Abstract: Epidemiologic evidence supporting a major chemopreventive role for vitamin D in various malignancies is strong. Likewise the use of the active metabolite of vitamin D, 1,25(OH)2D3, and its analogs to prevent and/or treat a wide variety of malignancies in animals is well established. The evidence has been less compelling for epidermal carcinogenesis perhaps because the same agent that produces vitamin D in the skin, UVB radiation (UVR), is also the same agent that results in most epidermal malignancies. However, recent studies indicate that the role of vitamin D and its receptor (VDR) in protecting against the development of epidermal tumors deserves a closer look. One such study found mice lacking the VDR were quite sensitive to epidermal tumor formation following the administration of the carcinogen DMBA. A more recent study showed that these mice were similarly more sensitive to tumor formation following UVR, results we have confirmed. The epidermis of the VDR null mouse is hyperproliferative with gross distortion of hair follicles, structures that may provide the origin for the tumors found in the skin following such treatment. Two interacting pathways critical for epidermal and hair follicle function, β-catenin and hedgehog (Hh), result in epidermal tumors when they are activated abnormally. Thus, we considered the possibility that loss of VDR predisposes to epidermal tumor formation by activation of either or both β-catenin and Hh signaling. We determined that all elements of the Hh signaling pathway are upregulated in the epidermis and utricles of the VDR null mouse, and that 1,25(OH)2D3 suppresses the expression of these elements in normal mouse skin. In addition we observed that the transcriptional activity of β-catenin was increased in keratinocytes lacking the VDR. These results lead us to the hypothesis that the VDR with its ligand 1,25(OH)2D3 functions as a tumor suppressor with respect to epidermal tumor formation in response to UVR by regulating Hh and β-catenin signaling.
Over 1 million skin cancers occur annually in the United States, 80% of which are basal cell carcinoma (BCC) [16% squamous cell carcinoma (SCC), 4% melanomas], making it by far the most common cancer (Greenlee et al., 2001). Ultraviolet radiation (UVR) is the major etiologic agent. UV wavelengths shorter than 280nm (UVC) are absorbed by the ozone layer and do not reach the earth. UV wavelengths longer than 320nm (UVA) have limited ability to induce the characteristic mutations in DNA seen in epidermal cancers. Thus UVB with a spectrum between 280-320nm is the major cause of these cancers (Freeman et al., 1989). The principal genotoxic lesions induced by UVR are cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PP), which, if not repaired, result in C to T or CC to TT mutations, the UVB “signature” lesion (Hussein, 2005). Such mutations in p53 are common (50-90%) in both BCC and SCC (Brash et al., 1991; Daya-Grosjean and Sarasin, 2005; Ziegler et al., 1994; Ziegler et al., 1993) as well as in actinic keratoses, the precursor lesions to SCC (Bito et al., 1995).
Precursor lesions for BCC have not been identified, but BCC are thought to arise from interfollicular basal cells, hair follicles, and sebaceous glands. Mutations in ras are much more common in SCC than BCC (Reifenberger et al., 2005), whereas mutations in the hedgehog (Hh) signaling pathway, in particular in patched 1 (Ptch 1), characterize BCC (Aszterbaum et al., 1999; Aszterbaum et al., 1998; Hahn et al., 1996; Johnson et al., 1996), but can also be found in SCC in patients who are also susceptible to BCC (Ping et al., 2001). β-catenin is also likely to play a role in at least some skin cancers. Overexpression of β-catenin leads to hyperproliferation of hair follicles, eventuating in hair follicle tumors called trichofolliculomas. However, nuclear localization of β-catenin has also been observed in BCC (Saldanha et al., 2004), suggesting that at least some BCC might also overexpress β-catenin. Mice lacking the vitamin D receptor (VDR) are predisposed to epidermal tumor development both from UVR and chemical carcinogens (Ellison et al., 2008; Zinser et al., 2002). In this report, after first describing the role of Hh and β-catenin signaling in epidermal tumor development, we will describe our studies showing that the VDR functions as a tumor suppressor in skin by modulating both the Hh and β-catenin signaling pathways.
The Hh Signaling Pathway in Epidermal Tumor Formation
Appreciation of the pivotal role of the Hh signaling pathway in BCC development began with the identification of the Ptch1 gene as the site of mutations underlying the rare autosomal dominant heritable basal cell nevus syndrome (BCNS) (Gorlin Syndrome), one of whose cardinal features is a high susceptibility to the development of BCCs (Aszterbaum et al., 1998; Hahn et al., 1996). The BCCs in these subjects frequently lose function of the inherited wildtype Ptch 1 allele, leaving the tumor cells functionally null of Ptch1. Subsequently it has become clear that essentially all BCCs, whether arising in patients with BCNS or sporadically, have mutations in Ptch 1 or other alterations in Hh signaling (Aszterbaum et al., 1998). This appreciation has resulted in the development of the Ptch1+/- (Gorlin) mouse as the first practical model of murine BCCs (Aszterbaum et al., 1999). Treatment of these mice (unlike treatment of Ptch 1 wildtype mice) with UVR or ionizing radiation produces BCC as well as SCC ( Aszterbaum et al., 1999). Ptch 1 is the membrane receptor for sonic hedgehog (Shh) (and other members of the hedgehog family, but Shh is the main family member in skin). In the absence of Shh, Ptch 1 inhibits the function of another membrane protein smoothened (Smoh). Shh reverses this inhibition freeing Smoh to enable the activation of a family of transcription factors Gli1, Gli2, and Gli3 (Figure 1), of which Gli1 and Gli2 are the principal activators of transcription in the skin. Suppressor of fused (Sufu) may maintain these transcription factors in the cytoplasm and/or limit their activity in the nucleus such that the loss of Sufu leads to increased Hh signaling (Barnfield et al., 2005; Svard et al., 2006). However, mutations in Sufu have not been clearly shown to result in BCC, although they are associated with medulloblastomas, a feature also of the Gorlin syndrome (Reifenberger et al., 2005). The role of Gli3 is likewise unclear, and unlike Gli1 and Gli2, it is not elevated in BCC. Overexpression of Gli1 and Gli2 in keratinocytes can increase the expression of each other as well as Ptch 1, the anti-apoptotic factor bcl2, cyclins D1 and D2, E2F1, and cdc45 (all of which promote proliferation) while suppressing genes associated with keratinocyte differentiation such as K1, K10, involucrin, loricrin, and the VDR (Grachtchouk et al., 2000; Nilsson et al., 2000; Regl et al., 2004a; Regl et al., 2004b; Regl et al., 2002). 1,25(OH)2D3 and VDR, on the other hand, have the opposite action on these genes. Mice overexpressing Gli1, Gli2, or Shh in their basal keratinocytes (Grachtchouk et al., 2000; Nilsson et al., 2000; Oro et al., 1997) or grafted with human keratinocytes overexpressing Shh (Fan et al., 1997) develop BCC like lesions. Furthermore, BCC show overexpression of Ptch1, Smoh, Gli1, and Gli2 (Bonifas et al., 2001; Tojo et al., 1999). Gli2 null mice resemble Shh null animals in phenotype, Gli2 deletion partially rescues Ptch 1 null animals, and Gli2 is required for Shh signaling in hair follicle development (Eichberger et al., 2004). Of relevance to our studies is that Shh, Ptch1, Ptch2, Gli1, and Gli2 have consensus sequences for vitamin D response elements (VDRE) in their promoters (Palmer et al., 2008; Wang et al., 2005), and their expression is increased in the epidermis and keratinocytes of VDR null mice but inhibited by 1,25(OH)2D3 in skin preparations in vitro (see below), suggesting that these VDREs are negative response elements.
The β-catenin Signaling Pathway in Epidermal Tumor Formation
The canonical wnt signaling pathway, of which β-catenin is an integral part, is shown in Figure 2. Wnt ligands bind to their seven-transmembrane frizzled receptors and an LRP5 or LRP6 co-receptor leading to phosphorylation of disheveled resulting in disruption of the axin/APC complex and inhibition of the kinase activity of glycogen synthase kinase 3β (GSK-3b). Dickkopf (Dkk) and soluble frizzled related protein (sFRP) inhibit this process either at the level of lipoprotein related protein (LRP) by Dkk or by binding wnt by sFRP. When active, GSK-3b phosphorylates the serine(s) within exon 3 of β-catenin facilitating its degradation by the E3 ubiquitin ligase. Thus wnt signaling increases the availability of β-catenin in the nucleus, which can then bind to transcription factors of the T-cell factor (TCF) and lymphoid enhancer factor (LEF) families to promote expression of genes such as cyclin D1 and c-myc (He et al., 1998) important for proliferation. β-catenin also forms part of the adherens junction complex with E-cadherin. Tyrosine phosphorylation of E-cadherin, as occurs after calcium administration to keratinocytes, promotes the binding of β-catenin and other catenins to the adherens junction complex (Bienz, 2005; Xie and Bikle, 2007) making it less available for transcriptional activity and where it may play a role in epidermal differentiation (Xie and Bikle, 2007). There are at least 15 different genes encoding wnts, and not all wnts work through the canonical pathway. In addition there are 10 genes encoding the frizzled receptors, six LRPs, multiple Dkks, sFRPs, TCFs, and LEFs, not all of which are expressed in all cells including keratinocytes. Thus, cells differ markedly in the components of the β-catenin signaling pathway utilized. This is well illustrated in the keratinocytes of the hair follicle and interfollicular epidermis. LEF 1 is the dominant transcription partner for β-catenin in the dermal portion of the hair follicle, which has little E-cadherin. The epidermal keratinocyte, on the other hand, has little LEF 1 but a lot of E-cadherin especially in the differentiated layers (Stenn and Paus, 2001). Overexpression of β-catenin in which exon 3 is deleted or mutated leads to hair follicle tumors (pilomatricomas, trichofolliculomas) (Chan et al., 1999; Gat et al., 1998) as previously mentioned, although the interfollicular epidermis is not much altered except in VDR null mice in which BCC form instead (Palmer et al., 2008). If both exon 3 and the transcription domain in the C-terminus are deleted, hair follicle cycling is disrupted with the appearance of cysts expressing markers of the interfollicular epidermis. This phenotype is similar to that of the VDR null mouse. This construct appears to block β-catenin signaling in the hair follicle but to increase endogenous β-catenin in the nucleus of the interfollicular epidermis. Thus this mutant has opposite actions in the keratinocytes of hair follicles (dominant negative) and epidermis (dominant positive) (DasGupta et al., 2002), which is reminiscent of the situation in the VDR null mouse. In human tumors Palmer et al. (2008) noted that trichofolliculomas have high nuclear levels of both β-catenin and VDR, whereas BCC have high levels of β-catenin but low levels of VDR, consistent with their animal data discussed below. Saldanha et al. (2004) likewise found nuclear localization of β-catenin in 20 of 86 BCC, which correlated with increased proliferative activity in these tumors, but they did not correlate these results with VDR levels.
The β-catenin and Hh pathways interact (Bienz, 2005; Palmer et al., 2008). Both are required for normal hair follicle development and cycling. Putative β-catenin/LEF response elements have been found in a number of Hh pathway genes (Palmer et al., 2008). Conditional deletion of β-catenin eliminates Shh expression from the hair follicle (Huelsken et al., 2001) and tongue (Iwatsuki et al., 2007), whereas Shh inhibits β-catenin transcriptional activity (Iwatsuki et al., 2007).
Vitamin D and Skin Cancer
1,25 Dihydroxyvitamin D3 [1,25(OH)2D3] has been evaluated for its potential anticancer activity for approximately 25 years (Eisman et al., 1979). The list of malignant cells that express the receptor for 1,25(OH)2D3 (VDR) is now quite extensive, and includes basal cell (BCC) and squamous cell (SCC) carcinomas (Kamradt et al., 2003; Ratnam et al., 1996) as well as melanomas (Colston et al., 1981). The accepted basis for the promise of 1,25(OH)2D3 in the prevention and treatment of malignancy includes its antiproliferative, prodifferentiating effects on most cell types. Epidemiologic evidence supporting the importance of adequate vitamin D nutrition (including sunlight exposure) for the prevention of a number of cancers including those of the colon, breast, and prostate (Bostick et al., 1993; Garland et al., 1985; Garland et al., 1990; Hanchette and Schwartz, 1992; Kearney et al., 1996) is strong. However, several large epidemiologic surveys have not shown such a correlation with skin cancers (Hunter et al., 1992; van Dam et al., 2000; Weinstock et al., 1992), although this issue is being reexamined. One potential complication is that UVB radiation (UVR) has the dual effect of promoting vitamin D3 synthesis in the skin (which can be further converted to 1,25(OH)2D3) and increasing DNA damage leading to skin cancer. Thus, although UVR may be the most efficient means of providing the nutritional requirement for vitamin D, the advantage to the skin may be countered by the increased risk of mutagenesis if the UVR is excessive. However, a threshold of UVR exposure may exist that would meet the nutritional requirements for vitamin D production without increasing the risk for epidermal tumor formation. Whether such a threshold exists remains unproven and controversial.
Recent studies indicate that VDR plays a protective role in the skin with respect to carcinogenesis. Zinser et al. (2002) treated VDR null mice bearing medroxyprogesterone pellets with two oral administrations of 7,12 dimethylbenzanthracene (DMBA) at 5.5 and 7 weeks, a protocol designed to induce breast cancers. No breast tumors were observed at least initially. Instead they found that 85% of the VDR null mice developed skin tumors within two months. No tumors (breast or skin) were found in the wildtype controls. The tumors were mostly sebaceous, squamous, and follicular papillomas, but several BCC were observed. No SCC were reported. These results have been confirmed using topical administration of DMBA/phorbol myristate acetate (TPA), although only papillomas were seen in the VDR null mice unlike retinoic X receptor α (RXRα) null mice which developed both BCC and SCC (Indra et al., 2007). A more recent study (Ellison et al., 2008) likewise confirmed the results by Zinser et al. (2002) but further demonstrated that the VDR null mice were also more susceptible to tumor formation induced by UVR. These tumors included SCC, results which we have confirmed (Figure 3).
Furthermore, acute UVR exposure markedly increases proliferation and retards CPD clearance in the VDR null epidermis compared to wildtype littermates (Figure 4) indicating a potential mechanism for the predisposition to malignancy in these mice.
Surprisingly, we did not find an increase in tumors in mice lacking the ability to make the VDR ligand 1,25(OH)2D3 (CYP27B1KO), suggesting that this is either a ligand independent action of VDR or another as yet unidentified ligand can substitute for 1,25(OH)2D3 in tumor protection by VDR.
VDR and Hh Interactions
The appearance of BCC in at least some of these studies (including our own) is surprising since the typical malignancy induced in mouse skin by UVR, ionizing radiation, or chemical carcinogens is SCC not BCC (Daya-Grosjean and Sarasin, 2005). The appearance of BCC is characteristic of tumors formed when hedgehog (Hh) signaling is amplified (Asterbaum et al., 1999). We have found that the epidermis and epidermal portion (utricles) of the hair follicles of adult VDR null animals overexpress elements of the Hh signaling pathway (Figure 5), suggesting that one of the causes of the increased susceptibility of the epidermis to malignant transformation is due to a loss of 1,25(OH)2D3 and/or VDR regulation of Hh signaling.
These observations raise the question whether 1,25(OH)2D3 regulates Hh signaling, and whether it is the loss of such regulation that predisposes to cancer in a variety of tissues including the skin when the vitamin D system is disrupted. Indeed, we found that, in epidermal sheets and full thickness explants of skin, 1,25(OH)2D3 inhibits the expression of Shh (Figure 6).
VDR and β-catenin Interactions
The appearance of hair follicle elements in many of the tumors forming in the VDR null mouse skin also suggests that the β-catenin pathway is disrupted. Like the Shh pathway, over expression and/or activating mutations in the β-catenin pathway lead to skin tumors, in this case pilomatricomas or trichofolliculomas (hair follicle tumors) (Chan et al., 1999; Gat et al., 1998; Xia et al., 2006). In colon cancer cells the VDR has been shown to bind to β-catenin, and reduce its transcriptional activity in a ligand dependent fashion (Palmer et al., 2001). Furthermore, in these cells 1,25(OH)2D3 has been shown to increase E-cadherin expression, such that β-catenin is redistributed from the nucleus to the plasma membrane where it forms a complex with E-cadherin and other catenins at adherens junctions (Shah et al., 2003). However, the suppression of β-catenin signaling by 1,25(OH)2D3 does not necessarily require E-cadherin (Shah et al., 2006). Rather β-catenin binds to VDR in its AF-2 domain, binding that enhances the ability of 1,25(OH)2D3 to activate the transcriptional activity of the VDR (Shah et al., 2006) but blocks the transcriptional activity of β-catenin. Similar processes are likely to be occurring in keratinocytes. A recent publication by Palmer et al. (2008) evaluated the interaction between VDR and β-catenin in transcriptional regulation, and identified putative response elements for VDR and β-catenin/LEF in a number of genes including Shh, Ptch1, Ptch2, Gli1, and Gli2, which are members of the Hh signaling pathway. Furthermore, they found that the ability of β-catenin overexpression to induce trichofolliculomas was blocked by an analog of 1,25(OH)2D3, and in the absence of VDR, BCC were induced rather than trichofolliculomas. We have shown in epidermal keratinocytes that knockdown of the VDR or its coactivator DRIP205 reduces expression of E-cadherin and formation of the β-catenin/E-cadherin membrane complex (Figure 7).
In mice transgenic for TOP Gal (a reporter gene for β-catenin transcriptional activity), increased activity of TOP Gal is observed when these mice are bred with mice null for VDR (not shown). On the other hand, overexpression of VDR and/or administration of 1,25(OH)2D3 blocks expression of a β-catenin target gene (cyclin D1) as well as that of TOP Glow, a reporter gene for β-catenin transcriptional activity (Figure 8).
Thus 1,25(OH)2D3 and VDR could serve to suppress tumor formation by limiting the hyperproliferative actions of β-catenin in the skin.
Working Model for the Tumor Suppressor Actions of VDR in Preventing Skin Cancer
Our working model by which 1,25(OH)2D3 and its receptor VDR regulate the Hh and β-catenin signaling pathways is shown in Figure 9.
The data supporting a role for 1,25(OH)2D3 and its receptor in preventing epidermal carcinogenesis via regulation of the Hh signaling and β-catenin pathways are intriguing but not conclusive. Furthermore, there are recent studies indicating that vitamin D itself, presumably by a non-receptor mediated action (binding to and suppressing Smoh), may inhibit Hh signaling directly (Bijlsma et al., 2006). As discussed above, most skin tumors induced by systemic DMBA in mice lacking the VDR contain hair follicle elements and/or are of basal cell origin, which are tumors characteristic of overexpression of the Hh and β-catenin pathways in keratinocytes. UVR induces mostly SCC but some BCC in VDR null animals (Ellison et al., 2008). The VDR is found in the outer root sheath of the hair follicle and basal layer of the interfollicular epidermis, postulated sources for BCC development (Bikle et al., 2006), but the VDR is also found in the upper layers of the epidermis where it promotes differentiation. Lack of VDR causes a hyperproliferative response in the keratinocytes of the outer root sheath of the hair follicle and basal layer of epidermis during catagen (days 15-19), with disruption of normal hair follicle cycling and decreased epidermal differentiation (Bikle et al., 2006; Xie et al., 2002).
During the first 3 weeks of life, components of the Hh signaling pathway (Shh, Ptch 1, Smoh, Gli 1, and Gli 2) are comparably expressed in both wildtype and VDR null mouse epidermis. However, by 7-11 weeks when proliferation levels are comparable between wildtype and VDR null mice, the keratinocytes in the epidermis from VDR null mice continue to express components of the Hh signaling pathway unlike that seen in wildtype epidermis as noted previously (Figure 5). In either case, 1,25(OH)2D3 suppresses the expression of these Hh pathway components in epidermal and full thickness skin preparations. Keratinocytes in which the VDR has been knocked down with siRNA directed against the VDR show increased proliferation, increased expression of components of the Hh signaling pathway, decreased E-cadherin expression, and increased β-catenin transcriptional activity as well as increased Hh pathway components, whereas 1,25(OH)2D3 and overexpression of VDR decrease proliferation, increase E-cadherin expression, and inhibit β-catenin transcriptional activity as well as expression of the Hh signaling pathway components. We postulate that increased Hh and β-catenin signaling in the keratinocytes of the VDR null animal predisposes the skin to the development of tumors in part by stimulating proliferation and reducing differentiation. At this point it is unclear the degree to which the role of VDR is ligand [i.e., 1,25(OH)2D3] dependent or independent. Disruption of hair follicle cycling in the VDR null mouse is accompanied by both hyperproliferation and increased apoptosis in the hair follicle (Drane et al., 2004); disruption of hair follicle cycling is not found in the CYP27B1 null mouse (Bikle et al., 2006). Furthermore, we were unable to show that mice lacking 1,25(OH)2D3 production (CYP27B1 null) had increased susceptibility to UVR induced tumor formation. Thus, 1,25(OH)2D3 may have only a synergistic role regarding UVR induced tumor formation in the epidermis. Conceivably the ability of VDR to regulate the Hh and β-catenin signaling pathways varies in a cell-specific and/or gene-specific fashion with respect to its requirement for 1,25(OH)2D3. We are currently investigating the extent to which 1,25(OH)2D3 or other potential ligands may modulate the tumor suppressor actions of VDR.
The authors report no conflicts of interest.
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[Discovery Medicine; ISSN: 1539-6509; Discov Med 11(56):7-17, January 2011.]