During EMT, epithelial cells acquire fibroblast-like properties and exhibit reduced cell-cell adhesion and increased motility. The plasticity afforded by the EMT is central to the complex remodeling of embryo and organ architecture during gastrulation and organogenesis. In pathological processes such as oncogenesis, the EMT may endow cancer cells with enhanced motility and invasiveness. Indeed, oncogenic transformation may be associated with signaling pathways promoting the EMT [22
]. Akt activation is frequent in human epithelial cancer. In our previous study [23
], Akt activation in OSCC was linked to aggressive clinical behavior and the loss of histological features of epithelial differentiation. These findings are consistent with Akt directly affecting epithelial cell morphology, cell motility, and invasiveness.
Grille et al
] demonstrated that OSCC cells engineered to express constitutively active Akt underwent EMT, characterized by downregulation of the epithelial markers desmoplakin, E-cadherin, and beta-catenin, and upregulation of the mesenchymal marker vimentin. The cells also lost their epithelial cell morphology and acquired fibroblast-like properties. In addition, the cells expressing constitutively active Akt exhibited reduced cell-cell adhesion, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals.
Because OSCC cells engineered to express constitutively active Akt have been known to undergo EMT, we tried to examine whether inhibition of Akt activity could restore epithelial characteristics and deplete mesenchymal features. In the present study, PIA treatment induced the expression and cytoplasmic localization of the epithelial markers (E-cadherin and β-catenin). In addition, it decreased the vimentin expression and localization, although the change was not as prominent as that in the epithelial markers. Also, the inhibition of Akt activity restored the polygonal epithelial morphology and reduced the migratory ability. This indicates that the inhibition of Akt activity could induce the MErT in OSCC cells, and that the gain of epithelial characteristic might earlier or more prominent event in the MErT of the OSCC than the loss of mesenchymal one.
Several EMT-inducing developmental regulators repress E-cadherin transcription via interaction with specific E-boxes of the proximal E-cadherin promoter [25
]. The Snail-related zinc-finger transcription factors (Snail and Slug), the (more distantly related) repressor SIP-1/ZEB-2, and the related Snail family member δ EF-1/ZEB1 are the most prominent [27
]. The Snail protein is one of the key molecules in the EMT and its expression is inversely correlated with E-cadherin expression in a number of cancers, including OSCC [31
]. Accordingly, inhibition of Akt activity induced downregulation of EMT-related transcription factor Snail. However, inhibition of Akt activity did not affect the expression level of the SIP-1/ZEB-2. These data suggest that Akt signaling could induce the EMT through activation of Snail, but not SIP-1/ZEB-2, in OSCC cells.
The basic helix-loop-helix transcription factor Twist, a protein known to be essential for initiating mesoderm development during gastrulation, was recently added to the growing list of developmental genes with a key role in E-cadherin repression and EMT induction [34
]. Yang et al
] demonstrated that knockdown of Twist expression by RNAi in a metastatic mammary tumor cell line prevented lung metastasis, and the high levels of Twist expression seen in 70% of invasive lobular breast carcinomas, which display many features of EMT, were inversely correlated with E-cadherin expression. However, there have been no reports on the relationship of Twist with the EMT in oral cancer cells. In the present study, inhibition of Akt activity induced downregulation of EMT-related Twist in OSCC cells. To our knowledge, this study is the first description of the participation of Twist in the EMT/MErT process in oral cancer.
Akt signaling has been deeply studied because Akt plays critical roles in regulating growth, proliferation, survival, metabolism, and other cellular activities [21
]. Chua et al
] showed that NF-κB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin in breast carcinoma cells. Similarly, Julian et al
] reported that activation of NF-κB by Akt upregulates Snail expression and induces EMT in OSCC cells, and expression of the NF-κB subunit p65 is sufficient for EMT induction. We investigated whether it could be possible in the reverse direction, which have been little known. In the present study, inhibition of Akt activity induced the MErT through interaction with NF-κB. Downregulation of NF-κB contributed to MErT. Huber et al
] showed that inhibition of NF-κB signaling prevents EMT in Ras-transformed epithelial cells, while activation of this pathway promotes the transition to a mesenchymal phenotype. Fig. shows a schematic representation of the proposed signaling mechanism that promotes MErT through the inhibition of Akt activity in KB and KOSCC-25B cells. Additional study using NF-κB inhibitors could be needed in order to verify this proposed pathway.
A schematic representation of the proposed signaling mechanism that promotes MErT through the inhibition of Akt activity in oral cancer cells.
In summary, we demonstrated that Akt inhibition by PIA treatment induced downregulation of Snail and Twist expression, upregulation of E-cadherin and β-catenin, downregulation of vimentin, and reduced cell migration, which led to the MErT in oral cancer cells. The MErT in oral cancer cells seems to be acquired through decreased NF-κB signaling. All of these findings suggest that Akt inhibition can induce the MErT through decreased NF-κB 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.