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Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells.

Hong KO, Kim JH, Hong JS, Yoon HJ, Lee JI, Hong SP, Hong SD - J. Exp. Clin. Cancer Res. (2009)

Bottom Line: Inhibition of Akt activity by PIA decreased NF-kappaB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells.In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression.All of these findings suggest that Akt inhibition could induce the MErT through decreased NF-kappaB signaling and downregulation of Snail and Twist in OSCC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea. hongko95@hanmail.net

ABSTRACT

Background: The Akt/PKB family of kinases is frequently activated in human cancers, including oral squamous cell carcinoma (OSCC). Akt-induced epithelial-to-mesenchymal transition (EMT) involves downregulation of E-cadherin, which appears to result from upregulation of the transcription repressor Snail. Recently, it was proposed that carcinoma cells, especially in metastatic sites, could acquire the mesenchymal-to-epithelial reverting transition (MErT) in order to adapt the microenvironments and re-expression of E-cadherin be a critical indicator of MErT. However, the precise mechanism and biologic or clinical importance of the MErT in cancers have been little known. This study aimed to investigate whether Akt inhibition would restore the expression of E-cadherin and beta-catenin, reduce that of Vimentin, and induce the MErT in OSCC cells with low or negative expression of E-cadherin. We also investigate whether inhibition of Akt activity would affect the E-cadherin repressors and signaling molecules like NF-kappaB, ERK, and p38.

Methods: We screened several OSCC cell lines in order to select suitable cell line models for inducing MErT, using immunoblotting and methylation specific-PCR. We examined whether Akt inhibitor phosphatidylinositol ether lipid analogues (PIA) treatment would restore the expression of E-cadherin and beta-catenin, reduce that of Vimentin, and induce the MErT in KB and KOSCC-25B cells using RT-PCR, immunoblotting, immunofluorescence analysis, and in vitro migration assay. We also investigated whether inhibition of Akt activity would affect the E-cadherin repressors, including Snail, Twist, and SIP-1/ZEB-2 and signaling molecules like NF-kappaB, ERK, JNK, and p38 using RT-PCR, immunoblotting, and immunofluorescence analysis.

Results: Of the 7 OSCC cell lines, KB and KOSCC-25B showed constitutively activated phosphorylated Akt and low or negative expression of E-cadherin. Inhibition of Akt activity by PIA decreased NF-kappaB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells. Akt inhibition led to downregulation of Snail and Twist expression. In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression. PIA treatment induced the expression of E-cadherin and beta-catenin, reduce that of Vimentin, restored their epithelial morphology of a polygonal shape, and reduced tumor cell migration in KB and KOSCC-25B cells, which was the corresponding feature of MErT.

Conclusion: All of these findings suggest that Akt inhibition could induce the MErT through decreased NF-kappaB signaling and downregulation of Snail and Twist in OSCC cells. A strategy involving Akt inhibition might be a useful therapeutic tool in controlling cancer dissemination and metastasis in oral cancer patients.

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Effects of Akt inhibition on the localization of the epithelial and mesenchymal markers. The inhibition of Akt activity induced the localization of E-cadherin and β-catenin, and decreased that of vimentin, as seen in the immunofluorescence analysis.
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Figure 5: Effects of Akt inhibition on the localization of the epithelial and mesenchymal markers. The inhibition of Akt activity induced the localization of E-cadherin and β-catenin, and decreased that of vimentin, as seen in the immunofluorescence analysis.

Mentions: KOSCC-25B cells had an elongated shape, assuming a fibroblast-like appearance. In contrast, PIA treatment of the cells seemed to restore their epithelial morphology of a polygonal shape (Fig. 4A upper panel). In phalloidin staining, KOSCC-25B cells demonstrated circumferential, cortical actin, and actin in elongated filopodia; however, no actin stress fibers were detected. In contrast, PIA-treated cells revealed an abudance of actin stress fibers (Fig. 4A lower panel). These results showed that PIA treatment of the cells induced actin cytoskeleton reorganization, which contributed to loss of the migratory phenotype. We examined whether PIA treatment could affect the expression and localization of E-cadherin and β-catenin, epithelial markers, and Vimentin, a mesenchymal marker. In accordance with the observed morphologic change, inhibition of Akt activity induced the expression in immunoblotting and RT-PCR (Fig. 4B) and localization of E-cadherin and β-catenin as seen in the immunofluorescence analysis (Fig. 5 upper and middle panel). Also, PIA treatment decreased the vimentin expression (Fig. 4B) or localization (Fig. 5 lower panel), although the change was not as prominent as that in the epithelial markers.


Inhibition of Akt activity induces the mesenchymal-to-epithelial reverting transition with restoring E-cadherin expression in KB and KOSCC-25B oral squamous cell carcinoma cells.

Hong KO, Kim JH, Hong JS, Yoon HJ, Lee JI, Hong SP, Hong SD - J. Exp. Clin. Cancer Res. (2009)

Effects of Akt inhibition on the localization of the epithelial and mesenchymal markers. The inhibition of Akt activity induced the localization of E-cadherin and β-catenin, and decreased that of vimentin, as seen in the immunofluorescence analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651854&req=5

Figure 5: Effects of Akt inhibition on the localization of the epithelial and mesenchymal markers. The inhibition of Akt activity induced the localization of E-cadherin and β-catenin, and decreased that of vimentin, as seen in the immunofluorescence analysis.
Mentions: KOSCC-25B cells had an elongated shape, assuming a fibroblast-like appearance. In contrast, PIA treatment of the cells seemed to restore their epithelial morphology of a polygonal shape (Fig. 4A upper panel). In phalloidin staining, KOSCC-25B cells demonstrated circumferential, cortical actin, and actin in elongated filopodia; however, no actin stress fibers were detected. In contrast, PIA-treated cells revealed an abudance of actin stress fibers (Fig. 4A lower panel). These results showed that PIA treatment of the cells induced actin cytoskeleton reorganization, which contributed to loss of the migratory phenotype. We examined whether PIA treatment could affect the expression and localization of E-cadherin and β-catenin, epithelial markers, and Vimentin, a mesenchymal marker. In accordance with the observed morphologic change, inhibition of Akt activity induced the expression in immunoblotting and RT-PCR (Fig. 4B) and localization of E-cadherin and β-catenin as seen in the immunofluorescence analysis (Fig. 5 upper and middle panel). Also, PIA treatment decreased the vimentin expression (Fig. 4B) or localization (Fig. 5 lower panel), although the change was not as prominent as that in the epithelial markers.

Bottom Line: Inhibition of Akt activity by PIA decreased NF-kappaB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells.In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression.All of these findings suggest that Akt inhibition could induce the MErT through decreased NF-kappaB signaling and downregulation of Snail and Twist in OSCC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea. hongko95@hanmail.net

ABSTRACT

Background: The Akt/PKB family of kinases is frequently activated in human cancers, including oral squamous cell carcinoma (OSCC). Akt-induced epithelial-to-mesenchymal transition (EMT) involves downregulation of E-cadherin, which appears to result from upregulation of the transcription repressor Snail. Recently, it was proposed that carcinoma cells, especially in metastatic sites, could acquire the mesenchymal-to-epithelial reverting transition (MErT) in order to adapt the microenvironments and re-expression of E-cadherin be a critical indicator of MErT. However, the precise mechanism and biologic or clinical importance of the MErT in cancers have been little known. This study aimed to investigate whether Akt inhibition would restore the expression of E-cadherin and beta-catenin, reduce that of Vimentin, and induce the MErT in OSCC cells with low or negative expression of E-cadherin. We also investigate whether inhibition of Akt activity would affect the E-cadherin repressors and signaling molecules like NF-kappaB, ERK, and p38.

Methods: We screened several OSCC cell lines in order to select suitable cell line models for inducing MErT, using immunoblotting and methylation specific-PCR. We examined whether Akt inhibitor phosphatidylinositol ether lipid analogues (PIA) treatment would restore the expression of E-cadherin and beta-catenin, reduce that of Vimentin, and induce the MErT in KB and KOSCC-25B cells using RT-PCR, immunoblotting, immunofluorescence analysis, and in vitro migration assay. We also investigated whether inhibition of Akt activity would affect the E-cadherin repressors, including Snail, Twist, and SIP-1/ZEB-2 and signaling molecules like NF-kappaB, ERK, JNK, and p38 using RT-PCR, immunoblotting, and immunofluorescence analysis.

Results: Of the 7 OSCC cell lines, KB and KOSCC-25B showed constitutively activated phosphorylated Akt and low or negative expression of E-cadherin. Inhibition of Akt activity by PIA decreased NF-kappaB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells. Akt inhibition led to downregulation of Snail and Twist expression. In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression. PIA treatment induced the expression of E-cadherin and beta-catenin, reduce that of Vimentin, restored their epithelial morphology of a polygonal shape, and reduced tumor cell migration in KB and KOSCC-25B cells, which was the corresponding feature of MErT.

Conclusion: All of these findings suggest that Akt inhibition could induce the MErT through decreased NF-kappaB signaling and downregulation of Snail and Twist in OSCC cells. A strategy involving Akt inhibition might be a useful therapeutic tool in controlling cancer dissemination and metastasis in oral cancer patients.

Show MeSH
Related in: MedlinePlus