Abstract 4755: Calpastatin phosphorylation regulates radiation response in glioblastoma

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

From genome integrity to cancer

“…This special issue of Genome Medicine highlights the advances made in understanding how compromised genome integrity impacts genome architecture, tumor biology, immune escape, and the mechanisms underlying differential response and resistance to cancer therapies, outlining new avenues for precision therapies and clinical decision-making. For example, at base pair resolution, a high frequency of SNVs with characteristic C > A and T > C mutations, together with a high degree of indel formation at polynucleotide repeat tracts (also termed microsatellite instability), has been found in tumors with mismatch repair deficiencies (MMRd) [7]. It is important to detect tumors that have MMRd because of their reported sensitivities to immunotherapy [8, 9], regardless of the cell of origin.…”