Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced “RNA disruption” is, the extent to which it is associated with drug response or what the underlying mechanisms are. Ovarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor. All chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3’-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption. Supporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.

The ribosome in eukaryotic cells is a macromolecular complex composed of four ribonucleic acids and over 80 proteins. This organelle facilitates protein synthesis in cells, and its activity is strongly upregulated in human cancers. Immune cells, a variety of cellular stressors and numerous structurally and mechanistically distinct anti-cancer agents have been shown to induce ribosomal RNA degradation in tumour cells in vitro and in vivo—a phenomenon we termed “RNA disruption”. RNA disruption can be quantified in cultured cell lines and patient samples using the RNA disruption assay (RDA). Unlike well-known high-throughput anti-cancer drug sensitivity assays, RDA can distinguish between dying and arrested tumour cells, making it an attractive assay for anti-cancer drug discovery and development. Low tumour RNA disruption during neoadjuvant chemotherapy (as measured using RDA) is strongly associated with residual disease and reduced disease-free survival, making it a potentially valuable chemo-resistance assessment tool. High RNA disruption may also indicate chemo-responsiveness. RDA holds the prospect of being a useful tool to escalate or de-escalate neoadjuvant chemotherapy in cancer patients. Moreover, the assay’s ability to predict treatment outcomes during neoadjuvant chemotherapy may permit its use in adaptive clinical trials and in drug approval by regulatory agencies. This review provides insight into the cellular processes involved in chemotherapy-induced RNA disruption. It also describes the results of clinical studies on tumour RNA disruption in cancer patients and suggests possible approaches that could be considered for the utilization of RDAs in the clinical management of breast cancer patients undergoing current neoadjuvant chemotherapy regimens.