Cancer Prevention Research Cyclic AMP-Responsive Element Binding Protein– and Nuclear Factor-κB–Regulated CXC Chemokine Gene Expression in Lung Carcinogenesis Hongxia Sun, Wen-Cheng Chung, Seung-Hee Ryu, Zhenlin Ju, Hai T. Tran, Edward Kim, Jonathan M. Kurie and Ja Seok Koo Abstract 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 angiogenicfactorsby IL-1 β 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. Con- ditioned medium from IL-1β–treated A549 cells markedly increased endothelial cell migra- tion, 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 abro- gated with the knockdown of cyclic AMP-responsive element binding protein (CREB) or nuclear factor κB (NF-κB). Moreover, the expression of the CXC chemokine genes as well as CREB and NF-κB 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-κB, 2-naphthol-AS-E-phosphate (KG-501), inhibited IL-1β–induced CXC chemokine gene expression and angiogenicactivity in NSCLC. We propose that targeting CREB or NF-κB using small-molecule inhibitors, such as KG-501, holds promise as a preventive and/or therapeuticapproachfor NSCLC. Lung cancer is the leading cause of cancer deaths both in the lines of evidence have shown that angiogenesis,the formation United States and worldwide (1). The growth and develop- of new blood vessels from the preexisting vasculature,is one ment of lung cancer as well as of other solid tumors is criti- of the critical steps in the entire process of cancer development cally dependent on a functional vascular supply. Numerous 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 neovasculariza- Authors' Affiliation: Department of Thoracic/Head and Neck Medical tion and would be unable to metastasize (6). For solid tumors Oncology, The University of Texas M. D. Anderson Cancer Center, to develop and metastasize,they need to secrete a number of Houston, Texas Received 12/08/2007; revised 06/10/2008; accepted 06/24/2008. proangiogenic factors to induce the formation of new blood Grant support: Department of Defense VITAL grant W81XW-04-1-0142 (J.S. vessels to overcome the physical limitations on the diffusion Koo); National Heart, Lung, and Blood Institute Grant R01-HL-077556 (J.S. Koo); of nutrients and oxygen within the tumor—a process known M. D. Anderson Cancer Center SPORE in Head and Neck Cancer (P50 CA097007) as angiogenic switch (3). Recognizing the importance of angio- (Principal Investigator: S.M. Lippman) Developmental Research Program Grant P50 CA097007 (J.S. Koo); and National Cancer Institute Cancer Center Support genesis in the growth of tumors has led to the development of Grant CA-16672 to The University of Texas M. D. Anderson Cancer Center. antiangiogenesis as a new strategy for cancer therapy and Note: H. Sun and W-C. Chung contributed equally to this work. prevention,a novel concept termed “angioprevention” (7–9). Current address for Z. Ju: Department of Bioinformatics and Computational Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas. Actually,a series of molecules proposed as chemopreventive Requests for reprints: Ja Seok Koo, Department of Thoracic/Head and agents have been shown to have potent antiangiogenic Neck Medical Oncology, Unit 432, The University of Texas M. D. Anderson properties when tested in in vitro and in vivo angiogenesis Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713- 792-6363; Fax: 713-794-5997; E-mail: [email protected]. models (7). ©2008 American Association for Cancer Research. Angiogenesis can be regulated by various growth factors doi:10.1158/1940-6207.CAPR-07-0002 and cytokines,including vascular endothelial growth factor Cancer Prev Res 2008;1(5) October 2008 316 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. CREB and NF-κB Regulate CXC Chemokine Gene Expression (VEGF),basic fibroblast growth factor,transforming growth regulated by IL-1β are still not clear. To elucidate these critical factors α and β,platelet-derived endothelial cell growth issues,we conducted a microarray analysis to determine the factors,chemokines,and interleukine (IL)-1 β (10–14). Recent effect of IL-1β on global gene expression in the NSCLC ade- studies have shown the importance of the tumor microenvi- nocarcinoma cell line A549 and in normal human tracheo- ronment in facilitating angiogenesis and promoting tumor bronchial epithelium (NHTBE) cells. We found that IL-1β invasion and metastasis (15–19). Once a tumor is vascularized, dramatically induced the expression of an array of proangio- the tumor-associated antigens can be recognized by the im- genic CXC chemokine genes and significantly augmented the mune system and the tumor is infiltrated by leukocytes. angiogenic activity of NSCLC. In addition,we found that tran- Although leukocyte infiltration in tumors is often considered scription factors cyclic AMP-responsive element binding pro- to be associated with better prognosis and overall survival, tein (CREB) and nuclear factor κB (NF-κB) both play critical studies have also shown that inflammatory cells can promote roles in the regulation of IL-1β–induced CXC chemokine gene tumor cell proliferation,angiogenesis,metastasis,and,hence, expression and angiogenic activity. tumor development (15,16). Leukocyte infiltration can influ- ence angiogenesis in tumors because some subsets of leuko- Materials and Methods cytes,especially the tumor-associated macrophages,can Cell cultures, chemicals, and conditioned media secrete both angiostatic and angiogenic factors (17,18). IL-1 is a proinflammatory cytokine produced mainly by monocytes NHTBE cells were purchased from Cambrex and cultured in six-well plates as described previously (24–28). Human umbilical vein and macrophages. There are two IL-1 agonistic proteins,IL-1 α β α endothelial cells (HUVEC) were purchased from Cambrex and main- and IL-1 . IL-1 is a precursor or membrane-associated mole- tained in EGM complete endothelial growth medium. HUVECs were cule and is primarily a regulator of intracellular events and a used at passage 4. The human NSCLC cell lines A549,H1734,H226, mediator of local reactions. On the other hand,IL-1 β acts as a and H2170 were obtained from the American Type Culture Collection systemic,hormone-like mediator and is only active in a se- and grown in RPMI 1640 containing 10% fetal bovine serum, creted mature form. However,once these two proteins bind 100 units/mL penicillin,and 100 μg/mL streptomycin. The BEAS- to their receptors,they have similar biological activities (20). 2B,1799,1198,and 1170-I human bronchial epithelial cell lines were Both IL-1α and IL-1β can promote tumor angiogenesis,but obtained from Dr. R. Lotan (The University of Texas M.D. Anderson theroleofIL-1β is more evident (14). IL-1 has been shown Cancer Center,Houston,TX) and Dr. A. Klein-Szanto (Fox Chase to contribute to the production of proangiogenic factors VEGF, Cancer Center,Philadelphia,PA) and grown in Keratinocyte Serum-Free Medium (Life Technologies,Inc.) containing epidermal hepatocyte growth factor,tumor necrosis factor,and CXC che- growth factor and bovine pituitary extract (29). All of the cells were mokines (14,21). Members of a subfamily of CXC chemokines cultured at 37°C in a humidified atmosphere of 95% air and 5% CO2. sharing a characteristic glutamate-leucine-arginine (ELR) mo- Conditioned media (CM) were generated as follows: NSCLC cells 5 tifneartheNH2 terminus of the molecule are chemoattrac- (3 × 10 /mL) were cultured in RPMI 1640 containing 0.5% fetal bovine tants for neutrophils and are important for wound repair. serum with or without IL-1β and/or 2-naphthol-AS-E-phosphate The ELR-positive chemokines,including CXCL1,CXCL2, (KG-501) at different concentrations for 24 h. Cell-free supernatants CXCL3,CXCL5,CXCL6,CXCL7,and CXCL8,are proangio- were collected and stored at −70°C until use. genic,whereas members of another subfamily lacking the Reagents and antibodies ELR motif—ELR-negative chemokines,such as CXCL4, IL-1β,Quantikine CXCL5 and CXCL8 ELISA kits,and neutralizing CXCL9,CXCL10,and CXCL11 —are in general IFN inducible antibodies against CXCL5,CXCL8,CXCR2,and VEGF were pur- and are potential inhibitors of angiogenesis. Generally,CXCR2 – chased from R&D Systems. KG-501 was purchased from Sigma- is the receptor for angiogenic CXC chemokine mediated an- Aldrich and dissolved in DMSO. Antibodies against NF-κBp65, giogenesis,and CXCR3 is the receptor for angiostatic IFN- CREB,and phospho-CREB (Ser 133) were purchased from Santa Cruz inducible CXC chemokine inhibition of angiogenesis (13). Biotechnology,Inc.,and Upstate,respectively. A monoclonal antibody CXC chemokine ligands and receptors have been shown to (mAb) against β-actin was from Sigma-Aldrich. Transwell chambers play important roles in mediating non–small cell lung cancer with polyethylene terephthalate membranes containing 8-μm pores (NSCLC)–associated angiogenesis and organ-specific metas- were obtained from BD Biosciences.