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The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

Kotelevets L, van Hengel J, Bruyneel E, Mareel M, van Roy F, Chastre E - J. Cell Biol. (2001)

Bottom Line: In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin.PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins.Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U410, Faculté de Médecine Bichat, 75018 Paris, France.

ABSTRACT
To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

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Effect of restoration of PTEN expression on the invasive phenotype of the PTEN-defective human melanoma cell line FM-45, the prostate carci-noma cell line PC-3, and the glioblastoma cell lines U87MG and U373MG. (A) Immunoblot analysis of E-cadherin, N-cadherin, and cadherin-11. Cell lysates (50 μg protein for all cell lines except for U87MGwt14 and U373MGwt16 lysates for which 30 μg was used) were immunoblotted with mAbs directed against E-cadherin (MB-2 and HECD-1), N-cadherin (Zymed), or cadherin-11 (113H), and probed using the ECL system. (B) Analysis of PTEN expression in U87MG and U373MG cells and their derivatives stably transfected by wild-type PTEN, and in FM-45 and PC-3 cell lines transiently expressing wild-type or mutant GFP–PTEN upon vaccinia infection. Cell lysates (100 μg protein) from parental and PTEN-transfected cells were immunoblotted with mAbs directed against PTEN. (C) Invasion of type I collagen by U87, U373, FM-45, and PC-3 cells and their derivatives transfected by wild-type or mutant PTEN. Cells were seeded on type I collagen gels at 37°C and the number and depth of cells inside the gel were measured after 24 h. The dependence of cell migration and invasion on N-cadherin was assessed by the blocking of N-cadherin using GC-4 antibodies (dilution 1:50).
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fig5: Effect of restoration of PTEN expression on the invasive phenotype of the PTEN-defective human melanoma cell line FM-45, the prostate carci-noma cell line PC-3, and the glioblastoma cell lines U87MG and U373MG. (A) Immunoblot analysis of E-cadherin, N-cadherin, and cadherin-11. Cell lysates (50 μg protein for all cell lines except for U87MGwt14 and U373MGwt16 lysates for which 30 μg was used) were immunoblotted with mAbs directed against E-cadherin (MB-2 and HECD-1), N-cadherin (Zymed), or cadherin-11 (113H), and probed using the ECL system. (B) Analysis of PTEN expression in U87MG and U373MG cells and their derivatives stably transfected by wild-type PTEN, and in FM-45 and PC-3 cell lines transiently expressing wild-type or mutant GFP–PTEN upon vaccinia infection. Cell lysates (100 μg protein) from parental and PTEN-transfected cells were immunoblotted with mAbs directed against PTEN. (C) Invasion of type I collagen by U87, U373, FM-45, and PC-3 cells and their derivatives transfected by wild-type or mutant PTEN. Cells were seeded on type I collagen gels at 37°C and the number and depth of cells inside the gel were measured after 24 h. The dependence of cell migration and invasion on N-cadherin was assessed by the blocking of N-cadherin using GC-4 antibodies (dilution 1:50).

Mentions: To further extend the effects of PTEN on the stabilization of junctional complexes and the reversion of the invasive phenotype, we restored PTEN expression in human cell lines known to be PTEN defective: glioblastoma cell lines U87MG and U373MG, melanoma cell line FM-45, and prostate carcinoma cell line PC-3 (Guldberg et al., 1997; Tamura et al., 1998; Whang et al., 1998). We first analyzed the expression patterns of epithelial E-cadherin, mesenchymal N-cadherin, and cadherin-11 (Fig. 5 A). All three cadherins were found to be expressed in PC-3 cells, which are known to be defective in Ca2+-dependent cell aggregation due to αE-catenin defects (Morton et al., 1993). In contrast, the U87MG and U373MG glioblastoma cells expressed mainly N-cadherin, and FM-45 melanoma cells were featured by weak expression of N-cadherin and major expression of cadherin-11. A small amount of the latter cadherin was also identified in U373MG cells. In agreement with the results obtained from MDCKts-src cells, wild-type PTEN reverted the invasive phenotype of U87MG and U373MG glioblastoma cells stably transfected by wild-type PTEN, and of FM-45 melanoma and PC-3 prostate carcinoma cells transiently transfected using vaccinia virus–mediated PTEN expression (Fig. 5, B and C). In the latter two cell lines, overexpression of mutant PTEN molecules did not revert the invasive phenotype (Fig. 5 C). These results confirm the critical role of the lipid phosphatase activity of PTEN in regulating invasiveness. Moreover, the invasive phenotype of the U87MG and U373MG glioblastoma cell lines was clearly dependent on N-cadherin activity, because it was inhibited after application of the N-cadherin blocking antibody GC-4 (Fig. 5 C), whereas it was noneffective on FM-45 cells that express mainly cadherin-11 (Fig. 5 A, and unpublished data). Nonetheless, PTEN-mediated invasion suppression was not associated with any significant change in the expression pattern of cadherins (Fig. 5 A, and unpublished data).


The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness.

Kotelevets L, van Hengel J, Bruyneel E, Mareel M, van Roy F, Chastre E - J. Cell Biol. (2001)

Effect of restoration of PTEN expression on the invasive phenotype of the PTEN-defective human melanoma cell line FM-45, the prostate carci-noma cell line PC-3, and the glioblastoma cell lines U87MG and U373MG. (A) Immunoblot analysis of E-cadherin, N-cadherin, and cadherin-11. Cell lysates (50 μg protein for all cell lines except for U87MGwt14 and U373MGwt16 lysates for which 30 μg was used) were immunoblotted with mAbs directed against E-cadherin (MB-2 and HECD-1), N-cadherin (Zymed), or cadherin-11 (113H), and probed using the ECL system. (B) Analysis of PTEN expression in U87MG and U373MG cells and their derivatives stably transfected by wild-type PTEN, and in FM-45 and PC-3 cell lines transiently expressing wild-type or mutant GFP–PTEN upon vaccinia infection. Cell lysates (100 μg protein) from parental and PTEN-transfected cells were immunoblotted with mAbs directed against PTEN. (C) Invasion of type I collagen by U87, U373, FM-45, and PC-3 cells and their derivatives transfected by wild-type or mutant PTEN. Cells were seeded on type I collagen gels at 37°C and the number and depth of cells inside the gel were measured after 24 h. The dependence of cell migration and invasion on N-cadherin was assessed by the blocking of N-cadherin using GC-4 antibodies (dilution 1:50).
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Related In: Results  -  Collection

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fig5: Effect of restoration of PTEN expression on the invasive phenotype of the PTEN-defective human melanoma cell line FM-45, the prostate carci-noma cell line PC-3, and the glioblastoma cell lines U87MG and U373MG. (A) Immunoblot analysis of E-cadherin, N-cadherin, and cadherin-11. Cell lysates (50 μg protein for all cell lines except for U87MGwt14 and U373MGwt16 lysates for which 30 μg was used) were immunoblotted with mAbs directed against E-cadherin (MB-2 and HECD-1), N-cadherin (Zymed), or cadherin-11 (113H), and probed using the ECL system. (B) Analysis of PTEN expression in U87MG and U373MG cells and their derivatives stably transfected by wild-type PTEN, and in FM-45 and PC-3 cell lines transiently expressing wild-type or mutant GFP–PTEN upon vaccinia infection. Cell lysates (100 μg protein) from parental and PTEN-transfected cells were immunoblotted with mAbs directed against PTEN. (C) Invasion of type I collagen by U87, U373, FM-45, and PC-3 cells and their derivatives transfected by wild-type or mutant PTEN. Cells were seeded on type I collagen gels at 37°C and the number and depth of cells inside the gel were measured after 24 h. The dependence of cell migration and invasion on N-cadherin was assessed by the blocking of N-cadherin using GC-4 antibodies (dilution 1:50).
Mentions: To further extend the effects of PTEN on the stabilization of junctional complexes and the reversion of the invasive phenotype, we restored PTEN expression in human cell lines known to be PTEN defective: glioblastoma cell lines U87MG and U373MG, melanoma cell line FM-45, and prostate carcinoma cell line PC-3 (Guldberg et al., 1997; Tamura et al., 1998; Whang et al., 1998). We first analyzed the expression patterns of epithelial E-cadherin, mesenchymal N-cadherin, and cadherin-11 (Fig. 5 A). All three cadherins were found to be expressed in PC-3 cells, which are known to be defective in Ca2+-dependent cell aggregation due to αE-catenin defects (Morton et al., 1993). In contrast, the U87MG and U373MG glioblastoma cells expressed mainly N-cadherin, and FM-45 melanoma cells were featured by weak expression of N-cadherin and major expression of cadherin-11. A small amount of the latter cadherin was also identified in U373MG cells. In agreement with the results obtained from MDCKts-src cells, wild-type PTEN reverted the invasive phenotype of U87MG and U373MG glioblastoma cells stably transfected by wild-type PTEN, and of FM-45 melanoma and PC-3 prostate carcinoma cells transiently transfected using vaccinia virus–mediated PTEN expression (Fig. 5, B and C). In the latter two cell lines, overexpression of mutant PTEN molecules did not revert the invasive phenotype (Fig. 5 C). These results confirm the critical role of the lipid phosphatase activity of PTEN in regulating invasiveness. Moreover, the invasive phenotype of the U87MG and U373MG glioblastoma cell lines was clearly dependent on N-cadherin activity, because it was inhibited after application of the N-cadherin blocking antibody GC-4 (Fig. 5 C), whereas it was noneffective on FM-45 cells that express mainly cadherin-11 (Fig. 5 A, and unpublished data). Nonetheless, PTEN-mediated invasion suppression was not associated with any significant change in the expression pattern of cadherins (Fig. 5 A, and unpublished data).

Bottom Line: In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin.PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins.Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U410, Faculté de Médecine Bichat, 75018 Paris, France.

ABSTRACT
To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell-cell aggregation, and suppressed the invasive phenotype in an E-cadherin-dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin-neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

Show MeSH
Related in: MedlinePlus