EGF-Mediated Transcriptional Coregulation of EpICD-LEF1 Alters Nanomechanical Properties of Endometrial Cancer Cells for Promoting Cell Migration
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE72948 | GEO | 2015/09/12
SECONDARY ACCESSION(S): PRJNA295390
REPOSITORIES: GEO
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Project description:Epithelial cell adhesion molecule (EpCAM), a membrane protein known to modulate cell-cell adhesion, is also a signaling molecule internalized into the nucleus for transcriptional regulation. Here we demonstrate that activated EGF/EGFR is a signaling factor to drive the proteolysis of EpCAM. Cleavage of the extracellular fragment EpEX results in topographic fading of cell-surface EpCAM detected by antibody-conjugated cantilevers of atomic force microscope (AFM). As a result, internalization of the cytoplasmic domain EpICD forms a transcription factor complex with LEF1 that regulates gene transcription for enhanced cell-mobility functions. Comprehensive probing of cell surface using AFM tip (without antibody) reveals increased elasticity and non-stickiness of these cells, promoting epithelial to mesenchymal transition. While EpCAM cleavage may contribute to the loss of cell-surface adhesiveness, its internalized EpICD additionally regulates targets for promoting cell migration. Thus, this EGF/EGFR-modulated action on structural EpCAM and regulatory EpICD can enhance invasion potential of transformed cells. RL95-2 were stimulated with EGF for 0,12,24,and 48 hr.Immunoprecipitation was carried out using antibodies against EpCAM and Lef-1, sequenced by Illumina HiSeq 2000
2015-09-12 | E-GEOD-72948 | biostudies-arrayexpress
Project description:To address early human placenta development, we aimed to characterize the cell surface proteomic profile of early trophoblast cells in an effort to understand trophoblast heterogeneity and population dynamics. We quantify cell surface protein expression in early first trimester human placentas using a high throughput flow cytometry screen of 370 Cluster of Differentiation (CD) antigens and confidently identified 23 increasing and 13 decreasing markers of dynamic populations between weeks 6 and 10 of human placenta development. We were particularly interested in transient trophoblast populations expressing either EpCAM (CD326) or CDCP1 (CD318) populations of trophoblasts. Immunohistochemistry analysis revealed these CD antigens are expressed by discrete cells with EpCAM localized to trophoblast in the proximal villi columns while CDCP1 is expressed by cells in the distal columns. Multi-color flow analysis confirmed independence of CDCP1 and EPCAM expressing cell populations and identified EpCAM cells as positive for EGFR. Microarray analysis of gene expression indicated EpCAM+/EGFR+ and EGFR+ cells showed high degree of gene expression similarities to villus cytotrophbolast but that loss of EpCAM expression was concomitant with exit from the cell cycle as EpCAM/EGFR cell were mitotically active while CDCP1 positive trophoblast are enriched in gene sets related to immune regulation functions and cell cycle/cell division and have significant similar gene expression to extravillous cytotrophoblast. Our study indicates at least two distinct subpopulations of cytotrophoblasts exit in the early first trimester and that the column likely maintains pools of actively dividing progenitor cells giving rise to the developing placenta villus tree.
2019-02-01 | GSE90646 | GEO
Project description:Epithelial cell adhesion molecule EpCAM is a single transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration, and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP-7) as a target gene of EpCAM signalling via EpICD nuclear translocation. EpCAM and MMP-7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP-7 is a novel target of EpCAM signalling. FaDu hypopharynx carcinoma cells were transiently transfected with a control siRNA or an EpCAM-specific siRNA. After two days, expression of EpCAM was assessed upon flow cytometry with antibodies, which specifically bind within the EGF-repeat in the extracellular domain of EpCAM (Balzar, et al., 1999). Compared to control siRNA, EpCAM siRNA induced a mean 43% reduction of EpCAM expression at the cell surface. Reduction of EpCAM expression upon siRNA treatment was confirmed by RT-PCR and qPCR. EpCAM down-regulation resulted in diminished cell proliferation assessed upon cell counting. The observed 43% decrease in cell surface expression of EpCAM translated in cell numbers diminished by 35%. This is a confirmation of earlier findings on the reduced proliferation of breast and colon cancer cell lines following siRNA-mediated knock-down of EpCAM (Maetzel, et al., 2009, Munz, et al., 2004, Osta, et al., 2004). Equal amounts of total RNA (RIN score = 10) from control- and EpCAM-siRNA-treated FaDu cells were next subjected to a single, explorative cDNA microarray analysis with the aim to identify potential target genes of the signal transduction cascade of EpCAM. All genes, which were up- or down-regulated by at least two-fold (>2-fold; 716 genes up-regulated, 552 genes down-regulated), were then subjected to a Panther classification software analysis (http://www.pantherdb.org/). Special emphasis was put on the regulation of genes associated with signalling pathways. Angiogenesis, Alzheimer's disease-presenilin pathway, Wnt signalling pathway, inflammation and cadherin signalling pathways were most strongly affected by EpCAM knock-down. mRNA levels of selected genes differently regulated following partial knock-down of EpCAM included the co-regulated genes for secreted frizzled-related protein 5, protocadherin 10, dickkopf homolog 2, MMP24, SPARC, Myst3, ErbB4, MMP12, WISP2, MMP7, Nanos1, WNT9A, and the counter-regulated genes for MMP11, fibronectin 1, DACT3, WNT2B, WNT5A, APC2, Spondin 1, and cateninM-NM-12. All genes except for protocadherin 10, Myst3, ErbB4, and WNT2B were validated in RT-PCR assays. Since several matrix metalloproteases were regulated after EpCAM knock-down and EpCAM is known to correlate with invasion, we chose this protein family for in-depth analysis. Matrix metalloprotease 7 (MMP-7) is a soluble factor, which, unlike other family members, is preferentially expressed in epithelial cells (Wilson, et al., 1995). Since EpCAM also displays highest expression in epithelia, we chose to proceed with the analysis of MMP7. SiRNA-mediated knock-down of EpCAM resulted in a comparable 50% down-regulation of MMP7 mRNA levels in RT-PCR and qPCR. Comparison of FaDu cells expressing EpCam vs FaDu cells with downregulated EpCam
2012-04-13 | E-GEOD-37257 | biostudies-arrayexpress
Project description:The cell surface receptor, Epithelial Cell Adhesion Molecule (EpCAM), is overexpressed in a variety of cancers and has been carefully examined for its functional role in carcinogenesis and its potential as a therapeutic target. However, little is known about the upstream molecular pathways that regulate EpCAM expression. To elucidate the mechanisms by which EpCAM expression is controlled, we designed a combinatorial screening strategy that utilized pooled shRNA gene knockdown with magnetically-activated cell separation for selection of cells with reduced EpCAM protein expression on the cell surface. The pooled RNAi screen was analyzed by comparing EpCAM-enriched and EpCAM-depleted cell populations for shRNA abundance using competitive hybridization to microarrays. Our RNAi screen and further validation efforts revealed that at least four genes (GRM1, SLITRK5, EFNA5, and IL23A) are important for EpCAM expression in the ovarian cancer cell line OVCAR8. Further investigations into the ephrin ligand, EFNA5, suggest that its activation by the Eph receptor, EPHA4, contributes to the regulation of EpCAM expression via a reverse signaling mechanism. Our screening strategy revealed novel mechanisms for the regulation of EpCAM expression and provides insight into the biology of this cancer-associated gene. Importantly, this study also demonstrates the utility of pooled RNAi screening in the identification of genes required for the regulation of cell surface protein expression. Two condition experiment; EpCAM-Bound or EpCAM-Unbound OVCAR8 cells vs Reference population of transduced cells without phenotypic selection; biological triplicate
2011-12-01 | E-GEOD-32985 | biostudies-arrayexpress
Project description:Aberrant mucin type O-linked glycosylation is a common occurrence in cancer. This type of O-linked glycosylation can occur on many cell surface glycoproteins where only a small number of sites may be present. EGFR is one such glycoprotein. Upon EGF ligation, EGFR induces a signaling cascade but can also translocate to the nucleus where it can directly regulate gene transcription. Here we show that upon EGF binding, breast cancer cells carrying different O-linked glycans respond by transcribing differential gene expression signatures. This is not a result of changes in signal transduction but due to the differential nuclear translocation of EGFR in the two glyco-phenotypes. This appears to be regulated by the formation of a EGFR/galectin-3/MUC1 complex at the cell surface that is present in cells carrying short core1-based O-glycans characteristic of tumour cells but absent in core 2 O-glycan carrying cells representative of normal mammary epithelial cells.
2020-12-04 | GSE107629 | GEO
Project description:The cell surface receptor, Epithelial Cell Adhesion Molecule (EpCAM), is overexpressed in a variety of cancers and has been carefully examined for its functional role in carcinogenesis and its potential as a therapeutic target. However, little is known about the upstream molecular pathways that regulate EpCAM expression. To elucidate the mechanisms by which EpCAM expression is controlled, we designed a combinatorial screening strategy that utilized pooled shRNA gene knockdown with magnetically-activated cell separation for selection of cells with reduced EpCAM protein expression on the cell surface. The pooled RNAi screen was analyzed by comparing EpCAM-enriched and EpCAM-depleted cell populations for shRNA abundance using competitive hybridization to microarrays. Our RNAi screen and further validation efforts revealed that at least four genes (GRM1, SLITRK5, EFNA5, and IL23A) are important for EpCAM expression in the ovarian cancer cell line OVCAR8. Further investigations into the ephrin ligand, EFNA5, suggest that its activation by the Eph receptor, EPHA4, contributes to the regulation of EpCAM expression via a reverse signaling mechanism. Our screening strategy revealed novel mechanisms for the regulation of EpCAM expression and provides insight into the biology of this cancer-associated gene. Importantly, this study also demonstrates the utility of pooled RNAi screening in the identification of genes required for the regulation of cell surface protein expression.
2011-12-01 | GSE32985 | GEO
Project description:Epithelial cell adhesion molecule EpCAM is a single transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration, and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP-7) as a target gene of EpCAM signalling via EpICD nuclear translocation. EpCAM and MMP-7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP-7 is a novel target of EpCAM signalling. FaDu hypopharynx carcinoma cells were transiently transfected with a control siRNA or an EpCAM-specific siRNA. After two days, expression of EpCAM was assessed upon flow cytometry with antibodies, which specifically bind within the EGF-repeat in the extracellular domain of EpCAM (Balzar, et al., 1999). Compared to control siRNA, EpCAM siRNA induced a mean 43% reduction of EpCAM expression at the cell surface. Reduction of EpCAM expression upon siRNA treatment was confirmed by RT-PCR and qPCR. EpCAM down-regulation resulted in diminished cell proliferation assessed upon cell counting. The observed 43% decrease in cell surface expression of EpCAM translated in cell numbers diminished by 35%. This is a confirmation of earlier findings on the reduced proliferation of breast and colon cancer cell lines following siRNA-mediated knock-down of EpCAM (Maetzel, et al., 2009, Munz, et al., 2004, Osta, et al., 2004). Equal amounts of total RNA (RIN score = 10) from control- and EpCAM-siRNA-treated FaDu cells were next subjected to a single, explorative cDNA microarray analysis with the aim to identify potential target genes of the signal transduction cascade of EpCAM. All genes, which were up- or down-regulated by at least two-fold (>2-fold; 716 genes up-regulated, 552 genes down-regulated), were then subjected to a Panther classification software analysis (http://www.pantherdb.org/). Special emphasis was put on the regulation of genes associated with signalling pathways. Angiogenesis, Alzheimer's disease-presenilin pathway, Wnt signalling pathway, inflammation and cadherin signalling pathways were most strongly affected by EpCAM knock-down. mRNA levels of selected genes differently regulated following partial knock-down of EpCAM included the co-regulated genes for secreted frizzled-related protein 5, protocadherin 10, dickkopf homolog 2, MMP24, SPARC, Myst3, ErbB4, MMP12, WISP2, MMP7, Nanos1, WNT9A, and the counter-regulated genes for MMP11, fibronectin 1, DACT3, WNT2B, WNT5A, APC2, Spondin 1, and cateninα2. All genes except for protocadherin 10, Myst3, ErbB4, and WNT2B were validated in RT-PCR assays. Since several matrix metalloproteases were regulated after EpCAM knock-down and EpCAM is known to correlate with invasion, we chose this protein family for in-depth analysis. Matrix metalloprotease 7 (MMP-7) is a soluble factor, which, unlike other family members, is preferentially expressed in epithelial cells (Wilson, et al., 1995). Since EpCAM also displays highest expression in epithelia, we chose to proceed with the analysis of MMP7. SiRNA-mediated knock-down of EpCAM resulted in a comparable 50% down-regulation of MMP7 mRNA levels in RT-PCR and qPCR.
2012-04-14 | GSE37257 | GEO
Project description:Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. Treatment with B6H12 antibody inhibited co-immunoprecipitation of EGFR with CD47 and inhibited EGF-induced EGFR tyrosine phosphorylation. B6H12 treatment of bCSC also suppressed asymmetric cell division and cell proliferation and up-regulated caspase 3/7 activity. Correspondingly, caspase-7 cleavage in human breast cancers correlated with CD47 expression. Our data shows that B6H12 specifically targets bCSCs but not differentiated cancer cells, and this CD47 signaling is independent of SIRPα. Three replicates of each condition were generated. Three replicates of each MDA-231 attached cells (differentiated), MDA-231 in suspension cells (bCSC), MDA-231 in suspension cells (bCSC) treated with Control Antibody and MDA-231 in suspension cells (bCSC) treated with B6H12 Antibody.
2016-04-01 | E-GEOD-67966 | biostudies-arrayexpress
Project description:Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signaling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g. HB-EGF) are known to bind to heparan sulfate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e. HB-EGF, CX3CL1) and 14 novel HSPG-binding putative ADAM17 substrates.
2024-10-15 | PXD055223 | Pride
Project description:Background: Maspin (SERPINB5) is a potential tumor suppressor gene with pleiotropic biological activities, including regulation of cell proliferation, death, adhesion, migration and gene expression. Several studies indicate that nuclear localization is essential for maspin tumor suppression activity. We have previously shown that the EGFR activation leads to maspin nuclear localization in MCF-10A cells. The present study investigated which EGFR downstream signaling molecules are involved in maspin nuclear localization and explored a possible role of cell-cell contact in this process. Methods: MCF-10A cells were treated with pharmacological inhibitors against EGFR downstream pathways followed by EGF treatment. Maspin subcellular localization was determined by immunofluorescence. To investigate the role of cell-cell contact these cells were either treated with chelating agents or plated on different cell densities. Maspin and E-cadherin subcellular localization was determined by immunofluorescence. In addition, proteomic and interactome analyses were conducted in order to identify molecular partners which interact with maspin in EGF-treated cells only. Results: EGFR activation regulates cell behavior via de extracellular signal–related kinase (ERK)/MAP kinase, phosphatidyl-inositol-3 (PI 3) kinase- AKT and Jak-STAT pathways. Using pharmacological inhibitors against key components of these signaling pathways we found that PI3K-AKT and Jak-STAT, but not MAP kinase pathway, regulate EGF-induced maspin nuclear accumulation in MCF-10A cells. Interestingly, we observed that maspin is predominantly nuclear in sparse cell culture, but it is redistributed to the cytoplasm in confluent cells even in the presence of EGF. Proteomic and interactome results suggest a role of maspin on post-transcriptional and translation regulation, protein folding and cell-cell adhesion.Conclusions: Maspin nuclear accumulation is determined by an interplay between EGFR (via PI3K-AKT and Jak-STAT pathways) and cell-to-cell contact.
2021-09-09 | PXD023224 | Pride