Purpose
Notch1, a trans-membrane receptor, has been recently shown to aid in the determination of thyroid cell fate associated with tumorigenesis. The present study aimed to investigate the clinical relevance of Notch1 and its role in the regulation of differentiated thyroid cancer (DTC) behavior.
Experimental design
We examined Notch1 expression level and its relationship with clinicopathologic features and outcomes of DTC. Notch1 intracellular domain (NICD) was further characterized both in vitro and in vivo by gain-of-function assays using an inducible system.
Results
Notch1 expression levels were down-regulated in primary DTC tissue samples compared with contralateral non-tumor and benign thyroid tissues. Decreased Notch1 expression in DTC was associated with advanced patient age (p=0.032) and the presence of extrathyroidal invasion (p=0.005). Patients with lower Notch1 expression had a significantly higher recurrence rate (p=0.038). Restoration of NICD in a stably doxycycline-inducible metastatic DTC cell line reduced cell growth and migration profoundly. Using an orthotopic thyroid cancer model, NICD induction significantly reduced the growth of the primary thyroid tumor and inhibited the development of lung metastasis. SERPINE1 was discovered by microarray as the most significant gene down-regulated by NICD. Further validation showed that induction of NICD reduced SERPINE1 expression in a dose-dependent manner while restoration of a relative higher level of SERPINE1was observed with NICD back to minimal level. Additionally, SERPINE1 knock-down inhibited DTC cell migration.
Conclusions
Notch1 regulates the aggressive phenotypes of DTC, which could be mediated by SERPINE1 inhibition. Notch1/SERPINE1 axis warrants further investigation as a novel therapeutic target for advanced DTC.
<p>RFP-positive FTC236-Notch1 cells for the establishment of orthotopic mouse model. (A). Over 26% of the cells from the initial transduced batch were positive for RFP and were sorted for further culturing. (B). The sorted cells were cultured for 3 passages and over 96% were RFP-positive. (C). The sorted cells were cultured for 6 passages and 97% were RFP-positive.</p>
<p>Scratch of a gap for wound healing assay was generated and closure of the gap was evaluated 24 hours later in FTC236-Notch1 cells with or without doxycycline treatment. The cells with NICD induction by doxycycline exhibited slower closure of the gap than those without NICD induction. Images were taken immediately after scratching the cultures 0 hr and 24hr later. Representative images at 0 hr and 24 hr are shown in the upper panel. The bar graph represents the average percentage of the gap closure. Each bar represents mean{plus minus} SEM; "Dox" indicates doxycycline; ** indicates p< 0.01; Scale bar:200µm.</p>
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