A novel function of the Wnt antagonist secreted frizzled-related protein 4 as a transcriptional regulator of Dickkopf-1, another Wnt antagonist, in glioblastoma cell line U87MG

Abstract

Wnt/β-catenin pathway dysregulation is associated with glioblastoma multiforme (GBM) pathogenesis and Wnt antagonists are downregulated in GBM. Wnt antagonist secreted frizzled-related protein 4 (sFRP4) has a tissue-specific, anti-metastatic and anti-stemness property. Our lab previously reported that gene silencing of sFRP4 in GBM cell line U87MG increases expression of another Wnt antagonist, Dickkopf-1 (Dkk1) and sFRP4 has a DNA binding ability. These findings in accordance with the nuclear localization of sFRP4 led to our present hypothesis that sFRP4 presumably negatively regulates Dkk1 and it probably interacts with the promoter region of Dkk1. Methylation-specific PCR (MSP), chromatin accessibility real-time PCR (ChART-PCR) assay, chromatin immunoprecipitation (ChIP), and quantitative DNA-protein interaction enzyme-linked immunosorbent assay (qDPI-ELISA) were carried out to test our hypothesis. We demonstrated that sFRP4 overexpression does not alter the methylation status of the Dkk1 promoter region. sFRP4 overexpression inhibits DNA-transcription factor interaction and enables chromatin accessibility to DNase I. Pertinently, sFRP4 has strong putative binding sites in the Dkk1 promoter region and its overexpression disrupts its interaction with the Dkk1 promoter. Interestingly, sFRP4 has the strongest affinity towards the ‐282 to +118 bp region. Downregulation of Dkk1 by overexpressed sFRP4 occurs by inhibition of the direct interaction of sFRP4 with the promoter region of Dkk1 as observed with low concentrations of sFRP4. We report for the first time a novel function of the Wnt antagonist sFRP4 acting as a transcription factor for another Wnt antagonist Dkk1, throwing open a new vista in the complex interplay between different antagonists of the Wnt pathway.