1α, 25-Dihydroxyvitamin D3 and the vitamin D receptor regulates ΔNp63α levels and keratinocyte proliferation

1α, 25-dihydroxyvitamin D3 (VD3), a secosteriod that has been explored as an anti-cancer agent, was also shown to promote cell survival. Its receptor, the Vitamin D Receptor (VDR), is a direct target of the proto-oncogene ΔNp63α, which is overexpressed in non-melanoma skin cancers. The interconnection between VDR/VD3 signaling and ΔNp63α, led us to examine whether VDR/VD3 signaling promotes keratinocyte proliferation by regulating ΔNp63α levels. Our data demonstrate that VDR regulates ΔNp63α expression at both the transcript and protein level. Interestingly, although low doses of VD3 led to an increase in ΔNp63α protein levels and keratinocyte proliferation, high doses of VD3 failed to increase ΔNp63α protein levels and resulted in reduced proliferation. Increased expression of ΔNp63α by low dose VD3 was shown to be dependent on VDR and critical for the proliferative effects of VD3. VD3-mediated increases in ΔNp63α protein levels occur via activation of both p38 MAPK and Akt kinases. Finally, analysis of samples from patients with squamous cell carcinoma (SCC), basal cell carcinoma and precursors to invasive SCC demonstrated a significant correlation between p63 and VDR levels when compared with healthy normal skin control samples. Delineation of the mechanisms by which VD3 exerts its effect on ΔNp63α and cell proliferation is critical for determining the future of VD3 in cancer therapies.


Introduction
The Vitamin D Receptor (VDR) is a member of the nuclear receptor family. In canonical VD 3 signaling, VDR bound to 1α, 25-dihydroxyvitamin D 3 (VD 3 ), the active form of Vitamin D 3 will heterodimerize with the retinoic X receptor, thereby modulating the expression of its target genes involved in cellular proliferation or apoptosis by binding to vitamin D response elements in their promoter region. 1,2 VDR is known to localize in the mitochondria via the permeability transition pore independent of its ligand. 3 VD 3 has been explored as an anticancer agent because of its role in apoptosis and inhibition of angiogenesis. 4-7 VD 3 has also been shown to promote cell survival via activation of the Akt pathway. 8,9 p38 was also activated in response to VD 3 . 10,11 Both increased Akt and p38 activity has been shown to increase the expression of ΔNp63α. 12,13 The transcription factor p63, a member of the p53 family, consists of six main isoforms due to alternative promoter usage and 3' splicing. 14 The P1 promoter drives the expression of the full-length N-terminal transactivation domain isoforms, TAp63, whereas the use of an internal P2 promoter yields the transcription of the ΔNp63 isoforms harboring a truncated N-terminal transactivation domain. Differential 3' splicing of p63 yields α, β and γ isoforms of both TAp63 and ΔNp63 proteins. 14 p63-null mice demonstrated that p63 is essential for the formation and proliferation of the epidermis along with other stratified epithelia. [15][16][17] The most abundant and physiologically relevant p63 isoform, ΔNp63α, is widely expressed in the basal layers of stratified epithelium where it primarily functions in maintaining epithelial integrity. 15,[18][19][20][21][22] ΔNp63α is overexpressed in many human cancers including non-melanoma skin cancers (NMSCs) such as basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). 18,[23][24][25][26][27][28] However, the loss of ΔNp63α leads to increased cell invasion. 29,30 Little is known about the mechanism underlying p63 regulation, particularly in the skin epithelium.
In this study, we examined whether VD 3 and VDR promotes keratinocyte proliferation via the regulation of ΔNp63α expression. We demonstrate that VDR positively regulates the expression of ΔNp63α. Furthermore, VD 3 has a dosedependent effect on ΔNp63α protein level. A direct correlation was observed between VD 3 -mediated increase in ΔNp63α levels and keratinocyte proliferation, which is dependent on VDR. Inhibition of both Akt or p38 activation led to a reduction in VD 3 -mediated increase in ΔNp63α protein levels. We observed significantly higher levels of both p63 and VDR expression in NMSCs when compared with normal skin indicating a possible correlation between p63 and VDR in these cancers. a direct target of p63, however, no studies to date have examined whether VDR in turn can regulate ΔNp63α. 30,31 Since, it has been suggested that both ΔNp63α and VDR/VD 3 can lead to increased keratinocyte proliferation, 8,9,32,33 we examined whether VDR was mediating cell proliferation by regulating ΔNp63α levels. We silenced VDR in two keratinocyte cell lines (HaCaT and HaCaT II-4) and examined whether ΔNp63α expression at both the protein and transcript levels were altered. To rule out p53-dependent effects, we also studied the effects of VDR silencing in primary neonatal human epidermal keratinocytes expressing wild-type p53. Cells transfected with siRNA against VDR showed a significant reduction in the transcript and protein levels of VDR (Figures 1a and b). Knockdown of VDR in HaCaT, HaCaT II-4 and neonatal human epidermal keratinocytes led to a concomitant reduction in ΔNp63α transcript and protein levels (Figures 1a and b). Similar results were observed in A431 cells, a SCC cell line (Supplementary Figure 1a). To further confirm that VDR is positively regulating ΔNp63α, we measured p63 transcript levels in total RNA obtained from skin of VDR knockout mice and wild-type littermates (obtained from Dr. Glendon Zinser at University of Cincinnati). Ablation of VDR significantly reduced the transcript levels of p63 in the skin of VDR knockout mice when compared with wild-type mice (Figure 1c). These data clearly demonstrate that VDR positively regulates ΔNp63α expression in vitro and in vivo.
ΔNp63α protein levels increased following treatment with low dose VD 3 . VDR can exert its effect in both a liganddependent or -independent manner. 34,35 Having demonstrated that VDR is essential for maintaining basal expression of ΔNp63α, we examined whether VDR exerts it effect on ΔNp63α in a ligand-dependent or -independent manner. We assessed the effects of increasing doses of VD 3 on ΔNp63α expression and observed a dose-dependent increase in ΔNp63α levels up to 10 nM (Supplementary Figure 2a). We focused on testing the effects of 10 nM and 100 nM of VD 3 on ΔNp63α expression in HaCaT, HaCaT II-4 and A431 cells for subsequent studies. Whereas treatment with low dose VD 3 increased ΔNp63α protein levels in HaCaT, HaCaT II-4 and A431 cells (Figure 2a and Supplementary Figure 1b), high dose VD 3 did not significantly affect ΔNp63α protein levels when compared with vehicle control treated cells (Figure 2a). Consistent with immunoblot analysis, quantitation of immunofluorescent staining of p63 and VDR in cells treated with VD 3 clearly demonstrated an increase in ΔNp63α expression by 10 nM VD 3 when compared with 100 nM VD 3 or vehicle-treated cells (Figure 2b). These results establish that only low doses of VD 3 leads to increased protein expression of ΔNp63α. VD 3 increases ΔNp63α transcript level. To understand the mechanism behind VD 3 -mediated regulation of ΔNp63α, we examined whether VD 3 treatment affects ΔNp63α transcription. To test this, we measured p63, VDR and CYP24A transcript levels in HaCaT ( Figure 3a) and HaCaT II-4 ( Figure 3b) cells following treatment with 10 nM or 100 nM VD 3 for 24 h. Both concentrations of VD 3 led to a modest but significant increase in p63 transcript levels when compared with vehicle-treated control samples. VD 3 did not significantly alter VDR transcript levels at 100 nM VD 3 in HaCaT and at both doses tested in HaCaT II-4. As a positive control, we measured the transcript levels of CYP24A, a known target of VD 3 , which showed a dose-dependent increase following VD 3 treatment. Taken together, both high and low dose of VD 3 increased p63 transcript levels.
Effects of VD 3 treatment on cell proliferation correlates with ΔNp63α protein levels. Previous studies have shown a correlation between increased ΔNp63α expression and cell proliferation in SCCs. [26][27][28] Because low dose VD 3 increased ΔNp63α levels (Figures 2 and 3), we examined whether low dose VD 3 treatment also leads to increased keratinocyte proliferation. We monitored cell proliferation at various time points following treatment of cells with VD 3 using MTS assay VDR and p63 are required for VD 3 -mediated cell proliferation. VD 3 was previously shown to function independent of its receptor, VDR. 36 To determine whether VD 3 -mediated effects on keratinocyte proliferation are dependent on VDR, we examined the effects of VDR knockdown on cell proliferation following VD 3 treatment.
HaCaT and HaCaT II-4 cells were transfected with nonsilencing control siRNA or VDR-specific siRNA followed by VD 3 treatment. Knockdown of VDR dramatically reduced cell proliferation in both HaCaT and HaCaT II-4 cells, regardless of VD 3 dose when compared to control siRNA-transfected cells (Figure 5a, upper panel). Immunoblot analysis of cells treated with VD 3 following knockdown of VDR showed a reduction in basal ΔNp63α levels and significantly impaired the induction of ΔNp63α protein by treatment with 10 nM VD 3 when compared with control cells (Figure 5a, bottom panel). Taken together, these data show that VD 3 requires VDR to increase ΔNp63α and keratinocyte proliferation.
To confirm that decreased ΔNp63α expression observed after knockdown of VDR was responsible for the concomitant reduced proliferation, p63 was silenced prior to VD 3 treatment of HaCaT and HaCaT II-4 cells. Silencing p63 reduced cell proliferation at all doses of VD 3 when compared with cells To confirm that the change in cell proliferation was not an artifact of the MTS assay, we also measured the change in cell viability following VD 3 treatment of HaCaT and HaCaT II-4 that were transfected with siRNA against p63 or VDR by trypan blue exclusion. Consistent with MTS assay, the loss of p63 or VDR led to a reduction in VD 3 -mediated increase in proliferation by trypan blue exclusion further confirming that both p63 and VDR are required for VD 3 -mediated increase in cell proliferation (Figure 5c).
ΔNp63α rescues VD 3 -mediated increase in proliferation following the loss of VDR. To verify that increased cell proliferation following low dose VD 3 treatment was dependent on VDR-mediated regulation of ΔNp63α expression, we generated HaCaT stable cell lines expressing ΔNp63α (HaCaT-ΔNp63α) or eGFP (HaCaT-eGFP) as a control. We confirmed increased expression of ΔNp63α in HaCaT-ΔNp63α when compared with HaCaT-eGFP and only HaCaT-eGFP expressed GFP protein ( Figure 6a). We next silenced VDR expression prior to VD 3 treatment of HaCaT-eGFP and HaCaT-ΔNp63α cells. As shown in Figure 6b, 10 nM VD 3 led to an increased cell number by trypan blue exclusion in both HaCaT-eGFP and HaCaT-ΔNp63α cells transfected with NSC when compared with vehicle treatment cells. As shown earlier ( Figure 5), the loss of VDR led to a reduction in HaCaT-eGFP cell number regardless of VD 3 dose ( Figure 6b). Importantly, ΔNp63α rescued the effects of VDR knockdown in HaCaT-ΔNp63α cells treated with 10 nM VD 3 (Figure 6b). Taken together, our results confirm that VD 3 mediated increase in ΔNp63α levels occurs via p38 and Akt activation. Previous studies have shown increased Akt activation following VD 3 treatment, particularly low doses of VD 3 . 8,9 The Akt pathway has been shown to increase ΔNp63α protein expression. 13 We therefore wanted to examine whether Akt activation upon VD 3 treatment was required for increased ΔNp63α protein levels. We measured Akt activation after treatment with VD 3 using an antibody directed against phosphorylated serine 473 of Akt (pAkt) and observed activation of Akt by low dose VD 3 preceded the increase in ΔNp63α (Figure 7a). To confirm that increased ΔNp63α levels and cell growth observed upon low dose VD 3 treatment is occurring because of activated Akt, we next examined the effects of VD 3 on ΔNp63α levels following the Inhibition of Akt combined with loss of VDR led to a further reduction in cell proliferation. Taken together, these results suggest that it is likely that in addition to Akt activation by VD 3 , increased ΔNp63α protein levels following low dose VD 3 treatment occurs via yet another pathway. We next examined the role of p38 mitogen-activated protein kinase (MAPK) signaling pathway in VD 3 -mediated increase in ΔNp63α levels. In addition to VD 3 treatment, a variety of other stimuli such as stress, growth factors or cytokines leads to the activation of p38 MAPK. 10,11 Interestingly, activated p38 MAPK was shown to increase ΔNp63α levels and cell proliferation in limbal epithelial cells. 12 To assess p38 MAPK activation by VD 3 in HaCaT cells following treatment with 10 nM and 100 nM of VD 3 , we monitored the phosphorylation of p38 MAPK and its downstream target MAPKAPK-2 at various time points post treatment. VD 3 induced the phosphorylation of both p38 MAPK and MAPKAPK-2 within 5 min of VD 3 treatment, which lasted longer following treatment with 10 nM VD 3 when compared with 100 nM VD 3 treatment (Figure 7d). Consistent with Figure 6a, we observed an increase in ΔNp63α protein levels following kinase activation with 10 nM VD 3 (Figure 7d). To confirm that VD 3 increased ΔNp63α protein levels via p38 MAPK activation, we examined whether pre-treatment with the p38 MAPK inhibitors SB202190 or BIRB-796 prior to VD 3 treatment led to a decrease in ΔNp63α protein levels. Inhibition of p38 activity led to a reduction in the ΔNp63α protein levels (Figures 7e and g). Furthermore, low dose VD 3 was unable to rescue ΔNp63α levels when p38 MAPK activation was inhibited (Figures 7e  and g), indicating that increased ΔNp63α levels in response to low levels of VD 3 occur via p38 MAPK activation.
Next, we studied the effects of SB202190 or BIRB-796 pretreatment on cell proliferation in the presence or absence of VDR in HaCaT cells. Inhibition of p38 MAPK led to a reduction in cell proliferation of cells treated with 10 nM VD 3 compared with cells treated with high dose VD 3 and DMSO (Figures 7f  and h). The loss of VDR alone or in combination with p38 MAPK inhibition reduced keratinocyte proliferation to similar levels regardless of VD 3 treatment (Figures 7f and h). Taken together, these data clearly indicated that p38 MAPK is required for low dose VD 3 -mediated increase in ΔNp63α protein levels and cell proliferation.
VDR and p63 expression are increased in NMSC. ΔNp63α is required for increased keratinocyte proliferation and has been shown to have a role in maintaining the proliferative capacity of basal keratinocytes in vivo. 15,[18][19][20][21][22] Both VDR and p63 have been shown to be overexpressed in NMSCs. [23][24][25][37][38][39][40] Therefore, we examined the expression levels of p63 and VDR in paraffin-embedded skin samples from individuals with normal skin (N = 49) and patients with BCCs (N = 54), SCC (N = 53) and the precursors to SCC (N = 59) by immunofluorescence to determine whether there is a correlation between p63 and VDR expression levels. We observed significantly higher levels of both p63 and VDR in BCC, SCC and the precursors to SCC when compared with normal non-cancerous skin as shown in representative images (Figure 8a). The levels of both p63 and VDR were highest in BCC followed by SCC and precursors to SCC (Figure 8b). Increased level of p63 and VDR might be responsible for increased proliferation upon exposure to VD 3 in NMSC. Interestingly, VD 3 production in the skin is a result of UV exposure and UV radiation is the most common cause of these cancers.

Discussion
An initial stage of proliferation followed by concomitant differentiation and inhibition of proliferation are important features of the skin, allowing for the development of each layer of the skin. 41 Delineating the mechanisms involved in triggering or preventing keratinocyte proliferation remains critical for a better understanding of NMSC. ΔNp63α is the most abundantly expressed p63 isoform in the basal layer of the skin where it is well known to upregulate genes involved in proliferation. 14, 16 We previously showed that ΔNp63α positively regulates VDR expression. 30 VDR is also expressed in the basal layer of the skin and is well known for its role in differentiation and calcium homeostasis. 35,42 Previous work showed that VDR functioned independently of VD 3 to activate  43 In support of the ligand-independent effects of VDR, we have shown that VDR is essential for basal levels of ΔNp63α transcription ( Figure 1) and could also increase the expression of ΔNp63α in the presence of its ligand VD 3 (Figure 2). Our findings suggest a new role for VDR in proliferation and cell survival through the regulation of ΔNp63α. Here, we show that VD 3 can regulate ΔNp63α and increase keratinocyte proliferation (Figures 2  and 4). The ligand requirement of VDR-mediated transcription might be context-specific allowing VDR to regulate genes necessary for cell homeostasis in the presence or absence of VD 3 , providing an additional layer by which keratinocyte proliferation is maintained in response to varying environmental conditions. VDR and VD 3 regulate many transcriptional targets, but their regulation of cellular processes is not limited to transcription; VDR and VD 3 also have non-genomic processes in the cell. 34 Several non-genomic functions of VD 3 have previously been determined, some of these functions included insulin secretion, smooth muscle migration and opening calcium and chloride channels. 34 We demonstrated that VD 3 enhances ΔNp63α expression in a non-genomic manner by increasing ΔNp63α protein within hours of VD 3 treatment (Figures 7a and d). This increase in ΔNp63α protein levels was shown to be dependent on VD 3 concentration as only low dose VD 3 was able to increase ΔNp63α levels ( Figure 2 and Supplementary Figures 1 and 2). Furthermore, this increase in ΔNp63α protein levels by low dose VD 3 correlated to an increase in cellular proliferation (Figure 4), whereas knockdown of ΔNp63α or VDR reduced cell proliferation at all doses tested ( Figure 5). Overexpression of ΔNp63α is seen in many NMSCs, such as SCC and VD 3 has been considered a chemotherapeutic adjuvant. 4,6,7,12,23,26,27 Our study indicates that reduction in ΔNp63α levels could improve the therapeutic potential of VD 3 in treating NMSCs. This is supported by previous studies, which have also established that ΔNp63α mediates resistance to cisplatin in head and neck SCCs and breast cancers, demonstrating the widespread benefit to controlling ΔNp63α in the treatment of epithelial cancers. [44][45][46] Prior studies have shown that VD 3 can activate p38 MAPK via non-genomic signaling by binding VDR complexed with caveolin-1 at the plasma membrane. 10,11 Once VD 3 is bound to membrane-associated VDR and caveolin-1, Src is recruited for the activation of p38 MAPK. 10 Activated p38 MAPK has been shown to increase ΔNp63α protein levels. 11 Our study shows that inhibition of p38 MAPK activation attenuates low dose VD 3 -mediated increases in ΔNp63α levels (Figure 7e and g). COX-2 a target of p38 MAPK is upregulated in NMSC, which further supports our findings that p38 MAPK is active in these cancers and can regulate its target genes, such as ΔNp63α. 47 Our data indicate that inhibition of p38 MAPK leads to a decrease in VD 3 -mediated increases in ΔNp63α, a marker of non-melanoma skin, suggesting that p38 MAPK is a potential pathway to target, in conjunction with chemotherapeutic regiment in these cancers.
We demonstrated that both p63 and VDR expression are increased in BCC, SCC and the precursor lesions to SCC (Figure 8). Given the role of p63 and VDR in the inhibition of cell invasion, it came as a surprise that both VDR and p63 show high expression in the more invasive cancer, SCC, when compared with the precursor of SCC (Figure 8). Consistent with previous reports, p63 show increased expression in BCC, 18 which correlates with an increased expression of VDR in BCC as shown in Figure 8. Our results imply that an elevated level of VDR does not indicate a good prognosis because we observed higher levels of VDR in more invasive cancers. p63 and VDR expression could be used as a predictor of patient response to VD 3 treatment as a chemotherapeutic agent. We have shown that VDR and VD 3 , known for their role in cancer prevention, also have a role in the regulation of oncogenic ΔNp63α, thus increasing keratinocyte proliferation at low VD 3 concentration, bringing to light a drawback of the use of VD 3 in cancer prevention and treatment. 48,49 Altogether these results suggest that VD 3 can have either a growth-suppressive or growth-stimulatory role in the presence of VDR in keratinocytes cells by regulating the expression of ΔNp63α. Monitoring VD 3 concentration in cancer treatment regimens will be critical to induce the proper signaling pathway to reduce cancer cell growth especially because our recent study clearly demonstrated that tumors from SKH-1 mice showed an increase in p63 expression with increasing concentration of dietary vitamin D. 50 Since, VD 3 action is dose-dependent, ensuring high doses of VD 3 are getting into cancer cells to reduce proliferation and ΔNp63α expression will be critical to its use as a chemotherapeutic agent. VDR and p63 siRNA used in this study were purchased from Qiagen (Valencia, CA, USA) and the target sequences used were described earlier. 30 The HaCaT-eGFP and HaCaT-ΔNp63α stable cell lines were generated by infecting parental HaCaT cells with lenti-virus plasmids expressing eGFP or ΔNp63α as described earlier. 30 At 72 h post infection, transduced cells were selected in blasticidin (10 mg/ ml) to obtain HaCaT stable cells expressing eGFP or ΔNp63α.
Reverse transcription PCR. Total RNA was extracted from human cells using the eZNA RNA isolation kit according to the manufacturer's protocol (Omega Bio-Tek, Norcross, GA, USA). TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA, USA) was used to synthesize cDNA from 1μg of total RNA. Total RNA extracted from the skin of VDR knockout mice and wild-type littermates were provided by Dr. Glendon Zinser (University of Cincinnati) in full accordance with the Institutional Animal Care and Use Committee of the University of Cincinnati.
VDR/1α, 25-dihydroxyvitamin D 3 regulates ΔNp63α NT Hill et al Quantitative real-time PCR analysis was performed as previously described using Assay on Demand specific for the genes of interest and normalized to endogenous GAPDH for human genes or to β-actin for murine genes of interest (PE Applied Biosystems, Foster City, CA, USA). 30,51 Human Assays on Demand used were GAPDH (4325792), VDR (Hs_0017213_ml) and pan p63 (Hs_00978340_ml). Murine Assays on Demand were pan p63 (Mm00495788_m1), VDR (Mm00437297_m1) and β-actin (Mm00607939_s1). Each experiment had an n = 3 independent experiments. Student's t-tests were used to determine significant difference.
Cell immunofluorescence assay. Cells were grown on sterile glass coverslips prior to fixation with 2% paraformaldehyde for 15 min. After three consecutive washes with PBS, cells were permeabilized with 0.2% triton X-100 diluted in PBS for 5 min. Cells were washed and blocked with 0.5% normal goat serum in PBS thrice before incubating with rat monoclonal anti-VDR 9A7 (Sigma-Aldrich) and rabbit polyclonal anti-p63 H129 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies primary antibodies for 1 h at room temperature. Excess primary antibody was removed with three consecutive 5-min washes in PBS-normal goat serum followed by incubation with AlexaFluor goat anti-rabbit 488 and goat anti-rat 568 antibodies at a dilution of 1 : 500 for 1 h at room temperature. Excess secondary antibody was removed with three consecutive 5-min washes in PBS-normal goat serum and one wash in PBS prior to mounting with Vecta-Shield plus DAPI Mounting Media (Vector Laboratories, Burlingame, CA, USA). Cells were visualized and imaged using a Leica CTR 6000 Microscope (Leica Microsystems, Wetzlar, Germany) and ImagePro 6.2 software (Media Cybernetics, Bethesda, MD, USA). Mean fluorescent intensity was determined with the ImagePro 6.2 software after normalizing to background fluorescence. At least 100 cells were measured for VDR and p63 staining intensity per condition for each experiment with an n = 3 independent experiments. Student's t-tests were used to determine significant difference.
Cell growth assays. Cells were seeded at 5000 cells per well in a 96-well flat bottom culture dish and at 24 h post-plating, cells were treated with 1 nM, 10 nM or 100 nM VD 3 as indicated. Proliferation was measured using Promega CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (MTS) at various time points posttreatment as described earlier. 20 Student's t-tests were used to determine significant difference. Cell viability was also measured by trypan blue exclusion and carried out in triplicate per condition per day post VD 3 treatment.
Tissue immunofluorescence assay. Formalin-fixed, paraffin-embedded human skin sections were stained for p63, and VDR as previously described 20,30 with one modification. Briefly, the human tissue were co-stained for p63 and VDR with two different antigen-retrieval processes, a heat base antigen-retrieval method was used for p63 followed by 20,30 an acid antigen-retrieval processes for VDR and finally neutralization with 0.1 M Borate Buffer pH8.5. 50 Human tissue samples consisted of normal skin (N = 49), precursor to SCC (N = 59) which included actinic keratosis, acantholytic actinic keratosis, acantholytic and hyperplastic actinic keratosis, hyperplastic actinic keratosis, squam in situ and bowenoid actinic keratosis, SCC (N = 53) which included superficial SCC, SCC arising from actinic keratosis background, SCC with perineural invasion, SCC, BCC (N = 54) which included infiltrative BCC, nodular BCC, nodular and infiltrative BCC and superficial BCC. Tissues were imaged using a Leica CTR 6000 Microscope (Leica Microsystems). Multiple measurements (at least nine), all of the same size, were taken of the epidermal tissue or cancerous tissues for each sample. Average mean fluorescence intensity was calculated following normalization to background by using the ImagePro 6.2 software.
Statistical analysis for stained tissue. Adjusted mean MFI and standard error of mean levels of p63 and VDR from normal skin samples, BCC samples, SCC samples and precursor to SCC samples were plotted. Repeated measures analysis of variance tests were conducted to account for the correlation between repeated measurements (nine measurements per sample). The comparisons of several covariance structures of repeated measures were performed to find the best covariance structure. The best covariance structure for the model was selected based on the smallest Akaike Information Criterion. 52 Post hoc multiple comparison procedures using Dunnett's test were performed to compare between mean MFI values of p63 and VDR between control samples (i.e., normal skin samples) and all other carcinoma samples. 53,54 PROC MIXED procedure (SAS/STAT, Ver 9.3, SAS Institute Inc., Cary, NC, USA) was used for analyses. Maximum experiment-wise error rates of 0.05 were set to consider whether differences were statistically significant.

Conflict of Interest
The authors declare no conflict of interest.