Doctoral Degrees (Physiological Sciences)
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Browsing Doctoral Degrees (Physiological Sciences) by Author "Du Plessis, Manisha"
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- ItemMolecular regulation of autophagy and metastasis in breast cancer: new insights into the role of serum amyloid A(Stellenbosch : Stellenbosch University, 2022-04) Du Plessis, Manisha; Engelbrecht, Anna-Mart; Davis, Tanja; De Villiers, Willem J. S.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.ENGLISH ABSTRACT: Introduction: Cytokines, growth factors and acute phase proteins present in the tumour microenvironment regulate inflammatory responses and alter crosstalk between various signalling pathways involved in the progression of cancer. It has recently been reported that serum amyloid A (SAA), an acute-phase protein, mainly produced by hepatocytes during an inflammatory response or infection, is also synthesized by cancer cells as well as other cells in the tumour microenvironment. SAA can activate several signalling pathways, including PI3K/Akt and MAPK signalling pathways, which are also known modulators of the intracellular degradation process, autophagy. However, no knowledge exists regarding the relationship between SAA and autophagy in breast cancer. Furthermore, it has been reported that SAA can promote the metastasis of cancer cells. Therefore, the aim of this study was to investigate the role of SAA in autophagy, metastasis and the activation of signalling pathways in in vitro and in vivo models of breast cancer. Methods: For the in vitro model the following cell lines were used, the triple-negative metastatic breast cancer cell line, MDA-MB-231, the oestrogen (ER+) and progesterone positive (PR+) epithelial-like cell line, MCF7, and the non-malignant breast epithelial cell line, MCF12A. These cell lines were transiently transfected with a control vector, pcDNA3, and an overexpression plasmid, pcDNA3-hSAA1, to overexpress SAA1. This study also investigated whether SAA1/2 is required for tumourigenesis in an in vivo tumour-bearing mouse model with double knockout of Saa1 and Saa2. We assessed autophagy, metastasis, proliferation, apoptosis and signalling pathway marker activation in these two models. Results: The overexpression of SAA1 in the MCF12A, MDA-MB-231 and MCF7 cell lines resulted in an increase in cell viability and increased the expression of the proliferation marker, MCM2, in the MCF12A and MCF7 cell lines. Furthermore, the overexpression of SAA in these cell lines resulted in the inhibition of autophagy, while the expression of the cargo recruiter, p62, was increased in the MCF7 cell line. SAA also promoted the migration of the MDA-MB- 231 and MCF7 cell lines, while no significant changes in the expression of the EMT markers were detected. The overexpression of SAA1 decreased the activation of the MAPK and PI3K signalling pathways in the MCF12A cell line, activated these pathways in the MDA-MB-231 cell line and inhibits Akt signalling in the MCF7 cell line. The overexpression of SAA1 resulted in a decrease in the colocalization of pERK and LC3-II in the MCF12A cell line and increased the colocalization of pERK and LC3-II in the MDA-MB-231 cell line. Furthermore, SAA1/2 knockout in vivo resulted in the induction of autophagy, while increasing the expression of p62. The knockout of SAA1/2 also promoted the resistance of these cancer cells to apoptosis, possibly through the regulation of autophagy. The knockout of SAA also inhibited the mesenchymal phenotype by downregulating the expression of vimentin. Lastly, the knockout of SAA1/2 resulted in the inhibition of the PI3K pathway protein, PKB/Akt, while increasing the activation of the MAPK, p38. Furthermore, knockout of SAA1/2 resulted in an altered inflammatory profile, evident in the decrease of plasma IL-1β, IL-6 and IL-10, while increasing the plasma levels of MCP-1 and TNF-α. Conclusions: We have determined for the first time a novel role for SAA in autophagy in breast cancer cells. SAA overexpression inhibited autophagy in breast cancer cells. Additionally, SAA promotes migration and proliferation through the cell-type specific regulation of the PI3K/Akt and MAPK, ERK1/2 and p38, signalling pathways. Furthermore, the double knockout of SAA1/2 induced autophagy, promoted tumour cell survival, inhibited metastasis and regulates the activation of the PI3K/Akt and p38 signalling pathways. Double knockdown of SAA1/2 also resulted in an altered inflammatory profile in vivo. Our results therefore suggest that SAA plays an important role in breast cancer tumourigenesis.