Dysferlin-deficient myotubes show tethering of different membrane compartments characterized by TMEM16E and DHPRα.

TMEM16E deficiency has been shown to be responsible for human limb-girdle muscular dystrophy LGMD2L. We found that endogenous TMEM16E co-localized with caveolin-3 at cytoplasmic vesicular compartments in a myotube from C2C12 cells (C2C12 myotube) without forming a molecular complex. In contrast, a myotube from murine myoblastic dysferlin-deficient GREG cells (GREG myotube) showed not only co-localization but also constitutive association of caveolin-3 and TMEM16E. GREG myotubes also displayed constitutive association of TMEM16E with DHPRα, which reside in different membrane compartments, indicating increased contact of the different vesicular membrane compartments. Τhese results suggest that a dynamic tethering of different membrane compartments might represent a distorted membrane damage repairing process in the absence of dysferlin.

TMEM16E (GDD1) exhibits protein instability and distinct characteristics in chloride channel/pore forming ability.

TMEM16E/GDD1 has been shown to be responsible for the bone-related late-onset disease gnathodiaphyseal dysplasia (GDD), with the dominant allele (TMEM16E(gdd) ) encoding a missense mutation at Cys356. Additionally, several recessive loss-of-function alleles of TMEM16E also cause late-onset limb girdle muscular dystrophy. In this study, we found that TMEM16E was rapidly degraded via the proteasome pathway, which was rescued by inhibition of the PI3K pathway and by the chemical chaperone, sodium butyrate. Moreover, TMEM16E(gdd) exhibited lower stability than TMEM16E, but showed similar propensity to be rescued. TMEM16E did not exhibit cell surface calcium-dependent chloride channel (CaCC) activity, which was originally identified in TMEM16A and TMEM16B, due to their intracellular vesicle distribution. A putative pore-forming domain of TMEM16E, which shared 39.8% similarity in 98 amino acids with TMEM16A, disrupted CaCC activity of TMEM16A via domain swapping. However, the Thr611Cys mutation in the swapped domain, which mimicked conserved cysteine residues between TMEM16A and TMEM16B, reconstituted CaCC activity. In addition, the GDD-causing cysteine mutation made in TMEM16A drastically altered CaCC activity. Based on these findings, TMEM16E possesses distinct function other than CaCC and another protein-stabilizing machinery toward the TMEM16E and TMEM16E(gdd) proteins should be considered for the on-set regulation of their phenotypes in tissues.

Expression and function of RIG-I in oral keratinocytes and fibroblasts.

BACKGROUND: Innate immune response by oral mucosal cells may be the first line of host defense against viral infection. Retinoic acid-inducible gene-I (RIG-I) recognizes viral dsRNA in the cytoplasm, and RIG-I-mediated signaling regulates antiviral type I IFN, and inflammatory chemokine production. Here, we tested the hypothesis that oral mucosal cell participation in host defense against viral infection via RIG-I. METHODS: RIG-I expression was detected in immortalized oral keratinocytes (RT7), oral fibroblasts (GT1) using and RT-PCR and immunohistochemistry. RT7 and GT1 were exposed to dsRNA virus mimic Poly I:C-LMW/LyoVec (PLV). Expression of IFN-ß and CXCL10 via RIG-I was examined by Real-time RT-PCR and ELISA. Phosphorylation of IRF3 and STAT1 were detected by western blotting. RESULTS: RT7 and GT1 constitutively expressed RIG-I in the cytoplasm. Furthermore, PLV increased IFN-ß and CXCL10 productions in both RT7 and GT1 via RIG-I concurrent with phosphorylation of IRF3 and STAT1. PLV-induced CXCL10 production was attenuated by neutralization of IFN-ß and blocking of IFN-α/ß receptor (IFNAR), indicating primal IFN-ß production via the RIG-I-IRF3 axis, which eventually induces CXCL10 production via the IFNAR -STAT1 axis. CONCLUSION: We propose that RIG-I in oral keratinocytes and fibroblasts may cumulatively develop host-defense mechanisms against viral infection in oral mucosa.

Maintenance of stem cell self-renewal in head and neck cancers requires actions of GSK3ß influenced by CD44 and RHAMM.

Cells sorted from head and neck cancers on the basis of their high expression of CD44 have high potency for tumor initiation. These cells are also involved in epithelial to mesenchymal transition (EMT) and we have previously reported that cancer stem cells (CSCs) exist as two biologically distinct phenotypes. Both phenotypes are CD44(high) but one is also ESA(high) and maintains epithelial characteristics, the other is ESA(low) , has mesenchymal characteristics and is migratory. Examining CD44-regulated signal pathways in these cells we show that CD44, and also RHAMM, act to inhibit phosphorylation of glycogen synthase kinase 3ß (GSK3ß). We show that inhibitory phosphorylation reduces the formation of both "tumor spheres" and "holoclone" colonies, functional indicators of stemness. GSK3ß inhibition also reduces the expression of stem cell markers such as Oct4, Sox2, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin in the CD44(high) /ESA(high) cell fraction. Transition of CSCs out of EMT and back to the epithelial CSC phenotype is induced by GSK3ß knockdown. These results indicate that GSK3ß plays a central role in determining and maintaining the phenotypes and behavior of CSCs in vitro and are likely to be involved in controlling the growth and spread of tumors in vivo.

Aesthetic recovery of alveolar atrophy following autogenous onlay bone grafting using interconnected porous hydroxyapatite ceramics (IP-CHA) and resorbable poly-L-lactic/polyglycolic acid screws: case report.

BACKGROUND: Onlay bone grafting techniques have some problems related to the limited volume of autogenous grafted bone and need for surgery to remove bone fixing screws. Here, we report a case of horizontal alveolar ridge atrophy following resection of a maxillary bone cyst, in which autogenous onlay bone grafting with interconnected porous hydroxyapatite ceramics (IP-CHA) and bioresorbable poly-L-lactic/polyglycolic acid (PLLA-PGA) screws was utilized. CASE PRESENTATION: A 51-year-old man had aesthetic complications related to alveolar atrophy following maxillary bone cyst extraction. We performed onlay grafting for aesthetic alveolar bone recovery using IP-CHA to provide adequate horizontal bone volume and PLLA-PGA screws for bone fixing to avoid later damage to host bone during surgical removal. During the operation, an autogenous cortical bone block was collected from the ramus mandibular and fixed to the alveolar ridge with PLLA-PGA screws, then the gap between the bone block and recipient bone was filled with a granular type of IP-CHA. Post-surgery orthopantomograph and CT scan findings showed no abnormal resorption of the grafted bone, and increased radiopacity, which indicated new bone formation in the area implanted with IP-CHA. CONCLUSION: Our results show that IP-CHA and resorbable PLLA-PGA screws are useful materials for autogenous onlay bone grafting.

AKT primes snail-induced EMT concomitantly with the collective migration of squamous cell carcinoma cells.

In this study, we found that wounding of a confluent monolayer of squamous cell carcinoma (SCC) cells induced epithelial-mesenchymal transition (EMT) specifically at the edge of the wound. This process required the combined stimulation of TGFß, TNFα, and PDGF-D. Such a combined cytokine treatment of confluent monolayers of the cells upregulated the expression levels of Snail and Slug via PI3K. The PI3K downstream effector, AKT, was dispensable for the upregulation of Snail and Slug, but essential for enabling EMT in response to upregulation of Snail and Slug.

Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies.

The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins.

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and ß5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.

Interleukin-8 and CXCL10 expression in oral keratinocytes and fibroblasts via Toll-like receptors.

Oral keratinocytes and fibroblasts may be the first line of host defense against oral microorganisms. Here, the contention that oral keratinocytes and fibroblasts recognize microbial components via Toll-like receptors (TLRs) and participate in development of oral inflammation was examined. It was found that immortalized oral keratinocytes (RT7), fibroblasts (GT1) and primary cells express mRNA of TLRs 1-10. Interleukin-8 (IL-8) production by RT7 cells was induced by treatment with TLRs 1-9 with the exception of TLR7 agonist, whereas GT1 cells were induced to produce IL-8 by all TLR agonists tested except for TLR7 and TLR9. GT1 cells showed increased CXCL10 production following treatment with agonists for TLR1/2, TLR3, TLR4, and TLR5, whereas only those for TLR3 and TLR5 increased CXCL10 production in RT7 cells. Moreover, TLR agonists differentially regulated tumor necrosis factor-alpha-induced IL-8 and CXCL10 production by the tested cell types. These findings suggest that recognition of pathogenic microorganisms in oral keratinocytes and fibroblasts by TLRs may have important roles in orchestrating host immune responses via production of various chemokines.

Overexpression of receptor for hyaluronan-mediated motility (RHAMM) in MC3T3-E1 cells induces proliferation and differentiation through phosphorylation of ERK1/2.

Receptor for hyaluronan (HA)-mediated motility (RHAMM) was first described as a soluble HA binding protein released by sub-confluent migrating cells. We previously found that RHAMM was highly expressed and plays an important role in proliferation in the human cementifying fibroma (HCF) cell line, which we previously established. HCF is a benign fibro-osseous neoplasm of the jaw and is composed of fibrous tissue containing varying amounts of mineralized material. However, the pathogenesis of HCF is not clear. In this paper, we examined the roles of RHAMM in osteoblastic cells. We generated RHAMM-overexpressing MC3T3-E1 cells and examined the cell proliferation and differentiation of osteoblastic cells. In MC3T3-E1 cells, overexpressing RHAMM was located intracellular and activated ERK1/2. Interestingly, the ERK1/2 activated by RHAMM overexpression promoted cell proliferation and suppressed the differentiation of osteoblastic cells. Our findings strongly suggest that RHAMM may play a key role in the osteoblastic differentiation process. The rupture of balance from differentiation to proliferation induced by RHAMM overexpression may link to the pathogenesis of bone neoplasms such as HCF.

Dexamethasone resets stable association of nuclear Snail with LSD1 concomitant with transition from EMT to partial EMT.

Cancer cells utilize epithelial to mesenchymal transition (EMT) during invasion and metastasis. This program has intermediate cell states with retained epithelial and gained mesenchymal features together, referred to as partial EMT. Histone demethylase LSD1 forms a complex with the EMT master transcription factor Snail to modify histone marks and regulate target gene expression. However, little is known about the formation of this complex during the Snail-dependent transition between partial EMT and EMT. Here we visualized the nuclear complex of Snail and LSD1 as foci signals using proximity ligation assay. We demonstrated that the nuclear foci numbers varied with the transition of exogenous Snail-dependent partial EMT to EMT. Furthermore, we found that long exposure to dexamethasone could revert exogenous Snail-dependent EMT to partial EMT. In this reversion, the nuclear foci numbers also returned to previous levels. Therefore, we concluded that Snail might select partial EMT or EMT by altering its association with LSD1.

RHAMM/ERK interaction induces proliferative activities of cementifying fibroma cells through a mechanism based on the CD44-EGFR.

We have previously established immortalized cells (HCF) from cementifying fibroma of the jaw bone. Here, we found that the receptor for hyaluronan (HA)-mediated motility (RHAMM) and epiregulin, a ligand for the epidermal growth factor receptor (EGFR), were highly expressed in HCF cells in comparison with osteoblasts by conducting a microarray analysis. The cell growth of HCF cells was significantly decreased by the knockdown of RHAMM using small interfering RNA (siRNA). RHAMM was associated with extracellular signal-regulated kinase (ERK) and essential for ERK phosphorylation. HCF cells had characteristic growth mechanisms in which epiregulin functions in an extracellular autocrine loop. Interestingly, exogenous HA induced the phosphorylation of EGFR, which was mainly dependent on CD44. The results raise the novel idea that the EGFR may activate Raf-MEK-ERK signaling in response to the binding of HA to CD44. Moreover, RHAMM was able to associate with TPX2 in the nucleus and was required for HA-induced activation of the Aurora A kinase. The results suggest that RHAMM has a predominant role in the cell cycle in HCF. Here, we report the new machinery by which RHAMM/ERK interaction induces the proliferative activity of cementifying fibroma cells via a specific signaling pathway through the CD44-EGFR axis.

The squamous cell carcinoma cell line OM-1 retains both p75-dependent stratified epithelial progenitor potential and cancer stem cell properties.

The low-affinity nerve growth factor receptor p75 is a stratified epithelial stem/progenitor marker of human epithelia. We found OM-1, a human squamous cell carcinoma (SCC) cell line, showed distinct cells with p75 cluster, especially located at the center of a growing colony in a monolayer culture. A cell with p75 cluster was surrounded by cytokeratin 14- and cytokeratin 13-expressing cells that settled at the outer margin of the colony. OM-1 cells were also capable of forming tumor spheres in a cell suspension culture, an ability which was attenuated by the inhibition of p75-signaling. Intriguingly, we also found a p75-negative cell population from a growing culture of OM-1 that re-committed to become p75-clustering cells. These results indicated the possibility that SCC with epithelial multi-layering capacity can exploit the p75-dependent stratified epithelial progenitor property for the cancer stemness.

Nodal lymphangiogenesis and metastasis: Role of tumor-induced lymphatic vessel activation in extramammary Paget's disease.

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget's disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

DeltaNp63alpha-dependent expression of Id-3 distinctively suppresses the invasiveness of human squamous cell carcinoma.

p63 is a member of the p53 family and DeltaNp63alpha is the dominant-expressing isoform of p63 in basal layer of normal stratified epithelium and human squamous cell carcinoma (SCC) cells. We have previously reported that down-regulation of p63 was accompanied with epithelial-to-mesenchymal transition (EMT) by Snail-expressing SCC cells, in which re-expression of DeltaNp63alpha diminished their invasiveness (Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N. Snail-induced down-regulation of DeltaNp63alpha acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 2007;67:9207-13). In this study, we found that DeltaNp63alpha positively regulated inhibitor of differentiation-3 (Id-3) expression. Id is a dominant negative regulator of E2A which is a transcriptional repressor of E-cadherin. Enforced expression of Id-3 was incapable of invoking E-cadherin expression in the SCC cells with EMT phenotype, whereas it significantly impaired their invasiveness with down-regulation of matrix-metalloproteinase-2 (MMP-2) expression. Reporter gene assay revealed that the Ets-1-induced MMP-2 promoter activity was suppressed by the Id-3, while the Id-3-dependent E-cadherin promoter activity was remarkably reduced in the presence of Snail. Furthermore, knockdown of p63 in SCC cells significantly decreased Id-3 expression, in which up-regulation of MMP-2 expression was concomitant with the acquired invasiveness. These findings propose a particular role of the off-signaling of the DeltaNp63alpha-Id-3 axis incident to Snail-mediated EMT for the MMP-2-dependent invasiveness in SCC cells.

Oncogenic role of nuclear accumulated Aurora-A.

Aurora-A, also known as Aik, BTAK, or STK15, is a centrosomal serine/threonine protein kinase, which is proto-oncogenic and is overexpressed in a wide range of human cancers. Besides gene amplification and mRNA overexpression, proteolytic resistance mechanisms are thought to contribute to overexpression of Aurora-A. However, it is not yet clear how overexpressed Aurora-A affects the expression of transformed phenotype. Here, we found that nuclear accumulation of Aurora-A was critical for transformation activity. Cellular protein fractionation experiments and immunoblot analysis demonstrated a predominance of Aurora-A in the nuclear soluble fraction in head and neck cancer cells. Indirect immunofluorescence using confocal laser microscopy confirmed nuclear Aurora-A in head and neck cancer cells, while most oral keratinocytes exhibited only centrosomal localization. The expression of nuclear export signal-fused Aurora-A demonstrated that the oncogenic transformation activity was lost on disruption of the nuclear localization. Thus, the cytoplasmic localization of overexpressed Aurora-A previously demonstrated by immunohistochemical analysis is not likely to correspond to that in intact cancer cells. This study identifies an alternative mode of Aurora-A overexpression in cancer, through nuclear rather than cytoplasmic functions. We suggest that substrates of Aurora-A in the cell nuclear soluble fraction can represent a novel therapeutic target for cancer.

Overexpression of the receptor for hyaluronan-mediated motility, correlates with expression of microtubule-associated protein in human oral squamous cell carcinomas.

Receptor for hyaluronan-mediated motility (RHAMM) has previously been characterized as a cell surface receptor for hyaluronan and a microtubule-associated intracellular hyaluronan binding protein. We examined the expression of RHAMM mRNA in 43 oral squamous cell carcinomas (SCCs) and 7 normal gingivae by real-time RT-PCR. The expression level of RHAMM mRNA was significantly higher in oral SCCs than normal gingivae (P=0.0047). Forty out of 43 oral SCCs showed expression of RHAMM splice variant (48 bp deletion). We immunohistochemically confirmed the protein expression of RHAMM in oral SCCs with higher levels of RHAMM mRNA. Patients with oral SCC who had high RHAMM expression had shorter survival rates than patients with low expression. However, it was not statistically significant. It has been reported that RHAMM interacts with spindle assembly factors such as microtubule-associated protein (TPX2). To investigate the expression of microtubule-associated protein in oral SCCs, mRNA expression of TPX2 was also examined by real-time RT-PCR. The expression level of TPX2 mRNA was significantly higher in oral SCCs than normal gingivae (P=0.046). Furthermore, a significant correlation between the mRNA expression levels of TPX2 and RHAMM was recognized (P=0.011). The results indicate that there is a strong correlation between the mRNA expression levels of TPX2 and RHAMM in oral SCCs. Our observations suggest that the up-regulations of human RHAMM and TPX2 gene correlate with the malignant condition and might be linked to the increased or abnormal cell proliferation in human oral SCCs.

Expression of TPX2 in salivary gland carcinomas.

TPX2 is a microtubule-associated protein and is required for microtubule formation at kinetochores in mammalian cells. The purpose of this study was to clarify the expression of TPX2 mRNA and correlation between TPX2 and clinicopathological factors in salivary gland carcinomas. The expression of TPX2 mRNA was investigated in 20 human salivary gland carcinomas (8 mucoepidermoid carcinomas, 7 adenoid cystic carcinomas, 5 acinic cell carcinomas) and 6 normal submandibular glands using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of TPX2 mRNA was higher in mucoepidermoid carcinomas (0.53+/-0.51) than in normal submandibular glands (0.047+/-0.029); a significant association was found (Mann-Whitney U test, P=0.0067). The mean expression levels of TPX2 were also higher in acinic cell carcinomas (0.45+/-0.49) and adenoid cystic carcinomas (0.28+/-0.22) than in normal submandibular glands. Statistical correlations were found (Mann-Whitney U test, P=0.028 and P=0.003, respectively). Correlation between expression of TPX2 and receptor for hyaluronan-mediated motility (RHAMM) was also investigated in this study. A significant association was found between the mRNA expression levels of TPX and RHAMM (Pearson's correlation coefficient by rank test, P=0.020). These results indicate that human TPX2 mRNA is closely linked to increased or abnormal cell proliferation in malignant salivary gland tumors.

Snail-induced down-regulation of DeltaNp63alpha acquires invasive phenotype of human squamous cell carcinoma.

p63 is a member of the p53 family and regulates crucial events in the formation of epithelial structures, but the role of p63 in tumor is unclear. We found that Snail-induced epithelial-to-mesenchymal transition (EMT) is accompanied by down-regulation of p63 in human squamous cell carcinomas (SCC). DeltaNp63alpha is the predominantly expressed p63 isoform in SCC cells. DeltaNp63 promoter activity required a CAAT/enhancer binding protein (C/EBP) binding element and was reduced remarkably by Snail. Down-regulation of DeltaNp63alpha and reduction of C/EBPalpha were observed in EMT phenotype cells, which exhibited invasive activity in vitro. p63 knockdown in cells enhanced invasive activity in the presence of E-cadherin. Conversely, forced expression of DeltaNp63alpha blocked invasive activity of cells with the EMT phenotype. These findings indicate that Snail down-regulates DeltaNp63alpha, leading to acquisition of the invasive phenotype by SCC. The invasive activity caused by down-regulation of DeltaNp63alpha does not require down-regulation of E-cadherin.

Gene expression profiling to identify genes associated with high-invasiveness in human squamous cell carcinoma with epithelial-to-mesenchymal transition.

The process of epithelial-to-mesenchymal transition (EMT) involves the acquisition of high-invasiveness by tumor. Snail represses target genes and induces EMT. In this study, we defined the signatures of gene expressions by cDNA microarray analyses in both human squamous cell carcinoma (SCC) cell lines with spontaneous EMT and with Snail-induced EMT, which exhibited high-invasive behavior in vitro. Of the 17,000 cDNA probes, 61 genes were found differentially expressed with >2- or <0.5-fold ratio and shared among the EMT phenotype cell lines, indicating candidates for invasion-associated genes regulated by Snail. Category analysis showed that these genes were mainly classified as development/differentiation, metabolism, apoptosis, angiogenesis and cell adhesion. These data illustrated that Snail regulates various molecular pathways for the establishment of EMT and the acquisition of high-invasiveness in SCC cells, yielding insight into the progression of SCC.