The Role of the Notch-3 Receptor in Stemness, EMT, and Tumorigenesis in Colorectal Cancer

Abstract

As the second leading cause of cancer death worldwide, colorectal cancer (CRC) poses a significant threat to both men and women alike. Although a small percentage of CRC cases are inherited, the majority of cases arise from environmental causes and are widely attributed to dysfunctional cellular pathways. Currently, the CRC progression model maintains that cancer growth, relapse and metastasis are due primarily to cancer stem cells (CSCs), a cell subpopulation that shares many properties with stem cells through epithelial to mesenchymal transition (EMT). We have recently reported that Notch-1 signaling is highly associated with promoting cancer stemness and EMT in CRC. Furthermore, we observed that expression of the Notch-1 receptor also upregulates Jagged-1, a ligand of the Notch receptors, which may affect stemness and EMT. However, this effect is reversed by treatment with DAPT, a [gamma]-secretase and Notch signaling inhibitor. Thus, we hypothesized that the effect of Notch-1 on stemness and EMT in CRC is mediated by Jagged-1 via another member of the Notch pathway, the Notch-3 receptor. To assess our hypothesis, we utilized the colon cancer cell line HCT-116. The parental HCT-116 cells were transduced with an IRES (internal ribosome entry site)-GFP retrovirus that expressed the human intracytoplasmic domain of Notch-1, thus producing the ICN1 cell line. The ICN1 cells were then transduced with a small hairpin RNA (shRNA) construct that effectively knocked down Notch-3, which created the ICN1-shN3 cell line. Growth ability, plating efficiency and colosphere formation were assessed in each cell line. In these experiments, cell culture and Western blot analysis were performed using standard methodology. We found that targeting Notch-3 via shRNA transduction in the colon tumor cell line ICN1-shN3 resulted in a significant decrease of Notch-3 expression. Further Western blot analysis indicated reduced expression of CD44 and Slug, markers for stemness and EMT, respectively. Further analyses showed that in the absence of functioning Notch-3, plating efficiency decreased by 60% and migration decreased by 22% when compared to the ICN1 cell line. These results were accompanied by significant changes in colosphere formation when the ICN1-shN3 cells were compared to ICN1 cells. ICN1-shN3 cells showed a 35% reduction in colosphere formation compared to the ICN cells. This difference was observed in colospheres of both high (33%) and medium/low (37%) proliferative capacity. These data indicate a key role for Notch-3 signaling in Notch-1-induced tumorigenesis mediated by Jagged-1. They also highlight the potential use of Notch-3 inhibitors to effectively target aggressive CRC tumors.