CCL20 expression has been described in a variety of human neoplasms, including colorectal, lung, pancreatic and breast human adenocarcinomas, malignant glioma, leukemia, lymphoma and melanoma However, the role of CCL20 in cancer development remains controversial. Since CCL20 is a potent chemoattractant for immature DCs, the most powerful antigen- presenting cells, it seems logical to attract iDCs to the tumor site to induce antitumor immune response. According to this approach, Fushimi et al., demonstrated in a mouse model that intratumor injection of adenovirus-mediated gene transfer of CCL20 could suppress tumor growth [35]. On the other hand, there is a growing body of evidence supporting the hypothesis that CCL20 production by cancer cells promotes tumor growth and invasive- ness. Contrary to the results of Fushimi et al., other groups of investigators have shown that transfection of rodent tumor cell line with CCL20 enhances tumor growth and decreases immunoge- nicity, despite the attraction of iDCs to the tumors [36]. In their model, as in human breast carcinomas which secrete high levels of CCL20 [37], attracted DCs to the tumor site did not mature. These may serve as one of the tumor escape mechanisms by which attracted immature DCs poorly stimulate T-cell antitumor response, and moreover can activate regulatory T cells which suppress specific anti-tumor immune responses. In our experimental models, we chose to evaluate the involvement of CCL20 in prostate and colon tumor growth, vascularization and invasiveness. Our results match the observa- tions of Kleef et al., who demonstrated that CCL20 is overexpressed in human pancreatic carcinoma cells and in infiltrating macro- phages adjacent to tumors, and stimulates the growth and invasion of neoplastic cells [38]. Up- regulation of CCL20 was demonstrated in human hepatocellular carcinoma tissues, and CCL20 expression was found to correlate with tumor grade [39]. Furthermore, we found that CXCR4, CCL20 and CCR6 are over- and coexpressed in human prostate cancer specimens (Fig. 5). In support of our findings it was recently published that the expression levels of CCR6 in prostate cancer are associated with clinical and pathologic features of more advanced and aggressive prostate cancer [40]. Here we show that, CCL20 promotes the growth and adhesion of CCR6-expressing cancer cell lines. Moreover, overexpression of CCL20 or CXCR4 in prostate and colon cancer cells promotes tumor growth that was neutralized with anti-CCL20 antibodies. These findings strongly emphasize the potential co-involvement of CCL20 and CXCR4 in cancer development. Altogether, our findings provide evidence for the existence of a putative CXCR4/CCL20 interaction that could be involved in cancer development processes. Furthermore, our data suggest a 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. Materials and Methods Cell culture and transduction of cell lines and cell clones The following human cell lines were used in the study: prostate cell lines PC3 (CRL-1435), LNCaP (CRL-10995), 22Rv1 (CRL- 2505), DU145 (HTB-81); acute promyelocytic leukemia cell lines NB4, HL-60 (CCL-240) and colon carcinoma cell line HT-29 (HTB-38). PC3 cell line stably overexpressing CCL20 (PC3- CCL20) was produced by transfection of PC3 cells pcDNA3- CCL20 construct. PC3-CCL20 single-cell clones were produced by limited dilutions. The level of secreted CCL20 protein was analyzed in supernatant of PC3-CCL20 clones using ELISA kit (R&D Systems, Minneapolis, MN). PC3-CXCR4 single-cell clones were produced by limited dilutions. CCL20 and CXCR4 Interaction RNA extraction and reverse transcription ad Semi- quantitative and Real-Time PCR Total RNA was extracted from prostate and leukemic cell lines using TRIzol reagent (Invitrogen Life Technologies). For cDNA synthesis, 2.5 microgram of total RNA were reverse-transcribed and the following primer pairs were used for PCR: b-actin sense 59 - CCCTGGACTTCGAGCAAGAG9 - 39, antisense 59 - TCTCCT- TCTGCATCCTGTCG - 39; CCL20 sense 59 – ATGTGCTGTA- CCAAGAGTTT - 39, antisense 59 - CAAGTCTGTTTTGGAT- TTGC - 39; CCR6 sense 59 – CCATTCTGGGCAGTGAGTCA - 39, antisense 59 - AGCAGCATCCCGCAGTTAA - 39; CXCR4 sense 59 - AGCTGTTGGCTGAAAAGGTGGTCTATG - 39, antisense 59 - GCGCTTCTGGTGGCCCTTGGAGTGTG - 39; CXCL12 sense 59 - ATGAACGCCAAGGTCGTGGTCG - 39, antisense 59 - TGTTGTTGTTCTTCAGCCG - 39. CCL20 quantitative PCR assay containing the primers and probe mix was purchased from Applied Biosystems, Foster City, CA, All reactions were run in triplicates using ABI Prism 7700 Sequence Detector System (Applied Biosystems). Gene expression of CCL20 gene was analyzed in relation to the levels of the housekeeping b-actin gene. Cell proliferation assay The effect of CCL20 on the viability of PC3, PC3-CXCR4.5, PC3-CCL20 clones and leukemia NB4 and HL60 cells was studied. In brief, PC3 and PC3-CXCR4.5 cells were seeded at 26104 cells/ 1 ml per well into a 24-well plate in medium supplemented with 0.1% FCS with or without various concentrations of CCL20 (PeproTech EC, London, UK). Following three days, the medium with or without CCL20 was renewed. On day six, the attached cells were harvested, stained with propidium iodide (Sigma, St. Louis, MO), and the number of viable cells was determined using FACS analysis. Optionally, PC3 and PC3-CXCR4.5 cells were labeled with 5-bromo-2-deoxyuridine (BrdU) (Sigma, St. Louis, MO) at a concentration of 10 mM during the last 16 hours of incubation and processed for BrdU detection using specific anti-BrdU antibody (eBioscience) and FACS analysis. Cell adhesion assay Prostate cancer cells (PC3, PC3-CXCR4.5 and PC3-CCL20 clones), leukemia NB4 and HL60 cells, and colon cancer cells HT- 29 (16105/500 ml) were allowed to adhere to 10 mg/ml fibronectin- coated or collagen type I-coated 24-well plates for 30 minutes at 37uC in serum-free RPMI supplemented with 0.1% bovine serum albumin (BSA). Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 ml FACS buffer (PBS6 1+0.1% BSA+0.01% NaNO3) with 5 mM EDTA and counted by FACScalibur (Becton Dickinson Immunocytometry Systems). ELISA assay Prostate cancer cells (PC3, PC3-CXCR4.5 and PC3-CCL20 clones), leukemia cells NB4 and HL60 and colon cancer cells HT-29 were seeded into a 12-well plate at 26105/1 ml of medium per well with various concentrations of CXCL12 (5–1,000 ng/ml) (Pepro- Tech EC, London, UK) or PTX (5 mg/ml) (List Biological Laboratories, Campbell, CA, USA). The cells were incubated for 48 hours, supernatants were collected and CCL20 protein levels were determined using ELISA kit (R&D Systems, Minneapolis, MN). For intracellular CCL20 detection, cells were harvested and lysed with lysis buffer containing 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM EDTA pH 8.0, 0.5% NP40 and protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany). Cell lysates were centrifuged at 14,000 g for 20 minutes at 4uC and equal amounts of protein extracts were applied for ELISA analysis. PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5125