Abstract P5-10-05: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. 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 (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in HER2+ BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes the protein NMIIA. NMIIA localizes to actin stress fibers and has been implicated in regulation of cell contractility and stress fiber organization.To test if there is an interaction between HER3 and NMIIA, we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using an NMIIA antibody. The products were analyzed using immunoblots. The results indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours.We next examined long term overall survival of primary breast cancer patients who have high and low levels of gene expression for MYH9 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.We next sought to examine overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in HER2+ breast cancer whole cell lysates. We observed that mRNA and protein levels of both HER3 and MYH9 increased upon HER2 inhibition with 24 hours of neratinib treatment. To further investigate the role of NMIIA, we knocked out NMIIA in BT474 and MDA-MB-453 using inducible lentiviral shRNA targeting MYH9. This system utilizes the Tet-On 3G induction system and is a highly controlled system that consists of an inducible RNA polymerase II promoter. In the presence of doxycycline, the TRE3G promoter is bound and activated by the constitutively expressed Tet-On 3G trans-activator protein. Non-targeting shRNA was used as control and cells were selected in 1-2 ug/ml puromycin. Western blot analysis demonstrated that cells infected with lentiviral shRNA targeting MYH9 treated with doxycycline had reduced protein expression of NMIIA. BT474 and MDA-MB-453 transduced with shMYH9 were used to perform growth, migration, and invasion assays. The results indicated that MYH9 knockdown + neratinib treatment suppresses BT474 and MDA-MB-453 growth, migration, and invasion. Currently, in vivo xenograft experiments examining BT474 and MDA-MB-453 tumor growth ±NMIIA shRNA in the presence or absence of neratinib treatment are underway. In conclusion, we have identified that NMIIA mRNA and protein levels are increased in the presence of neratinib treatment. Our data reveals that HER2 inhibition increases NMIIA to promote HER2+ breast cancer growth. Citation Format: Samar M Alanazi, Rosalin Mishra, Hima Patel, Long Yuan, Mary Kate Kilroy, Joan T Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [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 P5-10-05.