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Lgr6+ cells have been identified as a novel class of proliferating (Ki67+) stem cells in mouse epidermis. We investigated their response to UV exposure in Lgr6-EGFP-Ires-CreERT2/R26R-LacZ haired and hairless mice and whether they become initiating cells of UV- or chemically induced skin tumors. UV overexposure erased Lgr6+ cells (EGFP+) from the interfollicular epidermis (IFE), but - as after wounding - they apparently repopulated the IFE from the hair follicles. Under sub-sunburn chronic UV exposure, Lgr6+ cells and their progeny (LacZ+ after pulse of tamoxifen) diminished strongly in the IFE. Although the inter-tumoral IFE clearly showed Lgr6 progeny, none of the UV- or chemically induced tumors (n = 22 and 41, respectively) appeared to be clonal expansions of Lgr6+ stem cells; i.e. no Lgr6+ cells or progeny in the proliferating tumor bulk. In checking for promoter methylation we found it to occur stochastically for the EGFP-Cre cassette. Lgr6 mRNA measured by qPCR was found to be diminished in skin tumors (also in UV tumors from wt type mice). The ratio of Lgr6/Ki67 was significantly reduced, pointing at a loss of Lgr6+ cells from the proliferative pool. Our data show that Lgr6+ cells are not major tumor-initiating cells in skin carcinogenesis.

Actively proliferating Lgr5+ skin stem cells are found deep in the hair follicle (HF). These cells renew the HF and drive its expansion in anagen phase. Their long residence and continuous mitotic activity make them prime candidates to transform into skin tumor-initiating cells. This was investigated by subjecting Lgr5-EGFP-Ires-CreERT2/R26R-LacZ mice (haired and hairless) to chemical and UV carcinogenic regimens. In the course of these regimens Lgr5+ cells (EGFP+) remained exclusively located in HFs, and in deep-seated cysts of hairless skin. In haired mice, progeny of Lgr5+ stem cells (LacZ+ after a pulse of tamoxifen) appeared in the interfollicular epidermis upon UV-induced sunburn and in TPA-induced hyperplasia. In hairless mice the progeny remained located in deep-seated cysts and in HF remnants. Progeny in hairless skin was only detected interfollicularly at a late stage, in between outgrowing tumors. Lgr5+ stem cells were absent in the ultimate tumor masses, and no tumor appeared to be a (clonal) expansion of Lgr5+ cells (52 tumors with tamoxifen at the start of carcinogenesis, 42 tumors with tamoxifen late during tumor outgrowth). In contrast to CD34/K15+ quiescent bulge stem cells, actively proliferating Lgr5+ stem cells do therefore not appear to be tumor drivers in experimental skin carcinogenesis., Competing Interests: The authors declare no conflicts of interest.

Chronic ultraviolet (UV) exposure induces clones of cells overexpressing mutant p53 in the interfollicular (IF) epidermis and subsequently squamous cell carcinomas (SCCs) with similar p53 mutations. Mutated p53 may give cells growth advantage over neighbouring cells by impaired apoptosis. We tested this by UV overexposure of skin laden with p53-mutant clones and assessed the impact on subsequent tumour development. P53-mutant clones were induced in two groups of hairless SKH1 mice by daily exposures (500 J/m(2) UV from TL12 lamps) for 28 days. On day 29, one group was overexposed (to 10 kJ/m(2) UV), whereas the control group received the regular daily dose. After 1 week of recovery, the daily exposures were resumed in both groups to induce SCCs. UV overexposure forced the entire IF basal layer into caspase-3-driven apoptosis while leaving overlying layers with sunburn cells intact. No apparent regions were spared from apoptosis. Pulse-chase BrdU labelling showed the IF epidermis to be repopulated from the hair follicles (remaining p63 positive). One week after overexposure, the p53-mutant clones had virtually disappeared (0.6, 95% confidence interval 0.5-0.8 per mouse versus 102, 59-179, without overexposure). Tumour development was significantly delayed after UV overexposure (P < 0.0001) by an average of 27 days (standard error of the mean 3); a period matching that of daily exposures preceding the overexposure. Thus, we found that UV-induced ablation of the IF epidermal basal layer eliminates p53-mutant clones and resets UV carcinogenesis. Furthermore, and in contrast with earlier reports, our data show that UV-induced p53-mutant clones and SCCs originate from the IF epidermis.