Tspan15 plays a crucial role in metastasis in oral squamous cell carcinoma.

Tetraspanin 15 (Tspan15) is a member of the tetraspanin family, which is associated with various biological events and several diseases, however, its role in human oral squamous cell carcinoma (OSCC) remains unknown. The current study aimed to clarify the role of Tspan15 in OSCC. The mRNA and protein expression levels of Tspan15 were up-regulated in OSCC cases and OSCC-derived cell lines. Significant up-regulated Tspan15 expression was found in the advanced OSCC cases; primary tumoral size (P = 0.042), regional lymph node metastasis (P = 0.036) and TNM classification (P = 0.024). The decreased expression of Tspan15 did not significantly affect cellular proliferation, whereas tumoral invasion and migration activities were suppressed in Tspan15-down-regulated cells, suggesting that Tspan15 might activate metastasis-related signaling. Moreover, in the Tspan15-down-regulated cells, the expression of a disintegrin and metalloproteinase (ADAM) 10 was also down-regulated and the cells secreted less soluble N-cadherin compared with control cells. And weak immunoreactivity of ß-catenin in the nucleus was detected in Tspan15-down-regulated cells compared with the control cells. These findings suggested that overexpression of Tspan15 positively regulates development of OSCC, and that ADAM10, N-cadherin, ß-catenin might be involved in the Tspan15-mediated pathway. These unusual conditions of cell adhesion molecules may lead to high metastasis rate found in Tspan15-overexpressing cases.

Growth suppression of human oral cancer cells by candidate agents for cetuximab-side effects.

Cetuximab, an inhibitor of the epidermal growth factor receptor that is used widely to treat human cancers including oral squamous cell carcinoma (OSCC), has characteristic side effects of skin rash and hypomagnesemia. However, the mechanisms of and therapeutic agents for skin rashes and hypomagnesemia are still poorly understood. Our gene expression profiling analyses showed that cetuximab activates the p38 MAPK pathways in human skin cells (human keratinocyte cell line [HaCaT]) and inhibits c-Fos-related signals in human embryonic kidney cells (HEK293). We found that while the p38 inhibitor SB203580 inhibited the expression of p38 MAPK targets in HaCaT cells, flavagline reactivated c-Fos-related factors in HEK293 cells. It is noteworthy that, in addition to not interfering with the effect of cetuximab by both compounds, flavagline has additive effect for OSCC growth inhibition in vivo. Collectively, our results indicate that combination of cetuximab and these potential therapeutic agents for cetuximab-related toxicities could be a promising therapeutic strategy for patients with OSCC.

UNC93B1 promotes tumoral growth by controlling the secretion level of granulocyte macrophage colony-stimulating factor in human oral cancer.

Unc-93 homolog B1 (UNC93B1), a transmembrane protein, is correlated with immune diseases, such as influenza, herpes simplex encephalitis, and the pathogenesis of systemic lupus erythematosus; however, the role of UNC93B1 in cancers including human oral squamous cell carcinomas (OSCCs) remains unknown. In the current study, we investigated the UNC93B1expression level in OSCCs using quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunohistochemistry. Our data showed that UNC93B1 mRNA and protein expressions increased markedly (p < 0.05) in OSCCs compared with normal cells and tissues and that high expression of UNC93B1 in OSCCs was related closely to tumoral size. UNC93B1 knockdown (shUNC93B1) OSCC cells showed decreased cellular proliferation by cell-cycle arrest in the G1 phase with up-regulation of p21Cip1 and down-regulation of CDK4, CDK6, cyclin D1, and cyclin E. We also found that granulocyte macrophage colony-stimulating factor (GM-CSF) was down-regulated significantly (p < 0.05) in shUNC93B1 OSCC cells. Moreover, inactivation of GM-CSF using neutralization antibody led to cell-cycle arrest at the G1 phase similar to the phenotype of the shUNC93B1 cells. The current findings indicated that UNC93B1 might play a crucial role in OSCC by controlling the secretion level of GM-CSF involved in tumoral growth and could be a potential therapeutic target for OSCCs.

Critical role of deoxynucleotidyl transferase terminal interacting protein 1 in oral cancer.

Deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1) forms a complex with histone deacetylase (HDAC); however, the relevance of DNTTIP1 in cancer remains unknown. The aim of this study was to examine DNTTIP1 expression and its functional mechanisms in oral squamous cell carcinomas (OSCCs). DNTTIP1 expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting analysis, and immunohistochemistry. The expression of DNTTIP1 was upregulated significantly in vitro and in vivo, and in patients with OSCC in whom DNTTIP1 was overexpressed and the expression level was correlated significantly (P < 0.05) with tumoral growth. DNTTIP1 knockdown (siDNTTIP1) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with high acetylation of p53 and upregulation of p21Cip1. Moreover, resveratrol, a HDAC inhibitor, controlled not only acetylated p53 status but also DNTTIP1 expression, leading to a similar phenotype of siDNTTIP1 cells. A marked (P < 0.05) reduction of tumoral growth in mouse xenograft models was observed with lower DNTTIP1 expression under the presence of this chemical reagent. Taken together, our results suggested that DNTTIP1-HDAC interaction promotes tumoral growth through deacetylation of p53 and that DNTTIP1 might be a critical therapeutic target in OSCCs.

Evaluation of tryptophan-aspartic acid repeat-containing protein 34 as a novel tumor-suppressor molecule in human oral cancer.

Tryptophan-aspartic acid (WD) repeat-containing protein 34 (WDR34), one of the WDR protein superfamilies with five WD40 domains, inhibits a transforming growth factor-beta (TGF-ß) activated kinase 1 (TAK1)-associated NF-κB activation pathway. Nevertheless, little is known about the roles of WDR34 in cancer. The current study sought to elucidate the clinical relevance of WDRsfb34 in oral squamous cell carcinoma (OSCC). We found WDR34 down-regulation in OSCCs compared with normal control tissues using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry. Models of overexpression of WDR34 (oeWDR34) showed depressed cellular growth through cell-cycle arrest at the G1 phase. To investigate the inhibitory function of WDR34, we challenged oeWDR34 cells with interleukin (IL)-1, a ligand for activation of the TAK1-NF-κB pathway and assessed the expression of a target gene of the pathway. oeWDR34 strongly inhibited IL-6 expression, which is closely related to tumoral growth, compared with control cells, suggesting that WDR34 would be a critical molecule for control of tumoral progression. In addition to the in vitro experiments, WDR34 negativity was correlated with tumoral growth of OSCCs. Our findings suggested that WDR34 inhibits OSCC progression and might be a potential tumor-suppressor molecule in OSCCs.

UBE2S associated with OSCC proliferation by promotion of P21 degradation via the ubiquitin-proteasome system.

Ubiquitin-conjugating enzyme E2S (UBE2S), a family of E2 protein in the ubiquitin-proteasome system, is highly expressed in several types of cancers; however, its roles in oral squamous cell carcinoma (OSCC) have not yet been well elucidated. The purpose of this study was to clarify the functional activities of UBE2S in OSCCs. We analyzed the expression levels of UBE2S in nine OSCC cell lines and primary OSCC tissues by quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry (IHC). The correlations between UBE2S expression and clinical classifications of OSCCs were analyzed using the IHC scoring system. We also used UBE2S knockdown OSCC cells for functional assays (proliferation assay, flow cytometry, and Western blotting). UBE2S was overexpressed in OSCCs in vitro and in vivo and was correlated significantly (P < 0.05) with the primary tumoral size. The cellular growth was decreased and the cell-cycle was arrested in the G2/M phase in the UBE2S knockdown (shUBE2S) cells. The expression level of P21, a target of the ubiquitin-proteasome system, was increased in the shUBE2S cells because of lower anaphase activity that promotes complex subunit 3 (APC3), an E3 ubiquitin ligase, compared with shMock cells. These findings might promote the understanding of the relationship between UBE2S overexpression and oral cancer proliferation, indicating that UBE2S would be a potential biomarker of and therapeutic target in OSCCs.

Novel mechanism of aberrant ZIP4 expression with zinc supplementation in oral tumorigenesis.

Zrt-Irt-like protein 4 (ZIP4) is critical molecule for proper mammalian development and releasing zinc from vesicular compartments. Recent studies suggested that ZIP4 plays an important role of tumor progression in pancreatic, prostate, and hepatocellular cancers, however, little is known about the detail mechanism of ZIP4 in their cancers. In the present study, we examined the possibility of ZIP4 as a new molecular target for oral squamous cell carcinoma (OSCC). We evaluated ZIP4 expression in OSCC-derived cell lines and primary OSCC samples by quantitative RT-PCR, immunoblotting, and immunohistochemistry (IHC). We also analyzed the clinical correlation between ZIP4 status and clinical behaviors in patients with OSCC. In addition, ZIP4 knockdown cells (shZIP4 cells) and ZnCl2 treatment were used for functional experiments, including cellular proliferation assay, zinc uptake assay, and cell-cycle analysis. ZIP4 mRNA and protein were up-regulated significantly in OSCCs compared with normal counterparts in vitro and in vivo. IHC showed that ZIP4 expression in the primary OSCC was positively correlated with primary tumoral size. The shZIP4 cells showed decrease accumulation of intercellular zinc and decreased cellular growth by cell-cycle arrest at the G1 phase, resulting from up-regulation of cyclin-dependent kinase inhibitors and down-regulation of cyclins and cyclin-dependent kinases. Since cellular growth of OSCC cells after treatment with zinc was significantly greater than control cells, we speculated that intercellular ZnCl2 accumulation is an important factor for cellular growth. Consistent with our hypothesis, not only decreased zinc uptake by ZIP4 knockdown but also chelating agent, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), showed inhibitory effects of cellular proliferation. Therefore, our data provide evidence for an essential role of ZIP4 and intracellular zinc for tumoral growth in OSCC, suggesting that zinc uptake might be a potential therapeutic targeting event for OSCCs.

TEAD4-YAP interaction regulates tumoral growth by controlling cell-cycle arrest at the G1 phase.

TEA domain transcription factor 4 (TEAD4), which has critical functions in the process of embryonic development, is expressed in various cancers. However, the important role of TEAD4 in human oral squamous cell carcinomas (OSCCs) remain unclear. Here we investigated the TEAD4 expression level and the functional mechanism in OSCC using quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Furthermore, TEAD4 knockdown model was used to evaluate cellular proliferation, cell-cycle analysis, and the interaction between TEAD4 and Yes-associated protein (YAP) which was reported to be a transcription coactivator of cellular proliferation. In the current study, we found that TEAD4 expression increased significantly in vitro and in vivo and correlated with tumoral size in OSCC patients. TEAD4 knockdown OSCC cells showed decreased cellular proliferation resulting from cell-cycle arrest in the G1 phase by down-regulation of cyclins, cyclin-dependent kinases (CDKs), and up-regulation of CDK inhibitors. We also found that the TEAD4-YAP complex in the nuclei may be related closely to transcriptions of G1 arrest-related genes. Taken together, we concluded that TEAD4 might play an important role in tumoral growth and have potential to be a therapeutic target in OSCCs.

Long-term culture of human odontoma-derived cells with a Rho kinase inhibitor.

Because of cellular senescence/apoptosis, no effective culture systems are available to maintain replication of cells from odontogenic tumors especially for odontoma, and, thus, the ability to isolate human odontoma-derived cells (hODCs) for functional studies is needed. The current study was undertaken to develop an approach to isolate hODCs and fully characterize the cells in vitro. The hODCs were cultured successfully with a Rho-associated protein kinase inhibitor (Y-27632) for an extended period with stabilized lengths of the telomeres to sustain a similar phenotype/property as the primary tumoral cells. While the hODCs showed stable long-term expansion with expression of major dental epithelial markers including dentin sialophosphoprotein (DSPP) even in the three-dimensional microenvironment, they lack the specific markers for the characteristics of stem cells. Moreover, cells from dental pulp showed significant up-regulation of DSPP when co-cultured with the hODCs, while control fibroblasts with the hODCs did not. Taken together, we propose that the hODCs can be isolated and expanded over the long term with Y-27632 to investigate not only the development of the hODCs but also other types of benign human tumors.

Cavin-2 in oral cancer: A potential predictor for tumor progression.

Cavin-2 (CVN2) affects formation of large caveolae, which are membrane-rich cholesterol domains associated with several functions in signal transduction. Accumulating evidence suggests that CVN2 is present in many cellular types; however, the molecular mechanisms of CVN2 in cancers and its clinical relevance are unknown. We proposed a mechanism by which CVN2 regulates caveolin-1 expression leading to slow cellular proliferation by inactivation of the extracellular regulated kinase (ERK) pathway. Quantitative reverse transcriptase-polymerase chain reaction and immunoblot analyses were used to assess the CVN2 regulation mechanism in oral squamous cell carcinoma (OSCC). Immunohistochemistry (IHC) was performed to analyze the correlation between CVN2 expression and clinical behavior in 115 patients with OSCC. A CVN2 overexpressed model of OSCC cells (oeCVN2 cells) was used for functional experiments. CVN2 expression was down-regulated significantly (P < 0.05) in OSCCs compared with normal counterparts in vitro and in vivo. In addition to the findings that a serum deprivation culture induced up-regulation of CVN2 and slowed cellular proliferation, oeCVN2 cell growth decreased because of cell-cycle arrest at the G1 phase resulting from up-regulated cyclin-dependent kinase inhibitors (p21(Cip1) and p27(Kip1) ) and down-regulated cyclins (cyclin D1, cyclin E) and cyclin-dependent kinases (CDK2, CDK4, and CDK6). Interestingly, CVN2 overexpression facilitated caveolin-1 recruitment and colocalization with each other. We also found decreased ERK phosphorylation levels, an upstream event in cell-cycle arrest. Clinically, IHC data from primary OSCCs showed high tumoral progression in CVN2-negative patients with OSCC. CVN2 may be a possible key regulator of OSCC progression via the CVN2/caveolin-1/ERK pathway and a potential therapeutic target for developing new treatments for OSCCs. © 2015 Wiley Periodicals, Inc.

Aberrant GIMAP2 expression affects oral squamous cell carcinoma progression by promoting cell cycle and inhibiting apoptosis.

GTPases of immunity-associated protein 2 (GIMAP2) is a GTPase family member associated with T cell survival. However, its mechanisms of action in oral squamous cell carcinoma (OSCC) remain largely unknown. Therefore, the present study aimed to elucidate the possible role of GIMAP2 in OSCC development by investigating its expression levels and molecular mechanisms in OSCC. Reverse transcription quantitative PCR, immunoblotting and immunohistochemistry indicated that GIMAP2 expression was significantly upregulated (P<0.05) in OSCC-derived cell lines and primary OSCC specimens compared with that in their normal counterparts. GIMAP2-knockdown OSCC cells exhibited decreased cell growth, which was associated with cyclin-dependent kinase (CDK)4, CDK6 and phosphorylated Rb downregulation and p53 and p21 upregulation. In addition to cell cycle arrest, GIMAP2 affected anti-apoptotic functions in GIMAP2-knockdown cells by upregulating Bcl-2 and downregulating Bax and Bak. These findings indicated that GIMAP2 may significantly influence OSCC development and apoptosis inhibition and thus is a potential biomarker of OSCC.

Tumor Suppressive Circular RNA-102450: Development of a Novel Diagnostic Procedure for Lymph Node Metastasis from Oral Cancer.

Circular RNAs (circRNAs), which form as covalently closed loop structures, have several biological functions such as regulation of cellular behavior by adsorbing microRNAs. However, there is limited information of circRNAs in oral squamous cell carcinoma (OSCC). Here, we aimed to elucidate the roles of aberrantly expressed circRNAs in OSCC. CircRNA microarray showed that circRNA-102450 was down-regulated in OSCC cells. Clinical validation of circRNA-102450 was performed using highly sensitive droplet digital PCR in preoperative liquid biopsy samples from 30 OSCC patients. Interestingly, none of 16 studied patients with high circRNA-102450 had regional lymph node metastasis (RLNM), whereas 4 of 14 studied patients (28.5%) with low expression had pathologically proven RLNM. Overexpressed circRNA-102450 significantly inhibited the tumor metastatic properties of cell proliferation, migration, and invasion. Furthermore, circRNA-102450 directly bound to, and consequently down-regulated, miR-1178 in OSCC cells. Taken together, circRNA-102450 has a tumor suppressive effect via the circRNA-102450/miR-1178 axis and may be a novel potential marker of RLNM in OSCC patients.

Engineered exosomes delivering specific tumor-suppressive RNAi attenuate oral cancer progression.

Exosomes are involved in a wide range of biological processes in human cells. Considerable evidence suggests that engineered exosomes (eExosomes) containing therapeutic agents can attenuate the oncogenic activity of human cancer cells. Despite its biomedical relevance, no information has been available for oral squamous cell carcinoma (OSCC), and therefore the development of specific OSCC-targeting eExosomes (octExosomes) is urgently needed. We demonstrated that exosomes from normal fibroblasts transfected with Epstein-Barr Virus Induced-3 (EBI3) cDNA were electroporated with siRNA of lymphocyte cytoplasmic protein 1 (LCP1), as octExosomes, and a series of experiments were performed to evaluate the loading specificity/effectiveness and their anti-oral cancer cell activities after administration of octExosomes. These experiments revealed that octExosomes were stable, effective for transferring siLCP1 into OSCC cells and LCP1 was downregulated in OSCC cells with octExosomes as compared with their counterparts, leading to a significant tumor-suppressive effect in vitro and in vivo. Here we report the development of a new valuable tool for inhibiting tumor cells. By engineering exosomes, siLCP1 was transferred to specifically suppress oncogenic activity of OSCC cells. Inhibition of other types of human malignant cells merits further study.

Identification of cisplatin-resistance related genes in head and neck squamous cell carcinoma.

Resistance to cisplatin is a major obstacle to successful treatment of head and neck squamous cell carcinoma (HNSCC). To investigate the molecular mechanism of this resistance, we compared the gene expression profiles between the cisplatin-sensitive SCC cell lines (Sa-3, H-1 and KB) and the cisplatin-resistant cell lines established from them (Sa-3R, H-1R and KB-R) using Affymetrix U133 Plus 2.0 microarray. We identified 199 genes differentially expressed in each group. To identify important functional networks and ontologies to cisplatin resistance, the 199 genes were analyzed using the Ingenuity Pathway Analysis Tool. Fifty-one of these genes were mapped to genetic networks, and we validated the top-10 upregulated genes by real-time reverse transcriptase-polymerase chain reaction. Five novel genes, LUM, PDE3B, PDGF-C, NRG1 and PKD2, showed excellent concordance with the microarray data. In 48 patients with oral SCC (OSCC), positive immunohistochemical staining for the five genes correlated with chemoresistance to cisplatin-based combination chemotherapy. In addition, the expression of the five genes predicted the patient outcomes with chemotherapy. Furthermore, siRNA-directed suppressed expression of the five genes resulted in enhanced susceptibility to cisplatin-mediated apoptosis. These results suggested that these five novel genes have great potential for predicting the efficacy of cisplatin-based chemotherapy against OSCC. Global gene analysis of cisplatin-resistant cell lines may provide new insights into the mechanisms underlying clinical cisplatin resistance and improve the efficacy of chemotherapy for human HNSCC.

Cross-resistance of platinum derivatives in H-1R, a cisplatin-resistant cell line.

We previously established H-1R cells, a cisplatin (CDDP)-resistant cell line, from H-1 cells, a CDDP-sensitive oral carcinoma cell line. The aim of this study was to identify the molecular mechanism of cross-resistance to antitumor drugs containing a platinum agent in H-1R cells. The 3-(3,4-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay and clonogenecity assay indicated that H-1R cells showed strong cross-resistance to carboplatin, nedaplatin and oxaliplatin. The expression status of the copper transporter and organic cation transporters was confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction. The transporters ATP7A, ATP7B, hCtr1, hOCT1 and hOCT2 were up-regulated, whereas hOCT3 was down-regulated. The cellular glutathione level was elevated 2-fold in H-1R cells compared with H-1 cells. Our results suggested that H-1 and H-1R cells may be useful in searching for candidate genes responsible for cross-resistance to platinum derivatives and for further studies to understand the mechanism of platinum resistance.

Lin-7C/VELI3/MALS-3: an essential component in metastasis of human squamous cell carcinoma.

Using proteomic selection, functional verification, and clinical validation, we identified specific down-regulation of Lin-7C/VELI3/MALS-3 (Lin-7C), which marks oral squamous cell carcinoma (OSCC) metastasis. Despite a rarity of sequence variations in the Lin-7C gene in both primary OSCC and OSCC-derived cells, a high prevalence of hypermethylation was detected in the CpG island region that strongly correlated with its down-regulation. Inducible Lin-7C mRNA by experimental demethylation was found in all OSCC cells tested. Overexpression of the Lin-7C gene in an OSCC cell clone does not contribute to underproliferation but results in a noninvasive phenotype with elevated beta-catenin expression. Experimental metastases in multiple organs of immunodeficient mice were inhibited in cells expressing Lin-7C. Finally, the Lin-7C expression status in primary tumors afforded significantly (P<0.001) high accuracy for predicting lymph node metastasis. These results establish Lin-7C as a novel target of early detection, prevention, and therapy for OSCC metastasis.

SYT12 plays a critical role in oral cancer and may be a novel therapeutic target.

Synaptotagmin12 (SYT12) has been well characterized as the regulator of transmitter release in the nervous system, however the relevance and molecular mechanisms of SYT12 in oral squamous cell carcinoma (OSCC) are not understood. In the current study, we investigated the expression of SYT12 and its molecular biological functions in OSCC by quantitative reverse transcriptase polymerase chain reaction, immunoblot analysis, and immunohistochemistry. SYT12 were up-regulated significantly in OSCC-derived cell lines and primary OSCC tissue compared with the normal counterparts (P<0.05) and the SYT12 expression levels were correlated significantly with clinical indicators, such as the primary tumoral size, lymph node metastasis, and TNM stage (P<0.05). SYT12 knockdown OSCC cells showed depressed cellular proliferation, migration, and invasion with cell cycle arrest at G1 phase. Surprisingly, we found increased calcium/calmodulin-dependent protein kinase 2 (CAMK2) inhibitor 1 (CAMK2N1) and decreased CAMK2-phosphorylation in the knockdown cells. Furthermore, treatment with L-3, 4-dihydroxyphenylalanine (L-dopa), a drug approved for Parkinson's disease, led to down-regulation of SYT12 and similar phenotypes to SYT12 knockdown cells. Taken together, we concluded that SYT12 plays a significant role in OSCC progression via CAMK2N1 and CAMK2, and that L-dopa would be a new drug for OSCC treatment through the SYT12 expression.

Resveratrol Targets Urokinase-Type Plasminogen Activator Receptor Expression to Overcome Cetuximab-Resistance in Oral Squamous Cell Carcinoma.

Drug resistance to anti-cancer agents is a major concern regarding the successful treatment of malignant tumors. Recent studies have suggested that acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies such as cetuximab are in part caused by genetic alterations in patients with oral squamous cell carcinoma (OSCC). However, the molecular mechanisms employed by other complementary pathways that govern resistance remain unclear. In the current study, we performed gene expression profiling combined with extensive molecular validation to explore alternative mechanisms driving cetuximab-resistance in OSCC cells. Among the genes identified, we discovered that a urokinase-type plasminogen activator receptor (uPAR)/integrin ß1/Src/FAK signal circuit converges to regulate ERK1/2 phosphorylation and this pathway drives cetuximab-resistance in the absence of EGFR overexpression or acquired EGFR activating mutations. Notably, the polyphenolic phytoalexin resveratrol, inhibited uPAR expression and consequently the signaling molecules ERK1/2 downstream of EGFR thus revealing additive effects on promoting OSCC cetuximab-sensitivity in vitro and in vivo. The current findings indicate that uPAR expression plays a critical role in acquired cetuximab resistance of OSCC and that combination therapy with resveratrol may provide an attractive means for treating these patients.

Evidence for critical role of Tie2/Ang1 interaction in metastatic oral cancer.

Angiopoietin-1 (Ang1) is a binding partner of endothelial cell-specific tyrosine-protein kinase receptor (Tie2), which serves important roles in vascular development and angiogenesis. Tie2 is closely associated with the metastasis of oral squamous cell carcinomas (OSCCs) however, little is known about the correlation between Tie2 and Ang1. In the present study, the functional mechanisms of the Tie2/Ang1 interaction were investigated using Tie2 overexpressed (oeTie2) OSCC cells and recombinant Ang1 protein. oeTie2 cells had increased cell-cell and cell-extracellular matrix adhesions compared with the control cells. Additionally, the adhesive activities increased following treatment with exogenous Ang1, indicating that Ang1 directly enhances Tie2 functions. In the clinical OSCC data from 10 cases positive for regional lymph node metastasis, all cases were negative for Tie2 expression and eight cases (80%) were negative for Ang1 expression. These results suggest that Tie2 and Ang1 serve important roles in cancer metastasis and may be potential biomarkers and therapeutic targets for OSCC metastasis.

Overexpression of Translocation Associated Membrane Protein 2 Leading to Cancer-Associated Matrix Metalloproteinase Activation as a Putative Metastatic Factor for Human Oral Cancer.

Translocation associated membrane protein 2 (TRAM2) has been characterized as a component of the translocon that is a gated channel at the endoplasmic reticulum (ER) membrane. TRAM2 is expressed in a wide variety of human organs. To date, no information is available regarding TRAM2 function in the genesis of human cancer. The purpose of this study was to investigate the status of the TRAM2 gene in oral squamous cell carcinoma (OSCC) cells and clinical OSCC samples. Using real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting analysis, and immunohistochemistry, we detected accelerated TRAM2 mRNA and protein expression levels both in OSCC-derived cell lines and primary tumors. Moreover, TRAM2-positive OSCC tissues were correlated closely (P<0.05) with metastasis to regional lymph nodes and vascular invasiveness. Of note, knockdown of TRAM2 inhibited metastatic phenotypes, including siTRAM2 cellular migration, invasiveness, and transendothelial migration activities with a significant (P<0.05) decrease in protein kinase RNA(PKR) - like ER kinase (PERK) and matrix metalloproteinases (MMPs) (MT1-MMP, MMP2, and MMP9). Taken together, our results suggested that TRAM2 might play a pivotal role in OSCC cellular metastasis by controlling major MMPs. This molecule might be a putative therapeutic target for OSCC.