RNA disruption is a widespread phenomenon that is associated with cell death in tumor cells

Abstract

A recent clinical trial found that chemotherapy-induced tumor rRNA degradation (RNA disruption) was associated with both pathological complete response (complete tumor destruction) and extended disease-free survival (DFS) in breast cancer patients treated with neoadjuvant chemotherapy. The RNA Disruption Assay (RDA) is a useful tool to quantify the magnitude of RNA disruption occurring within cells or tumors, expressed as the RNA Disruption Index (RDI), and is currently the subject of an international clinical trial. The purpose of this study was to investigate the phenomenon of RNA disruption in response to both chemotherapy agents and cellular stressors in the ovarian carcinoma A2780 cell line and the breast adenocarcinoma MDA-MB-231 cell line. Also, we assessed the relationship between RNA disruption and both cell death and RNase L expression using multiple cell viability assays and CRISPR-Cas9-mediated RNase L knockout clones, respectively. RNA disruption was induced in response to multiple chemotherapy agents and several cellular stressors, including oxidative stress, ER stress, protein translation inhibition and starvation. However, the induction and magnitude of RNA disruption was found to be both dose- and stressor-specific, and dependent on the cell line studied. Mycoplasma contaminations of host cell lines were also found to potentiate a markedly different RNA disruption banding pattern in response to chemotherapy. RNA disruption was also found to be associated with cell death, as significant increases in RDI values were observed for chemotherapy doses that induce partial or complete cell death, as measured by cell counting, and both cellular recovery and flow cytometry DNA content analysis. Finally, knockout of RNase L expression in the A2780 cell line did not affect RNA disruption, demonstrating that RNase L in not involved in the RNA disruption mechanism(s). Overall, these findings support the novel role of RDA as a tool for quantifying tumor cell death in vitro and in vivo.