Hair follicle development is orchestrated by secreted signaling molecules that act on intracellular effector pathways in epithelial and mesenchymal progenitors10
. The canonical Wnt signaling pathway initiates hair bud formation, whereas Hh signaling subsequently promotes the proliferative expansion of follicle epithelium required to assemble a mature follicle7,8,11,12
. BCC is the most common type of cancer in light-skinned individuals, and several mutations leading to constitutive activation of the Hh pathway have been identified in these tumors, including loss-of-function mutations in PTCH1
and gain-of-function mutations in SMO2
. Nearly all human BCCs show elevated Hh pathway activity, and several animal models support the notion that uncontrolled Hh signaling is sufficient to drive BCC- or BCC-like tumorigenesis in mice2
. The morphological similarity between early superficial BCCs and hair germs was first noted over 70 years ago4,5
, suggesting the possibility that canonical Wnt signaling is involved in pathological responses to deregulated Hh signaling in skin. Although some prior studies have identified coordinate changes in the Hh and canonical Wnt pathways in BCCs and other neoplasms13–19
, direct evidence establishing the functional significance of Wnt signaling in Hh pathway-driven pathology in vivo
is lacking. In this report we combined analysis of human tissues with in-depth studies of genetic mouse models to test the role of Hh-Wnt crosstalk in the setting of constitutive Hh signaling in skin.
Both human superficial BCCs and embryonic hair buds () comprise a focal grouping of epidermal cells protruding into the underlying dermis () and express early-stage follicle lineage markers, including the outer root sheath markers K17 and Sox9, and the hair matrix/inner root sheath marker CDP (). Epithelial cells in both the superficial BCCs and embryonic hair buds were more proliferative, based on Ki67 immunostaining, than adjacent epidermis () and did not express the epidermal differentiation marker K1 (). In contrast to embryonic hair buds (, arrowhead), superficial BCCs did not show a morphologically recognizable mesenchymal papilla (), which is required for later stages of hair follicle morphogenesis.
Figure 1 Human superficial basal cell carcinoma (BCC) expresses hair bud lineage markers. (a, b) H&E-stained sections of early human superficial BCC and embryonic hair buds. Dashed line in b indicates epithelial component of embryonic hair follicle, with (more ...)
We then examined epithelial bud development in mice with focally activated Hh signaling in skin, achieved using the M2SMO
(). We restricted much of our analysis to a triangular region of volar skin completely devoid of follicles or other skin appendages, allowing us to study the effects of deregulated Hh signaling in a morphogenetically naive epidermis (). Constitutive activation of Hh signaling using M2SMO resulted in de novo
epithelial bud initiation in this normally hairless region (). Similar to human superficial BCC and embryonic hair germs (), immunophenotyping revealed multiple similarities between M2SMO-induced epithelial buds and embryonic mouse hair buds (). However, downregulation of E-cadherin, a characteristic alteration in embryonic hair buds that may contribute to a shift from membrane-bound to cytoplasmic and nuclear β-catenin, was not apparent in M2SMO-induced buds (), and M2SMO-induced ectopic buds (like human superficial BCCs) were not associated with a morphologically or biochemically detectable mesenchymal condensate ()).
Figure 2 Ectopic Hh signaling in M2SMO-expressing hairless mouse skin drives superficial BCC-like downgrowths resembling hair buds. Histological appearance, marker expression and increased proliferation rate of ectopic epithelial buds in volar skin from M2SMO-expressing (more ...)
The formation of de novo
epithelial buds in response to ectopic Hh signaling in epidermis was intriguing, given the compelling evidence pointing to the canonical Wnt pathway as the initiator of skin appendage development7,8,20–22
. We therefore determined whether pathological Hh signaling in this setting was associated with activation of the canonical Wnt/β-catenin pathway (). Increased levels of β-catenin in the cytoplasm and nucleus were seen in human embryonic hair buds (), consistent with the notion that Wnt/β-catenin signaling is activated during early stages of human follicle development, as already shown in mouse hair follicles (). We also detected increased levels of β-catenin in the cytoplasm and nucleus of neoplastic cells in human superficial BCCs and in M2SMO-induced mouse epithelial buds (), suggesting activation of the canonical Wnt pathway in response to ectopic Hh signaling. Furthermore, nonphosphorylated (active) β-catenin was detected in lysates from M2SMO
-transgenic mouse skin by immunoblotting, with little or no expression in control, nontransgenic volar skin (). Epithelial buds arising in M2SMO-expressing mice progress to form follicular hamartomas (Supplementary Fig. 1
), a type of benign tumor associated with deregulated Hh signaling in mice and humans3,23,24
, but not BCCs. Follicular hamartomas expressed the same set of lineage markers as buds, but with Ki67, β-catenin and cyclin D1 largely restricted to the outermost cell layers (Supplementary Fig. 1 and Supplementary Note
Figure 3 Canonical Wnt signaling is activated in human superficial BCC and epithelial buds in M2SMO-expressing hairless mouse skin. (a, b) β-catenin immunostaining reveals nuclear-cytoplasmic β-catenin localization in human superficial BCC and (more ...)
To determine if aberrant Hh signaling may be influencing the canonical Wnt/β-catenin pathway through alterations in Wnt ligand expression, we performed semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) (). As expected, mRNA encoding the Hh target genes Gli1
, indicating Hh pathway activation, was detected in samples from M2SMO volar skin, with negligible levels in controls. In addition, transcripts encoding multiple Wnt ligands (Wnt3, 4, 5a, 7b, 10a and 10b) and the transcriptional coactivator Tcf4, were coordinately induced in M2SMO volar skin. Expression of endogenous Wnt target genes25 Axin2
, and the indirect target Ccnd1
was also upregulated in M2SMO-expressing skin, indicating activation of a transcriptional program associated with canonical Wnt signaling.
Using a transgenic mouse model wherein doxycycline-regulated activation of the secreted Wnt inhibitor Dkk1 could be achieved in skin26
, we next tested whether canonical Wnt signaling was required for development of epithelial buds and their expansion to form hamartomas (). We first induced Dkk1 in M2SMO-expressing mice (M2SMO + Dkk1) during embryogenesis, by administering doxycycline to pregnant dams at embryonic day 16.5 (). Blockade of canonical Wnt signaling at this stage led to nearly complete inhibition of M2SMO-driven ectopic bud development in volar skin, as assessed by whole-mount analysis and histology () of skin on postnatal day 7 (P7). Dkk1 induction at P1 () also led to a marked inhibition of M2SMO-mediated hair bud development and a profound suppression of follicular hamartoma formation, both in volar () and in tail () skin examined at P35. Although expression of Wnt
genes was similarly elevated in M2SMO and M2SMO + Dkk1 skin (), translocation of β-catenin to the cytoplasm and nucleus was suppressed in M2SMO + Dkk1 mice (), and expression of the Wnt target genes Axin2
was negligible, indicating effective blockade of canonical Wnt signaling by Dkk1 (). In contrast, expression of the Hh target genes Gli1
was similar in M2SMO and M2SMO + Dkk1 skin, indicating that M2SMO-driven constitutive Hh signaling was unaffected by Wnt pathway inhibition (). Similarly, ectopic expression of the Hh-responsive keratin, K17 (ref. 27
) was seen in both M2SMO and M2SMO + Dkk1 volar skin (), whereas expression of hair bud and follicular hamartoma markers Sox9 and CDP, and increased cell proliferation, were no longer detected in M2SMO + Dkk1 mice (). The profound blockade of M2SMO-induced hair bud and follicular hamartoma development by Dkk1 establishes that biological responses to ectopic Hh signaling in skin are largely mediated indirectly, via the canonical Wnt/β-catenin signaling pathway.
Figure 4 Inhibition of canonical Wnt signaling with Dkk1 blocks M2SMO-induced development of epithelial buds and follicular hamartomas. (a) Experimental timeline for induction of Dkk1 expression in M2SMO + Dkk1 mice during embryogenesis (E16.5) with analysis at (more ...)
Notably, buds and hamartomas also developed in dorsal skin of M2SMO mice, but in this location they were not suppressed in M2SMO + Dkk1 mice, and β-catenin remained localized to the nucleus, suggesting that Dkk1 was not effectively inhibiting canonical Wnt signaling at this site. This may be due to insufficiently high expression of Dkk1 in dorsal skin or a relative deficiency in the level of Kremens 1 and 2 (ref. 28
), which facilitate Dkk1's ability to block Wnt signaling. Bud and hamartoma development was also not inhibited in small regions of volar skin near footpads (arrowhead in ), and here again the presence of nuclear β-catenin indicated inefficient blockade of canonical Wnt signaling. These results further underscore the tight correlation between inhibition of canonical Wnt/β-catenin signaling and suppression of Hh pathway-induced bud and hamartoma development.
There are multiple reports describing interactions between the Wnt and Hh pathways at various levels, and two of these are particularly noteworthy in light of our findings. Expression of Wnt5A
, and 11
has been reported in Xenopus
animal cap explants injected with Gli2 and Gli3 mRNA, and blockade of Wnt signaling inhibits the morphogenetic response to Gli2 in this system15
. Also, in E1A-immortalized RK3E rat kidney cells, expression of Wnt2b
was induced by GLI1, and dominant-negative TCF4 inhibited GLI1-mediated focus formation in cell culture19
. Although these data demonstrate that Hh-Wnt crosstalk is necessary for an embryonic process and in vitro
transformation, respectively, our findings are the first to establish a stringent requirement for canonical Wnt/β-catenin signaling in Hh pathway-driven neoplasia.
Previous studies in normal skin have shown that in developing hair buds, canonical Wnt signaling precedes, and is required for, subsequent activation of Hh signaling7,8,20,21
. Our data indicate that this temporal relationship is reversed in the setting of Hh-driven pathology in epidermis, where ectopic activation of Hh signaling leads to canonical Wnt signaling with resultant formation of de novo
epithelial buds and follicular hamartomas. Several earlier reports have described links between the Hh and Wnt pathways in BCC, including upregulation of one or more Wnt genes15
and localization of β-catenin to the cytoplasm and/or nucleus16–18
. Our results are in keeping with these observations and provide the first direct evidence that canonical Wnt signaling is essential for a tumorigenic response to deregulated Hh signaling in skin, but additional studies are required to test the significance of Hh-Wnt crosstalk in full-blown BCC. Interestingly, a recent report described a role for β-catenin in cutaneous squamous cell carcinoma29
, which is biologically and pathogenetically distinct from BCC and follicular hamartomas, and not linked to aberrations in the Hh pathway, but it is not known whether signaling in squamous tumors is driven by Wnt ligands.
Taken together, our findings suggest that blockade of canonical Wnt/β-catenin signaling may be a useful strategy for treatment of neoplasms currently considered to be caused by uncontrolled Hh signaling. Because deregulated Hh signaling influences β-catenin signaling primarily at the level of Wnt ligands, the range of potential therapeutic strategies is considerably greater than it is for colorectal and other cancers with mutational defects in APC or β-catenin, and would likely include antibodies or other recombinant proteins that antagonize the interaction of Wnt ligands with Frizzled and LRP receptors. Future work will better clarify the utility of targeting proximal Wnt pathway components for the prevention or treatment of Hh-dependent neoplasms and other disorders.