Abstract 5271: Chemotherapy-induced anastasis as a novel mechanism of therapeutic resistance

Abstract

Abstract Resistance to chemotherapy and targeted cytotoxic therapy is an important contributor to tumor recurrence and metastatic progression, and remains a major hurdle in the treatment of cancer. Evidence suggests that cancer cells can recover from apoptosis subsequent to activation of caspase 3 (Cas3) through “anastasis”, a recently discovered process which enables recovery of cells from the brink of cell death following transient exposure to a potentially lethal dose of an apoptotic stimulus. Since chemotherapy induces apoptosis and is often delivered at maximum tolerated doses to patients, anastasis is a novel potential survival mechanism used by cancer cells to enable recovery after chemotherapy, thereby facilitating disease recurrence. Here, we investigated the extent to which triple negative breast cancer (TNBC) cells undergo anastasis in response to chemotherapy and identified potential molecular pathways and mechanisms of anastasis used by these cells. Analysis by time-lapse microscopy of human TNBC cells expressing a GFP-tagged Cas3 reporter and exposed to lethal doses of cisplatin or paclitaxel overnight demonstrated the presence of GFP+ cells showing phenotypic hallmarks of apoptosis that recovered with normal morphology. Furthermore, TNBC cells transduced with a novel genetically encoded biosensor that permanently labels cells that survive transient caspase activation, called mammalian CasExpress, were observed to be labeled with GFP following exposure to cisplatin, whereas mock-treated control cells were GFP negative. GFP+ TNBC cells enriched by FACS sorting showed significantly increased levels of resistance to chemotherapeutic agents (P < .05). Analysis by qPCR and Western blot demonstrated that TNBC cells recovering from exposure to cisplatin or paclitaxel significantly increased time-dependent expression of an alternatively spliced short isoform of Cas3 (Cas3s; P < 0.001), which acts an antagonist of full length Cas3 and confers resistance to apoptosis by breast cancer cells, while levels of full length Cas3 were not altered. Recovered cells also expressed significantly increased levels of genes (P < .05) and proteins associated with epithelial mesenchymal plasticity and hypoxic stress, including snail, slug, twist and HIF-1A, and showed increased levels of migration and invasion (P < .05). Finally, post-anastasis cells grown orthotopically as xenografts in vivo showed increased metastatic burden, compared to controls. Collectively, these data suggest that anastasis is a novel mechanism of chemoresistance used by TNBC cells and identify potential molecular pathways and vulnerabilities in anastatic cancer cells that could be targeted to develop new therapeutic strategies to overcome cancer recurrence and metastasis. Citation Format: Jennifer Nagel, Paul C. McDonald, Denise J. Montell, Shoukat Dedhar. Chemotherapy-induced anastasis as a novel mechanism of therapeutic resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5271.