Doctoral Degrees (Physiological Sciences)

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Browsing Doctoral Degrees (Physiological Sciences) by Author "Davis, Tanja Andrea"

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    The role of the AHNAK protein in breast cancer : implications for tumour metastasis and chemoresistance
    (Stellenbosch : Stellenbosch University, 2016-12) Davis, Tanja Andrea; Engelbrecht, Anna-Mart; Loos, Benjamin; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Introduction – Cancer continues to have a significant impact on society. While there has been much success in characterising tumours and identifying targetable markers, two major problems are still faced today, namely therapeutic failure and advanced progression of the disease. The human AHNAK protein is a giant scaffold protein involved in multiple cellular processes and has now also been suggested to be associated with cancer, particularly with regards to tumour metastasis and chemoresponse. However, limited information and several contradicting findings have contributed to a poor understanding of the role of AHNAK in cancer. Thus, we aimed to characterise the AHNAK protein in cancer by determining the role of the protein in the chemotherapeutic response of breast cancer to doxorubicin (DXR) and also in cellular migration. Methods – For the in vitro model the non-metastatic DXR-sensitive epithelial-like MCF-7 and metastatic DXR-resistant mesenchymal-like MDA-MB-231 cell lines were used. We performed DXR treatments and assessed AHNAK’s protein expression and intracellular localisation. We also assessed these properties in a tumour-bearing mouse model. AHNAK knockdown and overexpression was achieved by means of transient plasmid transfections in both cell lines and following DXR treatments we assessed apoptotic marker expression, cell cycle modulation, epithelial-mesenchymal transition (EMT) marker expression and cellular migration. Results – DXR induced dose-independent and dose-dependent changes in AHNAK protein expression in MCF-7 and MDA-MB-231 cells, respectively, but it did not affect its intracellular localisation in these cells. In the tumour-bearing mouse model DXR also induced dose-dependent changes in AHNAK expression without affecting its localisation, similar to the MDA-MB-231 cells. In the MDA-MB-231 cells, DXR promoted apoptosis inhibition by decreasing cPARP and cCasp7 expression. Knockdown of AHNAK prevented this inhibition while overexpression induced a similar inhibitory effect. With cell cycle analyses we observed that DXR also resulted in S phase arrest in these cells. AHNAK knockdown completely prevented the DXR-induced cell cycle arrest while overexpression was sufficient to cause such an arrest on its own. No significant effects were observed with these experiments in the MCF-7 cells. DXR induced EMT in the MCF-7 cells but AHNAK knockdown or overexpression did not affect this. In MDA-MB-231 cells DXR treatment showed a trend of decreased EMT and while AHNAK knockdown had no effect on this, its overexpression showed clearer evidence of EMT reduction. AHNAK knockdown also had no major effects on cell migration in both cell lines, although its overexpression generally decreased cellular migration. Conclusions – We show that AHNAK plays a novel role in the DXR-response of breast cancer cells and this involved AHNAK’s expression, apoptosis inhibition and cell cycle modulation. Possible molecular mechanisms are proposed but require further investigation. Our results regarding the role of AHNAK in tumour cell migration is less clear and contradicting when compared to other studies. These results may have potential therapeutic implications with regards to the modulation of DXR response to improve treatment efficacy.