Deregulation of a c-myc-miR34a circuitry in tumorspheres from in vitro transformed cell lines

According to the cancer stem cell (CSC) hypothesis, only a subset of tumor cells, sharing features with normal stem cells, are endowed with tumorigenic potential. The origin of CSCs is elusive; however, evidence has been reported that they can originate form bulk tumor cells through a dedifferentiation process or, as shown in in vitro transformed cell lines, during neoplastic transformation of differentiated cells. To investigate the possible generation of CSCs during propagation of in vitro transformed cell lines, we exploited a cellular system derived in our laboratory from telomerase immortalized human fibroblasts, which recapitulates fibroblast neoplastic transformation. To isolate potential CSCs, we used the tumorsphere approach. Growing cells in the absence of serum and in the presence of growth factors, we obtained spheres with a frequency ranging between 2-10%. Sphere cells could be replated for at least six times and showed increased Sox2 expression, suggesting that they are endowed with self-renewal potential and stemness features. However, preliminary in vivo tumorigenic assays failed to show an increased tumorigenicity of sphere cells. Moreover, compared to adherently growing cells, sphere cells were characterized by a reduced expression of genes involved in tumorigenesis and stemness, as c-myc, GNL3 and Notch, as well as an increased expression of the tumorsuppressor microRNA miR34a. These observations suggest that tumorsphere formation might not always be an effective methods to isolate highly tumorigenic cells and that CSC properties and tumorigenicity could be dissociated features. Experiments are in progress to determine the c-myc downregulation mechanism and its possible functional meaning.