Abstract 1952: Notch-1 regulated stemness and EMT in colorectal cancer

Abstract

Abstract Colorectal cancer (CRC) is the second leading cause of cancer death nationwide. In 2013, nearly 156,000 patients will be diagnosed with CRC. Surgical resection for early stage disease offers the best chance for cure; however, up to 30% of patients will relapse. Current models of CRC progression hold that tumor development is driven by a small subpopulation of cancer initiating (CIC) or cancer stem (CSC) cells that share many properties of stem cells. These cells have the capacity to drive tumor growth, are largely responsible for relapse and have been implicated in metastasis via epithelial to mesenchymal transition (EMT), similar to normal counterparts in organogenesis. Notch-1 is the major receptor for a highly conserved signaling pathway that is well-documented to control stem cell and lineage commitment in various organs, including the colon. The function of Notch signaling in colorectal cancer, however, is not well-defined. Based on preliminary data in which colon tumor samples from CRC patients expressed Notch-1 but surrounding benign tissue did not, we sought to test the hypothesis that Notch-1 signaling in colon cells promotes tumorigenesis by enhancing stem cell and EMT characteristics of tumor cells. The human colon tumor line HCT-116 was transduced with a retroviral construct that expresses constitutively active Notch-1 (ICN1). Hes-1 protein levels were increased in the activated Notch-1 transduced cells, indicating that the construct was functional in activating Notch-signaling. These HCT116/ICN1 cells exhibited higher plating efficiency than the parental cell line. The average colony size of the HCT116/ICN1 cells was about two-fold smaller than the parental cell line (p<0.001). In addition, cells with constitutively active Notch-1 (HCT116/ICN1) had a longer duplication time than parental HCT-116 cells (p<0.03). Consistent with longer duplication time, HCT116/ICN1 cells were two-fold slower in completing wound healing as compared with the parental HCT-116 cell line (p<0.001). Transwell migration assays revealed a two-fold increase in migration of HCT116/ICN1 cells as compared with the parental control (p<0.03). Western blot analysis demonstrated that the HCT116/ICN1 cells with activated Notch-1 expressed highly increased levels of the EMT associated proteins Smad3, CD44 and Slug. This was accompanied by a four-fold decreased expression of E-cadherin. Meanwhile, E-cadherin was not altered in HCT116 cells transduced with a Notch-1 shRNA lentiviral construct that inactivated Notch-1 signaling. Finally, levels of the numb protein, a Notch-1 antagonist, appeared to be significantly reduced (five-fold decrease) by constitutive Notch-1 signaling. Alternatively, inactivation of Notch-1 resulted in a four-fold increased expression of the numb protein. Collectively, our data point to the Notch-1 pathway as being a potential key regulator of EMT in colorectal tumors and as such, may be of therapeutic or prognostic utility. Citation Format: Alex Fender, Makenzie Nutter, Timothy Fitzgerald, Fred Bertrand, George Sigounas. Notch-1 regulated stemness and EMT in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1952. doi:10.1158/1538-7445.AM2014-1952