Daisuke Aoyama

Involvement of the transcription factor twist in phenotype alteration through epithelial–mesenchymal transition in lung cancer cells†

Epithelial–mesenchymal transition (EMT), which involves the persistent loss of epithelial markers and expression of mesenchymal markers, is assumed to have a critical role in not only tissue development during embryogenesis but also central mechanisms that enhance the invasive and metastatic ability of cancer cells. Twist has been identified to play an essential role in EMT‐mediated tumor invasion and metastasis. Although recent studies suggest that twist expression levels in tissue specimens of lung cancer might be associated with prognosis, the expression of twist in lung cancer cells itself and its effect have not been fully evaluated. Here, we evaluated twist expression and its effect on phenotype alteration in lung cancer cell lines. Twist expression varied among human lung cancer cell lines. The lung cancer cell lines with high twist expression also tended to show a high vimentin/E‐cadherin ratio, which was supported by a migration assay, in which high twist expression gave rise to high cell motility. Furthermore, in comparison to control cells, the lung cancer cells with ectopic expression of twist showed a significant phenotype alteration through EMT and an increasing ability to migrate in vitro, in part, due to a tenfold increase in matrix metalloproteinases activity and almost a 60% increase in modulation of focal adhesion kinase activity, although a contribution of microRNA appeared unlikely in our study. Our present analysis of twist expression in lung cancer provide clues to comprehensive understanding of the mechanisms, by which metastasis often develops in lung cancer.

Differential modulation of surfactant protein D under acute and persistent hypoxia in acute lung injury

Hypoxia contributes to the development of fibrosis with epithelial-mesenchymal transition (EMT) via stimulation of hypoxia-inducible factor 1α (HIF-1α) and de novo twist expression. Although hypoxemia is associated with increasing levels of surfactant protein D (SP-D) in acute lung injury (ALI), the longitudinal effects of hypoxia on SP-D expression in lung tissue injury/fibrosis have not been fully evaluated. Here, the involvement of hypoxia and SP-D modulation was evaluated in a model of bleomycin-induced lung injury. We also investigated the molecular mechanisms by which hypoxia might modulate SP-D expression in alveolar cells, by using a doxycycline (Dox)-dependent HIF-1α expression system. Tissue hypoxia and altered SP-D levels were present in bleomycin-induced fibrotic lesions. Acute hypoxia induced SP-D expression, supported by the finding that Dox-induced expression of HIF-1α increased SP-D expression. In contrast, persistent hypoxia repressed SP-D expression coupled with an EMT phenotype and twist expression. Long-term expression of HIF-1α caused SP-D repression with twist expression. Ectopic twist expression repressed SP-D expression. The longitudinal observation of hypoxia and SP-D levels in ALI in vivo was supported by the finding that HIF-1α expression stabilized by acute hypoxia induced increasing SP-D expression in alveolar cells, whereas persistent hypoxia induced de novo twist expression in these cells, causing repression of SP-D and acquisition of an EMT phenotype. Thus this is the first study to demonstrate the molecular mechanisms, in which SP-D expression under acute and persistent hypoxia in acute lung injury might be differentially modulated by stabilized HIF-1α expression and de novo twist expression.

Involvement of TGFβ-Induced Phosphorylation of the PTEN C-Terminus on TGFβ-Induced Acquisition of Malignant Phenotypes in Lung Cancer Cells

Transforming growth factor β (TGFβ) derived from the tumor microenvironment induces malignant phenotypes such as epithelial-mesenchymal transition (EMT) and aberrant cell motility in lung cancers. TGFβ-induced translocation of β-catenin from E-cadherin complexes into the cytoplasm is involved in the transcription of EMT target genes. PTEN (phosphatase and tensin homologue deleted from chromosome 10) is known to exert phosphatase activity by binding to E-cadherin complexes via β-catenin, and recent studies suggest that phosphorylation of the PTEN C-terminus tail might cause loss of this PTEN phosphatase activity. However, whether TGFβ can modulate both β-catenin translocation and PTEN phosphatase activity via phosphorylation of the PTEN C-terminus remains elusive. Furthermore, the role of phosphorylation of the PTEN C-terminus in TGFβ-induced malignant phenotypes has not been evaluated. To investigate whether modulation of phosphorylation of the PTEN C-terminus can regulate malignant phenotypes, here we established lung cancer cells expressing PTEN protein with mutation of phosphorylation sites in the PTEN C-terminus (PTEN4A). We found that TGFβ stimulation yielded a two-fold increase in the phosphorylated -PTEN/PTEN ratio. Expression of PTEN4A repressed TGFβ-induced EMT and cell motility even after snail expression. Our data showed that PTEN4A might repress EMT through complete blockade of β-catenin translocation into the cytoplasm, besides the inhibitory effect of PTEN4A on TGFβ-induced activation of smad-independent signaling pathways. In a xenograft model, the tumor growth ratio was repressed in cells expressing PTEN4A. Taken together, these data suggest that phosphorylation sites in the PTEN C-terminus might be a therapeutic target for TGFβ-induced malignant phenotypes in lung cancer cells.

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

BackgroundPersistent 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.MethodsWestern 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.ResultsLung 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.ConclusionOur 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.

Direct regulation of transforming growth factor β-induced epithelial-mesenchymal transition by the protein phosphatase activity of unphosphorylated PTEN in lung cancer cells.

Transforming growth factor β (TGFβ) causes the acquisition of epithelial–mesenchymal transition (EMT). Although the tumor suppressor gene PTEN (phosphatase and tensin homologue deleted from chromosome 10) can negatively regulate many signaling pathways activated by TGFβ, hyperactivation of these signaling pathways is observed in lung cancer cells. We recently showed that PTEN might be subject to TGFβ‐induced phosphorylation of its C‐terminus, resulting in a loss of its enzyme activities; PTEN with an unphosphorylated C‐terminus (PTEN4A), but not PTEN wild, inhibits TGFβ‐induced EMT. Nevertheless, whether or not the blockade of TGFβ‐induced EMT by the PTEN phosphatase activity might be attributed to the unphosphorylated PTEN C‐terminus itself has not been fully determined. Furthermore, the lipid phosphatase activity of PTEN is well characterized, whereas the protein phosphatase activity has not been determined. By using lung cancer cells carrying PTEN domain deletions or point mutants, we investigated the role of PTEN protein phosphatase activities on TGFβ‐induced EMT in lung cancer cells. The unphosphorylated PTEN C‐terminus might not directly retain the phosphatase activities and repress TGFβ‐induced EMT; the modification that keeps the PTEN C‐terminus not phosphorylated might enable PTEN to retain the phosphatase activity. PTEN4A with G129E mutation, which lacks lipid phosphatase activity but retains protein phosphatase activity, repressed TGFβ‐induced EMT. Furthermore, the protein phosphatase activity of PTEN4A depended on an essential association between the C2 and phosphatase domains. These data suggest that the protein phosphatase activity of PTEN with an unphosphorylated C‐terminus might be a therapeutic target to negatively regulate TGFβ‐induced EMT in lung cancer cells.

Exogenous induction of unphosphorylated PTEN reduces TGFβ-induced extracellular matrix expressions in lung fibroblasts.

Transforming growth factor β (TGFβ) plays an important role in regulating aberrant extracellular matrix (ECM) production from alveolar/epithelial cells (AECs) and fibroblasts in pulmonary fibrosis. Although the tumor suppressor gene phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively control many TGFβ‐activated signaling pathways via the phosphatase activity, hyperactivation of the TGFβ‐related signaling pathways is often observed in fibrosis. Loss of PTEN expression might cause TGFβ‐induced ECM production. In addition, TGFβ was recently shown to induce loss of PTEN enzymatic activity by phosphorylating the PTEN C‐terminus. Therefore, we hypothesized that exogenous transfer of unphosphorylated PTEN (PTEN4A) might lead to reduce TGFβ‐induced ECM expression in not only epithelial cells but also fibroblasts. Adenovirus‐based exogenous PTEN4A induction successfully reduced TGFβ‐induced fibronectin expression and retained β‐catenin at the cell membrane in human epithelial cells. Exogenous unphosphorylated PTEN also attenuated TGFβ‐induced ECM production and inhibited TGFβ‐induced β‐catenin translocation in a human fibroblast cell line and in mouse primary isolated lung fibroblasts. Conversely, TGFβ‐induced α‐smooth muscle actin expression did not seem to be inhibited in these fibroblasts. Our data suggest that exogenous administration of unphosphorylated PTEN might be a promising strategy to restore TGFβ‐induced loss of PTEN activity and reduce aberrant TGFβ‐induced ECM production from epithelial cells and fibroblasts in lung fibrosis as compared with wild‐type PTEN induction.