The Roles of Ras and Neurofibromin 1 in Human Neuroblastoma Stem Cell Malignancy
Cancer stem or tumor-initiating cells are present in many human cancers. However, the molecular mechanisms responsible for the clinical aggressiveness of these cells are still unclear. Human neuroblastoma, originating from the embryonic neural crest, is characterized by cellular heterogeneity. Cells of three phenotypes (N-, I-, and S-type) have been isolated and characterized. Among these, the I-type cell – the cancer stem cell of neuroblastoma – is the most malignant.^ In the present study, I show that, although wild-type N-Ras protein is expressed at similar levels in all three neuroblastoma cell phenotypes, a significantly higher level of activation (>5-fold) is restricted to I-type cancer stem cells. To study the role of activated N-Ras in neuroblastoma cell malignancy, Ras-GTP levels were altered using dominant-negative (DN) or constitutively active (CA) N-Ras constructs. DN-N-Ras transfection led to >2-fold decrease in Ras-GTP levels in two I-type cell lines and caused a significant >3-fold decrease in their colony-forming efficiency (CFE) in soft agar. Conversely, when weakly malignant N-type cells were transfected with CA-N-Ras, Ras-GTP levels increased >10-fold and this was accompanied by a significant >7-fold increased CFE. Moreover, in cell survival studies, high level of Ras-GTP was shown to protect neuroblastoma cells from apoptosis. Thus, high level of N-Ras-GTP promotes the increased malignancy and survival of I-type neuroblastoma cancer stem cells.^ To reveal the possible mechanisms underlying the enhanced Ras activation found in neuroblastoma cancer stem cells, I showed that one important Ras-GTPase activating protein, neurofibromin, was specifically down-regulated (>3-fold) in I-type cells. To assess whether lower neurofibromin content directly results in elevated Ras activation, NF1-specific shRNA was expressed in N-type cells, leading to a significant >2-fold decrease in neurofibromin amount and a >3-fold increase in Ras-GTP level. Moreover, these cells showed significant >2-fold increases in CFE. These results suggest that high level of Ras-GTP - as a consequence of low amount of neurofibromin - is a cancer stem cell-specific property in neuroblastoma, and is instrumental in their malignancy. Furthermore, a MG132 study showed that the I-type cell-specific down-regulation of neurofibromin is due to increased ubiquitin-proteasome-dependent degradation of neurofibromin protein.^ These findings are the first to implicate wild-type N-Ras and neurofibromin in the malignancy of human neuroblastoma and to identify a cancer stem cell-specific regulatory mechanism underlying their highly malignant behavior. These findings may make possible new directions for future therapeutic treatment of this often fatal childhood cancer. ^
Biology, Molecular|Biology, Cell|Health Sciences, Oncology
"The Roles of Ras and Neurofibromin 1 in Human Neuroblastoma Stem Cell Malignancy"
(January 1, 2011).
ETD Collection for Fordham University.