Identification of VDR-Responsive Gene Signatures in Breast Cancer Cells

Abstract

Objectives: Defining transcriptional profiles which predict cancer cell anti-proliferative responsiveness towards 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] is required to improve and tailor the chemotherapeutic application of this seco-steroid hormone to individual cancer patients. Methods: We undertook a transcriptomic approach with Affymetrix human U133 GeneChips to determine responsive and resistant gene signatures in MCF-7 breast cancer cells and 1α,25(OH)₂D₃-resistant MCF-7_Res cells, respectively. Principal component and hierarchical clustering analyses demonstrated that the patterns of responsiveness between the 2 cell types differed clearly and were used to generate heat maps. Differentially regulated gene targets were validated with Q-RT-PCR and the biological impact upon proliferation measured. Results: In untreated MCF-7_Res cells, 163 genes were up-regulated and 274 down-regulated (with a log₂ ratio of >0.5) compared to the MCF-7 controls. Using the same gene expression threshold, 1α,25(OH)₂D₃ treatment (100 nM, 6 h) of MCF-7 cells up-regulated 91 genes and down-regulated 5, whereas in MCF-7_Res, despite their resistance to the anti-proliferative effects, 156 genes were modulated with 91 being down-regulated. Strikingly, CYP24 was the only induced gene that was common to the genetic profiles of the 2 sets of 1α,25(OH)₂D₃-treated cells. Heat map analyses defined 2 sub-clusters of genes: (1) basal expression patterns associated with insensitivity towards 1α,25(OH)₂D₃ and (2) regulated expression patterns associated with 1α,25(OH)₂D₃ sensitivity. This latter cluster contained BAX, GADD45α, IGFBP-3, EGFR, MAPK4 and TGF-β₂. Time course studies confirmed the 1α,25(OH)₂D₃ regulation of TGF-β₂ in MCF-7 and non-tumourigenic MCF-12A cells but not in MCF-7_Res cells. Co-treatment of MCF-7_Res cells with exogenous TGF-β₂ plus 1α,25(OH)₂D₃ enhanced anti-proliferative and vitamin D receptor transcriptional effects. Conclusions: Basal and regulated gene patterns can be used to predict and monitor the cellular response towards vitamin D₃ compounds and may possibly be applied as a further diagnostic tool.

Antiproliferative action of menadione and 1,25(OH)2D3 on breast cancer cells

“…Continuous exposure of MCF-7 cells to 1,25(OH) 2 D 3 produces apoptosis of those cells unable to differentiate, generating a vitamin D resistant cell population named MCF-7 DRes cells [17]. Towsend et al [18] have done transcriptomic analyses to determine responsive and resistant gene signatures in MCF-7 and MCF-7 DRes cells, respectively. The proapoptotic gene BAX was one of the most significantly upregulated genes (3.25-fold) by 1,25(OH) 2 D 3 in MCF-7 cells.…”

Vitamin D Actions in Mammary Gland and Breast Cancer

“…MCF-7 DRES cells can be sensitized to the growthinhibitory effects of 1,25(OH) 2 D 3 by co-treatment with low concentrations of the phorbol ester TPA, suggesting that phosphorylation pathways may be altered in this cell line [28,101]. Genomic and proteomic profiling supports the notion that pathways unrelated to VDR itself are disturbed during selection for 1,25(OH) 2 D 3 resistance [52,68]. Further studies with these interesting cell lines will be necessary to resolve the mechanism(s) of vitamin D resistance, in particular the relative contribution of genetic versus epi-genetic changes.…”

“…Comparative analysis of gene profiles in sensitive and resistant MCF-7 variants has yielded a number of candidates that confer sensitivity to vitamin-D-mediated growth inhibition, including BAX, GADD45a, IGFBP-3, EGFR, MAPK4, and TGFb [52]. Using a proteomics approach, Byrne and Welsh [68] profiled 270 apoptosis-related proteins in sensitive and resistant MCF-7 cells.…”

Nutrigenomics, Vitamin D and Cancer Prevention

“…For example, 1,25(OH) 2 D inhibits cell growth of both normal and tumor cells by inhibiting the transition for the G 1 to the S phase of the cell cycle [47]. This effect was mediated by increased expression of cyclin A1 in ovarian cancer cells [48], whereas breast cancer cells had increased expression of cyclin D2 [49]. …”