Expression of Wnt and Notch Signaling Pathways are Increased in Cancer Stem-Like Cells in Metastatic Melanoma Cell Line (A375)

Backgound and Cancer stem cells assume to be responsible for increasing cancer properties such as migration, and drug sensitivity. A lot of studies revealed signaling pathways are enhanced in cancer stem cells. Melanoma is well-known as heterogeneous cancer and its severity in treatment. These can be attributed to the existence of cancer stem cells. In this study, we first aimed to separate cancer stem cells (cancer stem-like cells) by sphere formation, characterized them, and second examine the expression of Wnt and Notch signaling pathway genes related to adherent cells.
Subjects and Adherent cell were cultured in the non-adherent condition with serum-free media; spheres obtained, then sphere formation, clonogenic assay, expression of CD133 and Nestin proteins, Nanog, NESTIN and Oct4 genes as stem related genes were assessed in comparison to adherent cells. In addition, Notch and Wnt signaling pathways genes in both adherent and spheres cells evaluated.
Sphere formation, clonogenic capacity, expression of Nestin protein, but not CD133 were increased in sphere cells in comparession to adherent cells. They also overexpressed β-catenin, cyclinD1, and c-Myc as Wnt down-stream genes, Notch1, HES1 as Notch down-stream genes, Nanog, and NESTIN as stem-related genes.
These results suggest that sphere culture model could be a proper experimental method to separate cancer stem-like cells. Our data also support two important pathways are overactivated in melanoma cancer stem-like cells which must be considered in targeting therapy.