Authors NA and JC contributed equally to this study and their names have been listed in alphabetical order of their last name.

Introduction: The purpose of the study is to examine the crosstalk between HER2/EGFR and Wnt signaling in HER2-positive (HER2+) breast cancer cells. Human epidermal growth factor receptors (HER) constitute a family of four transmembrane receptors (HER1-HER4). Ligand binding to HER1 (EGFR), HER3, and HER4 results in heterodimerization with other HER family members, including HER2. HER2+ breast cancer is characterized by an amplification of the HER2 gene, resulting in an increase in HER2 protein presence on the surface of cells, magnification of downstream intracellular signaling, and enhanced responsiveness to ligand stimulation (e.g., EGF). Approximately 20-30% of breast cancers have HER2 amplifications. The Wnt pathway is highly conserved in mammals and overexpression of some Wnt family members results in cancer. Wnts are ligands that bind to Frizzled/LRP receptors to initiate downstream signaling that results in the stabilization and nuclear translocation of β-catenin. Once in the nucleus, β-catenin associates with activators such as TCF/LEF, SMADs, ATF2, and KLF4 to promote the transcription of many target genes. EGFR and Wnt crosstalk has been observed in various cell types following EGF treatment. As an example, EGF treatment of human epidermoid carcinoma cells results in β-catenin nuclear translocation and activation of TCF/LEF dependent reporters. Mitogen-Activated Protein Kinases (MAPKs), which are activated following EGF stimulation, have been demonstrated to inhibit GSK3β, a negative regulator of β-catenin. However, no genome-wide analysis has been conducted to determine what genes are regulated by β-catenin following EGF treatment in HER2+ breast cancer cells. We sought to determine the modulation of gene expression following the stimulation of HER2+ breast cancer cells with EGF and to investigate the mechanisms that underlie the changes observed in gene expression. Our studies have revealed exciting and novel findings that elucidate the effects of EGFR signaling on the epigenetic landscape. Specifically, we have identified putative β-catenin targets that become activated following EGFR stimulation. We hypothesize that EGFR signaling promotes the activation of specific β-catenin genes in order to alter cellular identity. Methods: RNA-seq and ChIP-seq for H3K18ac and H3K27ac was conducted following an EGF treatment time course in SKBR3 cells. The levels of several proteins of interest were determined by Western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by Western blot analysis. Results: RNA-seq analysis following an EGF treatment time course revealed that approximately 2,200 genes are either upregulated or downregulated compared to untreated cells. Moreover, the expression profiles clearly demonstrated waves of transcription. Next, we determined the status of H3K18ac and H3K27ac using ChIP-seq following an EGF time course. We found that H3K18ac and H3K27ac increased globally within 1h post-EGF treatment compared to untreated cells. We conducted a motif discovery search for transcription factor binding sites contained from -1000bp to +200bp for all activated genes and determined that each wave of transcription had some unique putative regulators. As expected, the genes activated at 1h and 2h post-EGF treatment contained c-Jun and JunD binding sites. Surprisingly, TCF3, TCF5, and LEF1 motifs were enriched in some genes that peaked in expression at 6h, 16h, and 24h post EGF treatment. Lastly, we biochemically fractionated the cellular compartments and detected an increase in chromatin associated β-catenin following EGF treatment, suggesting a crosstalk between EGFR and Wnt signaling components. We plan to determine the genome-wide localization of β-catenin following EGF treatment. Conclusions: Our data suggest that a crosstalk between EGFR and Wnt signaling components may regulate β-catenin target genes and lead HER2+ cells' resistance to therapeutic treatment. Citation Format: Miguel Nava, Nwamaka Amobi, Nathan Zemke, Arnold Berk, Robin Farias-Eisner, Jay Vadgama, Yanyuan Wu. EGFR signaling mediated modulation of transcription and probable crosstalk with components of Wnt signaling [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B68.

Introduction: The HER2-positive breast cancer is characterized by an amplification of the HER2 gene, resulting in an increase in HER2 presence on the surface of cells and magnification of downstream intracellular signaling. Treatment of HER2+ breast cancer with trastuzumab decreases tumor volume, reduces metastasis and increases overall survival. Unfortunately, HER2-positive breast cancers develop resistance to trastuzumab. The mechanism of resistance may involve the activation of compensatory networks of different cell signaling pathways. The purpose of the study is to examine the crosstalk between HER2/EGFR and Wnt signaling in HER2-positive breast cancer cells. Methods: In order to address this, a HER2-positive breast cancer cell line (SKBR3) was used and induced with EGF. The phosphorylation of p38 and ERK1/2 was determined by Western blot analysis. The cellular localization of proteins of interest was examined using biochemically fractionated lysates followed by western blot analysis. Results: Our data demonstrated an early and transient peak of phospho-p38 at 30min post EGF treatment. Interestingly, both phospho-p38 and phospo-ERK1/2 were detected in the nuclear compartment at this time. β-catenin, the major effector molecule in Wnt signaling, was stabilized 30min post EGF treatment. β-catenin protein levels remained higher in EGF treated cells than untreated cells up to 4h post EGF treatment. It is likely that a stabilized β-catenin following EGF treatment potently modulates a subset of Wnt targets. We are assessing this possibility using chromatin immunoprecipitation based experiments for H3K18ac, H3K27ac, pol2 and β-catenin. Taken together, our data suggests that EGFR signaling promotes Wnt signaling in HER2-positive breast cancer cells. We hypothesize that this promotes cancer stem cell niche maintenance and an EMT phenotype. Citation Format: Miguel Nava, Nwamaka A. Amobi, Yanyuan Wu, Robin Farias-Eisner, Jay Vadgama. Cross talk between mitogen-activated protein kinase signaling cascades and Wnt/β-catenin in HER2+ overexpressing breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1333. doi:10.1158/1538-7445.AM2017-1333