Epidermal growth factor down-regulates the expression of neutrophil gelatinase-associated lipocalin (NGAL) through E-cadherin in pancreatic cancer cells

Authors

  • Zhimin Tong MD, PhD,

    1. Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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  • Subhankar Chakraborty MD,

    1. Department of Biochemistry and Molecular Biology, Eppley Cancer Institute, University of Nebraska Medical Center, Omaha, Nebraska
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  • Bokyung Sung PhD,

    1. Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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  • Pooja Koolwal MD,

    1. Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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  • Sukhwinder Kaur PhD,

    1. Department of Biochemistry and Molecular Biology, Eppley Cancer Institute, University of Nebraska Medical Center, Omaha, Nebraska
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  • Bharat B. Aggarwal PhD,

    1. Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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  • Sendurai A. Mani PhD,

    1. Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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  • Robert S. Bresalier MD,

    1. Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
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  • Surinder K. Batra PhD,

    1. Department of Biochemistry and Molecular Biology, Eppley Cancer Institute, University of Nebraska Medical Center, Omaha, Nebraska
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  • Sushovan Guha MD, PhD

    Corresponding author
    1. Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
    • Department of Gastroenterology, Hepatology, and Nutrition, Unit 436, 1515 Holcombe Boulevard, Houston, TX 77030
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    • Fax: (713) 563-4398


Abstract

BACKGROUND:

The authors previously reported that neutrophil gelatinase-associated lipocalin (NGAL) overexpression significantly blocked invasion and angiogenesis of pancreatic ductal adenocarcinoma (PDAC). They also demonstrated a loss of NGAL expression in the advanced stages of PDAC. However, little is known regarding the mechanisms of NGAL regulation in PDAC. Because the epidermal growth factor (EGF)-EGF receptor (EGFR) axis is up-regulated significantly in PDAC, they examined EGF-mediated NGAL regulation in these cells.

METHODS:

The NGAL-positive cell lines AsPC-1 and BxPC-3 were used as a model system. Quantitative real-time polymerase chain reaction (RT-PCR), Western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or small hairpin RNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay.

RESULTS:

NGAL expression was positively associated with tumor differentiation and was down-regulated significantly after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin down-regulation was partly through the EGFR-dependent mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) (MEK-ERK) signaling pathway. In addition, E-cadherin down-regulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued the inhibition of NGAL expression by EGF. Furthermore, EGF, in part through E-cadherin, reduced NGAL promoter activity by blocking nuclear factor κB (NF-κB) activation.

CONCLUSIONS:

The current study demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect was mediated in part through activation of the EGFR-MEK-ERK signaling pathway, which, in turn, down-regulated E-cadherin with a subsequent reduction in NF-κB activation. These findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC. Cancer 2011;. © 2010 American Cancer Society.

Neutrophil gelatinase-associated lipocalin (NGAL), also referred as lipocalin 2, is a 25-kD, secreted protein that belongs to the lipocalin family.1 NGAL binds to and transports a variety of lipophilic substances through a 3-dimensional β-barrel structure that is common to lipocalins.1 It also is considered an antimicrobial factor because of its ability to deplete iron, which is required for bacterial growth.2 In addition, increased NGAL expression has been observed in a variety of pathologic conditions, including inflammation,3, 4 acute ischemic renal injury,5 and various human cancers.3, 6, 7

There are substantial data to support a key role for NGAL in cancer progression. For instance, NGAL expression is up-regulated and associated with enhanced tumor growth and invasiveness in breast cancer8 and in esophageal squamous cell carcinoma.9 In contrast, NGAL acts as an antitumor and antimetastatic factor in ovarian,10 colon,11 and pancreatic cancers.12 Moreover, NGAL has emerged as a biomarker for detecting early stage cancer and monitoring the progression of established cancers, including ovarian10 and pancreatic cancers.12, 13 NGAL is not expressed by normal pancreatic ducts but is expressed aberrantly in the early preneoplastic stages of pancreatic ductal adenocarcinoma (PDAC), termed pancreatic intraepithelial neoplasias (PanINs).13 Furthermore, NGAL expression in established PDAC correlates inversely with the grade of differentiation.13 However, the mechanism by which NGAL expression is regulated in PDAC cells remains unknown.

It is well documented that growth factors, especially epidermal growth factor (EGF) and EGF receptor (EGFR)-driven signaling pathways, play an important role in cancer progression.14 The overexpression of EGF and EGFR has been reported in various cancer types, including PDAC.15, 16 It has been demonstrated that EGF treatment in vitro enhances the invasiveness and metastatic properties of several different cancer cells, including ovarian,17 cervical,18 epidermoid,19 and breast cancers.20 In PDAC, both EGF and EGFR are overexpressed and associated with increased tumor size, advanced clinical stage, and decreased patient survival.21, 22 In addition, it has been demonstrated that EGF promotes the invasiveness of PDAC cells by activating matrix metalloproteinase-2 and nuclear factor-κB (NF-κB).23, 24 However, the downstream signaling that mediates EGF-induced PDAC aggressiveness remains poorly understood.

In the current study, we investigated the possible role of EGF in regulating NGAL expression. We observed that EGF down-regulates NGAL and E-cadherin expression in PDAC cells in vitro, and this down-regulation was blocked by EGFR or extracellular signal-regulated kinase (ERK) pathway inhibitors. Down-regulation and up-regulation of E-cadherin resulted in decreased and increased NGAL expression in PDAC cells, respectively. EGF treatment also was associated with a significant decrease in NF-κB–mediated transcription of NGAL messenger RNA (mRNA), whereas mutation of the NF-κB binding site (in the NGAL promoter) blocked EGF-induced down-regulation of NGAL expression. Thus, our findings reveal that EGF inhibits NGAL expression through an EGFR-driven ERK pathway and involves the inhibition of NF-κB–mediated transactivation of NGAL. Furthermore, we demonstrated that E-cadherin modulates NGAL expression in PDAC cells, suggesting a possible mechanism for EGF-induced aggressiveness of PDAC cells.

MATERIALS AND METHODS

Cells Lines and Culture Condition

PANC-1, MIA PaCa-2, AsPC-1, and BxPC-3 cells were purchased from the American Type Culture Collection (Rockville, Md) and cultured as described previously.12 293FT cells were obtained from Invitrogen (Carlsbad, Calif) and maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and 500 μg/mL G418 antibiotic solution.

Reagents and Treatments

EGF was purchased from Sigma Chemical Company (St. Louis, Mo); PD98059, U0126, E-cadherin, and phosphorylated ERK antibodies were from purchased from Cell Signaling Technology (Danvers, Mass); AG1478 was purchased from Calbiochem (Gibbstown, NJ); and mouse monoclonal NGAL antibodies were purchased from Antibody Shop (Gentofte, Demark). A vector encoding E-cadherin short hairpin RNA (pLKO-shE-cadherin) and its control vectors were gifts from Dr. Sendurai Mani (The University of Texas M. D. Anderson Cancer Center, Houston, Tex). pCMV-SPORT6-E-cadherin was purchased from OPEN Biosystems (Huntsville, Ala). pGL3-luc/NGAL (−900) and pGL3-luc/NGAL (NF-κB mutant [mut]) were gifts from Dr. Tatsushi Muta (Tohoku University, Sendai, Japan). pGL3-luc/NF-κB was obtained from Dr. Bharat B. Aggarwal (The University of Texas M. D. Anderson Cancer Center, Houston, Tex).

Plasmid Construction, Lentivirus Production, and Transduction

Human E-cadherin combinational DNA (cDNA) was released from pCMV-SPORT6 with EcoRV and NotI and was subcloned into the lentiviral vector pCDH-VMV-MCS-EF1-Puro between EcoRI and NotI to create phE-cadherin. The identity and orientation of this construct were confirmed by DNA sequencing. To produce lentivirus that overexpressed shE-cadherin or E-cadherin, we cotransfected pLKO-shE-cadherin, or phE-cadherin and control vectors with their packaging and envelope plasmids into 293FT cells using lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen). Forty-eight hours later, the viral supernatant was collected and centrifuged at 3000 revolutions per minute for 15 minutes. For transduction with lentivirus, cells were infected with 2 times diluted virus media containing 6 μg/mL polybrene for 16 hours. The expression of target proteins was confirmed by quantitative real-time polymerase chain reaction (Q-PCR) and/or Western blot analysis.

Q-PCR

RNA isolation and cDNA synthesis were performed as described before.12 Q-PCR was performed using SYBR Green on a Bio-Rad thermocycler (Bio-Rad, Hercules, Calif). PCR primers were designed using Primer3 software. The expression level of a gene was measured using the 2−ΔΔCt method. Glyceraldehyde 3-phosphate dehydrogenase was used as the internal control.

Western Blot Analysis

Twenty micrograms of cell lysate were separated on a 10% to 12% sodium dodecyl sulfate-polyacrylamide electrophoresis gels. The separated proteins were transferred electrophoretically to Immobilon membranes (Millipore, Bedford, Mass) and incubated with 5% dry milk in Tris-buffered saline with Tween-20 (25 mM Tris-HCl, pH 7.6; 200 mM NaCl; and 0.15% Tween-20) for 1 hour at room temperature. Target protein levels were measured by immunoblotting with corresponding antibodies at 1:1000 dilutions for 2 hours at room temperature as previously described.12

Immunohistochemical Analysis

Tissue microarrays made from formalin-fixed, paraffin-embedded tissues were purchased from US Biomax (Rockville, Md) and comprised normal sections and PDAC tissue sections (catalog number PA1001). Immunohistochemistry was performed as described previously13 with a 1 in 1,000 concentration of primary antibody (NGAL rabbit polyclonal) in phosphate-buffered saline. NGAL expression in the tissues was expressed as either positive or negative.

Immunofluorescence Staining

Immunofluorescence staining was done as described previously.25 The NGAL antibody was used at a dilution of 1:100 in phosphate-buffered saline. Fluorescent images were viewed under an IX81 confocal microscope (Olympus, Tokyo, Japan).

Luciferase Assay

To assess the activity of the NGAL promoter, AsPC-1 cells were transfected with a promoter reporter construct that contained either an intact NF-κB binding site (pGL3-luc/NGAL [−900]) or a mutation that prevented NF-κB binding (pGL3-luc/NGAL [NF-κB mut]). After 16 hours of serum starvation, these cells were treated with 25 ng/mL EGF. For the NF-κB binding activity assay, cells were transfected with pGL3-luc/NF-κB after 16 hours of infection with lentivirus carrying either shE-cadherin or control lentivirus. pGL4.74 (hRluc/TK; Promega, Madison, Wis) was cotransfected as a control for transfection efficiency. Twenty-four hours after transfection, the cells were lysed, and luciferase activity was measured using the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Firefly luciferase activity was normalized to that of Renilla luciferase.

Electrophoretic Mobility Shift Assay

To determine NF-κB activation, an electrophoretic mobility shift assay (EMSA) was performed as described previously.26 The DNA-protein complex that formed was separated from the free oligonucleotide on 6.6% native polyacrylamide gels. The gels were dried and observed, and the radioactive bands were quantified using a PhosphorImager (GE Healthcare, Piscataway, NJ) loaded with an ImageQuant software (GE Healthcare).

Statistical Analysis

To analyze of the immunohistochemical results, each spot was considered as an individual sample. NGAL expression, the type of tissue, and the PDAC grade were considered as categorical variables and were compared using a chi-square test. Continuous variables were compared using a 2-tailed Student t test. A P value <.05 was considered as significant.

RESULTS

NGAL Expression Correlates With Tumor Differentiation

In our previous study,12 we demonstrated that NGAL expression is associated with a well differentiated phenotype in PDAC cell lines. In the current study, we used a larger pool of primary PDAC samples to analyze NGAL expression. In total, 73 tissue cores were available for immunohistochemical analysis. Figure 1A,B illustrates that NGAL was not expressed in any of the normal ducts (0 of 10) but was expressed in 2 of 10 (20%) normal tissues adjacent to cancer. Furthermore, NGAL was expressed in 50 of 73 PDAC tissue sections (68%), and its expression was associated with tumor differentiation (P < .0001). Of the 63 PDAC sections that had a defined tumor grade, 16 of 20 (80%) well differentiated, 20 of 26 (77%) moderately differentiated, and 5 of 21 (24%) poorly differentiated PDAC tissue sections expressed NGAL. These results suggest that NGAL promotes differentiation in PDAC cells.

Figure 1.

Neutrophil gelatinase-associated lipocalin (NGAL) expression is associated with cell differentiation. (A) Immunohistochemical (IHC) analysis revealed NGAL staining at different tumor stages in pancreatic cancer tissues. (B) Variation of NGAL expression in pancreatic cancer is listed by tumor grade. This summary of the IHC results indicates a correlation between NGAL expression and cell differentiation.

EGF Treatment Inhibits NGAL Expression in PDAC Cells

Studies have demonstrated that EGF and EGFR expression is associated with a loss of PDAC cell differentiation, whereas NGAL expression correlates with a well differentiated phenotype.27, 28 To investigate whether EGF is involved in the down-regulation of NGAL in advanced PDAC, we analyzed the effect of EGF on 2 NGAL-expressing PDAC cell lines: AsPC-1 and BxPC-3. Exposure to EGF in vitro significantly reduced the expression of NGAL mRNA (by 48.4% in AsPC-1 cells and by 44.7% in BxPC-3 cells) compared with the expression in untreated cells as determined by Q-PCR (Fig. 2A). Furthermore, the production of NGAL protein also was reduced significantly by EGF treatment as determined by both Western blot analysis (Fig. 2B) and immunofluorescence (Fig. 2C).

Figure 2.

Epidermal growth factor (EGF) reduces neutrophil gelatinase-associated lipocalin (NGAL) expression and its effect through the EGF receptor (EGFR). (A) Quantitative real-time polymerase chain reaction analysis was used to detect NGAL expression using total RNA extracted from cells that were treated with 25 ng/mL EGF for 24 hours. NGAL expression was determined using the 2−ΔΔCt method, and the results are expressed as the mean ± standard error of 3 independent experiments. An asterisks indicates P < .05. (B) Western blot analysis revealed NGAL expression in AsPC-1 and BxPC-3 cells that were treated for 24 hours with the indicated concentrations of EGF. (C) Immunofluorescent staining of NGAL is shown. Cells were treated with 25 ng/mL of EGF in serum-free media. Twenty-four hours later, the cells were stained for NGAL (green) and nuclei (4,6-diamidino-2-phenylindole [DAPI], blue) and were photographed using a confocal microscope (original magnification, ×60). (D) Cells were treated with or without the EGFR antagonist AG1478 (AG) for 1 hour followed by EGF treatment at the indicated doses for 24 hours. NGAL protein levels were determined by Western blot analysis.

EGF is a well known ligand of EGFR. The binding of EGF to EGFR activates downstream signals that stimulate cancer cell invasion and metastasis. To determine whether the EGF-mediated inhibition of NGAL is EGFR-dependent, we treated AsPC-1 and BxPC-3 cells with 10 μM of AG1478, an EGFR inhibitor, before stimulating them with EGF. AG1478, as expected, abolished the EGF-mediated down-regulation of NGAL expression (Fig. 2D).

EGF Exposure Leads to Down-Regulation of E-Cadherin

Accumulating evidence demonstrates a positive correlation between high EGF and EGFR expression and increased cancer invasion and metastasis in PDAC cells. It also has been demonstrated that loss of E-cadherin is a causal factor that promotes tumor progression. To determine whether E-cadherin contributes to EGF-mediated inhibition of NGAL expression, we examined the alteration in E-cadherin by Western blot analysis after exposing cells to EGF. EGF treatment led to remarkably reduced E-cadherin protein levels concurrent with a reduction in NGAL protein levels (Fig. 3A). Furthermore, E-cadherin expression was positively correlated with NGAL expression in all 4 cell lines (Fig. 3B), suggesting that E-cadherin may be involved in the down-regulation of NGAL by EGF.

Figure 3.

Down-regulation of E-cadherin reduces gelatinase-associated lipocalin (NGAL) expression. (A) Western blot analysis shows NGAL and E-cadherin expression using protein lysate from cells that were treated with epidermal growth factor (EGF) at the indicated doses for 24 hours. (B) Western blot analysis shows the relation between NGAL and E-cadherin expression using protein lysates from selected pancreatic ductal adenocarcinoma cell lines (AsPC-1, BxPC-3, Mia-2, and Panc-1). (C) RNA was extracted from cells that were infected for 48 hours with lentivirus carrying E-cadherin–specific small hairpin RNA (shE-Cad) or its controls and was used for quantitative real-time polymerase chain reaction analysis. Gene expression was calculated using the 2−ΔΔCt method, and the results are expressed as the mean ± standard error of 3 independent experiments. An asterisk indicates P < .05. (D) NGAL protein levels were determined by Western blot analysis using protein lysates from cells that were infected for 72 hours with lentivirus carrying shE-Cad or its controls.

EGF-Mediated Inhibition of NGAL Expression Involves E-Cadherin

To further confirm the role of E-cadherin in the EGF-mediated inhibition of NGAL expression, we generated a lentivirus designed to knock down or over express human E-cadherin. Knock down of E-cadherin by infection with shE-cadherin significantly reduced NGAL mRNA expression in AsPC-1 and BxPC-3 cells (by 36.4% and 44.8%, respectively, compared with vector control-transected cells) as measured by Q-PCR (Fig. 3C). Western blot analysis also demonstrated a reduction in NGAL protein expression in cells that were infected with shE-cadherin (Fig. 3D). Conversely, E-cadherin overexpression led to an increase in NGAL expression (Fig. 4A) and abolished the inhibition of NGAL expression by EGF (Fig. 4B). Taken together, these observations suggest that E-cadherin positively regulates NGAL expression in PDAC cells and that loss of E-cadherin expression by EGF stimulation may contribute to EGF-mediated inhibition of NGAL expression in PDAC cells.

Figure 4.

E-cadherin overexpression leads to increased gelatinase-associated lipocalin (NGAL) expression. (A) For Western blot analysis of NGAL protein levels, protein lysates were used from cells that were infected for 72 hours with lentivirus carrying human E-cadherin or its controls. (B) Cells were treated with epidermal growth factor (EGF) at the indicated doses for 24 hours after they were infected with lentivirus carrying human E-cadherin or its controls. NGAL protein levels were then evaluated by Western blot analysis.

EGF-Mediated Inhibition of NGAL and E-Cadherin Expression Involves the ERK Pathway

The binding of EGF to EGFR can activate a number of downstream signaling pathways (including the mitogen-activated protein kinase [MEK]-ERK pathway) that, in turn, regulate the expression of several genes. It was reported previously that the ERK pathway is involved in regulating E-cadherin expression.29, 30 To determine the possible role of the ERK pathway in EGF-mediated inhibition of E-cadherin and NGAL expression, we treated cells with either PD98059 or U0126 (inhibitors of ERK) for 24 hours, and evaluated NGAL expression by Western blot analysis. Both PD98059 treatment and U0126 treatment led to a significant dose-dependent increase in NGAL protein levels (Fig. 5A,B). In addition, PD98059 and U0126 pretreatment completely abolished the effect of EGF on E-cadherin and NGAL expression (Fig. 5C).

Figure 5.

These blots reveal effects of the extracellular signal-regulated kinase (ERK) pathway on gelatinase-associated lipocalin (NGAL) expression. (A,B) For Western blot analysis of NGAL expression, cell lysates were used from cells that were treated for 24 hours with the ERK pathway inhibitor PD98059 (PD) or U0126 at the indicated doses. P-ERK indicates phosphorylated ERK; T-ERK, total ERK. (C) Cells were treated with 25 μM of PD or with 10 μM of U0126 for 1 hour followed by treatment for 24 hours with 25 ng/mL of EGF. The cell lysates then were used for Western blot analyses of E-cadherin and NGAL expression. (D) Western blot analysis shows expression of zinc finger E-box-binding homeobox 1 (ZEB1) after treatment for 24 hours with 25 ng/mL EGF.

Several transcription factors, including Twist, Snail, Slug, and zinc finger E-box-binding homeobox 1 (Zeb1), are known to be involved in regulating E-cadherin expression. To determine whether EGF inhibits E-cadherin expression through these transcription factors, we evaluated the effect of EGF treatment on Zeb1 expression. Cells that were exposed to 25 ng/mL of EGF for 24 hours had higher Zeb1 protein levels than control cells (Fig. 5D), and this was accompanied by a concomitant reduction in E-cadherin and NGAL expression.

EGF Inhibits NGAL Expression by Repressing NF-κB–Mediated Transcription

NF-κB is one of the most important transcription factors that regulates NGAL expression. To determine whether it plays a role in EGF-mediated inhibition of NGAL expression, we transfected AsPC-1 cells with a luciferase reporter construct driven by either 900 base pairs of wild-type NGAL promoter or a mutant NGAL promoter that carried an inactivating mutation in its NF-κB binding site. Simulation of the transfected cells with 25 ng/mL of EGF for 24 hours led to a significant decrease in the activity of the wild-type NGAL promoter-driven luciferase reporter gene compared with untreated controls. In cells that were transfected with mutant NGAL promoter, basal transcription at the NGAL promoter was significantly decreased (compared with that in wild-type promoter-transfected cells), and the transcriptional activity did not change with EGF treatment (Fig. 6A). In addition, down-regulating E-cadherin by infecting AsPC-1 cells with lentivirus that carried shE-cadherin led to a significant reduction in luciferase activity in cells that were cotransfected with the wild-type NF-κB promoter reporter construct (Fig. 6B). Similar results were observed in cells that were treated with EGF in an EMSA analysis (Fig. 6C). Together; these data suggest that EGF down-regulates NGAL expression at the transcriptional level by inhibiting the binding of NF-κB to the NGAL promoter.

Figure 6.

Epidermal growth factor (EGF) down-regulates gelatinase-associated lipocalin (NGAL) expression by inhibiting nuclear factor κB (NF-κB) binding activity. (A) AsPC-1 cells were cotransfected with the vector pGL3-luc/NGAL (−900) or pGL3-luc/NGAL (NF-κB-mutant [mut]) and pGL4.74 (hRluc/TK) (a control for transfection efficiency) for 24 hours after treatment with 25 ng/mL EGF. Cell lysates then were used for the luciferase (Luc) activity assay. An asterisk indicates P < .05. AP-1 indicates activator protein 1; C/EBP, CCAAT/enhancer binding protein; TATA, DNA core promoter sequence involved in transcription. (B) AsPC-1 cells were infected for 16 hours with lentivirus carrying E-cadherin–specific small hairpin RNA (shE-cadherin) or its controls followed by cotransfection with pGL3-luc/NF-κB and pGL4.74 (hRluc/TK) for 24 hours. Thereafter, the cell lysates were used for luciferase activity assays. An asterisk indicates P < .05. (C) Nuclear extracts from AsPC-1 cells that were treated for 24 hours with EGF at the doses indicated and a positive control (p.c.) (nuclear extracts from KBM-5 cells that were stimulated with tumor necrosis factor-α) were analyzed by electrophoretic mobility shift assay as described in the text (see Materials and Methods). (D) This model illustrates the EGF-mediated down-regulation of NGAL. EGF activates extracellular signal-regulated kinase (ERK) signaling by activating the EGF receptor (EGFR), leading to increased zinc finger E-box-binding homeobox 1 (ZEB1) expression and reduced E-cadherin expression, which results in decreased NGAL expression by blocking NF-κB binding to the NGAL promoter. PM indicates plasma membrane.

DISCUSSION

NGAL plays an important role in PDAC cell biology by inhibiting PDAC cell invasion and metastasis. EGF is a critical mitogen involved in PDAC cell invasion and metastasis, but nothing is known about how it regulates NGAL expression to mediate this key biologic event in neoplastic progression. In the current study, we demonstrated that EGF down-regulates E-cadherin and NGAL by activating EGFR and the mitogen-activated protein kinase (MAPK)-ERK intracellular signaling pathway in PDAC cells.

Previous studies have demonstrated that the function of EGF depends on its binding to and activation of the EGFR. EGFR activation, in turn, regulates the expression of its target genes, which promote cell growth, invasion, and metastasis through downstream signaling.31, 32 NGAL appears to play a role opposite to that of EGF and EGFR in modulating PDAC cell invasion and metastasis.12 Given the opposing roles of NGAL and EGF in PDAC, we hypothesize that NGAL is an EGF-regulated gene in PDAC cells. Li et al33 reported that ErbB2 (EGFR type 2) overexpression resulted in increased NGAL expression in breast cancer cells. In contrast, studies in ovarian cancer cells indicated that EGF treatment led to reduce NGAL protein expression.10 These seemingly opposite results are consistent with the different biologic functions of NGAL in these 2 cancer types. In breast cancer, NGAL promotes malignant cell proliferation, invasion, and metastasis8; whereas it is an antitumorigenic and antimetastatic factor in ovarian cancer cells.10 In PDAC cells, exposure to EGF significantly decreased both mRNA and protein levels of NGAL. Blocking EGFR activation with an EGFR antagonist (AG1478) abolished EGF-mediated NGAL down-regulation. These findings suggest that EGF is a negative regulator of NGAL expression in PDAC cells and plays a role in EGF-mediated invasion and metastasis of these cells. In addition, the observed positive association between NGAL expression and differentiation in PDAC tissues may be caused at least in part by the negative association between EGF and EGFR expression and PDAC cell differentiation.27

It is widely accepted that E-cadherin plays a critical role in the epithelial-mesenchymal transition, an early event in cancer cell invasion and metastasis.34 E-cadherin expression frequently is lost in metastatic human cancers, including PDAC.35, 36 Loss of E-cadherin expression has been associated with activation of the EGF/EGFR cascade in several cancer types, including breast,19, 37 cervical,18 and pancreatic cancers.38 Our data confirmed those earlier reports, which described the EGF-mediated and EGFR-mediated down-regulation of E-cadherin expression in PDAC cells. In addition, we observed that the down-regulation of E-cadherin expression by EGF was paralleled by a down-regulation of NGAL expression in PDAC cells, suggesting that E-cadherin is a novel regulator of EGF-mediated NGAL down-regulation. Indeed, E-cadherin expression was correlated with NGAL expression in PDAC cells. Furthermore, the manipulation of E-cadherin expression by either up-regulation or down-regulation led to an up-regulation or down-regulation of NGAL expression, respectively. Moreover, E-cadherin overexpression blocked the EGF-mediated inhibition of NGAL expression, further supporting the role of E-cadherin as a positive regulator of NGAL expression in PDAC cells. Lim et al10 reported that EGF down-regulated both E-cadherin and NGAL expression in ovarian cancer cells. In immortalized human breast epithelial cells, E-cadherin down-regulation by RNA interference or dominant-negative E-cadherin resulted in reduced NGAL expression.39 It is noteworthy that NGAL overexpression in colon cancer cells resulted in altered subcellular localization of E-cadherin, thus suggesting a downstream effect of NGAL on E-cadherin.25 But the detailed mechanism of NGAL-mediated E-cadherin regulation remains unclear. However, in PDAC cells, we did not observe any downstream effects of NGAL overexpression or under expression on E-cadherin expression.12 Furthermore, we demonstrated that NGAL overexpression significantly reduced the invasion/metastasis of PDAC cells both in vitro and in vivo.12 Thus, the downstream effects of NGAL may be cell context-dependent. Together, our findings demonstrate that E-cadherin regulates NGAL expression in PDAC cells and contributes to EGF-mediated NGAL down-regulation in PDAC cells.

The transcriptional factors Snail, Slug, and Zeb1 are key factors that repress E-cadherin expression.34, 40 It also has been reported that Zeb1 expression is correlated inversely with E-cadherin expression in primary tumor samples from patients with PDAC and in PDAC cell lines.41 In the same studies, Zeb1 gene silencing by small interfering RNA led to increased E-cadherin expression. These data suggest that Zeb1 is an important negative regulator of E-cadherin expression in PDAC. We observed EGF-associated, reciprocal changes in Zeb1 and E-cadherin expression, further supporting the role of Zeb1 in EGF-induced E-cadherin down-regulation in PDAC cells.

Several of signaling pathways can be triggered by EGF/EGFR activation, including the MEK/ERK pathways. These pathways, in turn, modulate downstream gene expression.31, 42 It has been suggested that E-cadherin expression is regulated by the MEK/ERK signaling cascade in PDAC cells.29 In the current study, we observed that treatment with the ERK pathway inhibitors PD98059 or U0126 resulted in increased NGAL expression. Furthermore, pretreatment with PD98059 and U0126 abolished the inhibition of both NGAL and E-cadherin expression by EGF. It was reported previously that exposure of H1435 lung cancer cells to U0125 increased Zeb1 expression.43 Together with our results, these data suggest that ERK pathway activation is required for EGF-mediated inhibition of E-cadherin (through Zeb1) and NGAL expression in PDAC cells.

NF-κB is 1 of the most important transcription factors.33, 44, 45 Its activity may be linked to EGF/EGFR activation. For instance, EGF treatment prevents H2O2-induced NF-κB activity in intestinal Caco-2 cells.46 In contrast, Biswas et al47 reported that EGF activates NF-κB activity in estrogen receptor-negative breast cancer cells. Moreover, it was observed that EGF-induced cyclooxygennase-2 expression was NF-κB–independent in human oral squamous carcinoma cells.48 Thus, the relation between EGF/EGFR activation and NF-κB activity may depend on the cell types and conditions used. Our data suggest that the NF-κB binding site is 1 of the key cis-elements that regulate NGAL expression in response to EGF stimulation in PDAC cells. This is supported by the observation that a mutation of the NF-κB binding site in the NGAL promoter blocked the inhibition of NGAL promoter activity by EGF. Furthermore, E-cadherin down-regulation by an E-cadherin–specific small hairpin RNA (shRNA) resulted in decreased binding of NF-κB to its cis-cognate element. Moreover, EMSA analysis revealed that EGF stimulation reduces the binding of NF-κB to the corresponding cis-element in a dose-dependent manner. Thus, our data strongly suggest that EGF-mediated NGAL down-regulation occurs at the transcriptional level and through the interruption of NF-κB binding to the NGAL promoter in PDAC cells. However, how EGF and E-cadherin interrupt the binding of NF-κB to the NGAL promoter needs further study.

On the basis of the results from this study, we propose a model of the EGF-mediated NGAL expression inhibition in PDAC cells (Fig. 6D). EGFR activation by EGF binding triggered the MEK/ERK signaling pathway, which, in turn, up-regulated Zeb1 expression. The up-regulated Zeb1, in turn, reduced E-cadherin expression. The loss of E-cadherin led to reduced NGAL expression by inhibiting the binding of NF-κB to the NGAL promoter. MEK/ERK pathways activation also can inhibit this binding by pathways other than E-cadherin. Furthermore, because it is a secreted protein, NGAL can be detected in body fluids, including urine, blood, pancreatic juice, and saliva. Thus, NGAL may be a potential biomarker for the loss of E-cadherin expression in PDAC.

Acknowledgements

We thank Ann M. Sutton in the Department of Scientific Publications at The University of Texas M. D. Anderson Cancer Center for reviewing this article.

CONFLICT OF INTEREST DISCLOSURES

This work was supported in part by an M. D. Anderson Cancer Center (MDACC) Physician Scientist Program Award (to S.G.), an Institutional Research Grant, a Cyrus Scholar Award (to S.G.), a McNair Foundation Scholar Award (to S.G.), National Institutes of Health (NIH) Grant R01 CA69480 (to R.S.B.), NIH Grant R01 CA78590 (to S.K.B.), and NIH Grant 5P30CA16672 (a Cancer Center Support Grant to MDACC).

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