OBJECTIVE: It is known that Notch signaling is minimally active in neuroendocrine (NE) cancer cells and the induction of Notch isoforms alter the malignant neuroendocrine phenotype. The induction of Notch3 by Histone Deacetylase Inhibitors (HDAC) inhibitors appears to be the result of increased mRNA expression at the transcriptional level, which leads to suppression of cancer cell proliferation and to apoptosis. The aim of our study is to investigate the molecular mechanism of HDAC inhibitor activation on the Notch3 pathway.
METHODS: We functionally characterized the Notch3 promoter using deletion mapping. The mapping started with the truncated genomic DNA fragment fused with a luciferase reporter, transfected into BON cell, a carcinoid cell line, screened for luciferase activity. Protein-DNA binding was then performed by electrophoretic mobility shift assay (EMSA).
RESULTS: One HDAC inhibitor, Thailandepsin-A, was shown to induce luciferase activity controlled by a small distal region of Notch3 promoter, from -120 to +1 of the start codon ATG. Further, we identified a functional DNA motif that is responsible for HDAC inhibitor induction located at -120 to -100 of the Notch3 promoter region. Thus, an in vitro assay, EMSA revealed the transcription factor-DNA complex formed in the flanking sequence.
CONCLUSION: We have identified the DNA motif located in the Notch3 promoter region that is responsive to HDAC inhibitor. This understanding of how HDAC inhibitor act on the Notch3 promoter may lead to the development of novel therapies for neuroendocrine cancers.
