The anthracycline doxorubicin is a front line antineoplastic agent with activity against a wide range of solid and blood tumours. Its mode of action is to stabilise topoisomerase-II-DNA cleavable complexes, inducing double stranded DNA breaks and leading to cell death (1). In the presence of formaldehyde this mechanism can be subverted to preferentially form covalent drug-DNA adducts, a mechanism that exerts a more potent effect in killing cancer cells in culture (2).
To investigate the efficacy of doxorubicin-DNA adducts against an aggressive metastatic breast cancer model, highly metastatic 4T1.2 and non-metastatic 67NR cell lines were utilised. Single agent doxorubicin was used to induce topoisomerase II associated breaks (measured using the Comet assay) while combination with the formaldehyde-releasing prodrug AN-9 was used to induce DNA adducts (measured by covalent incorporation of 14C doxorubicin into DNA). Doxorubicin and AN-9 combinations reduced DNA breaks and promoted doxorubicin-DNA adduct formation in both non-metastatic and metastatic cells, consistent with a switch in mechanism of action of doxorubicin. Growth inhibition studies revealed that the metastatic cells were inherently resistant to doxorubicin (4-fold cf non-metastatic cells). The effects of the combination were markedly more pronounced on the metastatic cells where a strong synergistic growth inhibition was observed, indicating that a switch in mechanism of action overcame their inherent resistance to doxorubicin. To elucidate cellular changes in response to the combination, transcriptomics analysis was performed using 4T1.2 (murine) and MDA-231 (human) breast cancer cell lines. Cells were treated for 24 h with 1µM of Doxorubicin with and without 100µM AN-9 and libraries were prepared for RNA sequencing. KEGG pathways associated with Spliceosome, RNA transport and ribosome biogenesis were upregulated in combination treated cells relative to other treatments. Gastrin was highly upregulated in combination treated cells relative to other treatments in both cell lines (19 fold - MDA-231, 115 fold - 4T1.2) and this is being investigated further as a possible biomarker of drug response in vitro and in vivo.