Abstract
Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths in New Zealand. Treatment with targeted therapies, which target specific driver mutations in NSCLC, are initially effective. However, relapse often occurs due to drug resistance, which can be preceded by a reversible drug tolerant state.
MicroRNAs are small non-coding RNAs which regulate gene expression through repression and are dysregulated in many cancer types. miR-21 is an oncomiR which is upregulated in NSCLC. It has been linked to drug tolerance and drug resistance, with higher expression in drug resistant cells. miR-21 has two functional strands, the 5 prime (miR-21-5p) and 3 prime (miR-21-3p) strands, each with potentially varying functions. Most of the research on miR-21 has been on miR-21-5p, and less is known about miR-21-3p. Furthermore, isoforms of miR-21, called isomiRs, also have activity that is not well understood. We aimed to understand the contribution of each strand to drug sensitivity and drug tolerance, to determine whether modulation of miR-21 expression may prevent the emergence of drug resistance.
Investigating the role of miR-21 in drug tolerance was done by transfecting EGFR-mutant PC9 and KRAS-mutant H358 NSCLC cells with miR-21 mimics, inhibitors, and controls. Transfected cells were treated with EGFR and KRAS inhibitors, and growth assays were carried out. We found that miR-21-3p reduced the sensitivity of PC9 cells to EGFR inhibitors, while miR-21-5p reduced the sensitivity of H358 cells to KRAS inhibitors. In PC9 cells, transfection with miR-21-3p inhibitors partially reversed the effect of miR-21-3p on EGFR inhibitor activity. In H358 cells, transfection with miR-21-5p inhibitors partially reversed the effect of miR-21-5p on KRAS inhibitor activity. The functions of miR-21 were tested using western blotting, where miR-21-5p was found to target the tumour suppressor PDCD4. RT-qPCR was used to measure the expression of miR-21 and miR-21 isomiRs in drug-tolerant cells, and our results suggest that miR-21 expression is increased in drug-tolerant cells.
Our results indicate that miR-21 has an important role in drug tolerance in NSCLC and may be an effective therapeutic target for those undergoing targeted therapy. In particular, our results highlight the effect of miR-21-3p on EGFR inhibitors, which warrants further investigation into how miR-21-3p inhibition may improve responses to EGFR inhibitors in NSCLC. Further studies are needed to better determine the role of each arm of miR-21, as well as its isoforms, in drug tolerance. This will aid investigation into therapeutic strategies which will prevent drug resistance from occurring.