Abstract
DNA topoisomerase IIα (TOP2α/170; 170 kDa) and topoisomerase IIβ (TOP2β/180; 180 kDa) are targets for a number of anticancer drugs, whose clinical efficacy is attenuated by chemoresistance. Our laboratory selected for an etoposide-resistant K562 clonal subline designated K/VP.5. These cells exhibited decreased TOP2α/170 and TOP2β/180 expression. We previously demonstrated that a microRNA-9 (miR-9)-mediated posttranscriptional mechanism plays a role in drug resistance via reduced TOP2α/170 protein in K/VP.5 cells. Here, it is hypothesized that a similar miR-9 mechanism is responsible for decreased TOP2β/180 levels in K/VP.5 cells. Both miR-9-3p and miR-9-5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by microRNA (miRNA) sequencing and quantitative polymerase chain reaction. The 3'-untranslated region (3'-UTR) of TOP2β/180 contains miRNA recognition elements (MRE) for both miRNAs. Cotransfection of K562 cells with a luciferase reporter plasmid harboring TOP2β/180 3'-UTR plus miR-9-3p or miR-9-5p mimics resulted in statistically significant decreased luciferase expression. miR-9-3p and miR-9-5p MRE mutations prevented this decrease, validating direct interaction between these miRNAs and TOP2β/180 mRNA. Transfection of K562 cells with miR-9-3p/5p mimics led to decreased TOP2β protein levels without a change in TOP2β/180 mRNA and resulted in reduced TOP2β-specific XK469-induced DNA damage. Conversely, K/VP.5 cells transfected with miR-9-3p/5p inhibitors led to increased TOP2β/180 protein without a change in TOP2β/180 mRNA and resulted in enhancement of XK469-induced DNA damage. Taken together, these results strongly suggest that TOP2β/180 mRNA is translationally repressed by miR-9-3p/5p, that these miRNAs play a role in acquired resistance to etoposide, and that they are potential targets for circumvention of resistance to TOP2-targeted agents. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p play a role in acquired resistance to etoposide via decreased DNA topoisomerase IIβ 180 kDa protein levels. These findings contribute further information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. In addition, miR-9-3p and miR-9-5p overexpression in cancer chemoresistance may lead to future validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.