Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression.

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.

Mucoepidermoid carcinoma with extensive spindled morphology and melanocytic marker expression.

Mucoepidermoid carcinoma (MEC) is the most common malignant neoplasm of the salivary gland. Albeit common, histologic variants have rarely been noted in MEC. Here, we report a 49-year-old man with a sublingual gland tumor. Histologically, the tumor was composed of spindle cells arranged in interlacing fascicules or globular nests. A few bland small glands containing mucous cells were also scattered. The spindle tumor cells completely lacked immunoreactivity for cytokeratin, and exhibited immunoreactivity for vimentin, S-100, HMB-45, Melan A, and SOX10. The tumor was initially suspected to be clear cell sarcoma, malignant melanoma, or perivascular epithelioid cell tumor with a few entrapped nonneoplastic duct epitheliums. However, reverse-transcription polymerase chain reaction revealed the CRTC3-MAML2 fusion gene product diagnostic of MEC. In fact, a very minor component of the epithelial cells was reminiscent of conventional MEC, whereas major spindled tumor cells possessed markedly altered differentiation. This is the first case report of MEC with extensive spindled morphology and melanocytic marker expression.

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.

SIPA1 promotes invasion and migration in human oral squamous cell carcinoma by ITGB1 and MMP7.

Signal-induced proliferation-associated protein 1 (SIPA1) is known to be a GTPase activating protein. Overexpressed SIPA1 is related to metastatic progression in breast and prostate cancers; however, the relevance of SIPA1 in oral squamous cell carcinoma (OSCC) is still unknown. The aim of this study was to examine SIPA1 expression and its functional mechanisms in OSCC. SIPA1 mRNA and protein expressions were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry. The expressions of SIPA1 were up-regulated significantly in vitro and in vivo. Moreover, SIPA1 expression was correlated with regional lymph node metastasis. We next assessed the cellular functions associated with tumoral metastasis using SIPA1 knockdown (shSIPA1) cells and analyzed the downstream molecules of SIPA1, i.e., bromodomain containing protein 4(BRD4), integrin beta1 (ITGB1), and matrix metalloproteinase 7 (MMP7). The shSIPA1 cells showed decreased invasiveness and migratory activities, however cellular adhesion ability was maintained at a high level. In addition, ITGB1 expression was greater in shSIPA1 cells, whereas MMP7 expression was lower than in control cells. This research is the first to establish that SIPA1 promotes cancer metastasis by regulating the ITGB1 and MMP7. Therefore, SIPA1 might be a novel therapeutic target for patients with lymph node metastasis of OSCC.

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.

p62 modulates the intrinsic signaling of UVB-induced apoptosis.

BACKGROUND: UVB radiation is the main source of sunburn and skin cancers. Apoptosis eliminates photodamaged cells, and is thus important for preventing epidermal carcinogenesis. The cytoplasmic regulatory protein p62/A170/sequestosome 1 (p62) molecule is involved in a variety of cellular and signaling pathways. p62 is known to be and important in autophagy, but its role in UVB-induced apoptosis remains to be clarified. OBJECTIVE: To investigate the role of p62 against UVB-induced apoptotic changes, using mouse embryonic fibroblasts (MEFs) derived from p62 homozygous knockout (p62(-/-)) mice. METHODS: p62(-/-) and wild-type (p62(+/+)) mice and MEFs were subjected to UVB irradiation, and the resultant apoptosis was analyzed using flow cytometry, quantitative real-time PCR, and western blots. RESULTS: Apoptosis was decreased in the p62(-/-) MEFs compared to p62(+/+) MEFs in response to UVB treatment. Compared with p62(+/+) MEFs, p62(-/-) MEFs expressed significantly more Bcl-2 and less Bax, and showed increased Src and Stat3 phosphorylation. Our results show that p62 regulates apoptotic pathways by modifying critical signaling intermediates such as Src and Stat3. CONCLUSION: p62 deficiency [corrected] reduces UVB-induced apoptosis by modulating intrinsic apoptotic signaling through Src phosphorylation.

ARNT2 Regulates Tumoral Growth in Oral Squamous Cell Carcinoma.

Aryl hydrocarbon receptor nuclear translocator (ARNT) 2 is a transcriptional factor related to adaptive responses against cellular stress from a xenobiotic substance. Recent evidence indicates ARNT is involved in carcinogenesis and cancer progression; however, little is known about the relevance of ARNT2 in the behavior of oral squamous cell carcinoma (OSCC). In the current study, we evaluated the ARNT2 mRNA and protein expression levels in OSCC in vitro and in vivo and the clinical relationship between ARNT2 expression levels in primary OSCCs and their clinicopathologic status by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemistry. Using ARNT2 overexpression models, we performed functional analyses to investigate the critical roles of ARNT2 in OSCC. ARNT2 mRNA and protein were down-regulated significantly (P < 0.05 for both comparisons) in nine OSCC-derived cells and primary OSCC (n=100 patients) compared with normal counterparts. In addition to the data from exogenous experiments that ARNT2-overexpressed cells showed decreased cellular proliferation, ARNT2-positive OSCC cases were correlated significantly (P < 0.05) with tumoral size. Since von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase, a negative regulator of hypoxia-inducible factor (HIF1)-α, is a downstream molecule of ARNT2, we speculated that HIF1-α and its downstream molecules would have key functions in cellular growth. Consistent with our hypothesis, overexpressed ARNT2 cells showed down-regulation of HIF1-α, which causes hypofunctioning of glucose transporter 1, leading to decreased cellular growth. Our results proposed for the first time that the ARNT2 level is an indicator of cellular proliferation in OSCCs. Therefore, ARNT2 may be a potential therapeutic target against progression of OSCCs.

MicroRNA-155-5p is associated with oral squamous cell carcinoma metastasis and poor prognosis.

BACKGROUND: Abnormal miRNA expression was recently implicated in the metastasis of oral squamous cell carcinoma (OSCC) and with a poor prognosis. The initiation of the invasion-metastasis cascade involves epithelial-mesenchymal transition (EMT). Our aim was to clarify how miRNA, especially miR-155-5p misexpression contributes to OSCC metastasis through EMT. METHODS: We collected tumor samples from 73 subjects with OSCC. The samples were analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and correlations between miR-155-5p levels and clinical characteristics were investigated. OSCC cell lines were analyzed by miRNA microarray and by transfection with a miR-155-5p mimic or inhibitor, followed by proliferation and wound-healing migration assays. qRT-PCR analyses of EMT makers in cells transfected with miR-155-5p inhibitor were performed. RESULTS: We found high miR-155-5p expression in tissue samples from subjects with OSCC that had metastasized to cervical lymph nodes. HSC-3 cells also strongly expressed miR-155-5p. The epithelial marker E-cadherin was strongly expressed in HSC-3 cells transfected with miR-155-5p inhibitor, and we observed elevated SOCS1 and decreased STAT3 expression in these cells. CONCLUSIONS: Our results suggest that miR-155-5p causes OSCC to metastasize, and could serve as a novel therapeutic target for OSCC.

Kinesin family member 14 in human oral cancer: A potential biomarker for tumoral growth.

Kinesin family member 14 (KIF14), a microtubule-based motor protein, plays an important role in chromosomal segregation, congression, and alignment. Considerable evidence indicates that KIF14 is involved in cytokinesis, although little is known about its role in oral squamous cell carcinomas (OSCCs). In the current study, we functionally and clinically investigated KIF14 expression in patients with OSCC. Quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses were used to assess the KIF14 regulatory mechanism in OSCC. Immunohistochemistry (IHC) was performed to analyze the correlation between KIF14 expression and clinical behavior in 104 patients with OSCC. A KIF14 knockdown model of OSCC cells (shKIF14 cells) was used for functional experiments. KIF14 expression was up-regulated significantly (P<0.05) in OSCCs compared with normal counterparts in vitro and in vivo. In addition, shKIF14 cells inhibited cellular proliferation compared with control cells by cell-cycle arrest at the G2/M phase through up-regulation of G2 arrest-related proteins (p-Cdc2 and cyclin B1). As expected, IHC data from primary OSCCs showed that KIF14-positive patients exhibited significantly (P<0.05) more larger tumors compared with KIF14-negative patients. The current results suggest for the first time that KIF14 is an indicator of tumoral size in OSCCs and that KIF14 might be a potential therapeutic target for development of new treatments for OSCCs.

Negative expression of N-acetylglucosaminyltransferase V in oral squamous cell carcinoma correlates with poor prognosis.

N-acetylglucosaminyltransferase V (GnT-V), an enzyme with a key role in the branching of asparagine-linked oligosaccharides, is strongly linked to tumor invasion and metastasis of many solid tumors. Here we searched for correlations between the clinical features of patients with oral squamous cell carcinoma (OSCC) and GnT-V expression in the tumor, and we studied the feasibility of using GnT-V as a marker for oral cancer prognosis. Samples from 68 patients with OSCC were examined by immunohistochemistry using antibodies against GnT-V. Correlations between the expression level of GnT-V in the tumor and patient clinical features were statistically analyzed. Positive GnT-V expression was found in 48 cases (70.6%), and negative GnT-V expression was found in 20 cases (29.4%). Negative GnT-V expression was associated with mode of invasion by multiple logistic regression analysis (OR: 3.605; P = 0.048). Biological characteristics of tumors and the Ki-67 labeling index were higher in tumors with negative GnT-V expression than in those with positive GnT-V expression, although the difference was not significant (P = 0.176). Patients with negative GnT-V expression had significantly shorter survival than those with tumors having positive GnT-V expression (5-year survival rate, 58.2% and 86.5%, respectively; P = 0.025). Negative GnT-V expression was a significant unfavorable prognostic factor for OSCC (hazard ratio, 4.246; P = 0.045). The loss of GnT-V expression is a likely indicator of tumors with high potential of tumor invasion and poor prognosis in OSCC patients.

Prognosis for gingival carcinomas with a delayed diagnosis after dental extraction.

PURPOSE: In gingival squamous cell carcinoma (GSCC), the association between survival and previous dental extraction (DE) is controversial. The purpose of this study was to investigate the prognosis for patients in whom GSCC was detected after DE was performed. PATIENTS AND METHODS: DE for GSCC tumor symptoms was performed in 19 patients before diagnosis (DE group) and not in 58 patients (non-DE group). The clinical features, characteristics, and prognosis were evaluated statistically between the 2 groups. RESULTS: The interval from DE to the first hospital visit was 1.1 to 97 weeks (median, 7.3 weeks). There was no significant difference in tumor status, node status, local recurrence, pathologically positive lymph nodes, or distant metastasis between the DE and non-DE groups. Bone invasion was observed radiographically in 6 patients with mandibular GSCC in the DE group (100%) and 13 in the non-DE group (68.4%). There was a significant difference in bone invasion between the DE and non-DE groups (P < .01). Segmental mandibulectomy was performed in 11 patients (84.6%) in the DE group and 21 patients (61.8%) in the non-DE group. Extent of resection tended to be larger for the DE group. The 5-year overall survival rate was 84.6% for the DE group and 65.8% for patients with mandibular GSCC in the non-DE group. For maxillary GSCC, the survival rates differed significantly between groups (33.3% in DE group and 73.7% in non-DE group). CONCLUSIONS: For mandibular GSCC, the resection field was appropriate for the extent of bone invasion after DE and the prognosis was similar to that in the non-DE group. For maxillary GSCC, a broad surgical field is suggested because of the potential for rapid spread in cancellous bony trabeculae.

Circulating tumor-derived mutant mitochondrial DNA: a predictive biomarker of clinical prognosis in human squamous cell carcinoma.

While circulating tumor-derived molecules have been identified in a variety of malignant tumors, it is sometimes difficult to detect the molecular targets due to the lower serum concentration. We report that evaluation of circulating tumor-derived mitochondrial DNA (mtDNA) seems to have novel efficiency for detecting tumoral micrometastasis. In murine xenografting human oral cancer cells, human mtDNAs could be quantitatively detected from multiple organs and blood samples whereas human nucleic DNAs could not. We also determined if this mtDNA blood test was relevant for patients with oral cancer with no histologic evidence of tumoral cells in their surgical margins. For this, mtDNA from normal and tumorous tissues and serum mtDNA obtained pre and postoperatively was examined at three different regions including the displacement loop, 12S-rRNA, and 16S-rRNA. All non-recurring patients had significantly higher amounts of mutant mtDNAs in the tumoral tissues compared with the non-recurring group. More importantly, on the blood test with the cut-off point by receiver operating characteristic (ROC) curve analysis, while the vast majority of serum mtDNA samples obtained postoperatively in the recurring cases maintained significantly higher amounts of mutant mtDNAs, the non-recurring cases did not, and they showed good prognosis. This is the first report of this approach for managing patients after resection of oral tumors, and may have substantial diagnostic potential for other tumoral types.

State of heat shock factor 1 expression as a putative diagnostic marker for oral squamous cell carcinoma.

Heat shock factor 1 (HSF1) is responsible for expres-- sion of a large class of heat shock proteins that have been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of HSF1 on the behavior of oral squamous cell carcinoma (OSCC). In this study, we assessed the value of HSF1 for predicting clinical outcomes in OSCC. Quantitative reverse transcriptase-polymerase chain reaction and Western blotting showed that the expressions of HSF1 mRNA and protein in OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Sa3, Ca9-22, KON and Ho-1-u-1) were elevated compared with those in human normal oral keratinocytes (P<0.05). Similar to in vitro data, HSF1 mRNA expression in primary OSCCs (n=50) was significantly greater than in normal counterparts (P<0.05). Since HSF1 was observed in the nucleus and cytoplasm by immu-- nohistochemistry, we investigated the correlation between the HSF1 expression status at each subcellular location and the clinical behavior of OSCCs. Among the clinical classifications, higher nuclear HSF1 expression was closely related to tumor size and histopathologic types (P<0.05). These results showed for the first time that nuclear HSF1 expression may contribute to cancer progression and that HSF1 might be a potential diagnostic biomarker and a therapeutic target for OSCCs.

Dermatopontin: a potential predictor for metastasis of human oral cancer.

Dermatopontin (DPT), a component of the extracellular matrix (ECM), is involved in promotion of cellular adhesion and ECM assembly activities. However, the role of DPT in the pathogenesis of carcinoma is unclear. We evaluated DPT expression in human oral cancer and its possible roles including cellular adhesion and invasiveness. We first investigated the DPT mRNA and protein expression status in human oral squamous cell carcinoma (OSCC)-derived cells. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analysis detected frequent downregulation of DPT in OSCC-derived cells compared to human normal oral keratinocytes. To assess the epigenetic regulation of DPT, OSCC-derived cells were treated with a histone deacetylase inhibitor, sodium butyrate (NaB). NaB restored the DPT expression in OSCC-derived cells. DPT-overexpressed cells were examined whether DPT could contribute to cellular adhesion and invasiveness. Markedly, increased adhesion and decreased invasiveness in DPT-overexpressed cells were found compared to mock-transfected cells. Adhesion of DPT-overexpressed cells was inhibited by α3ß1 integrin functional blocking antibody. OSCC-derived cells treated with NaB also decreased invasiveness. The expression status of DPT in primary OSCCs (n = 97) was analyzed and compared to clinicopathological behavior. DPT expression in primary OSCCs was significantly lower (p < 0.05) than in the normal counterparts and was correlated significantly (p < 0.05) with regional lymph node metastasis. Our data provided strong evidence that downregulation of DPT is a characteristic event in OSCCs and that DPT was correlated with cellular adhesion and invasiveness. Therefore, DPT might play an important role in regulating tumor invasion and metastasis.

Dickkopf-1 in human oral cancer.

Dickkopf-1 (Dkk1), a negative regulator of the Wnt signaling pathway, is implicated in tumorigenesis in several types of cancer. The purpose of this study was to determine the involvement of Dkk1 in oral squamous cell carcinoma (OSCC). We found that Dkk1 is frequently upregulated in OSCC-derived cell lines and primary OSCCs compared with normal counterparts. Unexpectedly, Dkk1-positive cases were correlated significantly (P<0.05) with a low risk of regional lymph node metastasis. We also found that cellular migration and invasiveness increased in Dkk1 knockdown cells and decreased in Dkk1 overexpressed cells. Furthermore, we investigated the relationship between the expression of Dkk1 and distribution of ß-catenin in OSCC cells, since the Wnt signaling pathway is related closely to ß-catenin. Whereas alteration of the ß-catenin levels was not observed in each subcellular fractionation, the phosphorylated ß-catenin levels in nuclei increased in Dkk1 knockdown cells and decreased in Dkk1 overexpressed cells. These data indicated that the high phosphorylation level of ß-catenin in nuclei was correlated with a high risk of tumor invasiveness. The current study suggested that Dkk1 plays an important role in regulating cellular migration and invasiveness, making Dkk1 a potential biomarker for early detection of lymph node metastasis in OSCCs.

Antitumor activity of satraplatin in cisplatin-resistant oral squamous cell carcinoma cells.

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.

Expression status of Zic family member 2 as a prognostic marker for oral squamous cell carcinoma.

PURPOSE: To determine the involvement of ZIC2 in oral squamous cell carcinoma (OSCC). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

State of homeobox A10 expression as a putative prognostic marker for oral squamous cell carcinoma.

Homeobox (HOX) A10, the regulator of embryonic morphogenesis and differentiation, is aberrantly expressed in several cancer types. Our previous study using microarray technology showed that significant up-regulation of HOXA10 occurs in oral squamous cell carcinoma (OSCC)-derived cell lines compared to human normal oral keratinocytes (HNOKs). The aim of the current study was to examine the status of HOXA10 mRNA and protein expression in OSCC-derived cell lines and human primary OSCCs. HOXA10 mRNA was up-regulated in six OSCC-derived cell lines compared with HNOKs and in primary OSCCs by using real-time quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemistry data indicated that HOXA10 protein expression levels were consistent with mRNA expression status in OSCC-derived cell lines and primary OSCCs. Furthermore, HOXA10 expression status was correlated with the TNM stage (P<0.05). These results indicate that HOXA10 expression could contribute to cancer progression and prognosis and that HOXA10 may be a potential diagnostic marker and a therapeutic target for OSCCs.