Abstract
Lung adenocarcinoma frequently harbours specific oncogene driver mutations which can be treated with targeted therapies. The development of drug resistance commonly occurs following treatment however, leading to poor survival outcomes. Drug tolerance is a non-genetic and reversible adaptive state adopted by a small number of cells in response to treatment, where cells known as drug-tolerant persisters (DTPs) survive under treatment and can eventually lead to the development of genetic drug resistance. Non-coding RNAs, including the microRNAs miR-21, the miR-15/107 family, and miR-34a, as well as the long non-coding RNA MALAT1, are dysregulated in non-small cell lung cancer (NSCLC) and have been implicated in drug tolerance and drug resistance. How manipulation of these RNAs may affect therapy responses was investigated in in vitro EGFR-mutant and KRAS-mutant lung adenocarcinoma models.
Overexpression of miR-21 affected therapy response and potential mRNA targets in a cell line-specific manner. miR-21-3p affected EGFR-inhibitor response and increased the emergence of drug tolerance while miR-21-5p affected KRAS-inhibitor response. Exploratory investigation found miR-21-3p to decrease mismatch repair protein expression in PC9 (EGFR- mutant) cells, while miR-21-5p was found to increase AICDA expression in PC9 cells and increase APOBEC activity and expression in H358 (KRAS-mutant) cells. Expression of both strands of miR-21 was found to be high in PC9 DTPs, and low in H358 DTPs. Differences in targets, activity, and expression of miR-21 between cell lines may suggest signalling pathway regulation by miR-21 which is specific to oncogene driver mutations.
Drawing on preliminary evidence, the effect of miR-21 inhibition in combination with re- expression of tumour suppressor miRNAs on therapy responses and the development of drug resistance was investigated. miR-21-3p inhibition in combination with re-expression of the miR-15/107 family or re-expression of miR-34a effectively improved targeted therapy response and delayed the emergence of drug tolerance, as did re-expression of miR-15/107 in combination with MALAT1 knockdown.
Finally, the effect of fetal bovine serum in cell culture media on DTP characterisation, and whether DTP features are drug specific or characteristic of slow growing stress-adapted cells were investigated. How more physiologically relevant medium can affect DTPs was also investigated. Limited DTP marker changes in response to variable media conditions suggested that NSCLC and melanoma DTP models were robust, where most established DTP markers were primarily influenced by drug treatment rather than growth and nutrient factor supply in media. These experiments highlighted considerations for in vitro studies which are important due to how the surrounding environment shapes how DTPs emerge and adapt. Future in vivo studies are needed to better recapitulate patient residual disease to understand how best to target DTPs, and whether miRNA modulation in combination with targeted therapies has therapeutic potential.
In conclusion, miRNA manipulation using combinations of miRNA mimics and inhibitors of miRNAs and lncRNAs implicated in cell growth, drug tolerance, and drug resistance may be useful therapeutic tools to improve responses to targeted therapies in NSCLC. Establishing miRNA mechanisms, improving miRNA therapeutic delivery, and ensuring clinically relevant models remain important future directions for the study of miRNA manipulation to target DTPs and prevent drug resistance.