Consequence of Gastrin-Releasing Peptide Receptor Activation in a

Consequence of Gastrin-Releasing Peptide Receptor Activation in a Human Colon Cancer Cell Line: A Proteomic Approach Tom Ruginis,† Lauren Taglia,† Damien Matusiak,† Bao-Shiang Lee,‡ and Richard V. Benya*,† Department of Medicine, University of Illinois at Chicago and Chicago Veterans Administration Medical Center (West Side Division), Chicago, Illinois 60612, and Protein Research Laboratory, Research Resources Center, University of Illinois at Chicago, Chicago, Illinois 60612 Received January 6, 2006

Gastrin-releasing peptide (GRP) and its receptor (GRPR) are aberrantly up-regulated in colon cancer. When expressed, they act as morphogens, retaining tumor cells in a better differentiated state and retarding metastasis. To identify targets activated in response to GRPR signaling we studied Caco-2 and HT-29 cells, colon cancer cell lines that expresses GRPR as a function of confluence. Total cell protein was extracted from pre-confluent cells (expressing GRP/GRPR) cultured in serum-free media in the presence or absence of GRPR-specific antagonist; as well as from confluent cells that do not express GRPR. Overall, we identified 5 proteins that are specifically down-regulated after GRP/GRPR expression: Bach2, creatine kinase B, p47, and two that could not be identified; and 6 proteins that are up-regulated: gephyrin, HSP70, HP1, ICAM-1, ACAT, and one that could not be identified. These findings suggest that the mechanism(s) by which GRP/GRPR mediate its morphogenic effects in colon cancer involve the actions of a number of hitherto unappreciated proteins. Keywords: bombesin • morphogen • motogen

Introduction Gastrin-releasing peptide (GRP) is the primary member of the bombesin family of neuropeptides. GRP acts by binding to a specific member of the 7 transmembrane spanning, G protein-coupled receptor superfamily.1-3 Of all of GRP’s effects, arguably the most studied is its ability to increase the proliferation of human cancers, including those arising in the colon.4,5 Yet our observations indicate that the actions of GRP/GRPR may be subtler than simply acting to increase the proliferation of various tumors. Rather, we have shown that GRP is a modest mitogen in malignancy [reviewed in ref 6], with its proliferative effects subordinate to it acting as a morphogen when aberrantly expressed in colon cancer. As morphogens in the context of colon cancer, GRP/GRPR act primarily to promote the assumption of a better-differentiated phenotype.7,8 In the context of solid tumors, differentiation refers to the degree to which malignant cells approximate the normal tissues whence they originated. For all solid tumors, differentiation portends survival since better-differentiated cancer cells metastasize less frequently than those that are more poorly differentiated. We have previously shown that GRPR-induced phosphorylation of focal adhesion kinase (FAK), at least in part, enhances cell adhesion to the extracellular matrix (ECM) and * To whom correspondence should be addressed. Department of Medicine, University of Illinois at Chicago, 840 South Wood Street (M/C 716), Chicago, IL 60612. Tel: (312) 569-7439. Fax: (312) 569-8114. E-mail: [email protected]. † University of Illinois at Chicago and Chicago Veterans Administration Medical Center. ‡ University of Illinois at Chicago.

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decreases cell deformability, reducing their ability to metastasize; while promoting cell motility in the context of remodeling, contributing to their ability to maintain a better-differentiated phenotype.9,10 Yet the end targets of GRPR action mediating its morphogenic properties in colon cancer have yet to be fully elucidated. We recently showed that the human colon cancer cell lines Caco-2 and HT-29 variably express GRP/GRPR as a function of confluence.11 Whereas confluent cells do not express GRP/ GRPR, both proteins are rapidly up-regulated when confluent cells are wounded or disaggregated.11 When present, GRP/GRPR critically modulates tumor cell motility in the context of remodeling, and promotes adherence to the extracellular matrix, key features of morphogens in the context of colon cancer.10,11 The transient nature of GRP/GRPR expression by Caco-2 and HT-29 cells makes them good models for identifying the protein(s) mediating or effectuating their morphogenic properties. Thus, in this study, we systematically compared and contrasted protein expression in pre-confluent Caco-2 cells (expressing GRP/GRPR) with post-confluent Caco-2 cells (not expressing GRP/GRPR); and with pre-confluent cells in the presence and absence of the GRPR-specific antagonist DPhe6bombesin (6-13)methyl ester.12,13 We then used a twodimensional difference gel electrophoresis followed by MALDITOF mass spectroscopy to identify the proteins up- or downregulated after GRPR-specific signaling. The specificity of this up-regulation was confirmed by noting expression at the RNA and protein level in HT-29 cells. We herein show that GRPRinitiated signaling specifically results in the up-regulation of 10.1021/pr060005g CCC: $33.50

 2006 American Chemical Society

Proteomic Consequences of GRP Receptor Activation

gephyrin, heat shock protein 70, heterochromatin-associated protein 1R, and intracellular adhesion protein-1 expression. We also show that GRPR-initiated signaling down-regulates the expression of Bach2, p47, and creatine kinase B. These findings provide a number of novel and previously unrecognized mechanistic pathways by which GRP/GRPR might mediate its morphogenic effects in colon cancer.

Experimental Section Materials. All chemicals were from Fisher Scientific (Hanover Park, IL) unless otherwise specified. Tissue culture reagents and media were purchased from Gibco BRL Life Technologies (Paisley, UK). Western blot and 2D equipment was from BioRad (Hercules, CA). Protein purification and staining reagents were from Amersham Biosciences (Piscataway, NJ). All other 2D reagents were from Bio-Rad (Hercules, CA). D-Phe6bombesin (6-13)methyl ester (ME) was a kind gift of Dr. David Coy (Tulane University Medical Center, New Orleans, LA). Protease inhibitor cocktail was from Sigma (St. Louis, MO). BCA Protein Assay Kit was from Pierce (Rockford, IL). 30% Acrylamide/Bis solution and Precision Plus protein standards were from Bio-Rad Laboratories Inc. (Richmond, CA). PVDF membranes were from Fisher Scientific (Pittsburgh, PA). Gephyrin antibody was from BD Biosciences (San Jose, CA). Hsp70, ACAT, and HP-1R antibodies were from Abcam Inc. (Cambridge, MA). Secondary antibodies were from Santa-Cruz Technologies (Santa Cruz, CA). Cell Culture. Pre-confluent Caco-2 cells (expressing GRP/ GRPR) were generated as follows: cells were plated at a density of 3 × 106 cells per F-75, and maintained overnight in a 1:1 mixture of DMEM:Ham’s F12 media supplemented with 20% fetal bovine serum in a 5% CO2 atmosphere. Pre-confluent cells were then washed and incubated with 10 mL serum-free media for 24 h. Media was replaced with fresh serum-free media either containing, or not containing, 1 µM D-Phe6bombesin (613)methyl ester for an additional 18 h. Under these conditions, cells were ∼70% confluent. Prior to protein extraction, cells were washed ×2 with serum-free PBS (pH 7.4). Post-confluent Caco-2 cells (not expressing GRP/GRPR) were generated as follows: cells were plated at a density of 3 × 106 cells per F-75, and maintained in a 1:1 mixture of DMEM:Ham’s F12 media supplemented with 20% fetal bovine serum in a 5% CO2 atmosphere for 5 days, at which point they were 2 days post-confluent. Cells were washed ×2 and incubated with 10 mL serum-free media for an additional 18 h. Prior to protein extraction, cells were washed ×2 with serum-free PBS. Protein Isolation. Adherent cells were detached with a rubber scraper using a 20:1 solution of RIPA buffer (150 mM NaCl, 50 mM HEPES, 5 mM NaF, 5 mM EDTA, 1 mM sodium orthovanade, 0.50% sodium deoxycholate, 1.0% NP-40; pH 7.4) and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cells were transferred to a 1.5 mL microcentrifuge tube and freeze-thawed ×5. This was followed by 10 min centrifugation at 15 000 × g at 4 °C. The supernatant was transferred to a new 1.5 mL microcentrifuge tube. Protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL). 350 µg of protein was used for Coomassie blue stained gels, and 25 µg was used for silver stained gels. Protein purification was performed using the 2-D Clean Up Kit (Bio-Rad) according to the manufacturer’s instructions. Reverse Transcriptase Polymerase Chain Reaction. Cells were lysed using RNA STAT-60. Chloroform was added and the solution centrifuged at 12 000 × g for 15 min at 4 °C. The

research articles aqueous layer was extracted, added to 2-propanol (0.5 mL per 1 mL of RNA STAT-60 used) and centrifuged at 12 000 × g for 10 min. at 4 °C. The RNA pellet was subsequently washed with 75% ethanol by vortexing, centrifuged at 7500 × g for 5 min at 4 °C, air-dried, and resuspended in water. mRNA and cDNA were isolated using the QIAGEN and Invitrogen kits as described by manufacturers. Human ICAM-1 was amplified from cDNA using forward primer 5′-CGG CCA GCT TAT ACA CAA GA-3′ and reverse primer 5′-GTA ACC TCA GAC GAC CCT TA3′ (121bp). HP-1R was amplified using forward primer 5′-AGC GGA CAG CTG ACA GTT CT-3′ and reverse primer 5′-TTC CAG TCC TCT CTC AAA GC-3′ (350 bp). THIL/ACAT was amplified using forward primer 5′-TTC AGG GAG CCA TTG AAA AG-3′ and reverse primer 5′-GGC TTT CAT TCC TGA AGC AC-3′ (182 bp). HSP-70 was amplified using forward primer 5′-CGA CCT GAA CAA GAG CAT CA-3′ and reverse primer 5′-AAG ATC TGC GTC TGC TTG GT-3′ (213 bp). Gephyrin was amplified using forward primer 5′-CCA TGG GGG AAA AGG ACT AT-3′ and reverse primer 5′-GTG CAG GCA CAA CAA AGA GA-3′ (204 bp). GRP-R was amplified from cDNA using forward primer 5′-ATA CAA AGC CAT TGT CCG GCC-3′ and reverse primer 5′-CTG CTT CTT GAC ATG TAT ATT CCC TT-3′ (352 bp). Actin, used as a control, was amplified from cDNA using forward primer 5′-ATG GAA GAA GAG ATC GC-3′ and reverse primer 5′-GGA TGC CAC GCT TGC TC-3′ (245 bp). PCR amplification for HP-1 R, THIL, Gephyrin, and ICAM-1 was carried out for 40 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s. PCR amplification for HSP-70 and GRPR was carried out for 40 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s. PCR amplification for Actin was carried out for 40 cycles at 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 60 s. Western Analysis. Caco-2 cells: Approximately 50 µg of total cell protein was run alongside Precision Plus Protein Standards (Bio-Rad) on a 10% poly-acrylamide gel run at 100 V for 2 h. The resolved proteins were electrophoretically transferred to a PVDF membrane (Fisher-Scientific, Hanover Park, IL) at 100 V for 1 h. The membrane was blocked overnight at 4 °C using 20 mL of 5% nonfat milk in Tris Buffered Saline with Tween 20, pH 7.0 (TBST) (Sigma-Aldrich, St. Louis, MO), and then placed in a Mini-Protean II Multi-Screen apparatus, and which allows for identical amounts of protein to be probed simultaneously with different antibodies. Membranes were incubated with primary antibody in 350 µL of 2.5% nonfat milk in TBST for 2 h on a platform shaker at room temperature. Following two 10 min rinses with TBST, horseradish peroxidase conjugated goat anti-rabbit IgG were incubated in their respective lanes for 1 h at a concentration of 1:10 000 in 350 µL of 2.5% nonfat milk in TBST. Immunoreactive bands were visualized using the ECL Plus detection system (Amersham Biosciences). HT-29 cells: Approximately 200 µg of protein was loaded and electrophoresed across an 8% polyacrylamide gel under denaturing and reducing conditions. The resolved proteins were electrophoretically transferred to PVDF membranes. Membranes were incubated with primary antibody in a MiniProtean II Multi-Screen apparatus for 2 h at the following concentrations: GRPR 1:500, Hsp70 1:1000, HP-1R 1:500, ICAM 1:200, Gephyrin 1:100, ACAT 1:1000, and Actin 1:50, followed by 2 sequential 10 min washes with TBST. GRPR, Hsp70, HP1R, and actin immunoreactive bands were visualized using a horseradish peroxidase conjugated goat anti-rabbit IgG, whereas ICAM, Gephyrin, and ACAT were visualized using a horseradish peroxidase conjugated goat anti-mouse IgG and the ECL Plus detection system. Journal of Proteome Research • Vol. 5, No. 6, 2006 1461

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Isoelectric Focusing. Following purification, the pelleted protein was dissolved in 335 µl of rehydration buffer [7 M urea, 2 M thiourea, 4% CHAPS (w/v), 40 mM DTT, 0.5% Bio-Lyte ampholytes (v/v)]. ReadyStrip IPG strips (17 cm) were actively rehydrated for 12 h at 50 V using a Protean IEF Cell (Bio-Rad, Hercules, CA). Isoelectric focusing of IPG strips of pH range 3-6 was performed at 40 000 V-hr, pH 5-8 at 50 000 V-hr, and pH 7-10 at 45 000 V-hr using the Protean IEF Cell. Electrophoresis. After focusing, strips of defined pH were placed in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M Urea, 30% Glycerol, 2% SDS) supplemented with 2% (w/v) DTT for 15 min on a platform rocker. Strips were then placed into new lanes into equilibration buffer supplemented with 2.5% (w/v) iodoacetamide for 15 min. Following equilibration, IPG strips were placed onto a 10% poly-acrylamide gel (20 × 20 cm) and run at 25 mA for 20 min, at 50 mA for 45 min, and at 100 mA until the gel front reached the bottom of the gel. Analytical gels were stained using a Silver Staining kit according to the manufacturer’s instructions. Gels used for protein extraction were stained with Coomassie blue by fixing overnight in staining solution [50% (v/v) methanol, 0.1% (w/v) Coomassie brilliant blue R-250, and 10% (v/v) acetic acid], and destained by washing ×3 in destaining solution [15% (v/v) methanol and 10% (v/v) acetic acid] for 1 h each time. Gel Analysis and MALDI-TOF. Gels were scanned using a ChemiDoc Camera, and analyzed using PDQuest 2-D Analysis Software Version 7.1.1. All gels were also visually inspected to check for errors in spot detection. Although spot density can vary from gel to gel, it has previously been shown that the relative spot size and density remains relatively stable within known biological samples.14 Spots were picked by hand using a OneTouch Spot Picker (The Gel Company, San Francisco, CA). In-gel tryptic digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of tryptic peptides was performed as previously described.15 Briefly, gel plugs were washed in 50% acetonitrile ×1, reduced of sulfide bonds in 60 mM DTT, alkylated of free sulfhydryl groups in 4-vinylpyridine in 6 M guanidine-HCl, 50 mM ammonium bicarbonate (pH 8.0) and 5mM EDTA, and then incubated in trypsin [in 50 mM ammonium bicarbonate (pH8.0) solution at a concentration of 2 µg/100 µL] overnight. For MALDI-TOF, residual peptides were extracted, spotted onto a 96 × 2 position MALDI-TOF target, and analyzed by a positive-ion Voyager DE-PRO Mass Spectrometer (Applied Biosystems, Foster City, CA) equipped with a nitrogen laser. Peptide mass results were used to identify the proteins using the ProteinProspector version 4.0.5 and MSFit link (http://prospector.ucsf.edu). Conditions were set to allow for one missed cleavage, a mass tolerance of 100 ppm, and limited to occur within the proximity of the isoelectric point (pI) and the protein’s approximate molecular weight. Protein-Protein Interactions. PathwayAssist Software version 3.0 was used to determine protein-protein interactions. Proteins identified as up- or down-regulated after GRP/GRPR expression were entered and evaluated using MedScan, a textmining tool that has identified over 100 000 binding, regulatory, modifying interactions reported and stored in PubMed.16 The ‘shortest pathway’ algorithm was used to determine the simplest connections between all identified proteins, with the analysis performed without filters. 1462

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Figure 1. Western blot analysis showing GRPR expression in Caco-2 cells. Cell lysates were obtained from pre-confluent cells in the presence (Lane 1) or absence (Lane 2) of the GRPR-specific antagonist D-Phe6bombesin (6-13)methyl ester, or from postconfluent (Lane 3) cells, as described in Methods. Proteins from the cells of the indicated condition were evaluated separately using the Mini-Protean II Multi-Screen apparatus, with antibodies to GRPR and actin added simultaneously.

Results Conditions for GRP/GRPR Expression. We previously showed using an immunohistochemical approach that GRP and its receptor were expressed by pre-confluent, but not postconfluent, Caco-2 cells.11 To confirm that our conditions yielded cells expressing, or not expressing, GRP/GRPR as a function of confluence, we first performed Western analysis on total cell proteins obtained from Caco-2 cells 2 days post plating, at which point they were ∼70% confluentsin the presence or absence of specific antagonist D-Phe6bombesin (6-13)methyl ester as described in Methodssas well as on proteins isolated from post-confluent Caco-2 cells. Cells were washed and lysates prepared as described in Methods. In all instances, 50 mg protein was separated by 10% SDS-PAGE. Under these conditions pre-confluent cells expressed GRPR, irrespective of exposure to the specific antagonist D-Phe6bombesin (613)methyl ester (Figure 1). In contrast, post-confluent cells showed no evidence of GRP (data NS) or GRPR expression (Figure 1). Thus these data defined the conditions under which 2D gel electrophoresis was subsequently performed. Protein Expression Profiles in Caco-2 Cells. Over 1900 protein spots could be distinguished in Caco-2 cells by 2D gel electrophoresis after isoelectric focusing across all 3 pI ranges was performed (Figure 2). In comparing pre- and postconfluent Caco-2 cells, the expression of 31 proteins differed (Figure 3). Of these, the expression of 10 was increased in GRP/ GRPR-expressing pre-confluent Caco-2 cells as compared to post-confluent Caco-2 cells, while the expression of 21 was decreased. Yet the protein expression profile of pre- versus post-confluent Caco-2 cells is not due only to the presence or absence of GRP/GRPR. Thus, we also compared protein expression profiles of GRP/GRPR-expressing pre-confluent Caco-2 cells cultured in the presence and absence of the antagonist ME. Under these conditions the expression of 5 proteins was increased, and the expression of 6 proteins decreased, when GRP/GRPR-expressing pre-confluent Caco-2 cells were not exposed to receptor-specific antagonist (Figure 3). Although ME has been shown to specifically block agonist binding to the GRPR,12,13 it nonetheless remains possible that this antagonist incompletely acts at this receptor, or has


Figure 2. Caco-2 cell proteome as a function of GRPR expression and/or activation. Proteins were separated in the first dimension by pI and in the second dimension by molecular weight. Proteins were obtained from GRP/GRPR-expressing preconfluent Caco-2 cells in the absence (shown) or presence of antagonist D-Phe6bombesin (6-13)methyl ester, and from postconfluent Caco-2 cells that do not express GRP/GRPR. First dimension separation was achieved using pI strips between 4 and 7, 5-8 (shown), and 7-10; with spots that changed between experimental conditions identified. Labels for proteins indicated as (A), (C), and (D) identify proteins whose presence diminished significantly in the presence of the GRPR antagonist or postconfluence; whereas that for (H) identifies the location of the relevant protein whose presence became detectable in the presence of the GRPR antagonist or post-confluence.

Figure 3. Venn diagram showing the number of proteins whose expression was altered as a function of the indicated experimental conditions. Pre-confluent cells express GRP/GRPR whereas post-confluent cells do not; whereas ME refers to pre-confluent cells cultured in the presence or absence of the GRPR-specific antagonist D-Phe6bombesin (6-13)methyl ester.

unknown effects elsewhere. Thus, to identify proteins whose expression was altered after GRP/GRPR expression, we considered further only those proteins whose presence was consistently altered as both a function of confluence and exposure to the antagonist ME (Figures 2, 3). Thus, given the experimental objective of identifying proteins whose expression is specifically regulated in response to GRPR signaling, we focused only on those spots whose expression, as assessed densitometrically, was attenuated by >70%, or increased more than 4-fold, as appropriate (Figure 4). The expression of all 11 proteins whose expression was altered both as a function of cell confluence as well as exposure to ME satisfied those conditions (Figure 4).

research articles Protein Identification by MALDI-TOF MS. Individual spots whose presence was determined to be GRP/GRPR-specific, as determined above, were excised from the relevant 2D gel (Figure 5), subjected to in-gel trypsin digestion, and characterized by MALDI-TOF MS (Figure 6). Proteins were identified using ProteinProspector as outlined in Methods. Six proteins were present in pre-confluent Caco-2 cells not exposed to the antagonist ME that diminished significantly both in the presence of ME and when cells became post-confluent: ICAM-1, heterochromatic-associated protein 1 (HP1), gephyrin, THIL/acetyl co-A acetyltransferase (ACAT), heat shock protein 70 isoform 1 (HSP70), and one that could not be definitively identified (Figure 5; Table 1). ICAM-1, Geph and HP1 were identified in gels whose proteins underwent isoelectric focusing between pI 5 and 8; whereas HSP70 was focused between pI 3-6 and THIL/acetyl co-A acetyltransferase was identified in gels whose proteins underwent isoelectric focusing between pI 7 and 10. In contrast, 5 proteins were present in significant amounts under conditions when GRP/GRPR was not present and/or unable to initiate signaling: Bach2, p47, creatine kinase B, and two that could not be definitively identified (Figure 5; Table 1). Creatine kinase B was identified in gels whose proteins underwent isoelectric focusing between pI 5 and 8; while p47 was identified in gels whose proteins underwent isoelectric focusing between pI 7 and 10 and Bach2 between 3 and 6. Confirmation of GRP/GRPR-Dependent Protein Up-Regulation. To confirm that the proteins whose expression was upregulated in Caco-2 cells after GRP/GRPR expression were not specific to that cell line, we also studied the expression of ICAM-1, HP1, gephyrin, THIL/ACAT, and HSP70 in HT-29 cells. Since, as we have previously shown,11 GRP/GRPR expression varies as a function of confluence in this cell line, similar to what is observed in Caco-2 cells, we studied pre-confluent (expressing GRP/GRPR) and post-confluent (not expressing these proteins). Cells were obtained at either 80% confluence or 3 days post-confluence, the RNA and proteins extracted, and evaluated by RT-PCR or Western blot analysis. Similar to Caco-2 cells, pre-confluent HT-29 cells expressing GRP/GRPR also expressed ICAM-1, HP1, gephyrin, THIL/ACAT, and HSP70 mRNA and protein (Figure 7). In contrast, postconfluent HT-29 cells not expressing GRP/GRPR did not express ICAM-1, HP1, gephyrin, THIL/ACAT, and HSP70 mRNA or protein (Figure 7). Likewise, post-confluent HT-29 cells expressed Bach2, p47, creatine kinase B mRNA and protein whereas pre-confluent cells expressing GRP/GRPR did not (Figure 8). Possible Protein Interactions. To explore possible interactions between the proteins identified whose expression is GRP/GRPR-dependent as determined above, we used PathwayAssist.16 This program suggests interactions based on those reported in PubMed, and identified using the text-mining tool MedScan.16 Each interaction identified by this program was independently assessed and gauged according to likelihood and/or accuracy. Connectivity was determined based on the simplest connection between proteins, and provided the basis for prioritizing the putative protein-protein interactions (Figure 9). Five proteins not identified experimentally as a part of this study were identified by PathwayAssist to be involved GRP/ GRPR signaling, listed in decreasing order of connectivity: Jun (also known as AP-1; connectivity score 2506), interferon-γ (2161), PKT2 (800), peroxisome proliferator-activated receptor-γ Journal of Proteome Research • Vol. 5, No. 6, 2006 1463

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Figure 4. Histogram of proteins whose expression was altered at least 4-fold in the presence or absence of GRP/GRPR. Panel A: Expression of proteins whose expression was down-regulated in the presence of functional GRPR. Panel B: Expression of proteins whose expression was up-regulated in the presence of functional GRPR. For both panels the striped bars represent proteins obtained from pre-confluent (GRPR-expressing) cells, solid bars represent proteins obtained from pre-confluent cells exposed to the GRPRspecific antagonist D-Phe6(bombesin) methyl ester, whereas the open bars represent proteins obtained from post-confluent cells (not expressing GRPR). Data represent the means ( SEM of 10 separately performed conditions.

(PPAR-γ; 699), and Cyp2b20 (150), a member of the cytochrome P450 family. These degrees of connectivity compare with those identified experimentally in this study: ICAM-1 (connectivity score 1070), GRP (399), GRPR (52), HSP70 (192), ACAT (30), gephyrin (26), Bach2 (22), CK-B (12), and p47 (2).

Discussion

As morphogens GRP/GRPR inhibit processes associated with metastasis. Specifically, we have shown that GRP promotes tumor cell adherence to the extracellular matrix; decreases cell deformability thereby diminishing the ability of tumor cells to translocate into the vascular or lymphatic spaces; and enhances cell motility in the context of remodeling.10,11 However, the protein(s) mediating GRP’s morphogenic effects are not known.

Gastrin-releasing peptide regulates a variety of physiological processes by binding to a specific 7 transmembrane-spanning, G protein-coupled receptor. GRP and its receptor (GRPR) are transiently expressed during gut development where they contribute to villous growth.17 After this period of normal expression, epithelial cells lining the adult intestine do not express either protein.7 However, GRP/GRPR are aberrantly upregulated in colon cancer where we have shown that they act as morphogens,6,7,10,11 recapitulating their role during normal development by promoting the assumption of a better differentiated phenotype.

In this study, we identified 11 separate proteins whose expression is strongly linked to GRP/GRPR expression. To do this we took advantage of our recent observation that the human colon cancer cell line Caco-2 expresses GRP/GRPR as a function of confluence.11 We then compared and contrasted protein expression in pre- and post-confluent Caco-2 cells; and in pre-confluent Caco-2 cells expressing GRP/GRPR in the presence or absence of receptor-specific antibody. Using this approach, we found that GRP/GRPR expression is associated with the up-regulation of intracellular adhesion molecule 1; heat-shock protein 70; heterochromatin-associated protein 1;

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Figure 5. Detailed view of 2D gel regions of proteins up- and down-regulated as a function of GRP/GRPR expression and/or function. (A), Magnified view of relevant gels showing proteins whose expression decreased when GRP/GRPR signaling was not present. (B), Magnified view of relevant gels showing proteins whose expression increased when GRP/GRPR signaling was not present. The letters A-I refer to the proteins identified in Figures 2 and 4. Images are representative examples of 10 separately generated gels. Pre, pre-confluent Caco-2 cells; Pre+ME, preconfluent Caco-2 cells cultured in the presence of antagonist D-Phe6(bombesin) methyl ester; Post, post-confluent Caco-2 cells.

Figure 6. Matrix-assisted laser desorption/ionization-time-offlight peptide mass spectroscopy map of gel-excised protein from pre-confluent Caco-2 cells (protein A in Figure 4). The spot was picked from a Coomassie blue stained gel, digested with trypsin, and MALDI-TOF MS performed as described in Methods. Peptide mass results were used to identify the proteins using the ProteinProspector version 4.0.5 and MS-Fit link, with conditions set to allow for one missed cleavage, a mass tolerance of 100 ppm, and limited to occur within the proximity of the isoelectric point and the protein’s approximate molecular weight.

gephyrin; acyl-coenzyme A:cholesterol acyltransferase; and 1 other protein that we could not identify. Likewise, we found that GRP/GRPR signaling results in the down-regulation of p47; creatine kinase B; Bach2; and 2 other proteins that we could not identify. Since Caco-2 and HT-29 cells have been extensively used as models for colorectal cancer as well as for normal intestinal development, the expression profile for some of the proteins we identified as a function of confluence can serve to validate our experimental approach. Specifically, the previously described decrease in expression of ACAT,18 HSP70 and ICAM119s and the increase in expression of CK-B20swith increasing Caco-2 cell confluence is consistent with our observations

research articles herein, and thus supports the validity of our current observations. Yet it warrants observing that these findings alone do not indicate that our current findings are simply confirmatory. Rather, these earlier studies nonspecifically looked for alterations in Caco-2 cell protein expression as a function of confluence whereas our study identifies for the first time potential mediators of GRP’s morphogenic properties. Since GRP and its receptor are known to promote villus growth and development during gut organogenesis,17 it is not surprising that a number of the proteins whose presence are a function of GRP/GRPR expression are involved with proliferation and/or apoptosis. Yet the proteins identified appear to have conflicting effects on these processes. For instance, we show that GRP up-regulates HSP70 and down-regulates Bach2. Since HSP70 inhibits lysosomal membrane permeabilization,21 its up-regulation promotes cell survival. Likewise, downregulation of Bach2, a transcriptional repressor that decreases cell proliferation and increases apoptosis,22 also promotes cell survival. In contrast, the down-regulation of p47, important for proliferation,23 and CK-B, important for cellular ATP regeneration and typically found in cells with high metabolic requirements,24 would be suspected to impair tumor cell survival. Thus, the consequences GRP/GRPR-mediated alterations in the expression of these proteins, particularly in colon cancer, have yet to be determined. In contrast, ACAT, HP1, and ICAM-1 are either known to be expressed as a function of normal development and/or may have properties consistent with morphogens in cancer. Since fetal colon has the ability to synthesize lipids,25 ACAT expression in this tissue type is expected; in contrast, its role post malignant transformation is not clear and may simply reflect the disordered recapitulation of proteins otherwise important for organogenesis. In contrast, while polycomb proteins likewise are important in normal development [reviewed in ref 26], recent studies have shown that individual members of this family can have specific and opposite effects on cancer cell behavior when aberrantly expressed post malignant transformation. For example, while up-regulation of the polycomb protein EZH2 promotes the metastasis of prostate cancers,27 breast cancer cells with a metastatic phenotype show a downregulation of HP-1.28 This suggests that HP1 up-regulation might attenuate metastasis, and thus may be an important mediator of GRP/GRPR’s morphogenic effects in colon cancer. However, the process whereby HP1 acts to mediate GRP’s morphogenic effects in colon cancer remains to be elucidated. Particularly intriguing is our finding that ICAM-1 is upregulated after GRP/GRPR expression. ICAM-1 has previously been shown to be up-regulated in human colon cancer as compared to adjacent nonmalignant epithelial cells;29 with its expression regulated, at least in osteoblasts and maturing osteoclasts, by focal adhesion kinase.30 This is of particular interest since we have previously shown that FAK critically mediates a number of GRP’s morphogenic properties.10 Since ICAM-1 enhances the attachment of other cell types, particularly inflammatory cells, up-regulation of this protein may not only play a role in the inflammatory component of colon cancer, but may also play a role in enhancing the attachment of tumor cells to each other. This may occur as a consequence of ICAM-1’s ability to directly interact with the actin cytoskeleton and with integrins [reviewed in ref 31]. If so, GRPmediated up-regulation of ICAM-1 may be important to attenuating colon cancer metastasis as well as promoting the inflammatory response. Journal of Proteome Research • Vol. 5, No. 6, 2006 1465

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Table 1. MALDI TOF Mass Spectroscopy Search Resultsa expression in cells of defined condition (% maximum)

protein name

accession no.

entry name

% sequence coverage

theoretical MW/pI

experimental MW/pI

pre-confluent (no ME)

pre-confluent (plus ME)

post-confluent

ICAM-1 Gephryin HP-1 HSP70 Thil/ACAT Not ID p47 CK-B Bach2 Not ID Not ID

P05362 Q9NQX3 P83916 P11142 P24752

ICAM GEPH CBX1 HSP7C THIL

36 22 62 21 43

57.8/7.86 79.7/5.25 21.4/4.85 70.9/5.37 45.2/8.98

P08567 P12277 Q9BYV9

PLEK KCRB BACH2

12 32 18

40.1/8.32 42.6/5.34 92.5/5.00

60/7.5 80/6.8 22/5.8 73/4.0 40/8.5 95/7.5 36/5.0 40/6.0 88/5.0 70/5.5 100/4.5

100 100 100 100 100 100 6.8 ( 2.7 0.2 ( 0.1 27.1 ( 2.5 11.8 ( 3.3 17.3 ( 4.0

27.7 ( 1.7 27.6 ( 2.0 18.9 ( 3.0 3.7 ( 1.9 17.8 ( 3.8 23.3 ( 2.0 85.9 ( 8.4 93.4 ( 10.2 83.3 ( 4.0 100 100

21.0 ( 1.9 24.9 ( 2.4 14.1 ( 3.3 2.6 ( 0.9 23.4 ( 7.0 19.9 ( 2.7 100 100 100 90.4 ( 2.0 96.1 ( 1.2

a Accession number (#) and Entry Name identify the relevant protein’s Swiss-Prot designator; while % sequence coverage identifies the extent to which the indicated entity could be sequenced by MALDI-TOF MS. Expression summarizes the densitometry means(SE for the amount of relevant protein in Caco-2 cells cultured in the defined condition, with the condition showing greatest expression set to 100%. In all instances, data was generated in duplicate from 10 independent cell preparations. ME, D-Phe6bombesin (6-13) methyl ester; MW, molecular weight; pI, isoelectric point.

Figure 7. mRNA (Panel A) and protein (Panel B) expression in pre- and post-confluent malignant human colonic epithelial HT29 cells for proteins up-regulated by GRP/GRPR. Panel A: mRNA expression in pre-confluent (left) and post-confluent (right) HT29 cells. As described in Methods, RNA was extracted from HT29 cells at the indicated confluence and evaluated for GRPR, HSP70, HP-1R, ICAM-1, Gephyrin, ACAT, and Actin mRNA expression by RT-PCR using gene-specific primers. Panel B: Western blot analysis showing GRPR, HSP-70, HP-1R, ICAM-1, Gephyrin, ACAT, and Actin protein expression in pre-confluent (left) and post-confluent (right) HT-29 cells.

We used the program PathwayAssist16 to assess how the proteins identified herein might interact after GRP activation of its cognate receptor. Although this is a theoretical approach, a number of the interactions, such as GRP’s role in activating the transcription factor Jun [for example see ref 32] and the signaling enzyme FAK [reviewed in ref 33], have been previously described, enhancing the likelihood that the other proteins identified by PathwayAssist truly contribute to GRP/GRPRmediated signaling. Of particular interest then, in addition to 1466


Figure 8. mRNA (Panel A) and protein (Panel B) expression in pre and post confluent malignant human colonic epithelial HT29 cells for proteins down-regulated by GRP/GRPR. Panel A: mRNA expression in pre-confluent (left) and post-confluent (right) HT-29 cells. As described in Methods, RNA was extracted from HT-29 cells at the indicated confluence and evaluated for GRPR, Bach2, creatine kinase B (CKB), p47 and Actin mRNA expression by RT-PCR using gene-specific primers. Panel B: Western blot analysis showing GRPR, creatine kinase B (CKB), p47 and Actin protein expression in pre-confluent (left) and post-confluent (right) HT-29 cells.

identifying possible links between GRP/GRPR and the proteins identified herein, is the identification of interferon-γ and PPAR-γ as potentially important in GRP/GRPR signaling. Interferon-γ is known to have antitumorigenic effects [reviewed in ref 34]; whereas PPAR-γ is both anti-neoplastic and prodifferentiating in selected cell types [reviewed in ref 35]. Thus, additional yet hitherto unsuspected proteins may be involved in mediating GRP’s morphogenic effects in cancer. In summary, we herein demonstrate that GRP activation of its cognate receptor results in the specific up- and downregulation of a number of proteins. These proteins may play important roles in mediating GRP/GRPR’s morphogenic effect when aberrantly expressed in colon cancer. Specific mecha-


Figure 9. Potential interactions between GRP/GRPR-dependent proteins as identified by PathwayAssist. Proteins whose expression was up-regulated after GRP/GRPR signaling are shown by shaded diamonds whereas those that were down-regulated are shown by shaded ovals. Proteins whose expression was not shown to be altered in this study, but identified by PathwayAssist as integral to the interactions shown here are indicated by the nonshaded ovals. Binding interactions are indicated by solid circles, interactions relating to expression and/or regulation are shown by open squares; with stimulatory interactions indicated by the solid arrows and inhibitory interactions indicated by the dashed lines.

nisms regulating GRP/GRPR altering the expression of these proteins, as well as their exact contributions to modulating colorectal cancer differentiation and metastasis, awaits further study. Abbreviations. ACAT, acyl-coenzyme A:cholesterol acyltransferase; CK-B, creatine kinase B; FAK, focal adhesion kinase; GRP, gastrin-releasing peptide; GRPR, GRP receptor; HSP, heat shock protein; HP, heterochromatin-associated protein; ICAM, intracellular adhesion molecule; PPAR-γ, peroxisome proliferator-activated receptor-γ.

Acknowledgment. This work was supported by NIH Grant No. CA-094346 and a VA Merit Review award (to R.V. Benya). References (1) Battey, J. F.; Way, J. M.; Corjay, M. H.; Shapira, H.; Kusano, K.; Harkins, R.; Wu, J. M.; Slattery, T.; Mann, E.; Feldman, R. I. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 395-399. (2) Spindel, E. R.; Giladi, E.; Brehm, P.; Goodman, R. H.; Segerson, T. P. Cloning and functional characterization of a complimentary DNA encoding the murine fibroblast bomesin/gastrin-releasing peptide receptor. Mol. Endocrinol. 1990, 4, 1956-1963. (3) Corgay, M. H.; Dobrzanski, D. J.; Way, J. M.; Viallet, J.; Shapira, H.; Worland, P.; Sausville, E. A.; Battey, J. F. Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells. J. Biol. Chem. 1991, 266, 1877118779. (4) Frucht, H.; Gazdar, A.; Jensen, R. T. human colon cancer cell line NCI-H716 expresses functional bombesin receptors. Proc. Am. Assoc. Cancer Res. 1991, 32, 47. (5) Frucht, H.; Gazdar, A. F.; Park, J. A.; Oie, H.; Jensen, R. T. Characterization of functional receptors for gastrointestinal hormones on human colon cancer cells. Cancer Res. 1992, 52, 1114-1122.

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PR060005G

Consequence of Gastrin-Releasing Peptide Receptor Activation in a

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