Cell type dependent endocytic internalization of ErbB2 with an artificial peptide ligand that binds to ErbB2

Abstract

ErbB2, which is a member of the epidermal growth factor (erbB) receptor family, is frequently overexpressed in breast and ovarian cancers. Antibody and small molecule anti-tyrosine kinase inhibitors have been developed for targeted therapies for cancers overexpressing erbB2. Internalization and downregulation of erbB2, which is induced by a ligand, may be important for efficacious therapeutic effects. However, ligand-dependent erbB2 internalization has not been well characterized. Here we investigated the internalization of erbB2 in SKBr3 and SKOv3 cells, both overexpressing erbB2, using an EC-1 peptide fused to eGFP (EC-eGFP), which specifically binds to erbB2. ErbB2 was internalized in SKOv3 cells when the cells were treated with EC-eGFP. The accumulation of endosomal erbB2 was EC-eGFP dependent, which colocalized with transferrin implying endocytosis via clathrin-coated pits. In contrast, internalization of erbB2 was not observed in SKBr3 cells. As a result, two different mechanisms, which are cell type dependent for the internalization of erbB2, are proposed.

Micro-SPECT/CT with 111In-DTPA-Pertuzumab Sensitively Detects Trastuzumab-Mediated HER2 Downregulation and Tumor Response in Athymic Mice Bearing MDA-MB-361 Human Breast Cancer Xenografts

“…It was not possible to evaluate the effect of treatment with trastuzumab in vivo on the uptake of 111 In-DTPApertuzumab in SKBr-3 cells, because these cells are poorly tumorigenic in athymic mice. The differences between MDA-MB-361 and SKBr-3 cells in HER2 downregulation caused by trastuzumab are consistent with earlier reports that HER2 on SKBr-3 cells are internalization-impaired (17,18). Hashizume et al (17) proposed that coexistent HER3 on SKBr-3 cells may impair HER2 internalization; however, in our study, radioligand binding assays with 111 In-DTPApertuzumab and flow cytometry and immunofluorescence confocal microscopy demonstrated that HER2 on MDA-MB-361 cells was downregulated by trastuzumab, despite the fact that these cells have HER3 expression similar to that of SKBr-3 cells (19).…”

“…The differences between MDA-MB-361 and SKBr-3 cells in HER2 downregulation caused by trastuzumab are consistent with earlier reports that HER2 on SKBr-3 cells are internalization-impaired (17,18). Hashizume et al (17) proposed that coexistent HER3 on SKBr-3 cells may impair HER2 internalization; however, in our study, radioligand binding assays with 111 In-DTPApertuzumab and flow cytometry and immunofluorescence confocal microscopy demonstrated that HER2 on MDA-MB-361 cells was downregulated by trastuzumab, despite the fact that these cells have HER3 expression similar to that of SKBr-3 cells (19). Interestingly, although hIgG and rituximab caused no changes in HER2 expression levels in SKBr-3 and MDA-MB-361 cells in vitro, there was an apparent but statistically insignificant decrease in tumor uptake of 111 In- DTPA-pertuzumab mice treated with hIgG or rituximab in vivo.…”

A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells

“…Furthermore, ErbB2 bound to Trastuzumab was shown to undergo multiple cycles of ErbB2 recycling and to slowly enter the lysosomes, leading to ErbB2 down-regulation (10,46). Similarly, increased endocytosis and down-regulation of surface ErbB2 has been observed with artificial peptide and aptamer ligands (47,48).…”

Construction and Characterization of Novel, Recombinant Immunotoxins Targeting the Her2/neu Oncogene Product: In vitro and In vivo Studies

“…Immunofluorescence-based internalization studies were also done on SKOV3 and MCF7 cells (27). Cells treated with immunotoxins or rGel were subjected to immunofluorescent staining with anti-rGel antibody (FITC-conjugated secondary antibody).…”