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[Cancer Research 66, 2081-2088, February 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Patched1 Functions as a Gatekeeper by Promoting Cell Cycle Progression

Christelle Adolphe, Rehan Hetherington, Tammy Ellis and Brandon Wainwright

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

Requests for reprints: Brandon Wainwright, Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, Queensland, Australia 4072. Phone: 61-7-3346-2053; Fax: 61-7-3346-2101; E-mail: B.Wainwright{at}imb.uq.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of ß-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G1-S phase and G2-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a "gatekeeper." In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors. (Cancer Res 2006; 66(4): 2081-8)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Perturbation of Patched pathway activity has been observed in several tumor types, including medulloblastoma, pancreatic adenoma, digestive tract tumors other than colon cancer, prostate cancer, and small cell lung cancer (1), clearly indicating that the pathway plays an important role in regulating cell proliferation. The most common tumor associated with inappropriate Patched pathway activity is basal cell carcinoma (BCC) of the skin, originally identified through mutation of the human Patched1 (PTCH1) receptor in both familial and sporadic BCC (2–6). Ptch1 encodes a 12-pass transmembrane glycoprotein that functions as a negative regulator of the Sonic Hedgehog (Shh) signaling pathway. Binding of Shh ligand to the Ptch1 receptor liberates Smoothened activity, which ultimately leads to the release and nuclear translocation of activator and repressor forms of the Gli family of zinc finger transcription factors (Gli1, Gli2, and Gli3). Inactivation of Ptch1 via loss of function mutation leads to constitutive pathway activation due to unrestrained Smoothened activity. A diverse range of downstream targets have been identified in mammals, including members of other developmental pathways, such as Wnt, BMP, and Notch, in addition to genes involved in cell cycle progression, apoptosis, and migration. However, it is important to note that there are relatively few universal target genes across different developmental systems; hence, the molecular mechanism by which pathway activation functions to promote tumor progression remains a controversial issue. Mice null for Ptch1 die during the early stages of embryonic development (at E9.5) due to severe developmental anomalies in embryonic patterning (7). The biology of Ptch1-induced skin tumors therefore have predominately employed UV irradiation of Ptch1 heterozygous (Ptch1+/–) mice (8–10). UV irradiation, however, can induce other cancer promoting effects, such as activation of the p53 pathway.

To conclusively determine the effects of cell autonomous Patched pathway activation and specifically explore the mechanism by which Ptch1 functions as a tumor suppressor and gatekeeper in skin tumor formation, we generated mice homozygous for a conditional null Ptch1 allele (Ptch1neo/neo; ref. 11) and induced the conditional ablation of Ptch1 in the skin using the Keratin6-Cre (Krt6a-Cre) promoter (12). In normal adult skin, Keratin6 (K6) is constitutively expressed in a small region of the hair follicle known as the companion cell layer (CCL; ref. 13), located between the differentiated inner root sheath cells and the more primitive outer root sheath (ORS) cells (contiguous with the basal cell layer of the interfollicular epidermis or IFE). However, K6 expression is induced into the basal cell populations of the IFE and ORS in response to epidermal wounding and chemical challenge with retinoic acid (RA; refs. 13, 14). Hence Krt6a-Cre:Ptch1neo/neo double transgenic mice allow us to compare the effects of specifically ablating Ptch1 in the discrete CCL region of the hair follicle versus eliminating Ptch1 from the entire epidermal basal cell compartment (IFE and ORS) following topical RA application. Here, we show that Ptch1 expression in the CCL plays an important role in maintaining ORS homeostasis, whereas loss of Ptch1 in the basal cell compartment results in the development of BCC-like lesions within 4 to 16 weeks. These results further define the origin of BCC and confirm, in vivo, that loss of Ptch1 is sufficient to induce skin tumor formation, thereby supporting the role of Patched1 as a "gatekeeper" in tumor formation. To elucidate the mechanism of Ptch1-induced skin tumor formation, we screened for changes in the cellular and spatial distribution of several candidate genes, including ß-catenin, members of the Notch pathway and the cell cycle regulators cyclin B1 and D1. Although loss of Ptch1 has no effect on the spatial distribution of Notch signaling components and has no effect on the translocation of ß-catenin into the nucleus, basal cells lacking Ptch1 exhibit increased proliferative activity and have a higher incidence of nuclear cyclin D1 and cyclin B1 expression. These results suggest that the mechanism of Ptch1 induced tumorigenicity is unlikely due to perturbation of other developmental pathways (Notch or Wnt) and points towards cell cycle regulation as the primary mechanism of neoplastic transformation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation and induction of Krt6a-Cre:Ptch1neo/neo transgenic mice. Mice homozygous for the Patched1 conditional allele (Ptch1neo/neo; ref. 11) were mated to mice carrying the Cre recombinase gene under the control of the inducible K6 promoter (Krt6a-Cre; ref. 12). Double transgenic animals (Krt6a-Cre:Ptch1neo/neo) were screened by PCR to confirm homozygosity for the Ptch1 null modified allele (primer sequences as per Ellis et al. 11) and presence of the Krt6a-Cre transgene (primer sequences as per Smyth et al. 12). Krt6a-Cre:Ptch1neo/neo transgenic animals (n = 10) and Ptch1neo/neo littermate controls (n = 6) ages 32 days were anesthetized and a 2-cm2 region of pelage follicles shaved from the left and right ribcage area. The right-hand side of each mouse was challenged with an initial dose of 30 µg of RA diluted in 50 µL acetone and the left-hand side treated with acetone alone (control). Mice were exposed to a second 30 µg dose of RA or acetone 48 hours later and allowed to recover for 7 days.

Skin biopsy. Skin biopsies were done 4 weeks after treatment as published (15). Half the skin specimen was embedded in standard ornithine carbamyl transferase (Tissue-Tek) and processed for alkaline phosphatase and LacZ detection as described (16). In parallel, the other half of each specimen was fixed in 1% PFA for 4 hours and processed for paraffin embedding. Collecting biopsies at the border of normal untreated skin minimized adverse wounding effects. Mice were photographed 16 weeks after treatment, and the remaining treated skin samples were processed as above. Untreated Krt6a-Cre:Ptch1neo/neo epidermal samples from the base of the tail were collected to control for any adverse effects of acetone and/or RA treatment.

Histology and RNA in situ hybridization. Histologic analyses were done on 4-µm, paraffin-embedded skin sections stained with H&E. RNA in situ analyses were done on 14-µm, paraffin-embedded skin sections using Ptch1 and Gli1 DIG-labeled RNA probes as previously described (15).

Immunofluorescence and immunohistochemistry. Antibody markers were analyzed on 4-µm, paraffin-embedded skin sections via standard immunofluorescence and immunohistochemistry techniques using the following primary antibodies: K14, 1:10,000 (Covance, Princeton, NJ); K6hf(Bax-1), 1:1000 (gift from L. Langbein, German Cancer Research Centre, Heidelberg, Germany); Ptch1, 1:200 (17); ß-catenin, 1:1000 (Sigma, St. Louis, MO); anti–active ß-catenin, 1:100 (Upstate, Lake Placid, NY); cyclin D1, 1:75; Notch 1, 1:200; Notch 2, 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA); Hes1, 1:800 (gift from T. Sudo, Toray Industries, Kawasaki, Japan); cyclin B1, 1:100 (gift from B. Gabrielli, Centre for Immunology and Cancer Research, The University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia); proliferating cell nuclear antigen (PCNA), 1:100 (Zymed, South San Francisco, CA); Gli1, 1:200 (Abcam, Cambridge, MA). High-temperature unmasking (Vector Labs, Burlingame, CA) was done in a boiling water bath for 60 seconds (30 minutes; Gli1), and MOM detection was used to block nonspecific binding of mouse primary antibodies (Vector Labs). Evaluation of double-immunostaining: basal cells residing in the IFE (not associated with sites of hyperplasia or tumor invagination) were scored for the presence or absence of Ptch1 protein expression and subsequent nuclear localization of cyclin D1 and cyclin B1 (scored via 4',6-diamidino-2-phenylindole or methyl green colocalization respectively). A minimum of 50 Ptch1-positive or Ptch1-null cells were sampled in triplicate for each skin sample.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Epidermal expression profile of the Hedgehog receptor: Ptch1. The expression profile of the Ptch1 receptor defines the population of cells that are responsive to Hedgehog and thus identifies the likely origin of Patched-induced tumors. Relatively little data exist describing the endogenous expression of Ptch1 protein in the skin with current Ptch1 protein expression data derived from LacZ expression profiles of heterozygous Ptch1 mutant mice (7). Here, we describe the expression profile of Ptch1 in mouse epidermis using a recently characterized Ptch1-specific antibody (17). We observed constitutive Ptch1 expression in the basal layer of the IFE (Fig. 1A and F) and the "permanent" upper portion of the hair follicle ORS (Fig. 1B, dotted line). Ptch1 expression in the lower "cycling" portion of the hair follicle ORS (lower two thirds beneath the bulge) is high during anagen (growth phase; Fig. 1F) and decreases or is lost during catagen (regression phase) and telogen (resting phase; Fig. 1B, solid line), consistent with previously published data (18). We also note that Ptch1 (Fig. 1C) is expressed in the CCL of the hair follicle given Ptch1 and the CCL-specific marker K6hf (Bax-1; ref. 19; Fig. 1D) overlap in this region (Fig. 1E). Support for specificity of the Ptch1 antibody include ablation of Ptch1 protein expression in Krt6a-Cre:Ptch1neo/neo conditional Ptch1 mutants, similar distribution to that observed by Ptch1 mRNA in situ hybridization analysis (15), and extensive characterization of Ptch1 at the subcellular level both in vivo and in vitro (17).


Figure 1
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  		Figure 1. Expression profile of Ptch1 in wild-type and transgenic epidermis. Expression profile of Ptch1 in wild-type (A-E), Krt6a-Cre:Ptch1neo/neo double transgenic (F-I), and Krt6a-Cre:Ptch1neo/neo/RA–induced (J) skin samples. A, endogenous Ptch1 is expressed in the basal cells of the IFE and ORS of the hair follicle (x40). B, Ptch1 expression in the ORS of the permanent region of the hair follicle remains constant (x20, dotted line), but Ptch1 expression in the lower cycling portion of the ORS is absent during catagen and telogen (solid line). Immunofluorescence staining of Ptch1 (C, x60) and the CCL-specific marker K6hf (D, x60) and merge (E, x60) indicates Ptch1 is expressed in the CCL (E, arrow). Constitutive expression of the Krt6a-Cre promoter maintains IFE and ORS Ptch1 expression (F, x20) but drives the specific deletion of Ptch1 (G, x60) from the K6 (H, x60) expressing cells of the CCL (merge, I, x60). RA induces the activity of the Krt6a-Cre promoter and induces the deletion of Ptch1 from the IFE and ORS (J, x40, arrows).

 
Loss of Ptch1 in the companion cell layer of the hair follicle impairs outer root sheath homeostasis and predisposes lesion formation. Ptch1 expression in Krt6a-Cre:Ptch1neo/neo epidermis persisted in the basal cells of the IFE and permanent hair follicle ORS cells (Fig. 1F) but was no longer detected in the CCL (Fig. 1G-I). These data confirm restricted Cre-mediated excision of Ptch1 in the CCL. Krt6a-Cre:Ptch1neo/neo mice are viable and largely indistinguishable from control littermates during the first and second post-natal hair cycles. At about the time when small loci of hair loss become apparent (3-5 months of age; Fig. 2A), histologic analysis revealed that most hair follicles were affected by severe ORS hyperplasia (Fig. 2B), as characterized by an increase in the number of concentric ORS layers expressing keratin 14 (K14; refer to Fig. 2H). These data suggest that either periods of Patched pathway inactivation or lower levels of pathway activity are required in the CCL to ensure normal ORS proliferation and differentiation. The severity of Krt6a-Cre:Ptch1neo/neo hair loss increases with age, and by 9 to 12 months, small epidermal lesions become evident (Fig. 2C). Histologic analysis revealed the majority of epithelial basal cell proliferations were associated with hair follicle structures and sebaceous glands (Fig. 2D). One likely explanation, consistent with the late onset of the lesions, is that loss of Ptch1 in the CCL predisposes ORS cells to proliferative behavior and a tumorigenic fate is conferred upon accrual of age-related genetic "hits" in other cancer promoting pathways, such as p53.


Figure 2
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  		Figure 2. Epidermal phenotype of control and Ptch1 conditional mice. Krt6a-Cre:Ptc1neo/neo mice exhibit small foci of hair loss (A) and ORS hyperplasia (B, H&E stain, x20) from 3 months. Hair loss progresses with age (C), and small epithelial invaginations are observed via histologic analysis by 9 to 12 months (D, x20). No overt epidermal morphology was observed in control samples treated with acetone (E, x20) or RA (I, x20). The phenotype observed in response to acetone treatment of Krt6a-Cre:Ptc1neo/neo skin was indistinguishable from untreated Krt6a-Cre:Ptc1neo/neo skin at 4 weeks (F) and 16 weeks (G). ORS hyperplasia characterized by increased number of concentric hair follicle outer root sheath cells expressing keratin 14 (H, x40). Within 4 weeks of inducing loss of Ptch1 function in the basal cells of Ptch1 conditional mice via RA application, epidermal lesions were evident in 25% of samples (J). Lesions were evident in 100% of Ptch1 conditional samples by 16 weeks (K, arrows). Note the presence of epidermal cyst formation (J and K, asterisk). Ptch1 conditional tumors express high levels of keratin 14 (L, x20) and exhibit clefting (M, x60) and basal cells that palisade the tumor border (N, x60). Tumors express high levels of the proliferation marker PCNA (O, x20; inset, control). Up-regulation and nuclear translocation of Gli1 protein confirms pathway activation (P, x20; inset, control).

 
RA treatment induces Cre-mediated deletion of Ptch1 in the basal cell population. We have previously shown that in response to a single topical application of 30 µg of trans-RA, transcription of the Krt6a-Cre promoter is induced in ~10% of epidermal basal cells (12). To improve the frequency of Cre-mediated excision, we challenged adult Krt6a-Cre:Z/AP reporter double transgenics with two 30 µg doses of RA 48 hours apart and detected loss of LacZ and induction of alkaline phosphatase expression in large patches of the epidermis.1 Consistent with previous in vivo studies of topical application of retinoids onto rodent and human skin studies (20), RA treatment showed evidence of epidermal thickening and basal cell hyperplasia; however, the hyperplastic phenotype resolved 7 to 14 days after treatment, and no adverse side effects were observed in control skin (refer to Fig. 2I). Treatment of Krt6a-Cre:Ptch1neo/neo epidermis with a 60 µg dose of RA (Krt6a-Cre:Ptch1neo/neo/RA) resulted in loss of Ptch1 protein expression in 40% of IFE basal cells and multiple hair follicle ORS (Fig. 1J). A similar distribution of K6 and Cre expression confirmed induction of Krt6aCre promoter activity (data not shown).

Loss of Ptch1 in the epidermal basal cell compartment results in rapid skin tumor formation. Previous studies by Oro and Higgins (18) suggest that Shh responsiveness is restricted to the growth phase (anagen) of the mouse hair follicle cycle. We therefore induced constitutive Patched pathway activity during second post-natal anagen and screened for evidence of tumor formation at 4 and 16 weeks. No overt skin, hair follicle, or tumor phenotypes were observed in Ptch1neo/neo control epidermis in response to either acetone (Fig. 2E) or RA treatment (Fig. 2I). The majority of Krt6a-Cre:Ptch1neo/neo/acetone–treated samples (100% at 4 weeks and 75% at 16 weeks) presented with ORS hyperplasia (Fig. 2F-H) indistinguishable from untreated Krt6a-Cre:Ptch1neo/neo control skin (refer to Fig. 2B). Thus, acetone treatment seemed to have no adverse effect on Krt6a-Cre:Ptch1neo/neo epidermal morphogenesis. A small proportion (25%) of Krt6a-Cre:Ptch1neo/neo/acetone–treated samples, however, showed evidence of basal cell lesion formation (data not shown), similar to those observed in Krt6a-Cre:Ptch1neo/neo/RA–treated samples (see below). One likely explanation is induced Krt6aCre promoter activity and subsequent interfollicular deletion of Ptch1 in response to epidermal wounding (biopsy procedure). However, it is important to note that ectopic deletion of Ptch1 protein did not occur in 75% of acetone-treated samples.

RA treatment of Krt6a-Cre:Ptch1neo/neo mice resulted in rapid and extensive skin tumor formation. Basal cell invaginations were observed within 4 weeks in 25% of Krt6a-Cre:Ptch1neo/neo/RA (Fig. 2J). By 16 weeks, 100% (n = 8) of Krt6a-Cre:Ptch1neo/neo/RA epidermal samples presented with substantial skin tumors encompassing most of the IFE (Fig. 2K). These results clearly show that loss of Ptch1 function in the epidermal basal cell population of mouse skin confers a neoplastic phenotype within 4 to 16 weeks with 100% penetrance. The tumors evident in Ptch1 conditional skin express high levels of keratin 14 (Fig. 2L), lack differentiated epithelia markers keratin 10 (K10) and loricrin (data not shown), and show histologic features characteristic of human BCC, including basal cells that have a high nuclear to cytoplasmic ratio, basal cells that palisade the periphery of the basal invagination (Fig. 2N), and retraction sites where the tumor epithelium is separated from the mesenchyme (clefting; Fig. 2M). Large epidermal cysts were also evident (Fig. 2J and K, asterisk). Ptch1 conditional skin tumors exhibit high levels of Ki67 and PCNA (Fig. 2O), indicating an increased rate of basal cell proliferation upon Ptch1 deletion. We also observed high levels of nuclear Gli1 protein expression (Fig. 2P) and up-regulation of Ptch1 and Gli1 mRNA (data not shown), confirming ectopic activation of the Patched pathway.

Patched pathway-induced skin tumors originate from the basal cell compartment. It was apparent from immunohistochemical detection that several Ptch1 conditional skin tumors derived from Ptch1-null cells residing in the IFE with no obvious association with hair follicle structures (Fig. 3B and C). Other tumors clearly arose from Ptch1-null cells in the ORS of the hair follicle (Fig. 3D), indicating perturbation of normal hair follicle growth. The distribution of K14 expression confirmed that Ptch1 conditional skin tumors originated from expansion of the IFE basal cell population (Fig. 3F) and hair follicle ORS cells (Fig. 3G). We also note that Ptch1 conditional tumors are often associated with sebaceous gland structures (Fig. 3H) and regions of sebaceous hyperplasia (Fig. 3C, asterisk).


Figure 3
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  		Figure 3. Origin of Patched pathway skin tumors. Endogenous expression of Ptch1 protein in the basal cells of control skin (A, x40). A subset of Ptch1 conditional skin tumors clearly arose from Ptch1-ablated cells residing in the IFE, with no obvious association with hair follicle structures (B and C, x40, arrows). Other Ptch1 conditional tumors arose from Ptch1-null cells of the hair follicle (D, x40, arrow). K14 expression of control epidermis depicts single basal layer of the IFE and ORS cells of the hair follicle (E, x40). Ptch1 conditional epidermal tumors are of basal cell origin, express high levels of K14 (F-H), arise from within the IFE (F, x20) and ORS cells of the hair follicle (G, x40) and are often associated with sebaceous structures (H, x20).

 
Constitutive activation of the Patched pathway does not promote translocation of ß-catenin into the nucleus. Current data suggest an interaction between Patched and Wnt signaling pathways. In the absence of a Wnt ligand, cytoplasmic ß-catenin is phosphorylated and targeted for degradation. Wnt expression inhibits the degradation machinery, resulting in the cytoplasmic accumulation and subsequent translocation of ß-catenin into the where it forms a transcriptional complex with the lymphoid enhancer binding factor/T-cell factor family of DNA binding proteins and activates transcription of target genes (21). High levels of nuclear ß-catenin have been observed in infiltrative, superficial, and micronodular BCC (22–25), leading to the hypothesis that this is due to Patched pathway–mediated Wnt expression. Recent evidence has also shown that Suppressor of Fused (SuFu), a negative inhibitor of the Patched pathway (26–28), functions to repress Tcf-mediated Wnt transcription by binding ß-catenin and promoting its nuclear export (29). To determine whether constitutive Patched pathway activity can directly regulate the cellular distribution of ß-catenin, we screened Ptch1 conditional skin with antibodies specific for the phosphorylated (Pan-ß-catenin) and active ({alpha}ABC) forms of the protein. Although Ptch1-positive cells of the epidermis express significant levels of phosphorylated ß-catenin (Fig. 4A), the membrane/cytoplasmic distribution of ß-catenin is elevated in the absence of Ptch1 (Fig. 4B). Consistent with previously published data (22), we observed regions of intense nuclear ß-catenin expression in a subset of Ptch1 conditional skin tumors (data not shown). However, the incidence of nuclear ß-catenin in the remaining Ptch1-deficient cells of Krt6a-Cre:Ptch1neo/neo epidermis (Fig. 4D) was similar to that observed in control skin (Fig. 4C). These results show that translocation of ß-catenin into the nucleus is not a direct consequence of Ptch1 elimination and thus it is unlikely that the Patched pathway mediates tumor formation by inducing Wnt activity.


Figure 4
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  		Figure 4. Expression profile of ß-catenin and Hes1 in control and Ptch1 conditional epidermis. ß-Catenin expression was visualized with pan-ß-catenin (A and B) and {alpha}ABC (C and D) antibodies. A, normal level and distribution of pan-ß-catenin in control epidermis (x40). B, increased level of membrane/cytoplasmic ß-catenin immunostaining in Ptch1 conditional skin/tumors (x40). C, incidence of nuclear (active) ß-catenin in control epidermis (x60). D, similar incidence of nuclear ß-catenin is observed in the absence of Ptch1 function (x60). E, Hes1 expression evident in the basal cells of control e14.5 epidermis (x40). F, Hes1 is predominately expressed in suprabasal cells from e18.5 (x40). Double immunostaining of Ptch1 and Hes1 in control adult epidermis (G, x100) and double immunofluorescence of in control E18.5 epidermis (H, x40) clearly show that Hes1 and Ptch1 do not colocalize in the skin. I, no evidence of ectopic Hes1 expression in Krt6a-Cre:Ptch1neo/neo/RA skin tumors (x40).

 
Loss of Ptch1 in the basal cell population of the skin does not effect the distribution of Notch signaling components. Several lines of evidence suggest a link between Hedgehog and Notch signaling pathways. In particular, inactivation of the Notch1 receptor in mouse skin gives rise to epidermal lesions closely resembling human BCC and transcriptional up-regulation of Shh, Ptch1, and Gli1 RNA within the tumors (30). Proteolytic cleavage of Notch protein receptors results in their nuclear translocation where they interact with the DNA-binding protein RBP-Jk, which triggers the conversion of RBP-Jk from a repressor to an activator of gene transcription, thereby activating a cascade of downstream signaling events (31). Given that loss of Notch 1 receptor function leads to Patched pathway activation and BCC (30), we wanted to determine whether loss of Ptch1 function results in down-regulation of Notch protein receptors, thereby implicating cross-talk between the two pathways and identifying a potential mechanism of Hedgehog pathway augmentation. Consistent with previously published data, Notch 1 and Notch 2 expression were restricted to the suprabasal layers of the Ptch1neo/neo control epidermis, and we observed no change in Notch 1 or Notch 2 expression in response to Ptch1 deletion in the CCL (Krt6a-Cre:Ptch1neo/neo/acetone) or epidermal basal cell population (Krt6a-Cre:Ptch1neo/neo/RA; data not shown). We also describe the epidermal expression of Hes1, a Notch effector implicated in the regulation of neural stem cells (32). Hes1 expression is evident in the basal cells of E14.5 mouse epidermis (Fig. 4E) and predominately expressed in suprabasal cells of E18.5 (Fig. 4F) and adult epidermis (Fig. 4G). Consistent with this observation, we did not detect any colocalization between Hes1 and Ptch1 (Fig. 4H), clearly showing that Notch-responsive cells are distinct from Hedgehog-responsive cells. In particular, we did not observe ectopic Hes1 expression within the epidermal tumors of Ptch1 conditional mice (Fig. 4I). These data show that loss of Ptch1 function in the skin does not perturb the normal epidermal expression profile of Notch protein receptors and does not induce ectopic expression of a major Notch effector.

Loss of Ptch1 promotes the nuclear localization of cyclin D1 and cyclin B1. Recent reports have shown that the transcriptional regulator of the Drosophila Hedgehog pathway, cubitus interruptus, directly promotes the transcription of cyclin D1 (33). We screened Ptch1 conditional skin for the cellular distribution of cyclin D1 and observed a high rate of nuclear expression in Ptch1-null tumor cells (Fig. 5A and B). However, to determine any immediate effect that loss of Ptch1 may have on the localization of cyclins and to minimize any indirect tumor-associated proliferative defects, we calculated the nuclear distribution of cyclin D1 (and cyclin B1) in the interfollicular basal cell population expressing Ptch1 versus preneoplastic cells interfollicular cells lacking Ptch1 function. The number of cells expressing nuclear cyclin D1 in Ptch1+/+ interfollicular basal cells was 15% (Fig. 5C). This number rose significantly (P = 0.00016) in the absence of Ptch1, whereby nuclear cyclin D1 expression was evident in ~70% in Ptch1-null interfollicular cells (Fig. 5C). These data clearly show that constitutive Hedgehog pathway activation promotes the nuclear translocation of cyclin D1.


Figure 5
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  		Figure 5. Loss of Ptch1 promotes the nuclear localization of cyclin D1 and cyclin B1. To determine any immediate effect (rather than any indirect tumor associated defect), we calculated the nuclear distribution of cyclin D1 and cyclin B1 from Ptch1-positive and Ptch1-null basal cells residing in the IFE. A, a relatively small proportion of IFE basal cells (~15%) express nuclear cyclin D1 (red) in the presence of Ptch1 (green; x40). B, the number of IFE cells expressing nuclear cyclin D1 in the absence of Ptch1 protein is significantly higher (P = 0.00016, x40). C, graphical representation of the number of cells expressing nuclear cyclin D1 in the presence of Ptch1 (red column, 15%) and absence of Ptch1 (yellow column, 70%). D, the nuclear expression of cyclin B1 is observed in a subset of Ptch1-positive IFE cells (x60). E, prevalence of nuclear cyclin B1 expression increases in the absence of Ptch1 (x40). F, graphical representation of the number of cells expressing nuclear cyclin B1 in the presence of Ptch1 (red, 45%) and the absence of Ptch1 (blue, >70%).

 
Current in vitro evidence suggests that Ptch1 can directly bind to phosphorylated cyclin B1 and inhibit its nuclear translocation (34), thereby implicating Ptch1 function as a negative regulator of G2-M phase. The presence of Shh has been shown to disrupt this interaction and allow cyclin B1 to localize to the nucleus, thereby initiating entry into mitosis (34). Our Ptch1 conditional mouse model provides a model system to study the in vivo distribution of cyclin B1 in the presence and absence of Ptch1 function. We screened Krt6a-Cre:Ptch1neo/neo/RA epidermis for the cellular distribution of cyclin B1 and observed a high rate of nuclear expression in Ptch1-null tumor cells (Fig. 5D and E). However, to determine if changes to the localization of cyclin B1 was a direct result of loss of Ptch1 function, we specifically screened the subcellular localization of cyclin B1 in preneoplastic Ptch1-null interfollicular basal cells. In the presence of Ptch1, an average of 43% of interfollicular basal cells present with nuclear cyclin B1 immunoreactivity (Fig. 5F). In contrast, an average of 73% of Ptch1-null interfollicular basal cells expressed nuclear cyclin B1 expression (Fig. 5F). Thus, loss of Ptch1 protein function results in a significant increase (P = 0.021) in the nuclear localization of cyclin B1. These results clearly show that Ptch1 plays a role in regulating the cellular distribution of cyclin B1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Patched1 functions as a gatekeeper. The most common tumor associated with constitutive Patched pathway activity is the human skin cancer, BCC. Overexpression of Shh (15, 35, 36), Dhh (15) or the transcription factors Gli1 (37) or Gli2 (38), and mutation of Smoothened (39) or UV irradiation of Ptch1+/– mice (40, 41) all result in BCC formation and hyperproliferation defects. Although inactivation of Ptch1 has been shown to be a major contributing factor of familial and sporadic BCC formation, loss of Ptch1 function has also been implicated in the etiology of a wide range of other tumors (42). This line of evidence has led to the hypothesis that Ptch1 acts as a gatekeeper gene in tumor formation, whereby loss of function is a necessary and sufficient step in tumor formation and neoplastic behavior (43, 44). Here, we describe the effects of ablating Ptch1 function in discrete regions of the skin and hair follicle in a conditional in vivo mouse model system. We have shown that loss of Ptch1 in the basal cells of the IFE and ORS cells of the hair follicle is sufficient to induce the hallmark features of skin tumors resembling human BCC. The rapid progression of these tumors (from as little as 4 weeks of latency period) would argue against the accumulation of random genetic hits, indicating that ligand-independent, constitutive activation of the Patched pathway is sufficient to induce skin tumor formation. Tumors within Krt6a-Cre:Ptch1neo/neo:RA epidermis clearly arose from Ptch1-null cells residing in the IFE and the ORS of the hair follicle. However, no evidence of tumor formation was evident from Ptch1-null cells of the CCL (Krt6a-Cre:Ptch1neo/neo epidermis). These data clearly identify epidermal basal cells as the origin of Ptch1-induced skin tumors, an observation that is consistent with the origin of Ptch1+/– skin tumors (18) and further supports the notion that BCCs can originate outside of the hair follicle (15, 45). It is also important to note that although the ORS cells of Krt6a-Cre:Ptch1neo/neo epidermis are hyperplastic, they continue to express Ptch1, and this seems to be sufficient to prevent the onset of tumorigenesis.

Mechanism of Patched1-induced tumorigenicity. Current hypotheses suggest that Patched pathway activation predisposes a cell to proliferative and expansive behavior. For example, BCC lesions have been shown to express high levels of FoxM1 (46), a Forkhead box protein that functions as a transcriptional regulator of G1-S (47) and is crucial for progression into mitosis (48). Ectopic Shh activity has also been shown to down-regulate the expression of the cell cycle inhibitor, p21Waf1/Cip1 (49), which acts to induce cell cycle arrest. Recent evidence also suggests that Ptch1 directly interacts with cyclin B1 (34), a component of the M-phase promoting factor and whose nuclear accumulation is required for G2-M transition. Furthermore, mutation in Ptch1 (50) or the presence of Shh ligand (34) disrupts this interaction and allows cyclin B1 to localize in the nucleus. It is also important to note that constitutive Patched pathway activity seems to promote tumor formation by inhibiting the expression of Fas, an important molecule in mediating apoptosis (41). Direct injection of a potent Smoothened agonist, cyclopamine, into BCC lesions of UV-irradiated Ptch1+/– mice induces the expression of Fas and results in subsequent apoptosis of tumorigenic cells (41). Apoptotic regulation, however, seems to be a downstream response of Hedgehog pathway activation, rather than a direct consequence of Ptch1 inactivation. The focus of this study was to elucidate the molecular mechanism of Ptch1-induced tumorigenicity. We therefore focused our analysis on the phenotype or cellular distribution of candidate genes in preneoplastic Ptch1-null keratinocytes. In this study, we have shown that loss of Ptch1 function results in a substantial increase in the nuclear translocation of the G1-S phase inducing factor, cyclin D1, and the G2-M phase inducing factor, cyclin B1. These data suggest that loss of Ptch1 function is likely to induce a tumorigenic phenotype by promoting cell cycle progression through G1-S and G2-M phase, by directly regulating the nuclear localization of cell cycle regulators. In support of our data, recent studies have shown that the Ptch1 target gene Gli2 directly regulates cyclin D1 during epidermal development (51). Furthermore, in vitro analyses have shown that Ptch1 can directly bind to phosphorylated cyclin B1, suggesting that Ptch1 functions as a negative regulator of entry into mitosis by preventing the nuclear accumulation of cyclin B1. Our evidence supports the role of Ptch1 in vivo as a negative regulator of G1-S and G2-M phase transition.

Alternative hypotheses suggest that Patched pathway activity promotes tumor formation by perturbing the activity or expression of other developmental pathways known to play a role in cancer progression, such as Wnt and Notch. Although we observed an increase in the nuclear distribution of ß-catenin in a subset of tumor cells, we did not observe nuclear localization of ß-catenin in preneoplastic Ptch1-null basal cells. Hence, translocation of ß-catenin does not seem to be the primary defect responsible for predisposing Ptch1-null cells with proliferative behavior. These data do not refute previous studies, which have shown that perturbation of Wnt signaling activity regulates Shh expression in the skin (52, 53) but rather indicate that Wnt signaling may be upstream of Patched pathway activity. In addition, we have shown that loss of Ptch1 function does not induce the down-regulation of Notch protein receptors or the ectopic expression of the Hes1 effector. These data indicate that Patched pathway activation does not augment neoplastic transformation by inactivating the tumor suppressor activity of the Notch signaling pathway. Given loss of Notch 1 function results in Hedgehog pathway activity (30), we conclude that Notch may act upstream of the Shh pathway as a negative inhibitor of pathway activity.

Concluding remarks. The conditional ablation of Ptch1 function in the skin shows that Patched pathway activity is likely to induce proliferation defects by promoting cell cycle progression and not via perturbation of other developmentally important pathway, such as Notch or Wnt signaling. Although we await the analysis of Ptch1-null phenotypes in other organ systems, we hypothesize that the molecular mechanism of Ptch1-induced tumorigenicity is conserved, implicating cell cycle regulation as one of the major defects of Hedgehog-associated tumors. Recent research efforts have shown that Hedgehog pathway inhibitors are useful at inducing BCC (41) and medulloblastoma (54) regression in Ptch1+/– mice. These results support our observation that Ptch1 acts as a gatekeeper, whereby inhibition of Hedgehog pathway activity is sufficient to induce Ptch1-induced tumor regression.


   Acknowledgments
 
Grant support: ARC Special Research Centre for Functional and Applied Genomics, the Australian National Health and Medical Research Council, and John Trivett Foundation Fellowship (T. Ellis).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the anonymous peer reviewers for their comments.


   Footnotes
 
1 R. Hetherington, unpublished data. Back

Received 6/20/05. Revised 10/13/05. Accepted 12/ 6/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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