TNF-α-induced Inhibition of Protein Myristoylation Via Binding Between NMT1 and Sorbs2 in Osteoblasts.

BACKGROUND/AIM: Bone resolution due to tumor invasion often occurs on the surface of the jaw and is important for clinical prognosis. Although cytokines, such as TNF-α are known to impair osteoblasts, the underlying mechanism remains unclear. Protein myristoylation, a post-translational modification, plays an important role in the development of immune responses and cancerization of cells. A clear understanding of the mechanisms underlying this involvement will provide insights into molecular-targeted therapies. N-myristoyltransferase1 (NMT1), a specific enzyme involved in myristoylation, is expressed in cancer cells and in other normal cells, suggesting that changes in myristoylation may result from the regulation of NMT1 in cancer cells. MATERIALS AND METHODS: Using newly emerging state-of-the-art techniques such as the Click-it assay, RNA interference, mass spectrometry, immunoprecipitation, immunocytochemistry, and western blotting, the expression of myristoylated proteins and the role of TNF-α stimulation on NMT1 and Sorbs2 binding were evaluated in a murine osteoblastic cell line (MC3T3-E1). RESULTS: The expression of myristoylated proteins was detected; however, TNF-α stimulation resulted in their inhibition in MC3T3-E1 cells. The expression of NMT1 also increased. Immunoprecipitation and mass spectrometry identified Sorbs2 as a novel binding protein of NMT1, which upon TNF-α stimulation, inhibited myristoylation. CONCLUSION: The binding between NMT1 and Sorbs2 can regulate myristoylation, and NMT1 can be considered as a potential target molecule for tumor invasion.

Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells.

Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytes and cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.

Regulation of NF-kB Signalling Through the PR55ß-RelA Interaction in Osteoblasts.

BACKGROUND/AIM: Nuclear factor kappa B (NF-kB) signalling including the RelA subunit is activated upon fibroblast growth factor (FGF) stimulation. A clear understanding of the mechanisms underlying this action will provide insights into molecular targeting therapy. Furthermore, protein phosphatase 2A (PP2A) is involved in RelA dephosphorylation, but little is known about the underlying mechanism. MATERIALS AND METHODS: Because the regulatory subunits of PP2A drive NF-kB signalling via RelA, we used qRT-PCR and immunoblot analysis to investigate the expression of these subunits in MC3T3-E1 cells. We examined weather FGF2 interacts with NF-kB using immunocytochemistry (IC), immunoprecipitation (IP), and pull-down assay (PD) using recombinant proteins. RESULTS: PR55ß expression was increased, whereas activated RelA was dephosphorylated upon FGF2 stimulation. Further, the interaction of PR55ß with RelA was confirmed by IC, IP, and PD. CONCLUSION: FGF2-induced PR55ß directly interacts with RelA and regulates NF-kB signalling.

Regulation of ß-Catenin Phosphorylation by PR55ß in Adenoid Cystic Carcinoma.

BACKGROUND/AIM: Adenoid cystic carcinoma (AdCC) is a rare cancer of the salivary gland with high risk of recurrence and metastasis. Wnt signalling is critical for determining tumor grade in AdCC, as it regulates invasion and migration. ß-catenin dephosphorylation plays an important role in the Wnt pathway, but its underlying molecular mechanism remains unclear. MATERIALS AND METHODS: Because the regulatory subunits of protein phosphatase 2A (PP2A) drive Wnt signalling via target molecules, including ß-catenin, we used qRT-PCR and immunoblot analysis to investigate the expression of these subunits in an AdCC cell line (ACCS) and a more aggressive subline (ACCS-M). RESULTS: PR55ß was highly expressed in ACCS-M, suggesting its functional importance. In addition, PR55ß expression was associated with tumor grade, with ACCS-M exhibiting higher PR55ß levels. More importantly, knockdown of PR55ß in ACCS-M cells significantly reduced invasiveness and metastatic ability. Furthermore, dephosphorylation and total levels of ß-catenin were dependent on PR55ß in ACCS-M. Finally, we confirmed a correlation between PR55ß staining intensity and histopathological type in human AdCC tissues. CONCLUSION: Our study provides new insight into the interaction between PR55ß and ß-catenin and suggests that PR55ß may be a target for the clinical treatment of AdCC.

Deregulation of Nicotinamide N-Methyltransferase and Gap Junction Protein Alpha-1 Causes Metastasis in Adenoid Cystic Carcinoma.

BACKGROUND/AIM: Adenoid cystic carcinoma (AdCC) is a malignant tumor that occurs in the salivary glands and frequently metastasizes. The aim of this study was to identify factors mediating AdCC metastasis. MATERIALS AND METHODS: We established three AdCC cell lines by orthotropic transplantation and in vivo selection: parental, highly metastatic (ACCS-M-GFP), and lymph node metastatic (ACCS-LN-GFP) cells. RESULTS: We examined the three cell lines. DNA microarray indicated significantly altered processes in ACCS-LN-GFP cells: particularly, the expression of nicotinamide N-methyltransferase (NNMT) was enhanced the most. NNMT is associated with tumorigenesis and is a potential tumor biomarker. Concomitantly, we found-significant down-regulation of gap junction protein alpha-1. We suggest that ACCS-LN-GFP cells acquire cancer stem cell features involving the up-regulation of NNMT and the loss of gap junction protein alpha-1, leading to epithelial-mesenchymal transition and consequent AdCC metastasis. CONCLUSION: NNMT is a potential biomarker of AdCC.

The relationship between lateral displacement of the mandible and scoliosis.

OBJECTIVES: Idiopathic scoliosis is an orthopaedic disease of childhood, with onset and progress occurring until adolescence. Here, the relationship between lateral displacement of the mandible and scoliosis was analysed quantitatively. METHODS: Seventy-nine non-syndromic Japanese patients (18 men, 61 women), who were diagnosed with jaw deformities and underwent surgical orthognathic treatment at Kyushu University Hospital from January 2011 to August 2014, were enrolled. Their mean age at the time of radiography was 25.3 ± 8.7 years. Postero-anterior cephalometric radiographs and chest radiographs were examined. In postero-anterior cephalometric radiographs, a horizontal baseline (X-axis) was drawn as a straight line that intersects both the zygomatic bases, and a vertical line (Y-axis) was marked perpendicular to the X-axis, with an intersection at the anterior nasal spine (ANS). Point A was defined as the intersection of the X- and Y-axes, and line A was defined as the line connecting point A to the menton. The angle made by the X-axis and line A (i.e., lateral displacement of the mandible) was measured. We designated an absolute value even if the mandibular menton was located on the right or left side. In chest radiographs, Cobb's method was used to measure scoliosis curves; the direction of the curve was designated similarly. RESULTS: Nine (11.4%) individuals had a Cobb angle >10°. There was a positive correlation between the Cobb angle and the degree of mandibular deviation (p < 0.05). CONCLUSION: Lateral displacement of the mandible and scoliosis are related.

Introduction of ID2 Enhances Invasiveness in ID2-null Oral Squamous Cell Carcinoma Cells via the SNAIL Axis.

AIM: Inhibitor of DNA-binding (ID) proteins are negative regulators of basic helix-loop-helix transcription factors that generally stimulate cell proliferation and inhibit differentiation. However, the role of ID2 in cancer progression remains ambiguous. Here, we investigated the function of ID2 in ID2-null oral squamous cell carcinoma (OSCC) cells. MATERIALS AND METHODS: We introduced an ID2 cDNA construct into ID2-null OSCC cells and compared them with empty-vector-transfected cells in terms of cell proliferation, invasion, and activity and expression of matrix metalloproteinase (MMP). RESULTS: ID2 introduction resulted in enhanced malignant phenotypes. The ID2-expressing cells showed increased N-cadherin, vimentin, and E-cadherin expression and epithelial-mesenchymal transition. In addition, cell invasion drastically increased with increased expression and activity of MMP2. Immunoprecipitation revealed a direct interaction between ID2 and zinc finger transcription factor, snail family transcriptional repressor 1 (SNAIL1). CONCLUSION: ID2 expression triggered a malignant phenotype, especially of invasive properties, through the ID2-SNAIL axis. Thus, ID2 represents a potential therapeutic target for OSCC.

Involvement of the T-box transcription factor Brachyury in early-stage embryonic mouse salivary gland.

The mouse submandibular gland (SMG) is important organ for embryonic development, and branching morphogenesis is regulated by many molecules containing transcription factors. Real-time reverse transcriptase polymerase chain reaction revealed that the expression of Brachyury increased in the SMG and peaked between E12.5-E13.5, concomitant with the early stage of branching morphogenesis. The expression of Brachyury in SMG rudiments between E12.5-E13.5 was confirmed by western blotting. In addition, fibronectin and Btbd7 (regulated by fibronectin), which are both essential for cleft formation, were expressed strongly during the same period. The Sox2 and Wnt3a, which regulate cell growth, were also expressed strongly during E12.5-E13.5. On the other hand, cleft formation and branching morphogenesis was suppressed by knockdown of Brachyury gene, suggesting that Brachyury plays a central role in regulating cell growth and cleft formation in early-stage embryonic mouse salivary gland development.

Efficient regulation of branching morphogenesis via fibroblast growth factor receptor 2c in early-stage embryonic mouse salivary glands.

Salivary gland (SG) defects have a wide range of health implications, including xerostomia, bacterial infections, and oral health issues. Branching morphogenesis is critical for SG development. A clear understanding of the mechanisms underlying this process will accelerate SG regeneration studies. Fibroblast growth factor receptor 2 (FGFR2) interacts with multiple fibroblast growth factors (FGFs), which promote development. FGFR2 consists of two isoforms, FGFR2b and FGFR2c. FGFR2b is critical for SG development, but little is known about the expression and function of FGFR2c. We investigated the expression of all FGFR family members in fetal SGs between embryonic day 12.5 (E12.5) and E18.5. Based on RT-PCR, we observed an increase in the expression of not only Fgfr2b, but also Fgfr2c in early-stage embryonic mouse SGs, suggesting that FGFR2c is related to SG development. The branch number decreased in response to exogenous FGF2 stimulation, and this effect was suppressed by a mouse anti-FGFR2c neutralizing antibody (NA) and siRNA targeting FGFR2c, whereas FGFR2b signaling was not inhibited. Moreover, the expression of marker genes related to EMT was induced by FGF2, and this expression was suppressed by the NA. These results suggested that branching morphogenesis in SGs is regulated by FGFR2c, in addition to FGFR2b. Interestingly, FGFR2c signaling also led to increased fgf10 expression, and this increase was suppressed by the NA. FGFR2c signaling regulates branching morphogenesis through the activation of FGFR2b signaling via increased FGF10 autocrine. These results provide new insight into the mechanisms by which crosstalk between FGFR2b and FGFR2c results in efficient branching morphogenesis.

Expression levels of SOX2, KLF4 and brachyury transcription factors are associated with metastasis and poor prognosis in oral squamous cell carcinoma.

The prognosis of oral squamous cell carcinoma (OSCC) patients is affected by tumor recurrence and metastasis, and cancer stem cells are hypothesized to be involved in these processes. Thus, the aim of the present study was to determine whether the expression levels of five stem cell-related transcription factors, sex determining region Y-box 2 (SOX2), octamer-binding transcription factor 4 (Oct4), avian myelocytomatosis viral oncogene homolog (c-Myc), Krüppel-like factor 4 (KLF4) and brachyury, are associated with metastasis and survival in OSCC. Immunohistochemistry was performed to analyze the expression of these proteins in biopsy specimens obtained from 108 OSCC patients. The results revealed that the expression of SOX2, Oct4, KLF4 and brachyury were significantly associated with lymph node metastasis (P=0.002, P=0.031, P=0.003 and P=0.007, respectively). In addition, the expression of KLF4 and brachyury were significantly associated with distant metastasis (P=0.014 and P=0.012, respectively). Furthermore, multivariate analysis revealed that SOX2 and KLF4 are predictive factors for lymph node metastasis [odds ratios (ORs), 4.526 and 4.851, respectively], and KLF4 is also a predictive factor for distant metastasis (OR, 9.607). In addition, OSCC patients with low co-expression of SOX2, KLF4 and brachyury exhibited a significantly lower disease-specific survival rate (78.6 vs. 100%; P=0.025; χ2=5.033) and disease-free survival rate (60.7 vs. 90.9%; P=0.015; χ2=5.897) when compared with OSCC patients with high co-expression of these factors. The results indicate that SOX2, KLF4 and brachyury serve important roles in tumor progression, and these transcription factors may thus represent clinically useful prognostic markers for OSCC.

Biological characterization and analysis of metastasis-related genes in cell lines derived from the primary lesion and lymph node metastasis of a squamous cell carcinoma arising in the mandibular gingiva.

Controlling metastatic lesions is an important part of improving cancer prognosis, in addition to controlling the primary lesion. There have been numerous histological studies on primary and metastatic lesions, but little basic research has been performed using cell lines from primary and metastatic lesions belonging to the same patient. In this study, we successfully established a cell line derived from lower gingival carcinoma (WK2) as well as a line derived from secondary cervical lymph node metastasis (WK3F) through primary cultures of tissue from a patient with oral squamous cell carcinoma. We then investigated the biological characteristics of the cancer cell lines from these primary and metastatic lesions and analyzed metastasis-related genes. Comparison of the biological characteristics in vitro showed that WK3F had higher cell proliferation ability and shorter cell doubling time than WK2. WK3F also had increased cell migratory ability and higher invasive and self-replication abilities. Heterotransplantation into nude mice resulted in high tumor formation rates in the tongue and high metastasis rates in the cervical lymph nodes. Changes in WK2 and WK3F gene expression were then comprehensively analyzed using microarrays. Genes with increased expression in WK3F compared to WK2 were extracted when the Z-score was ≥2.0 and the ratio was ≥5.0, while genes with reduced expression in WK3F compared to WK2 were extracted when the Z-score was ≤-2.0 and the ratio was ≤0.2; differences were found in 604 genes. From these, MAGEC1 (88.0-fold), MMP-7 (18.6-fold), SNAI1 (6.6-fold), MACC1 (6.2-fold), and HTRA1 (0.012-fold) were selected as metastasis-related candidate genes. The results suggest that these molecules could be important for clarifying the mechanisms that regulate metastasis and provide new therapeutic targets for inhibiting tumor invasion.

In vitro induction of specific CD8+ T lymphocytes by tumor-associated antigenic peptides in patients with oral squamous cell carcinoma.

The aim of this study was to clarify candidate peptides for peptide-based specific immunotherapy of patients with oral squamous cell carcinoma (SCC). Thirteen peptides were examined for in vitro induction of peptide-specific CD8(+) T lymphocyte (CD8(+)TL) activity in peripheral blood mononuclear cells from 35 patients with oral SCC. A correlation between the induction ability of CD8(+)TL and in vivo immune response of host was carried out immunohistochemically in 23 patients. Peptide-specific activities of CD8(+)TL for at least one peptide were detectable in 21/35 patients (60.0%). The potent peptides were SART-1(690) in 9/35 (25.7%), SART-2(93), and ART4(75) in 7/35 (20.0%), respectively. In the 9 patients with SART-1(690)-specific activity, the whole of activities was significantly inducible for more number of other peptides compared to that in 26 patients without the activity (P=0.035). Cellular responses in 7 patients with SART-1(690)-specific activity were significantly stronger than those in 16 patients without the activity (P=0.027). Furthermore, the number of CD3(+) T cells around the SCC was also significantly different between the 2 groups of patients (P=0.041). In conclusion, SART-1(690), SART-2(93), and ART4(75) could be applicable as peptide-based specific immunotherapies for the majority of patients with oral SCC.

Multiple schwannomas in the oral floor: case report.

We present a case of multiple schwannomas of the oral floor in a 62-year-old man, which met the diagnostic criteria of schwannomatosis.

T-cell receptor Vbeta gene usage by T cells reactive with the tumor-rejection antigen SART-1 in oral squamous cell carcinoma.

We recently described that the SART-1(690-698) peptide could induce HLA-A24-restricted cytotoxic T lymphocytes (CTLs), which recognize the SART-1(259) (+) tumor cells from peripheral blood mononuclear cells (PBMCs) of HLA-A24(+) cancer patients. In our study, in 5 of 14 HLA-A24(+) patients with oral squamous cell carcinomas (SCCs), CTLs could be induced with the SART-1(690-698) peptide from the PBMCs. In 2 of the patients from whom the highest CTL activities were induced, the T-cell receptor (TCR) Vbeta repertoire expressed by the SART-1(690-698)-specific CTLs was found to be restricted and multiple Vbeta families were predominantly expressed in each patient. Although the predominant Vbeta families were different between the 2 patients, Vbeta7 was highly and commonly predominant. The same predominant Vbeta families were also detected in the tumor-infiltrating lymphocytes (TILs) from each patient, and each Vbeta family contained one or more unique T-cell clonotypes. The unique T-cell clonotypes were found to be common between the TILs and SART-1(690-698)-specific CTLs from each patient, and especially 2 T-cell clonotypes with Vbeta7 were identical even in the 2 patients. One of the 2 T-cell clonotypes with Vbeta7 was detected in the TILs from 11 of 14 HLA-A24(+) patients and another was found in those from 8 of HLA-A24(+) patients, while none of 10 HLA-A24(-) patients demonstrated both T-cell clonotypes. These results strongly suggest that the T-cell clonotypes with Vbeta7 are major TCR Vbeta genes expressed by SART-1(690-698)-specific CTLs. Furthermore, autologous tumor cells from one of the HLA-A24(+) patients stimulated the PBMCs and regional lymph node cells (LNCs) to expand the same T-cell clonotypes as those in the SART-1(690-698)-specific CTLs. These results strongly suggest that the SART-1(690-698)-specific CTLs clearly accumulate in vivo, especially in the TILs, as a consequence of in situ antigenic stimulation by autologous tumor cells. The identification of the unique TCR Vbeta genes used by SART-1(259)-specific CTLs should help to improve the diagnosis of the specific immune response in patients with SART-1(259) (+) cancers, especially during anticancer immunotherapy.

Possible function of salivary gland epithelial cells as nonprofessional antigen-presenting cells in the development of Sjögren's syndrome.

OBJECTIVE: To explore the potential of salivary gland epithelial cells to act as nonprofessional antigen-presenting cells (APC) in the development of Sjögren's syndrome (SS). METHODS: Expression of HLA-DR antigens, costimulatory molecules, and adhesion molecules on epithelial cells was immunohistochemically examined in labial salivary glands from patients with SS. An association with the expression of T cell derived cytokine messenger RNA (mRNA) was observed. The expression of these molecules was confirmed using cultured salivary gland epithelial cells. The ability of the salivary gland epithelial cells as nonprofessional APC was examined in a mixed culture system using the salivary gland epithelial cells and allogeneic lymphocytes. RESULTS: Expression of HLA-DR antigens, CD80, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and E-selectin was immunohistochemically detected on duct cells from all patients; however, the expression of CD86 was limited to only some patients. Concomitant expression of CD80 on duct cells and Th1 cytokine mRNA, and CD86 on duct cells and Th2 cytokine mRNA, was observed. Interferon-gamma (IFN-gamma) induced the cultured salivary gland epithelial cells to express HLA class I antigens, HLA-DR antigens, CD80, and ICAM-1, while tumor necrosis factor-alpha (TNF-alpha) induced the expression of HLA class I antigens, CD80, CD86, and VCAM. Cultured salivary gland epithelial cells treated with either IFN-gamma or TNF-alpha also caused allogeneic lymphocytes to proliferate. CONCLUSION: The ability of salivary gland epithelial cells to express HLA-DR antigens, costimulatory molecules, and adhesion molecules and thus to act as nonprofessional APC was suggested. CD80 and CD86 expression of these cells was also suggested to be involved in the activation of Th1 and Th2, respectively.