Notch1 signaling regulates the neuroendocrine phenotype of medullary thyroid cancer cells

Abstract

Introduction. The Notch1 pathway plays an essential role in regulating the development of neuroendocrine (NE) cells in the gut and lung. During development, induction of Notch1, a multi-functional trans-membrane receptor, leads to downstream suppression of the NE transcription factor hASH1, causing a reduction in the number of NE cells in the GI tract and lung. The role of Notch1 in the NE differentiation of the thyroid is unknown. However, based upon its role in the gut and lung, we hypothesized that activation of Notch1 signaling could potentially reverse NE differentiation and reduce hormone production from NE tumors such as medullary thyroid cancer (MTC). Methods. To determine the role of Notch1 in thyroid NE tumor cells, human MTC “TT” cells were stably transfected with a dose-dependent, doxycycline-inducible Notch1 vector creating TT-Notch cells. TT and TT-Notch cells were treated with various concentrations of doxycycline (0–1 μg/ml). Doxycycline-inducible Notch1 protein expression was confirmed by Western Blots. Notch1 activation and pathway induction were determined by Notch1 binding assays and Western analysis for its downstream targets HES-1 and hASH1. NE hormone production was measured by Western blots for chromogranin A. Results. TT cells had no detectable Notch1 protein and high levels of hASH1 and chromogranin A at baseline. Induction of Notch1 with doxycycline treatment of TT-Notch cells led to a dose-dependent increase in Notch1 protein and Notch1 binding activity. Furthermore, with increasing Notch1 induction, there was a progressive reduction in hASH1 protein and a dramatic corresponding decrease in chromogranin A levels. Conclusions. The Notch1 signaling pathway is functionally conserved in MTC cells. Activation of this important developmental pathway led to dose-dependent reductions in NE hormone levels. Therefore, the Notch1 pathway tightly regulates the NE phenotype of MTC cells. Moreover, activation of Notch1 could potentially serve as a novel target for the treatment of NE tumors including MTC.