title: Pathway-focused proteomic signatures in HER2-overexpressing breast cancer with a basal-like phenotype: new insights into de novo resistance to trastuzumab (Herceptin).
abstract: Pioneering clinical studies in de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab have suggested that HER2 gene-amplification can take place also in a basal-like molecular background to generate basal/HER2+ tumors intrinsically resistant to trastuzumab. Here, we first investigated the unique histogenesis of the basal/HER2+ phenotype in breast carcinomas. The presence of basal CK5/CK6 cytokeratin expression in HER2+ tumors revealed a significant overlap in the histological features of HER2+/CK5/6+ and basal-like breast carcinomas. Basal/HER2+ tumors were typically poorly differentiated, high-grade invasive ductal carcinomas with large geographic necrosis, pushing margins of invasion, syncytial arrangement of tumor cells, ribbon- or festoon-like architecture, squamous metaplasia, stromal lymphocytic infiltrates, high mitotic index and strong p53 positivity. Secondly, we performed low-scale proteomic approaches in JIMT-1 cells, a unique model of HER2-gene amplified trastuzumab-resistant breast carcinoma with a basal-like phenotype, to develop biomarker signatures that may differentiate trastuzumab-responsive from non-responsive tumors. When applying antibody-based array technology to the extracellular milieu of trastuzumab-refractory JIMT-1 and trastuzumab-sensitive SKBR3 cell cultures, JIMT-1 cells were found to secrete higher amounts of several growth factors including amphiregulin, EGF, IGFBP-6, PDGF-AA, neurotrophins, TGFbeta and VEGF. Semi-quantitative signaling node multi-target sandwich ELISAs revealed that JIMT-1 cells drastically overactivate RelA, the prosurvival subunit of NF-kappaB as compared to trastuzumab-sensitive luminal/HER2+ SKBR3 cells. When simultaneously assessing the activation status of 42 receptor tyrosine kinases (RTK) using a human phospho-RTK array, JIMT-1 cells were found to constitutively display hyperactivation of the insulin-like growth factor-I receptor (IGF-1R). High-content immunofluorescence imaging revealed that activated IGF-1R mainly localized at focal adhesion-like structures in JIMT-1 cells. In vitro wound healing assays suggested that this functional reorganization of the JIMT-1 cytoskeletal reorganization may account for an exacerbated trastuzumab-refractory 'migratogenic' phenotype. Forthcoming studies should validate the notion that identification of basal-like immunophenotypes and/or basal-like molecular signatures within HER2+ breast carcinomas may provide rapid means to define subgroups of breast cancer patients likely to display resistance to trastuzumab ab initio.
authors.affiliation: Catalan Institute of Oncology (ICO), 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: Begoña
authors.initials: B
authors.lastname: Martin-Castilló
authors.forename: Maria Carmen
authors.initials: MC
authors.lastname: Pérez-Martínez
authors.forename: Silvia
authors.initials: S
authors.lastname: Cufí
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Barco
authors.forename: Luis
authors.initials: L
authors.lastname: Bernado
authors.forename: Joan
authors.initials: J
authors.lastname: Brunet
authors.forename: Eugeni
authors.initials: E
authors.lastname: López-Bonet
authors.forename: Javíer A
authors.initials: JA
authors.lastname: Menendez
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000494:pharmacology*; D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D005260:Female; D005784:Gene Amplification; D006801:Humans; D016130:Immunophenotyping; D040901:Proteomics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D018719:Erb-b2 Receptor Tyrosine Kinases; D017526:Receptor, IGF Type 1; D000068878:Trastuzumab