Establishment and characterization of a carcinoma-associated fibroblast cell line derived from a human salivary gland adenoid cystic carcinoma.

To investigate the evolution pattern of the Runx3 gene 5'-CpG island ~3478 bp region methylation in human salivary gland adenoid cystic carcinoma (SGACC). Quantitative MSP method was used to detect the methylation status of CpG island in various regions (No.1-10) of Runx3 promoter region, and Western blot was used for detection of the expression of Runx3 protein in 41 salivary gland SGACC samples and corresponding non-neoplastic salivary gland tissues. A Logistic model was used to analyze the risk ratio between the methylation status of CpG island in Runx3 gene and development of salivary SGACC, meanwhile, the possible association among the methylation of Runx3 gene, the clinicopathological parameters of SGACCs, and Runx3 protein expression was compared. The results of qMSP showed that the hypermethylation initially occurred at the most 5' region of the Runx3 CpG island and spread to the transcription start site. The methylation rate was highest in region No. 1 and No. 2 among the successive ten regions ranging from the 5' region to the transcription start site within the Runx3 CpG island, and lowest in the transcription start site both in SGACCs and normal salivary glands. Furthermore, there was no methylation in the transcription start site in nomal salivary glands tissues. Together with the results of Logistic model analysis, those results indicate that the transcription start site within the Runx3 promoter CpG island is critical for gene silencing. Western blot results revealed that the Runx3 protein level in SGACC was significantly lower than that in normal salivary glands (P < 0.01). In combination of the results of qMSP, it is presumed that the Runx3 gene methylation is one of the reason inducing the down-regulation of Runx3 in SGACCs. Methylation of the Runx3 CpG island spreads from the most 5'-region to the transcription start site in human salivary gland adenoid cystic carcinoma, and the transcription start site may be a critical region for the methylation of Runx3. The evolution pattern of Runx3 gene methylation is related to the tumorigenesis of SGACCs.

Adenoid cystic carcinoma (ACC) is an uncommon salivary gland malignancy with a poor long-term prognosis. Clinical reports show the high rates of local recurrences and distant metastases. This study aimed to investigate the expression of MIF, Beclin1, and light-chain 3 (LC3) in salivary adenoid cystic carcinoma (SACC).Tissue specimens were obtained from 48 salivary glands adenoid cystic carcinoma (SACC) patients and 15 oral squamous cell carcinoma (OSCC) patients. Immunohistochemical staining was performed to estimate the level of LC3, Beclin1, and MIF. All SACC patients were followed up. The Kaplan–Meier method was used to compare the prognosis of patients after treatment.The 3-year, 5 year-, and 10 year-survival rates of the SACC patients were 83.9%, 69.9%, and 46.6%, respectively. MIF, LC3, and Beclin1 in SACC were all obviously over-expressed. MIF showed an increased tendency in cases with advanced TNM stages, and at the same time, there was an inversely proportional relationship between MIF and LC3, Beclin1.The long-term survival of SACC patients is poor. MIF might be a risk factor for SACC patients, whereas, LC3 and Beclin1 might be an effective strategy for treatment of SACC.

To examine the expression of Ezrin in human salivary gland adenoid cystic carcinoma and investigate the effects of Ezrin gene silence on cell proliferation, apoptosis and invasion of adenoid cystic carcinoma (ACC)-M. The expression of Ezrin was detected by immunohistochemistry in normal salivary gland tissue (n=15), pleomorphic adenoma (n=40) and salivary gland adenoid cystic carcinoma (n=43). The Ezrin Stealth RNAi Duplex, containing Stealth RNAi Negative Control Duplex were constructed and transfected into ACC-M cells by Lipofectamine 2000. The expression levels of Ezrin were detected by RT-PCR and immunohistochemistry. The cell cycle and apoptosis rate were analyzed by flow cytometry (FCM). The cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) and cell invasion by Transwell test. The positive rate of Ezrin expression in ACC was significantly higher than that in normal salivary gland tissue and pleomorphic adenoma (P<0.05). After transfection of Ezrin Stealth RNAi Duplex, the mRNA and protein expression of Ezrin were down-regulated, the cell proliferation activity was inhibited, the G0-G1 Phase cells were increased, and the apoptosis rate of Ezrin Stealth RNAi Duplex group was higher than that in control groups and cell invasion ability was decreased. Over expression of Ezrin in human salivary gland adenoid cystic carcinoma may promote genesis, development and metastasis of tumors. Ezrin Stealth RNAi Duplex could efficiently down-regulate the expression of Ezrin gene, and partly inhibited proliferation of ACC-M cells, induce apoptosis and decrease invasion ability of these cells in vitro.

Critical regions and spreading of runt-related transcription factor-3 C-phosphate-G (CpG) island methylation in human salivary gland adenoid cystic carcinoma

Carcinoma-associated fibroblasts (CAFs) are critical in determining tumor invasion and metastasis. However the role of CAFs in the invasion of salivary gland adenoid cystic carcinoma (ACC) is poorly understood. In this study, we isolated primary CAFs from two ACC patients. ACC-derived CAFs expressed typical CAF biomarkers and showed increased migration and invasion activity. Conditioned medium collected from CAFs significantly promoted ACC cell migration and invasion. Co-culture of CAFs with ACC cells in a microfluidic device further revealed that CAFs localized at the invasion front and ACC cells followed the track behind the CAFs. Interfering of both matrix metalloproteinase and CXCL12/CXCR4 pathway inhibited ACC invasion promoted by CAFs. Overall, our study demonstrates that ACC-derived CAFs exhibit the most important defining feature of CAFs by promoting cancer invasion. In addition to secretion of soluble factors, CAFs also lead ACC invasion by creating an invasive track in the ECM.

Adenoid cystic carcinoma (ACC), a rare and progressive salivary malignancy, is characterized by histogenetic, morphologic, and clinical heterogeneity. Extensive efforts to characterize the molecular events associated with these tumors have included the identification of biomarkers for prognostication and post-therapy assessment. In a previous study of genome-wide methylation screening, the authors of the current report identified a limited number of differentially methylated gene regions in ACC, and significant hypermethylation was observed at the transcriptional start sites of genes that encode for the transcription factor engrailed homeobox 1 (EN1). Clinicopathologic correlation analyses indicated that EN1 methylation status is correlated with histologic tumor grade, tumor location, and final patient outcome. To ascertain definitively whether aberrant EN1 expression accompanies human salivary ACC, the authors used an immunohistochemical technique to directly evaluate EN1 protein expression in ACC of the salivary gland. The data revealed increased EN1 protein expression in solid type ACC, which was correlated with a significantly lower survival rate. The current results validated EN1 as a potential biomarker in a large cohort of patients with salivary ACC. Immunohistochemical analysis of EN1 in biopsy specimens obtained for diagnostic purposes and/or surgically resected material may reveal that EN1 is a biologic predictor of poor prognosis in patients with salivary ACC.

The aetiology and pathogenesis of salivary gland malignancies remain unknown. To reveal novel molecular factors behind the development of salivary gland cancer, we performed gene expression analyses from Smgb-Tag mouse salivary gland samples. The overall purpose was to apply these results for clinical use to find new approaches for both possible therapeutic targets and more accurate diagnostic tools. Smgb-Tag mouse strain, in which salivary neoplasms arise through a dysplastic phase in submandibular glands, was investigated using genome-wide microarray expression analysis, ingenuity pathway analysis, RT-PCR, and immunohistochemistry. Thirty-eight human salivary gland adenoid cystic carcinoma samples were investigated using immunohistochemistry for validation purposes. Our genome-wide study showed that Ppp2r1b, a PP2A subunit encoding tumor suppressor gene, is underexpressed in submandibular gland tumors of Smgb-Tag mice. mTOR signaling pathway was significantly enriched and mTOR linked PP2A subunit gene B55 gamma was significantly underexpressed in the analyses. Furthermore, parallel immunohistochemical analysis of three PP2A inhibitors demonstrated that two PP2A inhibitors, CIP2A and SET, are highly expressed in both dysplastic and adenocarcinomatous tumors of the Smgb-Tag mice. In addition, all 38 investigated human salivary adenoid cystic carcinoma samples stained positively for CIP2A and most for SET. Finally, p-S6 staining showed activation of mTOR pathway in human adenoid cystic carcinoma samples. Our results suggest that PP2A inhibition either via PP2A subunit underexpression or PP2A inhibitor overexpression play an important role in the formation of salivary gland malignancy, potentially due to mTOR signaling activation.

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is been used in the clinic due to its pleiotropic effects, such as breast cancer, prostate cancer, pancreatic cancer. Simvastatin has recently been demonstrated to serve a potential role in the prophylaxis and therapeutics of a number of human cancers. The majority of reports concerning simvastatin treatment in the majority of human cancers have demonstrated that survivin is significantly decreased as a result and has been implicated in tumorigenesis. However, only a limited number of studies have investigated the use of simvastatin for the treatment of salivary gland adenoid cystic carcinoma (SACC). Therefore, this agent is a candidate for further investigation. The aim of the present study was to investigate the effects of simvastatin on the proliferation, invasion and apoptosis of the human salivary adenoid cystic carcinoma cell line, SACC-83, as well as survivin expression in the cells. The Cell Counting kit-8 assay results revealed that simvastatin inhibited the proliferation of SACC-83 cells in a dose-dependent (10 to 50 µM) and time-dependent (24 to 48 h) manner when compared with the untreated cells. Flow cytometry analysis indicated that simvastatin increased the percentage of cells in early and late apoptosis. Invasion assays revealed that simvastatin treatment inhibited the invasiveness of SACC-83 cells in a dose-dependent manner. In addition, simvastatin downregulated survivin expression in SACC-83 cells. In conclusion, simvastatin significantly inhibited the proliferation and invasion of SACC-83 cells, induced apoptosis, and reduced the expression of survivin, which suggests that simvastatin may be a novel target for SACC therapy.

SIKVAV, a Laminin α1-Derived Peptide, Interacts with Integrins and Increases Protease Activity of a Human Salivary Gland Adenoid Cystic Carcinoma Cell Line through the ERK 1/2 Signaling Pathway

BackgroundThis study aimed to investigate the expression level of the GATA6 gene in different oral cancer cells.MethodsIn this study, we sub-cultured normal oral epithelial cell lines HOK, human tongue squamous cell carcinoma cell lines CAL-27 and SCC-4, and human salivary gland adenoid cystic carcinoma cell lines SACC-LM and SACC-83. Subsequently, we used reverse transcription-polymerase chain reaction RT-PCR and Western blot methods to detect the mRNA and the protein expressions of GATA6 in normal oral epithelial cells, human tongue squamous cell carcinoma cells, and human salivary gland adenoid cystic carcinoma cells.ResultsThe results of this study showed that the mRNA expression levels of GATA6 in CAL-27, SCC-4, and SACC-LM cells were significantly increased when compared with the HOK cells. However, the mRNA expression level of GATA6 in the SACC-83 cells had no significant difference compared with the HOK cells. The protein expression levels of GATA6 in the SCC-4 and SACC-LM cells were, however, significantly increased whereas the protein expression levels of GATA6 in the CAL-27 and SACC-83 cells had no significant difference when compared with the HOK cells.ConclusionThe GATA6 gene may be related to the occurrence and progression of certain oral cancers.

The role of promoter methylation in the inactivation of E-cadherin (E-cad) in salivary gland adenoid cystic carcinoma (ACC) is unknown. The objective of this study was to determine the role and potential clinical implications of promoter methylation of E-cad in salivary gland ACC. The promoter methylation status of E-cad was determined by using methylation-specific polymerase chain reaction (PCR) analysis in 60 primary salivary gland ACC tissues and 3 ACC cell lines. The level of E-cad protein expression was determined by immunohistochemical analysis of each tumor. E-cad protein and messenger RNA (mRNA) expression levels were examined by immunohistochemical analysis and reverse transcriptase-PCR in 3 ACC cell lines. Associations between molecular alterations and patients' clinicopathologic characteristics were analyzed statistically. E-cad mRNA expression was examined in a 5-azacytidine-treated ACC-2 cell line. Promoter methylation of E-cad was detected in 34 of 60 tumors (57%). Of those 34 tumors, 18 tumors (53%) showed no E-cad protein expression, whereas only 5 of the remaining 26 tumors (19%) without E-cad promoter methylation showed no E-cad protein expression (P = .01). Tumors that had E-cad promoter methylation had a significantly higher histologic grade (P = .01) and more perineural invasion (P = .02) compared with tumors that did not have methylation. All 3 ACC cell lines exhibited E-cad promoter methylation and a lack of E-cad mRNA and protein expression, whereas 5-azacytidine restoredE-cad mRNA expression in the ACC-2 cell line. E-cad frequently is inactivated in salivary gland ACC through promoter methylation, and E-cad promoter methylation may play a role in tumor cell differentiation and perineural invasion.

Previous genomic studies revealed phosphotidylinositol-3-kinase (PI3K)/Akt pathway mutation in human salivary gland adenoid cystic carcinoma (ACC). No validation of its prognostic value has been reported. P-Akt, pan-Akt, phosphorylated-mammalian target of rapamycin (p-mTOR), PI3K, and insulin-like growth factor-1 receptor beta (IGF-1Rβ) were detected on 120 salivary gland ACC/adjacent salivary gland pairs immunohistochemically and were correlated with clinicopathological data. Expression of cytoplasmic and nuclear p-Akt, cytoplasmic p-mTOR, nuclear pan-Akt, and nuclear IGF-1Rβ were higher in ACC than in adjacent salivary glands. P-Akt, p-mTOR, PI3K, and IGF-1Rβ expression were correlated with one another in both cytoplasm and nucleus. Low p-mTOR expression in both subcellular compartments was associated with locoregional recurrence, poor disease-free survival (DFS), and overall survival (OS). Low nuclear p-Akt (Ser473) and p-mTOR expression were independent predictors for poor OS and DFS, respectively. High level of Akt/mTOR activation in ACC is correlated with a significantly improved survival. P-mTOR and nuclear p-Akt are prognostic biomarkers of salivary gland ACC. © 2017 Wiley Periodicals, Inc. Head Neck 39: 1145-1154, 2017.

The purpose of the study was to evaluate the long-term outcome of minor salivary and mucous gland (MiSG) adenoid cystic carcinoma (ACC) of the head and neck and to compare the results with earlier reports including our recently published series on major salivary gland (MaSG) ACC. The study comprised 68 MiSG ACCs operated during 1974-2012 at the Helsinki University Hospital, Helsinki, Finland. Medical records and histological samples were reviewed. Our previously published cohort comprising 54 MaSG ACCs during the years from 1974 to 2009 was used for comparison. The most common locations were the oral cavity and sinonasal cavities. Most patients presented stages IV (33.8%) and I (23.5%) disease. Primary treatment with curative intent, mainly surgery, was offered for 64 patients. Thirty-three (51.6%) of these patients developed a disease recurrence and 22 (66.7%) patients in less than 5years. The difference in the length of recurrence-free time (<5 vs. >5years) had an impact on OS and DSS (p<0.001) showing worse prognosis for the earlier recurring group. T classes 2-4 (p=0.005, p<0.001, and p=0.001, respectively) and stages II-IV (p=0.019, p<0.001, and p=0.002, respectively) were associated with worse OS, DSS, and DFS. MiSG ACC had a similar long-term survival compared to MaSG ACC. Patients with stage I MiSG ACC seem to carry a favourable prognosis compared with those with stages II, III, and IV tumours. It is thus noteworthy that stage II tumours represent a truly advanced disease entity warranting a more aggressive treatment approach.

BackgroundPim-1 (Provirus integration site for Moloney murine leukemia virus 1) belongs to the Ser/Thr kinase family and plays a pivotal role in occurrence and development of oncogenesis. Recent studies have demonstrated that Pim-1 phosphorylates RUNX3 and alters its subcellular localization. However, few studies have concerned the implications of Pim-1 in the salivary gland adenoid cystic carcinoma (ACC). In this study, we aimed to clarify the function of Pim-1 in ACC in vitro. Meanwhile, we measured the levels of Pim-1 and RUNX3 in the ACC tissues. The correlations between Pim-1/RUNX3 levels and clinical parameters were also analyzed.MethodsSACC-83 and SACC-LM cells were transfected with the Pim-1 siRNA. Pim-1 mRNA and protein expression were measured using real-time PCR and immnuoblot, respectively. Cell proliferation was analyzed by CCK-8 assay. Cell cycle, apoptosis, and mitochondrial membrane potential were detected by flow cytometry. Effects of Pim-1 on cells’ invasion were evaluated by transwell migration assay. Pim-1 and RUNX3 levels in ACC tissues were examined by immunohistochemistry.ResultsPim-1 siRNA reduces cell proliferation, induces apoptosis, causes cell cycle arrest through cell cycle related proteins (Cyclin D1 and CDK4), mitochondrial depolarization, and decreases invasive ability in SACC-83 and SACC-LM cells. Pim-1 and RUNX3 levels are significantly relevant and associated with T-stage and nerve invasion in the ACC tissues.ConclusionsThis study demonstrates the oncogenic role of Pim-1 in ACC. The findings also suggest that Pim-1 may serve as a neoteric therapeutic target and potential prognostic marker for ACC cancer.

Noxa, which is subset of the Bcl-2 family of proteins, was previously reported to have considerable therapeutic potential in diverse cancers. However, its expression and role in salivary gland adenoid cystic carcinoma (ACC) have not been well studied. This study aimed to elucidate the expression and role of Noxa in salivary gland ACC. The expression levels of NOXA and its association with overall survival in salivary gland ACC were analyzed by quantitative real-time PCR. We next examined the effects of Noxa overexpression or inhibition on colony formation, proliferation, apoptosis, and autophagy of salivary gland ACC cells. Furthermore, promoter analysis was performed to identify the potential transcriptional activator of NOXA. NOXA was markedly down-regulated and significantly correlated with a more aggressive phenotype and poor overall survival of salivary gland ACC. Ectopic expression of Noxa suppressed the viability and growth of ACC cells, which involved the induction of apoptosis and autophagy. Moreover, the transcriptional activity of NOXA gene could be enhanced by p53. The findings of this study indicate that Noxa, activated transcriptionally by p53, suppress the progression of ACC, whereby it regulates proliferation, apoptosis, and autophagy.