- Title
- The regulation, function and expression of Δ40p53 in breast cancer
- Creator
- Morten, Brianna Catherine
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Breast cancer is the most common malignancy in women and it is the second highest cause of cancer-related death. The tumour suppressor gene p53 is crucial for the prevention of cancer through its role in the maintenance of cellular growth and differentiation. Inactivation of p53 through mutations is the most common event in cancer. However, in breast cancer, p53 is mutated in only ~24% of cases and this suggests that other mechanisms are responsible for the loss of p53 function. Ascertaining the mechanisms responsible for its inactivation may lead to the identification of novel treatment targets or prognostic tools. The p53 isoforms were discovered over a decade ago and they may represent one mechanism that regulates p53 functionality. Currently, over 14 p53 isoforms have been discovered and their expression is deregulated in many human cancers. One such isoform, Δ40p53, has been found to inhibit p53, or support its tumour suppressive functions, depending on its ratio to full-length p53 (FLp53). Previous studies from our laboratory have shown that Δ40p53 is the most highly expressed p53 isoform in breast cancer, but its function and the clinical implications of its expression are yet to be elucidated. Furthermore, current methods of detecting Δ40p53 at the mRNA and protein level lack the sensitivity and specificity to analyse the endogenous expression of this isoform in clinical specimens. The aims described in this thesis were to elucidate the regulation and function of this isoform in breast cancer. Further studies aimed to design and evaluate novel methods for the detection of Δ40p53 mRNA and protein expression to aid in the clinical implementation of Δ40p53 as a breast cancer biomarker. Δ40p53 can be produced by alternative translation or alternative splicing, but our previous studies have indicated that alternative splicing is likely to be a major route of Δ40p53 production in breast cancer. G-quadruplex structures within intron 3 of p53 are important for the production of FLp53 mRNA and disruption of these structures by a polymorphism in intron 3 of p53 (PIN3) has been shown to lead to increased levels of Δ40p53 mRNA in vitro. The first part of this thesis reports on whether PIN3 was associated with the expression level of Δ40p53 in breast tumour tissues. PIN3 was shown to be associated with a low Δ40p53:FLp53 ratio, and that this was correlated with improved disease-free survival. This suggests that the Δ40p53:FLp53 ratio is modified by PIN3 in breast cancer and that the Δ40p53:FLp53 ratio and PIN3 may be potential prognostic indicators for breast cancer outcome. Following this, the function of Δ40p53 was investigated in estrogen receptor (ER)-positive breast cancer. p53 can interact with and regulate the expression of another important transcription factor in breast cancer, ER, and this interaction may be important in breast cancer development and progression. However, the role that Δ40p53 may play in this interaction is unclear. In this thesis, knockdown of Δ40p53 caused a reduction in the expression of ER and its target genes PR and pS2 in the presence of estrogen, and this was supported by overexpression studies examining Δ40p53. Furthermore, a high Δ40p53:FLp53 ratio was shown to be an indicator of worse disease-free survival in ER-positive breast cancers, suggesting that the level of Δ40p53 expression is related to treatment outcomes. After determining a role for Δ40p53 in mediating the expression and function of ER in breast cancer, the next chapter evaluated two new methods of detecting Δ40p53 expression in tumour tissues. Firstly, a method using branched DNA probes (QuantiGene 2.0 assay) was examined to determine if it was superior to real-time PCR for the detection of Δ40p53 mRNA in formalin-fixed paraffin-embedded (FFPE) tissues. The results were also compared in matched fresh frozen (FF) tissues. Δ40p53 was unable to be detected by either real-time PCR or the QuantiGene 2.0 assay in FFPE tissues, but FLp53 was detected. However, the expression of FLp53 in FFPE tissues was not correlated between the two different methods. This study confirmed the difficulties in quantitating mRNA from these archival specimens. Finally, the use of targeted mass spectrometry in the quantification of Δ40p53 protein expression was investigated in a small pilot study. While only preliminary, the results suggested that Δ40p53 endogenous expression was distinguishable from FLp53 and was accurately quantitated in breast cancer cells. This method provided a novel technique for determining the abundance of Δ40p53 protein compared to FLp53 protein. Taken together, the work portrayed in this thesis has demonstrated that Δ40p53 expression is associated with PIN3 in breast cancer. A novel functional role for Δ40p53 has also been identified, the regulation of ER expression in breast cancer which is associated with clinical outcomes, and a novel method for the quantification of Δ40p53 protein expression has been developed that may be useful for the analysis of clinical samples. These findings highlight the importance of high Δ40p53 expression in breast cancer, which has been previously reported on by our laboratory, and suggests that Δ40p53 may be an important prognostic marker in breast cancer.
- Subject
- breast cancer; Δ40p53; p53; ER; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1335583
- Identifier
- uon:27459
- Rights
- Copyright 2017 Brianna Catherine Morten
- Language
- eng
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| View Details Download | ATTACHMENT01 | Thesis | 27 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 752 KB | Adobe Acrobat PDF | View Details Download |