Dai Nakashima

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.

Epithelial cell transforming sequence 2 in human oral cancer.

Background Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor for Rho family GTPase, which has been implicated in the malignant phenotype of human cancers. Little is known about the effect of a high level of ECT2 in regulating oral cancer cell behavior. In this study, we investigated the involvement of ECT2 in oral squamous cell carcinoma (OSCC). Methodology/Principal Findings We analyzed ECT2 expression in OSCC-derived cell lines and primary OSCCs compared with matched normal tissue (n = 96) by quantitative reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. We then evaluated the correlation between the ECT2 expression status in primary OSCCs and the clinicopathological features. ECT2 expression was significantly up-regulated in OSCCs in vitro and in vivo (p<0.05). Among the clinical variables analyzed, higher ECT2 expression also was associated with the TNM stage grading (p<0.05). When we performed functional analyses of ECT2 in OSCC-derived cells using the shRNA system, the cellular proliferation of the ECT2 knockdown cells decreased significantly compared with the control cells (p<0.05). Cell cycle analysis by flow cytometry showed arrest of cell cycle progression at the G1 phase in the ECT2 knockdown cells. We also found up-regulation of the Cip/Kip family of the cyclin-dependent kinase inhibitors, p21cip1 and p27kip1, and down-regulation of cyclin D1, cyclin E, and CDK4. These data suggested that the elevated Cip/Kip family induced inhibition of the cyclin D1-CDK complex activity leading to cell cycle arrest at the G1 phase. Conclusions/Significance Our results proposed for the first time that ECT2 is an indicator of cellular proliferation in OSCCs and that ECT2 might be a potential therapeutic target for the development of new treatments for OSCCs.

Genes and molecular pathways related to radioresistance of oral squamous cell carcinoma cells

To identify genes associated with radioresistant oral squamous cell carcinoma (OSCC), we compared gene expression signatures between OSCC cell lines exhibiting radioresistance and cells with radiosensitivity after X‐ray irradiation in a dose‐dependent manner using Affymetrix GeneChip analysis with Human Genome‐U133 plus 2.0 GeneChip. The microarray data identified 167 genes that were significantly overexpressed in radioresistant cells after X‐ray irradiation. Among the genes identified, 40 were mapped to 3 highly significant genetic networks identified by the Ingenuity Pathway Analysis tool. Gene ontology analysis showed that cancer‐related function had the highest significance. The 40 genes included 25 cancer‐related genes that formed 1 network and were categorized by function into growth and proliferation, apoptosis, and adhesion. Furthermore, real‐time quantitative reverse transcriptase–polymerase chain reaction showed that the mRNA expression levels of the 25 genes were higher in radioresistant cells than in radiosensitive cells in a dose‐dependent manner and in a time‐dependent manner. Our results suggest that the identified genes help to elucidate the molecular mechanisms of the radioresistance of OSCC and could be radiotherapeutic molecular markers for choosing the appropriate radiotherapy for this disease.

Protein expression profiling identifies maspin and stathmin as potential biomarkers of adenoid cystic carcinoma of the salivary glands

Adenoid cystic carcinoma (ACC) is one of the most common malignant tumors of the salivary glands. It tends to grow slowly but is associated with a poor prognosis compared to other malignant salivary gland tumors. To identify specific markers of ACC, we examined protein expression profiling in ACC xenograft and normal salivary glands (NSG) using fluorescent 2‐dimensional differential in‐gel electrophoresis (2‐D‐DIGE), an emerging technique for comparative proteomics, that improves the reproducibility and reliability of differential protein expression analysis between the samples. To identify the proteins, matrix‐assisted laser desorption/ionization time‐of‐flight peptide mass fingerprinting was carried out. Using these strategies, we detected 4 upregulated proteins and 5 downregulated proteins in ACC xenograft. Maspin and stathmin were selected for further analyses. Western blotting and immunohistochemical staining showed a higher expression of these proteins in ACC xenograft and clinical ACC tissue compared to NSG. Furthermore, Expression of these proteins was correlated with the histologic grading of ACC (n = 10). Therefore, our data indicate that maspin and stathmin may be not only useful biomarkers of ACC but also markers of biologic behavior in this tumor.

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.

Lin-7C/VELI3/MALS-3: An Essential Component in Metastasis of Human Squamous Cell Carcinoma

Using proteomic selection, functional verification, and clinical validation, we identified specific down-regulation of Lin-7C/VELI3/MALS-3 (Lin-7C), which marks oral squamous cell carcinoma (OSCC) metastasis. Despite a rarity of sequence variations in the Lin-7C gene in both primary OSCC and OSCC-derived cells, a high prevalence of hypermethylation was detected in the CpG island region that strongly correlated with its down-regulation. Inducible Lin-7C mRNA by experimental demethylation was found in all OSCC cells tested. Overexpression of the Lin-7C gene in an OSCC cell clone does not contribute to underproliferation but results in a noninvasive phenotype with elevated beta-catenin expression. Experimental metastases in multiple organs of immunodeficient mice were inhibited in cells expressing Lin-7C. Finally, the Lin-7C expression status in primary tumors afforded significantly (P<0.001) high accuracy for predicting lymph node metastasis. These results establish Lin-7C as a novel target of early detection, prevention, and therapy for OSCC metastasis.

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.

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.