Tomohide Isobe

Functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy

Bio-hybrid artificial organs are an attractive concept to restore organ function through precise biological cooperation with surrounding tissues in vivo. However, in bio-hybrid artificial organs, an artificial organ with fibrous connective tissues, including muscles, tendons and ligaments, has not been developed. Here, we have enveloped with embryonic dental follicle tissue around a HA-coated dental implant, and transplanted into the lower first molar region of a murine tooth-loss model. We successfully developed a novel fibrous connected tooth implant using a HA-coated dental implant and dental follicle stem cells as a bio-hybrid organ. This bio-hybrid implant restored physiological functions, including bone remodelling, regeneration of severe bone-defect and responsiveness to noxious stimuli, through regeneration with periodontal tissues, such as periodontal ligament and cementum. Thus, this study represents the potential for a next-generation bio-hybrid implant for tooth loss as a future bio-hybrid artificial organ replacement therapy.

Sclerosing odontogenic carcinoma with benign fibro-osseous lesion of the mandible: an extremely rare case report.

A case of sclerosing odontogenic carcinoma (SOC) admixed with a benign fibro‐osseous lesion (BFOL) is reported herein. A 67‐year‐old male had paresthesia in the mental region. Computed tomography detected an intragnathic mass that was focally expansile with disappearance of cortical bone, and contained admixed radiolucency and radio‐opacity. Under the pathological diagnosis as benign fibro‐osseous lesion, it was surgically removed by curettage. Microscopic analysis showed that a few parts of the resected materials contained dispersed thin cords and small nests of epithelial cells accompanied by fibrous stroma. Cellular atypia and mitotic figures were not evident. The diagnosis of BFOL with hyperplastic and metaplastic odontogenic epithelia was ultimately made. Eight months after the operation, the lesion recurred and segmental mandibulectomy was carried out. Histologically, the lesion was predominantly occupied by the fibro‐osseous component with irregular‐shaped foci of epithelial component. The epithelial component exhibited mostly thin cord or small nest patterns and showed definite perineural infiltration. Immunohistochemically, the epithelial cells were positive for p63, cytokeratin (CK) 6 and CK19, and focally positive for CK7 but negative for vimentin. MIB‐1 positive nuclei were inconspicuous. To the best of our knowledge, this report is the first case of SOC with BFOL.

ANGPTL4 regulates the metastatic potential of oral squamous cell carcinoma.

Lymph node metastasis is a major factor for poor prognosis in oral squamous cell carcinoma (OSCC). However, the molecular mechanisms of lymph node metastasis are unclear. We determined that angiopoietin-like protein 4 (ANGPTL4) mRNA and protein expression were increased in OSCC cells established from the primary site in metastatic cases. In addition, ANGPTL4 expression in biopsy specimens was correlated with the presence of lymph node metastasis. Therefore, our initial findings suggest that OSCC cells expressing ANGPTL4 may possess metastatic ability. Furthermore, cell culture supernatants from OSCC cells that metastasized to the lymph node contain ANGPTL4 and promote invasive ability. These findings suggest that secreted ANGPTL4 may affect the invasive ability of OSCC. Moreover, the rates of positive ANGPTL4 expression at the primary site were significantly higher in the lymph node metastasis group. These results demonstrate that ANGPTL4 contributes to OSCC metastasis by stimulating cell invasion. Therefore, ANGPTL4 is a potential therapeutic target for preventing cancer metastasis.

Lin28a is a putative factor in regulating cancer stem cell-like properties in side population cells of oral squamous cell carcinoma.

Cancer stem cells (CSCs) are among the target cells of cancer therapy because they are uniquely involved in both cancer progression and sensitivity to chemotherapeutic agents. We identified side population (SP) cells, which are known to be an enriched population of CSC, in five oral squamous cell carcinoma (OSCC) cells (SCC9, SCC25, TOSCC7, TOSCC17, and TOSCC23). The percentages of SP cells ranged from 0% to 3.3%, with TOSCC23 cells showing the highest percentages of SP cells (3.3% of the total cell population). The SP cells isolated from TOSCC23 cells also showed greater cell proliferation and invasion compared to non-SP (MP) cells. Therefore, our initial findings suggested that SP cells were enriched for CSC-like cells. Furthermore, DNA microarray analysis revealed that the expression of cell proliferation-related and anti-apoptotic genes was greater in SP cells compared to MP cells. We focused on Lin28a, which showed the highest expression (approximately 22-fold) among the upregulated genes. The overexpression of Lin28a in TOSCC23 cells increased their proliferation, colony formation, and invasion. These findings suggest that Lin28a is an appropriate CSC target molecule for OSCC treatment.

Antitumor activity of suberoylanilide hydroxamic acid against human oral squamous cell carcinoma cell lines in vitro and in vivo

It has been reported recently that histone deacetylase inhibitors (HDACIs) can block the growth of a variety of malignant tumor cells by reversing the silencing of the tumor suppressor genes; these will be the anticancer agents of the next generation. In this study, we evaluated the antitumor effects of the HDACI suberoylanilide hydroxamic acid (SAHA) on oral squamous cell carcinoma (OSCC) and investigated its molecular mechanism. SAHA suppressed the in vitro proliferation of OSCC cell lines in a dose- and time-dependent manner. Flow cytometric analyses showed that treatment with SAHA led to G1 phase cell-cycle arrest of OSCC cells, accompanying a decrease in the percentage of S-phase cells. Western blot analyses demonstrated that the expression of p21 protein was remarkably augmented and hyperacetylation of p53 was induced after SAHA treatment. These results suggest that administration of SAHA suppresses OSCC growth through G1 phase arrest. Additionally, we observed that the growth of xenograft SAS tumors in nude mice was significantly blocked by the administration of SAHA without major adverse effects.

Potential role of hematopoietic pre-B cell leukemia transcription factor-interacting protein in oral carcinogenesis.

BACKGROUND Hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) is a corepressor of pre-B-cell leukemia homeobox (PBX) 1 and is known to play a role in hematopoiesis. Recently, HPIP was demonstrated to promote breast cancer cell proliferation and hepatocellular carcinoma growth. Moreover, it has been revealed that homeobox and PBX proteins, the expression of which is regulated by HPIP, play key roles in cancer of various organs, including oral squamous cell carcinoma (OSCC). Nevertheless, there has not been any study regarding the role of HPIP in OSCC. This study investigated the expression of HPIP in normal oral mucosa, epithelial precursor lesion (OEPL), and OSCC, and the functional roles of HPIP in OSCC cells and normal keratinocytes. MATERIALS AND METHODS Immunohistochemical analysis of HPIP, Ki-67, and involucrin was performed in OSCC specimens, and the change in involucrin expression following RNA interference treatment against HPIP was examined by quantitative RT-PCR and Western blot analysis in SCC9 and NHEK cells undergoing extracellular calcium-induced differentiation. Matrigel transwell and cell proliferation assays for both cell lines transfected with HPIP siRNA were also conducted. RESULTS HPIP expression increased in OEPL and OSCC specimens. In vitro analysis revealed that HPIP suppressed differentiation and proliferation of SCC9 cells and transwell migration of NHEK cells, while HPIP promoted invasion of SCC9 and proliferation of NHEK cells. However, HPIP has no significant effect on NHEK cell differentiation. CONCLUSION HPIP may play a critical role in oral carcinogenesis and is thus a potential target for anticancer therapy, with particular emphasis on its involvement in differentiation and migration/metastasis.

Expression of BMI1 and ZEB1 in epithelial-mesenchymal transition of tongue squamous cell carcinoma

Epithelial-mesenchymal transition (EMT) is a crucial event required for the invasion and progression of carcinogenesis, inducing stem-like properties in epithelial cells. In the present study, the expression of BMI1, which controls self-renewal in stem cells, as well as that of ZEB1, a transcription factor that regulates EMT, was evaluated for its role in EMT and the carcinogenic processes of tongue squamous cell carcinoma (TSCC). Collagen invasion assays using two TSCC cells and 64 tongue specimens (32 carcinomas and 32 dysplasias) were employed and analyzed in the present study. We assessed the protein and mRNA expression levels of BMI1, ZEB1, vimentin and E-cadherin in the two cell lines and tumor tissues. The protein and mRNA expression of BMI1 and ZEB1 occurred at the invasion of TSCC. The elevated levels of BMI1 and ZEB1 were accompanied by the downregulation of E-cadherin and upregulation of vimentin at the invasive front, indicative of EMT in vitro and in vivo. The results showed that BMI1 and ZEB1 are important factors in association with the promotion of EMT and invasion of TSCC.

Sclerosing odontogenic carcinoma

To the Editor: We were intrigued by the recent letter to the editor by Ide et al. The authors had previously introduced a case of primary intraosseous squamous cell carcinoma (PIOSCC) as sclerosing odontogenic carcinoma (SOC). After reviewing this letter to the editor, we believe that there are some major issues regarding their arguments. Our points of concern are as follows. First, in this letter to the editor and the previous report of PIOSCC, they did not provide sufficient evidence or reasoning to support their claim that this lesion was a carcinoma. Although the authors presented figures (eg fig. 2a) in their letter to the editor that demonstrate the invasion of ‘perivascular tissue’ by this tumor, which was composed of basaloid cells reminiscent of Malassez’ epithelial rests (as described in a previous report of PIOSCC), the major evidence of malignancy is still lacking because it is known that even benign odontogenic tumors such as intraosseous ameloblastoma exhibit infiltration of surrounding structures (e.g. mandibular tumors invade the perineural tissue of the inferior alveolar nerve). Thus, in the first case report of SOC, the authors’ claims that malignancy of SOC was based on the invasion of skeletal muscle (not the invasion of ‘perivascular tissue’ in the connective tissue that was located close to the tumor mass), and perineural invasion (not the invasion of ‘perineural tissue’), may not be valid, and more conclusive evidence of malignancy is needed. Second, it was not appropriate that the authors had previously reported this case as PIOSCC. Because the authors had earlier described that this tumor lacked a mature squamous phenotype in their previous report, based on the criteria of the 2001 Armed Forces Institute of Pathology (AFIP) publication and 2005 WHO classification, such a tumor without any conventional squamous cell carcinoma component should not have been diagnosed as PIOSCC or primary intraosseous carcinoma (PIOC); the authors should have diagnosed this tumor as ‘malignant odontogenic tumor’ or ‘odontogenic carcinoma’ during the initial diagnosis. Third, there was no description about the immunohistochemical analysis neither in this letter to the editor nor in the previous report of PIOSCC. In order to support their claim that this tumor was SOC, the immunohistochemical results of p63, CK5/6, CK7, CK8/18, CK19, CK20, MIB-1, S-100, SMA, etc., which have all been previously demonstrated as useful differential diagnostic markers of SOC, should have been presented as images for diagnosis of exclusion. Fourth, the authors claimed that they found two possible cases of SOC with benign fibro-osseous lesion (BFOL). In one of the papers cited by the authors from Jones et al. no definite finding of malignancy in the odontogenic epithelium was presented in the histopathological figure, nor did they offer any description about malignancy of this lesion. The authors should have clarified how to evaluate the basis of malignancy of the odontogenic epithelium in the case presented by Jones et al. which was compatible with SOC, based on the descriptions and figure in the paper. Moreover, in another paper cited by the authors from Bennett et al. the presented figures were only that of newly formed bone trabeculae, islands of epithelium, and typical conventional squamous cell carcinoma. The authors should have given more objective reasoning as to why they believed that this case of odontogenic squamous cell carcinoma with osseous metaplasia was similar to SOC on the basis of such few figures, whose findings were not sufficient enough to support the authors’ claims. Furthermore, in the previous report of SOC, no definite component of conventional squamous cell carcinoma was observed in the SOC. The authors should have provided strict descriptions on how to distinguish SOC from different odontogenic tumors (e.g. central odontogenic fibroma, ameloblastic carcinoma, clear cell odontogenic carcinoma, primary intraosseous carcinoma, etc.) based on the descriptions and figures in the papers of Jones et al. and Bennett et al. According to the issues described above, the authors’ various and numerous speculations based on the previous reports of two possible cases of SOC should not have been presented in this letter to the editor.

Expression of myelin and lymphocyte protein (MAL) in oral carcinogenesis.

In the pathological diagnosis of oral squamous cell carcinoma, we often confront the difficulty of determining whether it is invasive carcinoma or epithelial dysplasia. Recently, myelin and lymphocyte protein (MAL; T-cell differentiation-related gene) has been reported to be a candidate gene suppressed in esophageal carcinoma. When we performed cDNA microarray analysis, we found that gene expression of MAL was significantly downregulated in oral squamous cell carcinoma (OSCC). We evaluated the expression of the MAL gene by laser microdissection and real-time PCR methods and protein localization by immunohistochemistry. The gene expression of MAL was significantly decreased in OSCC compared with normal epithelium (P < 0.05). Furthermore, protein expression of MAL disappeared gradually in proportion to malignancy. The results suggest that MAL plays an important role during oral carcinogenesis and that the gene may have potential as a biomarker target for OSCC.

Abstract 4687: Gene expression of breast cancer with CD44+CD24-/low genotype

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Purpose: Breast cancer cells with a CD44+CD24-/low phenotype have been suggested to have tumor-initiating properties with stem cell-like. The purpose of this study is to clarify the gene expression profiling of cells with different CD44/CD24 genotypes within breast cancer. Methods: Laser-captured microdissection was used to select the isolation of cancer cells in 36 frozen tissues of breast cancer, and RNA extracted from these cells was examined by real-time RT-PCR to quantify CD44 and CD24 expressions. Human stem cell RT2 profiler PCR array was used for gene expression analysis in the groups of different CD44/CD24 genotype. Associations between different CD44/CD24 genotypes and clinical variables were assessed by Fishers exact test, and the Mann-Whitney U test was used for gene expression profiling. P<0.05 was considered significant. Results: Thirty-six tumors were divided into 3 groups. Group A was composed of CD44+CD24-/low genotype, in which the ratio of CD44/CD24 was >1.0. Group B was composed of CD44+CD24+ genotype, in which that was 0.1 < ratio ≤ 1.0. In group C of CD44-/lowCD24+ genotype, that was < 0.1. The number of tumors in group A, B and C was 5, 29 and 2, respectively. Regarding the correlation of CD44/CD24 status with tumor characteristics, tumors of group A were significantly associated with lymph node metastasis compared with those of group B (P=0.029). The number of tumor with HER 2 score 3 within group A and B was 0 and 11, respectively. There was no significantly difference in ER status and tumor size. As far as the signaling pathways, the number of expression genes for Notch pathway in group A was significantly greater than in group B (P=0.028), and that for Wnt pathway was not significantly different between group A and B. Overexpressed gene in group A was NUMB, which is related to the programmed cell death. MYST1 and SOX2 tended to show higher levels of group A than of group B. Conclusion: This study suggests that Notch pathway may be more important than Wnt pathway on breast cancer with CD44+CD24-/low genotype, and the CD44+CD24-/low status would be associated with low/negative HER2 expression. Moreover, the gene of self-renewal markers, such as MYST1, SOX2 and NUMB might be a target for therapy against breast cancer with low/negative HER2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4687.