Figure 5

Knockdown of the VDR in MDA-MD-231 breast cancer cells downregulates Wnt/β-catenin signaling pathway. (a) Hierarchical clustering of 121 differentially expressed genes (P<0.05) between MDA-VDR-KD compared to MDA-NT cell lines (microarray data set) showed distinctive expression patterns. The three lanes represent gene array patterns derived from three separate cultures of the same cell line (MD-NT or MDA-VDR-KD) cultured in identical conditions for the same amount of time. (b) From this list, candidate genes belonging to the Wnt/β-catenin pathway, downstream of β-catenin were shown to be downregulated. (c) qRT-PCR was used to validate these genes whereby reflecting processes in decreased proliferation and increased apoptosis. (d,e) Both cytoplasmic and nuclear β-catenin protein levels were reduced in MDA-VDR-KD compared to MDA-NT cells. Treatment with 10⁻⁸ mol·L⁻¹ 1,25(OH)₂D₃ induced nuclear β-catenin protein levels in MDA-NT but not in MDA-VDR-KD cells while GSK-3β expression remained unchanged in 1,25(OH)₂D₃-treated MDA-NT and MDA-VDR-KD cells (d). Reduced β-catenin protein levels in tumors derived from VDR knockdown cells relative to NT controls (IHC stain, e). (f–j) Inhibition of GSK-3β via treatment of VDR-NT and VDR-KD cells with 1 μmol·L⁻¹ BIO resulted in increased expression levels of β-catenin protein (f) as well as Axin2, cyclin D1 and Ki67 mRNA (g). Inhibition of GSK-3β was associated with increased growth of MDA-VDR-KD cells (h), significantly reduced apoptosis (i) but unchanged proliferation of VDR-KD cells, as assessed by Ki67 expression (j). Asterisks denote significance difference from controls (*P<0.05; **P<0.01). Data are mean±s.e.m.