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Exploring the role of chemotherapy-induced anastasis in triple-negative breast cancer

University of British Columbia

Triple-negative breast cancer (TNBC), is an aggressive and metastatic variant that lacks relevant treatment-targeted receptors. In addition, resistance to cytotoxic chemotherapeutic drugs is a common attribute of these cells, although little is known about how it is acquired. My hypothesis is that “Anastasis”, a reversal of end-stage apoptosis demarked by caspase-3 (Cas3) cleavage plays a role in drug resistance and cancer progression. Anastasis has been observed in many cell types, including cancer, however, its role in response to apoptotic stimuli is poorly understood, especially concerning its induction by clinically relevant chemotherapy agents. To test the above hypothesis, I used a GFP-tagged Cas3 reporter in order to measure DEVDase (pro-caspase) activity, indicating the level of Cas3 activation indirectly. This allowed for the selection of living, apoptotic cells using fluorescent activated cell sorting (FACS), whereby post-anastatic cells survived chemotherapy-induced apoptosis upon drug removal. While these cells do not appear phenotypically different, it was shown that they are more resistant to the original treatment, possess increased DNA damage, are more invasive, migratory, and metastatic, as well as more metabolically robust. Mechanistically, I determined that these cells produce a truncated caspase-3 isoform (Cas3s) that prevents apoptosome assembly early in the recovery stages of anastasis and long after its completion. Additionally, levels of native Cas3 remained unchanged during recovery and a decrease in Cas3 activation was observed upon treatment. Furthermore, a phenotypic characteristic of the post-anastatic cells revealed a significant up-regulation of epithelial-mesenchymal transition (EMT) and hypoxia stress markers. Inhibition of one of the up-regulated proteins, integrin-linked kinase (ILK) resulted in re-sensitization to chemotherapy treatment, a decrease in migration, as well as a dampening of the enhanced metabolic activity. These findings support a potential role for anastasis as a novel mechanism for resistance in TNBC and provides mechanistic insight into its role in tumor cell biology, that which has not been previously described.

Text

2019-09-09

Attribution-NoDerivatives 4.0 International

http://hdl.handle.net/2429/71641

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nd/4.0/