Introduction • Inflammatory breast cancer (IBC) is a form of breast cancer that is characterized by rapid proliferation and metastasis. • One of the therapeutic targets identified is E-cadherin however much research hasn’t been done to understand the role of E- cadherin and its soluble fragments in IBC. • The epithelial to mesenchymal transition (EMT) is thought to be one of the most important steps in cancer metastasis [4]. • EMT is initiated by EMT inducing signals like HGF, EGF, PDGF which activates transcription factors such as Snail, Slug, ZEB1, Twist and so on. Some of the major EMT regulators are Snail, Zeb and Twist families. These transcriptional factors regulate EMT and cancer metastasis [4]. • The role of soluble E-cadherin, an 80-kDa extracellular proteolytic fragment of full-length E-cadherin, in EMT is unknown. Results (Cont’d) Conclusions • EMT pathways were enriched in the sEcad overexpression versus control group by GSEA hallmark analysis. • By PCR, higher gene expression of Vim, Snail, and Ecad were observed in sEcad-expressing cells. However, FN1 and Twist showed lower expression in sEcad overexpressing SUM149 cells compare with control. • By western blot, we observed that EMT markers such as Vim, Twist and sEcad were upregulated in sEcad overexpressing SUM149 and SUM190 cells, with the downregulation of FN1 In SUM149 cells. • Additional experiments are warranted to evaluate how sEcad regulates the expression and function of EMT inducing transcription factors. Acknowledgement I would like to acknowledge the Partnership in Cancer Science and Medicine (PCCSM) program and UT MD Anderson for supporting and funding this research. I would like to thank Dr.Debeb for his guidance and support in conducting this research. I would also like to thank Drs Xiao Ding Hu and Emilly Villodre for their guidance and mentorship throughout this program. References 1. Villodre, E.S., et al., NDRG1 Expression Is an Independent Prognostic Factor in Inflammatory Breast Cancer. Cancers (Basel), 2020. 12(12). 2. Kalluri, R. and R.A. Weinberg, The basics of epithelial-mesenchymal transition. J Clin Invest, 2009. 119(6): p. 1420-8. 3. Patil, P.U., et al., Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR. J Cell Sci, 2015. 128(23): p. 4366-79. 4. Zheng, H. and Y. Kang, Multilayer control of the EMT master regulators. Oncogene, 2014. 33(14): p. 1755-63. Results Title of the Poster Presentation Goes Here Authors of the Poster Presentation Goes Here The University of Texas MD Anderson Cancer Center Role of Soluble E-Cadherin in the Epithelial to Mesenchymal transition of Inflammatory Breast Cancer Fikir Tadesse, Xiao Ding Hu, , Emilly Schlee Villodre, Bisrat Debeb DVM, Ph.D Department of Breast Medical Oncology, Partnership for Careers in Cancer Science and Medicine, University of Texas MD Anderson Cancer Center, Houston, TX Abstract Breast cancer is one of the most common cancers in women, and the second leading cause of cancer death in women. The majority of deaths occur due to metastasis. Inflammatory breast cancer (IBC) is a highly aggressive and rare form of breast cancer with a high propensity to metastasize. IBC is also known to have worse prognosis than other types of breast cancer [1]. The epithelial to mesenchymal transition (EMT), where the epithelial features in carcinoma cells are converted to mesenchymal phenotype, increases cells motility and invasion thereby confer metastatic properties upon cancer cells [2]. Various markers and regulators associated with EMT have been reported in breast cancer patients [3]. The purpose of this study is to determine the role of soluble E-cadherin (sEcad), an 80-kDa extracellular proteolytic fragment of full- length E-cadherin, in regulating EMT. We found that sEcad overexpression affects the expression of some of the known EMT markers such as Vimentin, Slug, Twist and E-cadherin. Research Question Does Soluble E-Cadherin regulate Epithelial to Mesenchymal transition (EMT)? Figure 1. GSEA Hallmark analysis of RNA Seq in the SUM149 control and sEcad overexpression group shows the enrichment of EMT pathways in the sEcad overexpression group. (Hu, Xiao Ding) Figure 4. Western blot results show that the protein expression level of EMT markers such as Vim, Twist, Fib and Ecad were upregulated, while Slug was downregulated in sE-cad overexpressing SUM190 cells. Figure 2. Real-time PCR was performed to analyze expression of different genes. EMT regulators (NNMT, Serpine2, HDAC1, RHOA, PLOD1), epithelial marker (Ecad), and mesenchymal markers (Vim, FN1, Twist, Snail and Slug) were used to compare between Control and sEcad overexpressing SUM149 cells. Methods Real-Time PCR: • The Total RNA was extracted from SUM149 control and sEcad overexpression cells using the TRIzol reagent. • The cDNA were obtained by using the High Capacity cDNA Reverse Transcription Kit. • Real-time PCR primers were designed using PRIMER 3 software for epithelial markers (E-cadherin-Ecad), mesenchymal marker (Vimentin- Vim, Fibronectin (FN1)- Fib, Twist, Snail and Slug) and other EMT regulators (NNMT, Serpine2, HDAC1, RHOA,PLOD1). • qRT-PCR analysis was conducted using a SYBR Green Supermix kit. The fold change in expression was calculated using the 2-ΔΔCt method with the GAPDH mRNA as an internal control. Experiments for each sample were performed in triplicate. Western Blot • Immunoblotting was used to evaluate protein expression of Vim, Fib, Ecad, Twist and Slug in control and sEcad overexpressing SUM149 and SUM 190 cell lines. Figure 3. Western blot results show that the protein expression level of EMT markers such as Vim, Slug, Twist and sEcad were upregulated while Fib was downregulated in sEcad overexpressing SUM149 cell lines compared to Control. A B A B Ecad FN1 Vim Snail Slug Twist HDAC1 RHOA Serpine2 NNMT PLOD1 0.0 0.5 1.0 1.5 2.0 2 3 4 100 200 mRNA fold change con sEcad