- Title
- Functional role of protein phosphatase 2A and its regulatory subunit B55α in development and breast cancer
- Creator
- Panicker, Nikita
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2022
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Protein Phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases that are involved in diverse signalling pathways regulating everything from normal mammalian development and physiology, to disease states such as cancer. This heterotrimeric enzyme consists of a structural A-subunit, a catalytic C-subunit and one of four families of regulatory B-subunits. The binding of a regulatory B-subunit to the core PP2A-A-C dimer, directs PP2A dephosphorylation activity to specific substrates and controls sub-cellular targeting. As such, several PP2A regulatory B-subunits play important roles during mammalian development and disease. PP2A inactivation, via mutation, reduced subunit expression or overexpression of endogenous PP2A-inhibitory proteins, commonly occurs in many human cancers, including breast cancer. PP2A activation using small molecules is an emerging and promising treatment option for breast cancer. The sphingosine analogue FTY720 (fingolimod; Gilenya (Novartis)) has shown pre-clinical anti-cancer efficacy as a PP2A activating compound. However, FTY720 it is not suitable for clinical development as an anti-cancer therapy due to its immunosuppressive side effects Therefore, the first aim of this thesis was to investigate the pre-clinical anti-breast cancer efficacy of novel PP2A activating analogues of FTY720 (Chapter 3) using in vitro cytotoxicity, migration, invasion and phosphatase activity assays, as well as an in vivo MDA-MB-231 xenograft mouse model of breast cancer. This chapter showed that PP2A activation and breast cancer cytotoxicity can be achieved without the immunosuppression associated with FTY720, and provides support for the development of these compounds as anti-breast cancer therapies. While PP2A is often considered a tumour suppressor, the specific role of most PP2A regulatory subunits in tumour development and progression is poorly understood. The PPP2R2A gene,encoding the regulatory subunit PP2A-B55α, is frequently deleted in breast tumours, and low PP2A-B55α expression is associated with poor patient outcome. However, the functional role of PP2A-B55α in breast cancer was not known. To address this, my laboratory developed the first Ppp2r2a knockout mouse using CRISPR/Cas9 gene editing. However, homozygous knockout of Ppp2r2a was embryonic lethal. Therefore, the second aim of this thesis was to investigate this embryonic lethality and the role of PP2A-B55α in embryonic development (Chapter 4). While homozygous Ppp2r2a deletion was confirmed to be embryonic lethal, I found that adult heterozygous mice were viable and appeared morphologically normal. Analysis of embryos at different developmental stages found a normal Mendelian ratio of Ppp2r2a-/- embryos at embryonic day (E) 10.5 (25%), but reduced Ppp2r2a-/- embryos at E14.5 (18%), and further reduced at E18.5 (10%). No live Ppp2r2a-/- pups were observed at birth. Ppp2r2a-/- embryos were significantly smaller than wild-type or heterozygous littermates and displayed a variety of neural defects such as exencephaly, spina bifida, and cranial vault collapse, as well as syndactyly and severe epidermal defects; all processes driven by growth and differentiation of the ectoderm. Ppp2r2a-/- embryos had incomplete epidermal barrier acquisition, associated with thin, poorly differentiated stratified epithelium with weak attachment to the underlying dermis. The basal keratinocytes in Ppp2r2a-/- embryos were highly disorganized, with reduced immunolabelling of integrins and basement membrane proteins, suggesting impaired focal adhesion and hemidesmosome assembly. The spinous and granular layers were thinner in the Ppp2r2a-/- embryos, with aberrant expression of adherens and tight junction associated proteins. The overlying stratum corneum was either absent or incomplete. Together, this data suggests that PP2A-B55α is an essential regulator of epidermal stratification, and is essential for ectodermal development during embryogenesis. Given the proposed tumour suppressor role for Ppp2r2a in breast cancer, the final two chapters of this thesis investigated the role of PP2A-B55α in mammary gland and mammary tumour development. In Chapter 5, adult wildtype and Ppp2r2a+/- mice were studied to characterise the effect of reduced PP2A-B55α expression on mammary gland development and the development of spontaneous neoplastic lesions with age. The final results chapter of this thesis (Chapter 6) further investigated the role of PP2A-B55 as a tumour suppressor versus promoter in the context of HER2+ breast tumours, using a transgenic model of HER2-driven breast cancer, MMTV-NIC (mouse mammary tumour virus – Neu (NDL2-5)-IRES (internal ribosome entry site)-Cre). Taken together, the work presented in this thesis greatly contributes to our understanding of the functions of PP2A, and particularly PP2A-B55α, in mammalian development and breast cancer, elucidating for the first time what these functions are in vivo. The mouse models developed in this thesis provide powerful tools for future investigations and pre-clinical testing of novel therapeutics.
- Subject
- breast cancer; phosphatase; embryonic development; PP2A; Ppp2r2a
- Identifier
- http://hdl.handle.net/1959.13/1508677
- Identifier
- uon:56144
- Rights
- Copyright 2022 Nikita Panicker
- Language
- eng
- Full Text
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