Endocytosis and Sorting of ErbB2 and the Site of Action of Cancer Therapeutics Trastuzumab and Geldanamycin

Abstract

ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…The present effort aims to test a pretargeted approach in a clinically relevant model that features an internalizing target. 49 Not only has HER2 been exploited as a signaling target via the therapeutic antibody trastuzumab, 50 it has been further utilized as a drug delivery vector in the antibody-drug conjugate trastuzumab emtansine 51 and in clinical imaging of HER2-positive breast cancer. 40–43 It should be noted, however, that HER2 is a somewhat unusual internalizing oncology target in that it exhibits extremely high expression levels and that trastuzumab binding does not down-regulate surface HER2, but instead passively recycles after HER2 endocytosis.…”

“…40–43 It should be noted, however, that HER2 is a somewhat unusual internalizing oncology target in that it exhibits extremely high expression levels and that trastuzumab binding does not down-regulate surface HER2, but instead passively recycles after HER2 endocytosis. 50 …”

“…The short incubation times, 2 hours for antibodies and 30 minutes for tetrazine, were used to minimize antibody internalization and were well below the internalization half-life for HER2 in KPL-4 cells (~12 h). 50 Cells were washed three times with warm HBSS and harvested following incubation with trypsin for 15 min at 37°C. Aliquots of media used for antibody and tetrazine incubation were counted alongside harvested cells using a 2480 Wizard 2 automatic gamma counter (Perkin Elmer).…”

Lapatinib, a HER2 tyrosine kinase inhibitor, induces stabilization and accumulation of HER2 and potentiates trastuzumab-dependent cell cytotoxicity

“…The cells with higher HER2 expression showed significantly higher trastuzumab-mediated cytolysis (data not shown). The results are expressed as mean of three independent experiments , others have not (Austin et al, 2004;Hommelgaard et al, 2004;Longva et al, 2005). Interestingly, in some of the models that have not observed trastuzumabmediated receptor downregulation (Austin et al, 2004;Longva et al, 2005), there is also a lack of trastuzumabinduced HER2 phosphorylation.…”

“…The results are expressed as mean of three independent experiments , others have not (Austin et al, 2004;Hommelgaard et al, 2004;Longva et al, 2005). Interestingly, in some of the models that have not observed trastuzumabmediated receptor downregulation (Austin et al, 2004;Longva et al, 2005), there is also a lack of trastuzumabinduced HER2 phosphorylation. It is therefore plausible that kinase activation is a required step for receptor ubiquitination and degradation and that, as a consequence, lapatinib and other receptor TKIs prevent receptor downregulation.…”