Abstract 2181: Tumor suppressor role of Notch3 in medullary thyroid carcinoma

Abstract

Abstract Introduction: NOTCH3 is a transmembrane receptor that appears to be absent in the neuroendocrine (NE) tumors. We have previously demonstrated that transient expression of the active portion of NOTCH3 (NICD3) alters the NE phenotype and inhibits the proliferation of carcinoid tumor cells in vitro. However, the long term effect of activation this pathway is not clearly understood in Medullary Thyroid Carcinoma (MTC). To elucidate the role of Notch3 in NE, we established a doxycycline inducible NOTCH3 intracellular domain in MTC TT cells. The aim of this study was to assess the long term effect of NOTCH3 and determine the mechanism of inhibition of TT cells growth. Methods: Doxycycline inducible TT-NOTCH3 cells were created by stable transfection of Tet-On vector followed by transfection of pRevTRE-NOTCH3 plasmid. Functional analysis of NICD3 was done by measuring the degree of luciferase activity by CBF binding assay. Quantification of HES and HEY gene expression (Notch signaling mediators) was done by real time RT-PCR. Effect of Notch3 on NE markers -chromogranin A (CgA) and achaete-scute complex-like1 (ASCL1) – was assessed by Western blot analysis. The influence of NOTCH3 overexpression on TT-NOTCH cell proliferation was measured by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay. The mechanism of growth inhibition was determined for apoptosis markers by Western blot and flow cytometry analyses. Results: Treatment of TT-NOTCH3 cells with doxycycline led to an induction of NOTCH3 protein in a dose-dependent manner. Increase in the CBF1 binding activity showed that the NOTCH3 protein is functional and this is associated with changes in transcriptional level of HES and HEY families. More importantly, NOTCH3 activation led to a dose dependant reduction of NE markers ASCL1 and CgA. In addition, NOTCH3 activity is required to suppress MTC cell proliferation and the level of growth regression depends on the amount of NOTCH3 protein expressed. Finally, Western blot analysis and flow cytometry experiments indicated that the growth inhibition is due to apoptosis. Conclusions: We demonstrate, for the first time, that overexpression of NOTCH3 in MTC cells suppresses tumor cells growth by promoting apoptosis. Moreover, Notch3 pathway is functional as active NOTCH3 triggers the CBF-dependent transcriptional network. Finally, the activation of Notch3 in the in vitro model resulted in down-regulation of NE markers indicated that this pathway is conserved in MTC. Therefore, activation of Notch3 could be a therapeutic strategy to treat patients with MTC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2181. doi:10.1158/1538-7445.AM2011-2181