Use of the Notch signaling pathway to predict disease progression and distant recurrence-free survival in early stage colon cancer

Abstract

14500 Background: The Notch pathway directs normal colonic development, but its aberrant activation may promote tumorigenesis. However, its expression in colon cancer is unknown. Therefore, we analyzed Notch receptors (1–4), ligands, and modifiers in colon cancers and suppressed this pathway with small interfering RNA (siRNA) and drug targeting. Methods: Gene chip microarrays were constructed from 308 normal and malignant colon tissues obtained at surgery, including normal mucosa (10%), polyps (15%), primary cancers (55%), and metastases from liver (13%) and lung (6%), using Affymetrix U133A arrays. Microarray data were normalized using the RMA method. Correlation analysis identified genes differentially expressed between sample classes, using Empirical Bayes t test, and genes associated with survival outcomes, using COX regression. In colon cancer cell lines (HCT116, SW620, and HT29), Notch receptors were downregulated by siRNA, and Notch activation was inhibited by gamma-secretase inhibitors (GSIs). Cell line viability was assessed by colony formation assays and apoptosis by DAPI staining. Results: Notch-3 expression increased with colon cancer progression and was highest in lung metastases (trend test, p<0.001). The Notch ligand JAG-2 increased significantly from polyps to Stage I cancers (p<0.001), whereas the negative Notch regulator Numb significantly decreased (p<0.001). The Notch targets cyclin D1 and survivin also increased with cancer progression. Overexpression of Notch-3 correlated with distant recurrence free survival in Stage III disease (p=0.074, HR=2.95); similar marginally significant correlation holds when adjusting for JAG-2 and NUMB expression. In vitro, siRNA to the Notch receptors inhibited growth but the greatest effect occurred with siRNA to Notch-3. GSIs significantly suppressed cell viability and induced apoptosis. Conclusions: Key elements of the Notch pathway are activated (Notch-3 and JAG-2) and suppressed (Numb) during early stage colon cancer progression. Targeting of this pathway could represent a new therapeutic strategy. Examination of the Notch pathway may have important prognostic significance in Stage III colon cancer and this requires prospective evaluation. No significant financial relationships to disclose.