Characterisation of the response by MDA-MB-231 breast cancer cells to amino acid starvation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand
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2020
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Massey University
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Abstract
Breast cancer, like many other types of cancer, exhibit abnormal amino acid metabolism and dependency on external sources of specific amino acids. The specific amino acids cancer cells rely on to support their growth and survival may provide potential targets for treating breast cancer patients. Moreover, targeting the specific pathways involved in overcoming amino acid starvation is another potential breast cancer therapy. Starvation of specific amino acids results in a reduction of breast cancer cell proliferation and activation of apoptosis. However, the involvement of the Amino Acid Response (AAR) mechanism during amino acid starvation conditions is poorly understood in breast cancers. In this study, we investigated the response of the triple negative breast cancer (TNBC) cell line - MDA-MB-231 - to glutamine and leucine starvation. We scored for the activation of the General Control Non-derepressible 2 (GCN2) protein and phosphorylation of alpha subunit of eukaryotic initiation factor 2 (eIF2α). We relate a higher activation and phosphorylation of GCN2 and eIF2α to a more severe AAR by MDA-MB-231 cells. In this study, we concluded that the MDA-MB-231 cells responded more strongly to glutamine starvation than leucine starvation by a greater activation of their AAR. Furthermore, the detection of the greater activation of apoptosis and a greater reduction in cell proliferation also suggests that glutamine supply is more critical than leucine to support MDA-MB-231 cell survival. We discuss
the roles and consumption rates of each amino acid as potential causes for this difference in AAR to glutamine and leucine starvation by MDA-MB-231 cells.
Furthermore, we inhibited GCN2 via siRNA mediated knockdown, before imposing glutamine and leucine starvation and screened for activation of apoptosis and a change in cell proliferation. We found that knockdown of GCN2 sensitised the MDA-MB-231 cells to both leucine and glutamine starvation, with a greater reduction of cell
proliferation and activation of apoptosis during glutamine starvation. Knockdown of GCN2 in unstressed MDA-MB-231 cells also led to a decrease in cell proliferation and increased apoptosis. In summary, these results suggest that, GCN2 appears to be important for MDA-MB-231 cell survival under unstressed conditions, and that GCN2 loss sensitises the cells to amino acid starvation, leading to a greater reduction in cell
viability.