Doctoral Degrees (Physiological Sciences)

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Browsing Doctoral Degrees (Physiological Sciences) by browse.metadata.advisor "De Villiers, Willem J. S."

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    The link between chronic inflammation, hypercoagulation, and a bacterial presence in colorectal carcinogenesis
    (Stellenbosch : Stellenbosch University, 2021-04) De Waal, Greta Marie; Pretorius, Etheresia; De Villiers, Willem J. S.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Colorectal cancer (CRC) leads to high rates of morbidity and mortality worldwide. CRC is a heterogeneous and highly complex disease, with factors such as genetic alterations, environmental risk factors, the gut microbiota, and inflammation contributing to the development and progression of colorectal tumours. A novel holistic approach was employed in this dissertation to investigate colorectal carcinogenesis, by analysing the systemic environment and local tumour environment of CRC patients. A close link exists between persistent infections, chronic (systemic) inflammation, and colorectal carcinogenesis. There is also an intricate relationship between gut dysbiosis and a pro- inflammatory profile in CRC patients. Alterations in the composition of the gut microbiota can contribute to the development of a dysfunctional gut barrier, thereby facilitating the translocation of bacteria and their highly potent inflammagenic molecules (specifically the presence of circulating lipopolysaccharide (LPS)). Such leaky gut conditions can promote systemic inflammation, of which a hallmark is increased hypercoagulability. Importantly, chronic inflammation and an activated (pathological) coagulation system are implicated in tumorigenesis. It was hypothesised that the presence of Helicobacter pylori, Escherichia coli, LPS, serum amyloid A (SAA), as well as structural changes in proteins are increased inside CRC tumour cells and/or their microenvironment, compared to control tissues. Furthermore, that circulating levels of LPS are elevated in CRC patients, compared to healthy subjects; and that the increased presence of circulating LPS and inflammatory molecules such as SAA (systemic inflammation) contributes to a hypercoagulable state and promotes hematological dysfunction in CRC patients. In this dissertation it was shown that there is an intratumour bacterial presence in CRC patients, together with significantly elevated levels of the bacterial wall component LPS in their circulation, compared to controls. It was also demonstrated that these patients have a pro-inflammatory profile, accompanied by coagulopathies. Moreover, it was found that structural protein changes are increased in CRC tumour tissues. The discovery of additional (novel) biomarkers for CRC screening, together with the development and employment of novel methods for the early detection of CRC risk, represent a growing research field and are crucial for the application of a true “personalized medicine” approach that can enable improved early CRC detection, diagnosis, and prognosis. Importantly, blood-based screening tools are an emerging research area of interest for CRC screening. Early detection of the presence of dysregulated circulating inflammatory markers and early diagnosis of an activated coagulation system, together with the detection of bacterial components in circulation and also in the local tumour environment, could be important, and may, in conjunction with modulation of the gut microbiota, serve as potential therapeutic targets. A holistic view enables us to have a better overall understanding of the link between chronic inflammation, hypercoagulation, and a bacterial presence in colorectal carcinogenesis. The hypothesis of this dissertation was supported in such a way that the findings can be applied in a clinical setting. The way forward is therefore to apply these (clinically relevant) novel research findings in practice, in order to aid in the early identification of individuals with an increased risk for CRC.
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    Molecular 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.
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    The role of Serum Amyloid A in NLRP3 inflammasome signalling in breast and colon cancer
    (Stellenbosch : Stellenbosch University, 2022-04) Fourie, Carla; Engelbrecht, Anna-Mart; Davis, Tanja; De Villiers, Willem J. S.; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction: Cancer is a complex disease with multiple interactions targeting the organism on cellular, tissue and systemic levels. The main research focus for the past decades has been on the genome and on the molecular level where signaling pathways were dissected for the development of targeted therapies. However, in order to develop more efficient therapeutic regimes, a better understanding on systemic level is required. Over the past few years, the role of serum amyloid A (SAA) has gathered significant evidence which highlights its role in the pathogenesis of several cancers, including breast and colorectum carcinomas. To date, SAA has been shown to bind to several pattern recognition receptors, which might suggest that inflammasomes play a role in the tumour-promoting properties of SAA. Inflammasomes are cytoplasmic multiprotein complexes characterized by a sensor protein, an adaptor protein, and inflammatory caspases. However, the role of inflammasomes in cancer remain controversial. The aim of this study was therefore to investigate the role of SAA in inflammasome signaling in breast and colon cancer. Methods: In this 3-part animal study, tissues were subjected to immunoblotting, real-time PCR, haematoxylin and eosin staining and immunohistochemistry. For the first inflammatory model, wild-type and SAA double knockout C57BL/6 mice received 2.5% dextran sulfate sodium, which was administered for a total of 5 days. To assess tumourigenesis, colitis-associated cancer and triple negative breast cancer models were used, respectively. For colitis- associated cancer, wild-type and SAA double knockout C57BL/6 mice received an intraperitoneal injection of 12.5 mg/kg azoxymethane. After one week, dextran sulfate sodium treatment was administered at a concentration of 2.5% for a total of 5 days, followed by a recovery period of 16 days. Dextran sulfate sodium treatment was administered for a total of 3 cycles. Triple negative breast tumours were established in wild-type and SAA double knockout C57BL/6 mice by injecting EO771 cells subcutaneously at the fourth mammary fat pad. The experimental endpoint was reached when tumour volumes reached 300-400 mm3. Results: In this study we have showed that in an in vivo model of dextran sulfate sodium induced colitis, SAA ablation exerted pro-inflammatory properties independent of the NLRP3 inflammasome. The ablation of serum amyloid A1/2 was associated with the increased expression of pro- inflammatory cytokines. In contrast, in an in vivo colitis-associated cancer and in a triple negative breast cancer model, the ablation of SAA suppressed canonical NLRP3 inflammasome activation, which was associated with anti-inflammatory properties. These findings suggest that during tumourigenesis, SAA functions as an endogenous damage associated molecular pattern in the tumour microenvironment. Conclusion: Here we show for the first time, in models of CAC and TNBC, the novel role of SAA in the activation of the NLRP3 inflammasome and the generation of pro-inflammatory cytokines, two mechanisms known to promote tumour development and metastasis. This study emphasizes the notion that the tumour-induced systemic environment acts as a critical regulator of cancer progression and metastasis. In conclusion, simultaneously targeting SAA and NLRP3 components could be beneficial for cancer treatments.