Abstract P4-01-12: Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models

Abstract

Abstract Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.