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Cell, Tumor, and Stem Cell Biology |
Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
Requests for reprints: M. Iqbal Parker, Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, 7925 Cape Town, South Africa. Phone: 27-21-4066335; Fax: 27-21-4066061; E-mail: mparker{at}curie.uct.ac.za.
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Abstract |
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, GROß, and CXCR2 are up-regulated in esophageal tumor tissue. Furthermore, GRO, GROß, and CXCR2 are constitutively expressed in WHCO1, an esophageal cancer cell line that was used as a model system here. GROß enhances transcription of EGR-1, via the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which can be blocked by a specific antagonist of CXCR2 (SB 225002) or specific antibody to GROß. WHCO1 cells treated with SB 225002 exhibited a 40% reduction in cell proliferation. A stable WHCO1 GRO RNA interference (RNAi) clone displayed a 43% reduction in GRO mRNA levels as determined by real-time RT-PCR, reduced levels of GRO by fluorescence microscopy, and a 60% reduction in the levels of phosphorylated ERK1/2. A stable clone expressing GROß RNAi displayed >95% reduction in GROß mRNA levels, reduced levels of GROß by fluorescence microscopy, and an 80% reduction in the levels of phosphorylated ERK1/2. Moreover, these GRO RNAi- and GROß RNAi-expressing clones displayed a 20% and 50% decrease in cell proliferation, respectively. Our results suggest that GRO-CXCR2 and GROß-CXCR2 signaling contributes significantly to esophageal cancer cell proliferation and that this autocrine signaling pathway may be involved in esophageal tumorigenesis. (Cancer Res 2006; 66(6): 3071-7) |
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and GROß, relative to adjacent normal tissues.2
Chemokines are a superfamily of small, cytokine-like proteins that selectively regulate the recruitment and trafficking of leukocyte subsets to inflammatory sites through chemoattraction (4). Members in this family have been divided into two major subfamilies, CXC and CC, based on the arrangement of the first two of the four conserved cysteine residues in the amino terminus of the proteins. The two cysteines are separated by a nonconserved amino acid in CXC chemokines and adjacent to each other in CC chemokines. Depending on the presence or absence of an ELR motif (Glu-Leu-Arg) immediately preceding the first cysteine, CXC chemokines are further subdivided into two groups, ELR+ CXC chemokines and ELR– CXC chemokines. ELR+ CXC chemokines, which are potent promoters of angiogenesis and bind to CXC chemokine receptor 1 or 2 (CXCR1 or CXCR2), include the GROs (GRO, GROß, and GRO
), interleukin-8 (IL-8), epithelial-neutrophil activating protein (ENA-78), granulocyte chemotactic protein (GCP-2), neutrophil-activating protein (NAP-2), and ß-thromboglobulin (5–7). ELR– CXC chemokines are potent inhibitors of the ELR+ CXC chemokine-mediated angiogenesis (6), and include monokine induced by IFN- (MIG), stromal cell-derived factor (SDF-1), IFN--inducible protein (IP-10), and platelet factor 4 (PF4; ref. 6). IP-10 and MIG bind to CXCR3 receptors, whereas SDF-1 only binds to the CXCR4 receptor (5). The biological functions of ELR+ CXC chemokines are primarily mediated via CXCR2, a 7-transmembrane G-protein coupled receptor. Signaling for this receptor is mediated via various signaling pathways, including the extracellular signal-regulated kinases (ERK; refs. 8, 9).
A large body of evidence indicates that CXC chemokines play a critical role in inflammatory reactions and wound healing (reviewed in ref. 10). Additional evidence suggests that certain chemokines are involved in tumorigenesis because a wide range of cancers express elevated levels of various chemokines, such as IL-8 in non–small cell lung carcinoma and gastric carcinomas (11–13), and GRO in melanoma and prostate cancer cells (14, 15). In melanoma cells, GRO acts as an autocrine/paracrine growth factor because these cells express both GRO and its receptor, CXCR2 (16, 17). IL-8 also functions as an autocrine growth factor in malignant melanoma cells (18) and other neoplasias, such as gastric (19), hepatic, and pancreatic cancers (20). Compared with GRO and IL-8, less is known about the role of GROß in tumorigenesis, although reports have implicated GROß in enhanced tumor growth of immortalized melanocytes (21).
Presently, the expression status and role of GRO and GROß in esophageal squamous cell carcinoma is unknown. We therefore examined the expression of these two chemokines and their receptor, CXCR2, in esophageal squamous cell carcinoma tissues and adjacent normal tissues. Our results show that tumor tissues overexpress GRO, GROß, and CXCR2 compared with adjacent normal esophageal epithelial tissue, and, furthermore, that the esophageal squamous cell carcinoma cell lines WHCO1, WHCO5, and WHCO6 coexpress GRO, GROß, and CXCR2. An antagonist of CXCR2 (SB 225002) reduced proliferation of WHCO1 cells, and knockdown of GRO and GROß in WHCO1 cells using RNA interference (RNAi) reduced cell proliferation by 20% and 50%, respectively. These results implicate GRO and CXCR2 in a proliferative autocrine loop in esophageal squamous cell carcinoma cells.
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Materials and Methods |
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-32P]dCTP using the Megaprime DNA labeling system (Amersham Pharmacia Biotech UK Limited, Little Chalfont, Bucks, United Kingdom). The membrane was stripped and rehybridized with a ß-actin probe to control for RNA loading. The probes included a 0.56 kb EcoRI fragment of EGR-1 cDNA and a 0.63 kb EcoRI fragment of ß-actin cDNA. After scanning the membranes using a phosphoimager, the membranes were exposed to X-ray film.
Real-time reverse transcription-PCR and reverse transcription-PCR. Total RNA was prepared from four paired normal and esophageal squamous cell carcinoma biopsies and esophageal squamous cell carcinoma cultured cells using TRIzol (Invitrogen) according to the instructions of the manufacturer and quantified using UV absorbance at 260 nm (DU650 spectrophotometer, Beckman Instrument, Inc., Fullerton, CA). cDNA was synthesized using 5 µg total RNA and oligo(dT)20 primer using the SuperScript III system for reverse transcription-PCR (RT-PCR; Invitrogen) following the instructions of the manufacturer. Target primers for amplifying GRO, GROß, and CXCR2 were designed using Primer Designer (Scientific & Educational Software Version 2.0). GRO forward primer: 5'-ACCTCCTCGCCAGCTCTT-3' and reverse primer: 5'-CTTCAGGAACAGCCACCAG-3'; GROß forward primer: 5'-AGCTCTCCTCCTCGCACA-3' and reverse primer: 5'-CTTCAGGAACAGCCACCAA-3'; CXCR2 forward primer: 5'-CTCCAATAACAGCAGGTCAC-3' and reverse primer: 5'-GGCTCAGCAGGAATACCA-3'. Each 50 µL reaction mixture for RT-PCR contained 2.5 mmol/L MgCl2, 0.5 µmol/L of each primer. Each 20 µL reaction mixture for real-time RT-PCR contained 2.5 mmol/L MgCl2, 0.5 µmol/L of each primer, and 1 µL LightCycler FastStart DNA Master SYBR Green I (Roche Diagnostics South Africa, Randburg, Gauteng, South Africa). RT-PCR analysis was done using GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) and real-time RT-PCR analysis was carried out with LightCycler II (Roche Diagnostics GmbH, Mannheim, Germany). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control to standardize and to test the RNA integrity. All experiments for real-time RT-PCR were done in triplicate, data was analyzed using the comparative Ct method, and results are shown as fold induction of mRNA.
Immunohistochemical analysis. Samples for immunohistochemical analysis were sectioned from archived, paraffin-embedded normal and tumor tissues obtained from esophageal cancer patients who had undergone esophagectomies. The dewaxed slides were incubated with 1:20 dilution of normal rabbit serum (for GRO and GROß, DakoCytomation Denmark A/S, Glostrup, Denmark) or normal goat serum (for CXCR2, DakoCytomation Denmark) for 30 minutes and then incubated with 1:50 dilution of anti-GRO polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), 1:100 dilution of anti-GROß polyclonal antibody (Santa Cruz Biotechnology) and 1:1,000 dilution of rabbit anti-human CXCR2 polyclonal antibody (a gift from Prof. Ann Richmond), respectively, at 4°C overnight. After rinsing in PBS, slides were incubated with 1:400 dilution of donkey anti-goat horseradish peroxidase (for GRO and GROß, DakoCytomation Denmark) or DAKO(R) EnVision+ peroxidase (for CXCR2, DakoCytomation Denmark) at room temperature for 30 minutes, and the color was developed using 3,3'-diaminobenzidine (DakoCytomation Denmark). Counterstaining was done with hematoxylin. The stained tissue sections were analyzed independently by two pathologists.
Cell culture. Three esophageal squamous cell carcinoma cell lines WHCO1, WHCO5, and WHCO6 originally established from surgical biopsies of primary esophageal squamous cell carcinomas (23) were cultured in DMEM containing 10% FCS at 37°C in a humidified atmosphere of 5% CO2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were carried out using the Cell Proliferation kit I (Roche Diagnostics South Africa) as described by the manufacturer. Briefly, 1.5 x 103 cells were plated in 96-well plates in a final volume of 180 µL DMEM per well. SB 225002 (antagonist of CXCR2, 400 nmol/L, Calbiochem, Darmstadt, Germany) was added to cells and 0.001% DMSO (solvent) was added as a control. After the indicated incubation period, 18 µL of the MTT labeling reagent (final concentration 0.5 mg/mL) was added to each well and incubated for 4 hours in a humidified atmosphere. One hundred eighty microliters of the solubilization solution were added to each well and the plates were left overnight at 37°C. The spectrophotometric absorbance of samples was measured at 595 nm using a microtiter plate reader.
Inhibition of CXCR2 activity. CXCR2 function was inhibited with either a specific antagonist (SB 225002) or GRO antibodies. For treatment with SB 225002, WHCO1 cells were plated at 1.5 x 105 per well in 2 mL DMEM in a six-well plate. Twenty-four hours later, the medium was changed, SB 225002 was added into the medium to the final concentrations of 25, 50, 100, 200, 400, and 800 nmol/L, and 0.001% DMSO was used as solvent control. Cells were incubated at 37°C for a further 48 hours. For treatment with antibodies, WHCO1 cells were plated into six-well plates at 1 x 105 per well. Three days later, GRO antibody or GROß antibody was added to the indicated concentration and incubated for the indicated times.
Western blot analysis. Treated WHCO1 cells were rinsed twice with ice-cold PBS and scraped off the plate in radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 25 mmol/L Tris-Cl (pH 7.5), 1% sodium deoxycholate, 20 µg/mL pepstatin, 5 µg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride], sonicated for 5 seconds on ice with a probe sonicator (Heat System-Ultrasonics, Inc., Plainview, NY), and centrifuged for 10 minutes at 13,000 x g in a microcentrifuge. The protein concentration of the lysates was determined using the BCA Protein Assay kit (Pierce, Rockford, IL). Fifteen micrograms protein per sample was electrophoresed on 10% SDS-PAGE and electrophoretically transferred to nitrocellulose membrane (Hybond-ECL, Amersham Pharmacia Biotech UK) at 4°C for 1.5 hours. Blots were incubated for 1 hour with 5% nonfat dry milk to block nonspecific binding sites and then incubated with goat polyclonal antibody against phosphorylated ERK1/2 (Santa Cruz Biotechnology) at 4°C overnight. The immunoreactivity was detected using peroxidase-conjugated antibody and visualized by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce). The blots were stripped before reprobing with antibody to ERK2 (Santa Cruz Biotechnology).
RNA interference. To create the small interfering RNA (siRNA) plasmid constructs, complementary strands of oligonucleotides specifically targeting GRO and GROß were synthesized. For GRO siRNA, oligo1: 5'-CTGTTTAGATGAATGTCAGTTCAAGAGACTGACATTCATCTAAACAGTTTTTT-3', oligo2: 5'-AATTAAAAAACTGTTTAGATGAATGTCAGTCTCTTGAACTGACATTCATCTAAACAGGGCC-3'; for GROß siRNA, oligo1: 5'-TCTACTTGCACACTCTCCCTTCAAGAGAGGGAGAGTGTGCAAGTAGATTTTTT-3', oligo2: 5'-AATTAAAAAATCTACTTGCACACTCTCCCTCTCTTGAAGGGAGAGTGTGCAAGTAGAGGCC-3'. After annealing, double-stranded oligonucleotides were cloned into ApaI and EcoRI linearized pSilencer 1.0-U6 vector (Ambion). The insert, together with U6 promoter, was released by NotI and KpnI digestion. The cytomegalovirus promoter in pcDNA 3.1(+) vector was released with NotI and MulI digestion and replaced with the U6 promoter/oligonucleotide insert and a linker to obtain pcDNA3.1-U6/GRORNAi and pcDNA3.1-U6/GROßRNAi. The inserts were confirmed by DNA sequence analysis. The U6 promoter-driven siRNAs express the sense and antisense strands of GRO or GROß siRNA that have a termination signal consisting of six thymidines.
Transfection and immunofluorescence. Stable transfection of WHCO1 cells with either pcDNA3.1-U6/GRORNAi or pcDNA3.1-U6/GRORNAi or pcDNA3.1-U6 (empty vector) using FuGENE 6 Transfection Reagent (Roche Diagnostics South Africa) was carried out as recommended by the manufacturer.
To detect the expression level of GRO or GROß in positive clones selected by G418, immunofluorescence staining was done. Briefly, cells were cultured on coverslips, fixed, and permeabilized with methanol and 4% paraformaldehyde, then incubated with 1:50 dilution of goat anti-GRO polyclonal antibody (Santa Cruz Biotechnology) or with 1:100 dilution of goat anti-GROß polyclonal antibody (Santa Cruz Biotechnology) in blocking buffer at 4°C overnight. After incubation with primary antibody, cells were washed five times in PBS (pH7.4) and incubated with 1:100 dilution of FITC-conjugated rabbit anti-goat antibody (Zymed Laboratories, Inc., South San Francisco, CA) for 2 hours at room temperature. After five washes in PBS, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich South Africa Ltd., Aston Manor, South Africa). Fluorescence was observed by using a x40 objective lens on an inverted microscope (Zeiss Axiovert 200 M, Jena, Germany). The specificity of the antibodies was tested using specific blocking peptides for either anti-GRO or anti-GROß using immunofluorescence staining.
Statistical analysis. The Student's t test was used for statistical significance of differences in the expression of GRO, GROß, and CXCR2 between groups. P < 0.05 was considered to be significant.
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Results |
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, GROß, and CXCR2 in esophageal squamous cell carcinoma. An initial study using cDNA microarray analysis indicated that both GRO and GROß were overexpressed by 1.7-fold in esophageal squamous cell carcinoma biopsies compared with adjacent normal tissue from the same individual (data not shown). Because the cDNA microarray was done with pooled RNA from three patients, using reciprocal labeling, further analysis was carried out to verify the difference in GRO and GROß expression between normal and tumor esophageal tissue. Specific primers were designed to quantitate GRO and GROß mRNA levels in tissue, and the PCR products generated (423 and 409 bp for GRO and GROß, respectively; Fig. 1A
) were sequenced to verify their identity. Real-time RT-PCR using total RNA isolated from esophageal squamous cell carcinoma and adjacent normal tissues confirmed that GRO was indeed overexpressed (by 2-fold to 5-fold, P < 0.05) in all four of the esophageal tumor tissues analyzed (Fig. 1B). Similarly, GROß mRNA levels were also overexpressed (by 1.5-8 fold, P < 0.05) in three of the four esophageal tumor tissues examined, relative to adjacent normal tissue (Fig. 1C). To establish whether GRO and GROß could potentially function as autocrine signaling molecules in esophageal squamous cell carcinomas, we determined the expression of CXCR2 (a receptor for GRO and GROß) in the same tissues. CXCR2 mRNA levels were also overexpressed (by 1.6-20 fold, P < 0.05) in the tumor tissues examined (Fig. 1D). To verify the expression of these two chemokines and their receptor, CXCR2, immunohistochemical analysis was done using esophageal tumor and matched normal tissue sections obtained from esophageal cancer patients that had undergone esophagectomies. The immunohistochemical analysis showed that GRO was overexpressed in 7 of 10 tumor sections analyzed, and that GROß was overexpressed in 11 of 14 tumor sections examined (Fig. 2A-D
). These results confirmed that both GRO and GROß were overexpressed in esophageal tumor tissue, and, furthermore, that these two chemokines were primarily located in the cytoplasm (Fig. 2A-D). CXCR2 levels were elevated in 88% of tumor sections examined (n = 65), whereas it was either absent or was present at very low levels in matched normal tissue (Fig. 2E and F). Our results also showed that the CXCR2 receptor was localized in the plasma membrane and cytoplasm of the cells examined. These findings demonstrating that esophageal squamous cell carcinomas coexpress GRO, GROß, and CXCR2, and suggest that these chemokines could play an autocrine role in this poorly understood cancer.
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, GROß, and CXCR2 in cultured esophageal cancer cell lines. Three cultured esophageal cancer lines (WHCO1, WHCO5, and WHCO6) were tested to identify a model system in which the role of CXCR2 and its ligands (GRO and GROß) could be explored in esophageal squamous cell carcinoma. RT-PCR analysis using total RNA isolated from the three cell lines revealed that GRO, GROß, and CXCR2 were expressed in all three cell lines examined (Fig. 3
). WHCO1 was selected as a model system to further explore the role of CXCR2 and its ligands in esophageal cancer because this cell line constitutively expresses GRO, GROß, and CXCR2.
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, GROß, and CXCR2 in WHCO1 cells constituted a functional autocrine loop in these cultured cells. Because recent evidence indicated that GRO could signal via the ERK1/2 pathway (24), we treated WHCO1 cells with SB 225002 (a specific inhibitor of CXCR2; ref. 25, 26), and measured levels of phosphorylated ERK1/2 in treated cells by Western blot analysis. Increasing concentrations of SB 225002 substantially reduced the levels of phosphorylated ERK1/2 in WHCO1 cells, leaving unphosphorylated ERK2 levels unaffected (Fig. 4A
). Densitometric analysis of the bands indicated that 100 nmol/L SB 225002 reduced p-ERK1/2 levels by 30%, similar to the IC50 value of 88 nmol/L reported by Catusse et al. (26). Although the results in Fig. 4A strongly suggest that the CXCR2 receptors are activated in WHCO1 cells (leading to phosphorylation of ERK1/2), the identity of the ligand/s responsible for activating CXCR2 is less clear. To address this question, WHCO1 cells were incubated with either anti-GRO or anti-GROß for the indicated times, followed by measurement of phosphorylated ERK1/2 levels. Treatment of WHCO1 cells with anti-GROß substantially reduced the amount of phosphorylated ERK1/2 in treated cells (Fig. 4B). In contrast, treatment of WHCO1 cells with anti-GRO had no effect on the levels of phosphorylated ERK1/2 measured in the cell lysates, regardless of the time of exposure to the antibody (Fig. 4C). As another marker of the functionality of the GROß/CXCR2 autocrine loop in WHCO1 cells, we also measured the effect of anti-GROß treatment on the expression of EGR-1 mRNA because previous reports have identified EGR-1 as a downstream target of ERK1/2 signaling (27–29). Depletion of GROß in the medium of WHCO1 cells (using anti-GROß) resulted in a time-dependent reduction of EGR-1 mRNA levels in treated cells (Fig. 5
). These results strongly suggested that GROß signals via the ERK1/2 pathway and constitutes an autocrine loop with CXCR2 in cultured WHCO1 cells.
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stimulates cell proliferation in melanoma cells through an autocrine loop (14, 16), we explored the possible contribution of the GRO/CXCR2 and GROß/CXCR2 loops to proliferation of WHCO1 cells. Blocking CXCR2 signaling in WHCO1 cells with 400 nm SB 225002 significantly decreased cell proliferation by 
40% to 50% (Fig. 6
), suggesting that CXCR2-mediated signaling contributed substantially to proliferation of WHCO1 cells in culture.
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and GROß expression in stably transfected WHCO1 cells. Using fluorescent immunocytochemistry, we showed that stably transfected WHCO1 cells containing siRNA against GROß expressed significantly lower levels of GROß than cells transfected with vector only (Fig. 7A, top
). Using commercially available competing ligands for GRO and GROß, we confirmed that >90% of the fluorescence signal observed for these antibodies was specific (data not shown). Real-time RT-PCR analysis confirmed >95% (P < 0.05) reduction in GROß mRNA levels in the GROß RNAi clone relative to the vector control (Fig. 7B, top). Furthermore, the stably transfected clone expressing siRNA against GROß displayed an 80% reduction in phosphorylated ERK1/2 levels relative to control cells (Fig. 7C), suggesting that secreted GROß contributed to the activation of this mitogen-activated protein kinase signaling pathway. Although WHCO1 cells stably transfected with pcDNA3.1-U6/GRORNAi expressed lower levels of GRO (Fig. 7A, bottom) than control cells, the reduction in GRO expression was less substantial than the knockdown observed for GROß (Fig. 7A, top). This was confirmed by real-time RT-PCR analysis that indicated that GRO mRNA levels had been knocked down by 50% (P < 0.05) in the GRO RNAi clone (relative to the vector control; Fig. 7B, bottom). The GRO RNAi clone only displayed a 60% reduction in the level of phosphorylated ERK1/2 relative to control cells (Fig. 7C). Reducing GROß expression levels in WHCO1 cells with GROß RNAi reduced the proliferation rate of these cells by 50% relative to wild-type or the vector control WHCO1 cells (Fig. 8
). WHCO1 cells stably transfected with GRO RNAi, however, displayed a 20% reduction in cell proliferation relative to vector control cells (Fig. 8). Cell cycle analysis by flow cytometry suggested that the reduced proliferation of GRO-RNAi and GROß-RNAi-expressing stable clones of WHCO1 was not due to severe disruption of the cell cycle or cell cycle block because GRO-RNAi and GROß-RNAi-expressing clones displayed cell cycle profiles similar to wild-type and vector control cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, GROß, and their receptor, CXCR2, which contributes substantially to the proliferation of esophageal cancer cells in culture. The immunohistochemical evidence presented here suggests that the GRO/CXCR2 and GROß/CXCR2 chemokine loop probably contributes to proliferation in most primary esophageal squamous cell carcinoma tissues in situ. Recent studies have shown an increasingly important role of chemokines in the biology of a wide array of cell types, including cancer cells, in addition to their role in regulating directional migration of leukocytes during inflammatory responses (31). Although this is the first description of a proliferative chemokine autocrine loop operating in esophageal cancer, other studies have implicated GRO and IL-8 in the progression of malignant melanoma, hepatic cancer, and pancreatic cancer via an autocrine loop involving CXCR2 (14, 16–18, 20).
Although the GROß/CXCR2 chemokine loop clearly contributes to proliferation in WHCO1 cells, the role of GRO in these cells is less obvious, despite our demonstration that esophageal cancer cells express both GRO and GROß (by immunohistochemistry and RT-PCR) and other studies showing that CXCR2 binds both GRO and GROß (9).
The modest reduction (20%) in cell proliferation observed in a GRO knockdown clone (Fig. 8) probably reflect an incomplete knockdown of GRO mRNA levels because fluorescent microscopy revealed a larger knockdown effect on GROß than GRO. The real-time RT-PCR results (Fig. 7B) also confirm that GRO was incompletely knocked down. Although only a 20% reduction of cell proliferation was observed in the GRO knockdown clone, our results still suggest that GRO signaling is mediated through ERK1/2, because a 60% reduction in phosphorylated ERK1/2 was shown in this clone. The apparent inability of anti-GRO treatment to reduce phosphorylated ERK1/2 levels in WHCO1 cells (Fig. 4C) could reflect the inability of the GRO antibody to remove secreted GRO from the culture medium.
Using cultured esophageal cancer cells as a model system, our results clearly suggest that GROß signaling is also mediated through ERK1/2, and that blocking GROß (either with an antibody, CXCR2 antagonist or RNAi) substantially reduced signaling through ERK1/2. This is consistent with previous evidence indicating that CXCR2 can signal through ERK1/2 in addition to other pathways (24). We also showed that GROß makes a major contribution to the elevated transcription of EGR-1 in cultured esophageal cancer cells. Preliminary immunohistochemical results obtained in our laboratory show that EGR-1 is significantly overexpressed in esophageal squamous cell carcinoma tissue compared with normal esophageal squamous epithelial tissue. The relationship between GROß and EGR-1 expression in esophageal cancer is presently being explored further in our laboratory. The implications of the relationship between GROß and EGR-1 are very significant because EGR-1 is known to regulate the expression of many genes involved in tumorigenesis, including insulin-like growth factor-II (32, 33) and vascular endothelial growth factor (34).
Because ELR-containing chemokines, such as GROß, GRO, and IL-8, are potent angiogenic factors (8), our observation that esophageal squamous cell carcinoma cells secrete GRO may facilitate the gain in these cells of yet another important phenotypic hallmark of cancer (30). In this context, the GRO/CXCR2 and GROß/CXCR2 loops may play an important role in the development and maintenance of squamous esophageal cancer, and consequently this chemokine system represents a significant therapeutic target that should be considered in the future treatment of esophageal cancer. Approaches targeting chemokine systems in other cancers have already shown considerable promise. A small molecular inhibitor of CXCR4 (AMD3100) reduced proliferation and induced apoptosis of intracranial xenograft brain tumors in mouse model studies (35). Progress made in the development of orally bioavailable, potent antagonists of CXCR2 (36) suggest that clinicians may soon have at their disposal a range of small molecule antagonists of CXCR2. The results of the present study (showing reduced proliferation of cultured cancer cells in response to CXCR2 antagonist or RNAi treatment) have highlighted the feasibility of disrupting the GROß/CXCR2 loop as a treatment strategy in squamous esophageal cancer. Studies showing that mouse gene knockouts of CXCR2 display minimal pathology (37) suggest that this treatment route warrants further investigation, despite the pleiotropic effects of chemokine systems.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Prof. Ann Richmond (Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN) for providing the CXCR2 antibody, Prof. Nor Hayati Othman (Universiti Sains Malaysia, Penang, Malaysia) and Dr. Ryan Soldin (University of Cape Town, Cape Town, South Africa) for pathologic evaluation of all the slides, Dr. Gabi Walther (Chris Bernard Division of Cardio-Thoracic Surgery, University of Cape Town, Cape Town, South Africa) for providing esophageal tissue samples, Heather McLeod and Nafiesa Allie for expert technical assistance in immunohistochemistry, Dr. Sharon Prince (Department of Human Biology, University of Cape Town, Cape Town, South Africa) for reagents, Dr. Virna Leaner for critically reading the paper, Catherine Arendse for expert assistance with the fluorescence-activated cell sorting analysis, and Dr. Dirk Lang and Liz van der Merwe for assistance with immunofluorescence analysis.
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Footnotes |
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Received 8/16/05. Revised 12/30/05. Accepted 1/10/06.
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References |
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, GROß, GRO, neutrophil-activating peptide-2, and epithelial cell-derived neutrophil-activating peptide-78 are potent agonist for the type B, but not the type A, human interleukin-8 receptor. J Biol Chem 1996;271:20545–50.-Chemokine-mediated signal transduction in human Kaposi's sarcoma spindle cells. Biochim Biophys Acta 2004;1691:129–39.[Medline] proteins. Int J Cancer 1997;73:94–103.[CrossRef][Medline]/KC, a chemokine receptor CXCR2 ligand, can be a potent trigger for neuronal ERK1/2 and PI-3 kinase pathways and for hyperphosphorylation—a role in Alzheimer's disease? J Neuroimmunol 2002;122:55–64.[CrossRef][Medline] (CXCL1) and a non-peptide antagonist (SB 225002) with the human CXCR2. Biochem Pharmacol 2003;65:813–21.[CrossRef][Medline]This article has been cited by other articles:
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  A. E. Wallace, K. J. Sales, R. D. Catalano, R. A. Anderson, A. R.W. Williams, M. R. Wilson, J. Schwarze, H. Wang, A. G. Rossi, and H. N. Jabbour Prostaglandin F2{alpha}-F-Prostanoid Receptor Signaling Promotes Neutrophil Chemotaxis via Chemokine (C-X-C Motif) Ligand 1 in Endometrial Adenocarcinoma Cancer Res., July 15, 2009; 69(14): 5726 - 5733. [Abstract] [Full Text] [PDF]
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H. Kawanishi, Y. Matsui, M. Ito, J. Watanabe, T. Takahashi, K. Nishizawa, H. Nishiyama, T. Kamoto, Y. Mikami, Y. Tanaka, et al. Secreted CXCL1 Is a Potential Mediator and Marker of the Tumor Invasion of Bladder Cancer Clin. Cancer Res., May 1, 2008; 14(9): 2579 - 2587. [Abstract] [Full Text] [PDF]
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