expression of E-cadherin is regulated by collagen type I and II (31). Based on these findings, we suspected that pro-HB-EGF expression might regulate the transcriptional repressor genes. Because the primary transcriptional repressors that suppress E-cadherin expression are zinc finger proteins including the members of Slug/Snail family, we attempted to detect the expression level of these repressors in response to mut-pro-HB-EGF expression. We did not observe any significant change of Snail, Slug, Twist, or SIP mRNA or protein in mut-pro-HB-EGF cells as evidenced by Western blotting or q-RT-PCR (data not shown). However, ZEB1 expression was suppressed by mut-pro-HB-EGF, as we observed a 50% inhibition of the endogenous ZEB1 expression in PANC-1/mut-pro-HB-EGF cells compared with PANC-1/vector control (Fig. 4A ). Transient transfection of siRNA against the endogenous ZEB1 gene in PANC-1 cells resulted in a 4.2-fold increase of the endogenous E-cadherin expression compared with scramble siRNA control transfection (Fig. 4B). We then transiently transfected cells with ZEB1-expressing construct and observed that ZEB1 dramatically down-regulated E-cadherin expression in both mut-pro-HB-EGF cells and vector control PANC-1 cells (Fig. 4C). Meanwhile, depletion of HB-EGF expression by use of siRNA resulted in the up-regulation of ZEB1 in PANC-1, BxPc-3, and Capan-2 cells as measured by q-RT-PCR (Fig. 4D). These data suggested the potential link of ZEB1 in pro-HB-EGF–mediated E-cadherin induction, although the mechanism underlying this regulation is unknown.


Figure 4
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  		Figure 4. Mut-pro-HB-EGF promotes E-cadherin expression via suppressing ZEB1 expression. A, mut-pro-HB-EGF expression down-regulated the transcriptional activity of ZEB1 by 50% as confirmed by q-RT-PCR in PANC-1/mut-pro-HB-EGF cells compared with PANC-1/vector cells. B, suppression of endogenous ZEB1 expression using siRNA increased the endogenous mRNA level of E-cadherin by 4.2-fold as supported by q-RT-PCR. C, exogenous expression of ZEB1 abrogated the E-cadherin expression in both PANC-1/vector and PANC-1/mut-pro-HB-EGF cells. D, PANC-1, BxPc-3, and Capan-2 cells were transiently transfected with siRNA against HB-EGF. Seventy-two hours later, the expression of ZEB1 mRNA was analyzed by q-RT-PCR. Suppression of endogenous HB-EGF expression using siRNA increased the transcriptional activity of ZEB1.

 
Inhibition of the ectodomain shedding by MMP inhibitor up-regulates E-cadherin expression. Lu et al. (32) showed previously that activation of EGFR inhibited E-cadherin expression through up-regulation of Snail. Therefore, we suspected that inhibition of HB-EGF shedding using MMP inhibitor might produce more pro-HB-EGF and abrogate EGFR activation, resulting in the up-regulation of E-cadherin. As we anticipated, treatment of GM6001, a broad range of MMP inhibitor that has been shown to inhibit HB-EGF shedding (10, 33, 34), resulted in a dramatic increase of E-cadherin in wt-pro-HB-EGF–expressing cells (Fig. 5A ). Although the endogenous pro-HB-EGF expression remains very low in PANC-1 cells, the treatment of GM6001 still had an effect on E-cadherin induction, as we observed the increased level of pro-HB-EGF along with E-cadherin up-regulation under GM6001 treatment (Fig. 5A). These data indicated the importance of endogenous pro-HB-EGF in the regulation of E-cadherin expression. Meanwhile, the phosphorylation of EGFR was dramatically diminished in the presence of GM6001, which led us to suspect that direct inhibition of EGFR activation may also augment E-cadherin expression. This was confirmed by the treatment of EGFR inhibitor AG1478, which resulted in the inhibition of EGFR phosphorylation and up-regulation of E-cadherin expression in both wt-pro-HB-EGF cells and vector cells (Fig. 5A). Inhibition of EGFR signaling transduction and shedding activity also had a dramatic effect on cell morphology, as we observed that treatment of AG1478 or GM6001 switched cells from fibroblast-like to epithelial-like morphology on collagen-coated culture surface (Fig. 5B).


Figure 5
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  		Figure 5. Inhibition of HB-EGF shedding by use of GM6001 up-regulates E-cadherin via suppressing the EGFR activation. A, PANC-1/vector and PANC-1/wt-pro-HB-EGF cells were treated with MMP inhibitor (GM6001, 10 µmol/L) or EGFR inhibitor (AG1478, 10 µmol/L) for indicated time. The expression of E-cadherin, phospho-EGFR, and total EGFR and the shedding of HB-EGF were detected using Western blotting. Inhibition of MMP activity abolished HB-EGF shedding as supported by the disappearance of tail fragment (10–15 kDa, representing the proteolytic COOH-terminal fragments) and the increased pro-HB-EGF expression (29 kDa). Treatment of GM6001 or AG1478 both augmented wt-pro-HB-EGF–induced E-cadherin expression via inhibiting EGFR activation. B, treatment of GM6001 or AG1478 induced the morphology changes. Phase-contrast images of cells (PANC-1/vector and PANC-1/mut-pro-HB-EGF) cultured for 3 d on collagen I–coated plate in the presence of GM6001 (10 µmol/L) or AG1478 (10 µmol/L). PANC-1/wt-pro-HB-EGF–expressing cells showed highly packed cuboidal epithelial-like morphology, whereas vector cells exhibited the fibroblastic morphology. C, mut-pro-HB-EGF expression inhibited cell migration. A cross-shaped wound was created in the monolayer of PANC-1 cells and the representative digital images of the wounded region before and after the incubation periods were visualized. D, effects of mut-pro-HB-EGF expression on motility of PANC-1 cells were assessed using BD Transwell chamber assays. Cells that migrated to the filter lower side were stained, photographed, and counted. Histogram showed the inhibition of motility in PANC-1/mut-pro-HB-EGF cells by 70% compared with PANC-1/vector cells. The cells in eight different fields were counted.

 
Pro-HB-EGF down-regulates cell migration and motility. To determine whether this up-regulation of E-cadherin was reflected in the change of cell motility, we first evaluated the ability of migration of PANC-1/vector cells and PANC-1/mut-pro-HB-EGF cells. Cells were grown to reach confluence and treated with hydroxyurea for 24 h in serum-free medium. A cross-shaped wound was created in the monolayer, and cells were cultured in the serum-free medium for another 18 h. Figure 5C showed the representative digital images of the wounded region before and after the incubation time. Our data showed that mut-pro-HB-EGF expression significantly reduced the migration ability compared with vector cells. Similar results were obtained when motility assay was done. The PANC-1/mut-pro-HB-EGF cells showed less ability to invade the basement membrane (70% decrease) even in presence of 10% FBS compared with PANC-1/vector cells (Fig. 5D).

Mut-pro-HB-EGF enhances gemcitabine/erlotinib–induced apoptosis. Studies have shown the correlation between sensitivity to gefitinib (EGFR inhibitor) and the expression of E-cadherin and ZEB1, suggesting their predictive value for the responsiveness to EGFR tyrosine kinase inhibitors. The up-regulation of E-cadherin expression increased the sensitivity to EGFR inhibition in vitro and in vivo in non–small cell lung carcinoma (35–37). Based on our findings, we suspected that mut-pro-HB-EGF expression may augment the sensitivity of chemotherapy and EGFR inhibition therapy. We treated PANC-1 cells with erlotinib (EGFR inhibitor) alone or combined with gemcitabine, which has been approved for locally advanced pancreatic cancer or metastatic cancer patients. The effect of mut-pro-HB-EGF on apoptosis in the presence of gemcitabine/erlotinib was analyzed by flow cytometry. As shown in Fig. 6A , we observed the increased apoptotic sensitivity in mut-pro-HB-EGF cells in all three treatments (a 1.73-fold increase in gemcitabine treatment, a 1.36-fold increase in erlotinib treatment, and a 1.81-fold increase in gemcitabine/erlotinib combination) compared with the same treatments in control groups. Pretreatment of GM6001 to inhibit shedding activity also achieved higher sensitivity in all three treatments (a 1.57-fold increase in gemcitabine treatment, a 1.87-fold increase in erlotinib treatment, and a 2.41-fold increase in gemcitabine/erlotinib combination) compared with the group without GM6001 pretreatment (Fig. 6A). These data indicated that up-regulation of E-cadherin by pro-HB-EGF enhanced the apoptotic response to gemcitabine/erlotinib.


Figure 6
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  		Figure 6. Mut-pro-HB-EGF modulates gemcitabine/erlotinib induced apoptosis (A) and the model of the roles of HB-EGF in the regulation of EMT (B). A, PANC-1/vector and PANC-1/mut-pro-HB-EGF cells were treated with gemcitabine (Gem; 1 µg/mL) and/or erlotinib (1 µmol/L) for 48 h. Cells were stained with Annexin V and 7-amino-actinomycin D. Chemotherapy-induced apoptosis was measured by flow cytometry. Mut-pro-HB-EGF expression increased the chemosensitivity of PANC-1 cells to gemcitabine/erlotinib–induced apoptosis compared with PANC-1/vector cells. Pretreatment of GM6001 also increased the chemotherapy sensitivity in all three treatments compared with the group without GM6001 pretreatment. B, model of the distinct roles of HB-EGF in the regulation of EMT. s-HB-EGF activates EGFR or other ErbB receptors to promote cell proliferation, tumorigenesis, and EMT. In contrast, pro-HB-EGF up-regulates E-cadherin via suppressing ZEB1 expression, which results in the negative regulation of EMT and sensitizes cells to chemotherapy. Therefore, the shedding activity of HB-EGF is a rather critical step in balancing the protumorigenesis or antitumorigenesis function of HB-EGF and other ligands of EGFR family. ADAMs, a disintegrin and metalloproteinases.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our interest in pro-HB-EGF stems from a previous study by Ongusaha et al. (8) and prior findings in our laboratory that suggested the dual roles of s-HB-EGF and pro-HB-EGF in the regulation of tumor progression and chemotherapy response (10). s-HB-EGF has been extensively studied as a growth factor of EGF family as it exerts the mitogenic function via activation of the EGFR signaling transduction. However, a considerable amount of pro-HB-EGF retains on the cell membrane, and the functional consequence of pro-HB-EGF is largely unknown. Our results as well as the work of others (8, 11, 12, 38) suggest that s-HB-EGF and pro-HB-EGF have distinct roles in tumor development, especially in the regulation of cell-cell contact and chemotherapy sensitivity.

In our study, we observed a significant increase of E-cadherin mRNA as well as protein level in mut-pro-HB-EGF–expressing cells. The induced E-cadherin seemed to localize on the cell membrane and promoted the E-cadherin expression. Inhibition of endogenous HB-EGF expression by siRNA clearly showed the down-regulation of E-cadherin expression. Moreover, the expression of E-cadherin was negatively associated with EGFR activation and inhibition of EGFR activation either by EGFR inhibitor (AG1478) or by MMP inhibitor (GM6001) significantly restored E-cadherin expression.

Multiple zinc finger repressors, including Snail, Slug, Twist, ZEB1, and SIP, negatively regulate E-cadherin transcription and this mechanism accounts for most cases of transcriptional repression of E-cadherin (14, 16, 17, 19, 20, 22, 23). Of those well-known transcription repressors, we found that only the ZEB1 gene expression was suppressed by mut-pro-HB-EGF, and the inhibition of endogenous HB-EGF expression by siRNA was accompanied by 1.6-fold increase of the endogenous ZEB1 mRNA and decreased E-cadherin expression. Exogenous ZEB1 restored the suppression of E-cadherin expression in mut-pro-HB-EGF cells, indicating that mut-pro-HB-EGF regulates E-cadherin expression via suppressing ZEB1 expression. However, the mechanism underlying such regulation is unknown yet.

The involvement of pro-HB-EGF in EMT was most evident in the morphologic change and its effect on cell migration and motility. Mut-pro-HB-EGF–expressing cells retained epithelial-like shape compared with vector cells with fibroblastic morphology when cultured on the collagen-coated surface. Cell motility was also suppressed by mut-pro-HB-EGF expression as supported by wound healing assay and cell motility assay. Most importantly, exogenous mut-pro-HB-EGF expression achieved higher sensitivity to gemcitabine/erlotinib–induced apoptosis compared with the vector group. Suppression of MMP activity by GM6001 pretreatment also dramatically enhanced gemcitabine/erlotinib–induced apoptosis compared with vector groups. These data provided strong evidence indicating the applicability of combining MMP inhibitors in pancreatic cancer treatment.

There are several clinical studies indicating the negative involvement of pro-HB-EGF and metastasis. In colonic neoplasm, hepatocellular carcinoma, breast cancer, and pancreatic adenocarcinoma, pro-HB-EGF expression was correlated with small size and early-stage tumor formation and inversely related to lymph node metastasis (26–29). In these studies, immunohistochemistry had been used to detect the expression of HB-EGF in patients with different cancers, and the expression of HB-EGF detected in tumor samples was predominantly membrane-bound HB-EGF. These data coincided with our immunohistochemical study of the pancreatic tumors and cell lines, which indicated that the expression of pro-HB-EGF was associated with differentiation status and might be negatively involved in the regulation of EMT. However, it remains unclear whether the transcription activity of HB-EGF gradually decreases during tumor progression or rather it is the shedding activity increased so that the pro-HB-EGF is not detectable at late stage of tumor development? Further studies are needed to address these questions.

Based on our findings, we propose a model of HB-EGF in tumor development (Fig. 6B). After shedding, the s-HB-EGF activates EGFR or other ErbB receptors to promote cell proliferation, tumorigenesis, and EMT. In contrast, pro-HB-EGF up-regulates E-cadherin via suppression of ZEB1 expression, which results in the negative control of EMT and sensitizes cells to chemotherapy. Therefore, the shedding activity of HB-EGF is a rather critical step in balancing the protumorigenesis or antitumorigenesis function of HB-EGF, and inhibition of shedding activity may represent a critical target to inhibit pancreatic metastasis and promote the efficacy of cancer therapy.


   Acknowledgments
 
Grant support: NIH grants CA77278-01A1 and CA98871-01 (J.C. Cusack).

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.

We thank Dr. Gregory Lauwers for his assistance with the histopathology.

Received 2/ 5/07. Revised 6/19/07. Accepted 6/29/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Izumi Y, Hirata M, Hasuwa H, et al. A metalloprotease-disintegrin, MDC9/meltrin-{gamma}/ADAM9 and PKC{delta} are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J 1998;17:7260–72.[CrossRef][Medline]
  2. Asakura M, Kitakaze M, Takashima S, et al. Cardiac hypertrophy is inhibited by antagonism of ADAM12 processing of HB-EGF: metalloproteinase inhibitors as a new therapy. Nat Med 2002;8:35–40.[CrossRef][Medline]
  3. Higashiyama S, Nanba D. ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk. Biochim Biophys Acta 2005;1751:110–7.[Medline]
  4. Freeman MR, Yoo JJ, Raab G, et al. Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder. J Clin Invest 1997;99:1028–36.[Medline]
  5. Elenius K, Paul S, Allison G, Sun J, Klagsbrun M. Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation. EMBO J 1997;16:1268–78.[CrossRef][Medline]
  6. Raab G, Klagsbrun M. Heparin-binding EGF-like growth factor. Biochim Biophys Acta 1997;1333:F179–99.[Medline]
  7. Iwamoto R, Yamazaki S, Asakura M, et al. Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function. Proc Natl Acad Sci U S A 2003;100:3221–6.[Abstract/Free Full Text]
  8. Ongusaha PP, Kwak JC, Zwible AJ, et al. HB-EGF is a potent inducer of tumor growth and angiogenesis. Cancer Res 2004;64:5283–90.[Abstract/Free Full Text]
  9. Miyamoto S, Hirata M, Yamazaki A, et al. Heparin-binding EGF-like growth factor is a promising target for ovarian cancer therapy. Cancer Res 2004;64:5720–7.[Abstract/Free Full Text]
  10. Wang F, Liu R, Lee SW, Sloss CM, Couget J, Cusack JC. Heparin-binding EGF-like growth factor is an early response gene to chemotherapy and contributes to chemotherapy resistance. Oncogene 2007;26:2006–14.[CrossRef][Medline]
  11. Singh AB, Tsukada T, Zent R, Harris RC. Membrane-associated HB-EGF modulates HGF-induced cellular responses in MDCK cells. J Cell Sci 2004;117:1365–79.[Abstract/Free Full Text]
  12. Iwamoto R, Handa K, Mekada E. Contact-dependent growth inhibition and apoptosis of epidermal growth factor (EGF) receptor-expressing cells by the membrane-anchored form of heparin-binding EGF-like growth factor. J Biol Chem 1999;274:25906–12.[Abstract/Free Full Text]
  13. Takemura T, Hino S, Murata Y, et al. Coexpression of CD9 augments the ability of membrane-bound heparin-binding epidermal growth factor-like growth factor (proHB-EGF) to preserve renal epithelial cell viability. Kidney Int 1999;55:71–81.[Medline]
  14. Kang Y, Massague J. Epithelial-mesenchymal transitions: twist in development and metastasis. Cell 2004;118:277–9.[CrossRef][Medline]
  15. Auerkari EI. Methylation of tumor suppressor genes p16(INK4a), p27(Kip1), and E-cadherin in carcinogenesis. Oral Oncol 2006;42:5–13.[CrossRef][Medline]
  16. Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol 2005;17:548–58.[CrossRef][Medline]
  17. Vernon AE, LaBonne C. Tumor metastasis: a new twist on epithelial-mesenchymal transitions. Curr Biol 2004;14:R719–21.[CrossRef][Medline]
  18. Cano A, Perez-Moreno MA, Rodrigo I, et al. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2000;2:76–83.[CrossRef][Medline]
  19. Hajra KM, Chen DY, Fearon ER. The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res 2002;62:1613–8.[Abstract/Free Full Text]
  20. Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H. NF-{kappa}B represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 2007;26:711–24.[CrossRef][Medline]
  21. Krishnamachary B, Zagzag D, Nagasawa H, et al. Hypoxia-inducible factor-1-dependent repression of E-cadherin in von Hippel-Lindau tumor suppressor-null renal cell carcinoma mediated by TCF3, ZFHX1A, ZFHX1B. Cancer Res 2006;66:2725–31.[Abstract/Free Full Text]
  22. Guaita S, Puig I, Franci C, et al. Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression. J Biol Chem 2002;277:39209–16.[Abstract/Free Full Text]
  23. Pena C, Garcia JM, Silva J, et al. E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations. Hum Mol Genet 2005;14:3361–70.[Abstract/Free Full Text]
  24. Comijn J, Berx G, Vermassen P, et al. The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 2001;7:1267–78.[CrossRef][Medline]
  25. Gambarotta G, Garzotto D, Destro E, et al. ErbB4 expression in neural progenitor cells (ST14A) is necessary to mediate neuregulin-1ß1-induced migration. J Biol Chem 2004;279:48808–16.[Abstract/Free Full Text]
  26. Ito Y, Higashiyama S, Takeda T, Okada M, Matsuura N. Bimodal expression of heparin-binding EGF-like growth factor in colonic neoplasms. Anticancer Res 2001;21:1391–4.[Medline]
  27. Ito Y, Takeda T, Higashiyama S, et al. Expression of heparin binding epidermal growth factor-like growth factor in hepatocellular carcinoma: an immunohistochemical study. Oncol Rep 2001;8:903–7.[Medline]
  28. Ito Y, Higashiyama S, Takeda T, Yamamoto Y, Wakasa KI, Matsuura N. Expression of heparin-binding epidermal growth factor-like growth factor in pancreatic adenocarcinoma. Int J Gastrointest Cancer 2001;29:47–52.[CrossRef][Medline]
  29. Ito Y, Takeda T, Higashiyama S, Noguchi S, Matsuura N. Expression of heparin-binding epidermal growth factor-like growth factor in breast carcinoma. Breast Cancer Res Treat 2001;67:81–5.[CrossRef][Medline]
  30. Sipos B, Moser S, Kalthoff H, Torok V, Lohr M, Kloppel G. A comprehensive characterization of pancreatic ductal carcinoma cell lines: towards the establishment of an in vitro research platform. Virchows Arch 2003;442:444–52.[Medline]
  31. Menke A, Philippi C, Vogelmann R, et al. Down-regulation of E-cadherin gene expression by collagen type I and type III in pancreatic cancer cell lines. Cancer Res 2001;61:3508–17.[Abstract/Free Full Text]
  32. Lu Z, Ghosh S, Wang Z, Hunter T. Downregulation of caveolin-1 function by EGF leads to the loss of E-cadherin, increased transcriptional activity of ß-catenin, and enhanced tumor cell invasion. Cancer Cell 2003;4:499–515.[CrossRef][Medline]
  33. Xu KP, Ding Y, Ling J, Dong Z, Yu FS. Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells. Invest Ophthalmol Vis Sci 2004;45:813–20.[Abstract/Free Full Text]
  34. Zhao Y, He D, Saatian B, et al. Regulation of lysophosphatidic acid-induced epidermal growth factor receptor transactivation and interleukin-8 secretion in human bronchial epithelial cells by protein kinase C{delta}, Lyn kinase, and matrix metalloproteinases. J Biol Chem 2006;281:19501–11.[Abstract/Free Full Text]
  35. Thomson S, Buck E, Petti F, et al. Epithelial to mesenchymal transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res 2005;65:9455–62.[Abstract/Free Full Text]
  36. Witta SE, Gemmill RM, Hirsch FR, et al. Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res 2006;66:944–50.[Abstract/Free Full Text]
  37. Yauch RL, Januario T, Eberhard DA, et al. Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin Cancer Res 2005;11:8686–98.[Abstract/Free Full Text]
  38. Nakamura K, Iwamoto R, Mekada E. Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria toxin receptor-associated protein (DRAP27)/CD9 form a complex with integrin {alpha}3ß1 at cell-cell contact sites. J Cell Biol 1995;129:1691–705.[Abstract/Free Full Text]



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