Abstract
Glioblastoma (GBM) is an aggressive, malignant brain tumor that inevitably develops resistance to conventional chemotherapy and radiation treatments. In order to identify signaling pathways involved in the development of radiation resistance, we performed mass spectrometry-based phospho-proteomic profiling of GBM cell lines and normal human astrocytes before and after radiation treatment. We found radiation induced phosphorylation of a number of proteins including calpastatin, specifically in GBM stem cells (GSCs). Herein, we focused on calpastatin, an endogenous inhibitor of calpain proteases. Radiation-induced phosphorylation of calpastatin at Ser-633 within the inhibitory domain was validated with a phospho-specific antibody. In order to test the functional significance of phosphorylated calpastatin, we utilized site-directed mutagenesis to generate phospho-inactive (Ser633Ala) and phospho-mimetic (Ser633Glu) mutant calpastatin. GBM cell lines stably expressing the mutant calpastatin showed that phosphorylation was necessary for radiation-induced calpain activation. We also showed that casein kinase 2, a pro-survival kinase overexpressed in many cancer types, phosphorylated calpastatin at Ser-633. Our results indicate that calpastatin phosphorylation promotes radiation resistance in GBM cells by increasing the activity of calpain proteases, which are known to promote survival and invasion in cancer.
Highlights
Glioblastoma (GBM) is the most prevalent and aggressive primary malignant brain tumor in adults, accounting for 60–70% of malignant gliomas
Our phospho-proteomic profiling data of normal human astrocytes and GBM cell lines demonstrated that www.impactjournals.com/oncotarget changes in protein phosphorylation could be detected immediately following radiation treatment, and some of these changes were specific to glioblastoma stem cell (GSC)
Because protein phosphorylation is one of the primary mechanisms regulating signaling transduction, we hypothesized that radiation-induced changes in phosphorylation occurring in GSCs contribute to treatment resistance mechanisms
Summary
Glioblastoma (GBM) is the most prevalent and aggressive primary malignant brain tumor in adults, accounting for 60–70% of malignant gliomas. GBM is a grade IV malignant glioma with 17,000 new cases diagnosed each year in the United States [1]. Patients diagnosed with GBM have a very poor prognosis and quality of life, with a median survival time of 12–15 months despite receiving the standard care that includes maximally safe resection followed by radiotherapy plus concomitant and adjuvant temozolomide chemotherapy [2]. Despite extensive studies on molecules regulating GBM radioresistance, the cellular mechanisms responsible for GBM treatment resistance remain largely unknown. This lack of progress is in part due to the fact that most of the data on these signaling pathways has been obtained pre-treatment, not accounting for how treatment-induced changes in signaling can impact therapeutic resistance. Certain signaling pathways appear to be associated with the phenomenon of radiation resistance; there is relatively sparse knowledge of the mechanisms by which radiation activates these pathways in GBMs, or how activation of these pathways leads to radiation resistance
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