title: Involvement of Rho GAP GRAF1 in maintenance of epithelial phenotype.
abstract: Adhesion of epithelial cell to each other and to extracellular matrix, as well as cell migration ability and cytoskeleton organization undergo significant alterations in the course of neoplastic transformation, but regulatory mechanisms involved in these processes are not fully understood. Here, we studied the role of a Rho GAP protein GRAF1 (GTPase Regulator Associated with Focal adhesion kinase-1) in the regulation of the epithelial phenotype in cells of breast derived, non-malignant, MCF10A cell line. GRAF1 was shown to be localized to cell-cell junctions, and its depletion resulted in accelerated cell migration velocity, elongation of the cells and cell colonies, impaired monolayer integrity and significant disruption of desmosomes with a loss of associated keratin filaments. These processes were accompanied by formation of larger focal adhesions, an increased number of contractile actin stress fibers, reduction in epithelial markers and increase in mesenchymal markers such as epithelial-mesenchymal transition (EMT)-specific transcription factors Snail-1 and Snail-2, as well as N-cadherin, and vimentin. Moreover, unlike control cells, GRAF1 knocked-down cells demonstrated anchorage-independent growth in soft agar. GRAF1 expression in several highly invasive breast cancer cell lines was low, as compared to the non-malignant MCF10A cells, while overexpressing of GRAF1 in the malignant BT-549 cell line led to a decrease of mesenchymal markers, especially the Snail-1 and 2. Altogether, our analysis suggests that GRAF1 plays a role in the maintenance of normal epithelial phenotype and its depletion leads to an EMT-like process that might be involved in neoplastic transformation.
authors: a Department of Molecular Cell Biology , Weizmann Institute of Science , Rehovot , Israel.|b The Chaim Sheba Medical Center, Tel-Hashomer Hospital , Israel.
authors: Miriam
authors: M
authors: Regev
authors: a Department of Molecular Cell Biology , Weizmann Institute of Science , Rehovot , Israel.
authors: Helena
authors: H
authors: Sabanay
authors: a Department of Molecular Cell Biology , Weizmann Institute of Science , Rehovot , Israel.
authors: Elena
authors: E
authors: Kartvelishvily
authors: a Department of Molecular Cell Biology , Weizmann Institute of Science , Rehovot , Israel.
authors: Zvi
authors: Z
authors: Kam
authors: a Department of Molecular Cell Biology , Weizmann Institute of Science , Rehovot , Israel.|c Mechanobiology Institute, National University of Singapore , Singapore.
authors: Alexander D
authors: AD
authors: Bershadsky
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000362:Agar; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D048430:Cell Shape; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology* / Q000648:ultrastructure; D058750:Epithelial-Mesenchymal Transition; D022001:Focal Adhesions / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D005782:Gels; D055785:Gene Knockdown Techniques; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism / Q000648:ultrastructure; D016086:Lentivirus / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010641:Phenotype; D034741:RNA, Small Interfering / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C115971:ARHGAP26 protein, human; D015415:Biomarkers; D020690:GTPase-Activating Proteins; D005782:Gels; D034741:RNA, Small Interfering; D000362:Agar; D054460:rho-Associated Kinases; D020830:rac1 GTP-Binding Protein
keywords: E-cadherin; EMT; MCF10A; anchorage dependence; cell migration; cell-cell junctions; focal adhesions