The effect of <i>NOTCH1</i> knockdown on the phenotype of human lung and colon cancer stem cells

Abstract

Introduction. Lung and colorectal cancers are the most common cancer types, characterized by a poor prognosis. Tumor progression is also caused by the aberrant activity of intercellular signaling pathways, which can arise due to mutations in genes encoding their components. In particular, the oncogenic role of NOTCH1 receptor of Notch signaling pathway has been proven for various cancer types, including lung and colorectal cancers. In this research, we delved deeper into the importance of NOTCH1 receptor expression for the progression of these malignancies. Aim. To investigate the importance of NOTCH1 expression in maintaining the cancer stem cell (CSC) pool and phenotype of human lung and colon cancers. Materials and methods. Experiments were performed on previously obtained NOTCH1 knockdown cell lines of human lung A549 and colon HCT116 carcinomas. First of all, we studied the effect of NOTCH1 knockdown on the metastatic ability of A549 cells and the tumorigenicity of A549 and HCT116 cells when injected to immunodeficient BALB/c nu/nu mice. Next, we carried out in vitro tests to determine CSC pool and phenotype in tumor cell culture: cytofluorimetric analysis of ABC-transporters activity to exclude dye to the external environment and analysis of colony formation in a semi-liquid medium. In conclusion, we assessed the proportion of cells in the culture producing the CSC marker – CD133 using flow cytometry and the expression level of some genes associated with CSC phenotype (NANOG, POU5F1, SOX2) using real-time polymerase chain reaction. Results. NOTCH1 knockdown decreased the number of experimental animals with metastases, the number of formed metastatic foci and increased in the minimum cell inoculation dose. The activity of ABC-transporters, the ability for unattached growth, the proportion of CD133-positive cells in culture, and the expression of genes associated with maintaining of CSC pool and phenotype decreased under NOTCH1 knockdown in both cell lines. Conclusion. NOTCH1 expression is important for maintaining CSC pool and phenotype of human lung and colon carcinomas. The obtained data may be valuable in the development of anticancer therapies.