Characterising the function of Tribbles Homolog 1 in breast cancer
|dc.contributor.author||McMillan, Hamish David|
|dc.identifier.citation||McMillan, H. D. (2021). Characterising the function of Tribbles Homolog 1 in breast cancer (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/12336||en|
|dc.description.abstract||Tribbles Homolog 1 (TRIB1) is a pseudokinase with a well characterised oncogenic capacity in acute myeloid leukaemia (AML). Although several transcriptomic studies have linked TRIB1 to the development and progression of several solid tumour types, including breast cancer, its role in the development of solid tumours is not well understood. This study aimed to evaluate the importance and mechanisms of action, of TRIB1 in breast cancer. Using siRNA and shRNA TRIB1 knockdown across three different breast cancer cell lines, revealed TRIB1 as an important regulator of cell proliferation. The knockdown of TRIB1 expression and protein levels inhibited the growth of MCF7, HCC1419 and MDA-MB-231 breast cancer cell lines. Each of these cell lines was derived from a different subtype of breast cancer and express TRIB1 at different levels, suggesting a subtype-independent role of TRIB1 in breast cancer cell proliferation. TRIB1 knockdown in MCF7 cells also results in decreased c-MYC mRNA and protein levels. One potential mechanism for subtype-independent regulation of proliferation, may be the regulation of the oncogene c-MYC, thought to be critical in up to 70% of cancers, as the overexpression of c-MYC can drive cell proliferation. Therefore, while the mechanism of c-MYC regulation remains unknown, the suppression of c-MYC expression, by TRIB1 knockdown, is likely to have a significant impact on cell proliferation. Two global analysis techniques were used to investigate the mechanisms underpinning the regulation of proliferation by TRIB1 in breast cancer. Quantitative Rapid Immunoprecipitation Mass Spectrometry of Endogenous proteins (qPLEX-RIME) was used to identify the TRB1 interactome in HCC1419 and MCF7 cells. The global changes in gene expression in response to TRIB1 knockdown in MCF7 cells were determined using RNAseq. The transcriptomic and proteomic datasets were analysed independently, and in conjunction, to elucidate the mechanisms underpinning the regulation of breast cancer cell proliferation. Interrogation of the TRB1 interactome by qPLEX-RIME, showed the conservation of the TRB1-COP1 interaction from AML, but also revealed association with a wider range of ubiquitination machinery. Association with an expanded range of ubiquitin machinery indicates TRB1 is important in ubiquitin signalling in breast cancer, as in AML, but suggests it may play a wider role than previously described. The qPLEX-RIME also revealed novel TRB1 associations with a number of transcription factors and a range of epigenetic regulators, including the key heterochromatin proteins CBX1, CBX3 and CBX5. Newly identified association with key epigenetic regulators, such as the CBX proteins, suggest a novel function for TRB1 in epigenetic regulation, in particular the regulation of chromatin accessibility. TRB1 association with the transcription factor ZBTB7A, which is known to regulate chromatin accessibility in promoter regions, further suggests TRB1 may regulate transcription in breast cancer through the control of chromatin accessibility. The RNAseq analysis of TRIB1 knockdown in MCF7 cells revealed the importance of TRIB1 across a range of proliferation associated processes. TRIB1 knockdown results in the down-regulation of cell cycle related signatures and the up-regulation of growth inhibiting signatures. TRIB1 knockdown also alters expression from specific transcription factors, including c-Myc, the oestrogen receptor and β-catenin. Wide reaching regulation of gene expression likely results in co-operative changes, allowing the regulation of proliferation across a range of genetic backgrounds. The direct integration of qPLEX-RIME and RNAseq analysis, through gene signatures, is supplemented with manual cross-referencing of observed gene expression changes with the known functions of TRB1 associated proteins. For instance integrated analysis allowed the importance of TRB1 associations with WNT/β-catenin regulators to FERMT2 and LRRFIP2 to be validated. A biochemical approach to evaluate the mechanism underpinning key TRB1 associations is presented as a pilot study of how qPLEX-RIME associations can be further characterised. The identification of direct TRB1 interactors and the characterisation of the interaction mechanism is critical in understanding the functional mechanisms of TRB1 in breast cancer, and will aid long term, in the design of drugs to inhibit or promote critical proliferation regulating TRB1 associations. In summary, TRIB1 has been shown to be important in the proliferation of a range of breast cancer cells and the regulation of a wide range of gene expression, including the expression of the potent oncogene c-MYC. The use of global approaches allowed the identification of a number of previously undocumented TRB1 associations, and enabled the identification of potential mechanisms for TRIB1 action in breast cancer. The integrated analysis of the qPLEX-RIME and RNAseq streamlined the identification of critical pathways and associations for further investigation. The work presented in this thesis identifies a number of novel functions of TRIB1 and provides a strong foundation for the continued investigation of TRIB1 in breast cancer.|
|dc.publisher||University of Otago|
|dc.rights||All items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.|
|dc.title||Characterising the function of Tribbles Homolog 1 in breast cancer|
|thesis.degree.name||Doctor of Philosophy|
|thesis.degree.grantor||University of Otago|
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