Investigating the Role of APOBEC3 in Chemoresistance in Non-Small Cell Lung Cancer Cell Lines

Non-small cell lung cancer (NSCLC) is the deadliest form of cancer, characterised by both intertumour and intratumour heterogeneity, which are critical factors influencing disease progression and therapeutic resistance. This study aimed to explore tumour heterogeneity before and after oxaliplatin treatment to identify potential intrinsic and/or extrinsic resistance mechanisms in non-small lung cancer cells, using single cell-derived clonal sublines from both A549 cell line and its oxaliplatin-resistant subline A549rOXALI 5000. By profiling the growth kinetics and drug responses of these clonal sublines-particularly to platinum-based compounds-we identified both pre-existing and treatment-induced resistant clonal sublines. Notably, oxaliplatin-resistant clonal sublines exhibited cross-resistance to cisplatin but remained relatively vulnerable to carboplatin and maintained sensitivity to paclitaxel. These findings suggest that the mechanisms of resistance to carboplatin and paclitaxel may differ from those of other platinum-based drugs, such as cisplatin and oxaliplatin, as well as other drugs tested in this study. This highlights carboplatin and paclitaxel as potential alternative treatment options in cases where resistance to other agents arises.

Concurrently, growing evidence supports the involvement of APOBEC3 enzymes (A3s), including A3A, A3B, A3C, A3D, A3F, A3G, and A3H, in both tumorigenesis and chemotherapy resistance across various cancer types. To investigate the potential role of APOBEC3 enzymes in contributing to intracellular heterogeneity-reflected in variations in growth kinetics, and drug responses-as well as to examine the impact of treatment on APOBEC3 expression, we assessed APOBEC3 expression profiles in A549, A549rOXALI5000, and their single cell-derived clonal sublines using RT-qPCR.

Overall, the results revealed a strong positive correlation between APOBEC3 mRNA expression levels-excluding APOBEC3H-and drug responses, with the exception of paclitaxel and carboplatin, across both the parental (A549) and oxaliplatin-resistant cell lines A549rOXALI5000 and their respective clonal sublines. Furthermore, the entire APOBEC3 locus, except for A3H, was upregulated in A549rOXALI5000 and its clonal sublines compared to A549 cell line, suggesting that oxaliplatin treatment may directly modulate APOBEC3 expression and contribute to acquired drug resistance. The diverse APOBEC3 expression patterns observed in A549 and its clonal sublines-marked by distinct enzyme profiles in resistant phenotypes-also suggest a potential role in intrinsic resistance.

This study highlights the potential involvement of APOBEC3 enzymes beyond the commonly studied A3A, A3B, and A3H, which have previously been linked to NSCLC tumorigenesis and drug resistance. Notably, A3D, A3F, and A3G were upregulated in highly resistant A549 phenotypes compared to the parental A549 cell line, as well as in A549rOXALI5000 and its clonal sublines. Interestingly, in A549rOXALI5000 sublines where oxaliplatin resistance was not maintained for three months, the expression levels of these enzymes declined, returning to levels comparable to those observed in A549 and its clonal sublines. These findings underscore potential the role of oxaliplatin treatment in driving APOBEC3 expression and its contribution to the development of chemoresistance.

Overall, this study provides new insights into intracellular heterogeneity before and after oxaliplatin treatment and suggests that APOBEC3 enzymes may serve as potential targets for developing therapies to overcome chemotherapy resistance in NSCLC. While the findings are based on mRNA expression levels, future studies should investigate APOBEC3 protein expression, deaminase activity, and associated mutational signatures to further validate their role in drug resistance and tumour progression.