Pathology - Theses
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ItemIdentification of CREB as a transcriptional regulator of glioblastoma biology( 2015)Glioblastoma (GBM) is both the most common and malignant type of brain tumour with a median survival of 7.4 months from the date of diagnosis; even following current treatment regimens of resection, radiation and chemotherapy, highlighting the need for a greater understanding of the molecular mechanisms driving tumour biology. The Cyclic-AMP Response Element Binding (CREB) protein is a kinase-inducible transcription factor which sits at a hub of pro-oncogenic signalling pathways. CREB is a critical regulator of normal brain development where it regulates proliferation, differentiation and survival. The phosphorylated form of CREB (pCREB) is overexpressed in several types of cancer where it regulates various aspects of tumour biology but little is known about the role of CREB in GBM. As such, this thesis hypothesises that pCREB is an oncogene in GBM where it is activated by multiple cancer signalling pathways and is essential for the maintenance of tumour biology. To investigate the pathways responsible for CREB activation in GBM and the potential of pCREB to serve as a prognostic biomarker, a combination of immunohistochemistry (IHC) and bioinformatics analysis were undertaken. pCREB was co-expressed in cells along with either pMAPK or pAKT in GBM specimens, implicating the PI3K/AKT and MEK/MAPK pathways in differentially activating CREB. Signalling through a MAPK-CREB axis was associated with cell proliferation whilst an AKT-CREB signalling axis was implicated in invasion. pCREB was not prognostic for survival in GBM however was associated with poor prognosis when analysed in the context of MEK/MAPK signalling. Investigation of the role of CREB in vitro confirmed the role of CREB in regulating proliferation. Knockdown of CREB resulted in slower cell cycle progression and this occurred through the transcriptional regulation of the cell cycle factor Cyclin D1. In addition, CREB was selectively regulated the activity of the PI3K/AKT pathway via a feedback mechanism which in turn regulated the expression of cell cycle factors Cyclin B1 and PCNA. In contrast to the PI3K/AKT and MEK/MAPK pathways, the cAMP/PKA pathway was not associated with CREB activation in GBM. Exogenous activation of the cAMP/PKA pathway resulted in BIM dependant apoptosis in selective GBM cell lines. CREB played a pro-survival role in this context where it inhibited BIM expression and apoptosis. To investigate the link between the PI3K/AKT pathway and CREB, hyperactivation of the PI3K/AKT pathway was induced in neural stem cells via an activating mutation of PIK3CA and loss of PTEN, leading to glioma initiation in vivo. Cells with these mutations expressed high levels of pAKT as well as CREB target genes Cyclin D1 and Cyclin B1 independently of exogenous growth factors. pCREB was expressed throughout PIK3CA/PTEN tumours and knockout of CREB in PIK3CA/PTEN tumours resulted in prolonged survival, demonstrating the key role of CREB in PI3K/AKT mediated tumour biology. Collectively this study establishes CREB as a pro-oncogenic factor in GBM where it plays a pivotal role in tumour growth and survival. These results form the basis for future research into the role of CREB in GBM and highlight the potential use of CREB as a target for future therapies.