Abstract
Recent studies suggest that epithelial–mesenchymal transition (EMT) correlates with cancer metastasis. In addition, there is growing evidence of the association of EMT with cancer stem cells (CSCs). Recently, we showed that the T-box transcription factor BRACHYURY could be a strong regulator of EMT and the CSC phenotype, which were effectively suppressed by a BRACHYURY knockdown in an adenoid cystic carcinoma cell line. In this study, we further tested whether BRACHYURY is a regulator of cancer stemness by means of forced expression of BRACHYURY in oral cancer cell lines. BRACHYURY, SOX2, or both were stably transfected into oral carcinoma cell lines. We analysed these transfectants with respect to self-renewal phenotypes using a sphere-formation assay, and we assessed the expression levels of EMT markers and stem cell markers using real-time reverse transcription-polymerase chain reaction (RT-PCR). Cell migration and invasiveness in vitro were evaluated using a wound healing assay and a tumour cell dissemination assay, respectively. Forced expression of BRACHYURY or SOX2 slightly increased expression of EMT and stem cell markers and the self-renewal phenotype. The expression levels, however, were much lower compared to those of cancer stem cell-like cells. Forced co-expression of BRACHYURY and SOX2 strongly upregulated EMT and stem cell markers and the self-renewal phenotype. Cell migration and invasiveness in vitro were also remarkably enhanced. These synergistic effects increased expression levels of FIBRONECTIN, SNAIL, SLUG, ZEB1, and TGF-β2. In particular, the effects on FIBRONECTIN and TGF-β2 were significant. We found that BRACHYURY and SOX2 synergistically promote cancer stemness in oral cancer cells. This finding points to the importance of gene or protein networks associated with BRACHYURY and SOX2 in the development and maintenance of the CSC phenotype.
Highlights
Metastasis is a multistep cascade involving the migration of tumour cells from their site of origin, evasion from host defence systems, subsequent seeding at distant organs, and growth of secondary tumours
We demonstrated that a cancer stem cells (CSCs)-like cell line, ACCS-M green fluorescence protein (GFP) undergoes epithelial–mesenchymal transition (EMT) [22], and that small hairpin RNA silencing of BRACHYURY downregulates EMT in that cell line and leads to a loss of CSC-like and EMT characteristics of this cell line [11]
We previously reported successful isolation of highly metastatic and tumourigenic CSC-like cells—the ACCS-M GFP cell line—from non-metastatic (0% incidence) and low tumourigenic (22.2% incidence) parental adenoid cystic carcinoma ACCS GFP cells using in vivo selection [22]
Summary
Metastasis is a multistep cascade involving the migration of tumour cells from their site of origin, evasion from host defence systems, subsequent seeding at distant organs, and growth of secondary tumours. The EMT or CSC program appears to be controlled by genes normally expressed in early embryos, including TWIST, SNAIL, SLUG, ZEB1, and SOX2 [12,13,14,15] These genes encode the transcription factors which enables the tumour cells to migrate and invade like mesenchymal cells. Tumourigenesis and metastasis of ACCS-M GFP in vivo were inhibited completely by BRACHYURY knockdown and partially by knockdown of SOX2, the conventional CSC regulator gene [11] These data suggest that EMT may be directly linked to CSCs, and that BRACHYURY controls EMT and the CSC phenotype (cancer stemness). We confirmed that expression of Brachyury protein strongly correlates with EMT and poor prognosis in oral cancer patients [5] In this regard, BRACHYURY silencing could effectively control cancer stemness and offer a new concept for the development of cancer treatments. We used forced expression of BRACHYURY and SOX2 in oral cancer cell lines to confirm that BRACHYURY and SOX2 are regulators of the CSC phenotype
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