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[Cancer Research 66, 7653-7660, August 1, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Epidermal Growth Factor Receptor–Related Protein Inhibits Cell Growth and Invasion in Pancreatic Cancer

Zhiwei Wang1, Radha Sengupta3, Sanjeev Banerjee1, Yiwei Li1, Yuxiang Zhang4, K.M. Wahidur Rahman1, Amro Aboukameel2, Ramzi Mohammad2, Adhip P.N. Majumdar3, James L. Abbruzzese5 and Fazlul H. Sarkar1

1 Departments of Pathology and 2 Internal Medicine, Division of Hematology and Oncology, Karmanos Cancer Institute, Wayne State University; 3 Department of Internal Medicine, Veterans Affairs Medical Center, Detroit, Michigan; 4 Department of Biochemistry and Molecular Biology, Capital University of Medical Sciences, Beijing, China; and 5 Department of Gastrointestinal Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Fazlul H. Sarkar, Department of Pathology, Wayne State University, 740 HWCRC, 110 East Warren Avenue, Detroit, MI 48201. Phone: 313-576-8327; Fax: 313-576-8389; E-mail: fsarkar{at}med.wayne.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The epidermal growth factor receptor (EGFR) signaling network plays critical roles in human cancers, including pancreatic cancer, suggesting that the discovery of specific agents targeting EGFR would be extremely valuable for pancreatic cancer therapy. EGFR-related protein (ERRP), a recently identified pan-erbB inhibitor, has been shown to inhibit growth and induce apoptosis of pancreatic cancer cells in vitro and tumor growth in a xenograft model. However, the precise molecular mechanism(s) by which ERRP exerts its antitumor activity remains unclear. The current investigation was undertaken to delineate the tumor growth inhibitory mechanism(s) of ERRP in pancreatic cancer cells. Using multiple molecular assays, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, apoptosis, gene transfection, real-time reverse transcription-PCR, Western blotting, invasion, and electrophoretic mobility shift assay for measuring DNA-binding activity of nuclear factor-{kappa}B (NF-{kappa}B), we found that ERRP caused marked inhibition of pancreatic cancer cell growth. This was accompanied by increased apoptosis and concomitant attenuation of Notch-1 and NF-{kappa}B and down-regulation of NF-{kappa}B downstream genes, such as matrix metalloproteinase-9 and vascular endothelial growth factor, resulting in the inhibition of pancreatic cancer cell invasion through the Matrigel. We also found that down-regulation of Notch-1 by small interfering RNA before ERRP treatment resulted in enhanced cell growth inhibition and apoptosis. Our data suggest that the ERRP-mediated inactivation of EGFR, Notch-1, NF-{kappa}B, and its downstream target genes contributed to the inhibition of cell growth and invasion. We conclude that ERRP could be an effective agent for inhibiting tumor growth and invasion for the treatment of pancreatic cancer. (Cancer Res 2006; 66(15): 7653-60)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pancreatic cancer is one of most common cancers and the fourth leading cause of cancer-related deaths in the United States with about 30,000 newly diagnosed cases per year (1). This could be due to its late diagnosis and a lack of effective treatment options. Presently, surgical resection provides the only option for better survival. Unfortunately, only 9% of patients with pancreatic cancer undergo resection; yet, the 5-year survival rate is only 15% to 25% after resection and only 5% for all pancreatic cancer (1). Therefore, there is a dire need for the development and evaluation of novel targeted therapeutic agents for improving the outcome of patients diagnosed with this deadly disease.

Pancreatic cancer like many other tumors has been shown to overexpress the epidermal growth factor receptor (EGFR) and/or its family members (2–4). Therefore, novel avenues by which EGFRs could be inactivated represent a promising strategy for the development of novel and selective anticancer therapies. EGFRs are a transmembrane tyrosine kinase protein. After ligand binding, EGFR dimerizes, either as a homodimer or heterodimer with other members of the EGFR family. EGFR is then auto-phosphorylated or trans-phosphorylated at specific tyrosine residues for its activation, resulting in the activation of multiple downstream signaling cascades, including phosphatidylinositol 3'-kinase and AKT, extracellular signal-regulated kinase, and the Notch pathway, ultimately leading to increased cellular proliferation and prevention of programmed cell death (2, 3, 5). Therefore, blockade of EGFRs activity should destroy EGFR-mediated signal transduction pathways and should result in the suppression of tumor growth (6, 7).

EGFR-related peptide (ERRP), a recently isolated pan-erbB inhibitor that targets multiple members of the EGFR family, seems to attenuate EGFR activation (8). We have found that recombinant ERRP inhibits the growth of a variety of cancer cells, including colon, prostate, and pancreas in vitro and tumor growth in vivo (9–12). Although there has been rapid progress for elucidating the mechanism of action of ERRP as an antitumor agent, the exact mechanism has not yet been fully established. We investigated whether ERRP-induced inhibition of pancreatic cancer cell growth could be attributed to EGFR and its downstream signaling, especially inactivation of Notch-1 and nuclear factor-{kappa}B (NF-{kappa}B) activity. Moreover, because cell migration and invasion are important processes that are involved in tumor development and metastasis, and because EGFR signaling is known to control these processes, we also examined the effect of ERRP on the processes of invasion of pancreatic cancer cells.

Overall in the present study, we investigated the role and mechanism(s) by which ERRP may lead to the attenuation of Notch-1, an EGFR downstream signaling, thereby inhibiting the growth of pancreatic cancer cells. We found that ERRP down-regulated the expression of Notch-1 and its downstream genes, including matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF), resulting in the inhibition of pancreatic cancer cell growth and invasion, which is believed to be mediated through the inactivation of the DNA-binding activity of NF-{kappa}B.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and experimental reagents. Human pancreatic cancer cell lines BxPC-3, HPAC, and PANC-1 were obtained from the American Type Culture Collection (Manassas, VA). Cell death ELISA kit was obtained from Roche (Indianapolis, IN). Primary antibodies for Notch-1, Hes-1, VEGF, MMP-9, Survivin, cyclooxygenase-2 (COX-2), and cyclin D1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All secondary antibodies were obtained from Pierce (Rockford, IL). Notch-1 small interfering RNA (siRNA) and control siRNA were obtained from Santa Cruz Biotechnology. LipofectAMINE 2000 was purchased from Invitrogen (Carlsbad, CA). Chemiluminescence detection of proteins was done with the use of a kit from Amersham Biosciences/Amersham Pharmacia Biotech (Piscataway, NJ). Protease inhibitor cocktail, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and all other chemicals were obtained from Sigma (St. Louis, MO).

Generation of recombinant ERRP. ERRP fusion protein was generated using the Drosophila expression system (Invitrogen) as described previously, and silver staining of the SDS-PAGE resulted in a predominant protein band of Mr 53 to 55 following purification (13, 14). The protein band of Mr 53 to 55 corresponded well with the calculated molecular mass of ERRP, which is composed of 479 amino acids. In the absence of CuSO4, no 53- to 55-kDa protein was detected. Immunoaffinity purified ERRP was used in all experiments.

Cell culture and growth assay. Human pancreatic cancer cell lines BxPC-3, HPAC, and PANC-1 were cultured in RPMI 1640 supplemented with 5% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO2 atmosphere at 37°C. In some experiments, cells were cultured in serum-free medium wherever indicated. Transforming growth factor-{alpha} (TGF-{alpha}; 7 nmol/L; Invitrogen) or heparin binding EGF (HB-EGF; 5 nmol/L, Sigma) was added to the medium whenever necessary as indicated under figure legend. To perform the growth assays, cells were incubated overnight at a density of 5,000 per well in 96-well plates and subsequently incubated with MTT reagent (0.5 mg/mL) at 37°C for 2 hours, and MTT assay was done as described earlier (14, 15).

Histone/DNA ELISA for detection of apoptosis. The Cell Death Detection ELISA kit was used for assessing apoptosis according to the manufacturer's protocol. Briefly, cells were treated with 5 µg/mL ERRP for different periods of time. After treatment, the cells were lysed, and the cell lysates were overlaid and incubated in microtiter plate modules coated with anti-histone antibody for detection of apoptosis as described earlier (16).

Real-time reverse transcription-PCR analysis for gene expression. The total RNA was isolated by Trizol (Invitrogen) and purified by RNeasy Mini kit and RNase-free DNase Set (Qiagen, Valencia, CA) according to the manufacturer's protocols. One microgram of total RNA from each sample was subjected to first strand cDNA synthesis using Taqman reverse transcription reagents kit (Applied Biosystems, Foster City, CA) in a total volume of 50 µL, including 6.25 units MultiScribe reverse transcriptase and 25 pmol random hexamers. Reverse transcription reaction and real-time PCR amplications were done as described earlier (17).

Western blot analysis. Cells were lysed in lysis buffer [50 mmol/L Tris (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 0.5% NP40, 0.5% Triton X-100, 2.5 mmol/L sodium orthovanadate, 10 µL/mL protease inhibitor cocktail, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF)] by incubating for 20 minutes at 4°C. The protein concentration was determined using the Bio-Rad assay system (Bio-Rad, Hercules, CA). Total proteins were fractionated using SDS-PAGE and transferred onto a nitrocellulose membrane for Western blotting as described earlier (17).

Electrophoretic mobility shift assay for measuring NF-{kappa}B activity. Cells exposed to ERRP or kept as control were washed with cold PBS and suspended in 0.15 mL of lysis buffer [10 mmol/L HEPES (pH 7.9), 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 2 µg/mL leupeptin, 2 µg/mL aprotinin, and 0.5 mg/mL benzamidine]. The nuclear protein was prepared and subjected to DNA-binding activity of NF-{kappa}B by electrophoretic mobility shift assay (EMSA) as described earlier (17).

Plasmids and transfections. The Notch-1 cDNA plasmid encoding the Notch-1 intracellular domain was a kind gift from L. Miele (Department of Biopharmaceutical Sciences and Cancer Center, University of Illinois at Chicago, Chicago, IL; ref. 18). BxPC-3 cells were transfected with Notch-1 siRNA and control siRNA, respectively, using LipofectAMINE 2000. BxPC-3 cells were stably transfected with human Notch-1 ICN or vector alone (pcDNA3) and maintained under neomycin selection. The transfected cells were treated with 5 µg/mL ERRP for 72 hours or kept as control. The proteins were extracted and measured by Western Blot. In addition, the cell growth and apoptotic cells in transfected cells with treatments were detected using MTT assay and Cell Apoptosis ELISA Detection kit, respectively, following the procedure described earlier.

VEGF assay. Human BxPC-3, HPAC, and PANC-1 pancreatic cancer cells were seeded in six-well plates (1.0 x 105 per well) and incubated at 37°C. After 24 hours, the cells were incubated in medium supplemented with 5 µg/mL ERRP for 72 hours. The cell culture supernatant was harvested, and cell count was done after trypsinization. After collection, the medium was spun at 800 x g for 3 minutes at 4°C to remove cell debris. The supernatant was either frozen at –20°C for VEGF assay later or assayed immediately using commercially available ELISA kits (R&D Systems, Inc., Minneapolis, MN).

MMP-9 activity assay. The pancreatic cancer cells were seeded in six-well plates and incubated at 37°C. After 24 hours, the complete medium was removed, and the cells were washed with serum-free medium. The cells were then incubated in serum-free medium supplemented with 5 µg/mL ERRP for 72 hours. MMP-9 activity in the medium was detected using Fluorokine E Human MMP-9 Activity Assay kit (R&D Systems) according to the manufacturer's protocol.

Invasion assay. The invasive activity of pancreatic cancer cells with different treatments was tested using BD BioCoat Tumor Invasion Assay System (BD Biosciences, Bedford, MA) according to the manufacturer's protocol with minor modifications. Briefly, pancreatic cancer cells (5 x 104) with serum-free medium supplemented with 5 µg/mL ERRP were seeded into the upper chamber of the system for invasion assay as described earlier (17).

BxPC-3 xenografts. Four-week-old female ICR/severe combined immunodeficient mice were obtained from Taconic Laboratory (Germantown, NY). The mice were adapted to animal housing, and BxPC-3 xenografts were developed as described earlier (14, 19). Tumor tissues were harvested for Western blot analysis. All studies involving mice were done under Animal Investigation Committee–approved protocols.

Densitometric and statistical analysis. The bidimensional optical densities of Notch-1 and ß-actin proteins on the films were quantified and analyzed with Molecular Analyst software (Bio-Rad). The cell growth inhibition by ERRP treatment was statistically evaluated using GraphPad StatMate software (GraphPad Software, Inc., San Diego, CA). Comparisons were made between control and ERRP treatment. P < 0.05 was used to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ERRP induced cell growth inhibition of BxPC-3, HPAC, and PANC-1 cells. We examined the growth inhibitory effects of ERRP using the MTT assay in three human pancreatic cancer cell lines, such as BxPC-3, HPAC, and PANC-1. The treatment of pancreatic cancer cells for 1 to 3 days with 2.5, 5, 10, and 20 µg/mL of ERRP resulted in cell growth inhibition in a dose- and time-dependent manner (Fig. 1 ). Next, we examined whether the inhibition of cell growth was also accompanied by the induction of apoptosis induced by ERRP. DNA/histone fragmentation analysis was employed to investigate the degree of apoptosis induced by ERRP.


Figure 1
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  		Figure 1. Effect of purified recombinant ERRP on pancreatic cancer cell growth. A, dose responses of purified recombinant ERRP on growth of pancreatic cancer cells. B to D, time-dependent cell growth inhibition by 5 µg/mL purified ERRP. Cells were seeded in 96-well plates at 5,000 per well and treated with varied concentrations of ERRP or for different time periods. After treatment, cell densities were determined by MTT assay. Points, mean (n = 6) of three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01, compared with the control.

 
ERRP induced apoptosis in pancreatic cancer cell lines. BxPC-3, HPAC, and PANC-1 cells were treated with 5 µg/mL ERRP for 24, 48, and 72 hours. After treatment, the degree of apoptosis was measured in all three cell lines. The induction of apoptosis was found to be time dependent (Fig. 2A ). These results provided convincing data showing that ERRP could induce apoptosis in pancreatic cancer cells. To further understand the molecular mechanism involved in ERRP-induced apoptosis of pancreatic cancer cells, alterations in the cell survival pathway were investigated.


Figure 2
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  		Figure 2. A, cell death assay for measuring apoptosis induced by ERRP. BxPC-3, HPAC, and PANC-1 cells were cultured in RPMI containing 5% FBS and exposed to 5 µg/mL ERRP for different periods of time. Apoptosis was measured by Histone DNA ELISA. Columns, mean; bars, SD. *, P < 0.05; **, P < 0.01, compared with the control. B to D, down-regulation of the Notch-1 at the mRNA and protein levels by ERRP. B, real-time RT-PCR showing changes in the levels of Notch-1 mRNA in pancreatic cancer cells following incubation without (control) and with recombinant ERRP (5 µg/mL) for 72 hours. C, Western blot analysis showing changes in the expression of Notch-1 and its target genes, including Hes-1 and cyclin D1, in pancreatic cancer cells following incubation without (control) and with recombinant ERRP (5 µg/mL) for 72 hours. C, control; T, ERRP treatment. D, effects of ERRP, Erbitux, or Herceptin on TGF-{alpha}– and HB-EGF–induced Notch-1 activation in pancreatic cancer cell line. BxPC-3, HPAC, and PANC-1 cells were plated using standard growth medium, and after 24 hours, the cells were transferred into serum-free RPMI for 48 hours to permit EGFR receptors to equilibrate to the cell surface. The cells were then treated for 90 minutes with the indicated concentrations of ERRP, Erbitux, or Herceptin before stimulation with 7 nmol/L TGF-{alpha} or 5 nmol/L HB-EGF for 15 minutes; whole-cell lysates were prepared, and extracts were electrophoresed and blotted for detection of Notch-1. Lane 1, control; lane 2, TGF-{alpha} (top) or HB-EGF (bottom); lane 3, TGF-{alpha} or HB-EGF + ERRP; lane 4, TGF-{alpha} or HB-EGF + Erbitux; lane 5, TGF-{alpha} or HB-EGF + Herceptin.

 
Down-regulation of the Notch-1 mRNA expression by ERRP. Notch-1 mRNA expression in all three cell lines treated with 5 µg/mL ERRP for 72 hours was assessed using real-time reverse transcription (RT-PCR). The expression of the Notch-1 gene at the mRNA level was down-regulated after ERRP treatment, suggesting transcriptional inactivation of Notch-1 gene expression in pancreatic cancer cells (Fig. 2B).

Down-regulation of Notch-1, Hes-1, and cyclin D1 protein expression by ERRP. To verify whether the down-regulation of Notch-1 by ERRP at the level of transcription ultimately results in alternations at the level of translation, we conducted Western blot analysis for the detection of Notch-1 protein. Western blot analysis showed that the protein level of Notch-1 was decreased in all three pancreatic cancer cell lines following treatment with ERRP (Fig. 2C). These results are in direct agreement with the RT-PCR data, showing that ERRP regulates the transcription and translation of the Notch-1 gene. In addition, we found that the expression of Notch-1 downstream target genes, including Hes-1 and cyclin D1, was also down-regulated in ERRP-treated cells (Fig. 2C).

Effects of ERRP, Erbitux, or Herceptin on TGF-{alpha}– and HB-EGF–induced Notch-1 activation in pancreatic cancer cell lines. To determine whether and to what extent ERRP affects Notch-1 function, BxPC-3 cells were serum starved for 48 hours and subsequently incubated with 5 µg/mL ERRP, Erbitux, or Herceptin for 90 minutes followed by exposure to 7 nmol/L TGF-{alpha} or 5 nmol/L HB-EGF for 15 minutes. Therapeutic monoclonal antibodies targeting the extracellular domains of EGFR and HER-2, such as Erbitux (cetuximab, ImClone Systems/Bristol-Myers Squibb, Somerville, NJ) and Herceptin (trastuzumab, Genentech, South San Francisco, CA), have been developed which showed efficacy in the treatment of patients with cancer. As a comparison with ERRP, we tested the effects of Erbitux and Herceptin on the Notch-1 activation. We observed a marked activation of Notch-1 in TGF-{alpha}– or HB-EGF–treated BxPC-3 cells, whereas pretreatment of cells with ERRP or Erbitux resulted in near complete inhibition of Notch-1 (Fig. 2D). Herceptin treatment reduced HB-EGF–induced Notch-1 activity but had no effect in TGF-{alpha}–induced Notch-1 activity (Fig. 2D). These results suggest that ERRP could be a better therapeutic agent for pancreatic cancer compared with either Erbitux or Herceptin.

Inhibition of NF-{kappa}B activation by ERRP. We have shown earlier that there is a crosstalk between Notch-1 and NF-{kappa}B (15); therefore, we investigated whether the downstream effect of ERRP induced by down-regulation of Notch-1 was mechanistically associated with NF-{kappa}B pathway. Nuclear extracts from control and ERRP-treated pancreatic cancer cells were subjected to analysis for NF-{kappa}B DNA-binding activity as measured by EMSA. We found that 5 µg/mL ERRP significantly inhibited NF-{kappa}B DNA-binding activity in all three pancreatic cancer cell lines compared with the control (Fig. 3A ). The specificity of NF-{kappa}B DNA binding to the DNA consensus sequence was confirmed by supershift assay using p65 antibodies. These results indicated that ERRP decreases NF-{kappa}B DNA-binding activity in pancreatic cancer cells. Although our results have shown by EMSA that ERRP inhibited NF-{kappa}B activation, DNA binding alone does not always correlate with NF-{kappa}B–dependent gene transcription. To confirm our results, we also determined the expression of NF-{kappa}B–dependent gene products, including VEGF, MMP-9, COX-2, and Survivin. Western blot analysis showed that ERRP inhibited the expression of these genes (Fig. 3B). These results further support the role of ERRP in blocking NF-{kappa}B–regulated gene products.


Figure 3
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  		Figure 3. Inhibition of NF-{kappa}B activation and the expression of its target genes by ERRP. A, EMSA analysis was done for pancreatic cancer cells. Nuclear extracts were prepared from control and ERRP-treated cells and subjected to analysis for NF-{kappa}B DNA-binding activity as measured by EMSA. Retinoblastoma protein level served as the nuclear protein loading control. Supershift assay showed that NF-{kappa}B band was shifted because of the formation of a bigger complex after addition of anti-NF-{kappa}B p65 antibody. This assay confirmed the specificity of NF-{kappa}B binding to the DNA consensus sequence. B, inhibition of NF-{kappa}B target gene expression by ERRP in pancreatic cancer cells. C, control; T, ERRP treatment. Western blot analysis showed that ERRP inhibited the expression of VEGF, MMP-9, and COX-2 genes in pancreatic cancer cells. C, ERRP inhibited the expression and activity of MMP-9. Top, real-time RT-PCR analysis of MMP-9 mRNA expression in pancreatic cancer cells treated with ERRP. Bottom, MMP-9 activity assay showing that MMP-9 was inhibited by ERRP. D, ERRP inhibited the expression and activity of VEGF. Top, real-time RT-PCR analysis of VEGF mRNA expression in pancreatic cancer cells treated with ERRP. Bottom, VEGF activity assay showing that VEGF level in the culture medium was inhibited by ERRP.

 
ERRP decreased MMP-9 gene transcription and its activity. To explore whether ERRP decreased MMP-9 gene at the transcriptional level, real-time RT-PCR was conducted to determine the alteration in MMP-9 mRNA. We found that MMP-9 mRNA was dramatically decreased in the ERRP-treated cells (Fig. 3C). Next, we examined whether ERRP could lead to a decrease in MMP-9 activity. There was about 2- to 3-fold decrease in the activity of MMP-9 in pancreatic cancer cell lines (Fig. 3C). These results are consistent with our observation of down-regulation of NF-{kappa}B activity that leads to transcriptional down-regulation of MMP-9 and its activity.

ERRP reduced VEGF gene transcription and its activity. To further investigate whether ERRP has any effect on VEGF reduction, whose expression is transcriptionally regulated by NF-{kappa}B, real-time RT-PCR was done to examine the transcription level of VEGF. We found that VEGF mRNA level was significantly reduced in the ERRP-treated cells (Fig. 3D). Most importantly, we also found that ERRP could lead to a decrease in the levels of VEGF secreted in the culture medium (Fig. 3D).

ERRP decreased pancreatic cancer cell invasion. MMP-9 and VEGF are thought to be critically involved in the processes of tumor cell invasion and metastasis. Because ERRP inhibited the expression and activity of MMP-9 and VEGF, we tested the effects of ERRP on cancer cell invasion. We used Matrigel invasion chamber assay to examine the invasive potential of ERRP-treated cells. As illustrated in Fig. 4 , ERRP-treated cells showed a low level of penetration through the Matrigel-coated membrane compared with the control cells. The value of fluorescence from the invaded pancreatic cancer cells was decreased about 2- to 3-fold compared with that of control cells (Fig. 4).


Figure 4
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  		Figure 4. ERRP decreased pancreatic cancer cell invasion. Top, invasion assay showing that ERRP-treated cells resulted in low penetration through the Matrigel-coated membrane compared with control cells. Bottom, value of fluorescence from the invaded cells. The value indicated the comparative amount of invaded cells.

 
Down-regulation of Notch-1 expression by siRNA promotes ERRP-induced cell growth inhibition and apoptosis in BxPC-3 cells. Because we observed similar effects of ERRP in all three pancreatic cancer cells, we conducted gene knockdown experiments in BxPC-3 cells. We used Western blot analysis to detect the protein level of Notch-1 and found that the intracellular Notch-1 was down-regulated in Notch-1 siRNA-transfected BxPC-3 cells compared with siRNA control-transfected cells (Fig. 5A ). Down-regulation of Notch-1 expression significantly inhibited cell growth in ERRP-treated cells (Fig. 6B ). Notch-1 siRNA-transfected BxPC-3 cells were significantly more sensitive to spontaneous and ERRP-induced apoptosis (Fig. 5B). However, overexpression of Notch-1 by cDNA transfection showed overexpression of Notch-1 protein as confirmed by Western blot analysis (Fig. 5C), and this overexpression in Notch-1 rescued ERRP-induced cell growth inhibition and abrogated ERRP-induced apoptosis to a certain degree (Fig. 5D).


Figure 5
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  		Figure 5. Pancreatic cancer cell growth inhibition and cell death induced by Notch-1 siRNA and ERRP. A, Notch-1 expression was down-regulated by ERRP and Notch-1 siRNA. Western blot analysis was used to detect the protein level of Notch-1. Intracellular Notch-1 was down-regulated in Notch-1 siRNA-transfected BxPC-3 cells compared with control. Lane 1, control; lane 2, 5 µg/mL ERRP; lane 3, Notch-1 siRNA; lane 4, Notch-1 siRNA + plus 5 µg/mL ERRP. B, top, down-regulation of Notch-1 expression significantly inhibited cell growth. ERRP + Notch-1 siRNA inhibited cell growth to a greater degree compared with ERRP alone. Bottom, pancreatic cancer cell death induced by Notch-1 siRNA and ERRP. Down-regulation of Notch-1 expression significantly increased apoptosis induced by ERRP. Notch-1 siRNA-transfected BxPC-3 cells were significantly more sensitive to spontaneous and ERRP-induced apoptosis. CS, control siRNA; NS, Notch-1 siRNA. Points, mean; bars, SD. *, P < 0.05; **, P < 0.01, compared with the control. C, Notch-1 expression was up-regulated by Notch-1 cDNA. Intracellular Notch-1 was increased in Notch-1 cDNA-transfected BxPC-3 cells compared with control-transfected cells. Lane 1, control; lane 2, control + 5 µg/mL ERRP; lane 3, Notch-1 cDNA; lane 4, Notch-1 cDNA + 5 µg/mL ERRP. D, top, overexpression of Notch-1 expression significantly promoted cell growth. Overexpression of Notch-1 rescued ERRP-induced cell growth inhibition. Bottom, overexpression of Notch-1 by Notch-1 cDNA transfection abrogated ERRP-induced apoptosis to a certain degree. CP, control plasmid; NP, Notch-1 plasmid. Points, mean; bars, SD. *, P < 0.05; **, P < 0.01, compared with the control.

 

Figure 6
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  		Figure 6. ERRP inhibits the expression of Notch-1 and NF-{kappa}B target genes in vivo. BxPC-3 xenografts were inoculated s.c. in severe combined immunodeficient mice as described previously (14, 19). Once transplanted, fragments developed into palpable tumors (about 80 mg), and groups of five animals were removed randomly and assigned to different treatment groups. ERRP was given at 1 mg/kg, as s.c. injection away from tumors for seven injections every other day. The control group received vehicle only (0.9% NaCl). Tumor xenografts were removed, and protein extracts were prepared. The expression of Notch-1, Hes-1, VEGF, MMP-9, COX-2, and Survivin was detected by Western blotting.

 
Effect of ERRP on Notch-1 expression in vivo. We have previously found that ERRP treatment significantly inhibited pancreatic tumor growth in vivo (14). To further investigate whether ERRP could down-regulate Notch-1 in vivo, we examined the Notch-1 expression in tumor tissues obtained from tumor-bearing mouse treated with ERRP as published earlier (14). Western blot analysis showed that the expression level of Notch-1 was significantly lower in tumors from the ERRP-treated mice than those from vehicle-treated control mice (Fig. 6), indicating that ERRP could down-regulate Notch-1 in vivo, similar to those observed in vitro. In addition, we found that the expression of Notch-1 downstream target gene Hes-1 was also down-regulated in ERRP-treated animal (Fig. 6). In our earlier report, we showed that ERRP could down-regulate the DNA-binding activity of NF-{kappa}B in vivo (14). To determine whether ERRP could affect the NF-{kappa}B-dependent gene products in vivo, we also examined the expression of VEGF, MMP-9, COX-2, and Survivin in tumor tissues using Western blot analysis. We found that the expression of VEGF, MMP-9, COX-2, and Survivin was also down-regulated in ERRP-treated animal (Fig. 6). These results are consistent with our in vitro data showing that ERRP is a powerful agent for the inhibition of pancreatic cancer cell growth and invasion.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EGFR signaling plays important roles in human cancers, including pancreatic cancer (20, 21). The activation of EGFR has been shown to enhance tumor growth, invasion, motility, tumor spreading and metastasis, and inhibition of apoptosis (3, 22). The overexpression of EGFR in pancreatic cancer has been correlated with shorter survival (22, 23). Therefore, identification of an inhibitor targeting EGFR is likely to provide a therapeutic benefit for pancreatic cancer. ERRP, a recently isolated pan-erbB inhibitor, possesses substantial homology with the extracellular ligand-binding domain of EGFR and its family members (8). Previously, we reported that ERRP inhibited cell growth of pancreatic cancer cells by attenuating EGFR activation in vitro and in vivo (13, 14). Our current data show that ERRP not only inhibits cell growth but also induces apoptotic cell death of pancreatic cancer cells, similar observations in colon and breast cancer cells (24).

In an earlier study, we also observed that treatment of pancreatic cancer cells with ERRP results in attenuation of tyrosine kinases activity and tyrosine phosphorylation of EGFR, suggesting that ERRP exerts its growth inhibitory effect by attenuating EGFR activation. Based on these observations, it would be logical to assume that the downstream signaling events of EGFR activation would also be affected. It has been shown that Notch-1 is activated as a direct consequence of EGFR activation in pancreatic cancer (5). It has been documented that the Notch genes and Notch target genes, including Hes-1 and cyclin D1, are overexpressed in human pancreatic cancer (5, 25, 26). Initial studies were done to examine the relative levels of EGFR in nine pancreatic cancer cell lines, such as BxPC-3, HPAC, PANC-1, AsPC-1, COLO-357, CAPAN1, CAPAN2, L3.6pl, and MIAPaCa, by Western blot analysis. We found that EGFR expression was negligible in four cell lines, such as CAPAN1, CAPAN2, L3.6pl, and MIAPaCa, whereas three cell lines, such as BxPC-3, HPAC, and PANC-1, expressed high levels of EGFR and Notch-1 (14, 16, 27). All these reports clearly suggest a possible link among EGFR, Notch overexpression, and pancreatic cancer. Indeed, we found that ERRP inhibits the activation of Notch-1 in vitro and in vivo in pancreatic cancer. We also found that ERRP inhibited the expression of Notch-1 target genes, Hes-1 and cyclin D1, and inhibited TGF-{alpha}– and HB-EGF–induced activation of Notch-1. The inhibition of EGFR, Notch-1, Hes-1, and cyclin D1 is noteworthy as they play a key role in pancreatic cancer cell growth (5, 15, 28). We also found that down-regulation of Notch-1 by siRNA together with ERRP treatment inhibited cell growth and induced apoptosis to a greater degree in pancreatic cancer cells compared with ERRP alone. Conversely, overexpression of Notch-1 by Notch-1 cDNA transfection abrogated ERRP-induced apoptosis to a certain degree. In view of this, we strongly believe that down-regulation of Notch-1 by ERRP is mechanistically linked to apoptotic processes due to inactivation of Notch-1 and its downstream target genes. Taken together, these results suggest that ERRP inhibited the activation of EGFR and also reduced the activity of Notch-1 signaling.

We also found that ERRP inhibited NF-{kappa}B activation in BxPC-3, HPAC, and PANC-1 cells. This could be another mechanism by which ERRP inhibits cell growth and induces apoptotic cell death. It has been reported that activation of EGFR leads to the activation of NF-{kappa}B, which has been shown to be activated in pancreatic cancer (28–30). Recent reports from our laboratory and others have shown that Notch-1 is an upstream target of NF-{kappa}B because overexpression of Notch-1 led to higher NF-{kappa}B activity (17, 31–33). These results indicate that there may be crosstalk between Notch and NF-{kappa}B pathways. EGFR, Notch, and NF-{kappa}B pathways are key regulators of numerous cellular events, such as proliferation and apoptosis. Therefore, inactivation of EGFR- and Notch-1-mediated cell growth inhibition and induction of apoptosis by ERRP could be partly mediated via inactivation of NF-{kappa}B activity.

NF-{kappa}B activation has been reported to regulate several genes, such as VEGF, COX-2, Survivin, and MMP-9, that are directly associated with metastatic processes (34–38). Indeed, in the present study, we showed that ERRP reduced NF-{kappa}B DNA binding activity and concomitantly inhibited the expression of VEGF, MMP-9, Survivin, and COX-2. We also found that ERRP reduced the secretion of VEGF in the culture medium and inhibited the activity of MMP-9 in pancreatic cancer cells. Because we observed that ERRP inhibited the expression of VEGF and MMP-9, we tested the effects of ERRP on the invasion of pancreatic cancer cells. We found that ERRP inhibited invasion of pancreatic cancer cells through the Matrigel. These results were consistent with inactivation of MMP-9 and VEGF, documenting that ERRP could inhibit cancer cell invasion partly through down-regulation of MMP-9 and VEGF due to inactivation of Notch-1 and NF-{kappa}B. These results are consistent with recent findings reported in other cancer cells (39).

Based on our results, along with the studies reported by other investigators, we speculate that one possible mechanism by which ERRP inhibits cell growth and cell invasion is due to inactivation of EGFR, leading to the down-regulation of Notch-1- and NF-{kappa}B-regulated genes, such as VEGF, COX-2, cyclin D1, Survivin, Bcl-2, and MMP-9. Based on our results, we propose a hypothetical pathway by which ERRP inhibits cell growth and invasion of pancreatic cancer cells, partly mediated through inactivation of Notch-1 and NF-{kappa}B signaling pathways (Fig. 7 ). However, further in-depth studies are needed to ascertain the precise molecular mechanism for establishing the cause and effect relationship between Notch-1 and NF-{kappa}B during ERRP-induced inhibition of cell growth and invasion of pancreatic cancer cells in animal models and in human pancreatic cancer.


Figure 7
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  		Figure 7. Schematic representation of ERRP-induced inhibition of NF-{kappa}B DNA-binding activity through Notch-1 signaling pathways and its downstream effects.

 
In summary, we presented experimental evidence which strongly supports the antitumor and antimetastatic effects of ERRP in pancreatic cancer. Thus, we believe that ERRP could potentially be an effective therapeutic agent for the inactivation of EGFR, Notch-1, NF-{kappa}B, and its downstream target genes, such as MMP-9 and VEGF, resulting in the inhibition of cell growth, invasion, and metastasis of pancreatic cancer.


   Acknowledgments
 
Grant support: National Cancer Institute/NIH grant 1R01CA101870-02 (F.H. Sarkar) and subcontract award (F.H. Sarkar) from the University of Texas M.D. Anderson Cancer Center through a Specialized Programs of Research Excellence grant S P20CA 101936-02 on pancreatic cancer awarded to James Abbruzzese.

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 Carrie Koerner for editorial assistance and the Puschelberg Foundation for their generous contribution.

Received 3/20/06. Revised 5/ 8/06. Accepted 5/24/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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