Why do most c-erbB-2/HER-2-positive breast cancer patients fail to respond to Herceptin?
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KeywordsEpidermal Growth Factor Receptor Breast Cancer Patient Human Epidermal Growth Factor Receptor Humanize Monoclonal Antibody Herceptin
The purpose of this study is to explore possible molecular and cellular mechanisms involved in the development of resistance to Herceptin in breast cancer patients.
Herceptin is a humanized monoclonal antibody targeted against the human epidermal growth factor receptor c-erbB-2 (HER-2) which is overexpressed in approximately 25–30% of invasive breast cancer. Herceptin recognizes an epitope on the extracellular domain of c-erbB-2 and blocks downstream signaling. Approximately 50% of patients respond to Herceptin therapy; however, the majority of these will demonstrate disease progression within 1 year of treatment initiation. Several molecular mechanisms contributing to Herceptin resistance have been proposed. This research aims to define the effects of Herceptin on subcellular c-erbB-2 receptor trafficking.
We have created a c-erbB-2 plasmid fused to Yellow Fluorescent Protein (c-erbB-2-YFP) and an epidermal growth factor receptor fused to Green Fluorescent Protein (EGFR-GFP). Both constructs were sequenced and the correct sequence obtained. Both constructs were shown to react with specific antibodies and to have the predicted molecular weight using western blotting.
Both EGFR-GFP and c-erbB-2-YFP plasmids were used to transiently transfect COS-7 cells. Time-course studies using low-light fluorescent microscopy revealed maximal membrane receptor expression between 18 and 24 hours after transfection. Herceptin immunoglobulin (Genentech Inc, South San Francisco, USA) was conjugated to Alexa Fluor 568 (Invitrogen Molecular Probes, Inc, USA) to allow visualization of the antibody. After 20 hours, c-erbB2-YFP transfected COS-7 cells were incubated with Alexa Fluor-labeled Herceptin for 2 hours. Serial fluorescent images were recorded over 12 hours allowing real-time visual localization of both the receptor and Herceptin.
These preliminary studies indicate that Herceptin induces receptor internalization. Further studies are planned whereby cells will be co-transfected with both c-erbB2-YFP and EGFR-GFP and exposed to an anti-EGFR antibody as well as Herceptin. Confocal microscopy will be utilized in mapping the fate of receptors and their antibodies. It may be that this dual targeting will exaggerate receptor internalization and degradation.
We demonstrate that both constructs can be expressed in mammalian cells and receptor trafficking can be observed using digital fluorescent microscopy. In addition, we have fluorescently labeled Herceptin and its ability to bind c-erbB-2 is retained. This study of receptor and antibody trafficking may lead to further knowledge of Herceptin's mechanism of action as well as that for drug resistance and the possible effects of the use of combined therapies.