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.

Preoperative chronic sinusitis as significant cause of postoperative infection and implant loss after sinus augmentation from a lateral approach.

OBJECTIVES: Among intra/postoperative complications of sinus augmentation from a lateral approach, postoperative infection and implant loss are particularly important because they have irreversible consequences. The purpose of this study was to determine the causes of postoperative infection and implant loss after a lateral approach and to determine the appropriate prophylaxis and therapy. MATERIALS AND METHODS: In total, 109 patients (121 sinuses, 252 implants) were included in this study. The correlation between postoperative infection and implant loss and clinical variables was assessed using logistic regression analyses. RESULTS: Postoperative infection and implant loss occurred in 8/121 sinuses (6.6%). Infection had the strongest correlation to preoperative chronic sinusitis (p = 0.007), followed by timing of implant insertion. Implant loss had the strongest correlation to preoperative chronic sinusitis (p = 0.007), followed by sex, diabetes, postoperative use of dentures, and intraoperative perforation of the sinus membrane. CONCLUSIONS: Preoperative chronic sinusitis could be a significant cause of postoperative infection and implant loss when using sinus augmentation from a lateral approach. For appropriate prophylaxis and therapy, it is necessary to diagnose the presence of chronic sinusitis that should be treated with proper methods prior to sinus augmentation.

The T-box transcription factor Brachyury regulates epithelial-mesenchymal transition in association with cancer stem-like cells in adenoid cystic carcinoma cells.

BACKGROUND: The high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) emphasize the need to better understand the biological factors associated with these outcomes. To analyze the mechanisms of AdCC metastasis, we established the green fluorescence protein (GFP)-transfected subline ACCS-GFP from the AdCC parental cell line and the metastatic ACCS-M GFP line from an in vivo metastasis model. METHODS: Using these cell lines, we investigated the involvement of the epithelial-mesenchymal transition (EMT) and cancer stem cell (CSCs) in AdCC metastasis by real-time RT-PCR for EMT related genes and stem cell markers. Characteristics of CSCs were also analyzed by sphere-forming ability and tumorigenicity. Short hairpin RNA (shRNA) silencing of target gene was also performed. RESULTS: ACCS-M GFP demonstrated characteristics of EMT and additionally displayed sphere-forming ability and high expression of EMT-related genes (Snail, Twist1, Twist2, Slug, zinc finger E-box binding homeobox 1 and 2 [Zeb1 and Zeb2], glycogen synthase kinase 3 beta [Gsk3ß and transforming growth factor beta 2 [Tgf-ß2]), stem cell markers (Nodal, Lefty, Oct-4, Pax6, Rex1, and Nanog), and differentiation markers (sex determining region Y [Sox2], Brachyury, and alpha fetoprotein [Afp]). These observations suggest that ACCS-M GFP shows the characteristics of CSCs and CSCs may be involved in the EMT of AdCC. Surprisingly, shRNA silencing of the T-box transcription factor Brachyury (also a differentiation marker) resulted in downregulation of the EMT and stem cell markers. In addition, sphere-forming ability, EMT characteristics, and tumorigenicity were simultaneously lost. Brachyury expression in clinical samples of AdCC was extremely high and closely related to EMT. This finding suggests that regulation of EMT by Brachyury in clinical AdCC may parallel that observed in vitro in this study. CONCLUSIONS: The use of a single cell line is a limitation of this study. However, parallel data from in vitro and clinical samples suggest the possibility that EMT is directly linked to CSCs and that Brachyury is a regulator of EMT and CSCs.

Involvement of epithelial-mesenchymal transition in adenoid cystic carcinoma metastasis.

The high frequencies of recurrence and distant metastasis of adenoid cystic carcinoma (AdCC) are significant obstacles for the long-term cure of patients with AdCC and emphasize the need for better understanding of the biological factors associated with these outcomes. To identify proteins that mediate AdCC metastasis, we established three AdCC cell lines expressing green fluorescent protein (GFP) from the ACCS cell line using orthotopic transplantation and in vivo selection in nude mice: Parental ACCS-GFP, highly tumorigenic ACCS-T GFP and metastatic ACCS-M GFP. ACCS-GFP and ACCS-M GFP were subjected to DNA microarray analysis and the results were used for data mining studies. DNA microarray analysis revealed significantly altered biological processes in the ACC-M GFP cells, including events related to cell adhesion (three categories) and signaling (three categories). In particular, a significant down-regulation of cell adhesion molecules, such as cadherins and integrin subunits was observed. The loss of E-cadherin and integrins and the gain of vimentin in ACCS-M GFP cells were confirmed by immunoblotting. These results suggest that epithelial-mesenchymal transition (EMT) is a putative event in AdCC metastasis that induces tumor cell dissemination from the primary tumor site. In summary, in this study we established a useful nude mouse metastasis model which will enable further AdCC metastasis research and clinical treatment trials and we also provide evidence that EMT is significantly involved in the AdCC metastatic process.

Expression of the urokinase receptor regulates focal adhesion assembly and cell migration in adenoid cystic carcinoma cells.

Adenoid cystic carcinoma (AdCC) cell lines (ACCS and ACCT) showed higher migration responses and adhesion to the extracellular matrix (ECM), especially types I and IV collagen, than did the oral squamous cell carcinoma (SCC) lines (NA and TF). The response to collagens was largely and exclusively inhibited by anti-alpha(2) integrin antibody. Moreover, AdCC cell lines expressed higher surface levels of urokinase-type plasminogen activator receptor (uPAR) than did SCC cell lines. When AdCC cells were plated on collagen, the surface level of uPAR was increased, and numerous focal adhesions consisting of uPAR, vinculin, and paxillin were assembled; whereas collagen-stimulated SCC cell counterparts or AdCC cells plated on other types of ECM, such as fibronectin, failed to assemble such definite focal adhesions. In order to elucidate the association of uPAR with collagen-induced events, an ACCS-AS cell line transfected with a vector expressing antisense uPAR RNA was established and shown to have reduced uPAR (about 10% that of parental ACCS at both the protein and mRNA levels). ACCS-AS showed a strong reduction of collagen-stimulated migration and focal adhesion assembly of alpha(2) integrin, vinculin, and paxillin. These findings suggest that AdCC has a proclivity for migrating to types I and IV collagens due to the overexpression of uPAR, which plays a key role in focal adhesion assembly and migration.

Involvement of hepatocyte growth factor in branching morphogenesis of murine salivary gland.

We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5-12 (E11.5-E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real-time reverse transcriptase-polymerase chain reaction showed that the expression of the c-met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14-E15.5. Although strong expression of the HGF and c-met transcripts was observed in the tongue muscles, this expression was limited at E13.5-E14.5. Serum-free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme-free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme-free epithelium failed to grow. When E13.5 or E14 mesenchyme-free epithelium and a recombinant human HGF (rh-HGF) -soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh-HGF and anti-HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh-HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh-HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c-met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c-Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme-derived HGF and c-met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage.