RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells

Abstract

We have previously shown that neoadjuvant chemotherapy can induce the degradation of tumour ribosomal RNA (rRNA) in patients with advanced breast cancer, a phenomenon we termed “RNA disruption”. Extensive tumour RNA disruption during chemotherapy was associated with a post-treatment pathological complete response and improved disease-free survival. The RNA disruption assay (RDA), which quantifies this phenomenon, is now being evaluated for its clinical utility in a large multinational clinical trial. However, it remains unclear if RNA disruption (i) is manifested across many tumour and non-tumour cell types, (ii) can occur in response to cell stress, and (iii) is associated with tumour cell death. In this study, we show that RNA disruption is induced by several mechanistically distinct chemotherapy agents and report that this phenomenon is observed in response to oxidative stress, endoplasmic reticulum (ER) stress, protein translation inhibition and nutrient/growth factor limitation. We further show that RNA disruption is dose- and time-dependent, and occurs in both tumourigenic and non-tumourigenic cell types. Northern blotting experiments suggest that the rRNA fragments generated during RNA disruption stem (at least in part) from the 28S rRNA. Moreover, we demonstrate that RNA disruption is reproducibly associated with three robust biomarkers of cell death: strongly reduced cell numbers, lost cell replicative capacity, and the generation of cells with a subG1 DNA content. Thus, our findings indicate that RNA disruption is a widespread phenomenon exhibited in mammalian cells under stress, and that high RNA disruption is associated with the onset of cell death.

Association of extensive RNA disruption with natural killer cell-mediated death of K562 chronic myelogenous leukemia cells

“…S6). These ndings are consistent with our previously published works showing that chemotherapeutic drug-dependent RNA disruption is associated with the onset of cell death 11,16 . Moreover, unlike the clonogenic, trypan blue exclusion and cell counting kit-8 assays, which respond to low, sub-lethal concentrations of drugs that typically only induce cell cycle arrest, the RDA responds solely to drug concentrations that cause cell destruction (as de ned by reduced cell numbers) 16 .…”

“…Our earlier work demonstrated that RNA disruption coincides with cytotoxicity and cell death in tumour cells treated with chemotherapeutic drugs 11 . We thus hypothesized that this relationship holds true in NK cell-treated K562 cells.…”

“…Pre-treatment of K562 cells with doxorubicin enhances NK cell-mediated RNA disruption in K562 cells. An earlier study looking at the effect of chemotherapeutic drugs on tumour cells found that doxorubicin triggers RNA disruption in K562 cells 11 . Given that immunotherapy drugs like pembrolizumab have been shown to improve outcome from chemotherapy in early and late stage triple negative breast cancer 48,49 and in metastatic non-small cell lung cancer 50 , and they are commonly prescribed along with chemotherapy, we next investigated whether we could improve NK cell-mediated RNA disruption by pretreating K562 cells with doxorubicin prior to addition of NK cells.…”

“…RNA disruption has been observed in vitro in a wide variety of tumour and non-tumour cell types, including breast epithelial cells, ovarian endometrioid cells, myeloid cells, melanocytes and vascular endothelial cells 11 . It is triggered by many structurally and mechanistically distinct chemotherapy agents as well as speci c cell stressors, including endoplasmic reticulum stress, oxidative stress and nutrient/growth factor limitation 11 . This phenomenon is associated with cell death in vitro, as measured by a reduction in cell numbers, loss of cell replicative capacity, and the generation of cells with a subG1 DNA content 11 .…”

“…It is triggered by many structurally and mechanistically distinct chemotherapy agents as well as speci c cell stressors, including endoplasmic reticulum stress, oxidative stress and nutrient/growth factor limitation 11 . This phenomenon is associated with cell death in vitro, as measured by a reduction in cell numbers, loss of cell replicative capacity, and the generation of cells with a subG1 DNA content 11 . Although RNA disruption was found to be associated with chemotherapy-induced tumour cell death in vitro 11,15,16 and clinical response to neoadjuvant chemotherapy in cancer patients in vivo 14,17,18 , its occurrence in response to an immunogenic challenge has yet to be explored.…”