An in-house peak-calling algorithm, BELT, was developed to map EpICD binding sites in the genome (21)

An in-house peak-calling algorithm, BELT, was developed to map EpICD binding sites in the genome (21). of 105 genes encoding functions for tight junction, adherent and cell migration. Furthermore, nanomechanical analysis by atomic force microscope (AFM) revealed increased softness and decreased adhesiveness of EGF-stimulated cancer cells, implicating acquisition of an epithelial-mesenchymal transition (EMT) phenotype. Thus, genome editing of could be associated with altering these nanomechanical properties towards a less aggressive phenotype. Using this integrative genomic-biophysical approach, we demonstrate for the first time an intricate relationship between EpCAM-regulated AZ876 transcription and altered biophysical properties of cells that promote EMT in advanced endometrial cancer. Introduction Epithelial cell adhesion molecule (EpCAM) is usually a cell-surface protein known to mediate cell-cell and cell-matrix interactions (1,2). The extracellular domain name AZ876 of EpCAM AZ876 (or EpEX) contains an N-terminal sequence, a thyroglobulin-like domain name, and a C-terminal domain name followed by a transmembrane domain name and an intracellular domain name (or EpICD) (3-5). The EpEX on the surface of one cell can bind to another EpEX on neighboring cells thereby holding these cells together (6). This EpCAM-mediated homophilic adhesion is usually further supported through inner interactions between EpICD and cytoplasmic fibers via -actinin that serves as an intracellular bridge to stabilize the entire adhesion unit (1,2,7). While EpCAM supports normal adhesion functions for epithelial cells, its transient down-regulation may promote epithelial-to-mesenchymal transition (EMT) for cancer cell migration and invasion (8). Also, circulating tumor cells SELL (CTCs) bound to seed metastases in cancers of epithelial origin display very diverse levels of EpCAM expression, possibly related to their stage of EMT and invasiveness (9). In endometrial cancer, malignant cells must undergo EMT to facilitate myometrial invasion (10). However, upregulated EpCAM is frequently observed in endometrial tumors and is known to promote invasion by preventing cell-cell adhesion (11). Conditional knockout of EpCAM in a murine model attenuates the motility and migration of epidermis-resident Langerhans cells, further suggesting the role of EpCAM as a negative regulator for cell adhesion (12). Therefore, these earlier studies indicate a paradoxical role of EpCAM for both cell adhesion and migration. On one hand, EpCAM mediates cell-cell contacts and thus prevents cell migration, but on the other hand the molecule can be switched to promote cell invasion. Recent studies reveal that regulated intra-membrane proteolysis (RIP) of EpCAM with -secretase results in shedding of the EpEX from the cell surface and release of EpICD into the cytoplasm (13). While the cleavage of EpEX may lead to a decrease in cell-cell AZ876 adhesion and thereby promote cell movement, the event alone is usually insufficient to explain multifaceted influences of EpCAM on advanced cancer invasion and metastasis. It has been suggested that internalized EpICD subsequently forms a complex with -catenin in the nucleus that regulate an oncogenic transcription program (13-15). Nevertheless, the molecular mechanisms underpinning this pleiotropic effect of EpCAM on advanced endometrial cancer development remain to be elucidated. Here we report that activation of epidermal growth factor receptor (EGFR) signaling by a ligand triggers EpCAM cleavage leading to nuclear internalization of EpICD in endometrial cancer cells. The internalized EpICD interacts with LEF1 in -catenin-mediated complexes that regulate gene transcription responsible for cell motility and migration. Atomic force microscopy (AFM) detected changes in nanomechanical properties of ligand-stimulated endometrial cancer cells, supporting the acquisition of an EMT phenotype. We also decided whether nanomechanical properties are reversed in cells carrying genome-edited were designed according to the instructions (19). These cells diluted into single cells were seeded into 96-well plates. Stable clones were cultured for two months under Puromycin selection. Selected clones were verified by sequencing to ensure the success of and for normalization. Primer sequences for RT-PCR AZ876 and BioMark system are listed in Supplementary Table S1. Nanomechanical imaging of cells with Atomic Force Microscopy (AFM) Cells cultured to keep confluence below 50% were imaged in Petri dishes using a Nanoscope Catalyst (Bruker) atomic force microscope mounted on a Nikon Ti inverted epi-fluorescent microscope. For scanning, individual cells without forming colonies and physically contacting other cells were selected. To achieve the highest consistency of the data under the applied conditions,.