Inducing HER2 endocytosis and downregulation via enhanced antibody-receptor clustering as a therapeutic strategy in breast cancer


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Jennifer Wymant1,Paul Moody1,Edward Sayers1,Oliver Castell1,William Jaimeson1,Paola Borri1,Peter Watson1,Arwyn Jones1
1Cardiff University

Abstract

Background

HER2 is over-expressed in 15-25% of breast cancers, driving development and progression of aggressive disease. Prognosis of HER2+ breast cancer is poor and the receptor is a priority therapeutic target. HER2 is resistant to endocytosis, which impedes targeting efforts with antibody drug conjugates (ADCs), e.g. trastuzumab-emtansine, where therapeutic function and lysosomal delivery are inextricably linked. Our research focuses on our recently published findings that HER2 endocytosis and downregulation can be enhanced in HER2+ breast cancer cells by an antibody-receptor clustering (ARC) strategy (Moody et al. 2015; Ogris and Sami 2015).

Method

Induction of ARC for HER2 was studied in SKBR3 and BT474 breast cancer cell lines and HER2-EGFP transfected HeLa cells. For downstream analysis: Western blotting was performed to examine changes in HER2 levels, activation and downstream signalling; live and fixed cell confocal microscopy was used to visualise endocytic trafficking; CellTitre Blue assay was used to measure cell viability and Total Internal Reflection Microscopy (TIRF) to examine ARC at the single-molecule level.

Results

We examined in breast cancer cell lines the effects of our ARC technique on HER2 levels at time points up to 48hr, finding that after an initial reduction in HER2, receptor levels recover. Cell viability studies revealed that after 72hr the viability of breast cancer cells is unaffected by ARC. Examination of HER2 activation and downstream signalling revealed ARC specific changes. Transfection of an HER2-EGFP construct into (HER2-) HeLa cells enabled surface binding of trastuzumab to HER2 overexpressing cells. Stimulation of ARC in this model resulted in extensive antibody internalisation compared with antibody alone. TIRF enabled visualisation of antibody-receptor dynamics at the plasma membrane.

Conclusion

Our data suggest that induction of ARC represents an effective approach for enhanced delivery of ADCs in HER2+ breast cancer and this strategy may extend to other receptor targets in a wide range of cancers.

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