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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

Total PTEN levels and phosphorylation levels of the PTEN C-terminus in lung cancer cells. The levels of total PTEN and p-PTEN expression were evaluated in five lung cancer cell lines. a Cell extracts were harvested for the analysis of p-PTEN, PTEN, and β-actin by western blotting. Equal amounts (20 μg) of protein were loaded for analysis. The ratio of total PTEN protein to β-actin, b and p-PTEN to total PTEN protein, c is presented as an intensity level relative to that in H441 cells. A representative blot is shown from three independent experiments. Asterisk indicates p < 0.05. (b, c), d Immunostaining for PTEN (green) was performed in H1299 (left) and H358 (right) cells. Nuclear staining was performed by Hoechst33342 (blue). Results are representative of three independent experiments. All images shown are × 200 magnification. e NIS-Elements AR software was used to compare the intensity level of PTEN expression between H1299 (left panels in d) and H358 (right panels in d) cells, compensated by cell numbers, which were counted by Hoechst33342 staining. Data represent the mean ± SE of two independent experiments. Asterisk indicates p < 0.05. To evaluate whether persistent hypoxia might modulate PTEN expression in lung cancer cells in vivo, we examined the tumor sections from Dox-treated nude mice at day 28 after inoculation with H358ON cells carrying GFP. f H–E staining was performed on tumor tissues. g Immunostaining for pimonidazole as a hypoxia marker was performed on a serial section of tumor tissues. An arrow indicates the cells positive for pimonidazole. h Control immunostaining for PTEN on a serial section of tumor samples is shown. i Immunostaining for PTEN was also performed on the serial section of tumor samples. An arrow indicates the cells positive for PTEN. Representative tumor sections are shown from three independent experiments. f–i All images shown are ×200 magnification
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Fig1: Total PTEN levels and phosphorylation levels of the PTEN C-terminus in lung cancer cells. The levels of total PTEN and p-PTEN expression were evaluated in five lung cancer cell lines. a Cell extracts were harvested for the analysis of p-PTEN, PTEN, and β-actin by western blotting. Equal amounts (20 μg) of protein were loaded for analysis. The ratio of total PTEN protein to β-actin, b and p-PTEN to total PTEN protein, c is presented as an intensity level relative to that in H441 cells. A representative blot is shown from three independent experiments. Asterisk indicates p < 0.05. (b, c), d Immunostaining for PTEN (green) was performed in H1299 (left) and H358 (right) cells. Nuclear staining was performed by Hoechst33342 (blue). Results are representative of three independent experiments. All images shown are × 200 magnification. e NIS-Elements AR software was used to compare the intensity level of PTEN expression between H1299 (left panels in d) and H358 (right panels in d) cells, compensated by cell numbers, which were counted by Hoechst33342 staining. Data represent the mean ± SE of two independent experiments. Asterisk indicates p < 0.05. To evaluate whether persistent hypoxia might modulate PTEN expression in lung cancer cells in vivo, we examined the tumor sections from Dox-treated nude mice at day 28 after inoculation with H358ON cells carrying GFP. f H–E staining was performed on tumor tissues. g Immunostaining for pimonidazole as a hypoxia marker was performed on a serial section of tumor tissues. An arrow indicates the cells positive for pimonidazole. h Control immunostaining for PTEN on a serial section of tumor samples is shown. i Immunostaining for PTEN was also performed on the serial section of tumor samples. An arrow indicates the cells positive for PTEN. Representative tumor sections are shown from three independent experiments. f–i All images shown are ×200 magnification

Mentions: To evaluate total PTEN expression levels and the p-PTEN/PTEN ratio in lung cancer cells, western blotting was performed in the following five lung cancer cell lines: H441, H358, A549, H157 and H1299 [20]. Western blotting analysis showed that H1299 cells involving the EMT phenotypes (20), had lower PTEN expression level than H441 and H358 cells; as consequence the p-PTEN/PTEN ratio was about threefold higher in H1299 cells than in H441 and H358 cells (Fig. 1a–c). These findings were supported by immunofluorescence images showing that PTEN protein was expressed in H358 cells, whereas H1299 cells showed low PTEN expression (Fig. 1d, e). To determine whether persistent hypoxia might modulate PTEN expression in lung cancer cells in vivo, we examined the H358ON cells expressing GFP that had been grown in the flank of Dox-treated nude mice after administration of pimonidazole, which detects hypoxia via immunohistochemical staining [6, 17]. H–E staining showed tumor growth in the subcutaneous lesion of nude mice (Fig. 1f). To detect tissue hypoxia in the tumor specimen in vivo, immunostaining for pimonidazole was performed (Fig. 1g). The tumor cells stained positive for intracellular pimonidazole, indicating that tissue hypoxia might be involved in the tumor lesions. Furthermore, to evaluate the association between tissue hypoxia and PTEN expression in tumor lesions in vivo, immunostaining for PTEN in a serial section of the tumor sample was undertaken. Although the immunostaining showed no or little PTEN expression in the tumor lesions (Fig. 1h, i), positive staining for PTEN was observed in the subcutaneous tissue of nude mice (Fig. 1h, i).Fig. 1


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)

Total PTEN levels and phosphorylation levels of the PTEN C-terminus in lung cancer cells. The levels of total PTEN and p-PTEN expression were evaluated in five lung cancer cell lines. a Cell extracts were harvested for the analysis of p-PTEN, PTEN, and β-actin by western blotting. Equal amounts (20 μg) of protein were loaded for analysis. The ratio of total PTEN protein to β-actin, b and p-PTEN to total PTEN protein, c is presented as an intensity level relative to that in H441 cells. A representative blot is shown from three independent experiments. Asterisk indicates p < 0.05. (b, c), d Immunostaining for PTEN (green) was performed in H1299 (left) and H358 (right) cells. Nuclear staining was performed by Hoechst33342 (blue). Results are representative of three independent experiments. All images shown are × 200 magnification. e NIS-Elements AR software was used to compare the intensity level of PTEN expression between H1299 (left panels in d) and H358 (right panels in d) cells, compensated by cell numbers, which were counted by Hoechst33342 staining. Data represent the mean ± SE of two independent experiments. Asterisk indicates p < 0.05. To evaluate whether persistent hypoxia might modulate PTEN expression in lung cancer cells in vivo, we examined the tumor sections from Dox-treated nude mice at day 28 after inoculation with H358ON cells carrying GFP. f H–E staining was performed on tumor tissues. g Immunostaining for pimonidazole as a hypoxia marker was performed on a serial section of tumor tissues. An arrow indicates the cells positive for pimonidazole. h Control immunostaining for PTEN on a serial section of tumor samples is shown. i Immunostaining for PTEN was also performed on the serial section of tumor samples. An arrow indicates the cells positive for PTEN. Representative tumor sections are shown from three independent experiments. f–i All images shown are ×200 magnification
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836157&req=5

Fig1: Total PTEN levels and phosphorylation levels of the PTEN C-terminus in lung cancer cells. The levels of total PTEN and p-PTEN expression were evaluated in five lung cancer cell lines. a Cell extracts were harvested for the analysis of p-PTEN, PTEN, and β-actin by western blotting. Equal amounts (20 μg) of protein were loaded for analysis. The ratio of total PTEN protein to β-actin, b and p-PTEN to total PTEN protein, c is presented as an intensity level relative to that in H441 cells. A representative blot is shown from three independent experiments. Asterisk indicates p < 0.05. (b, c), d Immunostaining for PTEN (green) was performed in H1299 (left) and H358 (right) cells. Nuclear staining was performed by Hoechst33342 (blue). Results are representative of three independent experiments. All images shown are × 200 magnification. e NIS-Elements AR software was used to compare the intensity level of PTEN expression between H1299 (left panels in d) and H358 (right panels in d) cells, compensated by cell numbers, which were counted by Hoechst33342 staining. Data represent the mean ± SE of two independent experiments. Asterisk indicates p < 0.05. To evaluate whether persistent hypoxia might modulate PTEN expression in lung cancer cells in vivo, we examined the tumor sections from Dox-treated nude mice at day 28 after inoculation with H358ON cells carrying GFP. f H–E staining was performed on tumor tissues. g Immunostaining for pimonidazole as a hypoxia marker was performed on a serial section of tumor tissues. An arrow indicates the cells positive for pimonidazole. h Control immunostaining for PTEN on a serial section of tumor samples is shown. i Immunostaining for PTEN was also performed on the serial section of tumor samples. An arrow indicates the cells positive for PTEN. Representative tumor sections are shown from three independent experiments. f–i All images shown are ×200 magnification
Mentions: To evaluate total PTEN expression levels and the p-PTEN/PTEN ratio in lung cancer cells, western blotting was performed in the following five lung cancer cell lines: H441, H358, A549, H157 and H1299 [20]. Western blotting analysis showed that H1299 cells involving the EMT phenotypes (20), had lower PTEN expression level than H441 and H358 cells; as consequence the p-PTEN/PTEN ratio was about threefold higher in H1299 cells than in H441 and H358 cells (Fig. 1a–c). These findings were supported by immunofluorescence images showing that PTEN protein was expressed in H358 cells, whereas H1299 cells showed low PTEN expression (Fig. 1d, e). To determine whether persistent hypoxia might modulate PTEN expression in lung cancer cells in vivo, we examined the H358ON cells expressing GFP that had been grown in the flank of Dox-treated nude mice after administration of pimonidazole, which detects hypoxia via immunohistochemical staining [6, 17]. H–E staining showed tumor growth in the subcutaneous lesion of nude mice (Fig. 1f). To detect tissue hypoxia in the tumor specimen in vivo, immunostaining for pimonidazole was performed (Fig. 1g). The tumor cells stained positive for intracellular pimonidazole, indicating that tissue hypoxia might be involved in the tumor lesions. Furthermore, to evaluate the association between tissue hypoxia and PTEN expression in tumor lesions in vivo, immunostaining for PTEN in a serial section of the tumor sample was undertaken. Although the immunostaining showed no or little PTEN expression in the tumor lesions (Fig. 1h, i), positive staining for PTEN was observed in the subcutaneous tissue of nude mice (Fig. 1h, i).Fig. 1

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