Yukinao Kouzu

Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis

Stathmin is an intracellular phosphoprotein that is overexpressed in a number of human malignancies. Our previous study using proteomic profiling showed that significant upregulation of stathmin occurs in oral squamous-cell carcinoma (OSCC)-derived cell lines. In the current study, to determine the potential involvement of stathmin in OSCC, we evaluated the state of stathmin protein and mRNA expression in OSCC-derived cell lines and human primary OSCCs. A significant increase in stathmin expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes. In immunohistochemistry, 65% of the OSCCs were positive for stathmin, and no immunoreaction was observed in corresponding normal tissues. Real-time quantitative reverse transcriptase–polymerase chain reaction data were consistent with the protein expression status. Moreover, stathmin expression status was correlated with the TNM stage grading. Furthermore, we found a statistical correlation between the protein expression status and disease-free survival (P=0.029). These results suggest that expression of stathmin could contribute to cancer progression/prognosis, and that stathmin may have potential as a biomarker and a therapeutic target for OSCC.

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

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

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.

Clinical significance of gelsolin-like actin-capping protein expression in oral carcinogenesis: an immunohistochemical study of premalignant and malignant lesions of the oral cavity.

BackgroundGelsolin-like actin-capping protein (CapG) is a ubiquitous gelsolin-family actin-modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis, and motility. CapG has generated great interest due to its oncogenic function in the control of cell migration or invasion in a variety of cancer cells. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous-cell carcinoma (OSCC) cells and detected significantly high expression levels of CapG in OSCC-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of CapG in OSCC, we evaluated the status of CapG protein and mRNA expression in human oral premalignant lesions (OPLs) and primary OSCCs and correlated the results with clinicopathologic variables.MethodsMatched normal and tumour tissue sections of 79 human primary OSCCs and 28 OPLs were analyzed for CapG expression by immunohistochemistry (IHC). Correlations between CapG-immunohistochemical staining scores of OSCCs and clinicopathologic features were evaluated by Fishers exact test. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to estimate CapG expression at the mRNA level.ResultsIn IHC, substantial up-regulation of CapG protein was observed in primary OSCCs (52%) and OPLs (64%), whereas corresponding normal tissues showed consistently weak or absent immunoreactivity of CapG. qRT-PCR data were consistent with the protein expression status. Moreover, CapG expression was correlated with the TNM stage grading of OSCCs.ConclusionOur finding of frequent dysregulated expression of CapG in premalignant and malignant lesions together with an association with an advanced clinical disease stage suggests that CapG could contribute to cancer development and progression and that CapG may have potential as a biomarker and a therapeutic target for OSCC.

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 and altered subcellular localization of autophagy-related 16-like 1 in human oral squamous-cell carcinoma: correlation with lymphovascular invasion and lymph-node metastasis

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.

Decreased expression of kallikrein-related peptidase 13: Possible contribution to metastasis of human oral cancer

The human kallikrein‐related peptidase family is comprised of 15 serine protease genes on chromosome 19q13.4 . Our previous microarray analyses showed that the gene kallikrein‐related peptidase 13 (KLK13) was down‐regulated in oral squamous cell carcinoma (OSCC) cell lines. We evaluated the expression status of KLK13 in primary OSCCs and performed functional molecular experiments in OSCC cell lines. In 102 primary tumors studied, KLK13 expression significantly (P < 0.05) decreased compared with matched normal counterparts. Interestingly, KLK13‐negative cases correlated significantly (P < 0.05) with regional lymph node metastasis. In vitro, cells overexpressing KLK13 (oeKLK13) had decreased invasiveness and motility and up‐regulation of adhesion molecules (E‐cadherin, α‐catenin, β‐catenin, junction plakoglobin, plakophilin4, desmocollin2, desmoglein3, and desmoplakin) compared with control cells. A rescue experiment that transfected oeKLK13 cells with siRNA against KLK13 restored invasiveness and migration activities with down‐regulated adhesion molecules. Based on our results, we concluded that KLK13 may play an important role in regulating cellular migration and invasiveness, making the loss of KLK13 a potential biomarker for early detection of lymph node metastasis in OSCCs.

Lipocalin-2 is associated with radioresistance in oral cancer and lung cancer cells.

The aim of the present study was to identify a target molecule that could predict the efficacy of radiotherapy in oral squamous cell carcinoma (OSCC). We used DNA microarray analysis to identify differences in gene expression after X-ray irradiation. We compared the gene expression profiles between X-ray (8 Gy)-irradiated Ca9-22 cells (an OSCC-derived cell line) and unirradiated Ca9-22 cells. A total of 167 genes with a 2-fold higher level of expression induced by X-ray irradiation were identified. Lipocalin-2 (LCN2) had the greatest increase in expression after X-ray irradiation, and it was categorized in a network that has cancer-related functions with the Ingenuity Pathway Analysis tool. Upregulated expression of LCN2 mRNA was validated by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. When the LCN2 gene was knocked down in OSCC cells (Ca9-22 and HSC-2) and lung cancer cells (A549) by using small interfering RNA, the radiosensitivity of these cells was enhanced. Our findings suggest that the overexpression of LCN2 is likely associated with radioresistance in oral cancer and lung cancer cells, and that LCN2 expression levels could be used to predict radioresistance. Thus, regulating the expression or function of LCN2 could enhance the radiation response, resulting in a favorable outcome of radiotherapy.

Network-based analysis of calcium-binding protein genes identifies Grp94 as a target in human oral carcinogenesis

To characterise Ca2+-binding protein gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with normal oral tissues. One hundred Ca2+-binding protein genes differentially expressed in OSCCs were identified, and genetic pathways associated with expression changes were generated. Among genes mapped to the network with the highest significance, glucose-regulated protein 94 kDa (Grp94) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs, and oral premalignant lesions (OPLs). A significant (P<0.001) overexpression of Grp94 protein was observed in all cell lines compared to normal oral epithelium. Immunohistochemical analysis showed highly expressed Grp94 in primary OSCCs and OPLs, whereas most of the corresponding normal tissues had no protein immunoreaction. Real-time quantitative reverse transcriptase-PCR data agreed with the protein expression status. Moreover, overexpression of Grp94 in primary tumours was significantly (P<0.001) correlated with poor disease-free survival. The results suggested that Grp94 may have potential clinical application as a novel diagnosis and prognostic biomarker for human OSCCs.

Glutamate acid decarboxylase 1 promotes metastasis of human oral cancer by β-catenin translocation and MMP7 activation.

BackgroundGlutamate decarboxylase 1 (GAD1), a rate-limiting enzyme in the production of γ-aminobutyric acid (GABA), is found in the GABAergic neurons of the central nervous system. Little is known about the relevance of GAD1 to oral squamous cell carcinoma (OSCC). We investigated the expression status of GAD1 and its functional mechanisms in OSCCs.MethodsWe evaluated GAD1 mRNA and protein expressions in OSCC-derived cells using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. To assess the critical functions of GAD1, i.e., cellular proliferation, invasiveness, and migration, OSCC-derived cells were treated with the shRNA and specific GAD1 inhibitor, 3-mercaptopropionic acid (3-MPA). GAD1 expression in 80 patients with primary OSCCs was analyzed and compared to the clinicopathological behaviors of OSCC.ResultsqRT-PCR and immunoblotting analyses detected frequent up-regulation of GAD1 in OSCC-derived cells compared to human normal oral keratinocytes. Suppression of nuclear localization of β-catenin and MMP7 secretion was observed in GAD1 knockdown and 3-MPA-treated cells. We also found low cellular invasiveness and migratory abilities in GAD1 knockdown and 3-MPA-treated cells. In the clinical samples, GAD1 expression in the primary OSCCs was significantly (P < 0.05) higher than in normal counterparts and was correlated significantly (P < 0.05) with regional lymph node metastasis.ConclusionsOur data showed that up-regulation of GAD1 was a characteristic event in OSCCs and that GAD1 was correlated with cellular invasiveness and migration by regulating β-catenin translocation and MMP7 activation. GAD1 might play an important role in controlling tumoral invasiveness and metastasis in oral cancer.