Kei-ichi Morita

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis.

Apoptosis signal‐regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H2O2, and activates c‐Jun NH2‐terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF‐ and H2O2‐induced sustained activations of JNK and p38 are lost in ASK1−/− embryonic fibroblasts, and that ASK1−/− cells are resistant to TNF‐ and H2O2‐induced apoptosis. TNF‐ but not Fas‐induced apoptosis requires ROS‐dependent activation of ASK1–JNK/p38 pathways. Thus, ASK1 is selectively required for TNF‐ and oxidative stress‐induced sustained activations of JNK/p38 and apoptosis.

Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress

Apoptosis signal‐regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H2O2). A yeast two‐hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1. PP5 directly dephosphorylated an essential phospho‐threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo. The interaction between PP5 and ASK1 was induced by H2O2 treatment and was followed by the decrease in ASK1 activity. PP5 inhibited not only H2O2‐induced sustained activation of ASK1 but also ASK1‐dependent apoptosis. Thus, PP5 appears to act as a physiological inhibitor of ASK1–JNK/p38 pathways by negative feedback.

The tumor suppressive microRNA miR-218 targets the mTOR component Rictor and inhibits AKT phosphorylation in oral cancer

The incidence of oral squamous cell carcinoma (OSCC) is rising rapidly in developed countries, posing a growing challenge due to the poor management of this type of malignancy at present. In this study, we profiled tumor suppressive microRNAs (miRNAs) that are silenced by DNA hypermethylation in OSCC using a function-based screening approach. This approach employed a cell proliferation assay for 327 synthetic miRNAs in two OSCC cell lines. Among the 110 miRNAs identified in this set that exhibited inhibitory properties, we compared DNA methylation and expression status in a wider panel of OSCC cell lines and primary tumor tissues, resulting in the identification of miR-218 and miR-585 as functionally significant miRNA genes that are frequently silenced in OSCC by DNA hypermethylation. Ectopic expression of miR-218 and miR-585 in OSCC cells lacking endogenous expression reduced cell growth in part through caspase-mediated apoptosis. Notably, miR-218 reduced levels of the rapamycin-insensitive component of mTOR, Rictor, in a manner associated with a suppression of Akt S473 phosphorylation. Together our findings define miR-585 as a tumor suppressive function that is often epigenetically silenced in OSCC, and they identify Rictor as a novel target of miR-218, suggesting that activation of the mTOR-Akt signaling pathway induced by Rictor contributes centrally to oral carcinogenesis.

Roles of Interleukin-6 and Parathyroid Hormone-Related Peptide in Osteoclast Formation Associated with Oral Cancers Significance of Interleukin-6 Synthesized by Stromal Cells in Response to Cancer Cells

We investigated the roles of interleukin-6 (IL-6) and parathyroid hormone-related peptide (PTHrP) in oral squamous cell carcinoma (OSCC)-induced osteoclast formation. Microarray analyses performed on 43 human OSCC specimens revealed that many of the specimens overexpressed PTHrP mRNA, but a few overexpressed IL-6 mRNA. Immunohistochemical analysis revealed that IL-6 was expressed not only in cancer cells but also in fibroblasts and osteoclasts at the tumor-bone interface. Many of the IL-6-positive cells coexpressed vimentin. Conditioned medium (CM) derived from the culture of oral cancer cell lines (BHY, Ca9-22, HSC3, and HO1-u-1) stimulated Rankl expression in stromal cells and osteoclast formation. Antibodies against both human PTHrP and mouse IL-6 receptor suppressed Rankl in ST2 cells and osteoclast formation induced by CM from BHY and Ca9-22, although the inhibitory effects of IL6 antibody were greater than those of PTHrP antibody. CM derived from all of the OSCC cell lines effectively induced IL-6 expression in stromal cells, and the induction was partially blocked by anti-PTHrP antibody. Xenografts of HSC3 cells onto the periosteal region of the parietal bone in athymic mice presented histology and expression profiles of RANKL and IL-6 similar to those observed in bone-invasive human OSCC specimens. These results indicate that OSCC provides a suitable microenvironment for osteoclast formation not only by producing IL-6 and PTHrP but also by stimulating stromal cells to synthesize IL-6.

Down-regulation of keratin 4 and keratin 13 expression in oral squamous cell carcinoma and epithelial dysplasia: a clue for histopathogenesis

Sakamoto K, Aragaki T, Morita K‐i, Kawachi H, Kayamori K, Nakanishi S, Omura K, Miki Y, Okada N, Katsube K‐i, Takizawa T & Yamaguchi A
(2011) Histopathology58, 531–542
Down‐regulation of keratin 4 and keratin 13 expression in oral squamous cell carcinoma and epithelial dysplasia: a clue for histopathogenesis

Reduction of NOTCH1 expression pertains to maturation abnormalities of keratinocytes in squamous neoplasms

Notch is a transmembrane receptor functioning in the determination of cell fate. Abnormal Notch signaling promotes tumor development, showing either oncogenic or tumor suppressive activity. The uncertainty about the exact role of Notch signaling, partially, stems from inconsistencies in descriptions of Notch expression in human cancers. Here, we clarified basal-cell dominant expression of NOTCH1 in squamous epithelium. NOTCH1 was downregulated in squamous neoplasms of oral mucosa, esophagus and uterine cervix, compared with the normal basal cells, although the expression tended to be retained in cervical lesions. NOTCH1 downregulation was observed even in precancers, and there was little difference between cancers and high-grade precancerous lesions, suggesting its minor contribution to cancer-specific events such as invasion. In culture experiments, reduction of NOTCH1 expression resulted in downregulation of keratin 13 and keratin 15, and upregulation of keratin 17, and NOTCH1 knockdown cells formed a dysplastic stratified epithelium mimicking a precancerous lesion. The NOTCH1 downregulation and the concomitant alterations of those keratin expressions were confirmed in the squamous neoplasms both by immunohistochemical and cDNA microarray analyses. Our data indicate that reduction of NOTCH1 expression directs the basal cells to cease terminal differentiation and to form an immature epithelium, thereby playing a major role in the histopathogenesis of epithelial dysplasia. Furthermore, downregulation of NOTCH1 expression seems to be an inherent mechanism for switching the epithelium from a normal and mature state to an activated and immature state, suggesting its essential role in maintaining the epithelial integrity.

Reduction of bone resorption by the application of platelet-rich plasma (PRP) in bone grafting of the alveolar cleft

OBJECTIVE We evaluated the effectiveness of platelet-rich plasma (PRP) on the regeneration of autogenous cancellous bone and marrow grafted in the alveolar cleft. DESIGN Twenty patients with alveolar clefts were examined; 6 were the control group and received cancellous bone and marrow grafts without PRP, while the remaining 14 comprised the PRP group and received grafts with PRP. Prior to surgery, 50 ml of blood was withdrawn and 5 ml of PRP gel produced through centrifugal separation. The bone graft mixed with PRP was then packed into the alveolar cleft. Postoperative bone density was assessed as the aluminium-equivalent value on occlusal X-ray films in a qualitative analysis. Quantitative evaluation of regenerated bone was made with computed tomography and panoramic radiographs at 1 month, 6 months and 1 year after surgery. RESULTS Satisfactory bone bridging formation was observed in all patients without any complications. The bone density of the PRP group was lower than that of the control group at 1 week, but the same after 1 month. The added PRP reduced the resorption of regenerated bone postoperatively. CONCLUSION Autogenous cancellous bone grafting with PRP, which significantly reduces postoperative bone resorption, is a reliable technique for alveolar bone grafting of cleft patients.

Recurrence patterns of oral leukoplakia after curative surgical resection: important factors that predict the risk of recurrence and malignancy.

BACKGROUND Oral leukoplakia can be treated using a variety of treatment procedures; however, the lesions recur in many cases irrespective of the treatment procedure used. The rate of recurrence was from 7.7% to 38.1%. This study aims to identify the important factors that can lower the risk of recurrence of oral leukoplakia treated by curative surgical resection. METHODS The clinical records of 52 patients with oral leukoplakia (53 lesions) who underwent curative surgical resection between 2004 and 2009 were retrospectively analyzed for the rate of recurrence, clinical outcome, epithelial dysplasia, lesion location, and resection margins. RESULTS The recurrence rate following curative surgical resection was 15.1%, with the most common site being the gingiva. Malignant transformation occurred in a single patient (1.9%). Minimal resection margins (<3 mm) were observed in many patients with recurrent disease, and recurrence was more likely in cases with positive margins (epithelial abnormalities at the resection margins) than in those with negative margins. There was no significant association between recurrence and the degree of epithelial dysplasia. CONCLUSIONS Our data suggest that surgical resection of oral leukoplakia is curative only if all areas of epithelial abnormalities are identified and resected. Moreover, an adequate resection margin may reduce the risk of recurrence.

DNA amplification and expression of FADD in oral squamous cell carcinoma

BACKGROUND The Fas-associated death domain-containing protein, FADD, is an adaptor for relaying apoptotic signals. However, recent studies have shown that FADD also plays an important role in the growth and regulation of the cell cycle. The purpose of this study was to elucidate the role of FADD in oral squamous cell carcinoma (SCC). METHODS The DNA amplification of FADD from 30 samples of tongue SCC was analyzed using real-time PCR and the protein expression of FADD from 60 samples of tongue SCC was analyzed using immunohistochemistry. RESULTS The DNA amplifications of FADD were observed in 13 cases (44.3%) and were significantly correlated with the histopathological differentiation grade of SCCs (P = 0.009). FADD expression levels compared with the matched adjacent epithelium increased significantly (P = 0.000). Additionally, the positive expressions of FADD were significantly correlated with lymph node metastasis of SCCs (P = 0.029) and the 5-year disease-specific survival rates (P = 0.049). A positive association between FADD expression level and the histopathological differentiation grade was found to be limited to T1 SCCs (P = 0.019). DNA amplification was moderately correlated (correlation coefficient = 0.406, P = 0.026) with expression of FADD in 30 samples of tongue SCC. CONCLUSION In tongue SCCs, the expression of FADD was higher when compared with that of adjacent areas, which might be determined via genomic amplification in 11q13.3. Thus, SCC cells with the expression of FADD are possibly more likely to become metastatic and to worsen survival rates.

Long-term outcome of non-surgical treatment in patients with oral leukoplakia

UNLABELLED The standard treatments for oral leukoplakia range from careful observation to complete resection. No surgical intervention is chosen for several supposable reasons. Surgical treatment and no surgical treatment for oral leukoplakia have no defined basis for comparisons, and few studies have reported on the long-term outcomes of oral leukoplakia without surgery. OBJECTIVES This study aimed to identify the important factors using a long-term wait-and-see policy in patients with oral leukoplakia. MATERIALS AND METHODS In total, 237 lesions from 218 patients selected for non-surgical therapy between 2001 and 2010 were analyzed. On the basis of long-term follow-up data, lesions were classified as unchanged, reduced, disappeared, expanded, and malignantly transformed. RESULTS In total, 135 (57.0%) lesions remained unchanged, 30 (12.7%) lesions were characterized by a reduction in size or clinical severity, and 44 (18.6%) lesions had disappeared. Another 17 (7.2%) lesions resulted in spread or clinical deterioration, and 11 (4.6%) lesions developed oral squamous cell carcinoma. CONCLUSIONS We demonstrated a cumulative malignant transformation rate of 11.6% in 10years without resection. The lesions that were nonhomogeneous, and higher degree of epithelial dysplasia, located on the tongue were likely to progress into cancer. In addition, 32.5% of lesions without surgical treatment were reduced or disappeared. There is a possibility that removal of considerable irritation for a long time contributes to the treatment of this disease. The development of appropriate treatments for oral leukoplakia is required, which will enable successful differentiation between surgical and observation cases.