The cellular origin of BCC has been approached largely through descriptive studies using human tissue specimens and has been rather poorly understood. In the past, experimental studies rigorously addressing this have been hampered in part by the fact that BCCs rarely develop in wild-type mice, even after exposure to physical or chemical carcinogens that are potent inducers of BCC development in rats (reviewed in ref. 53
). However, genetic models wherein different components of the Hh pathway are altered in cutaneous epithelia have yielded BCCs and related tumor types in mouse skin with high penetrance (reviewed in refs. 9
). Using multiple mouse models to control the timing and level of oncogenic Hh signaling activity and, most importantly, the particular cell populations where Hh signaling was altered, we have conclusively established that the transformation potential and varied BCC-like tumor phenotypes in skin are dictated by (a) the cell population being targeted, (b) timing (relative to the hair cycle), and (c) level of oncogenic Hh signaling.
We find that multiple epithelial compartments in skin are competent to form BCC-like tumors in response to GLI2ΔN, but the particular tumor subtype differs based on the cells in which GLI2ΔN is active, indicating that while an oncogenic Hh signal is sufficient to drive tumorigenesis, tumor phenotype is strongly influenced by its cell of origin. Postnatal induction of GLI2ΔN in telogen follicle stem cells leads to the development of nodular BCC-like skin tumors (Figures and ), while induction in interfollicular epidermis or epidermis from hairless skin yields superficial BCC-like tumors (Figure ); however, additional studies will be required to ascertain how closely these superficial tumors in mice mimic their human counterparts. An epidermal origin for superficial BCCs in humans is supported by histological studies examining microscopic tumors, which are first detected in interfollicular epidermis or the uppermost portion of the hair follicle, the infundibulum (54
). Collectively, both human and mouse data argue that hair follicles are not required for the development of superficial BCCs, most of which appear to arise from interfollicular epidermis.
In contrast to superficial BCCs, several previous reports point to the follicle as a potential site of origin for nodular BCC in mouse (25
) and rat (55
), but the involvement of follicle stem cells in the development of these tumors was not addressed. Using K15
promoter–driven Cre alleles, we show that progenitors of nodular, BCC-like tumors reside within the secondary hair germ and lower bulge stem cell compartments of the hair follicle (Figures , , and ). Our data are in keeping with recently reported lineage-tracing studies in irradiated Ptch+/–
mice that had developed basaloid skin tumors, nearly all of which appear to arise from K15-expressing cells (56
). The presence of mixed BCC growth patterns in up to nearly 40% of human BCC samples (22
) raises the possibility that nodular BCCs can expand upward to also involve epidermis as superficial BCCs, or vice versa, since hair follicle stem cell progeny migrate into the epidermis during wound healing (44
). Given the resistance of cells in much of the bulge to Hh-driven transformation (Figure and ref. 24
), it will be interesting to determine whether injury-induced mobilization and migration of these cells or their progeny into epidermis renders them competent to form tumors outside of the hair follicle stem cell niche.
In short-term induction studies using K14-rtTA;tetO-GLI2
mice, we found that cells in the telogen bulge were resistant to GLI2ΔN-induced transformation. This appeared to be due to (a) impaired accumulation of GLI2ΔN in this cellular compartment, (b) a blunted proliferative response in bulge cells that do express detectable levels of GLI2ΔN, and (c) activation of apoptosis in bulge cells (Figure ), which may eliminate potential tumor progenitors in this stem cell compartment. Induction of apoptosis has also been reported in neural stem cells engineered to express Gli1 (60
), suggesting that this response may serve as a protective mechanism against deregulated, high-level Hh/Gli signaling in other stem cell populations. Epithelial cells residing within the central isthmus, just above the follicle bulge, also appeared largely unaffected in these mice (Figure A). In this compartment, however, many cells did not contain detectable GLI2ΔN protein (Figure D), but additional studies will be required to assess whether this reflects focal deficiencies in transcription driven by the K14 and K5 promoters or modulation of Gli proteins at the post-translational level, which is likely to involve proteosome-mediated degradation (45
The timing of GLI2ΔN transgene induction relative to the hair cycle has a profound effect on BCC-like tumor development. This is best studied in dorsal skin, where the kinetics of hair cycling is well defined (Figure D and ref. 41
) and telogen follicles can be reliably induced to enter anagen by hair plucking or depilation. Although microscopic BCC-like tumors arose spontaneously from a subset of telogen hair follicles in dorsal skin by 2 weeks of GLI2ΔN expression, gross evidence of tumor development during the same time period was seen in skin containing anagen hair follicles (Figure ). Accelerated tumorigenesis does not appear to be related to markedly greater accumulation of GLI2ΔN protein in anagen, as might have been expected (32
), since telogen-derived tumors and anagen-derived tumors had comparable levels of immunostaining for GLI2ΔN (Supplemental Figure 2). In addition, the transcriptional activity of GLI2ΔN also appears to be similar in telogen-initiated and anagen-initiated tumors, based on Gli1
expression (Supplemental Figure 2).
Since anagen is characterized by robust proliferation of transit-amplifying stem cell progeny to reconstruct a mature follicle, accelerated tumorigenesis may reflect the ability of a small number of GLI2ΔN-expressing nascent tumor cells in telogen to rapidly expand into a detectable tumor mass under these growth-promoting conditions. This raises the possibility that in human BCC, quiescent follicle stem cells carrying mutations leading to Hh pathway activation may remain dormant until mobilized during early anagen or in response to another regenerative stimulus, when their selective growth advantage can lead to tumor formation. The rapid development of BCC-like nodules directly from the anagen outer root sheath (Figure D) is in keeping with this idea, and this finding identifies this transit-amplifying cell population as the likely cell of origin for BCCs that arise during anagen.
In addition to the important roles of cell of origin and hair cycling, we also show that GLI2ΔN expression levels define tumor phenotype, with low-level GLI2ΔN yielding basaloid hamartomas mimicking skin lesions in SMO-expressing mice (35
), rather than nodular BCC-like tumors (Figure ). These data provide direct evidence supporting the hypothesis that a sufficiently high level of Hh signaling must be achieved to drive development of nodular BCC-like tumors (35
). In addition, induction of oncogenic Smo in follicle stem cells, using the same strategy that yields multiple BCC-like tumors in response to GLI2ΔN, gives rise to a hyperplastic outer root sheath but no BCC-like tumors (Figure ). These results are in general agreement with a recent study in which clonal, postnatal activation of a Cre-inducible Smo mutant failed to produce tumors from follicle stem cells or their progeny, but did lead to the development of basaloid skin tumors derived primarily from interfollicular epidermis (24
). However, these lesions do not have the typical histological or gross appearance of nodular BCCs, which is in keeping with the idea that only high-level Hh signaling leads to BCC development (35
). Establishing a definitive diagnosis in mouse models of cancer that is based on comparisons with human pathology can be challenging, particularly in skin, where there are major differences in tissue architecture between these two species. Nevertheless, the pattern of lineage markers, robust proliferation, and occasional areas of central necrosis seen in GLI2ΔN-expressing tumors described in this report are in keeping with the diagnosis of nodular, BCC-like tumors. Interestingly, the weak transforming potential of activated Smo
alleles may be limited to skin, since Smo
is a potent oncogene in cerebellum, where it gives rise to medulloblastomas with high penetrance when targeted to neuronal progenitors (50
The results of our studies can be summarized as follows. (a) We have established the utility of a versatile new mouse model of cancer that can be readily adapted to study a variety of oncogenic pathways in different target organs. (b) We have shown that nodular BCC-like skin tumors in mice can arise from Lgr5+
stem cells in the resting hair follicle and from basal cell compartments of the epidermis, growing hair follicle (outer root sheath), and sebaceous gland. These findings are in keeping with the varied histological presentations of human BCC, which include epidermis-associated superficial BCCs, nodular BCCs (which we propose are derived from hair follicles), and BCCs with sebaceous differentiation (22
). (c) In contrast to other basal cell populations, stem cells within the bulge appear to be largely incapable of mounting a hyperplastic response to GLI2ΔN expression, although a fraction of these cells do activate expression of proliferation markers but appear destined to be eliminated via apoptosis. (d) We show that nodular BCC-like tumors do not form in mice expressing reduced levels of GLI2ΔN, but instead these mice develop basaloid hamartomas that are strikingly similar to lesions arising in mice expressing oncogenic Smo alleles. These findings provide direct evidence supporting the concept that full-blown nodular BCCs develop only in response to high-level Hh/Gli signaling activity, and may explain the reported lack of nodular tumors arising from follicle stem cells in Smo-expressing mice.