Defining the origins of Ras/p53-mediated squamous cell carcinoma

The precise identity of cancer cells of origin and the molecular events of tumor initiation in epidermal squamous cell carcinoma (SCC) are unknown. Here we show that malignancy potential is related to the developmental capacity of the initiating cancer cell in a genetically defined, intact, and inducible in vivo model. Specifically, these data demonstrate that SCCs can originate from inside the hair follicle stem cell (SC) niche or from immediate progenitors, whereas more developmentally restricted progeny, the transit amplifying (TA) cells, are unable to generate even benign tumors in the same genetic context. Using a temporal model of tumorigenesis in situ, we highlight the phenotypes of cancer progression from the hair follicle SC niche, including hyperplasia, epithelial to mesenchymal transition, and SCC formation. Furthermore, we provide insights into the inability of hair follicle TA cells to respond to tumorigenic stimuli.

The precise identity of cancer cells of origin and the molecular events of tumor initiation in epidermal squamous cell carcinoma (SCC) are unknown. Here we show that malignancy potential is related to the developmental capacity of the initiating cancer cell in a genetically defined, intact, and inducible in vivo model. Specifically, these data demonstrate that SCCs can originate from inside the hair follicle stem cell (SC) niche or from immediate progenitors, whereas more developmentally restricted progeny, the transit amplifying (TA) cells, are unable to generate even benign tumors in the same genetic context. Using a temporal model of tumorigenesis in situ, we highlight the phenotypes of cancer progression from the hair follicle SC niche, including hyperplasia, epithelial to mesenchymal transition, and SCC formation. Furthermore, we provide insights into the inability of hair follicle TA cells to respond to tumorigenic stimuli. mouse models of cancer | tumor initiating cells | epidermal stem cells S quamous cell carcinoma (SCC), a common type of nonmelanoma skin cancer, harbors significant risk of metastasis that can eventually lead to death (1). Permanent treatment and prevention of SCC requires an improved understanding of the unique cell types capable of initiating these malignancies and the mechanisms that underlie their transformation. Mutations in the Ras gene family are found in 30% of all human cancers and are often found in human cases of SCC (2)(3)(4). Numerous animal studies have extended the understanding of SCC by demonstrating a causative role for the Ras family in the development of SCC. In these mouse models, Ras signaling has been shown to be sufficient to drive epidermal tumorigenesis through either overexpression or targeted expression of an oncogenic form of Hras or Kras. These studies have targeted broad epidermal cell populations that include stem cells (SCs), as well as cells of the outer root sheath (ORS) and interfollicular epidermis (IFE) (4)(5)(6)(7)(8)(9). Among these, a landmark article indicated that the SCC cells of origin reside in the hair follicle rather than the IFE (5). However, the keratin promoter used to drive oncogenic Ras expression did not allow for a direct determination of the cancer cell of origin amongst the different cell types in the hair follicle. The candidates for an SCC cell of origin therefore remained follicular SCs, ORS cells, transit amplifying (TA) cells in the matrix, and the differentiated cells of the inner root sheath (IRS) and hair shaft. In addition, other studies have shown that lineagerestricted or differentiated cells of the IFE are sufficient to serve as cancer cells of origin when subjected to supraphysiological levels of Ras activity at sites of mechanical stress (10). These high levels of Ras expression, however, are not normally found in human cases of SCC (10,11). Between follicular SCs and TA cells, it is not clear whether one or both are capable of initiating tumorigenesis. Thus, we sought to directly compare the malignancy potential between follicular SCs and TA cells.
Recent evidence suggests that a range of cell types populate the SC niche of the hair follicle, each capable of reconstituting different portions of the hair follicle. These subpopulations each exhibit SC characteristics and have at least one unique expression marker (12)(13)(14)(15)(16). Lgr5 marks the bottom of the bulge during telogen (resting phase of the hair cycle) and the entire ORS during anagen (growth phase) (13). Lgr6 marks the top of the bulge, exclusive of the LGR5 population, and represents a slower dividing subset of cells during telogen (14). A fragment of the keratin 15 promoter has been shown to be active across the entire bulge in telogen and anagen and in hair germs during telogen (15,17). Because the K15CrePR allele seems to be specific to the bulge during anagen, whereas the LGR5-Cre-eGfp allele marks the entire ORS during anagen, we used the K15CrePR allele to manipulate gene expression specifically in the bulge at any point in the hair cycle.
The inducible mouse model used here allows for a direct spatial and temporal comparison of tumorigenic potential between SCs and TA cells of the hair follicle. This model also enables a precise characterization of the earliest steps of oncogenic Ras and Ras/ p53 (oncogenic Ras expression with p53 gene deletion) induced tumorigenesis, including stages of hyperplasia, epithelial to mesenchymal transition (EMT), and dedifferentiation. Finally, we analyze downstream signaling pathways of Ras in different target cells to highlight the role these pathways play in Ras and Ras/p53induced tumorigenesis.

Results
To determine the cellular requirements for SCC, we generated an inducible, in vivo model system that allows for expression of an oncogene and deletion of a tumor suppressor in either the SC or the TA cell populations of the intact hair follicle. To target the hair follicle SC compartment, an allele was used that comprises a fragment of the keratin 15 gene regulatory unit driving to the CrePR gene. This allele is active in the hair follicle bulge, where both LGR5 + and LGR6 + cells reside, and is known to be the primary site for multipotent follicular SCs ( Fig. 1 A and B and Fig. S1A) (17,18). To target the TA cell compartment, the ShhCreER knockin allele was used, because it has been shown to be active in a subset of hair follicle matrix cells and not in other lineages of the epidermis ( Fig. 1 A and B and Fig. S1C) (19,20). ShhCreER + matrix cells are both rapidly dividing and produce the differentiated IRS of the follicle and thus meet the definition of TA cells.
The K15CrePR and ShhCreER inducible alleles were used in combination with the Lox-Stop-Lox-Kras G12D (LSL-Kras G12D ) knockin and a floxed p53 allele to induce tumorigenic transformation in a cell type-specific manner during murine adulthood (21)(22)(23)(24). Importantly, this oncogenic form of the Kras gene is driven by the endogenous locus and therefore does not result in supraphysiological expression of a transgene. Because murine and human SCC have been linked to activating mutations in Ras family members (including both Hras and Kras) and mutations in p53 (2,4,5,(25)(26)(27)(28)(29)(30)(31), the LSL-Kras G12D allele alone or with floxed p53 alleles was used in conjunction with either K15CrePR or ShhCreER to determine the relative capacity of hair follicle SCs vs. lineage-restricted TA cells to serve as epidermal cancer cells of origin in SCC. Although this same LSL-Kras G12D allele has been used to induce tumorigenesis in the epidermis previously, a direct comparison of SC vs. TA was not performed (6,9).
First, to determine the effect of targeting oncogenic Kras G12D expression specifically to either the hair follicle SC niche or a more restricted TA population, the mating strategy outlined in Fig. 1B was used. Hair follicles undergo cycles of growth (anagen), rest (telogen), and degeneration (catagen) throughout the life of the animal. Animals were treated with either tamoxifen or mifepristone at the onset of anagen, as judged by regrowth after shaving. Six to 10 weeks after treatment with mifepristone (K15CrePR) or tamoxifen (ShhCreER) by i.p. injection, the epidermis of K15CrePR; LSL-Kras G12D (K15CrePR; Kras G12D ) mice consistently exhibited structures that appeared as keratin-filled cysts and displayed widespread hyperplasia of the hair follicle and sebaceous gland (n = 18 of 18 animals; Fig. 1C). Furthermore, affected follicles never returned to telogen. These phenotypes were found in both dorsal and ventral epidermis, although cysts appeared to be much more prevalent in the ventral epidermis (Fig. 1D). Some of these phenotypes were consistent with a recent report that also targeted this population with the same tumorigenic Kras allele (32).
In contrast, induction of the same oncogenic stimulus in a portion of the TA cells of the hair follicle matrix during anagen in ShhCreER; LSL-Kras G12D (ShhCreER; Kras G12D ) mice resulted in no abnormal epithelial structures, and follicles returned to telogen within a 10-wk timeframe (n = 9 of 9; Fig. 1C). These data suggest that when the Kras G12D oncogene is expressed in the hair follicle SC niche, benign tumor formation ensued, whereas the same stimulus delivered to Shh-expressing matrix TA cells was unable to generate any signs of tumorigenesis. These data indicate that bulge cells or their immediate descendants, before becoming lineage-restricted TA cells, can serve as cells of origin for epidermal tumorigenesis.
To confirm that Cre activity independent of mifepristone or tamoxifen administration did not confound this model system, untreated controls containing the appropriate tumorigenic alleles were examined. Many untreated K15CrePR; Kras G12D animals did eventually develop hair follicle hyperplasias and follicular cysts, but these were less severe and occurred only at timepoints well beyond those used for the analyses described in this report (Fig. 1D). This effect was caused by uninduced translocation of CrePR to the nucleus, not because of expression from the K15 promoter at sites other than the follicular bulge, as determined by lineage tracing (see below; Fig. S1 A and B).
To verify the fidelity of transgene expression specifically to bulge SCs and matrix TA populations, K15CrePR and ShhCreER mice were crossed to LSL-YFP mice to perform lineage tracing (33). Consistent with previously published work, these two methods generated YFP expression specifically in either the hair follicle SC niche and hair germ or in matrix TA cells after a brief pulse with mifepristone or tamoxifen ( Fig. S1 A and C) (20). ShhCreER; LSL-YFP animals further showed lineage tracing of YFP from the TA compartment to the precortex, the IRS, rarely to the cortex and cuticle, and never to the companion layer ( Fig.  S2 A and B).
To determine the rate of recombination induced by Cre in K15CrePR and ShhCreER skin, we used the YFP allele to calculate recombination efficiency on a per cell per hair follicle basis. A similar level of recombination was found in these two mouse lines: K15CrePR skin exhibited 17.4 ± 2.6 YFP + cells per bulge (34.0 ± 4.7%, across 17 follicles), and ShhCreER skin showed 18.2 ± 1.4 YFP + cells per Shh-expressing matrix (36.7 ± 2.1%, across 18 follicles). To confirm that the Kras G12D allele was In addition, a number of relevant cell types are shown (cp, companion layer; cx, hair cortex; ors, outer root sheath; dp, dermal papillae; mx, matrix; ge, hair germ). (B) Model of the targeting strategy to express oncogenic Kras G12D in either bulge SCs or matrix TA cells. (C) Histopathology of phenotypes induced by Kras G12D expression in the follicular bulge, including hyperplastic sebaceous glands (i), hyperplastic hair follicles (ii), and follicular cysts (iii). No apparent abnormal hair follicle phenotypes were detected in ShhCreER; Kras G12D skin. (Magnification, 10×.) (D) Quantification of phenotypes in treated and untreated mice. The indicated phenotypes were quantified on a per-follicle basis on the indicated genotypes with or without mifepristone treatment. The timecourse indicates the number of weeks after anagen, as measured by regrowth after shaving. The number of phenotypes found in treated animals was significantly increased over those found in untreated animals, even at timepoints far later than those used for phenotypic analysis in treated animals. *P ≤ 0.049). expressed in Cre-activated YFP + populations, we performed RT-PCR on YFP + and YFP − sorted cells to demonstrate that the HindIII restriction site introduced into the Kras allele by the G12D mutation was present (34). This Kras G12D -specific HindIII restriction site was enriched in the YFP + population but not found in the Kras G12D YFP − population or in wild-type Kras controls (Fig. S2C). Together, these analyses indicated that biased recombination frequency or efficiency could not account for drastic differences in tumorigenic potential of K15 + and SHH + cells.
Lineage tracing performed in other studies with this K15CrePR allele and a Rosa-LSL-LacZ reporter, or with K15-GFP mice, indicated that rare labeled cells can be found in the IFE (17,18). We were rarely able to detect YFP + cells (0.2%, n = 5,772 IFE cells) in the IFE of treated K15CrePR; LSL-YFP animals, even at extended timepoints (Fig. S1B). Furthermore, the phenotypes found in treated K15CrePR; Kras G12D and K15CrePR; Kras G12D ; p53 ff animals during initiation and progression distinctly arose from the hair follicle within the dermis (see below) and occurred at a much higher frequency relative to the leaky expression in the IFE. These results indicated that although the K15 promoter used here may have minor off-target expression in the IFE, these cells did not factor into the interpretation of these experiments.
Immunostaining for total Kras protein in both follicular SC (CD34 + ) and matrix populations also indicated very similar levels of expression in both control and induced animals ( Fig. S3 A  and B). This suggests that the difference observed in the tumorigenic sensitivity between SC and TA compartments is likely not due to a significantly higher level of oncogenic Kras expression in the SC population. Furthermore, mRNA analysis for all Ras family members indicated that whereas Kras was expressed at a similar level in hair follicle bulge and matrix cells, Hras was significantly different between these populations (Fig. S3C); thus, the use of Kras provided a fair comparison by expression levels.
To confirm that tumorigenesis was initiated from the follicular bulge and to better describe the phenotype generated by expression of Kras G12D in hair follicle bulges, broad sections of tissue were isolated and analyzed with a variety of markers of cell identity and proliferation. This approach enabled the visualization of both phenotypic initiation and progression. Temporal histopathological examination revealed that the first event in tumor formation is an expansion of the bulge and hair germ at the onset of telogen to anagen transition ( Fig. 2 and Figs. S3B and S4B). Within these expanded bulges, most cells were CD34 + and Sox9 + , confirming their bulge origin (Figs. S3A, S4B, and S5E). Furthermore, these hyperplastic CD34 + bulges also exhibited YFP fluorescence in many cells, representing allelic recombination from activated Cre expression (Fig. S4B). In full anagen, hyperplasia extended downward and was most apparent in the middle ORS (Fig. S4D). At this point in phenotype progression, CD34 was found to be aberrantly expressed throughout all of the cells of the hyperplastic ORS (Fig. S4D), consistent with previous reports suggesting that CD34 is up-regulated in the progression of SCC (35). These observations are significant because they suggest that SCs do not first travel to the matrix and become TA cells before generating hyperplasia, but that in fact the first phenotype observed is bulge expansion.
Expansion of the bulge region and then the ORS led to either a hyperplastic follicle or a keratin-filled cyst. Bulge and ORS expansion correlated with expression of markers typical of hyperproliferative epidermis such as keratin 6 (Krt6) and Ki67 (Fig. S5 C and D). CAATT displacement protein (CDP), a marker of matrix TA cells (36), excluded a matrix origin for these expanded hair follicles and keratin-filled cysts (Fig. S5F). Furthermore, none of the phenotypes generated seemed to be associated with the IFE across 18 experiments and hundreds of follicles, suggesting that Kras G12D expression and initiation of hyperplasias was confined to the SC niche of the hair follicle in K15CrePR; Kras G12D animals.
To determine the minimum cellular and molecular requirement for SCC in the epidermis, K15CrePR; Kras G12D and Shh-CreER; Kras G12D mice were crossed to mice bearing floxed alleles of the p53 tumor suppressor (6, 23). K15CrePR; Kras G12D ; p53 ff and ShhCreER; Kras G12D ; p53 ff mice were treated with mifepristone or tamoxifen as described for the "one-hit" (Kras G12Donly) model described above (Fig. 3A). In addition to the hyperplastic hair follicles and keratin-filled cysts found in one-hit animals, introduction of these two genetic hits into the SC compartment generated between one and six SCCs per animal (n = 9 of 9; Fig. 3 B and C). This occurred over a similar time course as the benign tumorigenesis observed in K15CrePR; Kras G12D mice. Notably, the IFE in these two-hit animals appeared normal. YFP was detected in the spindle-shaped cancer cells invading the dermis (Fig. 3D), indicating that the SCC was indeed generated by cells receiving the indicated genetic hits and not by genetically wild-type cells stimulated by the surrounding environment. To confirm that the second hit had occurred as expected, YFP + cells isolated from SCCs showed a complete lack of p53 expression (Fig. S6 A and B). This further demonstrated that the Cre allele used is highly efficient and that lineage tracing with the YFP allele is accurate for not only Kras G12D but also for p53 gene deletion.
To determine whether expression of Kras G12D and ablation of p53 in the TA compartment of the hair follicle could also initiate tumorigenesis, ShhCreER; Kras G12D ; p53 ff mice were generated and treated. No epidermal tumors of any kind were generated with this allelic combination (n = 6 of 6) (Fig. 3C). Additionally, to determine whether any changes in the developmental program of the hair follicle could be found in ShhCreER; Kras G12D ; p53 ff mice, markers for proliferation and differentiation of the matrix and its descendants were examined after a brief pulse of tamoxifen. No difference was found in Ki67 (proliferation), CDP (matrix and IRS), Krt31 (precortex, cortex, cuticle), or Krt6 (companion layer) staining (Fig. S6 C-G). Comparison of the histopathology of K15CrePR; Kras G12D ; p53 ff and ShhCreER; Kras G12D ; p53 ff skin demonstrated that bulge cells or their immediate descendants have a high malignancy potential and can serve as cancer cells of origin, whereas their lineage-restricted progeny, the Shh-expressing TA cells, cannot (Fig. 3C).
Examination of the epidermis outside of SCC formations in K15CrePR; Kras G12D ; p53 ff and in regions of K15CrePR; Kras G12D skin showed the presence of hair shafts within the dermis surrounded by spindle-like cells, instead of a distinct ORS (Fig. 3C and Fig. S7 A and B). EMTs are commonly found in developing epithelial cancers and are thought to be essential for invasion of the epithelial cells into an underlying stroma and development of carcinoma (37). We examined a number of markers for EMT to determine whether these spindle-like cells surrounding the displaced hair shafts could be epithelial cells undergoing EMT. Examination of the surrounding area around these cells demonstrated high Tenascin-C (TnC) (Fig. S7 A and B), an extra-cellular matrix protein deposited during EMT (38)(39)(40). TnC is not normally found in the dermis of the skin but was found adjacent to the ORS of anagen follicles and adjacent to the bulge and infundibulum in telogen follicles (Fig. S7A).
EMTs also exhibit decreased expression of epithelial-specific markers, such as keratin 5 (Krt5) and E-cadherin (E-cad) (37,41,42). Cells exhibiting high TnC also demonstrated very low or absent Krt5 and E-cad, consistent with the suggestion that these cells are undergoing EMT (Fig. S7A). Next, markers known to be upregulated during EMT were examined. Vimentin (Vim), one such marker found at low levels in the dermal cells of the skin, was found at very high levels in cells exhibiting high TnC (Fig. S7B). A similar pattern of expression was detected in definable SCCs during cancer progression (Fig. S8). Additionally, keratin 8 (Krt8), a marker of simple epithelium and of spindle cells of SCCs (43,44), was only detected in cells of SCCs (Figs. S6B, S7B, and S8D). This result indicates that Krt8 protein expression is found relatively late during SCC progression. To determine whether cells expressing EMT markers were also proliferating, Ki67 staining was used. We did find colocalization of Ki67 and markers of EMT in some cases, suggesting the possibility that these cells might be capable of driving formation of SCC. As expected, proliferation was abundant in SCCs (Figs. S7B and S8E). In summary, these data indicated that hair follicle SCs of K15CrePR; Kras G12D or K15-CrePR; Kras G12D ; p53 ff mice generate cells that undergo hyperplasia and EMT. Only K15CrePR; Kras G12D ; p53 ff transformed cells, however, were able to form SCCs.
To determine the molecular mechanisms underlying the ability of hair follicle SCs to act as cancer cells of origin in SCC, potential downstream signaling pathways were probed for activation by immunostaining. The Mek/Erk, Akt, and p38 pathways have been shown to be directly downstream of Ras activation in a variety of settings, including oncogenic transformation (45,46). In control skin, phosphorylated Erk (p-Erk) was found in basal cells of the IFE and in the bulges and ORS of hair follicles ( Fig.  4A and Fig. S9A). p-Erk was weakly expressed in bulges during the initial stages of hyperplasia, very strongly in hyperplastic anagen follicles, only weakly in the basal cells of cysts, and not within cells of the SCC (Fig. 4A and Fig. S9A). Aberrant expression of p-Erk was not found in ShhCreER; Kras G12D ; p53 ff induced matrix or IRS cells (Fig. S10B).
Akt activity is sufficient to induce skin tumors in vivo (47)(48)(49)(50)(51), and Akt activity is increased during SCC progression initiated by chemical carcinogenesis (54). Akt pathway activation was detected by p-Akt in the suprabasal cells of epithelial cysts and  was found in remnant follicular structures within SCCs (Fig. 4B). Control skin showed p-Akt in only a few cells in the telogen bulge and in a subset of non-ORS cells near the bulge during anagen (Fig. 4B and Fig. S9B). This p-Akt population was expanded in hyperplastic bulges but was not found in early anagen ORS hyperplasias (Fig. S9B). Furthermore, p-Akt was never detected in the hair follicle TA population, in any context (Fig.  S10B). To determine the status of further downstream signaling, a number of phosphorylated cell signaling proteins were examined for activity. In control skin, p-S6 was detected in the precortex and IRS of hair follicles but not in the SC or TA niche ( Fig. 4C and Figs. S9C and S10B). p-S6 was also present in hyperplastic cysts and at low levels in hyperplastic bulges, the ORS of hyperplastic anagen follicles, and SCCs of K15CrePR; Kras G12D ; p53 ff mice ( Fig. 4C and Fig. S9C). p-Ikkα/β and p-Nfκb, downstream of Akt, were also detected in hyperplastic hair follicles and cysts but were not found in control hair follicles (Fig. S10C). Taken together, it seems that mutant Kras expression activates Erk during follicular hyperplasia and Akt/S6 signaling during cyst formation. The fact that the hair follicle TA population seemed refractory to Ras signaling pathway stimulation upon induction suggests a mechanism used by TA cells to prevent tumorigenesis in this system.

Discussion
The results of this study have several important implications. First, these data reinforce previous studies suggesting that SCC can arise from the hair follicle (3) and demonstrate that SCCs probably arise from follicular SCs more often than from any other type of cell. Second, the data shown here demonstrate that cells of hair follicle bulge SC niche, or cells immediately exiting the bulge, can serve as the origin of SCC. Third, Shh-expressing TA cells of the hair follicle cannot serve as SCC cells of origin, at least not with the tumorigenic load provided by this model system. Fourth, although physiological expression of oncogenic Ras can drive formation of hyperplasia and EMT, a second hit seems to be required for the development of SCC. Fifth, this study provides evidence that both the Erk and Akt pathways are activated during Ras-induced tumorigenesis from hair follicle SCs. Finally, hair follicle TA cells seem to be refractory to stimulation by the Ras pathway, suggesting a molecular explanation for their inability to drive tumor formation in this model.
The data presented here directly compare the relative susceptibility of bulge cells and their more restricted progeny to serve as cancer cells of origin in SCC; these results are consistent with data from cancers of other tissues, such as the intestine, blood and brain (51)(52)(53). Similar to these other studies, SCs are more likely to serve as cancer cells of origin than their more restricted progeny.
Because TA cells are only found transiently, it is also possible that they are not present for a sufficient time to initiate hyperplasia before undergoing hair cycle-mediated apoptosis. This could indicate that TA cells in general do not persist long enough to serve as cancer cells of origin and would provide a simple explanation for the discrepancy between TA cells and SCs as tumor initiators. However, we cannot detect any effect of oncogenic Kras expression in the TA cells immediately after anagen initiation, at which time bulge cells have already initiated hyperplasia ( Fig. 2 and Figs. S3 and S4) and some weak activation of downstream kinase pathways (Fig. S9). Additionally, Shh-expressing TA cells are only found on one side of the matrix (Figs. S1C and S6A) and contribute to the IRS but not to the companion cell layer and only rarely to the cortex and cuticle layers (Figs. S2 and S6). Therefore, we cannot exclude the possibility that non-Shh-expressing matrix cells can act as cancer cells of origin.
These data also do not exclude the possibility that Shhexpressing TA cells of the hair follicle or other cells within the IFE could serve as cancer cells of origin in other contexts. Lapouge et al.(50) demonstrated that targeting of the same Kras allele to the IFE is capable of driving benign papilloma formation. It is possible that hair follicle TA cells could generate cancer upon introduction of different genetic lesions or the addition of environmental insults, such as inflammation. Along these lines, previous studies showed that transgenic expression of oncogenic Hras in the differentiated compartment of the IFE was sufficient to drive tumorigenesis in areas of high mechanical stress or wounding (10,54). Additionally, a recent study highlighted this tumorigenic mechanism in a model of pancreatic cancer. In that model, certain cell populations were refractory to transformation until exposed to an inflammatory environment generated by wounding (55). This raises the question of whether a tumorigenic load exists that can drive cancer formation in the Shh-expressing hair follicle TA population and represents a direction for future experiments.

Materials and Methods
Animals. Animals were acquired from Jackson Labs (K15CrePR and ShhCreER) or the National Cancer Institute Mouse Models of Human Cancers Consortium repository (LSL-Kras G12D and p53 ff ) and maintained under conditions set forth by the Institutional Animal Care and Use Committee and the Animal Research Committee (ARC) (University of California, Los Angeles). K15CrePR; Kras G12D and K15CrePR; Kras G12D ; p53 ff animals were treated by i.p. injections of mifepristone (10 mg/mL dissolved in sunflower seed oil, 2 mg per day) immediately before the onset of the second adult hair cycle (roughly 10 wk postnatal, as measured by regrowth after shaving), for 3-5 d. ShhCreER; Kras G12D and ShhCreER ;Kras G12D ; p53 ff animals were treated with tamoxifen for 3-5 d (10 mg/mL, 2 mg per day) at the beginning of the second adult hair cycle (anagen), as measured by regrowth after shaving. Phenotypes shown from treated animals were produced 6-10 wk after anagen. Because of Shh expression in tissues other than the skin, soft-tissue sarcomas did develop over longer periods of time and were similar to those described previously (62).