Pivotal Role of PDGFR alpha in anti-Notch1 and EGFR Resistant Cancer Stem Cells Derived from Peritumor Tissue of Glioblastoma Multiforme.

IntroductionNowadays there is need to develop new targeted therapies for brain tumors and therefore we aim to target transmembrane receptors such as Notch1, EGFR and PDGFR, which are already under investigation in clinical trials for the treatment of glioblastoma multiforme. Cancer stem cells represent a rare fraction of cancer cells characterized by resistance to chemotherapy and radiation. In this study, we investigated gene expression and function of Notch1, EGFR and PDGFR to address their role among tumor core- (c-CSC) vs. peritumor tissue-derived cancer stem cells (p-CSC) of six cases of GBM.MethodsMTS assay was performed to evaluate the cell response to pharmacological treatments. RT-PCR and Western blot experiments were performed to state the expression of Notch, EGFR and PDGFR?/? and to understand the biological effects exerted by single or combination of specific inhibitors in GBM CSC. GBM CSC invasive ability was tested in vitro in absence or presence of Notch1 and/or EGFR inhibitors.ResultsWe first evaluated changes in cellular response of GBM CSC undergoing to Notch inhibitor GSI-X, either alone or along with AG1478, an EGFR inhibitor. GSI-X induces a significant decrease of cell growth preferentially in c-CSC in comparison with p-CSC, and no effects were observed in cell cycle distribution, apoptosis and cell invasion assays. AG1478 significantly reduces cell growth of either c-CSC or p-CSC pools, in particular three cases were studied more thoroughly and showed a shifting in the G1 phase of cell cycle, sometimes associated with apoptosis and loss of cell invasion. Interestingly two cases, c-CSC pools were more sensitive to simultaneous anti-Notch1 and anti-EGFR treatment than either therapy alone respect to p-CSC, which resulted mostly resistant to treatment. Of note, all p-CSC of six Cases examined showed an inherent over expression of PDGFR? compared to c-CSC, the second most frequently mutated RTK in GBM, following EGFR. RNA interference experiments and pharmacological inhibition of the endogenous PDGFR? significantly reduced cell growth rate and survivability of p-CSC. The latter effects were maximized in combination with AG1478.ConclusionThese results suggest that simultaneous targeting of EGFR and PDGFR may be a better therapeutic option for a GBM therapy.