Abstract 5661: 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. It is known 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 neratinib. We immunoprecipitated HER3 using a HER3 antibody from BT474 cells treated ± neratinib. Fmass 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 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 a protein called non-muscle myosin of class II, isoform A (NMIIA). It localizes to actin stress fibers and has been implicated in many cell functions. To confirm the interaction between HER3 and NMIIA we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using a NMIIA antibody. Immunoblots indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours. We next examined overall survival of primary breast cancer patients who have high 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 with low levels of MYH9. Furthermore, we evaluated the overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in BT474 and MDA-MB-453 whole cell lysates. RT-qPCR and immunoblots showed increased HER3 and NM-IIA mRNA and protein levels upon neratinib treatment for 24 hours We examined the affect NMIIA loss has on HER3 signaling. Transduced MDA-MB-453 and BT474 cells with shMYH9 and doxycycline induction demonstrate a reduction in HER3 protein levels compared to cells transduced with a control sequence and doxycycline induction. We observed a concomitant reduction in P-HER3 (Y1289), downstream P-Akt (T308), and P-Erk1/2. In addition, NM-IIA knockdown suppresses cells growth, proliferation, migration, and invasion. In conclusion, there is a bidirectional relationship between NMIIA and HER signaling in HER2+ breast cancer cells. HER2 inhibition increases NMIIA and NMIIA promotes HER3 expression. Loss of NMIIA reduces HER3 protein and concomitant reductions in PI3K/Akt and MAPK signaling. Loss of NMIIA in combination with HER2 inhibition results in reduction in HER2+ breast cancer cell proliferation, growth on matrigel, migration, and invasion. Studies are ongoing to decipher the mechanisms for the bidirectional relationship between NMIIA and HER signaling. Citation Format: Samar M. Alanazi, Rosalin Mishra, Hima Patel, Mary K. Kilroy, Joan T. Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5661.