Involvement of CCR6/CCL20/IL-17 axis in NSCLC disease progression

Abstract

Objectives: Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.

Methods: A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.

Results: CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52-15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.

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.

Figure 1. Expression of CCR6/CCL20 in NSCLC tissue samples.

PCR signal for CCL20, CCR6 and beta-actin in three NSCLC tumor samples (A). CCL20 protein levels in NSCLC tumors and in tumor adjacent lung tissue (n = 3) (B). Representative immunohistochemistry staining for CCL20 and CCR6 in lung adenocarcinoma tissue sections. CCL20 - Low X10 (D) and high power X20 (E) magnification. CCR6 - Low power (X10) (F, G) and high power (X20) (H) magnification. Immune cell infiltrates located with in lung adenocarcinoma tumor stain positive for CCR6 (X40) (I). Negative control staining (X10) is shown (C). (*P<0.05)

Figure 2. CCR6/CCL20 expression and NSCLC disease progression.

Kaplan-Meier analysis of disease free survival interval (months) according to disease stage. (A). Kaplan-Meier analysis of disease free survival interval (months) according to percentage of CCR6 positive cells. (B). CCR6 staining index for lung adenocarcinoma and squamous cell carcinoma according to disease stage is shown (C).

Figure 3. Expression of CCR6/CCL20 in NSCLC-derived cell lines.

PCR signal for CCL20, CCR6 and beta-actin in L3, L4 and A549 cell lines (A). ELISA assay for CCL20 in the supernatant of L3, L4 and A549 cell lines (B). Flow Cytometry histogram analysis of CCR6 and CCL20 staining of L3, L4 and A549 cell lines, control antibody – purple, staining antibody – green line (C).

Figure 4. CCL20 induces NSCLC proliferation and ERK phosphorylation.

Colony formation by L3, L4 and A549 cells in response to stimulation with increasing concentrations of CCL20 (A, B, C). ERK phosphorylation in L3, L4 and A549 cells in response to CCL20 stimulation. Phosphorylated ERK upper panel and total ERK lower panel (D, E, F). (*P<0.05, **P<0.01)

Figure 5. ERK inhibition decreases CCL20-mediated NSCLC proliferation.

Colony formation by L3, L4 and A549 cells in response to stimulation with increasing concentrations of CCL20 in the presence or absence of the ERK inhibitor PD98059 (A, B, C).

Figure 6. IL-17 is expressed in NSCLC and induces CCL20 production by NSCLC-derived cell lines.

PCR signal for IL-17A, IL-17A receptor and b-actin in NSCLC tissue samples, in tumor adjacent lung tissue and in L3, L4 and A549 cell lines. Tumor adjacent lung tissue (A). NSCLC tumor sample (B). L3, L4 and A549 cell lines (C). Infiltrating immune cells were isolated from tumor tissue (TD-IC) and tumor adjacent lung tissue (LD-IC). Immune cells were incubated with or without anti CD3 antibody and production of IL-17 assessed by ELISA - two cases (D, E). Semi-quantitative PCR analysis for CCL20 in L3, L4 cells 549 cells in response to IL-17 and IL-22 stimulation (F, G, H). ELISA assays for CCL20 in the supernatant of L3, L4 cells 549 cells following stimulation with increasing concentrations of IL-17 (I, J, K).