Basal cell carcinomas in mice arise from hair follicle stem cells and multiple epithelial progenitor populations (original) (raw)
Conditional GLI2ΔN expression in hair follicle stem cells and their progeny. We generated a conditional, triple-transgenic model that combines Cre-lox and tet-regulated gene-switch technologies to achieve tight control of transgene expression, both spatially and temporally, in hair follicle stem cells or a wide variety of other cell populations. The 3 components of this model include a transgenic Cre driver; a ROSA26 promoter–based, Cre-inducible (lox-STOP-lox) reverse tet transcriptional activator (rtTA) strain (37), which we designate R26-LSL-rtTA; and a _tetO_-regulated effector strain, in which transgene expression can be induced by doxycycline when rtTA transactivator is present (Figure 1A). Targeting specificity is governed by the choice of Cre driver, while the oncogenic stimulus is provided by the specific tetO effector mouse, making this a versatile model for manipulating essentially any oncogenic pathway in any organ, if appropriate Cre and tetO lines are available. Importantly, because rtTA induction is Cre dependent and under the control of the ubiquitously expressed ROSA26 promoter, rtTA will be expressed in all cells that have undergone a Cre-mediated recombination event, as well as their progeny. In addition, by altering the doxycycline treatment regimen, transgene expression can be induced to different levels. For the experiments in this study, we generated tetO-GLI2_Δ_N mice, since we had previously shown that GLI2ΔN and the analogous mouse allele, Gli2ΔN2, are potent activators of oncogenic Hh signaling and drive BCC-like skin tumor development in mice (38, 39).
BCC-like skin tumors arise from hair follicle stem cells. (A) General scheme showing triple-transgenic model combining Cre-lox and tet/doxycycline-regulated technologies to achieve tight spatial and temporal control of transgene expression. The 3 components include mouse strains carrying: (i) a tissue-specific promoter (TSP) driving expression of a hormone-inducible Cre allele fused to a steroid binding domain (CreSBD); (ii) a Cre-inducible “tet on” rtTA, under the control of the ubiquitously expressed ROSA26 promoter (R26-LSL-rtTA) (37); and (iii) an oncogene downstream of multiple tetO sequences and minimal CMV promoter that is induced by rtTA when doxycycline (doxy) is present. (B) Cellular compartments in a quiescent (telogen) hair follicle. Most hair follicle stem cells are localized to the bulge and secondary hair germ compartments. (C) Compartments in which K15-CrePR1 mice can drive recombination in K15-CrePR1;R26-LSL-rtTA;tetO-GLI2_Δ_N (iK15;rtTA;GLI2_Δ_N) mice to activate rtTA expression. (D) Synchronized hair growth cycle in postnatal mouse skin, with approximate mouse ages and timing of doxycycline treatment/GLI2ΔN transgene induction indicated. (E) Spontaneous development of microscopic skin tumors from telogen hair follicle stem cells in dorsal skin of iK15;rtTA;GLI2_Δ_N mice, after 3 weeks of doxycycline treatment. Note tumor development from lowermost follicle in a region corresponding to the secondary hair germ. Original magnification, ×200 (left panel); ×400 (right panels). (F) Spontaneous tumor development from K15+ follicle stem cells at other body sites 3 weeks after GLI2ΔN induction. Original magnification, ×100 (tail, dorsal paw, and snout); ×200 (ear).
Nodular BCC-like skin tumors arise from resting hair follicle stem cells. To test whether hair follicle stem cells are competent to form skin tumors in response to GLI2ΔN, we used K15-CrePR1 mice, which have previously been shown to have Cre recombinase activity in stem cells in the bulge and secondary hair germ of the telogen follicle following treatment with the progesterone analog RU486 (ref. 40, Figure 1, B and C, and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI46307DS1). We generated K15-CrePR1;R26-LSL-rtTA;tetO-GLI2_Δ_N (iK15;rtTA;GLI2_Δ_N) triple-transgenic mice, which we treated with RU486 to activate Cre function, and induced rtTA following Cre-mediated excision of the elements inhibiting its expression. We then treated mice with doxycycline starting at 7 weeks of age to induce GLI2ΔN expression. Because the first few hair cycles in dorsal mouse skin are synchronized, essentially all hair follicles in this region are in a prolonged telogen (resting) phase of the hair cycle at this time (ref. 41 and Figure 1D), so any effects of GLI2ΔN on cell proliferation will not be obscured by physiologic hair follicle growth in anagen.
Although dorsal skin appeared grossly normal after 3 weeks of doxycycline treatment, multiple microscopic skin tumors were detected at this time. Tumor nodules were located within the dermis and comprised masses of basaloid-appearing cells with scant cytoplasm resembling human BCC (Figure 1E). The histology of early-stage tumors suggested that they are derived from the secondary hair germ, with more proximal regions of the bulge largely unaffected (Figure 1, B and E). In keeping with the expression pattern of the promoter in K15-CrePR1 mice (40), the sebaceous glands, upper hair follicle, and epidermis were largely unaffected (Figure 1E). Hair follicle–associated tumors were observed at multiple other sites, including the tail, ear, dorsal paw, and snout (Figure 1F). Tumors on the snout and dorsal paws developed more rapidly than at other sites, with grossly visible tumors seen at 3 weeks of treatment when only microscopic tumors were evident elsewhere. Large tumors sometimes contained regions of central necrosis (not shown), as occasionally seen in human BCCs.
In addition to a nodular BCC-like morphology, immunophenotyping revealed a pattern of lineage markers characteristic of human BCC. Tumors expressed keratins K5 and K17 and Sox9 (42); exhibited undetectable or negligible levels of keratins K1 and K6; and were hyperproliferative, based on Ki67 immunostaining (Figure 2A). Immunostaining for MYC revealed expression of epitope-tagged GLI2ΔN in tumor cells, with relatively few MYC-positive cells in normal-appearing hair follicles (Figure 2A), suggesting that the recombination efficiency using our protocol was relatively low. This was corroborated using RU486-treated K15-CrePR1;R26R mice carrying a Cre-inducible lacZ reporter allele (data not shown). In situ hybridization of early tumors revealed expression of GLI2ΔN transgene and Hh target genes Gli1 and Ptch1 (Figure 2B), which serve as molecular markers of deregulated Hh signaling in human BCC (9, 10). These results establish that _K15_-expressing stem cells in resting hair follicles of dorsal skin are competent to form nodular BCC-like skin tumors, which appear to arise preferentially from the secondary hair germ or lowermost bulge stem cell compartments.
Nodular skin tumors arising from K15+ stem cells express BCC markers. (A) Immunostaining showing BCC-like keratin profile (K5 and K17 expressed, K1 and K6 not expressed). Tumor cells also express the BCC marker Sox9 and are highly proliferative, based on Ki67 immunostaining. BCC-like tumors also express MYC-tagged GLI2ΔN transgene product. Original magnification, ×200. (B) In situ hybridization showing transcripts for Hh target genes Gli1 and Ptch1 in early-stage microscopic tumors, along with GLI2_Δ_N, detected using transgene-specific riboprobe against the SV40 poly(A) tail. Original magnification, ×200.
Nodular BCC-like skin tumors arise from the Lrg5-expressing subset of follicle stem cells. Although the location of early-stage tumors in iK15;rtTA;GLI2_Δ_N mice (Figure 1E) suggests that they are derived from cells in the secondary hair germ or lowermost bulge, given the expression pattern of K15-driven recombination (Figure 1C) we cannot rule out the possibility that tumor initiation occurs in a cell within the bulge compartment but proliferative expansion requires migration into the neighboring secondary hair germ. To define more precisely which stem cell population in the telogen follicle can give rise to GLI2ΔN-driven BCC-like skin tumors, we used Lgr5-CreER mice, which drive recombination in a more limited distribution comprising the secondary hair germ and a subset of cells in the lowermost portion of the bulge (ref. 43, Figure 3A, and Supplemental Figure 1), to generate Lgr5-CreER;R26-LSL-rtTA;tetO-GLI2_Δ_N (iLgr5;rtTA;GLI2_Δ_N) mice. Mice were treated with tamoxifen to activate Cre function, and doxycycline was again started at week 7 to activate GLI2ΔN expression at a time when hair follicles in dorsal skin are in telogen (Figure 3B).
Robust BCC-like skin tumor development from the Lgr5+ subset of follicle stem cells. (A) _Lgr5-CreER_–driven recombination is limited to the secondary hair germ and lowermost bulge region. (B) Hair growth cycle and timing of doxycycline treatment/GLI2ΔN expression. TAM, tamoxifen. (C) Spontaneous development of microscopic skin tumors from telogen hair follicle stem cells in dorsal skin of iLgr5;rtTA;GLI2_Δ_N mice after 3 weeks of doxycycline treatment. Note early tumor development from the secondary hair germ. Original magnification, ×200 (left panels); ×400 (right panels). (D) Spontaneous nodular BCC-like tumor development from Lgr5+ follicle stem cells at other body sites. Original magnification, ×200.
Similar to iK15;rtTA;GLI2_Δ_N mice, in which both the bulge and secondary hair germ compartment are targeted, iLgr5;rtTA;GLI2_Δ_N mice developed tumors on dorsal skin (Figure 3C) as well as tail, ear, dorsal paw, and snout (Figure 3D). The kinetics of tumor induction in different body sites was similar to that of tumors arising in K15-targeted mice, with tumors appearing first on the snout and dorsal paws. The morphology of tumors was also similar to that arising in K15-targeted mice, and analysis of early-stage tumors revealed their derivation from the secondary hair germ or lowermost bulge region, with other compartments of the hair follicle and epidermis unaffected (Figure 3, C and D). These results identify the Lgr5+ subset of follicle stem cells as a source of progenitors for GLI2ΔN-driven BCC-like skin tumors and suggest that stem cells in the secondary hair germ and lowermost bulge are preferentially susceptible to Hh pathway–driven tumorigenesis.
Stem cells in the follicle bulge are resistant to GLI2ΔN-induced hyperplasia. The nearly exclusive development of GLI2ΔN-expressing tumors from cells in secondary hair germs of iK15;rtTA;GLI2_Δ_N mice (Figure 1E), despite K15-driven targeting to stem cells in the bulge as well, suggested that bulge cells may be resistant to transformation by GLI2ΔN. To explore this possibility and to assess the responsiveness of other epithelial cell populations in skin, we targeted GLI2ΔN expression broadly to the entire basal layer compartment throughout the resting hair follicle, sebaceous gland, and epidermis using K14-rtTA;tetO-GLI2_Δ_N (K14;GLI2_Δ_N) bitransgenic mice. Doxycycline treatment was started at 7 weeks, and mice were euthanized and tissue was collected for analysis after 2 and 5 days of GLI2ΔN transgene induction. By day 5, there was prominent expansion of proliferating basaloid cells in nearly all epithelial compartments of skin, including the epidermis, hair follicle infundibulum, sebaceous gland progenitors, and secondary hair germ (Figure 4A). In striking contrast, basaloid hyperplasia was not apparent in hair follicle epithelium between the hair germ and sebaceous gland, which includes the bulge compartment and adjacent central isthmus (Figure 1B and Figure 4A).
Impaired responsiveness of follicle bulge stem cells to acute induction of GLI2ΔN. (A) Histology of epithelial compartments in tail skin of control and K14-rtTA;tetO-GLI2_Δ_N (K14;GLI2_Δ_N) bitransgenic mice reveals basaloid cell hyperplasia in epidermis, sebaceous gland, and secondary hair germ (yellow asterisks in right panels), but not in the bulge or central isthmus (marked with black and dotted yellow bars, respectively). Original magnification, ×200 (upper panels); ×600 (lower panels). (B) Immunostaining for MYC reveals GLI2ΔN transgene expression in basal epithelial compartments, with the exception of cells in the central isthmus (white brackets). Coimmunostaining for GLI2ΔN (MYC) and PCNA reveals increased proliferation in all compartments expressing GLI2ΔN, although the fraction of PCNA+ cells is lower in the bulge than other compartments (D). Original magnification, ×200 (upper panels); ×600 (lower panels). (C) Coimmunostaining for the bulge marker K15 and PCNA confirms proliferation in bulge stem cells in tail as well as dorsal skin. Original magnification, ×400. (D) Proliferative response to GLI2ΔN is approximately 50% lower in bulge cells than in epidermal or sebaceous gland cells. (E) Progressive increase in number of Ki67+ normally quiescent bulge cells 2 and 5 days after activation of GLI2ΔN transgene expression by using doxycycline. Data in D and E are presented as mean ± SEM; *P < 0.05, **P < 0.005. (F) Increased apoptosis in bulge compartment of GLI2ΔN-expressing mice compared with controls, based on immunostaining for activated caspase-3 (arrows). Original magnification, ×600.
GLI2ΔN was detected by MYC immunostaining in hyperplastic basaloid cells of the epidermis, sebaceous gland, hair follicle infundibulum, and secondary hair germ, and at each of these sites there was a striking increase in proliferative activity detected by proliferating cell nuclear antigen (PCNA) immunostaining, which increased between 2 and 5 days of treatment (Figure 4B). Cells in the bulge region also expressed GLI2ΔN (Figure 4, B and E), but the fraction of these cells that was PCNA- or Ki67-positive was lower than that of GLI2ΔN-expressing epithelial cells in other regions (Figure 4, B and D). Coimmunostaining for PCNA and the bulge cell marker K15 confirmed that cells induced to proliferate in GLI2ΔN-expressing mice were normally quiescent bulge keratinocytes, both in tail and dorsal skin (Figure 4C). Thus, although basaloid hyperplasia suggestive of neoplastic transformation was not observed in the bulge during the time frame of this experiment, at least some cells in this stem cell compartment activated expression of proliferation markers in response to GLI2ΔN. Given the lack of basaloid hyperplasia in bulge cells (Figure 4A) and the absence of bulge-derived microscopic tumors (Figure 1E), we performed immunostaining to test whether GLI2ΔN expression leads to apoptosis. In contrast to controls, bulge compartments of K14;GLI2_Δ_N mice treated with doxycycline for 5 days contained cells expressing activated caspase-3 (Figure 4F). Thus, the selective resistance of bulge cells to GLI2ΔN-induced hyperplasia and tumorigenesis may be due to elimination via apoptosis of aberrantly proliferating cells in this stem cell compartment.
In addition to bulge cells, the central isthmus (Figure 1B), which contains the recently described Lgr6+ stem cell compartment (44), was also relatively unaltered in K14;GLI2_Δ_N mice (Figure 4A). Remarkably, despite the predicted expression pattern of the K14 promoter, which is expected to target all basal layer compartments (Supplemental Figure 1), basal cells in the central isthmus frequently had strikingly reduced or undetectable levels of GLI2ΔN relative to other basal layer populations (Figure 4B, brackets). Similar results were seen in K5-rtTA;tetO-GLI2_Δ_N mice (data not shown), in which transgene expression is also targeted to basal cell compartments in skin. While these results are in keeping with the idea that Hh signaling is tightly regulated in different stem cell compartments of the resting follicle via modulation of Gli protein levels (45–47), they may also reflect a reduction in or loss of K14 and K5 promoter activity in this region of the resting hair follicle. Collectively, the results of short-term GLI2ΔN induction studies indicate that, with the notable exception of cells residing within the bulge and possibly the central isthmus, multiple epithelial progenitor or stem cell populations in skin can give rise to basaloid hyperplasia following induction of GLI2ΔN expression, and that cells in each of these compartments may be competent to form BCC-like skin tumors.
BCC-like skin tumors can arise from hair follicle, sebaceous gland, and epidermal lineages. Because the severe phenotype of K14;GLI2_Δ_N mice precluded analysis beyond 10 days of transgene induction, to identify cell populations in skin that are competent to form BCC-like tumors, we again used the triple-transgenic model (Figure 1A), but with a conditional K5-CreER driver that can be activated in basal cell compartments in skin (Figure 5A, Supplemental Figure 1, and ref. 48). Low-dose tamoxifen was administered to K5-CreER;R26-LSL-rtTA;tetO-GLI2_Δ_N (iK5;rtTA;GLI2_Δ_N) mice to drive recombination in a limited number of K5-expressing cells, and GLI2ΔN expression was induced with doxycycline. Using this approach, we first noted skin abnormalities after 14 days, and by 21 days of treatment, histology revealed BCC-like tumors in skin from multiple body sites, including the tail, snout, dorsal paws, ears, and hairless volar skin (Figure 5B). As predicted based on cell populations responding to short-term GLI2ΔN induction (Figure 4), tumors appeared to arise from secondary hair germs, sebaceous glands, and epidermis, with epidermis-associated tumors (Figure 5B) resembling human superficial BCC. Taken together, these data reveal that oncogenic Hh signaling can drive BCC-like skin tumor development from several different epithelial progenitor populations in skin, although the BCC subtype (superficial versus nodular) is defined by the cell of origin (epidermis versus hair follicle, respectively).
Induction of GLI2ΔN expression leads to BCC-like tumors derived from all 3 epithelial skin lineages. (A) Widespread potential recombination pattern for K5-CreER transgenic driver used to generate iK5;rtTA;GLI2_Δ_N mice. (B) Treatment with low-dose tamoxifen to drive recombination in a limited number of cells yielded BCC-like tumors in tail arising from epidermis (1), sebaceous gland (2), and lower follicle (3), with the bulge compartment (asterisk) unaffected. Superficial BCC-like tumor development in hairless skin confirms that these tumors can arise from interfollicular epidermis. Original magnification, ×200 (tail and ear); ×100 (dorsal paw and snout); ×400 (volar).
Accelerated BCC-like tumor development from anagen hair follicles. Follicles during the second, prolonged telogen phase can be induced into a synchronized and premature anagen phase by either chemical (Nair) or physical (hair plucking) depilation, providing a convenient method for triggering synchronized hair follicle growth with precise timing (Figure 6A). Previous studies suggested that anagen hair follicles are particularly susceptible to Hh pathway–driven tumorigenesis (29, 32), raising the possibility that activated follicle stem cells or transit-amplifying progeny are tumor progenitors in this setting. To examine this possibility, we induced GLI2ΔN expression in iK15;rtTA;GLI2_Δ_N or iLgr5;rtTA;GLI2_Δ_N transgenic mice in early anagen, rather than telogen (Figure 6A). Dorsal hair was clipped during the second telogen phase of the hair cycle; unscheduled anagen was induced using either Nair or hair plucking; and 1 day later, GLI2ΔN transgene was induced by administration of doxycycline. As expected, induction of anagen in control mice led to hair follicle elongation and grossly apparent hair growth, whereas anagen-induced regions of skin in iK15;rtTA;GLI2_Δ_N mice contained grossly evident tumors by 14–15 days of transgene expression (Figure 6, B and C). Telogen follicles in surrounding skin were largely unaffected (Figure 6C), with the exception of occasional microscopic tumors as described above (Figure 1E). Similar results were seen using iLgr5;rtTA;GLI2_Δ_N mice (data not shown). The differential response of telogen versus anagen follicles was highly consistent: telogen dorsal skin of all iK15;rtTA;GLI2_Δ_N mice examined (n = 7) appeared grossly normal after 2 weeks of GLI2ΔN induction, while 100% of mice (n = 18) with anagen-induced dorsal skin had gross evidence of tumor development at the same time point. The accelerated development of BCC-like tumors in anagen versus telogen hair follicles does not appear to be related to gross differences in GLI2_Δ_N transgene expression, protein accumulation, or transcriptional activity, based on in situ analysis of Hh/Gli target genes Gli1 and Ptch1 (Supplemental Figure 2). Together, these experiments provide evidence that BCC development occurs preferentially (but not exclusively) during the anagen phase of the hair cycle (29, 32) and are the first to our knowledge to directly test this concept using a conditional mouse model to activate oncogenic Hh signaling at different stages of the hair cycle.
Anagen accelerates GLI2ΔN-driven tumorigenesis. (A) Hair growth cycle in dorsal skin showing timing of depilation (to induce anagen) and doxycycline treatment (to activate GLI2ΔN expression in either early or mid-anagen). (B) Depilation of control mice activates hair follicle growth, with anagen follicles extending into the subcutaneous adipose layer. Tangential section (right panel) shows outer root sheath compartment of the anagen follicle (arrowheads) and pigmented hair shafts (asterisks). Original magnification, ×100 (left and middle panels); ×400 (right panel). (C) Induction of GLI2ΔN in iK15;rtTA;GLI2_Δ_N mice during early anagen leads to widespread tumor development from growing hair follicles in 2 weeks, at a time when spontaneous tumors in adjacent skin are rare (left panel). In some areas, tumors are contiguous with and appear to replace the outer root sheath compartment of the anagen follicle (arrowheads in right panel). Original magnification, ×40 (left panel); ×100 (middle panel); ×400 (right panel). (D) GLI2ΔN induction for 1 week in mature hair follicles (mid-anagen) shows tumor derivation directly from the outer root sheath. Immunostaining for transgene (right panel) reveals GLI2ΔN expression in all regions exhibiting BCC-like changes. Dashed lines delineate hair follicles. Typical BCC-like tumors develop after an additional week of transgene expression (lower panels). Original magnification, ×100 (upper-left panel); ×400 (upper-middle and -right panels); ×40 (lower-left panel); ×100 (lower-middle panel); ×400 (lower-right panel).
BCC-like tumors can arise from the anagen follicle outer root sheath. Tumor cells frequently appeared contiguous with the follicle outer root sheath (Figure 6, B and C), raising the possibility that this transit-amplifying progenitor cell population (32) is a potential source of tumor progenitors in anagen. To address whether tumor nodules could arise directly from outer root sheath cells, we delayed transgene activation until 7 days after anagen induction (mid-anagen, Figure 6A) to allow for the proliferative expansion needed to assemble a mature anagen hair follicle (41). One week after transgene induction in mature anagen hair follicles, microscopic, GLI2ΔN-expressing tumor nodules were detected arising directly from the outer root sheath (Figure 6D), supporting the idea that this compartment contains cells capable of transformation by deregulated Hh/Gli signaling and may provide an expanded pool of potential tumor progenitors that accounts for the increased incidence of BCCs during anagen (29, 32). After an additional week of GLI2ΔN expression, large, nodular tumors had formed that filled much of the dermis in regions where anagen hair growth had been activated (Figure 6D).
Activated SMO is a weak oncogene in mouse skin that can be mimicked by low-level GLI2ΔN. Our findings using GLI2ΔN as the oncogenic driver establish that hair follicle stem cells in telogen are competent to form nodular skin tumors and that tumorigenesis is accelerated during anagen. We had previously shown that oncogenic human SMO (M2SMO) is a weak Hh pathway activator in skin and yielded basaloid follicular hamartomas rather than nodular BCCs or other BCC-like tumors, which are associated with high-level Hh signaling activity in both mice and humans (35, 36, 49). To further explore this issue in the context of stem cell targeting, we used tetO-SmoA1 mice that we had previously generated (50) to produce K15-CrePR1;R26-LSL-rtTA;tetO-SmoA1 (iK15;rtTA;SmoA1) triple-transgenic mice, and again performed transgene induction studies during early anagen (Figure 7A). In striking contrast to the nodular tumors seen in anagen-activated dorsal skin of _GLI2_Δ_N_-expressing mice (Figure 6C), mice expressing SmoA1 exhibited modest outer root sheath hyperplasia (Figure 7B) but no evidence of frank tumor development. These findings indicate that SmoA1, using the same modeling strategy that yields robust tumors in response to GLI2ΔN, is not sufficient to drive BCC-like tumorigenesis from K15+ follicle stem cells or their progeny.
Oncogenic Smo drives outer root sheath hyperplasia but not nodular BCC-like tumor development. (A) Schematic depicting timing of depilation and transgene activation in iK15;rtTA;SmoA1 mice. (B) In contrast to analogous GLI2ΔN-expressing mice, which produce nodular BCC-like tumors when the same experimental approach is used (Figure 6C), activation of SmoA1 in early anagen leads to hyperplasia of the outer root sheath (compare distance between arrowheads in upper- and lower-right panels) but no evidence of BCC-like tumors. Original magnification, ×200.
We previously proposed that the absence of nodular BCC-like tumors in mice expressing activated SMO was due to relatively weak oncogenic signaling, based on the low expression level of Gli target genes in lesions resembling basaloid follicular hamartomas (arising in SMO mice) compared with BCCs (arising in Gli2 mice) (35). To directly test whether the level of Gli transcriptional activity is a determinant of tumor phenotype, we modified the doxycycline dosing regimen to induce different levels of GLI2ΔN in skin. Whereas GLI2ΔN-expressing mice treated with standard doses of doxycycline (GLI2ΔN-high) produced nodular and superficial BCC-like tumors, mice treated with low-dose doxycycline (GLI2ΔN-low) developed slow-growing basaloid tumors similar to those in mice expressing oncogenic SMO in skin (Figure 8A and refs. 35, 36). Although these lesions resemble human basaloid follicular hamartomas in several respects (49, 51, 52), as previously discussed (35), we favor a more conservative classification as basaloid hamartomas, with variable degrees of sebaceous hyperplasia.
Low-level expression of GLI2ΔN gives rise to basaloid hamartomas instead of nodular BCC-like tumors. (A) Histology showing nodular BCC-like tumors in iK5;rtTA;GLI2_Δ_N mouse treated with 1 gm/kg doxycycline in chow and 200 μg/ml doxycycline in drinking water (GLI2ΔN-high), compared with basaloid hamartomas in K5;rtTA;GLI2_Δ_N mice treated with 2 μg/ml doxycycline (GLI2ΔN-low) in drinking water, which resemble lesions that arise in mice harboring a Cre-inducible M2SMO allele (SMO). (B) Ki67 reveals limited proliferation at the periphery of basaloid hamartomas arising in GLI2ΔN-low and SMO mice, compared with diffuse proliferation in nodular BCCs in GLI2ΔN-high mice. Dashed lines in right panels outline the extent of epithelial cells comprising hamartomas. HF indicates an anagen hair follicle matrix with robust Ki67 immunostaining. (C) MYC immunostaining to detect GLI2ΔN confirms low-level expression in GLI2ΔN-low mice. Original magnification, ×200 (A–C).
Immunostaining for Ki67 revealed diffusely increased proliferation in BCC-like tumors in GLI2ΔN-high mice, with a low level of proliferation limited to the periphery of basaloid hamartomas in GLI2ΔN-low or SMO mice (Figure 8B), reflecting findings in human BCC and basaloid follicular hamartomas (49). MYC immunostaining to detect GLI2ΔN confirmed markedly reduced expression levels in hamartomatous proliferations arising in GLI2ΔN-low mice relative to uniformly robust expression in BCC-like tumors in GLI2ΔN-high mice (Figure 8C). These results support the concept that low-level oncogenic Hh/Gli signaling is an effective inducer of basaloid hamartomas, but not the nodular BCC-like skin tumors that are seen with higher-level signaling (25, 26, 31, 33, 35, 45).