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Hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.

Kohnoh T, Hashimoto N, Ando A, Sakamoto K, Miyazaki S, Aoyama D, Kusunose M, Kimura M, Omote N, Imaizumi K, Kawabe T, Hasegawa Y - Cancer Cell Int. (2016)

Bottom Line: Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes.The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system.PTEN4A did not affect stabilization of hypoxia-inducible factor 1α.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-Ku, Nagoya 466-8550 Japan.

ABSTRACT

Background: Persistent hypoxia stimulation, one of the most critical microenvironmental factors, accelerates the acquisition of epithelial-mesenchymal transition (EMT) phenotypes in lung cancer cells. Loss of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression might accelerate the development of lung cancer in vivo. Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes. Nevertheless, it is not known whether persistent hypoxia can modulate PTEN phosphatase activity or whether hypoxia-induced EMT phenotypes are negatively regulated by the PTEN phosphatase activity. We aimed to investigate hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.

Methods: Western blotting was performed in five lung cancer cell lines to evaluate total PTEN expression levels and the PTEN activation. In a xenograft model of lung cancer cells with endogenous PTEN expression, the PTEN expression was evaluated by immunohistochemistry. To examine the effect of hypoxia on phenotypic alterations in lung cancer cells in vitro, the cells were cultured under hypoxia. The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system.

Results: Lung cancer cells involving the EMT phenotypes showed a decrease in total PTEN expression and an increase in p-PTEN. In a xenograft model, loss of PTEN expression was observed in the tumor lesions showing tissue hypoxia. Persistent hypoxia yielded an approximately eight-fold increase in the p-PTEN/PTEN ratio in vitro. PTEN4A did not affect stabilization of hypoxia-inducible factor 1α. PTEN4A blunted hypoxia-induced EMT via inhibition of β-catenin translocation into the cytoplasm and nucleus.

Conclusion: Our study strengthens the therapeutic possibility that compensatory induction of unphosphorylated PTEN may inhibit the acquisition of EMT phenotypes in lung cancer cells under persistent hypoxia.

No MeSH data available.


Related in: MedlinePlus

Hypoxia-induced modulation of PTEN expression and EMT in H358 cells. a Cell extracts were analyzed for the levels of total and phosphorylated PTEN at the indicated time. A blot is representative of three independent experiments. b The ratio of p-PTEN to total PTEN is presented as an intensity level relative to that in H358 naïve cells at 0 min. Data were shown as the mean ± standard error (SE). The experiment was performed three times with similar results. Asterisk indicates p < 0.05. c Western blotting analysis for fibronectin and E-cadherin at the indicated time points was carried out. A blot is representative of three independent experiments. d The ratio of fibronectin to E-cadherin (F/E ratio) was evaluated, compared with that in cells treated under hypoxia at 0 min (bottom). Asterisk indicates p < 0.05. e, f By using confocal laser scanning microscopy and imaging software, we evaluated the fluorescence intensities of β-catenin (red) and E-cadherin (green) in the cells under normoxia or hypoxia. Hoechst33342 (blue) was utilized for nuclear staining. The left and right images in e shows cells cultured under normoxia and hypoxia, respectively. The upper and lower panels in f plot the fluorescence intensity of β-catenin red, E-cadherin green, and nucleus blue over a cross-section of cells cultured under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments
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Fig2: Hypoxia-induced modulation of PTEN expression and EMT in H358 cells. a Cell extracts were analyzed for the levels of total and phosphorylated PTEN at the indicated time. A blot is representative of three independent experiments. b The ratio of p-PTEN to total PTEN is presented as an intensity level relative to that in H358 naïve cells at 0 min. Data were shown as the mean ± standard error (SE). The experiment was performed three times with similar results. Asterisk indicates p < 0.05. c Western blotting analysis for fibronectin and E-cadherin at the indicated time points was carried out. A blot is representative of three independent experiments. d The ratio of fibronectin to E-cadherin (F/E ratio) was evaluated, compared with that in cells treated under hypoxia at 0 min (bottom). Asterisk indicates p < 0.05. e, f By using confocal laser scanning microscopy and imaging software, we evaluated the fluorescence intensities of β-catenin (red) and E-cadherin (green) in the cells under normoxia or hypoxia. Hoechst33342 (blue) was utilized for nuclear staining. The left and right images in e shows cells cultured under normoxia and hypoxia, respectively. The upper and lower panels in f plot the fluorescence intensity of β-catenin red, E-cadherin green, and nucleus blue over a cross-section of cells cultured under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments

Mentions: Next, to evaluate the effect of persistent hypoxia on modulation of PTEN expression in lung cancers, H358 cells were treated under hypoxia in vitro. Under persistent hypoxia stimulation, total PTEN levels decreased in a time-dependent manner but p-PTEN levels remained steady in H358 cells, leading to an approximately eightfold increase in the p-PTEN/PTEN ratio 72 h after hypoxia stimulation (Fig. 2a, b). To determine whether persistent hypoxia can induce the acquisition of EMT phenotypes in H358 cells [6], western blotting analysis for fibronectin and E-cadherin was carried out [21–23], which demonstrated that hypoxia induced EMT phenotypes in H358 cells in a time-dependent manner (Fig. 2c, d). Mounting evidence suggest that de novo expression of mesenchymal genes in epithelial cells could be induced by translocation of β-catenin into the cytoplasm and the nucleus [21, 24]. Therefore, we determined hypoxia-stimulated translocation of β-catenin in lung cancer cells. Because many studies suggest that E-cadherin is one of the most important constituents of adherens junction on cell membrane [21, 25–27], double immunostaing for β-catenin and E-cadherin was performed and then the localization was evaluated by using confocal microscopy. The results suggested that β-catenin was co-localized with E-cadherin on the cell membrane in H358 cells cultured under normoxia (Fig. 2e, f), but had translocated into the cytoplasm and the nucleus when the cells were cultured under hypoxia (Fig. 2e, f).Fig. 2


Hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.

Kohnoh T, Hashimoto N, Ando A, Sakamoto K, Miyazaki S, Aoyama D, Kusunose M, Kimura M, Omote N, Imaizumi K, Kawabe T, Hasegawa Y - Cancer Cell Int. (2016)

Hypoxia-induced modulation of PTEN expression and EMT in H358 cells. a Cell extracts were analyzed for the levels of total and phosphorylated PTEN at the indicated time. A blot is representative of three independent experiments. b The ratio of p-PTEN to total PTEN is presented as an intensity level relative to that in H358 naïve cells at 0 min. Data were shown as the mean ± standard error (SE). The experiment was performed three times with similar results. Asterisk indicates p < 0.05. c Western blotting analysis for fibronectin and E-cadherin at the indicated time points was carried out. A blot is representative of three independent experiments. d The ratio of fibronectin to E-cadherin (F/E ratio) was evaluated, compared with that in cells treated under hypoxia at 0 min (bottom). Asterisk indicates p < 0.05. e, f By using confocal laser scanning microscopy and imaging software, we evaluated the fluorescence intensities of β-catenin (red) and E-cadherin (green) in the cells under normoxia or hypoxia. Hoechst33342 (blue) was utilized for nuclear staining. The left and right images in e shows cells cultured under normoxia and hypoxia, respectively. The upper and lower panels in f plot the fluorescence intensity of β-catenin red, E-cadherin green, and nucleus blue over a cross-section of cells cultured under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments
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Related In: Results  -  Collection

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Fig2: Hypoxia-induced modulation of PTEN expression and EMT in H358 cells. a Cell extracts were analyzed for the levels of total and phosphorylated PTEN at the indicated time. A blot is representative of three independent experiments. b The ratio of p-PTEN to total PTEN is presented as an intensity level relative to that in H358 naïve cells at 0 min. Data were shown as the mean ± standard error (SE). The experiment was performed three times with similar results. Asterisk indicates p < 0.05. c Western blotting analysis for fibronectin and E-cadherin at the indicated time points was carried out. A blot is representative of three independent experiments. d The ratio of fibronectin to E-cadherin (F/E ratio) was evaluated, compared with that in cells treated under hypoxia at 0 min (bottom). Asterisk indicates p < 0.05. e, f By using confocal laser scanning microscopy and imaging software, we evaluated the fluorescence intensities of β-catenin (red) and E-cadherin (green) in the cells under normoxia or hypoxia. Hoechst33342 (blue) was utilized for nuclear staining. The left and right images in e shows cells cultured under normoxia and hypoxia, respectively. The upper and lower panels in f plot the fluorescence intensity of β-catenin red, E-cadherin green, and nucleus blue over a cross-section of cells cultured under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments
Mentions: Next, to evaluate the effect of persistent hypoxia on modulation of PTEN expression in lung cancers, H358 cells were treated under hypoxia in vitro. Under persistent hypoxia stimulation, total PTEN levels decreased in a time-dependent manner but p-PTEN levels remained steady in H358 cells, leading to an approximately eightfold increase in the p-PTEN/PTEN ratio 72 h after hypoxia stimulation (Fig. 2a, b). To determine whether persistent hypoxia can induce the acquisition of EMT phenotypes in H358 cells [6], western blotting analysis for fibronectin and E-cadherin was carried out [21–23], which demonstrated that hypoxia induced EMT phenotypes in H358 cells in a time-dependent manner (Fig. 2c, d). Mounting evidence suggest that de novo expression of mesenchymal genes in epithelial cells could be induced by translocation of β-catenin into the cytoplasm and the nucleus [21, 24]. Therefore, we determined hypoxia-stimulated translocation of β-catenin in lung cancer cells. Because many studies suggest that E-cadherin is one of the most important constituents of adherens junction on cell membrane [21, 25–27], double immunostaing for β-catenin and E-cadherin was performed and then the localization was evaluated by using confocal microscopy. The results suggested that β-catenin was co-localized with E-cadherin on the cell membrane in H358 cells cultured under normoxia (Fig. 2e, f), but had translocated into the cytoplasm and the nucleus when the cells were cultured under hypoxia (Fig. 2e, f).Fig. 2

Bottom Line: Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes.The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system.PTEN4A did not affect stabilization of hypoxia-inducible factor 1α.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-Ku, Nagoya 466-8550 Japan.

ABSTRACT

Background: Persistent hypoxia stimulation, one of the most critical microenvironmental factors, accelerates the acquisition of epithelial-mesenchymal transition (EMT) phenotypes in lung cancer cells. Loss of phosphatase and tensin homologue deleted from chromosome 10 (PTEN) expression might accelerate the development of lung cancer in vivo. Recent studies suggest that tumor microenvironmental factors might modulate the PTEN activity though a decrease in total PTEN expression and an increase in phosphorylation of the PTEN C-terminus (p-PTEN), resulting in the acquisition of the EMT phenotypes. Nevertheless, it is not known whether persistent hypoxia can modulate PTEN phosphatase activity or whether hypoxia-induced EMT phenotypes are negatively regulated by the PTEN phosphatase activity. We aimed to investigate hypoxia-induced modulation of PTEN activity and EMT phenotypes in lung cancers.

Methods: Western blotting was performed in five lung cancer cell lines to evaluate total PTEN expression levels and the PTEN activation. In a xenograft model of lung cancer cells with endogenous PTEN expression, the PTEN expression was evaluated by immunohistochemistry. To examine the effect of hypoxia on phenotypic alterations in lung cancer cells in vitro, the cells were cultured under hypoxia. The effect of unphosphorylated PTEN (PTEN4A) induction on hypoxia-induced EMT phenotypes was evaluated, by using a Dox-dependent gene expression system.

Results: Lung cancer cells involving the EMT phenotypes showed a decrease in total PTEN expression and an increase in p-PTEN. In a xenograft model, loss of PTEN expression was observed in the tumor lesions showing tissue hypoxia. Persistent hypoxia yielded an approximately eight-fold increase in the p-PTEN/PTEN ratio in vitro. PTEN4A did not affect stabilization of hypoxia-inducible factor 1α. PTEN4A blunted hypoxia-induced EMT via inhibition of β-catenin translocation into the cytoplasm and nucleus.

Conclusion: Our study strengthens the therapeutic possibility that compensatory induction of unphosphorylated PTEN may inhibit the acquisition of EMT phenotypes in lung cancer cells under persistent hypoxia.

No MeSH data available.


Related in: MedlinePlus