Increasing intratumor C/EBP-β LIP and nitric oxide levels overcome resistance to doxorubicin in triple negative breast cancer

Abstract

Abstract Background Triple negative breast cancer (TNBC) easily develops resistance to the first-line drug doxorubicin, because of the high levels of the drug efflux transporter P-glycoprotein (Pgp) and the activation of pro-survival pathways dependent on endoplasmic reticulum (ER). Interfering with these mechanisms may overcome the resistance to doxorubicin, a still unmet need in TNBC. Methods We analyzed a panel of human and murine breast cancer cells for their resistance to doxorubicin, Pgp expression, lysosome and proteasome activity, nitrite production, ER-dependent cell death and immunogenic cell death parameters. We evaluated the efficacy of genetic (C/EBP-β LIP induction) and pharmacological strategies (lysosome and proteasome inhibitors), in restoring the ER-dependent and immunogenic-dependent cell death induced by doxorubicin, in vitro and in syngeneic mice bearing chemoresistant TNBC. The results were analyzed by one-way analysis of variance test. Results We found that TNBC cells characterized by high levels of Pgp and resistance to doxorubicin, had low induction of the ER-dependent pro-apoptotic factor C/EBP-β LIP upon doxorubicin treatment and high activities of lysosome and proteasome that constitutively destroyed LIP. The combination of chloroquine and bortezomib restored doxorubicin sensitivity by activating multiple and interconnected mechanisms. First, chloroquine and bortezomib prevented C/EBP-β LIP degradation and activated LIP-dependent CHOP/TRB3/caspase 3 axis in response to doxorubicin. Second, C/EBP-β LIP down-regulated Pgp and up-regulated calreticulin that triggered the dendritic cell (DC)-mediated phagocytosis of tumor cell, followed by the activation of anti-tumor CD8+T-lymphocytes upon doxorubicin treatment. Third, chloroquine and bortezomib increased the endogenous production of nitric oxide that further induced C/EBP-β LIP and inhibited Pgp activity, enhancing doxorubicin’s cytotoxicity. In orthotopic models of resistant TNBC, intratumor C/EBP-β LIP induction - achieved by a specific expression vector or by chloroquine and bortezomib - effectively reduced tumor growth and Pgp expression, increased intra-tumor apoptosis and anti-tumor immune-infiltrate, rescuing the efficacy of doxorubicin. Conclusions We suggest that preventing C/EBP-β LIP degradation by lysosome and proteasome inhibitors triggers multiple virtuous circuitries that restore ER-dependent apoptosis, down-regulate Pgp and re-activate the DC/CD8+T-lymphocytes response against TNBC. Lysosome and proteasome inhibitors associated with doxorubicin may overcome the resistance to the drug in TNBC.

Keywords

• Triple negative breast cancer
• Doxorubicin
• P-glycoprotein
• Endoplasmic reticulum stress
• CAAT/enhancer binding protein (C/EBP)-β
• Calreticulin