Takaaki Tsunematsu

The FGFR1 inhibitor PD173074 induces mesenchymal–epithelial transition through the transcription factor AP-1

Background:Epithelial–mesenchymal transition (EMT) is a crucial process in cancer progression that provides cancer cells with the ability to escape from the primary focus, invade stromal tissues and migrate to distant regions. Cell lines that lack E-cadherin show increased tumorigenesis and metastasis, and the expression levels of E-cadherin and Snail correlate inversely with the prognosis of patients suffering from breast cancer or oral squamous cell carcinoma (OSCC). Moreover, recent studies have shown that most EMT cases are regulated by soluble growth factors or cytokines. Among these factors, fibroblast growth factors (FGFs) execute diverse functions by binding to and activating members of the FGF receptor (FGFR) family, including FGFR1–4. Fibroblast growth factor receptor 1 is an oncoprotein that is involved in tumorigenesis, and PD173074 is known to be a selective inhibitor of FGFR1. However, the roles of FGFR1 and FGFR1 inhibitors have not yet been examined in detail.Methods:Here, we investigated the expression of FGFR1 in head and neck squamous cell carcinoma (HNSCC) and the role of the FGFR1 inhibitor PD173074 in carcinogenesis and the EMT process.Results:Fibroblast growth factor receptor 1 was highly expressed in 54% of HNSCC cases and was significantly correlated with malignant behaviours. Nuclear FGFR1 expression was also observed and correlated well with histological differentiation, the pattern of invasion and abundant nuclear polymorphism. Fibroblast growth factor receptor 1 was also overexpressed in EMT cell lines compared with non-EMT cell lines. Furthermore, treatment of HOC313 cells with PD173074 suppressed cellular proliferation and invasion and reduced ERK1/2 and p38 activation. These cells also demonstrated morphological changes, transforming from spindle- to cobble stone-like in shape. In addition, the expression levels of certain matrix metalloproteinases (MMPs), whose genes contain activator protein-1 (AP-1) promoter sites, as well as Snail1 and Snail2 were reduced following PD173074 treatment.Conclusion:Taken together, these data suggest that PD173074 inhibits the MAPK pathway, which regulates the activity of AP-1 and induces MET. Furthermore, this induction of MET likely suppresses cancer cell growth and invasion.

RUNX3 has an oncogenic role in head and neck cancer.

Background Runt-related transcription factor 3 (RUNX3) is a tumor suppressor of cancer and appears to be an important component of the transforming growth factor-beta (TGF-ß)-induced tumor suppression pathway. Surprisingly, we found that RUNX3 expression level in head and neck squamous cell carcinoma (HNSCC) tissues, which is one of the most common types of human cancer, was higher than that in normal tissues by a previously published microarray dataset in our preliminary study. Therefore, here we examined the oncogenic role of RUNX3 in HNSCC. Principal Findings Frequent RUNX3 expression and its correlation with malignant behavior were observed in HNSCC. Ectopic RUNX3 overexpression promoted cell growth and inhibited serum starvation-induced apoptosis and chemotherapeutic drug induced apoptosis in HNSCC cells. These findings were confirmed by RUNX3 knockdown. Moreover, RUNX3 overexpression enhanced tumorsphere formation. RUNX3 expression level was well correlated with the methylation status in HNSCC cells. Moreover, RUNX3 expression was low due to the methylation of its promoter in normal oral epithelial cells. Conclusions/Significance Our findings suggest that i) RUNX3 has an oncogenic role in HNSCC, ii) RUNX3 expression observed in HNSCC may be caused in part by demethylation during cancer development, and iii) RUNX3 expression can be a useful marker for predicting malignant behavior and the effect of chemotherapeutic drugs in HNSCC.

MMP-10/Stromelysin-2 Promotes Invasion of Head and Neck Cancer

Background Periostin, IFN-induced transmembrane protein 1 (IFITM1) and Wingless-type MMTV integration site family, member 5B (Wnt-5b) were previously identified as the invasion promoted genes of head and neck squamous cell carcinoma (HNSCC) by comparing the gene expression profiles between parent and a highly invasive clone. We have previously reported that Periostin and IFITM1 promoted the invasion of HNSCC cells. Here we demonstrated that Wnt-5b overexpression promoted the invasion of HNSCC cells. Moreover, stromelysin-2 (matrix metalloproteinase-10; MMP-10) was identified as a common up-regulated gene among Periostin, IFITM1 and Wnt-5b overexpressing HNSCC cells by using microarray data sets. In this study, we investigated the roles of MMP-10 in the invasion of HNSCC. Methods and Findings We examined the expression of MMP-10 in HNSCC cases by immunohistochemistry. High expression of MMP-10 was frequently observed and was significantly correlated with the invasiveness and metastasis in HNSCC cases. Next, we examined the roles of MMP-10 in the invasion of HNSCC cells in vitro. Ectopic overexpression of MMP-10 promoted the invasion of HNSCC cells, and knockdown of MMP-10 suppressed the invasion of HNSCC cells. Moreover, MMP-10 knockdown suppressed Periostin and Wnt-5b-promoted invasion. Interestingly, MMP-10 overexpression induced the decreased p38 activity and MMP-10 knockdown induced the increased p38 activity. In addition, treatment with a p38 inhibitor SB203580 in HNSCC cells inhibited the invasion. Conclusions These results suggest that MMP-10 plays an important role in the invasion and metastasis of HNSCC, and that invasion driven by MMP-10 is partially associated with p38 MAPK inhibition. We suggest that MMP-10 can be used as a marker for prediction of metastasis in HNSCC.

Ameloblastin Regulates Osteogenic Differentiation by Inhibiting Src Kinase via Cross Talk between Integrin β1 and CD63

ABSTRACT Ameloblastin, the most abundant nonamelogenin enamel matrix protein, plays a role in ameloblast differentiation. Here, we found that ameloblastin was expressed in osteosarcoma cells; to explore the potential functions of ameloblastin in osteoblasts, we investigated whether this protein is involved in osteogenic differentiation and bone formation on the premise that CD63, a member of the transmembrane-4 glycoprotein superfamily, interacts with integrins in the presence of ameloblastin. Ameloblastin bound to CD63 and promoted CD63 binding to integrin β1. The interaction between CD63 and integrin β1 induced Src kinase inactivation via the binding of CD63 to Src. The reduction of Src activity and osteogenic differentiation mediated by ameloblastin were abrogated by treatment with anti-CD63 antibody and overexpression of constitutively active Src, respectively. Therefore, our results suggest that ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin β1.

Nuclear Survivin expression is correlated with malignant behaviors of head and neck cancer together with Aurora-B

Survivin belongs to the inhibitors of apoptosis (IAP) gene family and inhibits apoptosis. Besides its role as IAP, Survivin recently appears to function as a subunit of the chromosomal passenger complex (CPC) for regulating cell division with other CPC proteins including Aurora-B and INCENP. Nuclear Survivin is suspected to control cell division, whereas cytoplasmic Survivin is considered cytoprotective. Although there are several studies on Survivin expression and its function as inhibition of apoptosis, there is no study on Survivin function as a CPC and its correlation with other CPC proteins in head and neck squamous cell carcinoma (HNSCC). Here, therefore, we examined nuclear Survivin expression and its functional correlation with Aurora-B in HNSCC. High expression of Survivin was well correlated with Aurora-B expression in nuclear fraction of HNSCC cell lines and tissues. Moreover, nuclear Survivin expression was significantly correlated with Ki-67 and Aurora-B expression by immunohistochemistry. Notably, HNSCC cases with nuclear Survivin and Aurora-B expression exhibited marked malignant behaviors. Interestingly, both Survivin and Aurora-B knockdown inhibited cell growth and tumorsphere formation. Overall suggest that nuclear Survivin may be involved in tumor progression together with Aurora-B, and that Survivin and Aurora-B can be useful diagnostic markers and therapeutic targets.

Periostin Directly and Indirectly Promotes Tumor Lymphangiogenesis of Head and Neck Cancer

Background Metastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis. Methods and Findings Periostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed. Conclusions Our findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients.

Oncogenic role of RUNX3 in head and neck cancer

Cumulative evidences show that Runt‐related transcription factor 3 (RUNX3) has a tumor suppressive role in various cancers. In particular, RUNX3 appears to be an important component of the transforming growth factor‐β (TGF‐β)‐induced tumor suppression pathway. Contrary to reports on this tumor suppressive role of RUNX3, RUNX3 can also function as an oncogene when overexpressed. Recently, we found that RUNX3 overexpression was frequently observed and was well correlated with malignant behaviors in head and neck cancer, which is one of the most common types of human cancer. Moreover, it has been revealed that RUNX3 overexpression promoted cell growth and inhibited apoptosis in head and neck cancer cells. This review introduces the oncogenic role of RUNX3 in certain types of cancer including head and neck cancer. J. Cell. Biochem. 112: 387–393, 2011.

Ectomesenchymal chondromyxoid tumor of the tongue: insights on histogenesis

OBJECTIVE The objective of this study was to investigate the histogenesis of ectomesenchymal chondromyxoid tumors (ECTs) of the tongue. STUDY DESIGN The biochemical characteristics of a rarely occurring tumor of the tongue were analyzed by immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR), and its biological properties were assessed in primary culture in serum-free media. RESULTS Immunohistochemistry showed that the tumor cells were strongly positive for vimentin, S-100, and glial fibrillary acidic protein (GFAP), but negative for cytokeratin and epithelial membrane antigen. In primary cultures, the cells derived from the ECT were morphologically similar to neuronal cells and expressed Nanog, GFAP, and MAP2. RT-PCR analysis of the surgical specimen was positive for OCT3/4, Sox2, Nanog, MAP2, and CD105 mRNAs. CONCLUSIONS The results of the present study indicate that ECTs originate from the ectomesenchymal cells of the neural crest and are similar in their molecular and biological characteristics to undifferentiated mesenchymal stem cells.

Selective Enhancing Effect of Early Mitotic Inhibitor 1 (Emi1) Depletion on the Sensitivity of Doxorubicin or X-ray Treatment in Human Cancer Cells

Background: To improve the effectiveness of chemo- and radiotherapy only in cancer tissue is important for avoiding side effects. Results: Emi1 depletion enhanced the sensitivity of anticancer reagents and X-ray irradiation in cancer cells. Conclusion: Emi1 siRNA would be a useful new modality for enhancing the effect of chemo- and radiotherapy in various tumors. Significance: This work provides new insights regarding synergistic effect of Emi1 knockdown in combination therapies. Chemotherapy and radiation in addition to surgery has proven useful in a number of different cancer types, but the effectiveness in normal tissue cannot be avoided in these therapies. To improve the effectiveness of these therapies selectively in cancer tissue is important for avoiding side effects. Early mitotic inhibitor 1 (Emi1) is known to have the function to inhibit anaphase-promoting complex/cyclosome ubiquitin ligase complex, which ubiquitylates the cell cycle-related proteins. It recently has been shown that Emi1 knockdown prevents transition from S to G2 phase by down-regulating geminin via anaphase-promoting complex/cyclosome activation. At present, anticancer drugs for targeting DNA synthesis to interfere with rapidly dividing cells commonly are used. As Emi1 depletion interferes with completion of DNA synthesis in cancer cells, we thought that Emi1 knockdown might enhance the sensitivity for anticancer agents. Here, we confirmed that Emi1 siRNA induced polyploidy for preventing transition from S to G2 phase in several cancer cell lines. Then, we treated Emi1 depleted cells with doxorubicin. Interestingly, increased apoptotic cells were observed after doxorubicin treatment in Emi1 siRNA-treated cancer cells. In addition, Emi1 depletion enhanced the sensitivity of x-ray irradiation in cancer cells. Importantly, synergistic effect of Emi1 knockdown in these combination therapies was not observed in normal cells. These results suggest that Emi1 siRNA can be a useful tool for enhancing of sensitivity of cancer cells to anticancer reagents and radiation.

Deregulation of Anaphase-promoting Complex/cyclosome-dependent Proteolysis in Cancer

Abstract Abnormal regulation of the cell cycle is essential for oncogenic transformation and tumor progression. The cell cycle is driven by the activity of the Cyclin/Cyclin/dependent kinase (Cdk) complex. Recently, the protein level of various cell cycle regulators including Cyclins and Cdk inhibitors was found to be regulated by the ubiquitin-proteasome pathway. In particular, SCF (Skp1-Cullin-F-box) and the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase complex play an important role in the ubiquitin-mediated proteolysis of cell cycle regulators. It has recently been revealed that the overexpression of oncogenic cell cycle regulators and reduced expression of tumor suppressive cell cycle regulators are caused by the abnormal regulation of ubiquitin-mediated proteolysis in cancer. In this review, we introduce the deregulation of APC/C ubiquitin ligase complex-dependent proteolysis in cancer.