Masanobu Yamatoji

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 α3β1 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.

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.

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 β-catenin in OSCC cells, since the Wnt signaling pathway is related closely to β-catenin. Whereas alteration of the β-catenin levels was not observed in each subcellular fractionation, the phosphorylated β-catenin levels in nuclei increased in Dkk1 knockdown cells and decreased in Dkk1 overexpressed cells. These data indicated that the high phosphorylation level of β-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.

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.

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.

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.

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

ABSTRACT 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. HighlightsSIPA1 expression was up‐regulated in oral squamous cell carcinoma (OSCC).SIPA1‐positive OSCCs were correlated with regional lymph node metastasis.SIPA1 controlled BRD4 and influenced transcription of ITGB1and MMP7.SIPA1 induced cellular invasion and migration and decreased cellular adhesion.SIPA1 might be a potential biomarker of cancer metastasis for OSCC.

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

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.

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.

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

ABSTRACT 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. HIGHLIGHTSDAGLA was up‐regulated in human oral squamous cell carcinoma (OSCC) cells compared with human normal oral keratinocytes (HNOKs).Overexpression of DAGLA in OSCCs controlled cell proliferation through cell‐cycle progression.The tumoral volume of the orlistat‐treated group was clearly smaller than that of the control group in vivo.DAGLA expression level might be a useful diagnostic indicator and a new therapeutic target for OSCCs.