The atypical chemokine receptor D6 suppresses the development of chemically induced skin tumors

Abstract

A subset of CC chemokines, acting through CC chemokine receptors (CCRs) 1 to 5, is instrumental in shaping inflammatory responses. Recently, we and others have demonstrated that the atypical chemokine receptor D6 actively sequesters and destroys many of these proinflammatory CC chemokines. This is critical for effective resolution of inflammation in vivo. Inflammation can be protumorigenic, and proinflammatory CC chemokines have been linked with various aspects of cancer biology, yet there is scant evidence supporting a critical role for these molecules in de novo tumor formation. Here, we show that D6-deficient mice have increased susceptibility to cutaneous tumor development in response to chemical carcinogenesis protocols and, remarkably, that D6 deletion is sufficient to make resistant mouse strains susceptible to invasive squamous cell carcinoma. Conversely, transgenic D6 expression in keratinocytes dampens cutaneous inflammation and can confer considerable protection from tumor formation in susceptible backgrounds. Tumor susceptibility consistently correlated with the level of recruitment of T cells and mast cells, cell types known to support the development of skin tumors in mice. These data demonstrate the importance of proinflammatory CC chemokines in de novo tumorigenesis and reveal chemokine sequestration by D6 to be a novel and effective method of tumor suppression.

Figure 1. D6 deficiency confers susceptibility to skin tumors in B6/129 mice.

(A and B) Fifteen DMBA-treated D6-deficient (KO) or WT mice received TPA for 24 weeks according to protocols shown. Total papillomas and papillomas greater than 5 mm in diameter were counted once a week. Fraction of papilloma-free mice (left) and mean tumor burden per mouse (right) are shown. Log-rank tests were applied to data in left panel. See Results for results of statistical tests on tumor burden data. (C–F) D6-deficient and WT mice were treated with TPA on days 1, 5, and 10, and skin was harvested 2 or 4 days later and scored for skin thickness (dermis plus epidermis) (C), Ki67⁺ keratinocytes (basal and suprabasal layers) (D), epidermal CD3⁺ T cells (E), and MCs (F). Dorsal skin of “carrier” control mice was harvested 2 days after application of carrier (acetone) and similarly assessed. (C, E, and F) A total of 25–40 data points collected from stained sections from 5 TPA-treated mice or 4–5 carrier controls of each genotype per time point. (D) The number of Ki67⁺ cells per millimeter was determined by counting 5 randomly selected 400-μm regions of epidermis from stained sections from 5 TPA-treated mice or 4–5 carrier controls of each genotype per time point. Data are representative of 3 repeated experiments and were analyzed using a 2-tailed unpaired Student’s t test. (G) Seven D6-deficient and WT mice were treated with TPA on days 1, 5, and 10, skin was harvested 2 days later, chemokine concentrations in eluates were determined using Luminex technology, and data were analyzed using a 2-tailed unpaired Student’s t test. A repeat experiment generated a similar result. hpf, high-powered field. *P < 0.001; ^†P < 0.01.

Figure 2. Deletion of D6 increases tumor burden in DMBA/TPA-treated FVB/N mice.

(A–D) D6-deficient or WT FVB/N mice were treated on 3 consecutive days with TPA, and skin was harvested 2 or 4 days later. Skin was also prepared from shaved carrier mice 2 days after receiving carrier (acetone) only. After staining, sections were scored for skin thickness (dermis plus epidermis) (A), Ki67⁺ keratinocytes (basal and suprabasal layers) (B), epidermal CD3⁺ T cells (C), and MCs (D). (A, C, and D) 25–40 data points were collected from stained sections from 5 TPA-treated mice or 4–5 carrier controls of each genotype per time point. (B) The number of Ki67⁺ keratinocytes/mm was determined by counting 5 randomly selected 400-μm regions of epidermis from stained sections from 5 TPA-treated mice or 4–5 carrier controls of each genotype per time point. Data were analyzed using a 2-tailed unpaired Student’s t test. Two repeat experiments generated similar data. (E) Fifteen DMBA-treated D6-deficient or WT mice received TPA twice a week for 20 weeks. Total papillomas and papillomas greater than 5 mm in diameter were counted once a week. Fraction of papilloma-free mice (left) and mean tumor burden per mouse (right) are shown. Log-rank tests were used to analyze data in the left panels. See Results for results of statistical tests on tumor burden data. *P < 0.001; ^†P < 0.01.

Figure 3. Transgenic expression of D6 in the epidermis of FVB/N mice dampens cutaneous inflammation and suppresses papilloma formation.

(A) RNA from epidermal sheets from dorsal skin of WT and K14-D6 mice was subjected to RT-PCR (with or without reverse transcriptase [RT]) for mouse D6 or actin. Products were electrophoresed on an agarose gel and visualized with ethidium bromide under UV light. (B) Keratinocytes (2.5 × 10⁵) from neonatal K14-D6 or WT mice were cultured in 1 ml of medium containing 10 nM BioCCL3, and remaining BioCCL3 was detected by western blotting at the times indicated. Data are representative of 3 repeated experiments. (C–E) WT and K14-D6 mice (n = 5 per group) received TPA on 3 consecutive days, and dorsal skin was harvested 4 days later. Untreated mice of each genotype (n = 5/group) were shaved and dorsal skin immediately harvested. Processed sections were scored for skin thickness (dermis plus epidermis) (C), epidermal CD3⁺ T cells (D), and MCs (E). A total of 25–40 data points were collected for each parameter from sections from 5 mice of each genotype. Data were analyzed using a 2-tailed unpaired Student’s t test. A repeat experiment generated similar data. (F) Fifteen K14-D6 mice and 17 WT counterparts received DMBA, and then TPA on days 1, 5, and 10 every 2 weeks for 20 weeks. Papillomas greater than 5 mm in diameter were counted once a week. The fraction of papilloma-free mice (left) and the mean tumor burden per mouse (right) are shown. Log-rank tests were applied to the data in the left panel. See Results for results of statistical tests on tumor burden data. *P < 0.001.

Figure 4. The K14-D6 transgene can be overwhelmed by increasing the frequency of TPA application and does not affect conversion of papillomas to SCC.

DMBA-treated K14-D6 or WT mice received TPA for 20 weeks either 2 times per week (A; 13 mice per genotype) or 3 times per week (B and C; 29 mice per genotype). (A and B) Papillomas greater than 5 mm in diameter were counted once a week. The fraction of papilloma-free mice (left) and mean tumor burden per mouse (right) are shown. (C) Mice used to generate data shown in B were followed for 45 weeks and assessed every week for SCC formation. Tumors that had the morphological appearance of SCCs were processed for histology and scored as SCCs when invasion into the dermis was detectable. Log-rank tests were applied to data in the left panels. See Results for results of statistical tests on tumor burden data.