Yasuyuki Minakawa

Kinesin Family member 4A: A Potential Predictor for Progression of Human Oral Cancer

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 in vitro 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.

Overexpression of LIM and SH3 Protein 1 leading to accelerated G2/M phase transition contributes to enhanced tumourigenesis in oral cancer.

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.

Persephin: A potential key component in human oral cancer progression through the RET receptor tyrosine kinase-mitogen-activated protein kinase signaling pathway.

Persephin (PSPN) is a neurotrophic factor of the glial cell line‐derived neurotrophic factor (GDNF) family that promotes survival of multiple populations of neurons. Little is known about the relevance of PSPN in human malignancy including oral squamous cell carcinoma (OSCC). This study was undertaken to evaluate PSPN mRNA and protein expression by analyzing cellular proliferation and the cell cycle in PSPN knockdown cells in vitro. PSPN mRNA and protein were significantly (P < 0.05) up‐regulated in OSCC‐derived cells compared with human normal oral keratinocytes (n = 7). Cellular proliferation decreased significantly (P < 0.05) in PSPN knockdown cells with reduced receptor tyrosine kinase (RTK) signaling, and cell‐cycle arrest at the G1 phase resulted from up‐regulation of the cyclin‐dependent kinase inhibitors (p21Cip1, p27Kip1, p15INK4B, and p16INK4A). Furthermore, the PSPN protein expression in 101 primary OSCCs was significantly (P < 0.05) higher than in normal counterparts. Among the clinical variables analyzed, overexpression of PSPN also was related closely (P < 0.05) to tumoral size. Our results suggested that PSPN is a possible key regulator of OSCC progression via PSPN‐RET‐mitogen‐activated protein kinase activation and that PSPN overexpression may have diagnostic potential for OSCC.

Protein O-fucosyltransferase 1: A potential diagnostic marker and therapeutic target for human oral cancer

Protein O-fucosyltransferase 1 (POFUT1) is the enzyme that adds O-fucose through O-glycosidic linkage to conserved serine or threonine residues in the epidermal growth factor-like repeats of a number of cellular surface and secreted proteins. Our previous study using microarray technology showed that significant upregulation of POFUT1 occurs in oral squamous cell carcinoma (OSCC)-derived cell lines compared to human normal oral keratinocytes. The aim of the present study was to examine the status of POFUT1 mRNA and protein expression in OSCC-derived cell lines and human primary OSCCs. POFUT1 mRNA was upregulated significantly (P<0.05 for both comparisons) in five OSCC-derived cell lines and primary OSCCs using quantitative reverse transcriptase-polymerase chain reaction. Immunohistochemistry data indicated that POFUT1 protein expression levels were consistent with mRNA expression status in OSCC-derived cell lines and primary OSCCs. Furthermore, POFUT1 expression status was correlated significantly (P=0.048) with the primary tumor size. The proliferation of POFUT1 knockdown cells was inhibited significantly compared with that of control cells. These results indicated that POFUT1 expression can contribute to cancer progression and that POFUT1 may serve as a diagnostic marker and a therapeutic target for OSCCs.

ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

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.

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.

Decorin in human oral cancer: a promising predictive biomarker of S-1 neoadjuvant chemosensitivity.

We reported previously that decorin (DCN) is significantly up-regulated in chemoresistant cancer cell lines. DCN is a small leucine-rich proteoglycan that exists and functions in stromal and epithelial cells. Accumulating evidence suggests that DCN affects the biology of several types of cancer by directly/indirectly targeting the signaling molecules involved in cell growth, survival, metastasis, and angiogenesis, however, the molecular mechanisms of DCN in chemoresistance and its clinical relevance are still unknown. Here we assumed that DCN silencing cells increase chemosusceptibility to S-1, consisted of tegafur, prodrug of 5-fluorouracil. We first established DCN knockdown transfectants derived from oral cancer cells for following experiments including chemosusceptibility assay to S-1. In addition to the in vitro data, DCN knockdown zenografting tumors in nude mice demonstrate decreasing cell proliferation and increasing apoptosis with dephosphorylation of AKT after S-1 chemotherapy. We also investigated whether DCN expression predicts the clinical responses of neoadjuvant chemotherapy (NAC) using S-1 (S-1 NAC) for oral cancer patients. Immunohistochemistry data in the preoperative biopsy samples was analyzed to determine the cut-off point for status of DCN expression by receiver operating curve analysis. Interestingly, low DCN expression was observed in five (83%) of six cases with complete responses to S-1 NAC, and in one (10%) case of 10 cases with stable/progressive disease, indicating that S-1 chemosensitivity is dramatically effective in oral cancer patients with low DCN expression compared with high DCN expression. Our findings suggest that DCN is a key regulator for chemoresistant mechanisms, and is a predictive immunomarker of the response to S-1 NAC and patient prognosis.

Down-regulated expression of family with sequence similarity 3, member B (FAM3B), in oral squamous cell carcinoma

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 .

Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity

Resistance to anticancer medications often leads to poor outcomes. The present study explored an effective approach for enhancing chemotherapy targeted against human cancer cells. Real-time quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed overexpression of members of aldo-keto reductase (AKR) 1C family, AKR1C1, AKR1C2, AKR1C3, and AKR1C4, in cisplatin, cis-diamminedichloroplatinum (II) (CDDP)-resistant human cancer cell lines, HeLa (cervical cancer cells) and Sa3 (oral squamous cell carcinoma cells). The genes were downregulated using small-interfering RNA (siRNA) transfection, and the sensitivity to CDDP or 5-fluorouracil (5-FU) was investigated. When the genes were knocked down, sensitivity to CDDP and 5-FU was restored. Furthermore, we found that administration of mefenamic acid, a widely used non-steroidal anti-inflammatory drug (NSAID) and a known inhibitor of AKR1Cs, enhanced sensitivity to CDDP and 5-FU. The present study suggests that AKR1C family is closely associated with drug resistance to CDDP and 5-FU, and mefenamic acid enhances their sensitivity through its inhibitory activity in drug-resistant human cancer cells. Thus, the use of mefenamic acid to control biological function of AKR1C may lead to effective clinical outcomes by overcoming anticancer drug resistance.