Smad4 loss in mouse keratinocytes leads to increased susceptibility to UV carcinogenesis with reduced Ercc1-mediated DNA repair

Abstract

Smad4 loss occurs frequently in human skin squamous cell carcinoma (SCC), but it is unknown whether this loss increases UV-induced carcinogenesis, a major etiological factor in skin cancer. In the present study, mice with keratinocyte-specific Smad4 deletion (K14.Smad4(-/-)) and wild-type (WT) littermates were chronically UV-irradiated. Compared with WT, K14.Smad4(-/-) mice exhibited increased DNA damage and increased susceptibility to UV-induced skin cancer. Among genes involved in repairing UV-induced DNA damage, Excision repair cross-complementation group 1 (Ercc1) messenger RNA was significantly reduced in UV-treated K14.Smad4(-/-) skin compared with WT skin. Further analysis revealed that Smad4 loss confers reduced Snail binding to the Ercc1 regulatory elements, resulting in reduced Ercc1 transcription. Consistently, transient transfection of Snai1 into Smad4(-/-) keratinocytes led to increased repair of UV-induced DNA lesions. Transfection of Ercc1 into Smad4(-/-) keratinocytes restored repair of UV-induced DNA damage. Further, immunostaining revealed that the presence of Smad4 protein is associated with the presence of Snail and Ercc1 proteins in human skin SCC and precancerous actinic keratoses. Collectively, Smad4 loss-associated Snail reduction compromises Ercc1-mediated DNA repair, contributing to increased UV-induced skin carcinogenesis. Thus, we identified a role for Snail in UV-induced DNA repair.

Figure 1. Smad4^−/− mice developed UV-induced skin tumors

(a) IHC staining of Smad4 in skin samples from UV-irradiated WT and Smad4^−/− mice. (b) Kinetics of tumor formation: data points represent mice that developed tumors, expressed as a percentage of mice with tumors among the total number of mice in that genotype group. (c) H&E staining showing a typical UV-induced K14.Smad4^−/− SCC morphology. Keratin pearl is highlighted with an asterisk. Scale bars: 100μm.

Figure 2. Increased DNA damage in UV-irradiated K14.Smad4^−/− skin, and reduced repair of CPD in Smad4^−/− keratinocytes

(a) Immunofluorescence staining of p-H2AX in UV-irradiated mouse skin and tumors. Keratin 14 was used as counterstain. Scale bar is 100μm. (b) Quantification of p-H2AX staining, n=6, 8 and 4 for WT skin, Smad4^−/− skin, Smad4^−/− tumors, respectively. (c) Alkaline comet assay using WT and Smad4^−/− keratinocytes. (d) Quantification of % DNA in comet tail. n=44. (e) CPD levels in UVB-irradiated WT and Smad4^−/− keratinocytes was measured by ELISA. CPD level in Smad4^−/− keratinocytes at 10 minutes was set at 100%. P ≥ 0.1 at 5, 10 and 30 minutes, respectively. *denotes p ≤ 0.0002, and ** denotes p ≤ 0.003, n=3. Error bars are S.E.M. (all panels).

Figure 3. Reduced expression of DNA repair genes in Smad4^−/− keratinocytes, reduced Ercc1 in K14.Smad4^−/− skin and tumors

(a) qRT-PCR measured mRNA levels of indicated genes in WT and Smad4^−/− keratinocytes. The mRNA level of each gene in WT keratinocytes was set at 1. * p≤ 0.002. Error bars are S.E.M., n=3. (b) mRNA levels of Smad4 and Ercc1 in UV-irradiated WT and K14.Smad4^−/− skin. * p≤ 0.02. Error bars are S.E.M., n=3 for WT skin and n=7 for Smad4−/− skin. (c) Immunohistochemical staining of Ercc1 was performed on UV-irradiated WT and K14.Smad4^−/− skin and tumors.

Figure 4. Snail regulates Ercc1 expression, Snail and Ercc1 mediate CPD repair in keratinocytes

(a) Snai1 mRNA level in UV-irradiated keratinocytes, the level in untreated WT keratinocytes was set at 1. (b) Snai1 mRNA level in UV-irradiated mice skin, the level in UV-irradiated WT skin was set at 1, n=4 for WT, n=7 for Smad4^−/−. * denotes p= 0.027 (c) Ercc1 mRNA level in human Snai1-transfected keratinocytes, the level in empty vector transfected WT keratinocytes was set at 1. ** denotes p≤ 0.002. (d) Snail Chromatin Immunoprecipitation from WT and Smad4^−/− keratinocyte extracts with/without UV irradiation. * p<0.03, ** p< 0.005. (e) CPD level in UVB-irradiated Snai1 transfected keratinocytes *p = 5.09E⁻⁵, ** p=0.005, *** p=0.01. (f) CPD level in UVB-irradiated Ercc1 transfected keratinocytes, *=0.026, **=0.046, *** p=0.10, n=3. Error bars: S.E.M.

Figure 5. Expression of Smad4 is associated with expression of Snail and Ercc1 proteins in human skin SCC and AK

Smad4, Snail and Ercc1 were stained in a human skin SCC tissue array (a, b) and human actinic keratosis (c, d) Representative images of a Smad4, Snail and Ercc1 triple negative case (a, c) and triple positive case (b, d). Scale bar: 100μm for all panels.