Vitamin D3 inhibits hedgehog signaling and proliferation in murine Basal cell carcinomas

Abstract

Constitutive Hedgehog (HH) signaling underlies several human tumors, including basal cell carcinoma (BCC). Recently, Bijlsma and colleagues reported a new biologic function for vitamin D3 in suppressing HH signaling in an in vitro model system. On the basis of that work, we have assessed effects of vitamin D3 on HH signaling and proliferation of murine BCCs in vitro and in vivo. We find that indeed in BCC cells, vitamin D3 blocks both proliferation and HH signaling as assessed by mRNA expression of the HH target gene Gli1. These effects of vitamin D3 on Gli1 expression and on BCC cell proliferation are comparable to the effects of cyclopamine, a known inhibitor of the HH pathway. These results are specific for vitamin D3, because the precursor 7-dehydrocholesterol and the downstream products 25-hydroxy vitamin D3 [25(OH)D] and 1,25-dihydroxy vitamin D3 [1,25(OH)(2)D] are considerably less effective in reducing either Gli1 mRNA or cellular proliferation. Moreover, these effects seem to be independent of the vitamin D receptor (VDR) because short hairpin RNA knockdown of VDR does not abrogate the anti-HH effects of D3 despite reducing expression of the VDR target gene 24-hydroxylase. Finally, topical vitamin D3 treatment of existing murine BCC tumors significantly decreases Gli1 and Ki67 staining. Thus, topical vitamin D3 acting via its HH inhibiting effect may hold promise as an effective anti-BCC agent.

Fig. 1

Vitamin D3 is a potent inhibitor of cellular proliferation and Gli1 mRNA (n = 3 experiments). (A) Cellular proliferation studies were conducted in a BCC cell line (ASZ), non-tumorigenic keratinocytes (C5N), and a medulloblastoma cell line (Med1) incubated for 48 h with cyclopamine (CPN) versus 1,25(OH)₂D and vitamin D3. Mean±SEM, *P < 0.01 compared to C5N. (B) Cellular proliferation was assayed in BCC cell lines (ASZ, BSZ, CSZ) and non-tumorigenic keratinocytes (C5N) 48 h after treatment. Mean±SEM, *P < 0.01 compared to C5N. (C) 24-hydroxylase mRNA relative expression in ASZ cells treated with 7DHC (10 µM), 25(OH)D (10 µM), 1,25(OH)₂D (0.1 µM), vitamin D3 (10 µM), and CPN (10 µM) at 24 and 48 h. Mean±SEM, *P < 0.01 compared to control (DMSO or EtOH). (D) Gli1 mRNA relative expression in ASZ cells treated with 7DHC (10 µM), 25(OH)D (10 µM), 1,25(OH)₂D (0.1 µM), vitamin D3 (10 µM), and CPN (10 µM) at 24 and 48 h. Mean±SEM, *P < 0.01 compared to control (DMSO or EtOH).

Fig. 2

Anti-BCC effects mediated by vitamin D3 are intact when vitamin D3 receptor is blocked (n =3 experiments). (A) Phase contrast and fluorescence images for ASZ cells treated with control, shVDR, or no treatment for 72 h. (B) Upper panel: western blot showing VDR and β-actin protein levels in ASZ cells treated with shVDR or control. Lower panel: Quantitation of VDR protein level relative to β-actin in ASZ cells treated with shVDR or control. Mean±SEM. (C) Comparison of relative 24-hydroxylase and Gli1 mRNA in ASZ cells pretreated with control vector or shVDR, and then exposed to vitamin D3 (5 µM) for 24 h. Mean±SD. Note: the y-axis scale is different from Figure 1C, D. (D) Cellular proliferation in ASZ cells pretreated with no treatment, control vector, shVDR, and then exposed to vitamin D3 at 5µM for 24 h. Proliferation was assessed as % of control ASZ cells without vitamin D3 treatment. Mean±SD.

Fig. 3

Fig. 3.1. Histology of BCCs treated topically with acetone (control) for 30 days (A–F) or with topical 1.3 mg/kg vitamin D3 for 30 days (G–L). β-gal and hematoxylin and eosin staining for acetone or vitamin D3 treated BCC (Panel A, G). BCCs from Ptch1+/− K14-Cre-ER p53fl/fl mice stain blue due to β-galactosidase activity which is encoded by the lacZ gene that was inserted to replace the wildtype Ptch1 gene. Keratinocyte markers of differentiation, K14 (Panel B, H) and K10 (Panel C, I) are shown as well as CC3, a marker of apoptosis (Panel D, J) and Ki67 for proliferation (Panel E, F, K, L). Scale bars, 100 µm. Fig. 3.2. Levels of Ki67 staining in BCC tumors treated with either acetone (control, n = 7) or vitamin D3 (1.3 mg/kg, n = 10) for 30 days (P < 0.05).

Fig. 4

Vitamin D3 decreases Gli1 and increases 24-hydroxylase in murine BCCs in vivo. (A) Relative mRNA level of Gli1 in total RNA from BCC tumors treated with either acetone (n = 9) or vitamin D3 (2.6 mg/kg) (n = 24) for 4 days. (B) Relative mRNA level of 24-hydroxylase in total RNA from BCC tumors treated with either acetone (n = 12) or vitamin D3 (2.6 mg/kg) (n=24). (C) Relative mRNA level of Gli1 in total RNA from BCC tumors treated with either control cream (n = 9) or 1,25(OH)₂D analog cream, calcipotriene (n = 9) for 4 days. (D) Relative mRNA level of 24-hydroxylase in total RNA from BCC tumors treated with either control (n = 7) cream or 1,25(OH)₂D analog cream, calcipotriene (n = 7).