Fractionation of a tumor-initiating UV dose introduces DNA damage-retaining cells in hairless mouse skin and renders subsequent TPA-promoted tumors non-regressing

Abstract

Sunburns and especially sub-sunburn chronic UV exposure are associated with increased risk of squamous cell carcinomas (SCCs). Here we focus on a possible difference in tumor initiation from a single severe-sunburn dose (on day 1, 21 hairless mice) and from an equal dose fractionated into very low sub-sunburn doses not causing any (growth-promoting) epidermal hyperplasia (40 days daily exposure, n=20). From day 47 all mice received 12-O-Tetradecanoylphorbol-13-acetate (TPA) applications (2x/wk) for 20 weeks to promote tumor development within the lifetime of the animals. After the sub-sunburn regimen sparse DNA damage-retaining basal cells (quiescent stem cells, QSCs) remained in the non-hyperplastic epidermis. These cells were forced to divide by TPA. After discontinuation of TPA tumors regressed and disappeared in the 'sunburn group' but persisted and grew in the 'sub-sunburn group' (0.06 vs 2.50 SCCs and precursors ≥4 mm/mouse after 280 days, p=0.03). As the tumors carried no mutations in p53, H/K/N-Ras and Notch1/2, these 'usual suspects' were not involved in the UV-driven tumor initiation. Although we could not selectively eliminate QSCs (unknown phenotype) to establish causality, our data suggest that forcing specifically DNA damage-retaining QSCs to divide--with high mutagenic risk--gives rise to persisting (mainly 'in situ') skin carcinomas.

Keywords: CPD retaining basal cells; UV carcinogenesis; dose fractionation; hyperkeratotic tumors.

Figure 1. CPD retaining cells are only present after a fractionated sub-sunburn UV dose in epidermis (arrows) and in upper dermis (non-dividing fibroblasts)

Mouse skin after fractionated sub-sunburn UV dose before TPA treatment, day 47 A. and after TPA treatment, day 54 C. and correspondingly after the unfractionated sunburn UV dose B. and D. Scale bar 100 μm.

Figure 2. Kaplan-Meier plot of tumor-free fraction of mice for AK/SCC drops significantly faster after fractionated sub-sunburn UV exposure

The tumor-free fraction for AK/SCCs ≥2 mm A. and ≥4 mm B. and papillomas ≥2 mm C. and ≥4mm D. for mice in the sub-sunburn group (○) or in the sunburn group (■). The grey area in the graphs represent the period in which TPA was applied.

Figure 3. Tumor yields (average number of tumors per mouse) showed non-regressing carcinomas only after fractionated sub-sunburn UV exposure

The yield of AK/SCCs ≥2 mm A. and ≥4 mm B. for mice irradiated with the fractionated sub-sunburn UV dose (○) or with the unfractionated sunburn dose of UV (5.6 MED) (■). The grey area in the graphs represent the period in which TPA was applied. Error bars show SEMs. Virtually all AK/SCCs go into regression in the sunburn group after cessation of TPA applications.

Figure 4. No significant difference in tumor yields (average number of tumors per mouse) for papillomas between the fractionated sub-sunburn and unfractionated sunburn UV dose groups

The yield for papillomas ≥2 mm A. and ≥4 mm B. for mice irradiated with the fractionated sub-sunburn UV dose (○) or with the unfractionated sunburn dose of UV (5.6 MED) (■). The grey area in the graphs represent the period in which TPA was applied. Error bars show SEMs.