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A Novel Role of E-Cadherin-Based Adherens Junctions in Neoplastic Cell Dissemination.

Rubtsova SN, Zhitnyak IY, Gloushankova NA - PLoS ONE (2015)

Bottom Line: Studying interactions of IAR-6-1 transformed cells stably expressing GFP-E-cadherin with the IAR-2 epithelial monolayer, we found that IAR-6-1 cells established E-cadherin-based adhesions with normal epithelial cells: dot-like dynamic E-cadherin-based adhesions in protrusions and large adherens junctions at the cell sides and rear.IAR-6-1DNE cells expressing a dominant-negative mutant form of E-cadherin with the mutation in the first extracellular domain practically lost the ability to adhere to IAR-2 cells and invade the IAR-2 epithelial monolayer.The ability of cancer cells to form E-cadherin-based AJs with the surrounding normal epithelial cells may play an important role in driving cancer cell dissemination in the body.

View Article: PubMed Central - PubMed

Affiliation: Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia.

ABSTRACT
Using confocal microscopy, we analyzed the behavior of IAR-6-1, IAR1170, and IAR1162 transformed epithelial cells seeded onto the confluent monolayer of normal IAR-2 epithelial cells. Live-cell imaging of neoplastic cells stably expressing EGFP and of normal epithelial cells stably expressing mKate2 showed that transformed cells retaining expression of E-cadherin were able to migrate over the IAR-2 epithelial monolayer and invade the monolayer. Transformed IAR cells invaded the IAR-2 monolayer at the boundaries between normal cells. Studying interactions of IAR-6-1 transformed cells stably expressing GFP-E-cadherin with the IAR-2 epithelial monolayer, we found that IAR-6-1 cells established E-cadherin-based adhesions with normal epithelial cells: dot-like dynamic E-cadherin-based adhesions in protrusions and large adherens junctions at the cell sides and rear. A comparative study of a panel of transformed IAR cells that differ by their ability to form E-cadherin-based AJs, either through loss of E-cadherin expression or through expression of a dominant negative E-cadherin mutant, demonstrated that E-cadherin-based AJs are key mediators of the interactions between neoplastic and normal epithelial cells. IAR-6-1DNE cells expressing a dominant-negative mutant form of E-cadherin with the mutation in the first extracellular domain practically lost the ability to adhere to IAR-2 cells and invade the IAR-2 epithelial monolayer. The ability of cancer cells to form E-cadherin-based AJs with the surrounding normal epithelial cells may play an important role in driving cancer cell dissemination in the body.

No MeSH data available.


Related in: MedlinePlus

Transformed IAR-6-1 cells form E-cadherin-based AJs with underlying normal IAR-2 cells.GFP-E-cadherin-expressing IAR-6-1 cells were seeded onto the confluent monolayer of mKate2-expressing IAR-2 cells. (A-B) Immunofluorescent staining for GFP. (A) E-cadherin accumulates in dot-like adhesions at the leading edge and in prominent AJs encircling the IAR-6-1 cell. Left—green channel. Boxed region is enlarged. Arrowhead indicates dot-like adhesions. Right—green and red channels. Dotted line indicates the position of the Y-projection. Scale bar 10 μm. (B) Y-projection. Arrows mark lateral AJs between IAR-6-1 and IAR-2 cells. (C) Selected confocal slices from time lapse Z-stacks (S4 Video). The green channel is a “Z- projection” of all three slices in a confocal Z-stack, the red channel is the top slice. Asterisks indicate lateral AJs. Scale bar 10 μm. (D) A close-up view of the boxed region from (A). “Z-projection” of the green channel of the same video. At the leading edge of the IAR-6-1 cell, transient E-cadherin-based AJs are formed and quickly disassembled. Arrowheads mark spots where diffuse E-cadherin accumulates into dot-like adhesions, asterisks mark persisting E-cadherin dots, and arrows indicate disappearance of the dots. Scale bar 5 μm.
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pone.0133578.g003: Transformed IAR-6-1 cells form E-cadherin-based AJs with underlying normal IAR-2 cells.GFP-E-cadherin-expressing IAR-6-1 cells were seeded onto the confluent monolayer of mKate2-expressing IAR-2 cells. (A-B) Immunofluorescent staining for GFP. (A) E-cadherin accumulates in dot-like adhesions at the leading edge and in prominent AJs encircling the IAR-6-1 cell. Left—green channel. Boxed region is enlarged. Arrowhead indicates dot-like adhesions. Right—green and red channels. Dotted line indicates the position of the Y-projection. Scale bar 10 μm. (B) Y-projection. Arrows mark lateral AJs between IAR-6-1 and IAR-2 cells. (C) Selected confocal slices from time lapse Z-stacks (S4 Video). The green channel is a “Z- projection” of all three slices in a confocal Z-stack, the red channel is the top slice. Asterisks indicate lateral AJs. Scale bar 10 μm. (D) A close-up view of the boxed region from (A). “Z-projection” of the green channel of the same video. At the leading edge of the IAR-6-1 cell, transient E-cadherin-based AJs are formed and quickly disassembled. Arrowheads mark spots where diffuse E-cadherin accumulates into dot-like adhesions, asterisks mark persisting E-cadherin dots, and arrows indicate disappearance of the dots. Scale bar 5 μm.

Mentions: The presence of dynamic E-cadherin-based adhesions between neoplastic and normal epithelial cells was detected by live-cell imaging. For these experiments we used IAR-2 cells stably expressing mKate2. IAR-6-1 cells were stably transfected with the GFP-E-cadherin construct. GFP-E-cadherin did not affect the distribution of endogenous E-cadherin and colocalized with it in AJs (S2 Fig). In IAR-6-1 cells seeded onto the IAR-2 monolayer, E-cadherin accumulated in dot-like adhesions at the leading edge and in large AJs at the rear and at the sides of transformed cells (Fig 3A and 3B). Using live-cell confocal imaging, we found that dot-like E-cadherin-based adhesions constantly formed and disappeared at the leading edge of transformed cells (Fig 3C and 3D, and S4 Video). Larger, more stable, AJs at the sides could merge (Fig 3A and S4 Video). We hypothesize that transformed cells are capable of migrating over the normal epithelial monolayer by attaching to underlying cells with E-cadherin-based AJs and using these AJs as anchor points.


A Novel Role of E-Cadherin-Based Adherens Junctions in Neoplastic Cell Dissemination.

Rubtsova SN, Zhitnyak IY, Gloushankova NA - PLoS ONE (2015)

Transformed IAR-6-1 cells form E-cadherin-based AJs with underlying normal IAR-2 cells.GFP-E-cadherin-expressing IAR-6-1 cells were seeded onto the confluent monolayer of mKate2-expressing IAR-2 cells. (A-B) Immunofluorescent staining for GFP. (A) E-cadherin accumulates in dot-like adhesions at the leading edge and in prominent AJs encircling the IAR-6-1 cell. Left—green channel. Boxed region is enlarged. Arrowhead indicates dot-like adhesions. Right—green and red channels. Dotted line indicates the position of the Y-projection. Scale bar 10 μm. (B) Y-projection. Arrows mark lateral AJs between IAR-6-1 and IAR-2 cells. (C) Selected confocal slices from time lapse Z-stacks (S4 Video). The green channel is a “Z- projection” of all three slices in a confocal Z-stack, the red channel is the top slice. Asterisks indicate lateral AJs. Scale bar 10 μm. (D) A close-up view of the boxed region from (A). “Z-projection” of the green channel of the same video. At the leading edge of the IAR-6-1 cell, transient E-cadherin-based AJs are formed and quickly disassembled. Arrowheads mark spots where diffuse E-cadherin accumulates into dot-like adhesions, asterisks mark persisting E-cadherin dots, and arrows indicate disappearance of the dots. Scale bar 5 μm.
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Related In: Results  -  Collection

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pone.0133578.g003: Transformed IAR-6-1 cells form E-cadherin-based AJs with underlying normal IAR-2 cells.GFP-E-cadherin-expressing IAR-6-1 cells were seeded onto the confluent monolayer of mKate2-expressing IAR-2 cells. (A-B) Immunofluorescent staining for GFP. (A) E-cadherin accumulates in dot-like adhesions at the leading edge and in prominent AJs encircling the IAR-6-1 cell. Left—green channel. Boxed region is enlarged. Arrowhead indicates dot-like adhesions. Right—green and red channels. Dotted line indicates the position of the Y-projection. Scale bar 10 μm. (B) Y-projection. Arrows mark lateral AJs between IAR-6-1 and IAR-2 cells. (C) Selected confocal slices from time lapse Z-stacks (S4 Video). The green channel is a “Z- projection” of all three slices in a confocal Z-stack, the red channel is the top slice. Asterisks indicate lateral AJs. Scale bar 10 μm. (D) A close-up view of the boxed region from (A). “Z-projection” of the green channel of the same video. At the leading edge of the IAR-6-1 cell, transient E-cadherin-based AJs are formed and quickly disassembled. Arrowheads mark spots where diffuse E-cadherin accumulates into dot-like adhesions, asterisks mark persisting E-cadherin dots, and arrows indicate disappearance of the dots. Scale bar 5 μm.
Mentions: The presence of dynamic E-cadherin-based adhesions between neoplastic and normal epithelial cells was detected by live-cell imaging. For these experiments we used IAR-2 cells stably expressing mKate2. IAR-6-1 cells were stably transfected with the GFP-E-cadherin construct. GFP-E-cadherin did not affect the distribution of endogenous E-cadherin and colocalized with it in AJs (S2 Fig). In IAR-6-1 cells seeded onto the IAR-2 monolayer, E-cadherin accumulated in dot-like adhesions at the leading edge and in large AJs at the rear and at the sides of transformed cells (Fig 3A and 3B). Using live-cell confocal imaging, we found that dot-like E-cadherin-based adhesions constantly formed and disappeared at the leading edge of transformed cells (Fig 3C and 3D, and S4 Video). Larger, more stable, AJs at the sides could merge (Fig 3A and S4 Video). We hypothesize that transformed cells are capable of migrating over the normal epithelial monolayer by attaching to underlying cells with E-cadherin-based AJs and using these AJs as anchor points.

Bottom Line: Studying interactions of IAR-6-1 transformed cells stably expressing GFP-E-cadherin with the IAR-2 epithelial monolayer, we found that IAR-6-1 cells established E-cadherin-based adhesions with normal epithelial cells: dot-like dynamic E-cadherin-based adhesions in protrusions and large adherens junctions at the cell sides and rear.IAR-6-1DNE cells expressing a dominant-negative mutant form of E-cadherin with the mutation in the first extracellular domain practically lost the ability to adhere to IAR-2 cells and invade the IAR-2 epithelial monolayer.The ability of cancer cells to form E-cadherin-based AJs with the surrounding normal epithelial cells may play an important role in driving cancer cell dissemination in the body.

View Article: PubMed Central - PubMed

Affiliation: Institute of Carcinogenesis, N.N. Blokhin Russian Cancer Research Center, Moscow, Russia.

ABSTRACT
Using confocal microscopy, we analyzed the behavior of IAR-6-1, IAR1170, and IAR1162 transformed epithelial cells seeded onto the confluent monolayer of normal IAR-2 epithelial cells. Live-cell imaging of neoplastic cells stably expressing EGFP and of normal epithelial cells stably expressing mKate2 showed that transformed cells retaining expression of E-cadherin were able to migrate over the IAR-2 epithelial monolayer and invade the monolayer. Transformed IAR cells invaded the IAR-2 monolayer at the boundaries between normal cells. Studying interactions of IAR-6-1 transformed cells stably expressing GFP-E-cadherin with the IAR-2 epithelial monolayer, we found that IAR-6-1 cells established E-cadherin-based adhesions with normal epithelial cells: dot-like dynamic E-cadherin-based adhesions in protrusions and large adherens junctions at the cell sides and rear. A comparative study of a panel of transformed IAR cells that differ by their ability to form E-cadherin-based AJs, either through loss of E-cadherin expression or through expression of a dominant negative E-cadherin mutant, demonstrated that E-cadherin-based AJs are key mediators of the interactions between neoplastic and normal epithelial cells. IAR-6-1DNE cells expressing a dominant-negative mutant form of E-cadherin with the mutation in the first extracellular domain practically lost the ability to adhere to IAR-2 cells and invade the IAR-2 epithelial monolayer. The ability of cancer cells to form E-cadherin-based AJs with the surrounding normal epithelial cells may play an important role in driving cancer cell dissemination in the body.

No MeSH data available.


Related in: MedlinePlus