[CANCER RESEARCH 64, 8262–8270, November 15, 2004] BRAK/CXCL14 Is a Potent Inhibitor of Angiogenesis and a Chemotactic Factor for Immature Dendritic Cells Thomas D. Shellenberger,1 Mary Wang,1 Manu Gujrati,4 Arumugam Jayakumar,1 Robert M. Strieter,5 Marie D. Burdick,5 Constantin G. Ioannides,2 Clayton L. Efferson,2 Adel K. El-Naggar,3 Dianna Roberts,1 Gary L. Clayman,1 and Mitchell J. Frederick1 Departments of 1Head and Neck Surgery, 2Gynecologic Oncology, and 3Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston, Texas; 4Department of Otorhinolaryngology, University of Kentucky, Lexington, Kentucky; and 5Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California, Los Angeles, California ABSTRACT biological significance. Moreover, little is known regarding the phys- iologic functions of this gene. BRAK/CXCL14 is a CXC chemokine constitutively expressed at the To date, three groups of investigators have examined the chemo- mRNA level in certain normal tissues but absent from many established tactic properties of BRAK for leukocytes and reported disparate tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK results. Among cells proposed to respond to BRAK by various inves- or the reason for decreased expression in cancer. To understand the tigators are prostaglandin E2–treated monocytes (5), cell lines from possible significance associated with loss of BRAK mRNA in tumors, we B-cell and monocytic cell lineages (3), neutrophils, and dendritic cells examined the pattern of BRAK protein expression in normal and tumor (4). A plausible explanation for the lack of agreement could be the specimens from patients with squamous cell carcinoma (SCC) of the different sources of BRAK used by investigators, which included a tongue and used recombinant BRAK (rBRAK) to investigate potential synthetic polypeptide, a murine homologue, and unpurified condi- biological functions. Using a peptide-specific antiserum, abundant expres- tioned supernatants from transfected mammalian cells. The recent sion of BRAK protein was found in suprabasal layers of normal tongue availability of commercially purified recombinant human BRAK mucosa but consistently was absent in tongue SCC. Consistent with pre- (rBRAK) should allow for better study of the physiologic targets and vious in situ mRNA studies, BRAK protein also was expressed strongly by functions of the BRAK gene. stromal cells adjacent to tumors. In the rat corneal micropocket assay, Although there are few published articles focusing on BRAK, one BRAK was a potent inhibitor of in vivo angiogenesis stimulated by mul- tiple angiogenic factors, including interleukin 8, basic fibroblast growth common finding appears to be the persistent absence of BRAK factor, and vascular endothelial growth factor. In vitro, rBRAK blocked mRNA from established tumor cell lines despite constitutive expres- endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggest- sion in normal tissues. Consistent with these findings, we previously ing this was a major mechanism for angiogenesis inhibition. Although only showed by in situ hybridization that BRAK mRNA is abundantly low affinity receptors for BRAK could be found on endothelial cells, expressed in normal squamous mucosa but absent from a majority of human immature monocyte-derived dendritic cells (iDCs) bound rBRAK head and neck SCC tumors (1). A role for chemokines in cancer is ϳ with high affinity (i.e., Kd, 2 nmol/L). Furthermore, rBRAK was che- supported by evidence that these molecules can regulate fundamental motactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our biological processes, including tumor-associated angiogenesis, acti- findings support a hypothesis that loss of BRAK expression from tumors vation of host tumor-specific immunity, and autocrine stimulation of may facilitate neovascularization and possibly contributes to immunologic tumor growth (6–12). escape. Chemokines are classified into subfamilies based on variations in a structural motif of conserved aminoproximal cysteine residues and include the CXC, CC, CX INTRODUCTION 3C, and the C families. BRAK belongs to the CXC family, which can be further subdivided by the presence or To identify genes associated with the malignant phenotype of head absence of a conserved “Glu-Leu-Arg” (ELR) motif at the NH2 and neck squamous cell carcinoma (SCC), we previously used differ- terminus. ELR(ϩ) CXC chemokines, such as GRO-␣/CXCL1, IL-8/ ential display of matched tumor explants and normal oral squamous CXCL8, and ENA-78/CXCL5, are angiogenic, whereas ELR(Ϫ) epithelial cells. One of the genes down-regulated in tumor specimens CXC chemokines induced by interferon, such as PF-4/CXCL4, IP- was BRAK/CXCL14, which encodes a novel chemokine (1). BRAK 10/CXCL10, and MIG/CXCL9, are angiostatic (10, 13–16). Although was independently cloned by Hromas et al. (2) and was found in not induced by interferon (1), BRAK does lack an ELR motif similar normal kidney and breast tissues but was absent in the majority of to the angiostatic CXC chemokines. However, the role of BRAK in established tumor cell lines. The work of multiple investigators (1–4) the regulation of angiogenesis remains to be established. formed an early consensus that BRAK mRNA was constitutively Chemokine action is mediated via members of the seven-trans- expressed in normal tissues but was absent in a variety of transformed membrane domain G protein–coupled receptors, which bind mul- cells. The absence of BRAK from many tumor cell lines (1, 2) and tiple ligands within chemokine subfamilies (17, 18). However, the head and neck SCC tumor specimens (1) is of currently unknown receptors and mechanisms by which chemokines inhibit chemo- taxis of endothelial cells currently are unknown. Although the Received 6/10/04; revised 8/9/04; accepted 9/8/04. angiostatic chemokines ␥-interferon–inducible protein (IP-10), Grant support: The University of Texas M.D. Anderson Cancer Center SPORE in MIG, and I-TAC mediate chemotaxis of activated T cells through Head and Neck Cancer Development Award, P50 CA97007. The costs of publication of this article were defrayed in part by the payment of page binding the high affinity chemokine receptor CXCR3 (19), there is charges. This article must therefore be hereby marked advertisement in accordance with little evidence that CXCR3 is involved in inhibition of endothelial 18 U.S.C. Section 1734 solely to indicate this fact. cell chemotaxis. Although endothelial cells reportedly express low Note: Supplementary data for this article can be found at Cancer Research Online (http://cancerres.aacrjournals.org). levels of CXCR3 (20, 21), a recent publication suggests that only Requests for reprints: Mitchell J. Frederick, Department of Head and Neck Surgery, the mRNA splice variant termed CXCR3B is present in these cells University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail: [email protected]. (22). This putative receptor was reported to mediate growth arrest ©2004 American Association for Cancer Research. of endothelial cells in response to PF-4 and IP-10. However, a role 8262 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. NOVEL FUNCTIONS OF BRAK for CXCR3B in inhibition of endothelial chemotaxis has not been negative, CD83 negative, and 95% CD1a positive by flow cytometry, whereas investigated. The chemokine receptor that binds BRAK currently mature dendritic cells were Ͼ80% CD83/CD1a positive and CD14 negative. has not been identified. Chemotactic Assays. In vitro endothelial chemotaxis assays were per- On the basis of the loss of BRAK mRNA in head and neck SCC, formed in a 48-well chemotaxis chamber (Neuro Probe, Gaithersburg, MD) ␮ as well as a structural relationship to other angiostatic CXC chemo- using an 8- m pore-sized filter precoated with type IV collagen. Cytokines known to stimulate migration of endothelial cells (IL-8, bFGF, or VEGF) were kines, we hypothesized that BRAK might inhibit angiogenesis. In this added at 10 ng/mL to the bottom wells of chemotaxis chambers containing report, we examine the expression of BRAK protein in SCC of the assay medium, rBRAK, or IP-10. Following starvation in DMEM containing tongue and confirm the antiangiogenic effect of this chemokine. We 0.1% FBS for 2 hours, HUVECs or HMECs were trypsinized, seeded at 12,500 also show that rBRAK is a potent chemoattractant for human imma- cells per well in the upper chamber, and allowed to migrate for 4 hours at 37°C. ture monocytic-derived dendritic cells (iDCs) through a specific high Unmigrated cells were scraped from the tops of filters, which were fixed, affinity receptor for BRAK. stained with Dif Quik (Baxter Scientific, Deerfield, IL), and mounted under oil immersion. Migratory cells were counted from nine random high-power fields from each well. The mean counts of cells from multiple wells were averaged MATERIALS AND METHODS and plotted graphically along with the SE of the means. Chemotaxis assays were repeated at least three times. Results were analyzed for significance using Reagents. Recombinant human cytokines including rBRAK, interleukin 8 the honest statistical difference test of unequal Ns. (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth Chemotaxis of dendritic cells also was measured in a microchemotaxis factor (bFGF),