Abstract 1896: Patient-derived HER3 mutations transform ER+ and HER2+ breast cancer cells via MAPK pathway activation

Abstract

Abstract We sought to investigate the role of patient-derived HER3 mutations in HER2+ and ER+ breast cancer cells using ectopic expression of HER3 mutants. We aim to identify mechanism(s) by which HER3 mutants enhanced HER2-mediated or hormone-driven transformation and if HER3 mutations confer resistance to HER2 or ER+ inhibitors. A series of HER3 mutations (F94L, G284R, D297Y, D313H, K329T, T355I, L792V and E1261A) were introduced and stable cell lines were generated in MCF10AHER2 and ER+ MCF-7 and T47D cells using lentiviral transduction. HER3T355I mutant was transforming in ER+ cells. HER3T355I mutant had increased p-HER3 and p-ERK1/2 expression compared to controls expressing wild-type HER3. Receptor tyrosine kinase array results indicated that T47D and MCF-7 overexpressing HER3T355I had increased p-HER4 and p-HER1 expression, respectively. HER3T355I counteracted the effect of the ER inhibitor, 4-hydroxytamoxifen but not fulvestrant. Cells expressing HER3T355I were subjected to lapatinib in presence or absence of ER inhibitor fulvestrant and specific ERK1/2 inhibitor, SCH772984. The data indicated that induced transforming activity observed in HER3T355I is via HER4/MAPK pathway in T47D and HER1/MAPK signaling in MCF-7 cells. Immunoblotting data indicated that cyclin D1 and p-ERK1/2 expressions were altered in response to combined treatment of lapatinib with or without fulvestrant and SCH772984 indicating that cyclinD1 mediated signaling downstream of the MAPK pathway. HER3T355I and HER3WT demonstrated reduced cell proliferation and matrigel colony formation in presence of lapatinib and fulvestrant/SCH772984. We noted crosstalk between ERα and HER3 in T47D cells. Based on structural modelling, T355I likely disrupted the interactions between domain III and the hinge region pocket that stabilized the tethered conformation of HER3T355I, potentially making the hinge more flexible, shifting the equilibrium from the tethered to the untethered state. Laboratory studies indicated that several HER3 mutants acquired gain-of-function phenotype and were resistant to lapatinib and partially resistant to neratinib in MCF10AHER2 cells. These mutants increased HER2-HER3 hetero-dimerization. However, there was no significant alteration in AKT signalling in HER3 mutants. We are currently using a proteomics approach to identify HER3-interacting proteins, and determine how HER2 inhibition affects the repertoire of HER3 binding partners in HER2+ breast cancers. We will also mine for HER3 binding partners in ER+ breast cancers by using ReCLIP, a methodology in which reversible cross-linking precedes HER3 immunoprecipitation and mass spectrometry-based identification of HER3-interacting proteins. We aim to identify kinases (other than HER2) that phosphorylate HER3 upon therapeutic inhibition of HER2 and PI3K or anti-endocrine therapy. Citation Format: Rosalin Mishra, Samar Alanazi, Long Yuan, Thomas Solomon, Tarjani M. Thaker, Natalia Jura, Joan T. Garrett. Patient-derived HER3 mutations transform ER+ and HER2+ breast cancer cells via MAPK pathway activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1896.