Interaction between CXCR4 and CCL20 Pathways Regulates Tumor Growth Katia Beider1, Michal Abraham1, Michal Begin1, Hanna Wald1, Ido D. Weiss1, Ori Wald1, Eli Pikarsky2, Rinat Abramovitch1,3, Evelyne Zeira1, Eithan Galun 1 , Arnon Nagler4*, Amnon Peled 1 * 1 Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel, 2 Department of Pathology, Hadassah Hebrew University Hospital, Jerusalem, Israel, 3 MRI Lab, HBRC, Hadassah Hebrew University Hospital, Jerusalem, Israel, 4 Bone Marrow Transplantation Department, Chaim Sheba Medical Center, Tel- Hashomer, Israel Abstract The chemokine receptor CXCR4 and its ligand CXCL12 is overexpressed in the majority of tumors and is critically involved in the development and metastasis of these tumors. CXCR4 is expressed in malignant tumor cells whereas its ligand SDF-1 (CXCL12) is expressed mainly by cancer associated fibroblasts (CAF). Similarly to CXCR4, the chemokine CCL20 is overexpressed in variety of tumors; however its role and regulation in tumors is not fully clear. Here, we show that the chemokine receptor CXCR4 stimulates the production of the chemokine CCL20 and that CCL20 stimulates the proliferation and adhesion to collagen of various tumor cells. Furthermore, overexpression of CCL20 in tumor cells promotes growth and adhesion in vitro and increased tumor growth and invasiveness in vivo. Moreover, neutralizing antibodies to CCL20 inhibit the in vivo growth of tumors that either overexpress CXCR4 or CCL20 or naturally express CCL20. These results reveal a role for CCL20 in CXCR4-dependent and -independent tumor growth and suggest a therapeutic potential for CCL20 and CCR6 antagonists in the treatment of CXCR4- and CCL20-dependent malignancies. Citation: Beider K, Abraham M, Begin M, Wald H, Weiss ID, et al. (2009) Interaction between CXCR4 and CCL20 Pathways Regulates Tumor Growth. PLoS ONE 4(4): e5125. doi:10.1371/journal.pone.0005125 Editor: Neil Hotchin, University of Birmingham, United Kingdom Received October 20, 2008; Accepted March 2, 2009; Published April 2, 2009 Copyright: 2009 Beider et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Prostate Cancer Fund (PCF), USA, and the Israeli Science Foundation (ISF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: arnon.nagler@sheba.health.gov.il (AN); peled@hadassah.org.il (AP) Introduction Chemokines, a family of small (5–20 kDa) pro-inflammatory cytokines, are primarily responsible for the directional migration, or chemotaxis, of lymphocytes to specific lymphoid tissues, and the recruitment of leukocytes to the sites of infection or tissue damage. However, chemokines are also implicated in other biological events including angiogenesis, angiostasis, embryogenesis, hema- topoiesis, lymphopoiesis, and HIV pathogenesis [1]. More recently, it has been established that cancer cells exploit signaling through chemokine receptors via several key steps involved in initiation and progression of primary and metastatic cancer. Different types of cancers express different chemokine receptors [2,3], however, only the chemokine receptor CXCR4 appears to be expressed by the majority of cancer types. Tumor cells from at least 23 different types of cancers of epithelial, mesenchymal and haematopoietic origin express CXCR4 [4]. Moreover, CXCR4 expression was found to be increased in several malignancies including gliomas, breast tumors, certain leukemia cell lines, uterine cancer, Burkitt’s lymphoma, neuroblastomas, and pancre- atic cancer [5,6,7,8,9,10]. CXCR4 was also found to play a critical role in the progression and development of various tumors including breast, prostate and clear cell renal carcinoma [6,11,12,13]. The importance of the CXCR4/CXCL12 pathway in tumor development was further demonstrated by neutralizing the interaction between CXCL12 and CXCR4. [6,14,15,16]. The mechanisms by which CXCR4 regulate tumor progression are yet not clear. Our recent results provide a novel mechanism for CXCR4-mediated tumor growth and metastasis and establish a functional link between CXCR4/CXCL12 and CCR6/CCL20 pathways in tumor development. Results CXCR4 up-regulates CCL20 mRNA and protein expression in prostate cancer cells In our previous work, we provided the evidence that the CXCR4 receptor promotes prostate tumor growth, invasion and vascularization [17]. In this study, we generated single-cell clones of CXCR4-transduced PC3 cells with high and stable levels of CXCR4 expression (Fig. 1A). CXCR4-transduced PC3 cells with high and stable levels of CXCR4 expression demonstrated a high proliferation rate in vitro in response to stimulation with CXCL12, and increased sensitivity to low concentrations of CXCL12, compared to wild-type PC3 cells (Fig. 1A). Moreover, mice injected subcutaneously with these cells developed bigger tumors compared to mice injected with wild-type PC3 cells, similarly to our previous results (Fig. 1B,C). In order to identify genes that are regulated by CXCR4 and contribute to the aggressive phenotype of CXCR4-expressing prostate cancer, we compared the expression pattern of various chemokines between CXCL12-stimulated and non-stimulated PC3-CXCR4.5 cells. We found an increase in expression of the chemokine CCL20 following stimulation with CXCL12. To PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5125