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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

Effect of unphosphorylated PTEN on hypoxia-induced stabilization of HIF-1α expression, translocation of β-catenin, and the EMT-related gene twist expression in H358 cells. a The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 6 h in the absence or presence of Dox. Western blotting analysis for f HIF-1α was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The ratio of HIF-1α to β-actin was compared with that in the cells cultured under normoxia in the absence of Dox. *p < 0.05 NS not significant. b The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The expression levels of twist mRNA were analyzed by using real-time PCR and normalized to GAPDH mRNA (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The relative twist expressions were compared with that in cells treated under normoxia. Data represent the mean ± standard error of the mean (SEM) from three independent experiments. *p < 0.05, NS not significant. c–h The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The intensity of fluorescence of β-catenin and E-cadherin was evaluated in H358ON cells with Dox-dependent GFP (c, d), GFP-WildPTEN (e, f) or GFP-PTEN4A (g, h) was evaluated. The left and right images in c, e, g show cells under normoxia and hypoxia, respectively. The upper and lower panels in d, f, h plot the fluorescence intensity of β-catenin (red), E-cadherin (green), and nucleus (blue) over a cross section of cells under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments
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Fig4: Effect of unphosphorylated PTEN on hypoxia-induced stabilization of HIF-1α expression, translocation of β-catenin, and the EMT-related gene twist expression in H358 cells. a The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 6 h in the absence or presence of Dox. Western blotting analysis for f HIF-1α was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The ratio of HIF-1α to β-actin was compared with that in the cells cultured under normoxia in the absence of Dox. *p < 0.05 NS not significant. b The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The expression levels of twist mRNA were analyzed by using real-time PCR and normalized to GAPDH mRNA (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The relative twist expressions were compared with that in cells treated under normoxia. Data represent the mean ± standard error of the mean (SEM) from three independent experiments. *p < 0.05, NS not significant. c–h The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The intensity of fluorescence of β-catenin and E-cadherin was evaluated in H358ON cells with Dox-dependent GFP (c, d), GFP-WildPTEN (e, f) or GFP-PTEN4A (g, h) was evaluated. The left and right images in c, e, g show cells under normoxia and hypoxia, respectively. The upper and lower panels in d, f, h plot the fluorescence intensity of β-catenin (red), E-cadherin (green), and nucleus (blue) over a cross section of cells under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments

Mentions: To investigate the molecular mechanisms underlying PTEN4A activity, whether hypoxia-induced signaling pathways could be modulated by GFP-PTEN4A was evaluated. A recent study demonstrated that HIF-1α expression directly regulates de novo EMT-related gene expression, causing an alteration in phenotype through EMT in a cancer metastasis model [5]. Here, Dox-induced expression of de novo GFP, GFP-WildPTEN, or GFP-PTEN4A did not repress the increase in de novo HIF-1α expression in hypoxia-stimulated H358ON cells (Fig. 4a). Next, we used real-time PCR to investigate whether the inhibitory effect of unphosphorylated PTEN on hypoxia-induced acquisition of EMT phenotypes might depend on the altered expression of EMT-related genes (Fig. 4b). Our previous study showed that hypoxia stimulation induced the increasing levels of twist, but not snail, in H358 cells [6]. In the present study, although de novo GFP expression did not alter hypoxia-induced twist expression, the increasing twist mRNA levels in hypoxia-stimulated H358ON cells expressing Dox-dependent GFP-WildPTEN and GFP-PTEN4A were repressed when Dox was added (Fig. 4b). Furthermore, we determined whether Dox-induced PTEN4A expression can modulate β-catenin translocation in hypoxia-stimulated lung cancer cells. Immunocytochemistry findings showed that translocation of β-catenin was not observed in H358ON cells expressing Dox-dependent GFP, GFP-WildPTEN, or GFP-PTEN4A when cultured under normoxia (Fig. 4c–h). Although GFP or GFP-WildPTEN protein induced by Dox did not inhibit hypoxia-induced β-catenin translocation into the cytoplasm and nucleus in H358ON cells (Fig. 4c–f), β-catenin translocation induced by hypoxia stimulation was completely inhibited by de novo GFP-PTEN4A protein (Fig. 4g, h).Fig. 4


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)

Effect of unphosphorylated PTEN on hypoxia-induced stabilization of HIF-1α expression, translocation of β-catenin, and the EMT-related gene twist expression in H358 cells. a The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 6 h in the absence or presence of Dox. Western blotting analysis for f HIF-1α was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The ratio of HIF-1α to β-actin was compared with that in the cells cultured under normoxia in the absence of Dox. *p < 0.05 NS not significant. b The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The expression levels of twist mRNA were analyzed by using real-time PCR and normalized to GAPDH mRNA (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The relative twist expressions were compared with that in cells treated under normoxia. Data represent the mean ± standard error of the mean (SEM) from three independent experiments. *p < 0.05, NS not significant. c–h The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The intensity of fluorescence of β-catenin and E-cadherin was evaluated in H358ON cells with Dox-dependent GFP (c, d), GFP-WildPTEN (e, f) or GFP-PTEN4A (g, h) was evaluated. The left and right images in c, e, g show cells under normoxia and hypoxia, respectively. The upper and lower panels in d, f, h plot the fluorescence intensity of β-catenin (red), E-cadherin (green), and nucleus (blue) over a cross section of cells under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments
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Fig4: Effect of unphosphorylated PTEN on hypoxia-induced stabilization of HIF-1α expression, translocation of β-catenin, and the EMT-related gene twist expression in H358 cells. a The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 6 h in the absence or presence of Dox. Western blotting analysis for f HIF-1α was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The ratio of HIF-1α to β-actin was compared with that in the cells cultured under normoxia in the absence of Dox. *p < 0.05 NS not significant. b The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The expression levels of twist mRNA were analyzed by using real-time PCR and normalized to GAPDH mRNA (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). The relative twist expressions were compared with that in cells treated under normoxia. Data represent the mean ± standard error of the mean (SEM) from three independent experiments. *p < 0.05, NS not significant. c–h The indicated cells were treated with vehicle or Dox for 24 h before hypoxia stimulation. And then, the cells were cultured under normoxia or hypoxia for a further 24 h in the absence or presence of Dox. The intensity of fluorescence of β-catenin and E-cadherin was evaluated in H358ON cells with Dox-dependent GFP (c, d), GFP-WildPTEN (e, f) or GFP-PTEN4A (g, h) was evaluated. The left and right images in c, e, g show cells under normoxia and hypoxia, respectively. The upper and lower panels in d, f, h plot the fluorescence intensity of β-catenin (red), E-cadherin (green), and nucleus (blue) over a cross section of cells under normoxia and hypoxia, respectively. Data are representative of at least three independent experiments
Mentions: To investigate the molecular mechanisms underlying PTEN4A activity, whether hypoxia-induced signaling pathways could be modulated by GFP-PTEN4A was evaluated. A recent study demonstrated that HIF-1α expression directly regulates de novo EMT-related gene expression, causing an alteration in phenotype through EMT in a cancer metastasis model [5]. Here, Dox-induced expression of de novo GFP, GFP-WildPTEN, or GFP-PTEN4A did not repress the increase in de novo HIF-1α expression in hypoxia-stimulated H358ON cells (Fig. 4a). Next, we used real-time PCR to investigate whether the inhibitory effect of unphosphorylated PTEN on hypoxia-induced acquisition of EMT phenotypes might depend on the altered expression of EMT-related genes (Fig. 4b). Our previous study showed that hypoxia stimulation induced the increasing levels of twist, but not snail, in H358 cells [6]. In the present study, although de novo GFP expression did not alter hypoxia-induced twist expression, the increasing twist mRNA levels in hypoxia-stimulated H358ON cells expressing Dox-dependent GFP-WildPTEN and GFP-PTEN4A were repressed when Dox was added (Fig. 4b). Furthermore, we determined whether Dox-induced PTEN4A expression can modulate β-catenin translocation in hypoxia-stimulated lung cancer cells. Immunocytochemistry findings showed that translocation of β-catenin was not observed in H358ON cells expressing Dox-dependent GFP, GFP-WildPTEN, or GFP-PTEN4A when cultured under normoxia (Fig. 4c–h). Although GFP or GFP-WildPTEN protein induced by Dox did not inhibit hypoxia-induced β-catenin translocation into the cytoplasm and nucleus in H358ON cells (Fig. 4c–f), β-catenin translocation induced by hypoxia stimulation was completely inhibited by de novo GFP-PTEN4A protein (Fig. 4g, h).Fig. 4

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