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, 37 (1), 293

Inhibition of Skin Carcinogenesis by Suppression of NF-κB Dependent ITGAV and TIMP-1 Expression in IL-32γ Overexpressed Condition

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Inhibition of Skin Carcinogenesis by Suppression of NF-κB Dependent ITGAV and TIMP-1 Expression in IL-32γ Overexpressed Condition

Yong Sun Lee et al. J Exp Clin Cancer Res.

Abstract

Background: Interleukin-32 (IL-32) has been associated with various diseases. Previous studies have shown that IL-32 inhibited the development of several tumors. However, the role of IL-32γ, an isotype of IL-32, in skin carcinogenesis remains unknown.

Methods: We compared 7,12-Dimethylbenz[a]anthracene/12-O-Tetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis in wild type (WT) and IL-32γ-overexpressing mice to evaluate the role of IL-32γ. We also analyzed cancer stemness and NF-κB signaling in skin cancer cell lines with or without IL-32γ expression by western blotting, quantitative real-time PCR and immunohistochemistry analysis.

Results: Carcinogen-induced tumor incidence in IL-32γ mice was significantly reduced in comparison to that in WT mice. Infiltration of inflammatory cells and the expression levels of pro-inflammatory mediators were decreased in the skin tumor tissues of IL-32γ mice compared with WT mice. Using a genome-wide association study analysis, we found that IL-32 was associated with integrin αV (ITGAV) and tissue inhibitor of metalloproteinase-1 (TIMP-1), which are critical factor for skin carcinogenesis. Reduced expression of ITGAV and TIMP-1 were identified in DMBA/TPA-induced skin tissues of IL-32γ mice compared to that in WT mice. NF-κB activity was also reduced in DMBA/TPA-induced skin tissues of IL-32γ mice. IL-32γ decreased cancer cell sphere formation and expression of stem cell markers, and increased chemotherapy-induced cancer cell death. IL-32γ also downregulated expression of ITGAV and TIMP-1, accompanied with the inhibition of NF-κB activity. In addition, IL-32γ expression with NF-κB inhibitor treatment further reduced skin inflammation, epidermal hyperplasia, and cancer cell sphere formation and downregulated expression levels of ITGAV and TIMP-1.

Conclusions: These findings indicated that IL-32γ suppressed skin carcinogenesis through the inhibition of both stemness and the inflammatory tumor microenvironment by the downregulation of TIMP-1 and ITGAV via inactivation of NF-κB signaling.

Keywords: IL-32γ; ITGAV; NF-κB; Skin tumor development; TIMP-1.

Conflict of interest statement

Ethics approval and consent to participate

The experimental protocols were carried out according to the guidelines for animal experiments of the Institutional Animal Care and Use Committee (IACUC) of Laboratory Animal Research Center at Chungbuk National University, Korea (CBNUA-1146-18-01).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Effect of IL-32γ on skin tumor development. a Representative images of WT and IL-32γ mice with skin papillomas. b Average number of papillomas per mouse in WT and IL-32γ mice following TPA treatment. n = 8. *p < 0.05. c Tumor incidence, showing the percentage of tumor-bearing mice at time course. n = 8. d hematoxylin and eosin (H&E) staining of skin sections and epidermal thickness in WT and IL-32γ mice. n = 8. *p < 0.05. e PCNA staining of skin sections in WT and IL-32γ mice. Scale bar, 10 μm
Fig. 2
Fig. 2
IL-32γ effects on DMBA/TPA-induced local inflammation and inflammatory cell infiltration. WT and IL-32γ mice were treated with DMBA/TPA for 25 weeks. a Real-time PCR analysis of different inflammatory mediators, TNF-α, IL-1β, IL-6, IL-4, IL-10, IL-13, CXCL1, CXCL2, S100A8 and S100A9, on mRNA isolated from skin tissue extracts. n = 5. *p < 0.05; **p < 0.01; ***p < 0.001. b Representative immunohistochemistry images showing Ly6G+ (granulocytes), CD11b + (monocytes/phagocytes) and F4/80+ (macrophages) cells in the skin sections of WT and IL-32γ mice. Ly6G, CD11b and F4/80 stainings were quantified by counting the number of positive cells in the field. Scale bar, 10 μm. n = 5. c Real-time PCR analysis of mRNA expression of Ly6G, CD11b and F4/80. n = 6. *p < 0.05; **p < 0.01. d Production of PGE2 in the skin tissues measured by ELISA and mRNA expression of mPGES-1 measured by real-time PCR. n = 6. *p < 0.05
Fig. 3
Fig. 3
IL-32γ suppresses skin cancer stemness. a and b Effect of IL-32γ on the expression of CD44 and CD133 in skin tumor by western blotting (a) and immunohistochemical analysis (b). Scale bar, 10 μm. n = 4. c Effects of IL-32γ on skin cancer sphere formation. Control and IL-32γ-overexpressing A431 and SK-Mel-28 cells were subject to sphere assay for 10 days. Representative images (top) of skin cancer spheres are shown. n = 3. *p < 0.05. d Expression of CD44 and CD133 were detected in A431 and SK-Mel-28 skin CSCs by western blotting. e Expression of Sox2 was analyzed by real-time PCR. n = 3. *p < 0.05. f Effects of IL-32γ on skin cancer chemotherapy. Skin cancer cells, A431 and SK-Mel-28, were cultured with 5-FU for 24 h. The cell viability was determined by MTT assay. n = 3. *p < 0.05
Fig. 4
Fig. 4
ITGAV and TIMP-1 are associated with inhibition of IL-32γ-induced cancer stemness. a, b Western blotting (a) and immunohistochemical (b) analysis of ITGAV and TIMP-1 in DMBA/TPA-induced skin tissues from WT and IL-32γ. Scale bar, 10 μm. n = 4. c Western blotting analysis of ITGAV and TIMP-1 in control and IL-32γ-overexpressing cells (A431 and SK-Mel-28). d Knockdown of ITGAV and TIMP-1 on skin cancer sphere formation. A431 and SK-Mel-28 cells were transfected with ITGAV or TIMP-1 siRNA (20 nM). After 24 h, cells were subject to sphere assay for 10 days. n = 3. *p < 0.05. e, A431 and SK-Mel-28 cells were transfected with ITGAV or TIMP-1 siRNA. Expression of CD44 and CD133 were detected in ITGAV and TIMP-1 knockdonwed A431 and SK-Mel-28 cells by western blotting. f Expression of Sox2 mRNA was analyzed by real-time PCR. n = 3. *p < 0.05
Fig. 5
Fig. 5
IL-32γ decreases NF-κB activity in tumor tissues and skin CSCs. a and b Expression of phosphorylated IKKα/β in cytosol extracts and nuclear translocation of p50 and p65 in the nuclear extracts of DMBA/TPA-induced skin tissues or A431 and SK-Mel-28 CSCs were determined by western blotting. c Expression of p50 and p65 in DMBA/TPA-induced skin tissues or A431 and SK-Mel-28 CSCs were analyzed by immunohistochemistry. Scale bar, 10 μm. n = 4. d DNA-binding activity of NF-κB was determined by EMSA in nuclear extracts of A431 and SK-Mel-28 CSCs
Fig. 6
Fig. 6
Inhibition of NF-κB activity suppresses cancer stemness and skin inflammation. a Effects of BAY on TPA-induced epidermal hyperplasia. WT and IL-32γ mice were topically administrated with BAY and TPA application and then sacrificed after 24 h. Dorsal skin tissues were analyzed by H&E staining. Representative images of skin tissues are shown. The bar graph shows the average epidermal thickness of mice in each group. Scale bar, 10 μm. n = 3. *p < 0.05; #p < 0.05. b Western blotting of ITGAV, TIMP-1 and S100A8 in cytosolic extracts and p65 in nuclear extracts of skin tissues from single TPA-treated with BAY mice are shown. c Effects of NF-κB inhibitor Bay 11–7082 (BAY) on skin cancer cell sphere formation. Control and IL-32γ-overexpressing A431 and SK-Mel-28 cells were subject to sphere assay in the presence of Bay (5 μM) for 10 days. d Expression of CD44, CD133, ITGAV and TIMP-1 in A431 CSCs. Control and IL-32γ-overexpressing A431 CSCs were treated with BAY (5 μM) for 24 h. Protein levels of CD44, CD133, ITGAV and TIMP-1 were detected by western blotting
Fig. 7
Fig. 7
IL-32, ITGAV and p65 expressions in the progression of human skin cancer patients. Tissue microarray analysis showing the expression of IL-32 (top), ITGAV (middle) and p65 (bottom) during skin tumor progression in normal, clinical stage I, II and III-IV. a Representative immunohistochemical images of each groups. b Bar graphs showing the ratio of IL-32, ITGAV and p65 expressions scored. Tissue microarray contained of 10 samples from normal skin tissues, 16 samples from stage I, 48 samples from stage II and 5 samples from stage III-IV. *p < 0.05

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