Abstract 2308: MUC1 modulation of E-cadherin through miR-200c and ZEB1

Abstract

Abstract MUC1, a cell-surface glycoprotein implicated in tumorigenesis and metastasis, has a cytoplasmic tail (MUC1.CT) that is phosphorylated by different receptor tyrosine kinases and conducts these signals by binding and altering the function or stability of transcription factors, thereby influencing gene transcription. MicroRNAs (miRNAs), short RNAs (19-22 nucleotides), regulate gene expression at the transcriptional level and are differentially regulated in cancer and metastasis. We discovered that MUC1.CT influences expression of miRNAs that contribute to metastasis, including miR-200c. MiR-200c is a known regulator of epithelial-to-mesenchymal transition (EMT) through regulation of ZEB1, a transcriptional repressor of E-cadherin. In addition, ZEB1 is a repressor of miR-200c in a feedback loop. Overexpression of MUC1 results in a dramatic decrease in miR-200c levels, resulting in increased levels of ZEB1. Chromatin immunoprecipitation (ChIP) was used to demonstrate that MUC1.CT occupies the ZEB1 binding motif upstream of the miR-200c start site. In addition, we found that ZEB1 occupancy of the miR-200c promoter region was enhanced 3.5-fold in MUC1 overexpressing cells, indicating that MUC1.CT may influence the expression and stability of ZEB1 at the miR-200c promoter. Co-immunoprecipitation experiments determined that MUC1.CT directly interacts with ZEB1. We evaluated levels of proteins affected by the miR-200c pathway in MUC1 overexpressing cells and observed an increase in ZEB1 levels and a decrease in E-cadherin levels, consistent with induction of EMT. We also confirmed an increase of ZEB1 occupancy at the E-cadherin promoter in MUC1 overexpressing cells, further confirming our hypothesis. In addition, MUC1.CT is differentially phosphorylated by several receptor tyrosine kinases. Antibodies against different phospho-isoforms were used with ChIP to investigate the signaling cascades that influence localization of the MUC1.CT to the miR-200c promoter. The phospho-YEKV form of MUC1.CT, which is representative of signaling through epidermal growth factor/receptor (EGF/EGFR), was the only phospho-MUC1.CT detected at the ZEB1 binding motif of the miR-200c promoter. EGF signaling is linked to a loss of E-cadherin, and our work is the first to link EGF signaling through the MUC1.CT as a direct regulator of miR-200c expression. These data portray a novel pathway of EMT modulation. EGF signaling via MUC1 regulates levels of miR-200c, altering regulation E-cadherin. Grant support was provided by grants from the NCI (Training Grant CA09476, R01CA57362, U01 CA111294) and student assistantships from UNMC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2308. doi:1538-7445.AM2012-2308