Abstract 362: EGFR signaling activates S100 gene expression in HER2-positive breast cancer cells

Abstract

Abstract Introduction: Human Epidermal Growth Factor Receptor-2-positive (HER2+) breast cancer is one of the four major molecular types of breast cancer. HER2 is a classical receptor tyrosine kinase (RTK) and its kinase activity is stimulated by heterodimerization with other ligand bound HER family members, such as EGFR/HER1. Treatment for HER2+ breast cancer includes the use of Trastuzumab, a monoclonal antibody that binds to the HER2 extracellular domain and inhibits downstream signaling. Resistance to Trastuzumab involves several cellular and molecular alterations, including gain of function mutations in proteins that are downstream of HER2 activation. The purpose of this study is to examine EGFR signaling in HER2+ breast cancer cells to discover novel putative drug targets. Methods: HER2-positive breast cancer cell line (SKBR3) was used before and after EGF treatment. In order to determine whether EGFR signaling was activated, the levels of several proteins of interest were determined by western blot analysis. RNA-seq and ChIP-seq for H3K18ac and H3K27ac was conducted following an EGF time course in SKBR3 cells. Results: Treatment of SKBR3 cells with EGF resulted in a transient increase in pERK1/2 and pAKT, which returned to basal levels at 1h and 2h post-EGF treatment, respectively. This was surprising because pEGFR remained higher in treated cells compared to untreated cells throughout the 24h time course. Immediate early genes (IEGs) became activated in SKBR3 cells, as previously reported in other cell types, during the early phase of EGFR stimulation (<4h). Using mRNA-seq we determined expression patterns during a 24h period and found that SKBR3 cells treated with EGF resulted in the activation and repression by 2-fold or more of over 4000 transcripts. We examined the chromatin landscape following EGFR stimulation with chromatin immunoprecipitation for H3K18ac and H3K27ac followed by next generation sequencing (ChIP-seq). We determined that, regardless of transcript levels, H3K18ac and H3K27ac oscillated at all regulated genes and globally. A closer look at the genes that peaked in expression at 24h post-EGF treatment revealed that S100 genes steadily increased in expression. Moreover, the chromatin surrounding the locus that contains the majority of S100 genes increased in H3K18ac and H3K27ac 1h post-EGF treatment, suggesting that S100 genes are a direct target of EGFR/HER2 signaling. Summary: We hypothesize that S100 proteins, which act as Ca2+ sensors, could play a role in EGF induced tumor cell growth and metastasis, contribute to Herceptin resistance and are likely drug targets in HER2+ breast cancer. Citation Format: Miguel Nava, Nathan R. Zemke, Arnie Berk, Robin Farias-Eisner, Jay Vadgama, Yanyuan Wu. EGFR signaling activates S100 gene expression in HER2-positive breast cancer cells [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 362.