The role of Notch signaling in regulation of epithelial to mesenchymal transition in thyroid cancer.
Authors: Vasyl Vasko, Kirk Jensen, Aneeta Patel, Motoyasu Saji, Andrew Bauer
Journal: Cancer Research (2008)

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA

5367 

The role of Notch signaling in regulation of epithelial to mesenchymal transition in thyroid cancer.    Background. The activation of Notch signaling pathway is required for epithelial to mesenchymal transition (EMT) during embryogenesis. EMT is important for thyroid cancer, however the role of Notch signaling in thyroid cancer has not been defined.    Objective: to characterize the expression of Notch signaling molecules in thyroid cancer and to define the role of Notch signaling in regulation of EMT.    Material and Methods.    Thirty five human thyroid cancer samples to include 10 follicular (FC) and 20 papillary cancers (PC) and 5 anaplastic thyroid cancer (AC) were examined by immunostaining using antibodies against Jagged1, Notch1, Hey1 and Hey2. We also examined six thyroid cancer cell lines including four cells lines with mesenchymal-like characteristics (NPA, FTC 133, 236 and 238) and two cells lines with epithelial-like patterns of growth (WRO and ARO). The level of Notch ligand - Jagged1; Notch receptors - Notch 1, 2, 3; enzymes involved in Notch cleavage - ADAM17, Presenilin1; downstream target of Notch - Hey1 and Hey2 were tested by quantitative RT PCR, Western blot and or immunostaining. Hey1 silencing was performed using Lipofectamine 2000 method and RNA was examined on custom SuperArray real time PCR plate for expression of 100 genes with known function in EMT and angiogenesis. Migration and invasion experiments were performed using Boyden chamber assays.    Results.    Jagged 1 and Notch 1 were differentially expressed in PC and FC. Jagged 1 was up-regulated in 1/10 FC but in 8/20 PC; Notch 1 was up-regulated in 4/10 FC, but in 3/20 PC. PC with RET/PTC rearrangements and AC showed low level of Jagged1, Notch1 and Hey expression. In vitro studies showed that epithelial like WRO and ARO cells had high level of Notch 1 expression and prominent membranous staining with anti-Notch antibodies. Mesenchymal-like cells were characterized by increased level of Hey1 and Hey 2 expression. Jagged 1 and Hey 1 were over-expressed in cell lines derived from metastases (FTC 236 and FTC 238) compared to the cell line derived from primary tumor (FTC 133). Hey 1 silencing induced cell growth and motility in both NPA and ARO thyroid cancer cells. Inhibition of Hey 1 expression in epithelial-like ARO cells resulted in EMT and loss of membranous E Cadherin localization. The inhibition of Hey 1 expression was associated with inhibition of I-kappa-B proteins (NFKBIB) expression, down regulation of cell cycle inhibitor CDKNA1 and up-regulation of IGF1, IGFR2 and TEK tyrosine kinase.    Conclusion. Notch signaling molecules are differently expressed in human FC and PC; and in thyroid cancer cells with mesenchymal or epithelial characteristics. The inhibition of Notch signaling in AC suggested its possible role as tumor suppressor. In thyroid cancer cells, down-regulation of Notch downstream target Hey1 induces EMT through activation of NFKB signaling.