Oral Presentation ESA-SRB Conference 2015

Multiple epidemiological studies have shown that lower circulating levels of vitamin D are associated with an increased risk of breast cancer. The major plasma form of vitamin D is 25(OH)D₃ (25D), the pro-hormone, which can be activated by hydroxylation to the active hormone 1,25(OH)₂D₃ (1,25D). To provide a molecular explanation for the epidemiological observations we have utilised a short-term ex vivo incubation system of human normal and cancerous breast tissues. This approach preserves the tissues 3D structure and allows short-term treatment with 25D or 1,25D. In common with many tissues, breast epithelial cells can metabolise 25D to the active hormone as they can express the hydroxylase CYP27B1. In such tissues there is also rapid induction of CYP24A1 which converts 1,25D to an inactive form.

Using such ex vivo normal and breast cancer tissues treated with 1,25D we isolated RNA and used mRNA-Seq to define vitamin D regulated genes.The success of vitamin D treatment was shown by the induction of highly up-regulated CYP24A1.  Only 14 genes were differentially expressed in common between normal and cancer tissue. On considering expression levels and known/possible functions we selected the over-expressed genes KLK6, CLMN, SERPINB1, and EFTUD1 for further analysis. Analysis of expression by qRT-PCR in additional human breast samples treated with1,25D further confirmed their expression was up-regulated following 1,25D treatment.

Induction of these four target genes by 1,25D are VDR dependent as VDR knockdown in non-malignant and malignant breast cell lines resulted in loss of 1,25D up-regulation. From epidemiological studies high vitamin D levels are associated with a lower risk of breast cancer, and therefore target genes induced by 1,25D would be predicted to have tumour suppressor-like characteristics. The known functions of these genes are consistent with this proposed role. In addition, analysis of publically available expression data in breast cancer shows high expression of KLK6, CLMN and SERPINA1 is significantly associated with a more favourable prognosis, particularly in the poorer prognostic triple negative subgroup. These data suggests high levels of vitamin D in breast tissues maintain the expression of specific cancer tumour suppressor genes.