Possible Roles of Proinflammatory Signaling in Keratinocytes Through Aryl Hydrocarbon Receptor Ligands for the Development of Squamous Cell Carcinoma

Abstract

Aryl hydrocarbon receptor (AhR) provides a deeper insight into the pathogenesis of cutaneous squamous cell carcinoma (cSCC). AhR ligands, such as 6-formylindolo[3,2-b] carbazole (FICZ), and 7,12-Dimethylbenz[a]anthracene (DMBA), constitute major substrates for the cytochrome P450 (CYP) family, and influence the expression of various cytokine genes, including IL-17 and IL-23-related genes via the AhR. On the other hand, proinflammatory cytokines could drive tumor progression through the TRAF-ERK5 signaling pathway in cSCC. From the above findings, we hypothesized that AhR ligands might enhance the mRNA expression of proinflammatory cytokines via the AhR, leading to the development of cSCC. The purpose of this study was to investigate (1) the immunomodulatory effects of FICZ and DMBA on normal human keratinocytes (NHKCs), focusing on IL-17, and related cytokines/chemokines (IL-23, IL-36γ, and CCL20), (2) the expression of these factors in AhR-dependent pathways using a two-stage chemically induced skin carcinogenesis mouse model, and (3) the expression of these factors in lesion-affected skin in cSCC. Both FICZ and DMBA augmented the expression of CYP1A1, p19, CCL20, and IL-36γ mRNA in NHKCs in vitro. Moreover, the mRNA expression of these proinflammatory factors, as well as IL-17, in mouse cSCC is significantly decreased in the AhR-(fl/fl) Krt5-(Cre) mice compared to wild type mice, leading to a decrease in the number of developed cSCC lesions. Furthermore, CCL20, IL-23, as well as IL-17, are detected in the lesion-affected skin of cSCC patients. Our study demonstrates a possible mechanism for the development of cSCC involving AhR-mediated signaling by epidermal keratinocytes and recruitment of Th17 cells.

Keywords: IL-17; aryl hydrocarbon receptor; carcinogenesis; cutaneous SCC; proinflammatory cytokines.

FIGURE 1

Immunohistochemical analysis of CYP1A1 expression and AhR expression in lesion-affected skin of AK and cSCC. Sections of skin from lesion-affected areas of AK (A,B,G), cSCC (C,D,H), marginal areas around cSCC lesions (E,I), or normal skin ([F]; nevus pigmentosus located at back) were deparaffinized and stained using anti-CYP1A1 antibodies (A–F), or AhR antibodies (G–I). The sections were developed with liquid permanent red. Scale bars, 100 μm (B,D–F), 200 μm (A,C,G–I). Representative specimens from analyses of 5 cases of actinic keratosis, 12 cases of cSCC, and 10 cases of nevus pigmentosus are shown.

FIGURE 2

Expression of CYP1A1, cytokine, and chemokine mRNA in normal human keratinocytes (NHKCs) stimulated with FICZ, or DMBA. NHKCs were cultured and treated with FICZ (10 nM), or DMBA (1 μM) as described in the Materials and Methods. At 4 h after stimulation, total RNA was recovered from NHKCs and amplified, labeled, and analyzed. Quantitative real-time PCR was conducted to determine the number of cDNA copies for each factor, and the mRNA expression level relative to that of untreated cells was calculated for each gene and time point after normalization against glyceraldehyde 3-phosphate dehydrogenase using the ΔΔCt method (A). NHKC culture supernatant was harvested as described in the Materials and Methods and analyzed by ELISA (B). Data from each donor were obtained from triplicate assays, and the mean ± SD was calculated. The means of at least three independent experiments are shown. IL-23 and IL-36β production was analyzed by western blotting as described in the Materials and Methods (C). *p < 0.05, **p < 0.01.

FIGURE 3

AhR dependency in two-stage chemically induced skin carcinogenesis mouse model. Mouse cSCC in each mouse was induced as described in the Materials and Methods. The schematic representation of model of carcinogenesis (A). The representative figure of DMBA-induced tumor in 5 wild type (AhR+/Krt5+) mouse and 5 AhR-(fl/fl) Krt5-(Cre) mouse (B). The number of developed tumors were counted by two independent researchers, and the average number of induced tumors is calculated (B). Expression of chemokines and cytokines in DMBA-induced tumor was analyzed by quantitative RT-PCR using the ΔΔCt method (n = 5). The data from each donor were obtained by triplicate assays, and then the mean ± SD was calculated (C). *Marks a significant (p < 0.05) difference. **Marks a significant (p < 0.01) difference.

FIGURE 4

Immunohistochemical analysis of CCL20, IL-23, IL-17, IL-36γ, and IL-17R expression in lesion-affected skin of cSCC. Sections of cSCC lesions were deparaffinized and stained using anti-CCL20 (a–c), anti–IL-23 (d–f), anti–IL-17 (g,h), anti–IL-36γ (i,j), or anti–IL-17R (k,l) antibodies. Sections were developed with liquid permanent red. Scale bars, 100 μm. Representative specimens from 12 cases of cSCC are shown. Scale bars, 50 μm (b,i), 100 μm (c,e–l), and 200 μm (a,d).