Glioblastoma (GBM) is an aggressive, malignant brain tumor that often 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 5 GBM cell lines and normal human astrocytes before and after radiation treatment. Fold changes in phosphorylation were calculated for each peptide across all cell lines at 30 seconds and 4 hours post-radiation. We found that radiation induces phosphorylation of calpastatin, an endogenous inhibitor of calpain proteases, specifically in GBM stem cells (GSCs). Radiation-induced phosphorylation of calpastatin at a serine within the inhibitory domain was validated by western blot with a phospho-specific antibody. In order to test the functional significance of phosphorylated calpastatin, we utilized site-directed mutagenesis to generate non-phosphorylatable and phospho-mimetic calpastatin mutants. Western blot analyses of GBM cell lines stably expressing the calpastatin mutant proteins show that calpastatin phosphorylation leads to increased calpain activity following radiation treatment. Our results indicate that calpastatin phosphorylation promotes radiation resistance in GBM by increasing the activity of calpain proteases, which are known to promote survival and invasion in cancers.
Abstract Glioblastoma (GBM) is the most prevalent and aggressive primary malignant brain tumor in adults. 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 of care treatment. GBMs are highly heterogeneous, invasive, and resistant to both chemo- and radio-therapy. The overall goal of this study is to identify novel mechanisms of radioresistance in GBM, so that more effective therapies can be developed in the future. We performed quantitative mass spectrometry-based phosphoproteomic profiling of GBM cell lines before and after radiation treatment, and determined post-radiation fold-changes in phosphorylation for each phosphopeptide. Results show calpastatin as one of the statistically significant differentially regulated phosphopeptides following radiation treatment. Calpastatin is an endogenous inhibitor of calpains, which are calcium-dependent cysteine proteases that control a wide range of cellular processes including cell survival and apoptosis. We hypothesized that phosphorylation inactivates calpastatin resulting in increased calpain activity. Western blot analyses with phospho-specific calpastatin antibodies validated the mass spectrometry results. In order to test the functional significance of phosphorylated calpastatin, we utilized site-directed mutagenesis to generate non-phosphorylatable and phospho-mimetic calpastatin proteins. Western blot analyses of U87 cells stably expressing CAST-S633A or -S633E supported the hypothesis that calpastatin phosphorylation leads to increased calpain activity. In conclusion, we identified increased levels of calpastatin phosphorylation in multiple GBM cell lines following radiation treatment, and shown that this correlates with increased calpain activity. Modulation of calpastatin activity is a potential strategy to increase radiosensitivity of glioblastomas. Targeting calpastatin is advantageous over targeting calpain itself, since many calpain inhibitors exhibit limited selectivity. Results of this study will contribute to our understanding of how GBM tumors become resistant to radiation therapy. Funding: R01CA108633, R01CA169368, RC2CA148190, U10CA180850-01 (NCI), Brain Tumor Funders Collaborative Grant, and The Ohio State University CCC (all to AC). Citation Format: Emily A. Bassett, Mitchell Pearson, Kamalakannan Palanichamy, Saikh J. Haque, Arnab Chakravarti. Calpastatin phosphorylation regulates radiation response in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4755. doi:10.1158/1538-7445.AM2017-4755
AbstractStem Cell Factor (SCF), the ligand for the c-kit proto-oncogene, has been shown to synergistically interact with other cytokines, enhancing the responsiveness of haemopoietic precursors. In this study we have examined the effects of SCF, in combination with interleukin-3 (IL-3), on FDCP-Mix A4 cells, a murine, multipotent, haemopoietic progenitor cell line. Low concentrations of IL-3 act to enhance cell survival but do not stimulate proliferation in A4 cells. Similarly, SCF when added alone, acts as a good survival stimulus, but is a poor proliferative stimulus. However, in combination with low concentrations of IL-3, SCF stimulates a synergistic enhancement of proliferation leading to a large increase in cell number after seven days. The synergy observed was not due to SCF stimulated alterations in the mRNA, protein levels or affinity of the IL-3 receptors. Therefore, interactions between cytokines at the level of cytoplasmic signalling pathways were investigated. IL-3 stimulated the rapid and transient tyrosine phosphorylation of several proteins (including those of molecular weights 130, 110, 100, 95, 80, 65, 50 and 45 kDa). Some of these proteins were identified as the Src Homology Collagen (SHC) protein, Janus kinase (JAK-2) and the Mitogen Activated Protein Kinase isoforms ERK 1 and ERK 2. IL-3 also stimulated a transient increase in the activity of both ERK 1 and 2. SCF stimulated a rapid and transient increase in the tyrosine phosphorylation of ERK 1 and ERK 2 although, coaddition of SCF with IL-3, caused no gross differences in the phosphorylation of SHC, JAK-2 or ERKs compared to those observed with IL-3 alone. Coaddition of SCF and low concentrations of IL-3 stimulated a reproducible synergistic increase in the activity of ERK 2, whereas only an additive increase in the activity of ERK 1 was observed. These results suggest that ERK 2 activation represents a point at which the two signalling pathways, stimulated by IL-3 and SCF, interact synergistically.Key Words: Stem cell factorIL-3haemopoiesisERK stimulationtyrosine phosphorylationMAP kinase and synergy
// Emily A. Bassett 1 , Kamalakannan Palanichamy 1 , Mitchell Pearson 1 , Joseph P. McElroy 2 , Saikh Jaharul Haque 1 , Erica Hlavin Bell 1 and Arnab Chakravarti 1 1 Department of Radiation Oncology, The Ohio State University Wexner Medical Center, Columbus, OH, USA 2 Center for Biostatistics, Department of Biomedical Informatics, The Ohio State University, Columbus, OH, USA Correspondence to: Arnab Chakravarti, email: chakravarti.7@osu.edu Keywords: glioblastoma; phospho-proteomic profiling; radiation response; calpastatin; casein kinase 2 Received: December 23, 2017 Accepted: February 10, 2018 Epub: February 19, 2018 Published: March 06, 2018 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.
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