Histone Deacetylase Inhibitors Transcriptionally Regulate Notch1 Expression in Neuroendocrine Malignancies

Abstract

Introduction: Neuroendocrine (NE) cancers are characterized by their ability to secrete active hormones and growth factors, which have been shown to regulate tumor growth and differentiation through modulation of a number of cellular signaling pathways. Recently, our laboratory has identified that the Notch1 pathway plays an important role in controlling growth and hormone production by NE cancers. We have shown that induction of Notch1 by histone deacetylase (HDAC) inhibitors valproic acid (VPA) and suberoyl bis-hydroxamic acid (SBHA) in human carcinoid cells markedly suppresses tumor cell growth and NE hormone production in vitro, and tumor growth in vivo. Although HDAC inhibitors are in clinical use for a variety of human cancers including carcinoids, the mechanisms by which they exert these anti-cancer effects are not clearly understood. We hypothesized that HDAC inhibitors act on the Notch1 promoter and regulate its expression at the transcriptional level. Method: Human BON (gastrointestinal) and H727 (pulmonary) carcinoid cell lines were treated with VPA (0-3 mM) or SBHA (0-30 uM) and total RNA was isolated to detect changes in Notch1 mRNA level by real time polymerase chain reaction (PCR). To determine the Notch1 promoter activity, BON and H727 cells were transiently transfected with the N1PR-Luc construct (Luciferase expression under the control of Notch1 promoter [-1 to -961 proximal to the translation initiation site]) and then treated with VPA or SBHA for 24 hours. The cells were subsequently lysed and assayed for luciferase activity to detect changes in Notch1 promoter activity. To map out the HDAC response element site on the N1PR-Luc construct, we created a series of deletion in the Notch1 promoter sequence. Results: Notch1 mRNA was upregulated in both carcinoid cell lines in response to VPA and SBHA in a dose-dependent manner. Importantly, both drugs enhanced Notch1 promoter activity as evidenced by an increase in luciferase activity. We have mapped the HDAC responsive site to a 250 bp fragment in the proximal portion of the Notch1 promoter which induces Notch1 transcription to 30 times basal levels. Conclusion: We report for the first time that HDAC inhibitors upregulate Notch1 at the transcriptional level. Importantly, we have isolated an HDAC response element within the 5” Notch1 promoter region. Further elucidation of this element would potentially delineate the mechanisms by which HDAC inhibitors mediate their anti cancer effects.