Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) Regulates Growth, Radiation Sensitivity, and is a Novel Prognostic Factor for Glioblastoma

Purpose/Objective(s): The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is crucial to many aspects of cell growth and survival in glioblastoma. Phosphatidyl inositol bisphosphate (PIP2) is converted to the triphosphate (PIP3) by PI3K which leads to Akt activation. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is capable of sequestering PIP2 at the membrane via electrostatic interactions. We thus hypothesized that the MARCKS protein could regulate proliferation and resistance to radiation through modulation of the PI3K/Akt pathway; and therefore would have a positive impact on clinical outcome for glioblastoma patients. Materials/Methods: Knockdown and over-expression of the MARCKS protein were achieved with lentiviral mediated shRNA (Open Biosystems) or lentiviral mediated over-expression (Invitrogen). Cell proliferation assays were preformed with ATPlite (PerkinElmer). Radiation sensitivity was assessed with clonogenic assays. Western blotting was performed using anti-MARCKS antibody (Epitomics), anti-pAkt Ser473 (Cell Signaling), anti-Caspase 3 (Cell Signaling) and anti-phospho DNA-PKcs (Genetex). Gene expression data was downloaded for 192 out of the 202 patients that did not have prior glioma. Cox proportional hazards models were fit using the coxph routine in the Survival package (version 2.35-7) in R (version 2.10.0). Kaplan-Meier curves for MARCKS quartiles or other expression categories were generated by predicting the survival times from the Cox model at the median age value using the survfit function from the survival package. Results: Genetic silencing of the MARCKS protein in U251 glioma cells lead to increased proliferation (p<0.01) and increased radiation resistance (DER=0.80). This correlated with a 40% increase in activation of the PI3K/Akt pathway. The mechanism of increased radiation resistance was an increase in nonhomologous end joining (NHEJ) DNA repair as indicated by increased DNA-PK activation, and decreased apoptosis as indicated by decreases in cleaved caspase 3 after irradiation. Conversely, over-expression of the MARCKS protein in U87 glioma cells lead to decreases in proliferation (p<0.001), however; the cells unexpectedly adopted a senescent state (p<0.0001). Age-adjusted MARCKS gene expression inversely correlated with overall survival in glioblastoma patients in the TCGA database (p=0.004). This effect was found to be specific to the Proneural molecular subtype (median survival of 47.2 months vs. 12.2 months, p<0.0002), and most pronounced in tumors with unmethylated MGMT status (median survival 65.3 months vs. 10.7 months, p<0.001). Conclusions: We have demonstrated that MARCKS can regulate proliferation, radiation sensitivity and is associated with improved clinical outcomes. These findings suggest MARCKS as a novel target and biomarker for prognosis in the Proneural subtype of GBM. . Control