Although PTEN mutations and deficiencies might be prevalent at the advanced tumor stage and in therapeutic resistance in many types of human cancer [
12,
28‐
30], lung cancers rarely show mutation of the PTEN gene [
12,
31]. Furthermore, mounting evidence suggests that loss of the PTEN phosphatase activity via the increasing p-PTEN/PTEN ratio might be also crucial for many types of cancer to acquire malignant phenotypes [
17,
27,
32,
33]. Therefore, we first evaluated total PTEN expression level and the phosphorylation level of PTEN in lung cancer cells, by assessing five lung cancer cell lines in which the EMT phenotypes had been previously analyzed [
20]. The data suggested that, among the five lung cancer cell lines, those with low PTEN expression level and a high p-PTEN/PTEN ratio showed a tendency to have a high degree of the EMT phenotypes [
20]. In addition to the above findings, the importance of the tumor microenvironment was suggested by recent studies [
1,
2]. Because H358 cells were suitable for evaluating the underlying mechanisms, by which tumor microenvironment factors might induce EMT [
6,
17,
20,
27], we utilized H358 cells for further experiments in the present study. Evidence of persistent hypoxia has been shown in tumor implant models and pulmonary fibrosis by several reliable approaches based on the detection of hypoxia-related molecules, such as pimonidazole [
6,
34,
35]. Indeed, our immunohistochemical findings in vivo suggested that tumor lesions with persistent hypoxia might show no or little PTEN expression. Because H358ON cells retain endogenous PTEN expression before inoculation into mice (Fig.
1), the decreased expression of PTEN observed in cancer cells indicates that persistent tissue hypoxia might modulate PTEN expression in vivo. It should be noted that phosphorylated PTEN expression was not detected in tumor specimens, possibly owing to our use of an antibody that is not recommended for immunohistochemistry staining. Although the acquisition of EMT phenotypes is modulated by persistent hypoxia [
3‐
7], persistent hypoxia induces a decrease in total PTEN expression and increases the p-PTEN/PTEN ratio over time in H358 cells incubated under hypoxia. Our in vitro data suggested that persistent hypoxia markedly repressed the expression of total PTEN expression in a time-dependent manner, whereas phosphorylated PTEN levels remained persistent even after hypoxia stimulation, resulting in an approximately eightfold increase in the p-PTEN/PTEN ratio in vitro [
36‐
38]. These findings indicate that modulation of total PTEN expression and the increasing p-PTEN/PTEN ratio might be induced not only by TGFβ activation [
17] but also by persistent hypoxia stimulation in lung cancer cells. Mounting evidence show that β-catenin is located on the cell membrane via E-cadherin complexes [
21,
25,
26]. We previously demonstrated that although β-catenin and E-cadherin were co-localized on the cell membrane when the cells were not treated with TGFβ, TGFβ might induce translocation of β-catenin from the cell membrane to the cytoplasm and nucleus [
27]. Taken together with the previous studies [
21,
25], double immunostaining for β-catenin and E-cadherin suggested that persistent hypoxia might also induce translocation of β-catenin from the cell membrane to the cytoplasm and nucleus resulting in induction of mesenchymal gene expression. Therefore, a therapeutic strategy to comprehensively regulate various factors derived from tumor microenvironment might be warranted. Nevertheless, whether or not hypoxia-induced EMT might be directly affected by the increasing phosphorylation levels of the PTEN C-terminus has not been fully evaluated. In the present study, compensatory induction of unphosphorylated PTEN blunted hypoxia-induced EMT in lung cancer cells. Although persistent hypoxia-induced HIF-1α stabilization can cause the acquisition of malignant phenotypes [
5,
7], PTEN4A did not appear to modulate hypoxia-induced HIF-1α stabilization, indicating that the compensatory induction of PTEN4A inhibited the acquisition of hypoxia-induced EMT phenotypes by mechanisms other than HIF-1α stabilization [
39]. Although hypoxia is known to induce de novo twist expression [
5,
6], our data suggested that compensatory induction of either PTEN4A or WildPTEN induced the decreasing levels of hypoxia-induced twist gene. De novo expressed GFP-WildPTEN protein decreased the fibronectin/E-cadherin ratio and expression of twist in the cells under persistent hypoxia. Meanwhile, it did not block hypoxia-induced β-catenin translocation from the cell membrane into the cytoplasm and nucleus. A recent study suggests that fibronectin transcription is induced by β-catenin translocation from E-cadherin complexes at the cell membrane into the cytoplasm and nucleus [
21]. Indeed, WildPTEN did not appear to repress hypoxia-induced fibronectin expression, whereas repression of hypoxia-induced E-cadherin was restored by induction of WildPTEN. PTEN binds E-cadherin complex via the PDZ binding domain in the PTEN C-terminus [
40]. Previous studies suggested that total PTEN expression levels might affect the response to WildPTEN transduction in glioma cells [
28‐
30]. In the present study, we showed that PTEN protein was abundantly expressed in H358 cells under normoxia, whereas persistent hypoxia induced the repression of total PTEN expression in H358 cells to the same degree as that in H1299 cells. Taken together, compensatory induction of WildPTEN might restore the repression of E-cadherin expression in hypoxia-induced H358 cells showing little expression of PTEN. Thus, exogenous administration of the PTEN gene might be hopeful for treatment of lung cancer under persistent hypoxia. It should be noted that de novo GFP-PTEN4A protein expression showed an approximately 40 % greater potential to block hypoxia-induced EMT in comparison with de novo GFP-WildPTEN protein expression. We demonstrated that unphosphorylated PTEN, but not WildPTEN, inhibits TGFβ-induced EMT [
17]. Research into the role of unphosphorylated PTEN in EMT induced by tumor microenvironmental factors remains important [
27].
In summary, our studies strengthen the therapeutic possibility that compensatory induction of unphosphorylated PTEN may inhibit the acquisition of EMT phenotypes in lung cancers under tissue microenvironments involving persistent hypoxia stimulation.