Chemokine receptor expression by tumor cells enhances their survival, proliferation and metastatic potential. Both tumor cells and tumor stroma produce chemokines 
. Several groups examined the role of the chemokine/chemokine receptor pair, CCL20/CCR6, in malignancy. Pirus Ghadjar et al., have shown that CCR6 expression guide hepatic metastasis of colonic malignancies 
. Kimsey et al., reported that co-localization of CCL20 and CCR6 promotes pancreatic cancer cell invasion 
. Others and we have shown that CCL20/CCR6 auto-signaling occurs in prostatic cancerous cells and that tumor CCR6 expression correlates with disease aggressiveness 
, thus marking intra-tumoral CCL20/CCR6 interactions as a pro-carcinogenic axis. CCL20 is over-expressed by lung epithelial cells in response to smoke and particulate matter 
. The role of this chemokine in lung cancer pathogenesis has not been studied thus far. To test the potential pro-carcinogenic effects of CCL20 and CCR6 in lung cancer, we first aimed to characterize the expression and tissue localization of this chemokine/chemokine receptor pair in NSCLC tumors. Immunohistochemistry studies indicated that the majority of lung adenocarcinoma tumor samples highly stained for CCL20, while only a minority highly expressed CCR6. Using Kaplan-Meier analysis, we looked for associations between the clinical course of lung adenocarcinoma patients and the extent of CCL20/CCR6 staining in their tumor samples. We found that high CCR6 expression was associated with a shorter disease-free survival (P
0.008). Furthermore, Cox regression analyses showed that high CCR6 expression was associated with a 4.87-fold increased risk for disease recurrence (P
0.0076, CI 95% 1.52–15.563) and the effect of CCR6 was independent of pathological stage of disease. The strength of this study lies on focusing on an accurate pathological staging, a specific diagnosis (lung adenocarcinoma) and the long duration of clinical follow-up. The weakness of this study is its relatively small sample size (49 patients). In order to expand the sampled data, we performed an additional analysis of CCR6 expression using a tumor tissue array enclosing over 50 lung-adenocarcinoma samples homogenously spread among the different disease stages. In this array, we found a higher CCR6 staining index among advanced stage adenocarcinomas. This pattern was unique for adenocarcinoma, albeit not detected in squamous cell tumors. Shedden K. et al., have extensively examined the gene expression-based survival of lung adenocarcinoma patients 
. Their data, containing clinical parameters and gene profiling of over 400 samples from lung adenocarcinoma patients, is available for download from the NCBI. We examined the expression patterns of CCL20 and CCR6 in this database. The reported gene array signal detection was low for CCR6, ranging between 4 to 115 and high for CCL20, ranging from 2–5,597. In contrast to the immunohistochemistry results described above we have not found a correlation between CCR6 gene array signal intensities and lung adenocarcinoma disease parameters. The different methods of detection, degradation of CCR6 mRNA and reduced sensitivity of the specific probe for CCR6 may account for this discrepancy. Interestingly however, we have found highly significant correlations between CCL20 signal intensities and three clinical parameters: 1) Vital status of patients (Alive – 535+/−58 vs. dead – 786 +/− 72, P
0.008), 2) Tumor differentiation (Well differentiated – 334+/−83 vs. poorly differentiated – 793 +/− 88, P
0.004) and 3) T score of TNM staging system (T-1 – 552+/−68 vs. T-3/4 – 1055 +/− 207, P
0.005). Take together, this data suggest that CCL20/CCR6 interactions in the tumor microenvironment may stimulate NSCLC disease progression. CCR6 is identified as a potential disease marker. These observations add to the accumulating clinical data regarding the involvement of CCL20/CCR6 in malignancy and mark this axis as a potential therapeutic target.
To test in vitro the potential effects of CCL20/CCR6 interactions in NSCLC, three tumor cell lines were tested. L3, L4 and A549 cells expressed low levels of CCR6 and produced varying levels of CCL20. Stimulation of these lines with CCL20 induced colony formation in a dose-dependent manner. CCR6-dependent ERK phosphorylation mediated proliferation of colorectal cancer cells 
. Activation of ERK signaling was also considered as a key pathway in NSCLC development 
. We found that CCL20 induced ERK phosphorylation in L3, L4 and A549 cells. Furthermore, inhibition of ERK signaling by PD98059 decreased the potential of CCL20 to induce colony formation while it did not affect the basal numbers of colonies formed, thus demonstrating the pro-proliferative potential of CCL20/CCR6 auto-signaling in NSCLC and marking ERK as an intracellular mediator of these signals.
Autocrine and paracrine chemokines/chemokine receptor interactions link chronic inflammation to malignancy 
. CCL20 is a bi-functional peptide with both innate and adaptive immune properties. CCL20 is induced in human airway epithelia by various pro-inflammatory signals as well as by smoke and particulate matter 
. CCL20 is the only chemokine known to interact with the receptor CCR6 
. CCL20 promotes smoke- related lung inflammation by the recruitment of CCR6-expressing subset of immune cells into lung parenchyma 
. For example, chronic cigarette smoke exposure induced the accumulation of CD4+ CCR6+ Th17-type in mouse airways resulting in airspace enlargement 
. Numasaki et al. reported that NSCLC tumors contain infiltrates of CD3+ IL-17 expressing cells and suggested that the crosstalk among these cells and the neoplastic cells enhances local tumor progression 
. The trafficking of Th-17 cells is guided by CCR6 
. Here we show that intra-tumoral levels of CCL20 were elevated as compared to tumor adjacent lung tissue and that CCR6 positive immune cell infiltrates were found in the tumor parenchyma. PCR signal for IL-17 was detected in three out of four NSCLC tissue samples, however it was not found in any of the tumor cell lines and was only weakly expressed in two out of four tumor adjacent lung tissue samples. Activation of tumor infiltrating immune cells with anti CD3 antibody induced IL-17 production. In line with previous reports, these observations suggested that IL-17 producing cells infiltrate NSCLC tumors. All NSCLC tumors and cell lines expressed the IL-17 receptor. We found that stimulation of NSCLC cell lines with IL-17- induced CCL20 production in a dose-dependent manner. Immature dendritic cells, FoxP3 regulatory T cells and Th-17 cells all expressed CCR6 and were reported to accumulate in NSCLC tumors 
. We speculate that IL-17-induced CCL20 production in the tumor microenvironment may act in an autocrine manner to stimulate tumor cell growth and in a paracrine manner to enhance the tumor-associated inflammatory response.
In conclusion, our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.