PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment

Background: Cancer stem cells (CSC) represent a rare fraction of cancer cells characterized by resistance to chemotherapy and radiation, therefore nowadays there is great need to develop new targeted therapies for brain tumors and our study aim to target pivotal transmembrane receptors such as Notch, EGFR and PDGFR, which are already under investigation in clinical trials setting for the treatment of Glioblastoma Multiforme (GBM). Methods: MTS assay was performed to evaluate cells response to pharmacological treatments. Quantitative RT-PCR and Western blots were performed to state the expression of Notch1, EGFR and PDGFR alpha/beta and the biological effects exerted by either single or combined targeted therapy in GBM CSC. GBM CSC invasive ability was tested in vitro in absence or presence of Notch and/or EGFR signaling inhibitors. Results: In this study, we investigated gene expression and function of Notch1, EGFR and PDGFR to determine their role among GBM tumor core-(c-CSC) vs. peritumor tissue-derived cancer stem cells (p-CSC) of six cases of GBM. Notch inhibition significantly impaired cell growth of c-CSC compared to p-CSC pools, with no effects observed in cell cycle distribution, apoptosis and cell invasion assays. Instead, anti-EGFR therapy induced cell cycle arrest, sometimes associated with apoptosis and reduction of cell invasiveness in GBM CSC. In two cases, c-CSC pools were more sensitive to simultaneous anti-Notch and anti-EGFR treatment than either therapy alone compared to p-CSC, which were mostly resistant to treatment. We reported the overexpression of PDGFR alpha and its up-regulation following anti-EGFR therapy in GBM p-CSC compared to c-CSC. RNA interference of PDGFR alpha significantly reduced cell proliferation rate of p-CSC, while its pharmacological inhibition with Crenolanib impaired survival of both CSC pools, whose effects in combination with EGFR inhibition were maximized. Conclusions: We have used different drugs combination to identify the more effective therapeutic targets for GBM CSC, particularly against GBM peritumor tissue-derived CSC, which are mostly resistant to treatments. Overall, our results provide the rationale for simultaneous targeting of EGFR and PDGFR, which would be beneficial in the treatment of GBM.