EZH2 Blockade Reverses Doxorubicin Resistance by Disrupting Metabolic and DNA Repair Networks in Breast Cancer

Background: Doxorubicin (DOX) resistance remains a major obstacle to effective chemotherapy in breast cancer. However, the pharmacologically actionable regulators sustaining this resistant phenotype and its therapeutic vulnerabilities remain incompletely defined. Methods: DOX-resistant breast cancer cell models were established and treated with the EZH2 inhibitors tazemetostat or GSK126, alone or in combination with DOX. Cell viability, oxidative stress, DNA damage, and mitochondrial function were assessed in vitro. Transcriptomic profiling was performed to identify pathway alterations. A pH-responsive liposomal system for delivery of DOX and tazemetostat was developed and evaluated in vivo. Results: EZH2 was highly expressed in breast cancer and correlated with poor clinical outcomes. DOX treatment induced adaptive upregulation of EZH2 in both sensitive and resistant cells. Pharmacological inhibition of EZH2 markedly restored DOX sensitivity and exhibited strong synergistic cytotoxicity in resistant models. EZH2 blockade enhanced DOX-induced oxidative stress and DNA damage, with concomitant suppression of multiple DNA repair pathways and increased γH2AX accumulation. Transcriptomic and functional analyses revealed disrupted mitochondrial function and energy metabolism, characterized by loss of mitochondrial membrane potential and ATP depletion. In vivo, combined EZH2 inhibition and DOX significantly suppressed tumor growth, while pH-responsive liposomal delivery further enhanced antitumor efficacy and reduced systemic toxicity. Conclusion: EZH2 is a critical determinant of DOX resistance in breast cancer by sustaining DNA damage tolerance and metabolic homeostasis. Pharmacological targeting of EZH2 in combination with DOX represents a rational strategy to overcome chemoresistance in breast cancer.