Abstract 4205: The role of non-muscle myosin IIA in HER2+ breast cancers

Abstract

Abstract HER2 is amplified in about 20% of breast cancers. HER3 plays a critical role in HER2 mediated tumorigenesis. It is now clear that inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using the irreversible pan HER inhibitor neratinib. We immunoaffinity-purified HER3 from HER2+ BT474 cells treated ± neratinib. Following immunoprecipitation using a HER3 antibody, binding partners were released under reducing conditions. Mass spectrometry experiments identified non-muscle myosin IIA (NMMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To confirm the presence of NMMIIA, we performed immunoprecipitation experiments in BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein nonmuscle myosin of class II, isoform A (NMMIIA). NMMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization To further investigate the role of NMMIIA, we used blebbistatin, a myosin IIa inhibitor, in growth assays using BT474 and MDA-MB-453 breast cancer cells in vitro. The combination of blebbistatin and neratinib significantly decreased the proliferation in both cell lines compared to neratinib or blebbistatin treatment alone. Additionally, we knocked out myosin IIa in BT474 cells using myosin IIa specific siRNA to examine the effect of myosin IIa silencing on HER3 and Myosin IIa protein levels in the presence or absence of neratinib treatment. Results indicated that in BT474 cells knockdown of NMMIIA resulted in both decreased HER3 and Myosin IIa protein expression and that neratinib treatment rescued this effect. We next examined long term overall survival of primary breast cancer patients who have high and low levels of MYH9 gene expression from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients who express low levels of MYH9. Currently, further experiments are ongoing to determine the mechanism of action of the Myosin IIA/HER3 complex, which could be an adaptive response to HER2 inhibitors in HER2+ breast cancers and to evaluate the role of myosin IIa on proliferation, migration, and invasion of HER2+ breast cancer cells. In conclusion, we have identified that neratinib treatment results in increased NMMIIA bound to HER3. Further studies to determine if NMMIIA could play a role in invasion of HER2+ breast cancer cells and/or resistance to HER2 inhibitors are ongoing. Citation Format: Samar M. Alanazi, Rosalin Mishra, Long Yuan, Hima Patel, Joan Garrett. The role of non-muscle myosin IIA in HER2+ breast cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4205.