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.
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.


CONCLUSIONS
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.
Hong
Kyoung-Ok
KO
Department of Oral Pathology, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea. hongko95@hanmail.net
Kim
Ji-Hong
JH
Hong
Ji-Soo
JS
Yoon
Hye-Jung
HJ
Lee
Jae-Il
JI
Hong
Sam-Pyo
SP
Hong
Seong-Doo
SD
D015820:Cadherins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000096:biosynthesis; D006801:Humans; D007624:KB Cells; D008648:Mesoderm / Q000473:pathology; D009062:Mouth Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis; D009687:Nuclear Proteins / Q000096:biosynthesis; D010716:Phosphatidylinositols / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000096:biosynthesis; D051879:Twist-Related Protein 1 / Q000096:biosynthesis; D014746:Vimentin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000074165:Zinc Finger E-box Binding Homeobox 2
D015820:Cadherins; C108381:GEMIN2 protein, human; D018398:Homeodomain Proteins; D016328:NF-kappa B; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010716:Phosphatidylinositols; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D000071250:Snail Family Transcription Factors; C103173:TWIST1 protein, human; D014157:Transcription Factors; D051879:Twist-Related Protein 1; D014746:Vimentin; C492363:ZEB2 protein, human; D000074165:Zinc Finger E-box Binding Homeobox 2; D051057:Proto-Oncogene Proteins c-akt