We hypothesised that normal regulation of 25(OH)D3-24-hydroxylase (encoded by CYP24) determines the autocrine/paracrine action of 1,25(OH)2D3 in normal breast and prostate epithelial cells. Furthermore, these processes are compromised in malignancy.
We therefore studied the regulation of CYP24, amongst a panel of breast and prostate cancer cell lines (ZR-75-1, T47-D, MCF-7, MDA-MB-231 breast cells and LNCaP, PC-3 and DU-145 prostate cells) that display different sensitivities to the induction of growth arrest by 1,25(OH)2D3.
Quantitative real time RT-PCR and western blot analyses demonstrated that in exponential growth, each of the lines had elevated CYP24 expression and upon reaching confluence all cell lines downregulated CYP24 levels. For example in MCF-7 cells there was a 10 and 3 fold decrease in mRNA and protein respectively. Paradoxically, modulation of CYP24 in response to 1a,25(OH)2D3 (10nM) was greatest in cells least sensitive to 1a,25(OH)2D3. For example PC-3 cells demonstrated an approximate 10-fold greater induction than normal prostate epithelial cells of CYP24 mRNA despite being approximately 1000 times less sensitive to 1a,25(OH)2D3-mediated growth inhibition.
We therefore investigated the role of CYP24 using antisense oligonucleotides in MDA-MB-231 cells. Antisense-treated cells demonstrated approximately 40% reduction in 1a,25(OH)2D3-induced CYP24 protein levels and a significant increased sensitivity to the antiproliferative actions of 1a,25(OH)2D3 (p<0.0001). These findings indicated that CYP24 metabolism of 1a,25(OH)2D3 to 1a,24,25(OH)3D3 protects against the antiproliferative actions of the parent molecule. Consistent with this, all the cell lines demonstrated significantly reduced antiproliferative responses to 1a,24,25(OH)3D3 compared to 1a,25(OH)2D3, indeed at physiological doses (10-10 M) 1a,24,25(OH)3D3 actively stimulated proliferation of MDA-MB-231 cells.
In conclusion, the VDR signalling axis is dynamic but skewed in cancer cells to resist the antiproliferative actions of 1a,25(OH)2D3 Increased induction of CYP24 in response to 1a,25(OH)2D3 appears central to this process.
08 - 11 Apr 2002
British Endocrine Societies