CCL20 and CXCR4 Interaction Figure 5. CCL20, CCR6 and CXCR4 expression in prostate cancer cell lines, in primary prostate tumor tissue and in normal prostate tissue. (A) Expression of CCL20 and CCR6 in human prostate cancer tissue and in human normal prostate tissue. Immunohistostaining of prostate cancer and normal specimens using the polyclonal antibody for CCL20 and the monoclonal antibody 140706 for CCR6. Original magnification of 6400 is shown. CCL20 and CCR6 expression was observed in endothelial and fibromuscular cells of prostate samples (signed with black arrows). A commercially available array including 52 samples (CA2) from patients with prostate cancer was stained for CCL20 (A9) and CCR6 (A0). Expression was scored on two levels: low or negative expression, and high expression. Statistical analysis of the immunohistochemical staining was performed using two-tailed Mann-Whitney test. (B) Comnmon expression pattern of CCL20 and CXCR4 in human prostate cancer. Immunohistostaining of prostate cancer specimens using the polyclonal antibody for CCL20 and the monoclonal antibody 12G5 for CXCR4. Original magnification of 6400 is shown. CCL20 and CXCR4 expression was observed in endothelial and fibromuscular cells of prostate samples. (B9) A commercially available array including 48 samples (CA3) from patients with prostate cancer was stained for CCL20 and CXCR4. Expression was scored on three levels: low, intermediate, and high expression. Samples that demonstrated the same scoring in CCL20 and CXCR4 expression levels were classified as having similar expression pattern. Samples where scores in CCL20 and CXCR4 expression differed in one level or more were classified as different expression patterns. doi:10.1371/journal.pone.0005125.g005 only six samples out of 48 (12.5%) demonstrated different expression levels of CXCR4 and CCL20. These results suggest that in human prostate cancer CCR6, CCL20, and CXCR4 are commonly overexpressed and that there is a correlation between CXCR4 and CCL20 expression. More studies are needed to determine the potential diagnostic role for CCL20 and CCR6. Altogether, our findings for the first time provide evidence for existence of a putative CXCR4/CCL20 interaction that could be involved in cancer development processes. Furthermore, our data suggest an in vivo role of the chemokine CCL20 in CXCR4 dependent and independent regulation of cancer growth and point to CCR6 and CCL20 as novel therapeutic targets in cancer. Discussion Recent studies have revealed the critical multifunctional role of CXCR4-CXCL12 in cancer progression. Here we show that CXCR4 up-regulates the expression of the chemokine CCL20 in various cancer cells, and that CCL20 in turn promotes in an autocrine manner the survival, proliferation and adhesion of cancer cells in vitro and enhances xenograft tumor growth, vascularization and invasion in vivo. CCL20 (also known as macrophage inflammatory protein-3a (MIP3a); liver and activa- tion-regulated chemokine (LARC) or exodus-1) is a 9 kDa CC- type chemokine, which is expressed constitutively at low levels by keratinocytes in epidermal layers of skin [28] intestinal mucosa [29], liver [30], epithelial crypts of tonsils [31], as well as in the epithelium of Peyer’s patches in the intestine [32]. While there is redundancy in the human chemokine network, CCL20 is the unique chemokine ligand of its receptor CCR6. Expression of CCL20 is strongly enhanced by pro-inflammatory stimuli, such as IL-1b, TNF-a, and LPS [33,34]. CCL20 selectively attracts CCR6-expressing natural killer cells, memory T cells, TH17 cells, and immature dendritic cells (DC) to the sites of inflammation to encounter the invading pathogens. Recent studies suggested that CCL20 is an important player in the tumor chemokine network and similarly to other chemokines, CCL20 may promote cancer progression by two mechanisms: acting directly as a growth factor on cancer cells or establishing a microenvironment that suppresses specific anticancer responses. PLoS ONE | www.plosone.org 9 April 2009 | Volume 4 | Issue 4 | e5125