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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Nucleolar and spindle-associated protein 1 (NUSAP1) is an important mitotic regulator. In addition to its crucial function in mitosis, NUSAP1 has recently received attention due to the interesting roles in carcinogenesis. The aim of this study was to reveal functional mechanisms of NUSAP1 in oral squamous cell carcinoma (OSCC). Methods: mRNA and protein expression levels of NUSAP1 in 9 OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. The correlation between the NUSAP1 expression profile and the clinicopathological factors was evaluated by immunohistochemistry (IHC) in clinical OSCC samples (n = 70). The NUSAP1 knockdown cells were established with short hairpin RNA (shRNA) in OSCC cells, and functional assays were performed using these cells. In addition to the evaluation of cellular proliferation and cell cycle, we also investigated the potential role of NUSAP1 in paclitaxel (PTX)-induced cellular responses. Results: mRNA and protein expression of NUSAP1 were significantly up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (P < 0.05). IHC revealed that NUSAP-1 expression is closely associated with primary advanced T stage (P<0.05). Suppression of NUSAP1 expression levels led to significant (P < 0.05) inhibition of cellular proliferation. Furthermore, apoptosis induced by PTX was enhanced in NUSAP1 knockdown OSCC cells. Conclusions: NUSAP1 may be a crucial biomarker for OSCC. Moreover, down-regulated NUSAP1 expression suppresses tumor proliferation and also enhances anti-tumor effect of PTX by activating apoptotic pathways. Thus, the present study strongly suggests that regulating NUSAP1 expression should contribute to the therapy for OSCC. [ABSTRACT FROM AUTHOR]

Angiopoietin-like 3 ( ANGPTL3), which is involved in new blood vessel growth and stimulation of mitogen-activated protein kinase ( MAPK), is expressed aberrantly in several types of human cancers. However, little is known about the relevance of ANGPTL3 in the behavior of oral squamous cell carcinoma ( OSCC). In this study, we evaluated ANGPTL3 mRNA and protein in OSCC-derived cell lines ( n = 8) and primary OSCCs ( n = 109) and assessed the effect of ANGPTL3 on the biology and function of OSCCs in vitro and in vivo. Significant ( P < 0.05) ANGPTL3 upregulation was detected in the cell lines and most primary OSCCs (60%) compared with the normal counterparts. The ANGPTL3 expression level was correlated closely ( P < 0.05) with tumoral size. In patients with T3/T4 tumors, the overall survival rate with an ANGPTL3-positive tumor was significantly ( P < 0.05) lower than that of ANGPTL3-negative cases. In vitro, cellular growth in ANGPTL3 knockdown cells significantly ( P < 0.05) decreased with inactivated extracellular regulated kinase ( ERK) and cell-cycle arrest at the G1 phase resulting from upregulation of the cyclin-dependent kinase inhibitors, including p21Cip1 and p27Kip1. We also observed a marked ( P < 0.05) reduction in the growth in ANGPTL3 knockdown-cell xenografts with decreased levels of phosphorylated ERK relative to control-cell xenografts. The current data indicated that ANGPTL3 may play a role in OSCCs via MAPK signaling cascades, making it a potentially useful diagnostic/therapeutic target for use in patients with OSCC. [ABSTRACT FROM AUTHOR]

Background: The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs. Methods: We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression. Results: LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression. Conclusion: LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer. [ABSTRACT FROM AUTHOR]

Background: LIM and SH3 protein 1 (LASP-1) is a specific focal adhesion protein involved in several malignant tumors. However, its role in oral squamous cell carcinoma (OSCC) is unknown. The aim of this study was to characterize the role and molecular status/mechanism of LASP-1 in OSCC. Methods: We evaluated LASP-1 mRNA and protein expressions in OSCC-derived cell lines and primary OSCCs. Using an shRNA system, we analyzed the effect of LASP-1 on the biology and function of the OSCC cell lines, HSC-3 and Ca9-22. The cells also were subcutaneously injected to evaluate tumor growth in vivo. Data were analyzed by the Fisher’s exact test or the Mann-Whitney U test. Bonferroni correction was used for multiple testing. Results: Significant up-regulation of LASP-1 was detected in OSCC-derived cell lines (n = 7, P<0.007) and primary OSCCs (n = 50, P<0.001) compared to normal controls. LASP-1 knockdown cells significantly inhibited cellular proliferation compared with shMock-transfected cells (P<0.025) by arresting cell-cycle progression at the G2 phase. We observed dramatic reduction in the growth of shLASP-1 OSCC xenografts compared with shMock xenografts in vivo. Conclusion: Our results suggested that overexpression of LASP-1 is linked closely to oral tumourigenicity and further provide novel evidence that LASP-1 plays an essential role in tumor cellular growth by mediating G2/M transition. [ABSTRACT FROM AUTHOR]

Purpose: Sec8, a component of the exocyst complex, has been implicated in tethering of secretory vesicles to specific regions on the plasma membrane. To investigate the involvement of Sec8 in oral squamous-cell carcinoma (OSCC), we evaluated the expression status and effect of Sec8 in OSCC cell lines. Methods: Sec8 mRNA and protein expressions in human OSCC cell lines were assessed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting. Functional analyses, proliferation assay, invasiveness assay, and gelatin zymography in Sec8 knockdown cells were performed. Also the correlation between Sec8 expression and the clinicopathological features in 98 primary OSCCs samples was evaluated by immunohistochemistry. Results: Sec8 mRNA and protein expression were significantly up-regulated in all cell lines ( p < 0.05). Sec8 knockdown cells were characterized by reduced cellular proliferation, invasiveness, and secretion of matrix metalloproteinases (MMPs) (MMP-2, proMMP-2, and proMMP-9). Sec8 protein expression in primary OSCCs also was significantly ( p < 0.05) greater than in normal counterparts, and higher Sec8 expression was correlated with tumor size ( p = 0.03). Conclusions: Our results suggested for the first time that Sec8 might play a specific role in OSCC progression by mediating MMP secretion. [ABSTRACT FROM AUTHOR]

Purpose: ALY, an essential mRNA export factor, is dysregulated in a wide variety of human malignancies. However, little is known about the relevance of ALY to oral squamous cell carcinoma (OSCC). The purpose of this study was to investigate ALY expression and its functional mechanisms in OSCCs. Methods: ALY mRNA and protein expression in seven OSCC-derived cell lines (Sa3, HO-1-u-1, KON, Ca9-22, HSC-2, HSC-3, and HSC-4) and primary OSCCs were analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemistry. We evaluated cellular invasiveness, migration, and the expression levels of metastasis modulators, ribosomal RNA processing 1 homolog B (RRP1B) and CD82, in ALY knockdown cells. Results: ALY was frequently up-regulated in OSCC-derived cell lines and primary OSCCs compared with normal counterparts at both the mRNA and protein expression levels. ALY-positive expression was correlated significantly ( P < 0.05) with a higher risk of regional lymph node metastasis. Furthermore, ALY knockdown cells caused a significant ( P < 0.05) decrease in cellular invasiveness and migration with up-regulation of RRP1B and CD82 compared with the control cells. Conclusion: Our results showed that ALY is linked to regional lymph node metastasis by regulating cellular invasiveness and migration. Therefore, ALY might be a potential biomarker for early detection of lymph node metastasis in OSCCs. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Family with sequence similarity 3, member B (FAM3B), also known as PANcreatic DERived factor (PANDER), is an islet-specific cytokine predominantly expressed in both pancreatic α- and β-cells. FAM3B is known to induce apoptosis in pancreatic cells, and may therefore be able to regulate apoptosis in other cell types, including cancer cells. However, the role of FAM3B in carcinogenesis is unknown. We previously performed global gene screening using DNA microarray analysis to identify crucial molecules in oral squamous cell carcinoma (OSCC). The results showed significant down-regulation of FAM3B in OSCC-derived cell lines, as compared with human normal oral keratinocytes (HNOKs). The aim of the present study was to clarify the association between FAM3B and OSCCs. Results: Expression profiles of FAM3B in OSCC-derived cell lines and human primary OSCCs were examined by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blot analysis. Significantly decreased expression of FAM3B mRNA and FAM3B protein were observed in five OSCC-derived cell lines, as compared with HNOKs. Moreover, immunohistochemical analysis revealed down-regulated FAM3B protein expression in primary OSCC samples. In order to examine whether microRNA (miRNA) potentially regulates FAM3B expression, miRNA microarray analysis was performed with OSCC-derived cell lines. miRNA microarray data showed that miR-181a, miR-181b, miR-200b, and miR-200c, which can induce imperfect base pairing with the 3′-untranslated region of FAM3B mRNA, were up-regulated in all cell lines examined. Conclusions: These results indicate that decreased FAM3B expression is able to promote OSCC progression and that certain miRNAs may be correlated with the altered expression of FAM3B. [Copyright &y& Elsevier]

Abstract: The development and progression of oral squamous cell carcinoma (OSCC) is a multistep process, which involves many genetic factors. Among them, loss of heterozygosity (LOH) studies have been used to identify regions on chromosomes that may contain putative tumor suppressor genes (TSGs). Here, we searched PubMed for relevant publications including our previous studies and compared results of LOH in OSCCs from the articles. LOHs in OSCCs were observed at various loci on almost all chromosomes, except X and Y. In this review, the LOH in patients with OSCC and the interrelationship between TSGs and OSCC initiation and progression are discussed. [Copyright &y& Elsevier]

Background. The aim of the current study was to identify the antitumor activity of satraplatin in paired cisplatin (CDDP)-resistant oral squamous cell carcinoma (OSCC) cell line and its parental cell line. Methods. CDDP-resistant (KB-R) cells and parental cells (KB) pair were used. Viability was assessed using the MTT and clonogenic assay. Real-time polymerase chain reaction (PCR), glutathione (GSH) assay, and flow cytometric analysis were used for further assessment. Results. KB-R cells did not show cross-resistance to satraplatin. The expression status of almost all transporters was upregulated in the KB-R cells. There was no difference in the GSH levels between the KB and KB-R cells. Flow cytometric analysis indicated that with satraplatin the G2/M phase was arrested in the KB-R cells. KB-R cells contain enriched side population cells. Conclusion. These data suggested that satraplatin has antitumor activity against the CDDP-resistant OSCC cells. The mechanism of cross-resistance to platinum agents seems to be multifactorial. [ABSTRACT FROM AUTHOR]

To determine the involvement of ZIC2 in oral squamous cell carcinoma (OSCC). ZIC2 mRNA and protein expression in primary OSCCs ( n = 74), oral premalignant lesions (OPLs, n = 20) and five OSCC-derived cell lines (HSC-2, HSC-3, OK-92, H1, and Sa3) were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry (IHC). In addition, we evaluated the correlation between ZIC2 IHC scores in OSCCs and the clinicopathologic status. Significant up-regulation of ZIC2 was detected in OSCC-derived cell lines ( P < 0.05), primary OSCCs ( P < 0.05) and OPLs ( P < 0.05) compared with normal counterparts. Among the clinical variables analyzed, ZIC2 expression was associated with the histopathologic types of OSCC. Furthermore, the survival rates differed significantly between ZIC2-positive cases and ZIC2-negative cases. These results suggested that ZIC2 expression is correlated with the differentiation type of OSCC and diagnosis and might be a potential prognostic indicator and therapeutic target for OSCCs. [ABSTRACT FROM AUTHOR]

The purpose of this study was to characterize changes in the expression of copper–zinc superoxide dismutase (Cu/Zn-SOD) and manganese SOD (Mn-SOD) in oral squamous-cell carcinoma (OSCC). Real-time quantitative reverse transcriptase-polymerase chain reaction analysis of Cu/Zn-SOD and Mn-SOD mRNA expression was carried out in 50 pairs of OSCC tissue specimens and corresponding normal tissues. Mn-SOD protein expression was evaluated further in 65 OSCC tissue samples and 33 oral premalignant lesions (OPLs) using immunohistochemistry. Significant ( P < 0.001) upregulation of Mn-SOD mRNA expression was observed in OSCC tissues compared with the normal tissue counterparts, whereas no significant difference was detected in Cu/Zn-SOD expression. Significant increases in Mn-SOD protein expression were seen in both OPLs ( P < 0.001) and OSCC tissue ( P < 0.001) together with a high incidence of lymph node metastasis ( P = 0.04). Our findings suggested that Mn-SOD overexpression is a frequent and early event during oral carcinogenesis and could contribute to aggressive OSCC. [ABSTRACT FROM AUTHOR]

To determine the potential involvement of ANXA1 in oral squamous-cell carcinoma (OSCC), we evaluated the ANXA1 protein expression in oral premalignant lesions (OPLs) and OSCCs and correlated the results with clinicopathologic variables. Matched normal and tumour specimens of 44 primary OSCCs and 28 OPLs were analyzed for ANXA1 subcellular localization and protein expression level by immunohistochemistry (IHC). Correlations between ANXA1-IHC staining scores of OSCCs and clinicopathologic features were evaluated by Fisher’s exact test. Markedly down-regulation of ANXA1 protein expression was identified on the plasma membrane of epithelial cells in OSCCs ( P < 0.001) and OPLs ( P = 0.001) compared with normal counterparts. Moreover, loss of plasma membranous ANXA1 expression was significantly correlated with the poorly differentiated status of OSCC cells ( P = 0.012). Our findings suggest that loss of ANXA1 is frequent and early event during oral carcinogenesis and that ANXA1 could contribute to maintaining epithelial differentiation in OSCC. [ABSTRACT FROM AUTHOR]

Objective: To identify genes associated with therapeutic targets of oral squamous cell carcinoma (OSCC), we compared gene expression profiles in OSCC-derived cell lines with human normal oral keratinocytes. Methods: We analyzed the gene expression profiles of OSCCs using Affymetrix GeneChip analysis. The identified genes were analyzed by an Ingenuity Pathway Analysis tool to identify networks of interacting genes. A candidate gene was further evaluated for the expression status of the mRNA and protein in OSCC-derived cell lines and primary OSCCs. Results: The microarray data identified 188 genes downregulated in OSCC-derived cell lines, and the genetic pathways associated with expression changes were generated. Among the genes mapped to the network with the highest significance, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was analyzed further. CEACAM1 mRNA and protein were frequently downregulated in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemical analysis showed that primary OSCCs were significantly decreased in CEACAM1. Moreover, CEACAM1 expression was correlated with the TNM staging. We also found that CEACAM1-negative expression was significant both for disease-free (p = 0.036) and overall survival (p = 0.032). Conclusion: Repression of CEACAM1 could contribute to cancer progression and may indicate a poor prognosis for patients with OSCC. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Summary: Autophagy is a dynamic process of subcellular degradation, which has recently sparked great interest because it is involved in various developmental processes and various diseases including cancer. Autophagy-related 16-like 1 is a component of a large protein complex essential for autophagosome formation. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous cell carcinoma cells and detected significantly high expression levels of autophagy-related 16-like 1 in oral squamous cell carcinoma–derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of autophagy-related 16-like 1 in oral squamous cell carcinoma, we evaluated the state of autophagy-related 16-like 1 protein expression in human oral premalignant lesions and primary oral squamous cell carcinomas, and correlated the results with clinicopathologic variables. Autophagy-related 16-like 1 immunoreaction was predominant in a variety of subcellular components of oral squamous cell carcinoma tissues, including the cytoplasm and plasma membrane of malignant cells (45% and 39%, respectively) and peritumoral and intratumoral stroma (52%), whereas all of the components in normal tissues had no or faint autophagy-related 16-like 1 expression. In addition, high stromal expression of autophagy-related 16-like 1 was associated significantly with lymphovascular invasion of tumor cells (P = .037) and positive lymph node status (P = .015). Furthermore, cytoplasmic and plasma membranous autophagy-related 16-like 1 were also expressed in abundance in the oral premalignant lesion cells (74% and 32%, respectively). Our finding suggests that dysregulation of autophagy-related 16-like 1 protein expression is a frequent and early event during oral carcinogenesis and could affect the malignant behavior of oral squamous cell carcinoma cells. [Copyright &y& Elsevier]

Purpose: Cisplatin (CDDP) is widely used for chemotherapy of oral squamous cell carcinoma (OSCC). However, the mechanism of resistance to CDDP is unclear. Recently, caveolin-1 was identified as being associated with both metastasis and multidrug resistance. In the present study, we showed that caveolin-1 expression is significantly related to chemosensitivity in OSCC. Methods: We established a CDDP-resistant cell line, H-1R, from the parental OSCC cell line, H-1. Caveolin-1 expression was determined by reverse transcriptase–polymerase chain reaction (RT-PCR) in both cell lines. We analyzed expression of caveolin-1 in 30 OSCC biopsy specimens and investigated the relationship between expression of caveolin-1 and patients‘ clinicopathological parameters and chemotherapeutic responses. Results: The 3-(3,4-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that H-1R has a ten-times greater resistance to CDDP than H-1 has. The level of caveolin-1 expression in H-1R was significantly decreased in comparison with that in H-1 by real-time RT-PCR analysis. Positive caveolin-1 immunostaining correlated positively with a complete response (16/20, 80.0%). However, negative immunostaining was found in 6/7 (85.7%) cases with no response. Positive immunohistochemical staining of caveolin-1 correlated positively with chemosensitivity to CDDP-based combination chemotherapy ( P=0.02). Conclusions: These results suggest that overexpression of the caveolin-1 gene may provide novel diagnostic markers associated with CDDP sensitivity in OSCC. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Recent cytogenetic and molecular studies with highly polymorphic microsatellite markers have implicated allele loss involving chromosome 4 in several human cancers, which suggests the presence of multiple tumor suppressor gene (TSG) loci. However, there has been no detailed analysis of loss of heterozygosity (LOH) on chromosome 4 in oral squamous cell carcinoma (OSCC). To determine the location of a putative TSG associated with OSCC on chromosome 4, polymerase chain reaction (PCR) analysis of microsatellite polymorphisms corresponding to 17 loci was performed to screen 32 patients with OSCC. LOH was observed in the majority of the tumors (75%) in at least one of the loci. The loci on the long arm exhibited a significantly higher frequency of deletions (66%) than those of the short arm (25%). Among the loci tested, frequent LOH was centered at D4S1573 on 4q25, which represents a region of about 4 centimorgans (cM). However, no commonly deleted regions were found on the short arm of the chromosome. We detected microsatellite instability (MI) in 31% of the cases. MI was also observed more frequently on the long arm (28%) than the short arm (6%). Thus, our data indicate that alterations of chromosome 4 regions, especially the long arm, are associated with OSCC tumorigenesis and that the 4q25 region may harbor at least one putative TSG. [ABSTRACT FROM AUTHOR]

Background:Kinesin family member 4A (KIF4A), a microtubule-based motor protein, was implicated in regulation of chromosomal structure and kinetochore microtubule dynamics. Considering the functions of KIF4A, we assumed that KIF4A is involved in progression of oral squamous cell carcinomas (OSCCs) via activation of the spindle assembly checkpoint (SAC). However, little is known about the relevance of KIF4A in the behavior of OSCC. We investigated the KIF4A expression status and its functional mechanisms in OSCC. Methods:The KIF4A expression levels in seven OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Using a KIF4A knockdown model, we assessed the expression of (SAC)-related molecules (BUB1, MAD2, CDC20, and cyclin B1), cell-cycle, and cellular proliferation. In addition to invitro data, the clinical correlation between the KIF4A expression levels in primary OSCCs (n = 106 patients) and the clinicopathologic status by immunohistochemistry (IHC) also were evaluated. Results:KIF4A mRNA and protein were up-regulated significantly (P < 0.05) in seven OSCC-derived cells compared with human normal oral keratinocytes. In the KIF4A knockdown cells, SAC activation was observed via increased BUB1 expression on the kinetochores, appropriate kinetochore localization of MAD2, down-regulation of CDC20, up-regulation of cyclin B1, and cell-cycle arrested at G2/M phase. The results showed that cellular proliferation of KIF4A knockdown cells decreased significantly (P < 0.05) compared with control cells. IHC showed that KIF4A expression in primary OSCCs was significantly (P < 0.05) greater than in the normal oral counterparts and that KIF4A-positive OSCCs were correlated closely (P < 0.05) with tumoral size. Conclusions:Our results proposed for the first time that KIF4A controls cellular proliferation via SAC activation. Therefore, KIF4A might be a key regulator for tumoral progression in OSCCs. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Unc-93 homolog B1 (UNC93B1) plays a significant role in tumoral growth. • UNC93B1 regulates cell-cycle arrest at the G1 phase in oral squamous cell carcinoma. • Down-regulated UNC93B1 stops granulocyte macrophage colony-stimulating factor. • UNC93B1 may control tumors via granulocyte macrophage colony-stimulating factor. • UNC93B1 may be a therapeutic target for oral squamous cell carcinoma. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Cetuximab activates the p38 MAPK pathways in human skin cells. • Cetuximab inhibits c-Fos-related signals in human embryonic kidney cells. • SB203580 inhibited the expression of p38 MAPK targets and tumor growth in vivo. • Flavagline reactivated c-Fos-related factors and tumor growth in vivo. [ABSTRACT FROM AUTHOR]

Multiple coagulation factor deficiency protein 2 (MCFD2), a binding partner of lectin mannose binding 1 (LMAN1), causes combined deficiencies of coagulation factors V and VIII. MCFD2 function in inherited hematologic disorders is well elucidated; however, little is known about its role in human tumorigenesis. The aim of the current study was to investigate the states of MCFD2 in oral squamous cell carcinoma (OSCC). The expression of MCFD2 was up-regulated significantly in all cell lines examined. Evaluation of the cellular functions associated with tumoral metastasis showed that MCFD2 knockdown (shMCFD2) cells exhibited significantly lower cellular invasiveness and migration and higher cellular adhesion compared with shControl cells. Of note, shMCFD2 cells also showed weak immunoreactivity of LMAN1 and a lower secretion level of galactoside-binding soluble 3 binding protein (LGALS3BP). In addition to in vitro validation, clinical data on 70 patients with OSCC indicated that state of MCFD2 expression level is associated with regional lymph node metastasis. Altogether, we have demonstrated that MCFD2 promotes cancer metastasis by regulating LMAN1 and LGALS3BP expression levels. Hence, MCFD2 may represent a promising candidate for a novel therapeutic target for patients with metastatic OSCCs. [ABSTRACT FROM AUTHOR]

Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Oncolytic virotherapy utilizes viruses that can selectively destroy cancer cells without harming normal tissues. Clinical trials of oncolytic viruses show that most oncolytic agents are well tolerated and safe. The virotherapeutic agents currently in use have limited potency when administered alone; however, combination therapy using virotherapeutic agents and conventional anticancer agents, such as chemotherapeutics, radiation, and gene therapy, exhibits encouraging levels of efficacy. Advances in recombinant DNA technology have allowed the development of viruses that are tumor-selective and armed with transgenes, increasing the application potential and efficacy of this novel anticancer therapy. Here, we review the development of oncolytic viruses and the clinical trials of oncolytic virotherapy for oral cancers. We discuss current issues and perspectives of this evolving anticancer therapy, highlighting the potential applications of a unique, naturally occurring oncolytic virus, Sindbis virus. [Copyright &y& Elsevier]

Abstract: Background and purpose: Heavy ion beams are high linear energy transfer (LET) radiation characterized by a higher relative biologic effectiveness than low LET radiation. The aim of the current study was to determine the difference of gene expression between heavy ion beams and X-rays in oral squamous cell carcinoma (OSCC)-derived cells. Materials and methods: The OSCC cells were irradiated with accelerated carbon or neon ion irradiation or X-rays using three different doses. We sought to identify genes the expression of which is affected by carbon and neon ion irradiation using Affymetrix GeneChip analysis. The identified genes were analyzed using the Ingenuity Pathway Analysis Tool to investigate the functional network and gene ontology. Changes in mRNA expression in the genes were assessed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Results: The microarray analysis identified 84 genes that were modulated by carbon and neon ion irradiation at all doses in OSCC cells. Among the genes, three genes (TGFBR2, SMURF2, and BMP7) and two genes (CCND1 and E2F3), respectively, were found to be involved in the transforming growth factor β-signaling pathway and cell cycle:G1/S checkpoint regulation pathway. The qRT-PCR data from the five genes after heavy ion irradiation were consistent with the microarray data (P <0.01). Conclusion: Our findings should serve as a basis for global characterization of radiation-regulated genes and pathways in heavy ion-irradiated OSCC. [Copyright &y& Elsevier]

Purpose: The purpose of this study was to assess the gene expression changes in oral squamous cell carcinoma (OSCC) cells after carbon ion irradiation.Methods and Materials: Three OSCC cell lines (HSC2, Ca9-22, and HSC3) were irradiated with accelerated carbon ion beams or X-rays using three different doses. The cellular sensitivities were determined by clonogenic survival assay. To identify genes the expression of which is influenced by carbon ion irradiation in a dose-dependent manner, we performed Affymetrix GeneChip analysis with HG-U133 plus 2.0 arrays containing 54,675 probe sets. The identified genes were analyzed using the Ingenuity Pathway Analysis Tool to investigate the functional network and gene ontology. Changes in mRNA expression in the genes were assessed by real-time reverse transcriptase-polymerase chain reaction.Results: We identified 98 genes with expression levels that were altered significantly at least twofold in each of the three carbon-irradiated OSCC cell lines at all dose points compared with nonirradiated control cells. Among these, SPHK1, the expression of which was significantly upregulated by carbon ion irradiation, was modulated little by X-rays. The function of SPHK1 related to cellular growth and proliferation had the highest p value (p = 9.25e-7 to 2.19e-2). Real-time reverse transcriptase-polymerase chain reaction analysis showed significantly elevated SPHK1 expression levels after carbon ion irradiation (p < 0.05), consistent with microarray data. Clonogenic survival assay indicated that carbon ion irradiation could induce cell death in Ca9-22 cells more effectively than X-rays.Conclusions: Our findings suggest that SPHK1 helps to elucidate the molecular mechanisms and processes underlying the biologic response to carbon ion beams in OSCC. [ABSTRACT FROM AUTHOR]

Abstract: Chromosome 1 open reading frame 10 (C1orf10) is a recently identified gene encoding a protein with an S100 EF-hand calcium-binding motif, and its expression is known to be down-regulated in esophageal squamous cell carcinoma. In this study, to determine whether the loss of C1orf10 gene function could contribute to the development of oral squamous cell carcinoma (OSCC), we have evaluated the expression status of this gene by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative real-time PCR analysis. A high frequency of decrease in C1orf10 gene was detected not only in OSCC-derived cell lines but also in tumor tissues. Next, to define biological function of this gene in oral carcinogenesis, we transfected Clorf10 with an Ecdysone-inducible system in OSCC cell lines and analyzed the effects of its overexpression. Induction of C1orf10 expression resulted in a significant decline in the rate of cell proliferation, and in an arrest in the G1 phase of the cell cycle, with a down-regulation of Cyclin D1 expression. However, we could not detect significant difference in the percentage of apoptotic cells. Thus, our results suggest that the down-regulation of C1orf10 gene plays a role in oral carcinogenesis, and that its expression may negatively regulate OSCC cell proliferation by arresting the cell cycle. [Copyright &y& Elsevier]

To evaluate the role of chromosome 2 deletions in human oral squamous cell carcinoma (SCC) progression and to define the precise location of putative tumor suppressor genes, we examined 40 primary tumors and seven lymph node metastatic tumors from 40 patients with oral SCC by the polymerase chain reaction (PCR)–loss of heterozygosity (LOH) assay, using 10 different polymorphic loci on the long arm of chromosome 2. LOH was observed in 67.5% of the patients at one or more loci on the chromosome 2q. Two commonly deleted regions with high frequency of LOH, D2S1327 region at 2q32–35 (31.6%) and D2S206 region at 2q36 (36.7%), were identified by the deletion mapping of chromosome 2q, suggesting the presence of putative tumor suppressor genes associated with oral SCC. Examination of seven metastatic tumors also revealed four commonly deleted regions, D2S436, D2S1327, D2S155, and D2S164. Of these four regions D2S1327 region has no significant increase in the frequency of LOH between in primary tumors and in metastatic tumors. However, at other three regions the frequencies were much increased in metastatic tumors, comparing the results in primary tumors. Especially, very high frequencies of LOH in metastatic tumors were detected at two regions on 2q35, 100.0% at D2S155 and 57.1% at D2S164, suggesting the significant relationship between lymph node metastasis and LOH at these two regions. Our results indicate that LOH on chromosome 2q is a common event in oncogenesis and/or progression of oral SCC, and also suggest that the LOH at 2q35 play a significant role in the lymph node metastasis. [Copyright &y& Elsevier]

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 β-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, β-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. • Tspan15 expression is upregulated in OSCC cell lines and advanced OSCC cases. • Tspan15 and ADAM10 knockdown cells show suppressed invasion and migration activities. • Tspan15 knockdown induces decreased expression of ADAM10 and N-cadherin, but β-catenin. • Tspan15 and ADAM10 knockdown induces decreased the amount of soluble N-cadherin. • Tspan15 knockdown suppresses nuclear localization of β-catenin. [ABSTRACT FROM AUTHOR]