Association of Notch signaling pathway expression in liposarcomas with outcome, and targeting with gamma-secretase inhibitors

Abstract

10526 Background: The Notch pathway directs normal fat cell development, but its aberrant activation may promote the development of sarcomas. The expression of the Notch pathway in liposarcoma (LPS), however, is unknown. We examined Notch signaling components in LPS's and suppressed Notch activation with drug targeting in LPS cell lines. Methods: RNA was isolated from 18 normal fat and 140 LPS tissue samples from five LPS subtypes: well-differentiated (33%), de-differentiated DD (25%), myxoid (12%), round cell (6%), and pleomorphic (13%), and were hybridized to Affymetrix U133A arrays. Microarray data were normalized with the RMA method. Correlation analysis identified genes expressed between sample classes, using Empirical Bayes t-test, and genes associated with survival, using Cox regression. The Notch pathway in two LPS lines, DDLS and LS141, was suppressed with a novel gamma-secretase inhibitor (GSI) or with siRNA to Notch receptors. Viability was assessed by colony formation, apoptosis by DAPI staining, and Notch expression by immunoblotting. Results: Expression of Notch-3 and its targets, Hes-1, Hey-1, and survivin, was increased in LPS subtypes, compared to fat tissue (p<0.001). Inhibition of Notch signaling with GSI's or siRNA to Notch-1 suppressed the viability of both DD LPS lines (p<0.05), inducing a G1/S arrest followed by apoptosis. Transfection of siRNA to each Notch receptor, especially Notch-3, also suppressed the viability of DD LPS's (p<0.05). Expression of Notch-3 (p=0.027, HR=2.64), Notch-4 (p=0.026, HR=2.70), the ligand JAG-2 (p=0.049, HR=2.32), and Hey-1 (p=0.001, HR=4.25) was associated with reduced distant recurrence free survival in patients with DD LPS's. Expression of the negative Notch regulator Fbxw7 was associated with improved overall survival in patients with LPS (p=0.008, HR=-1.42). Conclusions: Elements of the Notch pathway (receptors, ligands, targets, and modifiers) are overexpressed in LPS's compared to normal fat tissue and associate with outcome. Suppression of Notch signaling decreased DD LPS cell line viability and induced apoptosis. Notch inhibition may represent a new therapeutic strategy for patients with LPS's and deserves further validation in a clinical trial. No significant financial relationships to disclose.