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Nuclear Factor I and Calpain Signaling in Glioblastoma
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- Author / Creator
- Vo, Minh The
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Glioblastoma (GBM), classified as WHO grade IV astrocytoma, is the most common and lethal adult primary brain tumour. Despite recent advances in treatment, GBM prognosis remains largely unchanged with most patients succumbing to the disease within ~15 months of diagnosis. This dismal outcome can be attributed in part to its highly infiltrative nature, with tumour cells spreading into normal brain parenchyma at early stages of the disease. We and others have shown that the expression of brain fatty acid-binding protein (FABP7) correlates with increased GBM cell migration in vitro and a worse prognosis. In GBM, FABP7 is regulated by the Nuclear factor I (NFI) family of four transcription factors: NFIA, B, C, X. These transcription factors, specifically NFIA and NFIB, have been shown to play key roles in regulating neural cell migration and gliogenesis. We have also demonstrated that NFI transcriptional activity is dependent on its phosphorylation state, with hypophosphorylated NFI associated with FABP7 expression. NFIs are dephosphorylated by the calcineurin phosphatase in GBM cells. Calcineurin, in turn, is cleaved and activated by calpain proteases. Ubiquitously-expressed calpain 1 and calpain 2, the best characterized members of the calpain family, have been shown to be master regulators of cell migration, with functions spanning all major steps of this process. Importantly, calpain 2 is essential for the infiltration of GBM cells in a zebrafish model. Calpain proteolytic activity is tightly regulated by the cell, with the most well-known mechanisms being autoproteolysis and regulation by its endogenous inhibitor, calpastatin (encoded by the CAST gene). Previous work from our lab has identified CAST as a putative target of NFI in GBM cells. Here, we confirm NFI binding to a de novo CAST alternative promoter. We show that binding of hypophosphorylated NFI to CAST intron 3 results in increased utilization of the alternative promoter and a higher ratio of class 2 (encoding for XL-less calpastatin isoforms) to class 1 (encoding for full-length calpastatin) CAST variants, which ultimately leads to altered subcellular distribution of calpastatin. Our findings provide a foundation for further investigations into the possibility of regulatory crosstalk between NFI and the calpain pathway. To this end, we identify two NFI-calpain positive feedback loops with opposing effects in GBM: NFIB-calpain 1 and NFIA-calpain 2. We provide evidence for two distinctive mechanisms by which differentially phosphorylated NFIB can increase calpain 1 activity. In turn, calpain 1, via calcineurin, promotes NFIB dephosphorylation, a process that is accompanied by altered subnuclear distribution and transcriptional activity of NFIB. We also show that the NFIB-calpain 1 positive feedback loop acts to suppress cell migration while having little to no effect on GBM cell viability. In contrast, we found only one mechanism by which NFIA can increase calpain 2 activity. Like calpain 1, calpain 2 cleaves and activates calcineurin, which dephosphorylates NFIA and causes an increase in FABP7 expression. The NFIA-calpain 2 crosstalk acts to increase GBM cell migration and survival. These NFI-calpain positive feedback loops can be exploited with calpain inhibitors to reduce GBM cell survival.
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- Subjects / Keywords
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- Graduation date
- Fall 2019
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- License
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.