The Human Cytomegalovirus–Encoded Chemokine Receptor US28 Promotes Angiogenesis and Tumor Formation Via Cyclooxygenase-2

Published OnlineFirst March 24, 2009; DOI: 10.1158/0008-5472.CAN-08-2487

Research Article

The Human Cytomegalovirus–Encoded Chemokine Receptor US28 Promotes Angiogenesis and Tumor Formation via Cyclooxygenase-2

David Maussang,1 Ellen Langemeijer,1 Carlos P. Fitzsimons,1 Marijke Stigter-van Walsum,2 Remco Dijkman,3 Martin K. Borg,3 Erik Slinger,1 Andreas Schreiber,4 Detlef Michel,4 Cornelis P. Tensen,3 Guus A.M.S. van Dongen,2 Rob Leurs,1 and Martine J. Smit1

1Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam; 2Department of Otolaryngology/Head and Neck Surgery, Vrije Universiteit Medical Center, Amsterdam, the Netherlands; 3Department of Dermatology, Leiden University Medical Center, Leiden, the Netherlands; and 4Institute of Virology, University Clinic Ulm, Ulm, Germany

Abstract considered an oncogenic herpesvirus (5), but it has been suggested The human cytomegalovirus (HCMV), potentially associated to act as an oncomodulator (6). The presence of HCMV proteins with the development of malignancies, encodes the constitu- has been detected in several malignancies, such as colon cancer (7), tively active chemokine receptor US28. Previously, we have malignant glioblastoma (8), and breast cancer (9). Importantly, shown that US28 expression induces an oncogenic phenotype HCMV preferably infects tumor cells, leading to enhanced cell both in vitro and in vivo. Microarray analysis revealed proliferation, angiogenesis, and resistance to apoptosis (6, 10). differential expression of genes involved in oncogenic signal- Of interest, most herpesviruses contain one or more genes that ing in US28-expressing NIH-3T3 cells. In particular, the encode for constitutively active G-protein coupled receptors (1). expression of cyclooxygenase-2 (COX-2), a key mediator of These viral G-protein coupled receptors show highest homology inflammatory diseases and major determinant in several to the class of chemokine receptors, known to be involved in the forms of cancer, was highly up-regulated. US28 induced control of the immune system but also the development of various increases in COX-2 expression via activation of nuclear types of cancer (11). As such, these viral G-protein coupled factor-KB, driving the production of vascular endothelial receptors likely contribute to viral pathogenesis. The KSHV- growth factor. Also, in HCMV-infected cells, US28 contributed encoded G-protein coupled receptor ORF74 is believed to act as to the viral induction of COX-2. Finally, the involvement of a viral oncogene and is considered a key determinant in the COX-2 in US28-mediated tumor formation was evaluated pathology of Kaposi’s sarcoma. ORF74 possesses proliferative, using the COX-2 selective inhibitor Celecoxib. Targeting COX-2 angiogenic, and antiapoptotic properties, and drives the cell- in vivo with Celecoxib led to a marked delay in the onset of transforming properties of KSHV (12, 13). Recently, we have shown tumor formation in nude mice injected with US28-transfected that the HCMV-encoded chemokine receptor US28, which can bind NIH-3T3 cells and a reduction of subsequent growth by several chemokines (e.g., CCL2, CCL5, CX3CL1), constitutively repressing the US28-induced angiogenic activity. Hence, the activates various inflammatory and proliferative signaling path- n n development of HCMV-related proliferative diseases may ways such as nuclear factor- B (NF- B) and induces tumor partially be ascribed to the ability of US28 to activate COX-2. formation in vivo (14). Expression of US28 activates Gaq-linked [Cancer Res 2009;69(7):2861–9] signaling pathways, e.g., production of inositol phosphate (15), resulting in an increase in cyclin D1 expression, DNA synthesis, and secretion of vascular endothelial growth factor (VEGF; ref. 14). Introduction Mice injected with US28-expressing NIH-3T3 cells develop tumors with high VEGF expression. Moreover, an increase in VEGF Herpesviruses are widespread pathogens, which establish a life- promoter activity is also apparent in HCMV-infected glioblastoma long latent and persistent infection. In immunocompetent hosts, cells, which can be attributed to the expression of US28 (14). infection is often asymptomatic, whereas reactivation can lead to Interestingly, US28 expression has also been detected during serious pathologic conditions (1). In particular, g-herpesviruses primary and secondary HCMV infection in immunosuppressed possess oncogenic potential, as they are able to transform cells patients (16). Considering the pathogenic potential of HCMV in upon infection (2). Kaposi’s sarcoma-associated herpesvirus these patients, US28 expression at this stage may play a role in the (KSHV) is the etiologic agent of Kaposi’s sarcoma (3), whereas progression of HCMV-linked proliferative diseases. Epstein-Barr virus (EBV) is associated with lymphoproliferative Profiling of the expression of thousands of genes by means of diseases such as Burkitt’s lymphoma and Hodgkin’s disease (4). DNA microarrays has served to discover new oncogenes and Unlike KSHV and EBV, the human cytomegalovirus (HCMV) is not potentially new targets for the treatment of cancer (17). It has also been used to understand molecular mechanisms underlying the development of herpesvirus associated diseases (18–20). To gain Note: Supplementary data for this article are available at Cancer Research Online mechanistic insight into the oncogenic behavior of the HCMV- (http://cancerres.aacrjournals.org/). encoded chemokine receptor US28, we performed microarray D. Maussang and E. Langemeijer contributed equally to this work. Requests for reprints: Martine J. Smit, Vrije Universiteit Amsterdam, De Boelelaan analysis on US28 and mock-transfected cells. Various proteins 1083, Amsterdam, 1081 HV, the Netherlands. Phone: 310-205987572; Fax: 310- involved in oncogenesis were found to be modulated by the 205987610; E-mail: [email protected]. I2009 American Association for Cancer Research. expression of US28. In particular, cyclooxygenase-2 (COX-2) was doi:10.1158/0008-5472.CAN-08-2487 highly up-regulated upon US28 expression. Also, in HCMV-infected www.aacrjournals.org 2861 Cancer Res 2009; 69: (7). April 1, 2009

Cancer Research cells, US28 contributed to the viral induction of COX-2. Finally, NF-nB activation was measured by transfecting cells with the NF-nB the COX-2–specific inhibitor Celecoxib did not only inhibit the luciferase reporter gene 24 h before infection. up-regulation of VEGF in US28-expressing cells but also markedly Western blot analysis. Quantification of COX-2 protein level was decreased US28-induced tumor formation rate in nude mice. As performed by Western blot on total cell lysates with a rabbit polyclonal COX-2 antibody (Cell Signaling Technology) and a mouse monoclonal such, US28 up-regulates COX-2 expression to promote tumor h-actin antibody (Sigma). formation. In vivo experiment. All animal experiments were conducted according to the NIH principles of laboratory animal care and Dutch national law [‘‘Wet op de Dierproeven’’ (Stb 1985, 336)], approved by the Dierexper- Materials and Methods imentencommissie from the Vrije Universiteit Medical Center and performed in compliance with the protocol FaCh 05-02. US28 stably Cell culture. The mock (empty pcDEF3 expression vector) and US28 transfected NIH-3T3 cells (2 Â 106) were injected s.c. into the flanks of 8- to stably transfected NIH-3T3 cell lines were cultured as previously described 10-wk-old female nude mice (Hsd, athymic nu/nu, 25–32 grams; Harlan (14). The human embryonic kidney cell line HEK 293T was cultured in Laboratories Cambridge Research Biochemicals). The control group was fed DMEM containing 10% fetal bovine serum (FBS) and transfected using with regular mouse chow (Teklad Harlan), whereas the Celecoxib group was polyethylenimine. The human foreskin fibroblast BJ cell line was maintained fed 1 d before the cell injection with mouse chow containing 1,500 ppm in MEM (Eagle) with 2 mmol/L L-glutamine and Earle’s balanced salt Celecoxib (Pfizer) ad libitum. solution adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mmol/L Immunohistochemistry. Cryosections of US28-induced xenografts were nonessential amino acids, 1.0 mmol/L sodium pyruvate, and 10% FBS. stained for the presence of CD31 using a rat anti-mouse CD31 antibody Chemokine binding and inositol phosphate accumulation experi- (BD Pharmingen) with a horseradish peroxidase–conjugated mouse anti-rat ments. Stably transfected NIH-3T3 cells and HCMV-infected BJ cells were antibody (Zymed Laboratories). analyzed for radiolabeled chemokine binding and inositol phosphate formation as previously described (14). Microarray analysis. NIH-3T3 cells stably transfected with either mock Results or US28 were serum starved overnight with 0.5% calf serum containing medium. Total RNA was isolated from two independent clones for both Microarray analysis of US28-expressing cells. Expression of cell lines with the RNeasy kit (QIAgen) followed by cDNA synthesis and the HCMV-encoded chemokine receptor US28 in NIH-3T3 cells overnight biotin-labeled cRNA amplification (MessageAmp II aRNA induces increased cell growth and a proangiogenic phenotype (14). Amplification; Ambion). Twenty micrograms of biotin-labeled cRNA was To gain insight into the underlying mechanisms of US28-related hybridized to Affymetrix Mouse Genome 430 2.0 arrays and processed oncogenic transformation, we analyzed gene expression profiles according to Affymetrix procedures. Arrays were normalized (RMA) and of US28-expressing NIH-3T3 cells and the corresponding mock- analyzed using LIMMA statistical package (21). The data have been transfected NIH-3T3 cells by cDNA microarray analysis. US28 deposited in National Center for Biotechnology Information’s Gene WT–expressing cells showed increases in [125I]-CX3CL1 binding Expression Omnibus (22) and are accessible through GEO Series accession (Fig. 1A) and inositol phosphate production (Fig. 1B), which was not number GSE13567.5 Genes differentially expressed between mock and US28- transfected cells were selected based on stringent false discovery rates apparent in mock-transfected cells. To reproducibly identify (false discovery rate, V0.02). The retrieved gene lists were analyzed with differentially expressed genes in our microarray analysis, two DAVID and EASE softwares (23, 24) and compared with lists of cancer- independent clonal cell lines, with similar receptor expression and related genes6 and to NF-nB transcriptional targets.7 functional characteristics (Fig. 1A and B) were used for both Quantitative real-time-PCR. For biological validation of microarray mock and US28-transfected NIH-3T3 cells. Analysis of the overall data by quantitative real-time-PCR (qPCR) analysis, total RNA was isolated microarrays intensities showed highest correlation between biolog- from NIH-3T3 cells and from US28 xenografts using the RNeasy kit (Qiagen) ical duplicates (i.e., mock to mock, US28 to US28; data not shown). and reverse transcribed using iScript cDNA synthesis kit (Bio-Rad), Expression data of the 45,001 Affymetrix probe sets on the according to the manufacturers’ instructions. qPCR primers (from Mouse Genome 430 2A Array were normalized (RMA) and analyzed Invitrogen or Isogen) used are described in Supplementary Table S4. PCR with LIMMA (21). Using a false discovery rate lower or equal to reactions were performed using SYBR Green mix with MyiQ Real-Time PCR detection system (Bio-Rad). qPCR on HCMV-infected cells was performed 0.02, we obtained 577 probe sets representing 556 genes with the ABI-Prism 7700 instrument and the SYBR Green PCR Master Mix (Supplementary Table S1). The most highly modulated genes are (Applied Biosystems). Data were evaluated with the sequence detection depicted in Fig. 1C. As can be seen in this figure, the observed software version 1.9.1 (Applied Biosystems) and the second derivative changes in gene expression were comparable in the two different maximum algorithm. In addition to melting curve analysis, specificity of clonal mock and US28-expressing cell lines. The involvement of the PCR products were confirmed by running controls on agarose gel differentially expressed genes in biological pathways was analyzed electrophoresis and subsequent DNA sequence analysis. using DAVID (23) for testing Kyoto Encyclopedia of Genes and n Luciferase reporter gene experiments. The NF- B and VEGF promoter Genomes pathways (27). The 556 genes did not present statistically luciferase reporter genes were previously described (14, 15), and the wild- enriched signaling pathways (after Benjamini correction for type (WT) and mutated COX-2 promoter luciferase reporter gene multiple testing). Annotation of the genes to the Kyoto Encyclo- plasmids were obtained from Dr. M. Fresno (Universidad Auto´noma de Madrid, Madrid, Spain; ref. 25). Luciferase activity was measured pedia of Genes and Genomes pathways using the EASE software 24 h posttransfection. (24) highlighted pathways involved in focal adhesion, actin Viral experiments. BJ cells were infected with the different HCMV virus cytoskeleton regulation, cell cycle, and several forms of cancer strains (AD169-WT and AD169-DUS28; ref. 26) with a multiplicity of but also signaling pathways such as p53, mitogen-activated protein infection of 3, and the different assays were performed 8 h postinfection. kinase (MAPK), Wnt, and transforming growth factor h (Supple- mentary Table S2). To focus on molecular mediators that contribute to the oncogenic potential of US28, we used the CancerGenes resource 5 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE13567 6 http://cbio.mskcc.org/CancerGenes (28), identifying among others, Akt1, Fus, Klf6, Mdm2,and 7 adapted from http://jura.wi.mit.edu/young_public/nfkb/literature_targets.xls Ptgs2/Cox-2 (Table 1). In addition, because US28 has previously

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Role of COX-2 in US28-Induced Tumor Formation

Figure 1. Characterization and microarray analysis of US28-expressing NIH-3T3 cells. A, independent stable clonal NIH-3T3 cell lines transfected with US28-WT and the G-protein uncoupled mutant US28-R129A bind [125I]-CX3CL1. Unlabeled CX3CL1 displaces specifically bound [125I]-CX3CL1. B, US28-WT constitutively induces the formation of inositol phosphate (InsP), whereas US28-R129A does not. C, affymetrix Mouse Genome Array data from US28 and mock-transfected cells were analyzed with the LIMMA software using a false discovery rate V0.02. The 35 most up-regulated (up) and down-regulated (down) probe sets are represented with a heatmap. Colors indicate log2 intensity values of normalized probe sets.

been shown to constitutively activate NF-nB signaling pathways expressing cells, but not cells expressing the G-protein uncoupled (15), the list of modulated genes was compared with known mutant US28-R129A, have been shown to present a transformed transcriptional targets of NF-nB7 (Table 1). Ptgs2/Cox-2 also seemed phenotype in vitro (14). NIH-3T3 cells expressing US28-R129A to be one of the most strongly up-regulated NF-nB target genes. showed comparable receptor expression levels to US28-WT– This finding of Ptgs2/Cox-2 in both cancer and NF-nB gene lists was expressing cells as measured by [125I]-CX3CL1 binding (Fig. 1A) of particular interest because COX-2 protein is highly up-regulated but did not show increases in inositol phosphate accumulation in various forms of cancers (29) but also induced upon HCMV- (Fig. 1B). Analysis of COX-2 mRNA expression by qPCR showed a infection (20). 19.7- F 1.8-fold increase in US28-WT–expressing cells compared To validate the microarray expression data, a few genes with with mock-transfected cells (Fig. 2A). Cells expressing US28-R129A high fold changes were analyzed by means of qPCR. As observed revealed no significant difference (1.6- F 0.6-fold) in COX-2 mRNA in the microarray experiment, Ptgs2/Cox-2 as well as other highly levels compared with mock-transfected cells (Fig. 2A). Similarly, differentially expressed genes (Mef2c, Cxcl12, and Tgfb2) showed a US28-WT–transfected NIH-3T3 cells showed a marked increase similar degree of up or down-regulation upon expression of US28 in in COX-2 protein expression compared with mock-transfected and NIH-3T3 cells (Supplementary Table S3). Because of the previously US28-R129A–expressing cells (Fig. 2B). reported oncogenic potential of US28 in vivo, we also determined US28 induces COX-2 and VEGF transcription via activation expression levels of these genes in RNA extracted from five of NF-KB. To understand the molecular mechanisms resulting independent US28-induced tumors derived from our xenograft in the up-regulation of COX-2, signaling studies with a COX-2 model (14). Expression of US28 was confirmed in all mouse tumors promoter reporter (25) were performed in HEK 293T cells. US28 by qPCR and Ptgs2/Cox-2 mRNA was highly up-regulated in US28- induced the human COX-2 promoter activation in a dose- induced tumors, highlighting a potential important role for COX-2 dependent manner, but no increase in COX-2 promoter activity during tumorigenesis. was observed in US28-R129A–expressing HEK 293T (Fig. 3A). US28 constitutive activity up-regulates COX-2 expression. Because the transcription of the COX-2 gene is under the control COX-2 is highly up-regulated in a variety of cancers and is known of NF-nB (31), we investigated the contribution of NF-nBinthe to drive expression of cyclin D1 and VEGF (30). Because COX-2 is COX-2 promoter reporter gene. US28-WT, but not US28-R129A, also up-regulated in HCMV-infected cells (20) and expression of constitutively activated the NF-nB transcription factor in trans- US28 results in induction of cyclin D1 and VEGF expression (14), fected HEK 293T cells (Fig. 3A). Moreover, inhibition of NF-nB we decided to further focus on COX-2 and examine its role in US28- activation with the InB phosphorylation inhibitor Bay 11-7082 induced proliferative signaling and tumor formation. US28-WT– (5 Amol/L) resulted in a severe reduction of US28-induced COX-2 www.aacrjournals.org 2863 Cancer Res 2009; 69: (7). April 1, 2009

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Table 1. US28-expressing cells modulate the expression of oncogenes and NF-nB transcriptional targets

Gene symbol Gene name Fold change

Cancer genes resource Net1 Neuroepithelial cell transforming gene 1 11.5 Ptgs2/Cox-2 Prostaglandin-endoperoxide synthase 2 6.6 Kpnb1 Karyopherin (importin) h 16.1 Cebpb CCAAT/enhancer binding protein (C/EBP), h 10.9, 2.9, 2.2 Actb Actin, h 5.1, 3.5, 2.4 Klf6 Kruppel-like factor 6 4.1, 3.3 Cttn Cortactin 3.4, 3.0 Fus Fusion (involved in t(12;16) in malignant liposarcoma) 3.5 Runx1 Runt-related transcription factor 1 2.9 Akt1 V-akt murine thymoma viral oncogene homologue 1 2.7 Araf V-raf murine sarcoma 3611 viral oncogene homologue 2.2 Mdm2 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) 2.2 Gli3 GLI-Kruppel family member GLI3 À3.1 Maf V-maf musculoaponeurotic fibrosarcoma oncogene homologue (avian) À3.1 Timp3 TIMP metallopeptidase inhibitor 3 À6.3, À9.2 Aff3 AF4/FMR2 family, member 3 À9.8

NF-nB transcriptional targets Ptgs2/Cox-2 Prostaglandin-endoperoxide synthase 2 6.6 Hgf Hepatocyte growth factor 4.9, 4.8, 3.1 Serpine1 Serine (or cysteine) peptidase inhibitor, clade E, member 1 4.2 Nr4a2 Nuclear receptor subfamily 4, group A, member 2 3.8, 3.6, 2.8 Gadd45b Growth arrest and DNA-damage–inducible 45 h 2.8 Csf1 Colony stimulating factor 1 (macrophage) 2.6 Cflar CASP8 and FADD-like apoptosis regulator 2.3 C3 Complement component 3 À2.8 Mmp9 Matrix metallopeptidase 9 À2.8, À6.8 Sox9 SRY-box containing gene 9 À2.9

Abbreviations: C/EBP, CAAT/enhancer binding protein; TIMP, tissue inhibitor of metalloproteinase.

promoter activation (Fig. 3B). To further assess the role of NF-nBin US28 mRNA (Fig. 4A). As expected, the deletion mutant (D) did not the US28-induced COX-2 expression, we used two truncated COX-2 induce the expression of US28 mRNA. Radioligand binding studies promoter reporter genes that do not contain sequential NF-nB showed that also on the protein level, only cells infected with the binding sites (25). 5¶-Deletions of the distal and both distal and AD169-WT virus expressed US28 on their cell surface (Fig. 4B). In proximal NF-nB binding sites reduced the US28-induced constitu- accordance with earlier findings (32), COX-2 mRNA levels were tive activation of the COX-2 promoter by respectively 30% and 80% clearly up-regulated in cells infected with the WT virus 8 h compared with the nondeleted promoter (Fig. 3C). Because COX-2 is known to drive expression of VEGF (30), shown to be up- regulated upon expression of US28 (14), we investigated the role of NF-nB in VEGF promotor activity using the NF-nB inhibitor Bay 11-7082. As seen for the US28-induced COX-2 promotor activity, the US28-induced VEGF promoter activation was markedly inhibited by Bay 11-7082, highlighting the involvement of NF-nB in this process (Fig. 3D). US28 induces COX-2 expression in HCMV-infected cells. Infection of cells with HCMV is associated with the activation of various signaling pathways linked to inflammation, including increased expression of COX-2 (32). Because US28 induced COX-2 expression in transfected cells, we examined the role of US28 in a viral setting. To this end, we used a HCMV deletion virus derived from the AD169 strain that does not contain the sequence Figure 2. US28 constitutively up-regulates COX-2 expression. A, encoding for US28 (AD169-DUS28). Human foreskin fibroblasts US28-WT–expressing NIH-3T3 cells (filled bar) present higher COX-2 mRNA were infected with either mock, AD169-WT, or the deletion mutant levels than mock-transfected cells. US28-R129A–expressing cells (open bar) do not show any modulation of COX-2 mRNA expression. B, COX-2 protein AD169-DUS28 (multiplicity of infection, 3). Eight hours postinfec- expression is higher in US28-WT–expressing cells than in mock- and tion, only cells infected with the WT virus presented expression of US28-R129A–transfected cells.

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Role of COX-2 in US28-Induced Tumor Formation postinfection, compared with mock-infected cells (Fig. 4C). In cells Celecoxib was able to inhibit in vitro an important angiogenic infected with AD169-DUS28, COX-2 mRNA levels were markedly factor involved in US28-induced tumor formation, it was also used reduced, highlighting a role for US28 in the viral induction of in vivo to examine the contribution of COX-2 during tumorigenesis. COX-2. The observed decrease in COX-2 mRNA in AD169-DUS28– Celecoxib treatment was started 1 day before the injection of US28- infected cells was not attributed to impairment in viral IE stably transfected NIH-3T3 cells into nude mice by feeding the expression of this mutant strain (data not shown), indicating that animals with chow containing 1,500 ppm Celecoxib (33). As the viral replication abilities of both viruses were similar. Because previously described, all control mice injected with US28-express- US28 is able to constitutively activate NF-nB (15), we also tested ing cells developed tumors larger than 50 mm3 within 3 weeks. Yet, whether this was the case in the HCMV context. Infection of cells at this time point, Celecoxib-fed mice showed a severely delayed with AD169-WT was clearly associated with increased activation of onset in tumor formation (Fig. 5B). Developing tumors reached the NF-nB as measured using a NF-nB reporter gene (Fig. 4D). When size of 50 mm3 between 21 and 39 days postinjection, whereas for infecting cells with the US28 deletion mutant, we found that the the control group, tumor formation occurred between 14 and HCMV-induced NF-nB activation was markedly impaired com- 21 days postinjection. Celecoxib also increased the tumor doubling pared with cells infected with the WT virus (Fig. 4D). This indicates time. Untreated mice injected with US28-expressing cells displayed that upon HCMV infection, US28 activates NF-nB, regulating in an average tumor doubling time of 3.4 F 0.3 days, whereas turn expression of its transcriptional target COX-2. Celecoxib-treated mice presented a longer doubling time of 6.4 F COX-2 plays an important role in US28-mediated tumor 0.4 days. When xenografts had reached their maximum size and formation. We have previously shown that US28 up-regulates animals were sacrificed, no significant changes in VEGF mRNA VEGF expression in stably transfected NIH-3T3 cells (14). Because levels were apparent between the untreated and Celecoxib-treated VEGF expression is in part regulated by COX-2 (30), we tested tumors (data not shown). To measure effects of Celecoxib on the in vitro the effect of the COX-2–specific nonsteroidal anti- angiogenic activity in US28-induced tumors, we examined CD31 inflammatory drug (NSAID) Celecoxib on the US28-induced expression by immunohistochemistry. Xenografts derived from secretion of VEGF. When culturing US28-expressing NIH-3T3 cells stably transfected NIH-3T3 cells lines show highest growth activity with Celecoxib (25 Amol/L), VEGF secretion was significantly in the outer cell layers rather than at the center of the tumor, which reduced (52% F 2%; P < 0.001), indicating the involvement of can become necrotic when tumors become large. As expected, the COX-2 in the US28-induced proangiogenic phenotype (Fig. 5A). As angiogenic activity in the untreated US28-derived tumors was

Figure 3. US28 activates the COX-2 and VEGF promoters via NF-nB. A, HEK 293T cells transiently transfected with increasing amounts of US28 show a dose-dependent activation of the human COX-2 promoter luciferase reporter gene. The human COX-2 promoter and the transcription factor NF-nB are activated after transfection of US28-WT but not with the G-protein uncoupled mutant US28-R129A. RLU, relative light units. B, US28-WT–induced COX-2 promoter activation is inhibited by the NF-nB inhibitor Bay 11-7082 (5 Amol/L). C, the US28-WT–induced COX-2 promoter activity is reduced when one and in particular both NF-nB binding sites are removed in 5¶-deletion mutants of the human COX-2 promoter. D, US28-WT–induced VEGF promoter activation is inhibited by the NF-nB inhibitor Bay 11-7082 (5 Amol/L). www.aacrjournals.org 2865 Cancer Res 2009; 69: (7). April 1, 2009

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Figure 4. US28 mediates COX-2 expression in HCMV-infected cells. A, human foreskin fibroblasts infected with mock (Mk), AD169-WT, or the deletion mutant AD169-DUS28 (D) viruses only express US28 mRNA with the WT virus 8 h postinfection. B, [125I]-CCL5 binding shows US28 protein expression in AD169-WT–infected cells but not in cells infected with AD169-DUS28 mutant. C and D, cells infected with AD169-WT present a higher expression level of COX-2 mRNA (C) and an increased NF-nB activation (D) compared with mock-infected cells. Deletion of US28 gene from the viral genome impairs COX-2 expression and NF-nB transcriptional activity in infected cells. clearly apparent in the outer area of the malignancies with a dense Because US28 constitutively activates NF-nB signaling, it is not localization of CD31-positive blood vessels (Fig. 5C). Interestingly, surprising to observe modulation of NF-nB target genes in US28- the Celecoxib-treated tumors presented a lower staining intensity expressing cells (Fig. 1D). One of such genes under control of of CD31 and a less dense distribution of newly formed blood NF-nB is COX-2 (31), which seems to be highly up-regulated in vessels compared with the untreated group. As such, COX-2 US28-expressing cells and tumors (Table 1). COX-2 is of specific inhibition by Celecoxib delayed the onset of US28-induced tumors interest because it is a key mediator of inflammation and it is now and slowed their overall growth by repressing angiogenic activity. well-established that it contributes to the pathogenesis of several forms of cancer. This enzyme is commonly expressed in both premalignant lesions and malignant tumors of e.g., colon, lung, head, neck, and breast (29). In view of the role of COX-2 in tumor Discussion development and its up-regulation in herpesvirus-infected cells, Infection of cells with herpesviruses is known to alter cellular this enzyme has been suggested to participate in neoplasia induced gene expression and cell function. Functional genomic analyses of by some of the oncogenic herpesviruses, including KSHV and EBV herpesvirus-infected cells have led to the identification of genes (39). Of interest, HCMV possesses oncomodulatory properties (6) that contribute to viral pathogenesis, including induction of and also up-regulates COX-2 expression within a few hours after tumorigenic events (18–20). In view of the reported oncomodula- infection (32). tory potential of HCMV (6) and particularly the observed Because our microarray studies reveal marked increases in COX- oncogenic properties of the HCMV-encoded chemokine receptor 2 expression in US28-expressing cells, and US28 was shown earlier US28 (14), we subjected US28-expressing cells to a detailed to up-regulate expression of VEGF and cyclin D1 (14), both known microarray analysis. As anticipated, US28 and mock-transfected to be regulated by COX-2 (30), COX-2 is a likely determinant in NIH-3T3 cells differentially express genes involved in oncogenic the US28-induced tumor formation. Our studies show that US28 events (e.g., renal cell carcinoma, prostate cancer, and melanoma). expression is associated with up-regulation of COX-2 at both the Significantly modulated genes are implicated in cell cycle, p53, and mRNA and protein levels via activation of NF-nB (Figs. 2 and 3). MAPK pathways (Supplementary Table S2), all associated with As expected, NF-nB is also implicated in the US28-induced VEGF proliferation (34–37). Also, modulation of genes involved in focal promoter activity (Fig. 3D). The G-protein uncoupled mutant US28- adhesion and actin cytoskeleton rearrangement, both of impor- R129A does not activate NF-nB nor up-regulate COX-2 expression tance in tissue invasion and metastasis (38), is observed. Although (Fig. 2 and 3), indicating a role for G proteins in the US28-mediated these pathways are not statistically enriched in a DAVID analysis, COX-2 up-regulation (15). they are correlated to the modulated genes using EASE, providing To further show the contribution of COX-2 in the US28-induced mechanistic insights on how US28 may induce transformation and tumor formation, we performed in vitro assays and intervention tumor development. Another analysis of the microarray data, studies in our US28 tumor xenograft model using the nonsteroidal focusing on cancer-related genes and NF-nBtranscriptional anti-inflammatory drug COX-2 inhibitor Celecoxib (Fig. 5). Several targets, reveals modulation of genes involved in several forms of clinical studies have provided encouraging evidence of preventive cancer that have previously been linked to HCMV infection such as effects of nonsteroidal anti-inflammatory drugs in cancers of colon, colorectal and breast cancers (Ptgs2/Cox-2, Nr4a2, Serpine1, Csf1, lung, breast, and prostate (40). Although some of these drugs were and Gadd45b). Genes participating in multiple forms of cancer associated with cardiovascular toxicity, recent studies presented (e.g., Net1, Cttn, Runx1, Mdm2, Timp3, and Hgf ) are also modulated Celecoxib as one of the coxibs possessing least side effects (41). by US28 expression in NIH-3T3 cells. Although a more systematic Various animal studies reported effective inhibition of cell growth approach is required to obtain insight in the oncogenic signaling in vitro and tumor growth in vivo upon Celecoxib treatment networks activated by US28, this study points toward several (33, 42, 43). In vivo, Celecoxib treatment of mice injected with oncogenic targets that are modulated by US28. US28-expressing cells severely delays and impairs tumor formation.

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Role of COX-2 in US28-Induced Tumor Formation

Figure 5. Celecoxibimpairs US28 oncogenic potential. A, release of VEGF in the culture medium of US28 stably transfected NIH-3T3 cells is partly inhibited by Celecoxib(25 Amol/L). ***, P < 0.0001. B, Celecoxib-treated mice injected with US28-expressing cells (o) develop tumors bigger than 50 mm3 later and at a slower rate than control mice (.). Twenty days postinjection of US28-expressing cells into nude mice, control mice present extensive tumor formation, whereas the Celecoxib-treated group (1,500 ppm) is still devoid of tumors. C, representative CD31 staining in US28-induced tumors from control and Celecoxib-treated mice. D, schematic representation of US28-induced COX-2 up-regulation and role during tumor formation. US28 is expressed at the cell surface of HCMV-infected cells and increases COX-2 expression via NF-nB. COX-2 activity leads to PGE2 release that can bind to its cognate receptors EP1-4 and subsequently induces VEGF and Cyclin D1 transcription. This results in angiogenesis and tumor formation that can be inhibited by Celecoxib. www.aacrjournals.org 2867 Cancer Res 2009; 69: (7). April 1, 2009

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After 3 weeks, no tumors are formed in the Celecoxib-treated mice, We have previously shown that US28 constitutive activity is whereas tumors are apparent in the control group, indicating that mediated by Gaq and Ghg proteins (14, 15). The constitutive COX-2 expression is of importance in early tumorigenic events activation of COX-2 or VEGF promoter by US28 could not be induced by US28. But in the long term, Celecoxib-treated mice also modulated by any of the CC and CX3C chemokines (CCL2, CCL5, present tumors, indicating that COX-2 is not the sole factor and CX3CL1) known to bind this viral G-protein coupled receptor responsible for US28-induced tumor formation. Importantly, (data not shown). Ligand-stimulation of US28 has however been Celecoxib treatment reduces the growing rate of the tumor shown to promote cell migration via activation of Ga12/13 proteins compared with untreated mice (P < 0.001 in Log-rank test), (47). As such, the Gaq pathway may be preferentially activated confirming the involvement of COX-2 in tumor development. In constitutively by US28 during oncogenic transformation, whereas addition, Celecoxib impairs the angiogenic phenotype induced by the ligand-induced signaling may be favorable for the migration US28, both in vitro by significantly (P < 0.0001) decreasing the of US28-expressing cells. We therefore postulate that upon HCMV production of VEGF in US28-expressing cells, and in vivo,by infection, US28 is expressed and constitutively activates NF-nBina reducing angiogenic activity in US28-derived xenografts. As such, ligand-independent manner. US28 potentially activates Gaq and the impairment of the COX-2/VEGF axis by Celecoxib could be Ghg (15) to induce the expression of inflammatory proteins such accounted for the decreased angiogenesis and subsequent slower as COX-2 (31). This enzyme is responsible for the synthesis of development of the tumors in vivo. It is important to note that our PGE2, which through activation of its cognate receptors EP1-4 microarray analysis indicates that other oncogenes beside COX-2 leads to a subsequent enhancement of cell proliferation (Cyclin D1) are likely to be implicated in US28-induced tumor progression. and promotion of angiogenesis (VEGF; ref. 30). US28-dependent The observed inhibitory effect of Celecoxib on tumor formation increases of at least COX-2 early after infection might be sufficient are most likely attributed to the functional inhibition of COX-2, to catalyze inflammatory processes, which may contribute to or although some COX-2 independent effects by Celecoxib have been enhance tumor formation (Fig. 5D). Hence, the development of reported (44). Other targets of Celecoxib include, e.g., Cyclin D1 HCMV-related proliferative diseases might in part be ascribed to (45) and NF-nB (46), both up-regulated in US28-transfected cells. the ability of US28 to modulate expression of COX-2. Thus, impairment of US28-induced tumorigenesis by Celecoxib, either through direct or indirect COX-2 inhibition, involves inhibition of proliferative signaling proteins that are constitutively Disclosure of Potential Conflicts of Interest activated by US28. No potential conflicts of interest were disclosed. Cells infected with the AD169 strain devoid of US28 (AD169- DUS28) show a reduced activation of NF-nB and up-regulation of COX-2 compared with cells infected with the WT virus (Fig. 4). Acknowledgments In vivo, US28 expression has been shown in lung transplant Received 7/3/08; revised 12/12/08; accepted 1/28/09; published OnlineFirst 3/24/09. immunosuppressed patients presenting HCMV primary infection The costs of publication of this article were defrayed in part by the payment of page or reactivation (16). Because of the US28-mediated increases in charges. This article must therefore be hereby marked advertisement in accordance NF-nB activation and COX-2 up-regulation in infected cells in vitro, with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. M. Fresno and Pfizer for providing respectively the human COX-2 US28 may be implicated in virus-associated pathologies by further promoter luciferase reporter gene plasmids and the COX-2 inhibitor Celecoxib, and enhancing and/or contributing to increases in COX-2 expression. Dr. H.F. Vischer for the creation of the Fig. 5D artwork.

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www.aacrjournals.org 2869 Cancer Res 2009; 69: (7). April 1, 2009

The Human Cytomegalovirus−Encoded Chemokine Receptor US28 Promotes Angiogenesis and Tumor Formation via Cyclooxygenase-2

David Maussang, Ellen Langemeijer, Carlos P. Fitzsimons, et al.

Cancer Res 2009;69:2861-2869. Published OnlineFirst March 24, 2009.

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