In 1941, Apperly (1
) noted that the incidence of colon cancer in the United States is considerably higher in the North than in the South, and the data favoring this “latitudinal gradient” remain strong for several cancers including in particular those of the colon, breast, and prostate. Four decades later, Garland and Garland noted that the differences in cancer incidence in different locales are inversely related to the amount of sunlight they receive and proposed that this gradient might be explained by an anti-cancer effect of varying amounts of vitamin D made in sun-exposed skin (2
). This proposal has been seminal, and 6000 papers have been published touching on vitamin D and cancer. Approaches taken to investigate this putative relationship include comparisons between cancer risk and sun exposure and/or dietary vitamin D intake; assessments of circulating 25(OH)D, the most readily available measurement of body vitamin D status, in patients with cancers and controls; comparisons of DNA polymorphisms in the genes encoding the vitamin D receptor (VDR) and the enzyme responsible for the catabolism of the VDR ligand: 1,25(OH)2
D; and studies of the cancer preventive effects of supplemental dietary vitamin D. The latter include one very large prospective study of the effects of 400 IU of vitamin D3/day, which showed no effects on cancer incidence or mortality (4
), albeit the compliance rate in this study was poor, and one considerably smaller prospective study of 1,100 IU vitamin D3/day, which found a statistically significant reduction of cancer incidence in those taking the vitamin supplement (5
). Taken together, the positive correlation of cancer incidence and latitude of residence seems strong and the inverse correlation of sunlight exposure and cancer seems moderately strong, but the mechanistic importance of any inverse correlation of cancer incidence and vitamin D3 and the anti-cancer efficacy of vitamin D3 supplementation remain uncertain (6
The most studied mechanism of the effect of vitamin D3 is the 1,25(OH)2
D induced transcriptional activation of the VDR with resultant changes in cell behavior including enhanced differentiation and reduced proliferation of skin keratinocytes (9
). By contrast, Bijlsma and colleagues (12
) recently proposed a new biologic function for unhydroxylated vitamin D3 - the inhibition of hedgehog (HH) signaling. They found that D3 binds to Smo specifically and thereby inhibits Gli reporter activity in C3H/10T1/2 fibroblasts in vitro
. In addition, D3 treatment of zebrafish in vivo
mimicked the Smo−/−
phenotype. In fact, Bijlsma and colleagues (12
) propose that Ptch1 protein accomplishes its inhibition of HH signaling by transporting vitamin D3 to Smo protein.
HH signaling was identified initially as a pathway crucial to development but more recently has come to be seen as a potentially important stimulator of carcinogenesis when dysregulated. This can occur via mutations in the genes encoding components of the pathway or by excess production of HH ligand by the tumor or stromal cells (13
). Indeed, the first in man inhibitor of HH signaling, GDC-0449, is now in clinical trials for at least eight human cancers [clinicaltrials.gov
], and several other HH inhibitors are in varying stages of clinical development.
Of the human cancers with mutations in HH signaling pathway components, the best studied tumor-HH relationship in humans and mice is that found in basal cell carcinomas (BCCs), and inhibition of HH signaling with small molecule drugs can have dramatic inhibitory effects on human BCCs (14
). BCCs are the most common of all human cancers, affecting approximately 1 million Americans per year (15
). The pivotal molecular abnormality in BCCs is constitutive activation of the HH signaling pathway, in 10–20% of tumors by mutational activation of SMO and in the great majority of the others associated with mutational inactivation of PTCH1 (16
). In addition to mutational activation of the HH pathway, human BCCs also frequently have mutations in p53 (22
+/− mice develop BCCs after mutational insults, and the addition of conditional loss of keratinocyte p53 greatly accelerates murine BCC carcinogenesis
Based on the known role of HH signaling in BCC carcinogenesis and on this newly reported HH-inhibitory function of vitamin D3, we have studied whether vitamin D3, its precursor, and its hydroxylated derivatives can inhibit cellular proliferation and down-regulate HH signaling in established murine BCC cell lines and in murine BCCs in vivo. We then investigated whether the anti-BCC effects of vitamin D3 are mediated via the classic VDR pathway.