Epithelial-mesenchymal Transition In Oral Squamous Cell Carcinoma Research Articles

M6A-Mediated IRS1 Regulates the Development of Oral Squamous Cell Carcinoma through p53/Line-1 Signaling.

The importance of N6-methyladenosine (m6A) modification in tumorigenesis and progression have been highlighted. This study aimed to investigate the modification of insulin receptor substrate 1 (IRS1) by m6A and its role in oral squamous cell carcinoma (OSCC). Bioinformatics was employed to predict differential genes related to epithelial-mesenchymal transition (EMT) in OSCC. Seventeen pairs of OSCC and paracancerous tissue samples were collected. The impact of IRS1 on OSCC cell growth and EMT was evaluated. The fluctuations in IRS1 enrichment and the involvement of p53/Line-1 were investigated. IRS1 was highly expressed in OSCC. IRS1 silencing decreased OSCC cell proliferation and increased apoptosis. IRS1 silencing hindered EMT by regulating related markers. IRS1 silencing upregulated p53 and downregulated Line-1 ORF1p. The p53 inhibition reversed the effects of IRS1 silencing and induced EMT in OSCC cells. Furthermore, the m6A modification of IRS1 was increased in OSCC cells. IRS1 were positively regulated by the m6A regulators methyltransferase-like 14 (METTL14) and YTH domain-containing protein 1 (YTHDC1). IRS1 bound to YTHDC1, and YTHDC1 knockdown inhibited the IRS1 nuclear export. The obesity-associated protein (FTO) negatively regulated IRS1, and FTO overexpression reversed the IRS1-induced OSCC tumor growth. m6A methylation-mediated IRS1 regulated EMT in OSCC through p53/Line-1. These findings provide potential therapeutic strategies for managing OSCC.

Abstract P50: PAX9 Activates Cell Death via Autophagic Degradation of EGFR in Oral squamous Cell Carcinoma

Abstract PAired boX 9 (PAX9) gene belongs to the PAX family and is critically essential for physiology and development. It has a significant role in maintaining squamous cell differentiation, and its downregulation is associated with tumor initiation and malignant transformation. In this study, we have analyzed the tumor suppressive potential of PAX9 by activating autophagy-dependent apoptosis in oral squamous cell carcinoma (OSCC). PAX9 overexpression inhibited cell growth and triggered apoptosis in OSCC. Moreover, PAX9 restricted orosphere formation and epithelial-mesenchymal transition in OSCC. Similarly, overexpression of PAX9 in OSCC induced autophagy activation and displayed enhanced lysosomal function in the transcriptional-dependent mechanism. Interestingly, PAX9-activated autophagy caused EGFR degradation leading to OSCC inhibition, thus establishing that PAX9 could induce lethal autophagy. In addition, PAX9 involves chemosensitizing OSCC to anticancer drugs, which exhibited profound growth inhibitory potential in OSCC. To check the status of PAX9 expression in OSCC in patient tissue, we performed IHC, and surprisingly, the expression of PAX9 was found to decreased in a higher grade of cancer tissues compared to the normal tissues. Further, we established a DMBA-induced oral carcinoma in hamster model, and it was observed that the expression of PAX9 was decreased in the 12th and 16th weeks of DMBA-exposed tissue compared to the normal tissue. On a mechanistic view, PAX9 expression was reduced with enhanced expression of DNMT1 in exposure to carcinogen in OSCC in vitro, indicating a potential association of methylation in controlling the expression of PAX9 during carcinogenesis. Citation Format: Chandra Sekhar Bhol, Gautam Sethi, Sujit Kumar Bhutia. PAX9 Activates Cell Death via Autophagic Degradation of EGFR in Oral squamous Cell Carcinoma [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P50.

Molecular expression of Forkhead Box C2 gene (FOXC2) and Prospero homeobox gene (PROX-1) in oral squamous carcinoma and their correlation with clinicopathological parameters: A prospective cohort study.

Forkhead box C2 gene (FOXC2) acts as an epithelial-mesenchymal transition (EMT) inducer while Prospero homeobox 1 gene (PROX-1) function as a regulator of lymphangiogenesis and angiogenesis in oral squamous cell carcinoma (OSCC). It is presumed that PROX-1 has both tumour-suppressive and oncogenic effects. The main aim of this study is to evaluate the role of PROX-1 and FOXC2 in the invasion and progression of OSCC cases and to correlate their expression with various histopathological parameters. A prospective cohort study was conducted in a total sample size of 52 OSCC tissues and histologically tumour-free margins of 20. mRNA expression and protein levels of FOXC2 and PROX-1 were evaluated using real-time PCR and sandwich enzyme-linked immunosorbent assay techniques. Chi-square analysis and correlation analysis were done. Kaplan-Meier analysis evaluated the survival rate. Mean Ct values of FOXC2 were 1.915 ± 0.519 and PROX-1 was 0.061 ± 0.173. There was a significant 2-fold increase in the FOXC2 expression and a 0.5-fold decrease in the PROX-1 expression in OSCC tissue. Increased levels of FOXC2 protein and decreased levels of PROX-1 with a mean difference of 1.64 ± 0.73 ng/ml and 1.27 ± 0.33 ng/ml were observed in OSCC compared to histologically tumour-free margins. A significant positive correlation was found between the FOXC2 expression and clinicopathological parameters such as staging, perineural invasion (PNI) and lymphovascular invasion (LVI) whereas PROX-1 showed a significant negative correlation with histopathological parameters such as staging, PNI, LVI and tumour staging. There was a significant positive correlation between the PROX-1 and histologically tumour-free margins in disease-free survival patients (P-value = 0.03). FOXC2 and PROX-1 expressions were correlated with lymphovascular invasion, OSCC tumour staging and PNI. Thus, FOXC2 and PROX-1 could be possible therapeutic targets in the treatment of OSCC that can inhibit the EMT in OSCC and thereby favouring a better prognosis.

RUNX1/miR-429 feedback loop promotes growth, metastasis, and epithelial-mesenchymal transition in oral squamous cell carcinoma by targeting ITGB1.

This study aimed to explore the role of miR-429 on the progression of oral squamous cell carcinoma (OSCC). OSCC cell lines were transfected with miR-429 mimic, pcDNA3.1-RUNX1, or pcDNA3.1-ITGB1, and their cell viability, apoptosis, migration, and invasion abilities were analyzed by cell counting, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, wound healing, and transwell assays, respectively. Furthermore, luciferase reporter assay, RNA pull-down, and ChIP were used to assess the regulation of miR-429, RUNX1, and ITGB1 expression in OSCC. Lastly, the biological role of the RUNX1/miR-429 feedback loop was explored in nude mice. The results revealed that miR-429 level was down-regulated, while RUNX1 and ITGB1 levels were up-regulated in OSCC tissues and that miR-429 was negatively correlated with RUNX1 and ITGB1 in OSCC tissues. Transfection of miR-429 mimic suppressed OSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Moreover, we found that miR-429 participated in OSCC progression by directly targeting ITGB1. Additionally, we found that RUNX1 negatively regulated miR-429 expression by binding to its promoter. Our results also revealed that the RUNX1/miR-429 feedback loop regulated ITGB1 expression and that RUNX1 overexpression rescued the inhibitory effects of miR-429 mimic on OSCC cells. In addition, miR-429 mimic significantly suppressed tumor growth, inflammatory cell infiltration, EMT, and ITGB1 expression in vivo, which were inhibited by RUNX1 overexpression. Altogether, these results indicate that the RUNX1/miR-429 feedback loop promoted growth, metastasis, and EMT in OSCC by targeting ITGB1.

Resveratrol is an inhibitory polyphenol of epithelial-mesenchymal transition induced by Fusobacterium nucleatum

ObjectiveResveratrol is a natural phytoalexin that has anti-inflammatory properties, reverses doxorubicin resistance, and inhibits epithelial-mesenchymal transition (EMT) in many types of cancer cells. Fusobacterium nucleatum is reportedly enriched in oral squamous cell carcinoma (OSCC) tissues compared to adjacent normal tissues, sparking interest in the relationship between F. nucleatum and OSCC. Recently, F. nucleatum was shown to be associated with EMT in OSCC. In the present study, we aimed to investigate the effects of the natural plant compound resveratrol on F. nucleatum-induced EMT in OSCC. DesignF. nucleatum was co-cultured with OSCC cells, with a multiplicity of infection (MOI) of 300:1. Resveratrol was used at a concentration of 10 μM. Cell Counting Kit-8 and wound healing assays were performed to examine the viability and migratory ability of OSCC cells. Subsequently, real-time RT-PCR was performed to investigate the gene expression of EMT-related markers. Western blotting and immunofluorescence analyses were used to further analyze the expression of the epithelial marker E-cadherin and the EMT transcription factor SNAI1. ResultsCo-cultivation with F. nucleatum did not significantly enhance cell viability. The co-cultured cells displayed similarities to the positive control of EMT, exhibiting enhanced migration and expression changes in EMT-related markers. SNAI1 was significantly upregulated, whereas E-cadherin, was significantly downregulated. Notably, resveratrol inhibited F. nucleatum-induced cell migration, decreasing the expression of SNAI1. ConclusionsResveratrol inhibited F. nucleatum-induced EMT by downregulating SNAI1, which may provide a target for OSCC treatment.

GATA6 regulates expression of annexin A10 (ANXA10) associated with epithelial–mesenchymal transition of oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) can disturb oral function and quality of life and is associated with poor survival, likely due to the development of cervical lymph node metastases. Epithelial-mesenchymal transition (EMT) is a process in which cells acquire molecular alterations that facilitate cell motility and invasion, and has been associated with tumor metastasis. EMT changes also play important roles in the induction of lymph node metastasis in OSCC. GATA6 is known as the earliest marker of the primitive endoderm lineages. GATA6 inhibits de-differentiation and EMT in human pancreatic ductal adenocarcinoma cells and promotes EMT. However, in OSCC, the expression and function of GATA6 in EMT and lymph node metastasis remains unclear. Therefore, this study aimed to clarify the targets of GATA6 in OSCC cells and whether the change in GATA6 expression affects EMT in OSCC cells, as well as the association between GATA6 and lymph node metastasis. The results showed that GATA6 knockdown OSCC cells promoted EMT and increased lymph node metastasis compared with control cells, whereas the overexpression of GATA6 inhibited the induction of EMT and reduced lymph node metastasis. In addition, annexin A10 (ANXA10) which is the largest type of Ca2+-regulated phospholipid-binding protein in eukaryotic cells was detected as a target gene for GATA6 and ANXA10 suppressed Vimentin expression in EMT in OSCC. Therefore, the GATA6/ANXA10 cascade may be a potential therapeutic approach for the treatment of lymph node metastases in OSCC patients.

Metformin inhibits epithelial-mesenchymal transition of oral squamous cell carcinoma via the mTOR/HIF-1α/PKM2/STAT3 pathway.

Epithelial-mesenchymal transition (EMT) serves an important role in the formation and development of various types of cancer, including oral squamous cell carcinoma (OSCC). Metformin, used for treating type 2 diabetes, has been revealed to exert an anticancer effect in various types of cancer, including liver, breast and colorectal cancer. However, its role in the EMT of OSCC has been rarely reported. Therefore, the present study aimed to investigate the effects of metformin on EMT and to identify its underlying mechanism in OSCC. Firstly, EMT was induced in CAL-27 cells using CoCl2. Subsequently, the effects of metformin on cell viability, migration and xenograft growth were evaluated in vitro and in vivo. Reverse transcription-quantitative PCR was performed to detect the expression levels of E-cadherin, vimentin, snail family transcriptional repressor 1, mTOR, hypoxia inducible factor 1α, pyruvate kinase M2 and STAT3. The results demonstrated that metformin abolished CoCl2-induced cell proliferation, migration, invasion and EMT. Moreover, metformin reversed EMT in OSCC by inhibiting the mTOR-associated HIF-1α/PKM2/STAT3 signaling pathway. Overall, the present findings characterized a novel mechanism via which metformin modulated EMT in OSCC.

Curcumin inhibits epithelial-mesenchymal transition in oral cancer cells via c-Met blockade.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. OSCC cells are highly invasive, a characteristic that involves epithelial-mesenchymal transition (EMT); the conversion of immotile epithelial cells into motile mesenchymal cells. EMT is involved in the progression of various types of cancer by promoting tumour cell scattering and conferring to these cells cancer stem cell (CSC)-like characteristics, such as self-renewal. Hepatocyte growth factor (HGF) signalling plays an important role in EMT induction and, therefore, contributes to cell invasion and metastasis in cancer. Due to its potential chemopreventative and anti-tumour activities, curcumin has attracted much interest and has been shown to act as a potent EMT inhibitor in various types of cancer. However, at present, the potential effects of curcumin on HGF-induced EMT in OSCC have not been investigated. Here, we demonstrated that HGF signalling could induce EMT in the HSC4 and Ca9-22 OSCC cell lines via the HGF receptor c-Met and downstream activation of the pro-survival ERK pathway. Notably, curcumin inhibited HGF-induced EMT and cell motility in HSC-4 and Ca9-22 cells via c-Met blockade. Therefore, these findings establish curcumin as a candidate drug for OSCC treatment. Furthermore, curcumin was able to effectively inhibit the HGF-induced increase in the levels of vimentin by downregulating the expression of phosphorylated c-Met, an ERK. In conclusion, the results of the present study demonstrated that curcumin was able to reverse HGF-induced EMT, possibly by inhibiting c-Met expression in oral cancer cells, providing a strong basis for the development of novel approaches for the treatment of oral cancer.

Epithelial-mesenchymal transition in oral squamous cell carcinoma: An insight into molecular mechanisms and clinical implications.

Epithelial–mesenchymal transition (EMT) is an important event in embryonic development, fibrosis and cancer invasion. During cancer progression, the activation of EMT permits cancer cells to acquire migratory, invasive and stem-like properties. Despite recent advances in treatment, there is no improvement in the 5-year overall survival rate of oral squamous cell carcinoma (OSCC). Local recurrence and lymph node metastasis are considered to be mainly responsible for the low survival rate in OSCC. EMT plays a major role in local recurrence and lymph node metastasis of oral cancer. This review article addresses the clinical implications of EMT in OSCC and explains the molecular mechanisms of EMT, highlighting the cadherin switching and signaling pathways involved.

Comprehensive Bioinformatics Analysis Reveals that N6-Methyladenosine (m6A) Methylation Reader HNRNPC Facilitates Progression of OSCC Through Promoting Epithelial-Mesenchymal Transition(EMT)

Background: M6A is the most widespread RNA internal modification that contributes to the transcription of RNA-mediated genes. Increasing evidence suggest that m6A has vital role in cancer initiation and progression. However, little is known about its roles in oral squamous cell carcinoma(OSCC). Therefore, this study aimed to explore the prognostic relevance of m6A related genes in OSCC through comprehensive bioinformatics analysis. Materials: Transcriptome expression profile were downloaded from The Cancer Genome Atlas (TCGA) following extracting m6A related genes and long non-coding RNA (lncRNA) based on expression level. The differentially expressed genes between OSCC samples and control samples was analyzed using R software. Furthermore, cluster analysis and principal component analysis (PCA) were used to analyze m6A related genes, and a risk model was constructed on basis of m6A related genes. In addition, co-expression network was established according to m6A related genes and differentially expressed lncRNA (DElncRNA) expression profile and crucial m6A genes were selected based on univariate, multivariate cox regression and survival analysis. Then, CCK-8, colony formation assays, transwell assay and scratch wound healing were performed to explore the biological effects of identified m6A-related genes in OSCC. Furthermore, Western blot assay was conducted to detect the EMT-associated genes expression level. Findings: A total of 13 m6A related genes were extracted and 8 genes were differentially expressed in OSCC. Subsequently, 418 DElncRNA were selected. M6A-Based Clustering Shows 2 Subtypes of OSCC with different clinical outcome.Then, a risk model including HNRNPC, METTL14, YTHDF2, ALKBH5 genes was established on basis of m6A expression profile, which can indicate the survival time and differentiation grade and may be an independent prognostic biomarkers. Furthermore, a co-expression network including 7 m6A related genes and 21 DElncRNA was constructed based on DElncRNA and m6A related genes and HNRNPC was identified as an independent prognostic biomarker in OSCC. Functional studies revealed that HNRNPC knockdown inhibited cell proliferation, migration and invasion of OSCC cells in vitro. Conversely, HNRNPC overexpression showed the opposite biological effects. Moreover, we identified that HNRNPC regulated epithelial-mesenchymal transition in OSCC. Interpretation: This study revealed that m6A may play a significant role in OSCC initiation and progression through integrated bioinformatics analysis. And co-expression network may be helpful for revealing potential correlation of lncRNA and m6A in OSCC. In addition, a risk model was established to predict the prognosis of OSCC.At last, HNRNPC was proved to promote OSCC carcinogenisis and be an independent prognostic biomarker of OSCC, suggesting that it may be a new therapeutic target of OSCC. Funding Statement:This study was supported by the National Natural Science Foundation of China (81472536); the Science and Technology Planning Project of Guangdong Province (No. 2017A020215181 and No. 2014A020212440); Project of Educational Commission of Guangdong Province of China (2018KTSCX026); the Scientific Research Planning Project of Southern Medical University (CX2018N016, PY2018N031) and the Presidential Foundation of the Nanfang Hospital (2014027, 2019Z030). Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: The study protocol was approved by the Ethics Committees of Nanfang Hospital of Guangdong Province (NO: NFEC-2018-027).

LncRNA GAS5 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition in oral squamous cell carcinoma by regulating the miR-21/PTEN axis

Growth-arrest-specific transcript 5 (GAS5) functions as a tumor suppressor in a variety of cancers. GAS5 has been reported to be down-regulated in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the mechanisms of how GAS5 acts as a tumor suppressor in OSCC. qRT-PCR, cell viability, wound-healing, and transwell assays showed that knockdown of GAS5 increased miR-21 expression and promoted proliferation, migration, invasion, and epithelial-mesenchymal transition of OSCC cells. In contrast, overexpression of GAS5 showed the opposite effects. Furthermore, miR-21 overexpression reversed the effect of GAS5. Western blot showed that knockdown of GAS5 suppressed PTEN, while phosphorylation of Akt was promoted. PCNA, cyclinD1, and Ki-67 were up-regulated, indicating enhanced proliferation. E-cadherin was down-regulated, while N-cadherin, vimentin, and snail1 were increased, indicating augmented epithelial-mesenchymal transition. Overexpression of GAS5 regulated these proteins inversely. Overexpression of miR-21 reversed the effect of GAS5 on these proteins. Taken together, GAS5 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition in OSCC through the miR-21/PTEN axis and might be a novel therapeutic target for OSCC.

Targeted silencing of CXCR4 inhibits epithelial-mesenchymal transition in oral squamous cell carcinoma.

Aberrant overexpression of C-X-C chemokine receptor type 4 (CXCR4) is a critical event during tumor metastasis. It has been previously reported that the expression of CXCR4 is linked with epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC) tissues derived from patients. The present study addresses the role of CXCR4 in EMT in tongue squamous cell carcinoma (TSCCA) cells in vitro and in xenograft models. Small interfering (si) RNA sequences targeting the CXCR4 gene were transfected into TSCCA cells. Cell migration, invasion, apoptosis and EMT markers were determined in TSCCA cells using wound healing and Transwell assays, Annexin V/propdidum iodide double staining and western blot analysis, respectively. In vivo, tumor growth was assessed by subcutaneous inoculation of cells into BALB/c nude mice. Phenotypic EMT markers and regulatory factors were detected in the tumor tissues derived from the mice. In vitro, silencing of CXCR4 expression suppressed cell migration and invasion, and induced apoptosis. The protein expression of the EMT-associated markers N-cadherin and matrix metalloproteinases 2/9 were attenuated, while E-cadherin was increased. In vivo, CXCR4 siRNA inhibited tumor growth, and EMT-associated proteins had similar expression patterns to the experimental results observed in vitro. In conclusion, the present study demonstrated that CXCR4 silencing suppressed EMT in OSCC, thus affecting tumor metastasis.

Hypoxia-induced epithelial-mesenchymal transition is regulated by phosphorylation of GSK3-β via PI3 K/Akt signaling in oral squamous cell carcinoma

Epithelial-mesenchymal transition (EMT) plays an important role in cancer invasion and metastasis induced by hypoxia. Here, we examined whether phosphorylation of GSK3-β via phosphoinositide 3-kinase (PI3 K)/Akt signaling is involved in enhancing the hypoxia-induced EMT in oral squamous cell carcinoma (OSCC). Experiments were performed in OSCC cell lines (HSC-2, HSC-3, HSC-4, SAS, and HO-1-U-1) under normoxic or hypoxic conditions. The EMT was assessed by Matrigel invasion assays and wound healing assays. OSCC cell lines (HSC-2 and HSC-4) overexpressing hypoxia-inducible factor (HIF)-1α were established to examine the effects of HIF-1α on EMT-related factors. Immunohistochemical staining was performed to examine phosphorylation of GSK3-β in 33 cases of tongue squamous cell carcinoma. Under hypoxic conditions, OSCC cell lines exhibited HIF-1α expression and showed evidence of the EMT. In cells overexpressing HIF-1α, the levels of phospho-Akt and phospho-GSK3-β were increased, resulting in induction of the EMT. Inhibition of GSK3-β phosphorylation suppressed these effects. Moreover, the intensity of pGSK3-β staining was significantly increased with cN stage and cTNM stage in patients with tongue squamous cell carcinoma. Our data showed that the hypoxia-induced EMT in OSCC was enhanced by GSK3-β phosphorylation, suggesting that GSK3-β may be important in the invasion and metastasis of OSCC.

P120ctn may participate in epithelial-mesenchymal transition in OSCC.

Oral squamous cell carcinoma (OSCC) is the eighth most common cause of cancer death. OSCC cells can easily invade tissues and metastasize to the cervical lymph nodes. Understanding the molecular basis of OSCC metastasis would facilitate the development of new therapeutic approaches to the disease. To identify the potential role of catenin (P120ctn) in the progression of OSCC, HN4 cells (derived from a primary OSCC) and HN12 cells (derived from a lymph node metastasis in the same patient) were used in the present study. The samples of 28 patients with OSCC were examined to determine the expression of P120ctn and E-cadherin (E-cad; E: Epithelial) in vivo. Then, P120ctn was subsequently knocked down in HN4 cells (HN4/shP120ctn) and overexpressed in HN12 cells (HN12/P120ctn). Invasion and migration capacity, as well as the expression of the epithelial-to-mesenchymal transition (EMT) markers N-cadherin and vimentin were detected. The results showed a positive correlation between the expression of P120ctn and E-cad in OSCC samples. The overexpression of P120ctn led to a decrease in both invasion and migration capacity of HN12 cells accompanied by a decrease in EMT markers. In contrast, knockdown of P120ctn led to an increase in both invasion and migration capacity accompanied by an increase in EMT markers. Considered together, we concluded that P120ctn might regulate EMT in OSCC through E-cad. The proper expression of P120ctn might therefore serve as a therapeutic goal for the inhibition of OSCC progression.

Loss of VHL expression contributes to epithelial–mesenchymal transition in oral squamous cell carcinoma

Loss of Von Hippel-Lindau (VHL) gene expression has been implicated in the development of human cancers. However, its function in oral squamous cell carcinoma (OSCC) remains undefined. The aim of this study was to clarify the VHL expression in OSCC and to explore the underlying mechanisms of VHL in modulating the epithelial-mesenchymal transition (EMT) in OSCC. The expression of VHL, HIF-1α and EMT related proteins in OSCC tissues were evaluated by immunohistochemistry. The correlation of VHL with clinico-pathological characteristics, prognosis and EMT related proteins were analyzed. The roles of VHL on the cell morphology, proliferation, migration, and invasion were determined by MTT, scratch and transwell invasion assay in Tscca and Tca8113P160 cells. The EMT related proteins were determined by Western blot and immunofluorescence (IF) methods. Loss of VHL expression was closely associated with pathologic grading, lymph node metastasis, poor prognosis, and EMT in OSCC. After re-expression of VHL, there was a cell morphologic change and motivation, proliferation, invasion of the cells were inhibited. The expression of Snail, N-cadherin and MMP-2/9, HIF-1α and VEGF were down-regulated in both the cell lines after transfection with VHL plasmid, while E-cadherin was up-regulated. Moreover, the effect of VHL suppressing β-catenin accumulation in nucleus was proved by Western blot and IF. VHL was significantly correlated with EMT process of OSCC. β-Catenin was an important downstream gene of VHL besides HIF-1α, which could induce the EMT process in OSCC.

T-box transcription factor Brachyury expression is correlated with epithelial-mesenchymal transition and lymph node metastasis in oral squamous cell carcinoma

The prognosis of patients with oral squamous cell carcinoma (SCC) is influenced by the presence of lymph node metastasis. Epithelial-mesenchymal transition (EMT), a process that involves events that convert adherent epithelial cells into individual migratory cells that can invade the extracellular matrix, is critical for cancer progression. Recently, the T-box transcription factor Brachyury was reported to promote EMT in human carcinoma cell lines. We analyzed the relationship between EMT (assessed by staining for E-cadherin and Vimentin) and the expression of Brachyury in association with lymph node metastasis in oral SCC. Oral SCC biopsy specimens (152 cases) were examined immunohistochemically for the expression of E-cadherin, Vimentin and Brachyury. Expression of Brachyury was correlated with EMT (p=0.035) and was significantly associated with lymph node and distant metastasis (p<0.05). Logistic regression analysis showed that Brachyury and EMT were predictive factors for lymph node metastasis (odds ratio 4.390 and 5.936, respectively) and that EMT was a predictive factor for distant metastases (odds ratio 11.786). Our findings present clinical evidence for an important role of Brachyury in EMT in oral SCC, and suggest that Brachyury and EMT patterns are useful prognostic markers.

Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma

Oral cancer is one of the drastic human cancers due to its aggressiveness and high mortality rate. Of all oral cancers, squamous cell carcinoma is the most common accounting for more than 90%. Epithelial-mesenchymal transition (EMT) is suggested to play an important role during cancer invasion and metastasis. Recently, emerging knowledge on EMT in carcinogenesis is explosive, tempting us to analyze previous studies on EMT in oral squamous cell carcinoma (OSCC). In this paper, we have first addressed the general molecular mechanisms of EMT, evidenced by alterations of cell morphology during EMT, the presence of cadherin switching, turning on and turning off of many specific genes, the activation of various signaling pathways, and so on. The remaining part of this paper will focus on recent findings of the investigations of EMT on OSCC. These include the evidence of EMT taking place in OSCC and the signaling pathways employed by OSCC cells during their invasion and metastasis. Collectively, with the large body of new knowledge on EMT in OSCC elaborated here, we are hopeful that targeting treatment for OSCC will be developed.

EGF/TGFβ1 co-stimulation of oral squamous cell carcinoma cells causes an epithelial-mesenchymal transition cell phenotype expressing laminin 332

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFβ1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFβ1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and β1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

414 POSTER An inhibitory effect of the hexamer fragment of HLDF differentiation factor on the development of experimental hemoblastosis

Epithelial–mesenchymal transition (EMT) plays an important role in cancer invasion and metastasis induced by hypoxia. Here, we examined whether phosphorylation of GSK3-β via phosphoinositide 3-kinase (PI3 K)/Akt signaling is involved in enhancing the hypoxia-induced EMT in oral squamous cell carcinoma (OSCC).Experiments were performed in OSCC cell lines (HSC-2, HSC-3, HSC-4, SAS, and HO-1-U-1) under normoxic or hypoxic conditions. The EMT was assessed by Matrigel invasion assays and wound healing assays. OSCC cell lines (HSC-2 and HSC-4) overexpressing hypoxia-inducible factor (HIF)-1α were established to examine the effects of HIF-1α on EMT-related factors. Immunohistochemical staining was performed to examine phosphorylation of GSK3-β in 33 cases of tongue squamous cell carcinoma.Under hypoxic conditions, OSCC cell lines exhibited HIF-1α expression and showed evidence of the EMT. In cells overexpressing HIF-1α, the levels of phospho-Akt and phospho-GSK3-β were increased, resulting in induction of the EMT. Inhibition of GSK3-β phosphorylation suppressed these effects. Moreover, the intensity of pGSK3-β staining was significantly increased with cN stage and cTNM stage in patients with tongue squamous cell carcinoma.Our data showed that the hypoxia-induced EMT in OSCC was enhanced by GSK3-β phosphorylation, suggesting that GSK3-β may be important in the invasion and metastasis of OSCC.