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. 2015 Oct 15;26(20):3606-14.
doi: 10.1091/mbc.E15-05-0282. Epub 2015 Aug 26.

Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis

Jill Goldstein  1 , Eve Roth  1 , Natalie Roberts  1 , Rachel Zwick  1 , Samantha Lin  1 , Sean Fletcher  1 , Ana Tadeu  1 , Christine Wu  1 , Amanda Beck  2 , Caroline Zeiss  2 , Mayte Suárez-Fariñas  3 , Valerie Horsley  4
Affiliations

Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis

Jill Goldstein et al. Mol Biol Cell. .

Abstract

Immunosuppressive therapies using calcineurin inhibitors, such as cyclosporine A, are associated with a higher incidence of squamous cell carcinoma formation in mice and humans. Calcineurin is believed to suppress tumorigenesis in part through Nfatc1, a transcription factor expressed primarily in hair follicle bulge stem cells in mice. However, mice overexpressing a constitutively active Nfatc1 isoform in the skin epithelium developed increased spontaneous skin squamous cell carcinomas. Because follicular stem cells can contribute to skin tumorigenesis, whether the endogenous expression of Nfatc1 inhibits or enhances skin tumorigenesis is unclear. Here we show that loss of the endogenous expression of Nfatc1 suppresses the rate of DMBA/TPA-induced skin tumorigenesis. Inducible deletion of Nfatc1 in follicular stem cells before tumor initiation significantly reduces the rate of tumorigenesis and the contribution of follicular stem cells to skin tumors. We find that skin tumors from mice lacking Nfatc1 display reduced Hras codon 61 mutations. Furthermore, Nfatc1 enhances the expression of genes involved in DMBA metabolism and increases DMBA-induced DNA damage in keratinocytes. Together these data implicate Nfatc1 in the regulation of skin stem cell-initiated tumorigenesis via the regulation of DMBA metabolism.

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Figures

FIGURE 1:
FIGURE 1:
Epidermal Nfatc1 deletion decreases DMBA/TPA tumorigenesis. (A) Schematic of K14-specific Nfatc1 deletion. (B) DMBA/TPA tumorigenesis regime. (C) Percentage of tumor-free cKO/control mice during DMBA/TPA tumorigenesis (16 mice/genotype). *p = 0.03; log-rank test. (D) Average tumor number in cKO/control mice. Data: mean ± SEM. The profiles (p = 0.03) and several time points were significantly different (*); mixed-effect model. (E) Tumor formation rate between cKO/control mice is significantly different, p = 0.0006; mixed-effect model; time: continuous variable. (F) Real-time PCR for K14 and K10 in cKO/control tumors. Data: mean ± SEM (3 mice/genotype). (G, H) Immunostaining for (G) BrdU (red) and (H) K14 (red) and K10 (green) in cross sections of cKO/control tumors 8–10 wk post-DMBA. DAPI, blue. Scale bar, 50 μm.
FIGURE 2:
FIGURE 2:
Nfatc1 deletion decreases the rate of tumor initiation but not tumor promotion. (A) Schematic of inducible K19-specific Nfatc1 deletion. (B) Nfatc1 immunostaining (green) in iKO mice 5 d after tamoxifen/vehicle. (C) DMBA/TPA initiation (ODT) and promotion (DOT) regimes. (D, G) Percentage of tumor-free iKO/control mice in (D) ODT or (G) DOT regime (n = 16 mice/genotype). (E, H) Average tumor number during (E) ODT or (H) DOT regime. Data: mean ± SEM (seven mice/genotype). Several time points were significantly different (*); mixed-effect model. (F, I) Tumor formation rate during (F) ODT regime is significantly different but not during (I) DOT regime (mixed-effect model; time: continuous variable). (J) Real-time PCR of Nfatc1 in tumor cells relative to FACS-sorted bulge cells. Data: mean ± SD (six mice/group). Immunostaining for (K) CD45 (red) and (L) Tcrδ (red) in cKO/control tumors 8–10 wk post-DMBA. (M) Quantification of Tcrδ+ cells in cKO/control tumors. Data: mean ± SEM (three mice/genotype). DAPI, blue; dotted line, epithelial–stromal barrier; white dots, hair shaft autofluorescence. Scale bar, 25 μm (B), 50 μm (K, L).
FIGURE 3:
FIGURE 3:
Nfatc1 deletion decreases stem cell contribution to papillomas during tumor initiation. (A) Schematic of inducible K19-specific Nfatc1 deletion and mTmG labeling. (B) Representative images of K19CreERT;mTmG follicles after tamoxifen. Images show unlabeled (only mT+ cells) or labeled (containing mG+ cells) follicles. (C) Percentage of mG+ hair follicles 8–12 wk post-DMBA (initiation, ODT; promotion, DOT). Data are mean ± SEM for 50–100 hair follicles from four individual mice/group. (D) Representative sagittal section of a tumor containing mG+ cells derived from K19-expressing bulge cells. (E) Percentage of tumors containing mG+ cells in iKO mice and littermate controls from sagittally sectioned tumors during ODT and DOT regimes. Data are mean ± SEM of three or four individual mice/genotype for 8–15 tumors from each mouse. Tumors were harvested 8–12 wk post-DMBA. DAPI, blue. White dots denote hair shaft autofluorescence. Scale bar, 25 μm.
FIGURE 4:
FIGURE 4:
Epidermal Nfatc1 deletion reduces DMBA-induced Hras codon mutations. (A) MHC class II immunostaining (red) from cKO/control epidermal sheets. (B) γH2A.X immunostaining (green) in cKO/control mice 24 h post-DMBA/vehicle. (C) Percentage of BrdU+ epidermal cells normalized to total basal cells 24 post-TPA/vehicle. Data: mean ± SEM (three or four mice/group; >4000 cells quantified/ mouse). (D) Epidermal thickness 24 h post-TPA/vehicle. Data: mean ± SEM (four mice/group). (E) Hras codon 61 mutations from cKO/control tumors 8–10 wk post-DMBA. +, CarC DNA. Optical density of CTA mutant bands. Chi-squared test. DAPI, blue. Scale bar, 50 μm.
FIGURE 5:
FIGURE 5:
Nfatc1 regulates expression of DMBA-metabolizing enzymes. (A) Real-time PCR analysis of mRNA expression for DMBA-metabolizing enzymes in FACS-purified sorted cKO bulge cells compared with control bulge cells. Data are mean ± SD (three or four mice/genotype). *p < 0.05; one-way ANOVA. (B) Real-time PCR for DMBA-metabolizing enzymes in caNFATc1 compared with control-infected keratinocytes. *p < 0.05; one-way ANOVA. (C–F) Real-time PCR for (C) Ahr, (D) Cyp1a1, (E) Cyp1b1, and (F) Nqo1 mRNA in caNFATc1- and control-infected keratinocytes at 0, 1, 4, and 24 h. The p value reflects two-way ANOVA for the effect of Nfatc1 expression. (G) γH2A.X immunostaining (red) in GFP- and caNFATc1-infected keratinocytes 24 h post-DMBA/vehicle. DAPI, blue. Scale bar, 50 μm. (H) Quantification of γH2A.X+ GFP- and caNFATc1-infected keratinocytes after 24 h in vehicle or DMBA. Data: mean ± SEM (three or four replicates for each treatment in two independent experiments; >179–1435 cells analyzed). One-way ANOVA.
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