Our findings demonstrate that the VDR promoter is hypermethylated in breast cancer, and provide evidence that its demethylation in breast cancer cell lines results in re-expression of the VDR transcripts. Similar epigenetic suppression of VDR mRNA expression has been demonstrated in placental and choriocarcinoma cell lines, which also showed VDR mRNA re-expression after AZA treatment.18
Our initial proliferation assays confirmed the relative Calcitriol insensitivity in breast cancer cells (see ), and an additive antiproliferative response to combined Calcitriol and AZA treatment. Treatment of breast cell lines with AZA resulted in near complete demethylation of the VDR promoter, whereas Calcitriol had almost no effect. Changes in VDR transcript levels mirrored these results (see ). These results were validated in clinical samples, where we show clear hypermethylation of a CpG island in the VDR promoter region in breast cancer tissue by both bisulfite sequencing and a specific MSP assay. Since this CpG island includes Sp1 binding sites, its methylation would be expected to affect Sp1 binding and transcriptional activation,19,20
and although methylation-induced transcriptional silencing most frequently involves CpG sites in the vicinity of the transcriptional start site (TS), there are many examples of critical methylation sites both upstream and downstream of the TS.21
Furthermore, demethylation results in re-expression of the VDR transcripts. Thus, much of the unresponsiveness of the VTD pathway in breast cancer is likely due to epigenetic silencing of VDR transcription, and this may be reversible by pharmacological intervention using demethylating agents.
Numerous conflicting studies have made it difficult to arrive at definitive conclusions regarding the mechanism of Calcitriol insensitivity and the biological impact of this mechanism in cancer, and the data on VDR expression levels in cancer have been particularly inconsistent. Quantitative RT-PCR of VDR has shown that its expression was modestly downregulated in endometrial cancer cells22
and in human colon and lung tumor samples, compared to their normal counterparts, while it was strongly overexpressed in ovarian cancer tissue.8
Reports of VDR expression in breast cancer tissue compared to adjacent normal or independent control tissue have been contradictory,8,23
which may be due to differences in the specific assays used. Our results suggest that a significant proportion of transcripts detected in breast cancer tissue may be N-terminally truncated and may not yield functional peptides, while full-length transcripts are relatively depressed, compared to normal breast epithelial tissue.
Very little is known about the possible roles in breast cancer of the various isoforms of VDR. Initial reports describing several VDR promoters and multiple transcripts with tissue-specific abundance variation12,17
were followed by more detailed biochemical analyses documenting functional differences between the VDRA, VDRB1 and VDRB2 isoforms.15,16,24
There are currently few data, however, on how this regulatory complexity affects breast cancer epidemiology or pathogenesis, let alone what role the potentially untranslated, N-terminally truncated variants we detected in our breast cancer samples might have. A detailed analysis of VDR splice variants, the encoded proteins, and their functional significance is beyond the scope of this report, but our data on VDR expression levels, which strongly depended on the specific amplicons used, suggest that studies failing to take into account the heterogeneity of transcripts in breast cancer tissue could be misleading. In this study, demethylation treatment of breast cancer cell lines with AZA induced the re-expression of active variant transcripts of VDR. These results suggest a correlation between VDR methylation and active transcript variant expression.
Further complicating the role of VDR in breast carcinogenesis, Calcitriol metabolism is controlled by a complex interplay of genetic, nutritional and environmental factors. For example, the dietary intake of Vitamin D only contributes about 10% to Calcitriol synthesis, while the UV-initiated cutaneous conversion of 7-dehydrocholesterol to Vitamin D accounts for about 90%.25
Vitamin D then undergoes a two-step activation process. The initial 25-hydroxylation is performed predominantly in the liver, and the resulting 25OH-VTD is bound to the D-binding transport protein (DBP), constitutes a reservoir, and is the most commonly measured Vitamin D metabolite. 25OH-VTD levels vary considerably depending on access to sunlight and skin pigmentation.26
The production of active Calcitriol by the rate-limiting 1α-hydroxylase CYP27B1 is under tight control in the kidney. It has recently become apparent, however, that CYP27B1 is expressed in a wide range of cell types, including breast epithelial cells, where it is subject to post-transcriptional27
negative feedback inhibition.28–30
Antiproliferative and differentiating effects of CYP27B1 have been detected in many different tissues.31
Similarly, there is a positive feedback loop with CYP24A1, which is induced by ligand-activated VDR. CYP24A1 hydoxylates Calcitriol at the 24-position, the first step in its degradation pathway.32
Our investigation of the expression levels of VDRE-containing, Calcitriol-metabolizing p450 hydroxylases showed that, consistent with a low Calcitriol pathway activity, the rate limiting activating 1α-hydroxylase and the main catabolic 24-hydroxylase mRNAs are underexpressed in breast cancer tissues (). Data from clinical specimens have been contradictory, however, with reports of up or downregulation of the opposing 1- or 24-hydroxylases.8,23,33
Some of these findings could be attributed to expression of splice-variants encoding non-functional peptides, particularly of 1α-hydroxylase (CYP27B1), as reported in several cancers, including breast cancer.34,35
Interestingly, as reported by others,36
we found consistently increased levels of CYP3A4, which has a strong 24-hydroxylase activity and is involved in the metabolism of several steroid hormones and in xenobiotic metabolism.37,38
CYP3A4 polymorphisms have been identified as potential risk factors for predisposition to breast and prostate cancer, and may have pharmacogenetic implications in the tumor response to several chemotherapeutic agents as well.39
In the context of a downregulated Calcitriol pathway in breast cancer, CYP3A4's expression may be less influenced by its VDRE than by triggers related to its role in steroid hormone and xenobiotic metabolism, and its Calcitriol catabolic actions may further depress already low levels of Calcitriol in breast tissue.
Decreased expression of p21 (CDKN1A) has been reported in breast cancer40
and in ovarian cancer.41
p21 contains three VDREs, and is known to be regulated by Calcitriol-induced cyclical chromatin looping.42
In agreement with these reports, we found decreased expression of the p21 tumor suppressor gene in our breast cancer samples, as would be expected in the context of an inactive Calcitriol pathway (see ).
Furthermore, we also demonstrated that VDR-responsive genes, including the tumor suppressor C/EBP, could be re-induced in vitro by demethylation treatment, while Calcitriol alone generally had little effect, except in the T47D cell line and on the expression of the CYP3A4 catabolic hydroxylase.
In summary, this report provides correlative evidence for the importance of the Calcitriol/VDR axis in breast cancer, and suggests that potentially reversible epigenetic silencing may be at the center of its inactivation.