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Factors regulating the nuclear localization of β-catenin in acute myeloid leukaemia

Ridsdale, Jenna 2018. Factors regulating the nuclear localization of β-catenin in acute myeloid leukaemia. PhD Thesis, Cardiff University.
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Abstract

Acute myeloid leukaemia (AML) is a highly heterogenous haematological malignancy that accounts for approximately 34% of newly diagnosed leukaemia cases in the United Kingdom. It is a disease that primarily affects the elderly, and the prognosis for AML is generally poor, with a 5-year survival rate of 25% in adults. Identifying factors that contribute to the pathogenesis of AML is important in developing new targeted treatments and improving patient outcome. The Wnt signalling pathway is one of the most commonly dysregulated signalling pathways in AML, and overexpression of its principal effector, β-catenin, is associated with a poor prognosis. β-catenin is a transcription factor that regulates key cellular processes, including proliferation and cell survival, by binding in complex with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins to Wnt target gene promoters. Nuclear localization of β-catenin is fundamental for its role as a transcription factor. Despite this, little is known about the mechanisms regulating this process in AML; previous work has shown AML blasts and cell lines are highly variable in their ability to translocate β-catenin from the cytosol to the nucleus. This study used a mass spectrometric approach to identify candidate β-catenin nuclear localization factors in AML. This approach was based on comparisons of β-catenin binding partners in the nucleus and cytoplasm of leukaemia cell lines that i) freely translocate β-catenin to the nucleus (K562 and HEL), or ii) resist the nuclear localization of β-catenin (THP-1, U937 and NOMO-1). Cytoplasmic/nuclear fractionation and immunoprecipitation of β-catenin were optimized in K562 cells, before being used to prepare samples of each cell line for mass spectrometry. Following mass spectrometric analysis, eight candidate β-catenin nuclear localization factors were identified, of which two proteins, RUNX1 and LEF-1, were verified by western blotting. Since LEF1 has been previously implicated as a β-catenin nuclear import factor in other contexts and because of its known role in AML, LEF-1 was chosen for further analysis. To determine the relevance of LEF-1 mediated β-catenin nuclear localization in AML pathogenesis, knockdown and overexpression studies were conducted. Knockdown of LEF-1 was successful in leukaemia lines. In cells stimulated with the Wnt agonists (Wnt3a and BIO), LEF-1 knockdown decreased the nuclear translocation of β-catenin and impacted β-catenin mediated transcription. Additionally, knockdown of LEF-1 appeared to reduce proliferation of leukaemia cells, but did not impact their migration or survival. Ectopic nuclear overexpression of LEF1 proved difficult to achieve in myeloid cells due to protein instability; making reciprocal demonstration of the role of LEF1 difficult to demonstrate. Overall this study has identified candidate β-catenin nuclear localization factors and has validated the role of one of these proteins (LEF1). This work provides insight into the potential mechanisms governing β-catenin nuclear localization in AML, and in the longer term, may lead to novel approaches for the treatment of AML.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 29 May 2018
Last Modified: 29 May 2018 08:13
URI: http://orca.cf.ac.uk/id/eprint/111743

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