Impact of p120-catenin isoforms 1A and 3A on epithelial mesenchymal transition of lung cancer cells expressing E-cadherin in different subcellular locations

Abstract

The epithelial mesenchymal transition (EMT) is an important process in tumor development. Despite previous investigations, it remains unclear how p120-catenin (p120ctn) isoforms 1A and 3A affect the EMT of tumor cells. Here we investigated expression of p120ctn, E-cadherin and vimentin in 78 human non-small cell lung cancer (NSCLC) samples by immunohistochemistry and found that p120ctn membrane expression positively correlated with E-cadherin expression (P<0.001) and negatively correlated with vimentin expression and lymph node metastasis (P<0.05). Meanwhile, p120ctn cytoplasmic expression negatively correlated with E-cadherin expression (P<0.001) and positively correlated with vimentin expression and lymph node metastasis (P<0.05). Cells expressing high (H460 and SPC) and low (H1299 and LK2) levels of p120ctn were screen to investigate its impact on EMT. E-cadherin was restricted to the cell membrane in H460 and H1299 cells, whereas it was expressed in the cytoplasm of SPC and LK2 cells. Ablation of endogenous p120ctn isoform 1A in cells expressing high levels of the protein resulted in decreased E-cadherin expression, increased N-cadherin, vimentin and snail expression and enhanced invasiveness in H460 cells. Meanwhile, completely opposite results were observed in SPC cells. Furthermore, transfection of in H1299 cells expressing low p120ctn levels with the p120ctn isoform 1A plasmid resulted in increased E-cadherin expression, decreased N-cadherin, vimentin and snail expression and weakened invasiveness, while LK2 cells showed completely opposite results. Both cell lines expressing low p120ctn levels and transfected with the p120ctn isoform 3A plasmid appeared to have increased E-cadherin expression, decreased N-cadherin, vimentin and snail expression and weakened invasiveness. In conclusion, in cells with membrane E-cadherin, both p120ctn isoforms 1A and 3A inhibited EMT and decreased cell invasiveness. In cells with cytoplasmic E-cadherin, p120ctn isoform 1A promoted EMT and increased cell invasiveness, while p120ctn isoform 3A inhibited the EMT and decreased cell invasiveness.

Figure 1. Immunohistochemical analysis of p120ctn, E-cadherin and vimentin localization in NSCLC.

(A) E-cadherin and p120ctn were membrane positive, and vimentin was negative in normal bronchial epithelial cells. (B) E-cadherin was membrane positive, and vimentin was negative in p120ctn membrane-positive lung cancer cells. (C) E-cadherin was negative, and vimentin was positive in p120ctn cytoplasmic-positive lung cancer cells.

Figure 2. Expression and localization of p120ctn and E-cadherin in H460, SPC, H1299 and LK2 cells.

(A) Western blot analyses showed expression of p120ctn and E-cadherin in nine lung cancer cell lines and HBE. (B) By immunofluorescence analysis, the expression of E-cadherin and p120ctn were observed restricted to the cell membrane at cell-cell adherens junctions in H460 and H1299 cells, whereas they both were confined to the cytoplasm in SPC and LK2 cells.

Figure 3. p120ctn isoform 1A plays a different role in regulating EMT in H460 and SPC cells.

(A) Ablation of p120ctn isoform 1A decreased E-cadherin expression and increased N-cadherin, snail and vimentin expression in H460 cells. (B) SPC cells were treated as in (A) and the opposite results were obtained. (C) Ablation of p120ctn isoform 1A enhanced the invasiveness of H460 cells (**P<0.01). (D) Ablation of p120ctn isoform 1A decreased the invasiveness of SPC cells (**P<0.01).

Figure 4. p120ctn isoform 3A maintains the role of inhibitiing EMT independently of E-cadherin localization.

(A, B) Both H1299 (E-cadherin membrane localization) and LK2 cells (E-cadherin cytoplasmic localization) transiently transfected with the p120ctn isoform 3A plasmid showed increased E-cadherin expression and decreased N-cadherin, vimentin and snail expression. (C, D) Transient transfection of p120ctn isoform 3A plasmids into H1299 and LK2 cells resulted in decreased cell invasiveness (**P<0.01). (E) E-cadherin remained localized on the membrane in H1299 cells and in the cytoplasm of LK2 cells after transfection of the p120ctn isoform 3A plasmid.