Triple Negative Breast Cancer (TNBC), an aggressive subtype of breast cancer, accounts for approximately 15% of cases. Radiation resistance remains a major therapeutic challenge, necessitating a deeper understanding of its underlying mechanism. The present study investigates the synergistic therapeutic potential of a FDA approved histone deacetylase inhibitor (HDACi), Romidepsin, an epigenetic modulator, in combination with the ABC drug efflux inhibitor, Verapamil, in radioresistant TNBC cells, aiming to elucidate their mechanism of action.

Radiation resistant TNBC cells (RR) were generated by exposing MDA-MB-468 cells to a clinical fractionated radiation dose of 40 Gray. These cells were characterized using colony-forming, migration scratch assay and FACS analysis. Cell viability assay was performed to generate dose response matrices and synergy score plot for RR cells in combination with romidepsin and verapamil. qRT-PCR and immunoblotting assays confirms the mechanistic insights into the radiation resistance and mode of drug action were analysed through apoptotic assays, FACS based assays.

RR cells exhibited increased radiation resistance, proliferation, and migration potential. Among the tested HDACis, (Romidepsin, SAHA, Belinostat), Romidepsin displayed a promising cytotoxic effect but was less effective in RR cells as compared to the parent radiation-sensitive (RS) cells. Gene expression analysis demonstrated upregulation of Endothelial to Mesenchymal Transition (EndMT) markers (N-Cad, ZEB1, TWIST, FIBRONECTIN), Cancer Stemness regulatory markers (OCT4, SOX2, NANOG) and ABC efflux transporters (ABCC1, ABCC2, ABCG2) in the RR cells. Hoechst efflux assays confirmed increased transporter activity in TNBC-RR cells. Romidepsin treatment alone did not significantly alter H3K9ac/K27ac acetylation levels in RR cells. However, Inhibition of efflux transporters by verapamil lowered romidepsin efflux, restored H3K9/K27 acetylation, and resensitized RR cells to Romidepsin. The combination of Romidepsin and Verapamil inhibited cell viability of TNBC-RR in a dose dependent synergistic manner. Additionally, an increase in the sub-G1 population upon combinatorial treatment indicated activation of cell death pathways in TNBC-RR cells. Ongoing in vivo studies aim to validate these findings in mouse models of radiation-resistant TNBC.

The synergistic effect of Romidepsin and Verapamil shows promising cytotoxic response in radiation-resistant TNBC cells, highlighting the potential of ABC transporter efflux inhibitors to overcome HDACi resistance. This study suggests a promising therapeutic strategy for improving treatment outcomes in radiation-resistant TNBC.