Regulation of RUNX2 transcription factor–DNA interactions and cell proliferation by vitamin D3 (cholecalciferol) prohormone activity

Abstract

The fat-soluble prohormone cholecalciferol (Vitamin D3) is a precursor of the circulating 25-OH Vitamin D3, which is converted by 1a-hydroxylase to the biologically active 1,25-OH Vitamin D3. Active Vitamin D3 interacts with the Vitamin D receptor (VDR), a transcription factor that plays an important role in calcium mobilization and bone formation. RUNX2 is a DNA-binding transcription factor that regulates target genes important in bone formation, angiogenesis, and cancer metastasis. Using computer-assisted drug design (CADD) and a microtiter plate-based DNA-binding enzyme-linked immunosorbent assay (D-ELISA) to measure nuclear RUNX2 DNA binding, we have found that Vitamin D3 prohormones can modulate RUNX2 DNA binding, which was dose-dependent and sensitive to trypsin, salt, and phosphatase treatment. Unlabeled oligonucleotide or truncated, dominant negative RUNX2 proteins were competitive inhibitors of RUNX2 DNA binding. The RUNX2 heterodimeric partner, Cbfb, was detected in the binding complexes with specific antibodies. Evaluation of several RUNX2:DNA targeted small molecules predicted by CADD screening revealed a previously unknown biological activity of the inactive Vitamin D3 precursor, cholecalciferol. Cholecalciferol modulated RUNX2:DNA binding at nanomolar concentrations even in cells with low VDR. Cholecalciferol and 25-OH Vitamin D3 prohormones were selective inhibitors of RUNX2-positive endothelial, bone, and breast cancer cell proliferation, but not of cells lacking RUNX2 expression. These compounds may have application in modulating RUNX2 activity in an angiogenic setting, in metastatic cells, and to promote bone formation in disease-mediated osteoporosis. The combination CADD discovery and D-ELISA screening approaches allows the testing of other novel derivatives of Vitamin D and/or transcriptional inhibitors with the potential to regulate DNA binding and biological function. ß

RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion

“…The inactive vitamin D3 precursor cholecalciferol has been shown to enhance RUNX2 DNA binding in vitro at a dose of 1 nM, while doses greater than 10 nM inhibited RUNX2 DNA binding [23]. In order to determine whether cholecalciferol was able to inhibit RUNX2 transcriptional activity in melanoma cells, we performed luciferase assays in the presence or absence of micromolar concentrations of cholecalciferol.…”

“…ShRNA-mediated knock down of RUNX2 in melanoma cell lines negatively affected cell growth and inhibited their migration and invasion in conjunction with a reduction in the levels of the kinase FAK/PTK2 involved in motility and adhesion. The RUNX2 DNA binding inhibitor Cholecalciferol [23] inhibited the activity of the RUNX2-responsive MMP13 promoter, and also decreased melanoma cell growth and their ability to migrate. Furthermore, we addressed the relevance of RUNX2 expression to human melanomagenesis using a melanoma tissue microarray and confirmed overexpression of RUNX2 in melanoma specimens as compared with benign nevi.…”

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

“…RUNX2 and its cofactor Cbf ß were found to be associated with the biotin-labeled oligonucleotides 6 , thus validating the specificity of the assay and also emphasizing that it is possible to identify cofactors that might associate with specific DNA-binding transcription factors. With continuous kinetic monitoring, incubation can be extended and less nuclear protein may be needed to detect changes in DNA binding.…”

“…2. Treat subconfluent cells with nocodazole (0.2 μg/ml for 16 hr) to arrest cells at the G2/M cell cycle boundary 6 . Under these conditions, the RUNX2 protein is stabilized and maximal DNA binding is achieved.…”

“…Therefore, kinetic monitoring is expected to be more sensitive than stop reaction methods. An important application of this kinetic method includes screening for drugs that inhibit or activate transcription factor DNA binding 6 .…”