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Cyclic AMP-Responsive Element Binding Protein– and Nuclear Factor-B–Regulated CXC Chemokine Gene Expression in Lung Carcinogenesis

Authors' Affiliation: Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas

Requests for reprints: Ja Seok Koo, Department of Thoracic/Head and Neck Medical Oncology, Unit 432, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363; Fax: 713-794-5997; E-mail: jskoo{at}mdanderson.org.

	Abstract

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The recognition of the importance of angiogenesis in tumor progression has led to the development of antiangiogenesis as a new strategy for cancer treatment and prevention. By modulating tumor microenvironment and inducing angiogenesis, the proinflammatory cytokine interleukine (IL)-1β has been reported to promote tumor development. However, the factors mediating IL-1β–induced angiogenesis in non–small cell lung cancer (NSCLC) and the regulation of these angiogenic factors by IL-1β are less clear. Here, we report that IL-1β up-regulated an array of proangiogenic CXC chemokine genes in the NSCLC cell line A549 and in normal human tracheobronchial epithelium cells, as determined by microarray analysis. Further analysis revealed that IL-1β induced much higher protein levels of CXC chemokines in NSCLC cells than in normal human tracheobronchial epithelium cells. Conditioned medium from IL-1β–treated A549 cells markedly increased endothelial cell migration, which was suppressed by neutralizing antibodies against CXCL5 and CXCR2. We also found that IL-1β–induced CXC chemokine gene overexpression in NSCLC cells was abrogated with the knockdown of cyclic AMP-responsive element binding protein (CREB) or nuclear factor B (NF-B). Moreover, the expression of the CXC chemokine genes as well as CREB and NF-B activities was greatly increased in the tumorigenic NSCLC cell line compared with normal, premalignant immortalized or nontumorigenic cell lines. A disruptor of the interaction between CREB-binding protein and transcription factors such as CREB and NF-B, 2-naphthol-AS-E-phosphate (KG-501), inhibited IL-1β–induced CXC chemokine gene expression and angiogenic activity in NSCLC. We propose that targeting CREB or NF-B using small-molecule inhibitors, such as KG-501, holds promise as a preventive and/or therapeutic approach for NSCLC.

Key Words: Angiogenesis • chemoprevention • CREB • NF-B • CXC Chemokine • lung cancer

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Lung cancer is the leading cause of cancer deaths both in the United States and worldwide (1). The growth and development of lung cancer as well as of other solid tumors is critically dependent on a functional vascular supply. Numerous lines of evidence have shown that angiogenesis, the formation of new blood vessels from the preexisting vasculature, is one of the critical steps in the entire process of cancer development from tumor growth to distant metastasis (2–5). In addition, a study showed that a solid tumor would remain dormant at a volume of only 2 to 3 mm³ in the absence of neovascularization and would be unable to metastasize (6). For solid tumors to develop and metastasize, they need to secrete a number of proangiogenic factors to induce the formation of new blood vessels to overcome the physical limitations on the diffusion of nutrients and oxygen within the tumor—a process known as angiogenic switch (3). Recognizing the importance of angiogenesis in the growth of tumors has led to the development of antiangiogenesis as a new strategy for cancer therapy and prevention, a novel concept termed "angioprevention" (7–9). Actually, a series of molecules proposed as chemopreventive agents have been shown to have potent antiangiogenic properties when tested in in vitro and in vivo angiogenesis models (7).

Angiogenesis can be regulated by various growth factors and cytokines, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, transforming growth factors and β, platelet-derived endothelial cell growth factors, chemokines, and interleukine (IL)-1β (10–14). Recent studies have shown the importance of the tumor microenvironment in facilitating angiogenesis and promoting tumor invasion and metastasis (15–19). Once a tumor is vascularized, the tumor-associated antigens can be recognized by the immune system and the tumor is infiltrated by leukocytes. Although leukocyte infiltration in tumors is often considered to be associated with better prognosis and overall survival, studies have also shown that inflammatory cells can promote tumor cell proliferation, angiogenesis, metastasis, and, hence, tumor development (15, 16). Leukocyte infiltration can influence angiogenesis in tumors because some subsets of leukocytes, especially the tumor-associated macrophages, can secrete both angiostatic and angiogenic factors (17, 18). IL-1 is a proinflammatory cytokine produced mainly by monocytes and macrophages. There are two IL-1 agonistic proteins, IL-1 and IL-1β. IL-1 is a precursor or membrane-associated molecule and is primarily a regulator of intracellular events and a mediator of local reactions. On the other hand, IL-1β acts as a systemic, hormone-like mediator and is only active in a secreted mature form. However, once these two proteins bind to their receptors, they have similar biological activities (20). Both IL-1 and IL-1β can promote tumor angiogenesis, but the role of IL-1β is more evident (14). IL-1 has been shown to contribute to the production of proangiogenic factors VEGF, hepatocyte growth factor, tumor necrosis factor, and CXC chemokines (14, 21). Members of a subfamily of CXC chemokines sharing a characteristic glutamate-leucine-arginine (ELR) motif near the NH₂ terminus of the molecule are chemoattractants for neutrophils and are important for wound repair. The ELR-positive chemokines, including CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8, are proangiogenic, whereas members of another subfamily lacking the ELR motif—ELR-negative chemokines, such as CXCL4, CXCL9, CXCL10, and CXCL11—are in general IFN inducible and are potential inhibitors of angiogenesis. Generally, CXCR2 is the receptor for angiogenic CXC chemokine–mediated angiogenesis, and CXCR3 is the receptor for angiostatic IFN-inducible CXC chemokine inhibition of angiogenesis (13). CXC chemokine ligands and receptors have been shown to play important roles in mediating non–small cell lung cancer (NSCLC)–associated angiogenesis and organ-specific metastases (13). Recently, it has been reported that CXCL5 and CXCL8 protein levels were elevated in tumor specimens freshly isolated from patients with NSCLC and that these two ELR-positive CXC chemokines are important mediators of angiogenesis during NSCLC tumorigenesis (22, 23). Compared with CXCL8, CXCL5 was reported to have a higher degree of correlation with NSCLC-derived angiogenesis (23). In a model system of human NSCLC tumorigenesis in severe combined immunodeficiency mice, CXCL5 expression was found to be directly correlated with tumor growth, tumor-derived angiogenesis, and metastatic potential. Depletion of CXCL5 in this model system resulted in attenuation of both tumor growth and spontaneous metastasis due to the inhibition of angiogenesis (23).

Being a product of tumor infiltrated macrophages, IL-1β is known to increase angiogenesis. However, in NSCLC, what angiogenic factors are induced by IL-1β and how they are regulated by IL-1β are still not clear. To elucidate these critical issues, we conducted a microarray analysis to determine the effect of IL-1β on global gene expression in the NSCLC adenocarcinoma cell line A549 and in normal human tracheobronchial epithelium (NHTBE) cells. We found that IL-1β dramatically induced the expression of an array of proangiogenic CXC chemokine genes and significantly augmented the angiogenic activity of NSCLC. In addition, we found that transcription factors cyclic AMP-responsive element binding protein (CREB) and nuclear factor B (NF-B) both play critical roles in the regulation of IL-1β–induced CXC chemokine gene expression and angiogenic activity.

	Materials and Methods

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Cell cultures, chemicals, and conditioned media
NHTBE cells were purchased from Cambrex and cultured in six-well plates as described previously (24–28). Human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex and maintained in EGM complete endothelial growth medium. HUVECs were used at passage 4. The human NSCLC cell lines A549, H1734, H226, and H2170 were obtained from the American Type Culture Collection and grown in RPMI 1640 containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. The BEAS-2B, 1799, 1198, and 1170-I human bronchial epithelial cell lines were obtained from Dr. R. Lotan (The University of Texas M.D. Anderson Cancer Center, Houston, TX) and Dr. A. Klein-Szanto (Fox Chase Cancer Center, Philadelphia, PA) and grown in Keratinocyte Serum-Free Medium (Life Technologies, Inc.) containing epidermal growth factor and bovine pituitary extract (29). All of the cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Conditioned media (CM) were generated as follows: NSCLC cells (3 x 10⁵/mL) were cultured in RPMI 1640 containing 0.5% fetal bovine serum with or without IL-1β and/or 2-naphthol-AS-E-phosphate (KG-501) at different concentrations for 24 h. Cell-free supernatants were collected and stored at –70°C until use.

Reagents and antibodies
IL-1β, Quantikine CXCL5 and CXCL8 ELISA kits, and neutralizing antibodies against CXCL5, CXCL8, CXCR2, and VEGF were purchased from R&D Systems. KG-501 was purchased from Sigma-Aldrich and dissolved in DMSO. Antibodies against NF-B p65, CREB, and phospho-CREB (Ser¹³³) were purchased from Santa Cruz Biotechnology, Inc., and Upstate, respectively. A monoclonal antibody (mAb) against β-actin was from Sigma-Aldrich. Transwell chambers with polyethylene terephthalate membranes containing 8-µm pores were obtained from BD Biosciences.

Microarray analysis
After confluence, NHTBE and A549 cells were treated with control medium or the same medium containing 2.5 ng/mL IL-1β for 8 h before total RNA extraction. Total RNA was isolated with the RNeasy Mini Kit (Qiagen). The integrity of mRNA and the relative rRNA contamination were analyzed with the RNA 6000 Nano LabChip (Agilent Technologies) and the Agilent 2100 bioanalyzer (Agilent Technologies). The RNA from control group was amplified and labeled with cyanine 3, and that from IL-1β–treated cells with cyanine 5. Equal amounts of the differently labeled RNAs were then mixed and hybridized with 44K whole human genome oligonucleotide microarrays (Agilent Technologies). After hybridization, the arrays were scanned and the resulting images were analyzed using the Agilent feature extraction software program (GE2, version 5.91, Agilent Technologies).

Quantitative reverse transcription-PCR
Validation of the differentially expressed genes in NHTBE and NSCLC cells was done using an iCycler real-time PCR detection system (Bio-Rad) with gene-specific primers. Primers for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the reference gene, were 5'-TGCACCACCAACTGCTTAGC (forward) and 5'-GGCATGGACTGTGGTCATGAG (reverse); for CXCL1, 5'-AGTGACAAATCCAACTGACC (forward) and 5'-GATGCTCAAACACATTAGGC (reverse); for CXCL2, 5'-CCCAAGTTAGTTCAATCCTG (forward) and 5'-TTCCTCAGCCTCTATCACAG (reverse); for CXCL3, 5'-CTTGTCTCAACCCCGCATCC (forward) and 5'-TCTGGTAAGGGCAGGGACCA (reverse); for CXCL5, 5'-TCCAATCTCCGCTCCTCCAC (forward) and 5'-AGCAGCAGCAGCACCAACAG (reverse); for CXCL6, 5'-GTTTGTCTGGACCCGGAAGC (forward) and 5'-TCCGCTGAAGACTGGGCAAT (reverse); for CXCL8, 5'-GCATAAAGACATACTCCAAACC (forward) and 5'-ACTTCTCCACAACCCTCTG (reverse); and for CREB, 5'-AAGCTGAAAACCAACAAATGACAGTT (forward) and 5'-TGAACTGTCTGCCCATTGG (reverse). Single-stranded cDNAs were synthesized in 50 µL of reverse transcription (RT) mix containing 1 µg of total RNA using the GeneAmp RNA PCR Core Kit (Applied Biosystems) according to the manufacturer's instructions. PCR analysis was done using 25-µL volumes with SYBR Green PCR Core Reagents (Applied Biosystems). Primers (200 nmol/L) and RT mix (2 µL) were used in each PCR. Each sample was assayed in triplicate per PCR run and the experiment was repeated thrice. The cycling conditions were an initial denaturation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and elongation at 60°C for 60 s. The real-time PCR data were analyzed using the comparative C_t method.

Transfection of small interfering RNA against NF-B p65 and transduction of lentiviral short hairpin RNA against CREB
SMARTpool-sequenced small interfering RNA (siRNA) targeting human NF-B p65 (GenBank accession no. NM_021975) and nonspecific control pool siRNA were purchased from Dharmacon RNA Technologies and diluted to 20 µmol/L. NSCLC cells at 50% confluence were transfected with siRNA for NF-B p65 or control siRNA at final concentrations of 50 and 100 nmol/L using the LipofectAMINE 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Seventy-two hours after transfection, the cells were treated with IL-1β for 8 h. Total protein and RNA were collected from each sample.

For CREB-targeting viral short hairpin RNA (shRNA) delivery, the lentiviral plasmid pLKO.1 with a shRNA clone against CREB (clone ID: TRCN0000011085) was purchased from Open Biosystems. The pLKO.1 plasmid with a scrambled shRNA sequence and virus packaging plasmids (psPAX2 and pseudo-typing plasmid pMD2.G) were obtained from Addgene. HEK 293T cells were obtained from the American Type Culture Collection. HEK 293T cells at 50% confluence were cotransfected with pLKO.1 and virus packaging plasmids using FuGENE 6 transfection reagent (Roche Applied Science). After transfection for 16 h, the medium containing the transfection agent was replaced with fresh growth medium. Viral particles were then collected from the medium every 24 h twice. The virus titer in the pooled suspension was determined by counting the puromycin-resistant colonies in the virus-transduced culture. For knockdown of CREB, cells at 50% confluence were incubated with viral suspension at a multiplicity of infection of about 50 for 16 h. Four days after transduction, the cells were treated with IL-1β for 24 h and total protein and RNA were collected from the cells.

Migration assay
HUVECs (5 x 10⁴) were suspended in serum-free RPMI 1640 and seeded in transwell chambers coated with gelatin. CM from NSCLC cells were applied in the outer chambers. HUVECs were incubated at 37°C in 5% CO₂ for 16 h. Following incubation, cells were fixed in 90% ethanol and stained with 0.1% crystal violet. Nonmigrated cells on the upper surface of the chamber filters were removed by swabbing, and the cells that had migrated through the filter were photographed under a microscope and quantified using the ImageJ software program (NIH, Bethesda, MD).¹

Western blot analysis
Western blot analysis of target proteins was done as described previously (30). Equal amounts of protein (30 µg) were resolved using 10% SDS-PAGE. The mouse mAb against human NF-B p65 was diluted in 5% nonfat milk at a ratio of 1:200 and rabbit polyclonal antibodies against human CREB and p-CREB were diluted at a ratio of 1:1,000 and incubated with the membranes overnight at 4°C. Proteins reactive with the primary antibody were visualized with a horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit secondary antibody and enhanced chemiluminescence reagents (Amersham Bioscience).

Measurement of CXC chemokine protein secretion
The levels of secreted CXC chemokines in the CM were measured with immuno-dot blotting and ELISA. Immuno-dot blotting was done as described before (24, 28). Briefly, the CM were applied to a nitrocellulose membrane using the Manifold I Dot-Blot System (Scheleicher & Schuell). The membrane was then probed with antihuman CXCL5 and CXCL8 antibodies and the target proteins were detected by chemiluminescence and quantified by densitometry. ELISA was done with Quantikine CXCL5 and CXCL8 ELISA kits (R&D Systems) according to the manufacturer's instruction. Both measurements were normalized against the cell number.

Chromatin immunoprecipitation analysis
Chromatin immunoprecipitation assay was done using EZ ChIP kits (Upstate) according to the manufacturer's instruction. Briefly, NHTBE, BEASE-2B, 1799, 1198, and 1170-1 cells were grown in plate with normal media and chromatins were cross-linked by reaction with 1% formaldehyde for 10 min. The cross-linked chromatins were fragmented by sonication and subsequently immunoprecipitated with anti-CREB (Upstate) or anti–NF-B p65 (Santa Cruz Biotechnology). The DNA in the precipitate was purified and used as the template for PCR. The primers for CXCL5 promoter with NF-B binding site were 5'-TAGAGGTGCACGCAGCTCCT (forward) and 5'-GAGCACTGTGGCTTCCTCGT (reverse); for CXCL5 promoter with CREB binding site, 5'-CTGGACACACGTATACTTGC (forward) and 5'-GGCAGGTCATTCTAGGTTTC (reverse); and for CXCL8 promoter with both CREB and NF-B binding sites, 5'-AAAACTTTCGTCATACTCCG (forward) and 5'-AAAGTTTGTGCCTTATGGAG (reverse). PCR products were then separated in 1.2% agarose gel and stained with GelRed (Biotium).

Statistical analysis
Each experiment presented in the figures was repeated three or more times. The data are presented as the mean ± SE. Comparisons between groups were evaluated using ANOVA and a two-tailed Student's t test. P < 0.05 was considered statistically significant.

	Results

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Microarray analysis revealed an array of CXC chemokine genes up-regulated by IL-1β in NHTBE and A549 cells
To identify IL-1β–responsive genes involved in angiogenesis and tumorigenesis in NSCLC, we used total RNA isolated from NHTBE and A549 cells treated with or without 2.5 ng/mL IL-1β for 8 hours to generate cRNA for microarray hybridization and analysis using 44K whole human genome oligonucleotide microarrays. Analysis of the resulting microarray images using the Agilent feature extraction software program showed that the expression of six CXC chemokine genes, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8, was commonly up-regulated in response to IL-1β in both NHTBE and A549 cells (P < 0.001). As expected, we also saw that the expression of another angiogenic gene, VEGF, was up-regulated by IL-1β in both NHTBE and A549 cells (Table 1). To confirm that these microarray data reflect the IL-1β–induced gene expression, we examined the expression of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 genes in NHTBE and A549 cells using quantitative reverse transcription-PCR (RT-PCR). Indeed, the expression of all of these CXC chemokine genes was strongly induced after stimulation by IL-1β, and the quantitative RT-PCR results agreed with the microarray analysis results (Table 1).

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. IL-1β differentially regulates CXCL5 and CXCL8 protein secretion in NHTBE and NSCLC cells. NHTBE, H226, H2170, H1734, and A549 cells were treated with 2.5 ng/mL IL-1β for 24 h. CM were collected from these cell lines. The concentrations of CXCL5 (A) and CXCL8 (B) in CM were measured by ELISA. Results were normalized according to the cell number. Columns, mean; bars, SE.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. IL-1β stimulates CXCL5 and CXCL8 gene expression and protein secretion in A549 cells in a time- and dose-dependent manner. A, A549 cells were incubated with control medium or with medium containing 2.5 ng/mL IL-1β for the indicated times. Total RNAs were collected from the cells and expression of the indicated genes was measured by quantitative RT-PCR. GAPDH was used as an internal control for normalizing the RNA loading. B, A549 cells were incubated with IL-1β at the indicated concentrations for 8 h. Total RNAs were collected from the cells for quantitative RT-PCR. C, culture media were collected from A549 cells with treatment as in B and the concentrations of CXCL5 and CXCL8 were measured with ELISA. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with untreated control.

View larger version (75K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. IL-1β significantly augments the angiogenic activity of NSCLC by inducing the expression of angiogenic CXC chemokine genes. A, CM were collected from A549 cells treated with either control medium (a) or medium containing 2.5 ng/mL IL-1β (b) for 24 h, and CM from IL-1β–treated A549 cells were preincubated with 10 µg/mL of nonimmune IgG (c), an anti-CXCL5 mAb (d), an anti-CXCL8 mAb (e), an anti-CXCR2 mAb (f), or an anti-VEGF mAb (g) at 37°C for 1 h. A migration assay was done by stimulating HUVECs with the indicated CM as described in Materials and Methods. Migrated cells were photographed after 16 h of incubation at 37°C. B, image analysis of HUVEC migration was quantified using the ImageJ software program. Columns, mean; bars, SE. *, P < 0.05.

NF-B and CREB mediate IL-1β–induced CXC chemokine gene expression

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. NF-B and CREB mediate IL-1β–induced CXC chemokine gene expression. A, transcription factor binding domains located in the gene promoters of CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8. B, A549 cells were transfected with siRNA for NF-B p65 or transduced with shCREB lentivirus; nontransduced cells and cells transfected with control siRNA or transduced with scrambled shRNA were used as controls. Proteins were extracted from the cells 72 h after transfection or 96 h after transduction. NF-B and CREB protein expression levels were detected by Western blot analysis. β-Actin protein was probed as a loading control. C, A549 and H1734 cells were transfected with siRNA for NF-B p65, and cells transfected with control siRNA were used as control. Seventy-two hours later, the cells were treated with either control medium or medium containing 2.5 ng/mL IL-1β for 8 h.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. The expression of CXC chemokine genes and CREB is associated with the development of lung cancer. A and B, total RNAs from NHTBE and subconfluent cultures of BEAS-2B, 1799, 1198, and 1170-I were extracted and the expressions of designated genes were quantitated with real-time PCR using specific primers. Each specific gene expression was normalized with the expression level of GAPDH. Columns, mean; bars, SE. C, whole-cell lysates (20 µg/lane) were isolated from NHTBE and IVLCM cell lines, and the levels of phospho-CREB (p-CREB) and total CREB were measured by immunoblot analysis. D, CREB and NF-B binding on CXCL5 and CXCL8 promoter. Chromatin immunoprecipitation assay was done with chromatins prepared from NHTBE, BEASE-2B, 1799, 1198, and 1170-1 cells. The binding of CREB or NF-B to the CXCL promoter was detected by visualization of the PCR product. The single bands detected in input samples indicate the specificity of the PCR primers.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. KG-501 suppresses NSCLC/IL-1β CM–induced migration of HUVECs by regulating IL-1β–induced CXC chemokine gene expression in NSCLC cells. A, HUVEC migration was induced with CM from A549 cells treated with control medium (a) or with medium containing 2.5 ng/mL IL-1β (b), 10 µmol/L KG-501 (c), 2.5 ng/mL IL-1β plus 2.5 µmol/L KG-501 (d), 2.5 ng/mL IL-1β plus 5 µmol/L KG-501 (e), or 2.5 ng/mL IL-1β plus 10 µmol/L KG-501 (f) in serum-free medium for 24 h. B, quantification of image analysis for the HUVEC migration assay. C, total RNAs were isolated from cells with the treatment described in A, and real-time PCR was done to measure the expression level of the indicated genes. D, the concentration of CXCL5 and CXCL8 protein in the CM was measured with ELISA. Results were normalized according to the cell number. Columns, mean; bars, SE. **, P < 0.01; ***, P < 0.001.

	Discussion

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Our data showing that IL-1β up-regulates the expression of angiogenic CXC chemokine genes and augments the angiogenic activity of NSCLC are consistent with previous reports on the role of CXCR2 and its ligands in promoting tumor-associated angiogenesis and early development of NSCLC (22, 23, 37–40). In an in vivo study using murine Lewis lung cancer heterotopic and orthotopic tumor model systems with CXCR2^–/– versus CXCR2^+/+ mice, researchers showed that the tumors in CXCR2^–/– mice exhibited reduced growth, increased necrosis, inhibited tumor-associated angiogenesis, and reduced metastatic potential (37). Similar to our finding that a neutralizing antibody against CXCR2 blocked CM-induced endothelial cell migration, the report showed that a specific neutralizing antibody against CXCR2 inhibited tumor growth, increased necrosis, and reduced tumors vessel density in CXCR2^+/+ mice (37). Furthermore, studies showed that CXCL5 and CXCL8 play a dominant role in promoting angiogenesis in patients with NSCLC (22, 23, 40). Whereas CXCL8 was the first angiogenic ELR-positive CXC chemokine discovered in NSCLC, CXCL5 reportedly has a higher degree of correlation with NSCLC-derived angiogenesis (23). In our study using the neutralizing antibodies, we observed that CXCL5 neutralization inhibited the migration of endothelial cells to the same degree as did CXCR2 neutralization. We failed to see this inhibitory effect using the CXCL8-neutralizing antibody, indicating that CXCL8 produced by A549 cells in response to IL-1β may not be sufficient to induce endothelial cell migration. Our experiment using KG-501 further supported such observation, as this small molecule blocked endothelial cell migration without affecting the CXCL8 level in CM. In addition, another angiogenic factor, VEGF, may play only a minor role in inducing endothelial cell migration in NSCLC, as neutralization of which could not inhibit the migration of HUVECs. Because high levels of CXCL5 and CXCL8 protein expression were detected in IL-1β–treated NSCLC cells but only a very low level of CXCL5 protein was induced in NHTBE cells, we speculated that IL-1β may induce an angiogenic response only in NSCLC tumor cells but not in surrounding normal cells.

It is well documented that IL-1β up-regulates the expression of the proangiogenic CXC chemokine genes and that NF-B is the common transcription factor that mediates this effect (31, 41, 42). All of the angiogenic CXC chemokine gene promoters contain a putative cis-element that is recognized by the NF-B family of transcriptional factors (31, 33, 43). Consistent with these findings, our results showed that after knockdown of NF-B p65 by siRNA transfection, both basal and IL-1β–induced expression of CXC chemokine genes decreased dramatically in A549 and H1734 cells. IL-1β can also regulate gene expression through the transcription factor CREB. Previously, we reported that IL-1β activates the mitogen-activated protein kinase (ERK1/2)/mitogen- and stress-activated protein kinase/CREB pathway and regulates MUC5AC gene expression in human airway epithelial cells (30). Sequence analysis of the CXC chemokine gene promoters identified a CRE or CRE-like domain in the gene CXCL1, CXCL2, CXCL3, CXCL5, and CXCL8 (31, 32, 34). In the present study, we showed that NSCLC cells with CREB knockdown were much less responsive to IL-1β than cells without such knockdown in terms of the induction of angiogenic CXC chemokine gene expression. We further confirmed the binding of CREB to the CXCL5 and CXCL8 promoters with chromatin immunoprecipitation assay. These data suggested that CREB can also regulate the expression of CXC chemokine genes. It has been reported that cyclooxygenase-2 is critical for IL-1β–induced angiogenesis both in vitro and in vivo through the production of prostanoids such as prostaglandin E₂ and thromboxane A₂ (44). In addition, Pold et al. (40) reported that cyclooxygenase-2 contributes to the progression of NSCLC tumorigenesis by enhancing the expression of CXCL5 and CXCL8. Because cyclooxygenase-2 expression is also regulated by CREB in many cell types, including lung cell lines (45–48), CREB may regulate CXC chemokine gene expression through multiple mechanisms that need to be further delineated. In addition, the interaction between CREB and NF-B in regulating the expression of these chemokine genes should be further investigated because these two transcription factors may compete with each other for the binding to CBP or work synergistically to affect the outcome of gene regulation (49). Additionally, the response elements for CREB and NF-B on the promoter of CXCL8 gene are consecutively located, which further complicated the interaction of these two transcription factors and might have also contributed to the perplexing pattern of CXCL8 expression in response to KG-501 treatment.

In the IVLCM cell lines, we detected that the expression of the angiogenic CXC chemokine genes CXCL5 and CXCL8 increased progressively from normal (NHTBE), immortalized (BEAS-2B and 1799), and transformed (1198) to tumorigenic (1170-I) human bronchial epithelial cells. This is not unexpected because the angiogenic CXC chemokines CXCL5 and CXCL8 have been reported to be elevated in NSCLC tissues and their expression is related to tumor progression (23, 50, 51). Recently, accumulating evidence shows that CXC chemokines induce tumorigenesis by stimulating cell proliferation, mediating cell survival, promoting angiogenesis, and facilitating tumor cell migration and invasion (13, 52). In addition, we detected that the expression and activation of CREB increased correspondingly with the tumorigenicity in these IVLCM cell lines. The consistency of the expression and activation of these factors with the expression of angiogenic CXC chemokine genes in IVLCM confirmed the regulation of CXC chemokine genes by CREB and NF-B, and lent support to their involvement in lung carcinogenesis. CREB has been known for its role in cell proliferation and survival (53–55). We have recently reported that the basal activity and expression level of CREB are commonly higher in a number of NSCLC cell lines versus normal cells, and the inhibition of CREB transcription activity induces apoptosis in these NSCLC cells by suppressing the expression of CREB-regulated genes that are involved in cell proliferation (56). Moreover, we have accumulated data from archived tumor tissue specimens showing that the CREB and p-CREB levels are commonly higher in the lung tumor tissues versus the adjacent normal tissues (57). By showing the role of CREB in tumor angiogenesis, the current study further suggests that CREB can be an effectual target for therapy as well as prevention of NSCLC.

KG-501 is a small molecule that binds to the KIX domain of CBP (35). It disrupts the interaction of CBP with CREB and inhibits the CREB-dependent activation of cellular genes. KG-501 can also disrupt the interaction of other factors with CBP, such as NF-B (35). Our finding that KG-501 reduced the NSCLC/IL-1β conditioned medium–induced migration of HUVECs by down-regulating the expression of the ELR-positive CXC chemokine genes in NSCLC cells indicates that KG-501 can be used as a therapeutic and/or preventive agent for inhibiting tumor-associated angiogenesis in NSCLC. Although the CXCL8 promoter contains both a NF-B-binding site and a CRE-like motif, and our knockdown experiments show that the depletion of either factor reduced IL-1β–induced CXCL8 expression (Fig. 4C and D), KG-501 was not able to effectively inhibit CXCL8 gene expression and protein secretion. Because KG-501 disrupts CREB-CBP or NF-B-CBP interaction, the regulation of CXCL8 gene by these factors may be mediated via a CBP-independent mechanism. A recent study showed that the transcriptional activity of CREB on CXCL8 promoter requires a different coactivator, termed transducer of regulated CREB (TORC1; ref. 34), suggesting a different regulatory mechanism beyond the binding of CREB to CRE. Nevertheless, CXCL8 did not seem to play a critical role in the induction of HUVEC migration as evidenced by the results of CXCL8 neutralization; therefore, KG-501 can still effectively inhibit NSCLC/IL-1β CM–induced HUVEC migration.

Our findings also implicated a positive association between chronic obstructive pulmonary disease (COPD) and lung cancer. Chronic obstructive pulmonary disease is a product of chronic inflammation that leads to tissue damage and physiologic adaptations (58, 59). It has been known for years that local inflammation in the lungs plays an important role in airway remodeling and parenchymal destruction, which are effects typified by chronic obstructive pulmonary disease (59). It is now well recognized that, in addition to lung inflammation, patients with chronic obstructive pulmonary disease frequently show persistent low-grade systemic inflammation, with the characteristic release of proinflammatory mediators such as IL-1β and tumor necrosis factor into the circulation (60). Considerable evidence has associated chronic inflammation with cancer development. Our finding that CREB and NF-B regulate proangiogenic CXC chemokines in response to proinflammatory cytokine IL-1β may provide a novel mechanistic linkage between chronic obstructive pulmonary disease and the development of lung cancer.

In summary, IL-1β increases the angiogenic activity of NSCLC by up-regulating the expression of an array of proangiogenic CXC chemokine genes, which subsequently induces endothelial cell migration. The transcription factors CREB and NF-B both can mediate this effect, suggesting that these two transcription factors are involved in tumor-associated angiogenesis and, therefore, could be potential targets for the angioprevention in NSCLC. We also conclude that the small molecule KG-501 neutralizes the effect of NSCLC/IL-1β–conditioned medium on endothelial cell migration by inhibiting CREB and NF-B transcriptional activity, which results in the down-regulation of CXC chemokine gene expression in NSCLC cells, suggesting that KG-501 may be used as a therapeutic and angiopreventive agent for NSCLC.

	Disclosure of Potential Conflicts of Interest

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No potential conflicts of interest were disclosed.

	Acknowledgments

TOP Abstract Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We thank Don Norwood for critical editing of the manuscript.

	Footnotes

 
Grant support: Department of Defense VITAL grant W81XW-04-1-0142 (J.S. Koo); National Heart, Lung, and Blood Institute Grant R01-HL-077556 (J.S. Koo); M.D. Anderson Cancer Center SPORE in Head and Neck Cancer (P50 CA097007) (Principal Investigator: S.M. Lippman) Developmental Research Program Grant P50 CA097007 (J.S. Koo); and National Cancer Institute Cancer Center Support Grant CA-16672 to The University of Texas M. D. Anderson Cancer Center.

Note: H. Sun and W-C. Chung contributed equally to this work.

Current address for Z. Ju: Department of Bioinformatics and Computational Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas.

¹http://rsb.info.nih.gov/ij

Received for publication December 8, 2007. Revision received June 10, 2008. Accepted June 24, 2008

	References

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