Abstract
BACKGROUND: Tumors are heterogeneous in nature, composed of different cell populations with various mutations and/or phenotypes. Using a single drug to encounter cancer progression is generally ineffective. To improve the treatment outcome, multiple drugs of distinctive mechanisms but complementary anticancer activities (combination therapy) are often used to enhance antitumor efficacy and minimize the risk of acquiring drug resistance. We report here the synergistic effects of salinomycin (a polyether antibiotic) and dasatinib (a Src kinase inhibitor). METHODS: Functionally, both drugs induce cell cycle arrest, intracellular reactive oxygen species (iROS) production, and apoptosis. We rationalized that an overlapping of the drug activities should offer an enhanced anticancer effect, either through vertical inhibition of the Src-STAT3 axis or horizontal suppression of multiple pathways. We determined the toxicity induced by the drug combination and studied the kinetics of iROS production by fluorescence imaging and flow cytometry. Using genomic and proteomic techniques, including RNA-sequencing (RNA-seq), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western Blot, we subsequently identified the responsible pathways that contributed to the synergistic effects of the drug combination. RESULTS: Compared to either drug alone, the drug combination showed enhanced potency against MDA-MB-468, MDA-MB-231, and MCF-7 human breast cancer (BC) cell lines and tumor spheroids. The drug combination induces both iROS generation and apoptosis in a time-dependent manner, following a 2-step kinetic profile. RNA-seq data revealed that the drug combination exhibited synergism through horizontal suppression of multiple pathways, possibly through a promotion of cell cycle arrest at the G1/S phase via the estrogen-mediated S-phase entry pathway, and partially via the BRCA1 and DNA damage response pathway. CONCLUSION: Transcriptomic analyses revealed for the first time, that the estrogen-mediated S-phase entry pathway partially contributed to the synergistic effect of the drug combination. More importantly, our studies led to the discoveries of new potential therapeutic targets, such as E2F2, as well as a novel drug-induced targeting of estrogen receptor β (ESR2) approach for triple-negative breast cancer treatment, currently lacking of targeted therapies.