title: Growth and molecular interactions of the anti-EGFR antibody cetuximab and the DNA cross-linking agent cisplatin in gefitinib-resistant MDA-MB-468 cells: new prospects in the treatment of triple-negative/basal-like breast cancer.
abstract: Three prominent hallmarks of triple-negative/basal-like breast carcinomas, a subtype of breast cancer gene phenotype associated with poor relapse-free and overall survival, are overexpression of the epidermal growth factor receptor (EGFR), hyperactivation of the MEK/ERK transduction pathway and high sensitivity to DNA-damaging agents. The cytotoxic interaction between EGFR inhibitors (monoclonal antibodies such as cetuximab and small molecule tyrosine kinase inhibitors such as gefitinib) and DNA cross-linking agents (e.g. platinum derivatives) might represent a promising combination for the treatment of triple-negative/basal-like breast tumors that are dependent upon EGFR/MEK/ERK signaling. We evaluated the growth and molecular interactions of the anti-EGFR antibody cetuximab (erbitux) and the DNA cross-linking agent cisplatin (cis-diammedichloroplatinum; CDDP) in the gefitinib-resistant MDA-MB-468 breast cancer cell line, an in vitro model system that shows many of the recurrent basal-like molecular abnormalities including ER-PR-HER2-negative status, TP53 deficiency, EGFR overexpression, PTEN loss and constitutive activation of the MEK/ERK pathway. Unlike other basal-like breast cancer models, MDA-MB-468 cells do not carry mutations of the key DNA repair gene BRCA1. Concurrent treatment with sub-optimal doses of cetuximab significantly enhanced CDDP-induced apoptotic cell death. However, an isobologram-based mathematical assessment of the nature of the interaction revealed a loss of synergism when employing a high-dose of cetuximab. Since BRCA1 depletion has been found to decrease DNA damage repair and cell survival in MDA-MB-468 cells when treated with DNA-damaging drugs, we employed ELISA-based quantitative analyses to measure BRCA1 protein levels in CDDP+/- cetuximab-treated cells. Cetuximab as single agent was as efficient as CDDP at increasing BRCA1 protein expression. Interestingly, cetuximab co-exposure significantly antagonized the ability of CDDP to up-regulate BRCA1 expression. Low-scale phosphor-proteomic approaches [i.e. phospho-receptor tyrosine kinase (RTK) and phospho-mitogen-activated protein kinases (MAPKs) Array Proteome Profiler capable of simultaneously identifying the relative levels of phosphorylation of 42 different RTKs and 23 different MAPKs and other serine/threonine kinases, respectively] revealed the ability of Cetuximab, as single agent, to paradoxically induce hyper-phosphorylation of EGFR while concomitantly deactivating p42/44 (ERK1/ERK2) MAPK. Unexpectedly, ELISA-based quantitative analyses of EGFR protein content demonstrated that simultaneous exposure to cetuximab and optimal doses of CDDP completely depleted EGFR protein in MDA-MB-468 cells. Although these findings preclinically support, at least in part, ongoing clinical trials for 'triple-negative/basal-like' metastatic breast cancer patients who are receiving either cetuximab alone versus cetuximab plus carboplatin (http://www.clinicaltrials.gov/ct/show/NCT00232505), the unexpected ability of CDDP to promote a complete depletion of the cetuximab target EGFR further suggests that treatment schedules, cetuximab/CDDP doses and BRCA1 status should be carefully considered when combining anti-EGFR antibodies and platinum derivatives in triple-negative/basal-like breast carcinomas.
authors.affiliation: Catalan Institute of Oncology (ICO), Dr Josep Trueta University Hospital of Girona, E-17007 Girona, Catalonia, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Oliveras-Ferraros
authors.forename: Alejandro
authors.initials: A
authors.lastname: Vazquez-Martin
authors.forename: Eugeni
authors.initials: E
authors.lastname: López-Bonet
authors.forename: Begoña
authors.initials: B
authors.lastname: Martín-Castillo
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Barco
authors.forename: Joan
authors.initials: J
authors.lastname: Brunet
authors.forename: Javier A
authors.initials: JA
authors.lastname: Menendez
mesh_terms: D000911:Antibodies, Monoclonal / Q000008:administration & dosage; D061067:Antibodies, Monoclonal, Humanized; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D019313:BRCA1 Protein / Q000378:metabolism; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D000068818:Cetuximab; D002945:Cisplatin / Q000008:administration & dosage; D003432:Cross-Linking Reagents / Q000008:administration & dosage; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D000077156:Gefitinib; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D051059:PTEN Phosphohydrolase / Q000172:deficiency; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage; D011799:Quinazolines / Q000494:pharmacology*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000172:deficiency; D011960:Receptors, Estrogen / Q000172:deficiency; D011980:Receptors, Progesterone / Q000172:deficiency; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency; D015854:Up-Regulation
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D019313:BRCA1 Protein; D000068818:Cetuximab; D002945:Cisplatin; D003432:Cross-Linking Reagents; D066246:ErbB Receptors; D000077156:Gefitinib; D020929:Mitogen-Activated Protein Kinase Kinases; D051059:PTEN Phosphohydrolase; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D016159:Tumor Suppressor Protein p53; C492913:BRCA1 protein, human; C495901:TP53 protein, human; C512478:EGFR protein, human; C508053:ERBB2 protein, human; C494929:PTEN protein, human