Acquired resistance to trastuzumab, a rationally designed HER-2 targeting antibody, remains a major hurdle in management of HER-2 positive breast cancer (HER-2+ BC) patients.
More...Acquired resistance to trastuzumab, a rationally designed HER-2 targeting antibody, remains a major hurdle in management of HER-2 positive breast cancer (HER-2+ BC) patients. Potential resistance mechanisms are numerous, derived primarily from studies where HER-2 positive cell lines are chronically exposed to trastuzumab. Recent evidence suggests a role for epithelial-mesenchymal transition (EMT) in trastuzumab resistance, but a definitive link between the two has been difficult to establish because relevant model systems are lacking. When sub-populations of trastuzumab sensitive SKBR-3 cells were isolated using cloning rings, an (EMT) occurred spontaneously in several (3/8) clones. SKBR-3 EMT-clones featured increased spindle morphology, expressed N-glycosylated β1-integrin, and decreased HER-2, all characteristics shared by JIMT-1, a cell lines with intrinsic resistance to trastuzumab. SKBR-3 EMT-clones were characterized by gene expression profiling and mammosphere formation. The N-glycosylated isoform of β-itnegrin was targeted with β-integrin inhibiting antibody, AIIB2. Transcriptional profiling revealed that SKBR-3 EMT-clones underwent a shift from a luminal molecular subtype to a more aggressive mesenchymal/ basal phenotype. Isolating clones from SKBR-3 cells with enforced expression of a β-integrin isoform lacking extensive N-glycosylation failed to increase the likelihood for spontaneous EMT in SKBR-3. However, specific inhibition of the heavily N-glycosylated variant of β1-integrin expressed by SKBR-3 EMT-clones restored epithelial morphology and impaired mammosphere formation. Furthermore, when SKBR-3 EMT-clones were treated with relevant doses of trastuzumab and lapatinib, they showed “spontaneous” resistance. In this study we describe a model of spontaneous EMT following clonal selection in HER-2+ cell line, SKBR-3. Using this model we establish the first direct link between EMT and resistance to HER-2 targeted therapies. We also identify the N-glycosylated isoform of β-integrin as a potential biomarker and target in HER-2+ BC refractory to HER-2 targeted therapies.
Overall design: RNA was isolated from 10 breast cancer cell lines in triplicate. Pairwise gene expression differences were compared between each of the SKBR-3 cell lines (SKBR-3/ b1, SK-EV-C4, and SK-B1-C1). Features selected had at least a 2X difference in at least one comparison, with a one-way ANOVA corrected p-value of >0.05. In addition, expression profiles from SKBR-3/ EV and SK-B1-C1 cell were compared and a differential gene list was generated to include genes that differed by at least 1.5X, with a t-test p-value of >0.02 (unpaired, Bonferroni corrected). A total of 1940 entities met these criteria and the expression of these genes was investigated in all breast cancer cell lines.
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