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SASH1 is an adapter and signaling protein that contains SH3 and SAM domains responsible for protein-protein interactions. SASH1 downregulation has been observed in many tumors. We examined localization of SASH1 in cultures of normal IAR-20 epithelial cells and HT-29 colorectal cancer cells using immunofluorescence staining and confocal microscopy. IAR-20 normal epithelial cells and HT-29 cells with epithelial phenotype formed stable linear adherens junctions (AJs) associated with circumferential actin bundles. In both IAR-20 and HT-29 cells, SASH1 was co-localized with zones of circumferential actin bundles and linear AJs. SASH1 was not detected in lamellipodia. IAR-20 and HT-29 cells treated with Epidermal Growth Factor underwent epithelial-mesenchymal transition (EMT). We observed significant differences in the course of EMT between IAR-20 and HT-29 cultures. IAR-20 cells underwent partial EMT acquiring migratory phenotype but retaining E-cadherin in unstable radial AJs. SASH1 was present in these contacts. Disappearance of AJs was observed in HT-29 cell undergoing a complete EMT, which also resulted in disruption of stable cell-cell adhesion. SASH1 was lost from the zones of cell-cell interaction. SASH1 depletion by means of RNA interference in IAR-20 cells led to destruction of stable linear AJs and acquisition of mesenchymal phenotype by some of the cells. These data indicate involvement of SASH1 in the maintenance of stable cell-cell adhesion.

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphological and migration characteristics of epithelial cells of IAR1162 and IAR1170 clones derived from a mixed culture of N-RasV12 oncogene-transformed IAR-2 cell line. It was found that the oncogenic RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, assembled E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the oncogenic RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of cell motility in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the motility of transformed epithelial cells and plays an important role in their collective migration. [ABSTRACT FROM AUTHOR]