Loss of hairless confers susceptibility to UVB-induced tumorigenesis via disruption of NF-kappaB signaling - PubMed (original) (raw)

Loss of hairless confers susceptibility to UVB-induced tumorigenesis via disruption of NF-kappaB signaling

Hyunmi Kim et al. PLoS One. 2012.

Abstract

In order to model squamous cell carcinoma development in vivo, researchers have long preferred hairless mouse models such as SKH-1 mice that have traditionally been classified as 'wild-type' mice irrespective of the genetic factors underlying their hairless phenotype. The work presented here shows that mutations in the Hairless (Hr) gene not only result in the hairless phenotype of the SKH-1 and Hr(-/-) mouse lines but also cause aberrant activation of NFκB and its downstream effectors. We show that in the epidermis, Hr is an early UVB response gene that regulates NFκB activation and thereby controls cellular responses to irradiation. Therefore, when Hr expression is decreased in Hr mutant animals there is a corresponding increase in NFκB activity that is augmented by UVB irradiation. This constitutive activation of NFκB in the Hr mutant epidermis leads to the stimulation a large variety of downstream effectors including the cell cycle regulators cyclin D1 and cyclin E, the anti-apoptosis protein Bcl-2, and the pro-inflammatory protein Cox-2. Therefore, Hr loss results in a state of uncontrolled epidermal proliferation that promotes tumor development, and Hr mutant mice should no longer be considered merely hairless 'wild-type' mice. Instead, Hr is a crucial UVB response gene and its loss creates a permissive environment that potentiates increased tumorigenesis.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Hairless mutants have lower levels of Hr, an early a UVB response gene

. A) Hr expression was measured in whole skin extracts by quantitative RT-PCR using primers designed to several locations in the Hr coding sequence. The Hr−/− and SKH-1 Hairless mutant strains have significantly decreased levels of Hr compared to WT. B) Hr expression was also measured in WT (Hr+/+) epidermis before and after UVB (100 mJ/cm2) exposure at 1, 8 and 24 hrs. Hr expression significantly increased at the 8 hr time point.

Figure 2

Figure 2. Mutations in Hr render animals susceptible to UVB-induced tumorigenesis.

A) Female Hr−/− (n = 10) and age/sex matched WT littermates (Hr +/+) were irradiated using 180 mJ/cm2 UVB three times a week for a total of 50 weeks and the number of tumors per animal were recorded on a weekly basis. WT littermates never developed tumors or papillomas, while Hr−/− animals developed tumors by 30 weeks. B) After 50 weeks of irradiation, Hr−/− mice had obvious tumors and histological analysis revealed that the majority of the tumors were aggressive SSCs. In contrast, WT animals did not develop any tumors even at the microscopic level.

Figure 3

Figure 3. Hairless mutant epidermis is highly proliferative and exhibits a defective UVB response.

A) Assessment of epidermal thickness using epidermal morphometry on H&E stained sections indicates that Hr−/− mice have significantly greater thickness prior to UVB (180 mJ/cm2) as compared to WT controls. Epidermal thickness remains elevated after UVB exposure. B) Skin sections were stained with Ki-67 to identify proliferating basal keratinocytes both at baseline and after UVB exposure at the specified time points. Hr−/− mice had a significantly higher percentage of Ki-67 positive cells at baseline as compared to WT and continued to have an elevated amount of Ki-67 positive cells after UVB. C) Skin sections were stained with TUNEL to identify apoptotic cells both at baseline and at the designated time points. The number of TUNEL positive cells was plotted per 2.5 mm section of skin. Hr−/− mice have a significantly lower number of TUNEL positive cells at 1 hr and 24 hr post UVB compared to WT.

Figure 4

Figure 4. Hairless mutant epidermis shows constitutive activation of the NFκB signaling pathway.

A) Western Blot and ImageJ densitometry analysis using anti-p65 antibody on isolated Hr−/− epidermal extracts after 1, 8 and 24 hrs post UVB (180 mJ/cm2) irradiation. B) Immunohistochemistry using p-IκBα antibody to determine the extent of NFκB signaling activity in the epidermis of WT (Hr+/+) and Hr−/− animals before and after a single, acute UVB (180 mJ/cm2) irradiation. IκBα is increased constitutively and after irradiation throughout the epidermal layer. C) Immunohistochemistry using p-IκBα antibody to determine the extent of NFκB signaling activity in the epidermis of WT (Hr+/+) and Hr−/− animals at the indicated time points during chronic UVB irradiation (180 mJ/cm2 UVB three times a week for a total of 50 weeks). IκBα is increased constitutively and after irradiation throughout the epidermal layer.

Figure 5

Figure 5. Administration of the NFκB inhibitor PDTC abrogates UVB-induced skin carcinogenesis in SKH-1 mice.

SKH-1 mice (n = 30 per treatment) were chronically irradiated with UVB (180 mJ/cm2, twice a week for 35 weeks) and either given PDTC (0.5%) or no treatment in drinking water (ad libitum). Non-irradiated SKH-1 mice (n = 12) were used as controls. A) PDTC treatment decreased the average number of tumors per mouse. B) PDTC treatment decreased the average tumor volume per mouse. C) Western blot analysis for the p50, p52, and p65 subunits of NFκB were performed on nuclear extracts prepared from non-irradiated epidermal samples and chronically irradiated epidermal and tumor samples from mice treated with PDTC or untreated. While PDTC treatment prevented the upregulation of p50 in both skin and tumor samples, it had no effect on the p52 expression in either sample. PDTC treatment specifically abrogated upregulation of p65 in non-tumor bearing epidermis. D) RT-PCR for the NFκB transcriptional targets TRAIL, IL-1β, TNF-α and Mn SOD was performed on non-irradiated epidermal samples and chronically irradiated epidermis and tumor samples from mice treated with PDTC or untreated. PDTC prevented upregulation of the NFκB targets in irradiated skin samples but not in tumor samples. E) Western blot analysis for cyclin D1, cyclin E, and PCNA (which are involved in cell cycle regulation) was performed on non-irradiated epidermal samples and chronically irradiated epidermis and tumor samples from either untreated mice or mice treated with PDTC. PDTC treatment abrogates the overexpression of these proteins in non-tumor bearing skin samples. F) Western blot analysis of caspase-3 (pro-apoptosis), Bcl-2 (anti-apoptosis), and COX-2 (inflammation) was performed on non-irradiated epidermal samples and chronically irradiated epidermis and tumor samples from mice treated with PDTC or untreated. PDTC inhibited Bcl-2 and COX-2 in both epidermis and tumor samples while it did not affect caspase-3.

Similar articles

Cited by

References

    1. Rogers HW, Weinstock MA, Harris AR, Hinckley MR, Feldman SR, et al. Incidence estimate of nonmelanoma skin cancer in the United States, 2006. Arch Dermatol. 2010;146:283–287. - PubMed
    1. Weinberg AS, Ogle CA, Shim EK. Metastatic cutaneous squamous cell carcinoma: an update. Dermatol Surg. 2007;33:885–899. - PubMed
    1. Kanoh M, Amoh Y, Sato Y, Katsuoka K. Expression of the hair stem cell-specific marker nestin in epidermal and follicular tumors. Eur J Dermatol. 2008;18:518–523. - PubMed
    1. Abbassi MS, Torres C, Achour W, Vinue L, Saenz Y, et al. Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia. Int J Antimicrob Agents. 2008;32:308–314. - PubMed
    1. Green CL, Khavari PA. Targets for molecular therapy of skin cancer. Semin Cancer Biol. 2004;14:63–69. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources