Epidermal growth factor receptor inhibition potentiates chemotherapeutics-mediated sensitization of metastatic breast cancer stem cells.

BACKGROUND: Metastasis has been a cause of the poor prognosis and cancer relapse of triple-negative breast cancer (TNBC) patients. The metastatic nature of TNBC is contributed by the breast cancer stem cells (CSCs) which have been implicated in tumorigenesis. Higher expression of epidermal growth factor receptor (EGFR) in breast CSCs has been used as a molecular target for breast cancer therapeutics. Thus, it necessitates the design and generation of efficacious EGFR inhibitors to target the downstream signaling associated with the cellular proliferation and tumorigenesis of breast cancer. AIM: To generate efficacious EGFR inhibitors that can potentiate the chemotherapeutic-mediated mitigation of breast cancer tumorigenesis. METHODS AND RESULTS: We identified small molecule EGFR inhibitors using molecular docking studies. In-vitro screening of the compounds was undertaken to identify the cytotoxicity profile of the small-molecule EGFR inhibitors followed by evaluation of the non-cytotoxic compounds in modulating the doxorubicin-induced migration, in-vitro tumorigenesis potential, and their effect on the pro-apoptotic genes' and protein markers' expression in TNBC cells. Compound 1e potentiated the doxorubicin-mediated inhibitory effect on proliferation, migration, in-vitro tumorigenesis capacity, and induction of apoptosis in MDA-MB-231 cells, and in the sorted CD24+-breast cancer cells and CD24-/CD44+-breast CSC populations. Orthotopic xenotransplantation of the breast CSCs-induced tumors in C57BL/6J mice was significantly inhibited by the low dose of Doxorubicin in the presence of compound 1e as depicted by molecular and immunohistochemical analysis. CONCLUSION: Thus, the study suggests that EGFR inhibition-mediated sensitization of the aggressive and metastatic breast CSCs in TNBCs toward chemotherapeutics may reduce the relapse of the disease.

ZEB1 potentiates chemoresistance in breast cancer stem cells by evading apoptosis.

Chemoresistance renders a challenge to the clinics to treat breast cancer patients. Current treatment strategies are effective in mitigating tumor growth but remain largely ineffective against cancer-initiating cells or breast Cancer Stem Cells (CSCs). Epithelial-to-mesenchymal-transition (EMT) regulates breast CSC physiology. Zinc finger E-box binding homeobox 1 (ZEB1) is a key EMT-transcription factor that regulates breast CSC - differentiation and metastasis. However, its potential role in modulating tumor chemoresistance has not yet been fully understood. In-silico analysis revealed a higher ZEB1 expression in breast cancer patients that leads to decreased overall and relapse-free survival. We generated sorted breast CSC with stable ZEB1 overexpression (CD24-/CD44+GFP-ZEB1) and/or silencing (CD24-/CD44+ZEB1 shRNA) as well as breast cancer cells with stable ZEB1 overexpression (CD24+GFP-ZEB1) and/or silencing (CD24+ZEB1 shRNA). An increased colony-forming efficiency and doxorubicin accumulation correlated with decreased promoter activity and expression profile of ABCC1 drug-efflux ABC transporter in CD24-/CD44+GFP-ZEB1. Additionally, CD24-/CD44+GFP-ZEB1 demonstrated doxorubicin-induced higher anti-apoptotic and lower pro-apoptotic protein expressions in the mitochondrial and cytosolic fractions. Chemoresistant CD24-/CD44+GFP-ZEB1 cells depicted 1000-fold higher IC-50 values of doxorubicin and decreased activation of JNK-p38 stress kinase molecular signaling-dependent mammosphere forming efficiency to evade apoptosis. Thus, ZEB1 and its downstream effectors are plausible therapeutic targets for the mitigation of breast cancer chemoresistance in patients.

Cell density modulates chemoresistance in breast cancer cells through differential expression of ABC transporters.

BACKGROUND: Breast cancer patients undergoing chemotherapy encounter a significant challenge of chemoresistance because of drug efflux by ATP-binding cassette (ABC) transporters. Breast cancer cell density alters considerably throughout the early stages of primary and secondary tumor development. Although cell density in culture influences kinetics, the effects of varying cell densities on the chemoresistance of breast cancer cells remains largely unexplored. METHODS AND RESULTS: We observed chemotherapeutics-induced differential gene and protein expression of ABC transporters in luminal and basal breast cancer cells cultured at low and high seeding densities. Low-density cultures depicted a significant increase in the mRNA expression of ABC transporters-ABCG2, ABCG1, ABCC4, ABCA2, ABCA3, ABCC2, ABCC3, ABCC6, ABCC7, and ABCC9 as compared with high-density cultures. Next, cells at both low and high seeding densities when pre-treated with cyclosporine A (CsA), a pan-inhibitor of ABC transporters, resulted in increased sensitization to chemotherapeutics-doxorubicin and tamoxifen at markedly low IC50 concentrations suggesting the role of ABC transporters. Finally, markedly high doxorubicin-drug accumulation, significantly increased expression of N-cadherin, and a significant decrease in chemotherapeutics-induced in vitro tumorigenesis was observed in low-density seeded breast cancer cells when pre-treated with CsA suggesting ABC transporters inhibition-mediated increased efficacy of chemotherapeutics. CONCLUSION: These findings suggest that breast cancer cells grown at low seeding density imparts chemoresistance towards doxorubicin or tamoxifen by a differential increase in the expression of ABC transporters. Thus, a combinatorial treatment strategy including ABC transporter inhibitors and chemotherapeutics can be a way forward for overcoming the breast cancer chemoresistance.