Mechanisms of resistance towards the vitamin D receptor in breast cancer

• Main Author: Townsend, Kelly
• Published: University of Birmingham 2005
• Subjects: 616.994490610724
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423420

Although best known for its role in maintenance of calcium homeostasis and bone metabolism, 1,25(OHhD3 also regulates proliferation, differentiation and apoptosis of epithelial cells from a range of tissues, including breast epithelial cells. However, reflecting the growth in this knowledge, over the last twenty years there has been significant interest in the antiproliferative actions of 1,25(OH)2D3 in normal physiology and their potential impact as a novel chemotherapy. The therapeutic impact of 1,25(OHhD3 and its synthetic analogs in cancer has been limited, not only because of the calciotropic side-effects, but also because of the variable sensitivity to the hormone. A number of studies have demonstrated that some breast cancer cell lines exhibit extreme resistance to the antiproliferative action of 1,25(OHhD3. For example, MDA-MB-231 cells require concentrations of greater than 1 ).lM to inhibit cell or colony proliferation by more than 50%. Therefore the aim of this thesis was to identify possible mechanisms for the apparent insensitivity to the antiproliferative actions of 1,25(OH)2D3 in breast cancer. To investigate this the mRNA expression and activity levels of the components of the 1,25(OHhD3 signalling axis (CYP27Bl, VDR and CYP24) were examined in both a panel of breast cancer cell lines and a cohort of 41 matched breast tumour and normal biopsy pairs (Chapter 3). Furthermore, a transcriptomic approach using an Affymetrix human V133 gene expression array (Affymetrix, UK), was utilised to study the gene expression patterns in MCF-7 and MCF-7Res cells, with and without 1,25(OH)2D3 treatment, in order to identify novel genes involved in both 1,25(OHhD3 sensitivity and resistance (Chapter 4). The data demonstrated that all components of the vitamin D signalling axis were expressed at the mRNA level and that both 1a-hydroxylase and 24-hydroxylase enzymes were active in a number of breast cancer cell lines, and in normal breast and the paired breast tumour tissue. Furthermore, all expression levels were elevated in the breast tumours and CYP24 expression was deregulated in breast tumours. Moreover, the increase in CYP27B 1 expression was possible associated with an immune infiltrate of tumour associated macrophages indicated by the fact that with the increased tumour expression of CYP27B 1 mRNA there was a corresponding increase in both TLR-4 and CD14 mRNA expression, which together form part of the macrophage endotoxin response system (Chapter 3). Furthermore, the transcriptomic approach identified a number of possible critical VDR targets involved in 1,25(OH)2D3 resistance including, CD44, ILIl, GADD45a, IGFBP-3, EGFR, ZNF38 and TGF-~2. The role of TGF-~2 was investigated further and a 24 hour time course demonstrated that 1,25(OHhD3 resistant cells did not upregulate TGF-~2 in response to l,25(OHhD3 treatment whereas both the sensitive MCF-7 and non tumourogenic MCF12A cells did. Furthermore treatment with TGF-~2 sensitises resistant cells to the antiproliferative effect of 1,25(OH)2D3 and there was evidence to suggest that TGF-~2 and 1,25(OHhD3 coregulate the expression of VDR target genes as indicated by the increased upregulation of the strongly responding VDR target gene CYP24 following co-treatment (Chapter 4). In conclusion, the data presented in this thesis has identified possible mechanisms of resistance towards the VDR in breast cancer cells, including deregulated autocrine metabolism 1,25(OH)2D3, and the inability of cancer cells to upregulate TGF-~2 following l,25(OHhD3 treatment.