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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 modulating phosphorylation sites in the PTEN C-terminuson hypoxia-induced EMT 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 24 h in the absence or presence of Dox. Western blotting analysis for total PTEN (top) and p-PTEN (bottom) was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). b Western blotting analysis for fibronectin and E-cadherin in the indicated cells cultured under normoxia or hypoxia for 48 h was carried out. A blot is representative of three independent experiments. c F/E ratio in the treated cells was compared with that in cells treated with vehicle in the absence of Dox. Asterisk indicates p < 0.05
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Fig3: Effect of modulating phosphorylation sites in the PTEN C-terminuson hypoxia-induced EMT 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 24 h in the absence or presence of Dox. Western blotting analysis for total PTEN (top) and p-PTEN (bottom) was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). b Western blotting analysis for fibronectin and E-cadherin in the indicated cells cultured under normoxia or hypoxia for 48 h was carried out. A blot is representative of three independent experiments. c F/E ratio in the treated cells was compared with that in cells treated with vehicle in the absence of Dox. Asterisk indicates p < 0.05

Mentions: We previously demonstrated that unphosphorylated PTEN inhibits TGFβ-induced acquisition of the EMT phenotypes in lung cancer cells [17]. Nevertheless, whether or not phosphorylation of the PTEN C-terminus might directly play a critical role in hypoxia-induced EMT remains elusive. Here, therefore, we evaluated the effect of unphosphorylated PTEN on hypoxia-induced EMT phenotypes, by using a Dox-dependent gene expression system (Fig. 3a). To evaluate whether hypoxia-induced EMT can be modulated by Dox-induced GFP-PTEN4A expression, western blotting analysis for fibronectin and E-cadherin was carried out. There was no or little reduction in the fibronectin/E-cadherin ratio in GFP-expressing cells; however, de novo GFP-PTEN4A protein expression by Dox yielded a significant decrease of about 88 % in the fibronectin/E-cadherin ratio (Fig. 3b, c). There was a limited repression in the fibronectin/E-cadherin ratio in GFP-WildPTEN-expressing cells (Fig. 3b, c); in particular, GFP-WildPTEN did not appear to repress hypoxia-induced fibronectin expression.Fig. 3


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 modulating phosphorylation sites in the PTEN C-terminuson hypoxia-induced EMT 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 24 h in the absence or presence of Dox. Western blotting analysis for total PTEN (top) and p-PTEN (bottom) was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). b Western blotting analysis for fibronectin and E-cadherin in the indicated cells cultured under normoxia or hypoxia for 48 h was carried out. A blot is representative of three independent experiments. c F/E ratio in the treated cells was compared with that in cells treated with vehicle in the absence of Dox. Asterisk indicates p < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836157&req=5

Fig3: Effect of modulating phosphorylation sites in the PTEN C-terminuson hypoxia-induced EMT 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 24 h in the absence or presence of Dox. Western blotting analysis for total PTEN (top) and p-PTEN (bottom) was carried out. A blot is representative of three independent experiments (GFP, left; GFP-WildPTEN, middle; GFPPTEN4A, right). b Western blotting analysis for fibronectin and E-cadherin in the indicated cells cultured under normoxia or hypoxia for 48 h was carried out. A blot is representative of three independent experiments. c F/E ratio in the treated cells was compared with that in cells treated with vehicle in the absence of Dox. Asterisk indicates p < 0.05
Mentions: We previously demonstrated that unphosphorylated PTEN inhibits TGFβ-induced acquisition of the EMT phenotypes in lung cancer cells [17]. Nevertheless, whether or not phosphorylation of the PTEN C-terminus might directly play a critical role in hypoxia-induced EMT remains elusive. Here, therefore, we evaluated the effect of unphosphorylated PTEN on hypoxia-induced EMT phenotypes, by using a Dox-dependent gene expression system (Fig. 3a). To evaluate whether hypoxia-induced EMT can be modulated by Dox-induced GFP-PTEN4A expression, western blotting analysis for fibronectin and E-cadherin was carried out. There was no or little reduction in the fibronectin/E-cadherin ratio in GFP-expressing cells; however, de novo GFP-PTEN4A protein expression by Dox yielded a significant decrease of about 88 % in the fibronectin/E-cadherin ratio (Fig. 3b, c). There was a limited repression in the fibronectin/E-cadherin ratio in GFP-WildPTEN-expressing cells (Fig. 3b, c); in particular, GFP-WildPTEN did not appear to repress hypoxia-induced fibronectin expression.Fig. 3

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