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A Pooled RNAi Screening Strategy Reveals Novel Regulators for EpCAM

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE32985 | GEO | 2011/12/01

SECONDARY ACCESSION(S): PRJNA146441

REPOSITORIES: GEO

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A Pooled RNAi Screening Strategy Reveals Novel Regulators for EpCAM

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

Homo sapiens

Project description:A Pooled RNAi Screening Strategy Reveals Novel Regulators for EpCAM

| PRJNA146441 | ENA

A genome-wide screen identifies competitive growth advantage for human epidermal stem cells with activated YAP

Project description:We performed genome-wide pooled RNAi screens for genes that confer a clonal growth advantage or disadvantage on epidermal stem- and cutaneous squamous cell carcinoma (SCC) cells What we know from our cell biology is that a shRNA underrepresented at t=14 targets a gene that confers growth advantage.

2017-03-31 | GSE79560 | GEO

Screening for genes that promote copper toxicity in a cell model of Wilson disease [HepG2 cells ATP7B-KO]

Project description:To find new genes that might be targeted for reduction of Copper (Cu) toxicity in Wilson disease we employed genome-wide shRNA screening in ATP7B-KO HepG2 cells, a bona fide WD cell model. Cells were screened using the Dharmacon™ Decode™ Pooled Lentiviral shRNA Screening Library targeting 18205 human genes (Dharmacon #RHS6083). The Lentiviral RNAi screening library is made of 10 pools of 9570 GIPZ short hairpin RNAs (shRNAs) packaged into high-titer lentiviral particles. Genomic DNA was isolated from the cells and read counts for each shRNAs were obtained by deep sequencing to determine the abundance of each shRNA in the Cu-treated cells compared to controls. This analysis allowed the identification of genes whose downregulation promoted survival of ATP7B-KO cells upon Cu overload. To exclude that some shRNAs would have been responsible for ATP7B-KO survival by suppressing generic proapoptotic genes, we run the same screening with the proapoptotic drug vinblastine instead of CuCl2. Genes, whose suppression allowed cell to resist Cu but not vinblastine were considered as Cu-specific hits and through this approach we identified PrP as a suitable target for reduction of Cu toxicity in WD. Indeed, we found that Cu accumulation in ATP7B-KO cells stimulates expression of PrP, which brings to toxic Cu overload while PrP suppression significantly reduces Cu accumulation and toxicity in ATP7B-deficient cells. In sum our data highlight an important regulatory role of PrP in copper metabolism and as a therapeutic tool for Wilson disease.

2024-12-09 | GSE277865 | GEO

Screening for genes that promote copper toxicity in a cell model of Wilson disease

2024-12-09 | GSE260454 | GEO

EGF-Mediated Transcriptional Coregulation of EpICD-LEF1 Alters Nanomechanical Properties of Endometrial Cancer Cells for Promoting Cell Migration

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

EGF-Mediated Transcriptional Coregulation of EpICD-LEF1 Alters Nanomechanical Properties of Endometrial Cancer Cells for Promoting Cell Migration

2015-09-12 | GSE72948 | GEO

SWI/SNF Chromatin-Remodeling Factor Smarcd3/Baf60c Controls Epithelial-Mesenchymal Transition by Inducing Wnt5a Signaling

Project description:We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM-) breast cancer cells to an epithelial EpCAM+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM- breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.

2013-08-19 | GSE40145 | GEO

Project description:High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

| PRJEB2680 | ENA

Cellular systems biology identifies dynamic trophoblast populations in early human placentas

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

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