Tumorspheres from in vitro transformed cell lines show deregulation of tumor-related molecular pathways

Evidence indicates that a subset of cells endowed with high tumorigenic potential and stemness features (cancer stem cells: CSCs) is responsible for tumor initiation and maintenance in several cancers. In this study, we used a tumor cellular model developed in our laboratory from telomerase immortalized human fibroblasts (named cen3tel) and the tumorsphere assay to possibly isolate and characterize CSCs from in vitro transformed cells. We found that cen3tel cells were able to form spheres (frequency ~ 2-10%) and sphere cells showed self renewal capacity, suggesting that in these populations there is a subset of cells with CSC-like features. Compared to adherent cells, sphere cells displayed a reversible and concerted downregulation of some tumor and stem cell-related genes, such as c-MYC, GNL3, Notch, ?-Catenin, and the overexpression of the tumor suppressor miR-34a, suggesting that these genes are connected in a circuitry and epigenetic mechanisms possibly modulate their expression. The deregulation of these genes in sphere cells is likely unrelated to stemness features but could be functional to protect cells from apoptosis, activating cell survival mechanisms during sphere growth. To this regard, we demonstrated that sphere cells do not show evidence of apoptotic death and overexpress the apoptosis negative regulator Bcl2A1. The pro-survival mechanisms observed in sphere cells could also play a role in favoring survival of cancer cell growing after detachment from the extracellular matrix. Here, we will also present gene expression profiles of sphere cells relatively to adherent cells, which indicate an extensive genetic reprogramming involving several cancer-related genes.