- Title
- The function of Δ40p53 in breast cancer
- Creator
- Zhang, Xiajie
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Breast cancer is the most commonly diagnosed female malignancy and the second leading cause of cancer-related death. Nearly all deaths from breast cancer are a result of resistance to DNA-damaging therapies and the subsequent development of metastases. The tumour suppressor, p53, is a master regulator of cell fate outcome following DNA damage, by regulating the expression of target genes involved in cell cycle arrest, DNA repair or apoptosis. Mutation of the tumour suppressor gene TP53 occurs frequently in other solid cancers, but in breast cancer, it is mutated in <25% of all cases, suggesting other mechanisms are accountable for dysregulation of the canonical p53 function. Twelve truncated p53 protein variants (isoforms) are generated from the TP53 gene. Studies have revealed their regulatory role toward the full-length p53 (p53α) and their prognostic value in cancer. The Δ40p53 isoform has been identified in our previous studies to be the mostly highly expressed p53 isoform in breast cancer and a high Δ40p53:p53 ratio is associated with worse-disease survival. This has led to the hypothesis that that high levels of Δ40p53 (as observed in breast cancer) will have a negative impact on p53 functional activities resulting in increased proliferation, migration and invasion, contributing to p53-mediated DNA methylation, as well as inhibition of apoptosis in response to DNA damaging therapies; and that this is responsible for the association of worse outcomes in breast cancer patients with high Δ40p53/p53 ratios. However, the endogenous role of Δ40p53 in the acquisition of an aggressive breast cancer phenotype and in the response to DNA damage is unclear. The overall aim of the studies described in this thesis is to investigate the molecular and functional consequences of a high Δ40p53/p53α ratio in regulating breast cancer growth, metastasis-related processes and in the response to DNA-damaging therapies. Gene expression analysis performed on breast cancer specimens with high or low Δ40p53 expression level highlighted a role for Δ40p53 in epithelial-mesenchymal transition, extracellular matrix communication and immune responses. Breast cancer cell line models derived from MCF-7 and ZR75-1 cells (with wild-type p53) were established using shRNA-transduction to generate stable and specific Δ40p53 or p53α knockdown, simulating the naturally occurring isoform-imbalance in breast cancers. A series of functional assays investigating the role of Δ40p53 in EMT, cell proliferation/migration/ invasion at the basal level were performed on these sublines as well as on previously established Δ40p53-overexpressing MCF-7 cell models. Results showed p53α-knockdown fundamentally contributed to increased cancer cell mobility and invasiveness, while Δ40p53-knockdown had limited and cell-dependent impact. Molecular characterisation by RNA-seq analysis further confirmed a link between Δ40p53 and immunity, and showed that Δ40p53 was an influencing factor in cell differentiation and tissue development. Following this, DNA methylation signatures were obtained by performing Illumina EPIC 850k methylation arrays. Differentially methylated probes (DMPs) and differentially methylated regions (DMRs) were identified comparing to the respective controls. An isoform-associated cytoband region containing pro-inflammatory factors and regulating extracellular components was shown to be differentially methylated following p53 isoform alteration. In addition, multiple transcription factor binding sites, micro-RNAs and cancer-associated genes were differentially affected depending on which p53 isoform was knocked down. These results imply that p53 isoforms are capable of regulating gene expression through both transcriptional and epigenetic means and this has not been demonstrated previously. In addition to the intrinsic differences caused by isoform-alteration, DNA-damaging treatment triggered similar responses across all cell lines. A high ratio of Δ40p53/p53α was associated with enhanced G2 arrest and attenuated apoptosis. These results were confirmed by RNA-seq analysis comparing doxorubicin-treated cells to untreated cells, identifying critical molecular markers such as CCNB1 and GADD45 that were altered by the p53 isoforms and that govern cell cycle progression. The comprehensive characterisation of these cell lines provides novel insights and potential biomarkers for further investigation of Δ40p53 in breast cancer as well as other cancers. The novel finding of Δ40p53 regulation of genes involved in immune responses contributes to the current knowledge of p53 isoform research and provides a new perspective accounting for breast cancer heterogeneity and treatment possibilities. Importantly, the finding that the Δ40p53/p53α ratio was a causative factor in the decreased response to DNA-damaging therapies may contribute to the discovery of novel treatment strategies with decreased toxicity and increased efficacy in breast cancers.
- Subject
- breast cancer; p53; isoforms; Δ40p53
- Identifier
- http://hdl.handle.net/1959.13/1412992
- Identifier
- uon:36560
- Rights
- Copyright 2020 Xiajie Zhang
- Language
- eng
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| View Details Download | ATTACHMENT01 | Thesis | 9 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 400 KB | Adobe Acrobat PDF | View Details Download |