Endophilin A2 promotes HER2 internalization and sensitivity to trastuzumab-based therapy in HER2-positive breast cancers

Tomas Baldassarre, Peter Truesdell, Andrew W. Craig

Human epidermal growth factor receptor-2 (HER2) is amplified and a clinical target in a subset of human breast cancers with high rates of metastasis. Targeted therapies involving the antibody trastuzumab and trastuzumab-emtansine (T-DM1) have greatly improved outcomes for HER2-positive (HER2+) breast cancer patients. However, resistance to these targeted therapies can develop and limit their efficacy. Here, we test the involvement of the endocytic adaptor protein endophilin A2 (Endo II) in HER2+ breast cancer models, and their responses to treatments with trastuzumab and T-DM1. Endo II expression in human breast tumors and lymph node metastases were analyzed by immunohistochemistry. Stable silencing of Endo II was achieved in HER2+ cancer cell lines (SK-BR-3 and HCC1954) to test Endo II effects on HER2 levels, localization and signaling, cell motility and tumor metastasis. The effects of Endo II silencing on the responses of HER2+ cancer cells to trastuzumab or T-DM1 treatments were tested using real-time cell motility and cytotoxicity assays. High Endo II protein expression was detected in HER2-positive tumors, and was linked to worse overall survival in node-positive HER2+ breast cancers at the mRNA level. Stable silencing of Endo II in HER2+ cell lines led to elevated levels of HER2 on the cell surface, impaired epidermal growth factor-induced HER2 internalization, and reduced signaling to downstream effector kinases Akt and Erk. Endo II silencing also led to decreased migration and invasion of HER2+ cancer cells in vitro, and impaired lung seeding following tail vein injection in mice. In addition, Endo II silencing also impaired HER2 internalization in response to Trastuzumab, and led to reduced cytotoxicity response in HER2+ cancer cells treated with T-DM1. Our study provides novel evidence of Endo II function in HER2+ cancer cell motility and trafficking of HER2 that relates to effective treatments with trastuzumab or T-DM1. Thus, differential expression of Endo II may relate to sensitivity or resistance to trastuzumab-based therapies for HER2+ cancers.