Breast Cancer Gene Expression Profile in Post-Menopausal Patients Supplemented with Vitamin D.

Abstract

Abstract Vitamin D supplementation is indicated for post-menopausal women to prevent osteoporosis and lower 25(OH)2D3 or 1,25(OH)2D3 serum levels have been associated with breast cancer incidence or prognosis (metastasis). The antiproliferative effects of vitamin D are observed in breast cancer cell lines exposed to phamacological doses of calcitriol (1,25(OH)2D3, 100nM) but whether physiological doses are sufficient to produce growth inhibition in vivo is not known. The aim of our study was to investigate gene expression profile changes of breast cancer samples from patients supplemented with calcitriol, presenting an anti-proliferative effect on the tumor. Post-menopausal women diagnosed with breast cancer were instructed to take one (0.25ug/day, n=8) or two (0.50ug/day, n=8) tablets of calcitriol after tumor biopsy. Median time of supplementation was 30 days. Sixteen tumor samples were collected during biopsy (before supplementation) and breast surgery (after supplementation). Proliferation index was evaluated by tumor Ki-67 immunohistochemistry (IHC) expression in breast cancer samples before and after calcitirol supplementation and 1000 cells were counted by three observers (p < 0,001, Pearson's correlation). After establishing a counting error between observers, the cut-off value was fixed in 26.53% (percentile 60%) to define response. Patients were then categorized as responsive (a reduction of more than 26.53% on positive Ki-67 positive cells) and non-responsive (difference between samples before and after supplementation not exceeding 26.53%). Among our sixteen patients, all were categorized as responsive to calcitriol supplementation except for two. So far, gene expression profile of two patients (both categorized as responsive) has been analyzed using the U133 Plus 2.0 Affymetrix Gene Chips from 100ng of total RNA. To verify signalling pathways possibly involved in response to vitamin D exposure, additional five samples from a parallel study, in which breast cancer samples from post menopausal patients were collected at surgery and treated in vitro with a low concentration of calcitriol, 0.5nM (that can be attained with subcutaneous administration of doses of 8ug calcitriol, without hypercalcemia) for 24h, were included in the analysis. All samples had RNA hybridized to the same gene chips. Results were normalized and analyzed using RMA and Mev.TM4 softwares. CYP24A1, a target gene of vitamin D, presented a positive regulation after calcitriol supplementation in all samples analyzed. Differentially expressed genes were involved in the regulation of cell cycle [SMAD2, cyclin E, YWHAQ (14-3-3 family] and calcium signalling (HTR7, PTGER1 and PTGER2). Our results indicate that the tumor proliferation index is reduced upon calcitriol supplementation. Moreover, potentially regulated pathways in breast cancer specimens after administration of low doses of calcitriol are regulation of cell cycle and calcium signaling.Supported by FAPESP 2007/01111-0 – 2007/04799-2 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6128.