Study of DNA Damage-Induced MSI1 Serine-347 Phosphorylation in Regulation of Radiosensitivity in Glioblastoma Multiforme

Abstract

Despite the growing therapeutic techniques, Glioblastoma Multiforme (GBM) patients have limited survival due to the location and malignancies of tumor burdens. Radiotherapy is one of the common treatments with maximal surgery and chemotherapy. However, the often radioresistance occurred in GBM results in recurrence and marginal disease control. Therefore, the medical need remains unmet to treat GBM efficiently. Musashi-1 (MSI1) is one of the RNA-binding proteins known for its high oncogenicity in many cancers, including GBM. MSI1 is highly expressed in tumors and abundant in tumorigenic cancer cells. However, the relationship between MSI1 and GBM radioresistance is still largely unknown. Three human GBM cell lines, U87-MG, 05MG, and S1R1, were used in vitro, and an orthotopic nude mouse GBM model was employed in vivo. Phos-Tag blotting and phospho-MSI1 (Ser 347) antibody helped to detect the phosphorylation level of MSI1. Ultra-low attachment dishes and 3D cell culture were performed for the tumor initiation study. Flow cytometry, immunohistochemistry staining, and confocal microscope with immunofluorescent staining provide respective biomarker expressions. We initiated our study by inducing DNA damage on GBM cell lines using ionizing radiation and an alkylating agent. With Phos-Tag and NetPhos3.1 prediction, we hypothesized that a novel phosphorylation site on MSI1 serine-347 (S347) may sense DNA damage events. To unveil the molecular mechanism, we found DNA-dependent protein kinase catalytic subunits (DNA-PKcs), one of the DNA damage repair components, physically interacted with MSI1 in the role of a kinase. We suggest that activated DNA-PKcs may phosphorylate MSI1-S347, leading to enhanced oncogenicity and subsequent malignancies. Wild-type MSI1-overexpressing GBM cells (MSI1-WT) showed better colony and sphere-forming ability, whereas loss-of-function mutation S347A deprived the oncogenic behaviors. Besides, S347A limited the cancer initiation in the orthotopic animal model compared with MSI1-WT, suggesting that phosphorylation of MSI1-S347 promotes GBM tumorigenicity both in vitro and in vivo. In addition, we noticed that CD133 (Prominin-1), a well-characterized cancer stem-like cell marker, accompanied MSI1-WT introduction but was not seen in S347A cells. Intriguingly, we identified a few members of the let-7 microRNA family, including let-7b, 7e, and 7i, who were rescued by the S347A mutation and correspondingly expressed with CD133 levels and tumorigenic abilities. This study explains a vital relationship between GBM radioresistance and a novel oncogenic post-translational modification on MSI1 S347. IR-induced DNA damage triggers DNA-PKcs activation that subsequently phosphorylates MSI1-S347, enhancing GBM tumorigenicity via let-7 pathway. Further studies on the potential regulators, either inhibiting DNA-PKcs or antagonizing agents, may provide GBM patients with an optimistic outcome.