Invasion Of Salivary Adenoid Cystic Carcinoma Research Articles

Silencing geranylgeranyltransferase I inhibits the migration and invasion of salivary adenoid cystic carcinoma through RhoA/ROCK1/MLC signaling and suppresses proliferation through cell cycle regulation.

Geranylgeranyltransferase type I (GGTase-I) significantly affects Rho proteins, such that the malignant progression of several cancers may be induced. Nevertheless, the effect and underlying mechanism of GGTase-I in the malignant progression of salivary adenoid cystic carcinoma (SACC) remain unclear. This study primarily aimed to investigate the role and mechanism of GGTase-I in mediating the malignant progression of SACC. The level of GGTase-I gene in cells was stably knocked down by short hairpin RNA-EGFP-lentivirus. The effects of GGTase-I silencing on the migration, invasion, and spread of cells were examined, the messenger RNA levels of GGTase-I and RhoA genes of SACC cells after GGTase-I knockdown were determined, and the protein levels of RhoA and RhoA membrane of SACC cells were analyzed. Moreover, the potential underlying mechanism of silencing GGTase-I on the above-mentioned aspects in SACC cells was assessed by examining the protein expression of ROCK1, MLC, p-MLC, E-cadherin, Vimentin, MMP2, and MMP9. Furthermore, the underlying mechanism of SACC cells proliferation was investigated through the analysis of the expression of cyclinD1, MYC, E2F1, and p21CIP1/WAF1 . Besides, the change of RhoA level in SACC tissues compared with normal paracancer tissues was demonstrated through quantitative reverse-transcription polymerase chain reaction and western blot experiments. Next, the effect after GGTase-I silencing was assessed through the subcutaneous tumorigenicity assay. As indicated by the result of this study, the silencing of GGTase-I significantly reduced the malignant progression of tumors in vivo while decreasing the migration, invasion, and proliferation of SACC cells and RhoA membrane, Vimentin, ROCK1, p-MLC, MMP2, MMP9, MYC, E2F1, and CyclinD1 expression. However, the protein expression of E-cadherin and p21CIP1/WAF1 was notably upregulated. Subsequently, no significant transform of RhoA and MLC proteins was identified. Furthermore, RhoA expression in SACC tissues was significantly higher than that in paracancerous tissues. As revealed by the results of this study, GGTase-I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway. GGTase-I is expected to serve as a novel exploration site of SACC.

Hypoxia promotes vasculogenic mimicry formation by vascular endothelial growth factor A mediating epithelial-mesenchymal transition in salivary adenoid cystic carcinoma.

ObjectivesTo investigate the role of hypoxia in vasculogenic mimicry (VM) of salivary adenoid cystic carcinoma (SACC) and the underlying mechanism involved.Materials and methodsFirstly, wound healing, transwell invasion, immunofluorescence and tube formation assays were performed to measure the effect of hypoxia on migration, invasion, EMT and VM of SACC cells, respectively. Then, immunofluorescence and RT‐PCR were used to detect the effect of hypoxia on VE‐cadherin and VEGFA expression. And pro‐vasculogenic mimicry effect of VEGFA was investigated by confocal laser scanning microscopy and Western blot. Moreover, the levels of E‐cadherin, N‐cadherin, Vimentin, CD44 and ALDH1 were determined by Western blot and immunofluorescence in SACC cells treated by exogenous VEGFA or bevacizumab. Finally, CD31/ PAS staining was performed to observe VM and immunohistochemistry was used to determine the levels of VEGFA and HIF‐1α in 95 SACC patients. The relationships between VM and clinicopathological variables, VEGFA or HIF‐1α level were analysed.ResultsHypoxia promoted cell migration, invasion, EMT and VM formation, and enhanced VE‐cadherin and VEGFA expression in SACC cells. Further, exogenous VEGFA markedly increased the levels of N‐cadherin, Vimentin, CD44 and ALDH1, and inhibited the expression of E‐cadherin, while the VEGFA inhibitor reversed these changes. In addition, VM channels existed in 25 of 95 SACC samples, and there was a strong positive correlation between VM and clinic stage, distant metastases, VEGFA and HIF‐1α expression.ConclusionsVEGFA played an important role in hypoxia‐induced VM through regulating EMT and stemness, which may eventually fuel the migration and invasion of SACC.

Upregulation of lncRNA ADAMTS9-AS2 Promotes Salivary Adenoid Cystic Carcinoma Metastasis via PI3K/Akt and MEK/Erk Signaling.

Neurotropic infiltrative growth and distant metastasis are the main causes of death in salivary adenoid cystic carcinoma (SACC) patients. Long noncoding RNAs (lncRNAs) are involved in many human neoplasms, however, their potential roles in SACC are unclear. In our study, we found that ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9) antisense RNA 2 (ADAMTS9-AS2) was significantly upregulated in SACC patients with metastasis and SACC-lung metastasis (LM) cells. Moreover, ADAMTS9-AS2expression was closely associated with the prognosis anddistant metastasis in SACC patients. Next, we found thatc-myc could specifically bind to the promoter of ADAMTS9-AS2 and activated its transcription. Knockdown of ADAMTS9-AS2 significantly inhibited migration and invasion of SACC cells invitro and distant lung metastasis invivo. Furthermore, ADAMTS9-AS2, which mainly expressed in the cytoplasm, shared microRNA (miRNA) response elements with Integrin α6 (ITGA6). Overexpression of ADAMTS9-AS2 competitively bound to miR-143-3p that inhibited ITGA6 from miRNA-mediated degradation, and thus it activated the activity of PI3K/Akt and MEK/Erk signaling and facilitated SACC metastasis. In summary, ADAMTS9-AS2 promotes migration and invasion in SACC by competing with miR-143-3p. This sheds a new insight into the regulation mechanism of ADAMTS9-AS2, and it provides a possible application for the SACC treatment.

Potential biological functions of microvesicles derived from adenoid cystic carcinoma.

Microvesicles (MVs) are secreted by multiple types of tumor cell and are involved in tumor progression and metastasis. The aim of the present study was to explore the effects of MVs derived from salivary adenoid cystic carcinoma (SACC) and to investigate their potential involvement in the pathogenesis of perineural invasion of SACC. MVs were isolated from ACCs cells, and differential gene expression profiles of these MVs were compared with their donor cells to speculate on their biological functions. Several candidate genes were validated using reverse transcription-quantitative polymerase chain reaction analysis. The effects of ACCs MVs on rat Schwann cells (RSC96 cells), which are the principal glia of the peripheral nervous system, were then evaluated by phospho-antibody array performed on RSC96 cells transduced with ACCs MVs. The results indicated that ACCs cells may produce MVs. Microarray-based expression profiles between ACCs cells and their MVs identified 1,355 genes involved in cell adhesion, development and the regulation of apoptosis. In addition, the extracellular signal-regulated protein kinase signal pathway in RSC96 cells may be induced by ACCs-derived MVs. These results may help to elucidate the mechanisms underlying perineural invasion in SACC, and to determine a promising anti-tumor biological therapeutic target.

MicroRNA-98 regulates invasion and migration of salivary adenoid cystic carcinoma by targeting N-Ras

Objective To investigate the effect of microRNA (miRNA, miR)-98 on suppression of proliferation, migration and invasion in salivary adenoid cystic carcinoma (SACC). Methods The expression of miR-98 was compared between SACC tissues and the adjacent tissues of 3 patients, as well as between SACC cell lines by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). Bioinfornatics analysis predicted miR-98 targeting site in gene N-Ras. N-Ras expression was detected in SACC tissues using immunohistochemistry. And the associations between N-Ras expression and clinicopathological features were analyzed. The effects of miR-98 on the proliferation, migration and invasion of SACC cell lines were also determined. Finally, the target relationship between miR-98 and N-Ras was identified by Dual-Luciferase Assay system, RT-qPCR and Western blotting. Results The expression of miR-98 in SACC tissues and ACC-M cells were both significantly lower than those in adjacent tissues and SACC-83 cells. N-Ras was overexpressed in SACC tissues and its overexpression was associated with the clinical stage and tumor size. After transfection of miR-98 mimics to ACC-M cells, the ability of cell proliferation reduced; invasion cell number reduced from 158.0±5.5 of control group to 92.0±4.7 of experimental group (t=15.800, P=0.000); and the migration ability reduced from (0.42±6.30)mm to (0.25±6.00) mm (t=4.630, P=0.001). While the downregulation of miR-98 increased the ability of proliferation. Invasion cell number increased from 88.0±4.0 to 123.0±10.6 (t=5.254, P=0.006) and migration distance increased from (0.15±7.50) mm to (0.38±8.80) mm after transfected with miR-98 inhibitor (t=9.109, P=0.000). At last, the target relationship between miR-98 and N-Ras was indentified by Dual-Luciferase Assay System. Conclusion MiR-98 can regulate invasion and migration of SACC via targeting N-Ras. Key words: MicroRNA-98; Salivary adenoid cystic carcinoma; Invasion; Migration

Substrate stiffness regulated migration and invasion ability of adenoid cystic carcinoma cells via RhoA/ROCK pathway.

Human salivary adenoid cystic carcinoma (SACC) is one of the most common malignant tumours of the salivary gland and has strong migratory and invasive ability, which often lead to poor prognosis and lower survival rate. Tumour tissue tends to stiffen during solid tumour progression. This study aimed to investigate the influence of various substrate stiffness on the migration and invasion of SACC. Salivary adenoid cystic carcinoma cell line ACC2 cells were cultured on polydimethylsiloxane substrates (PDMS) with varying stiffness for investigating the effects of substrate stiffness on the activities of MMPs and TIMPs. The underlying mechanism was also explored. When ACC2 cells were cultured on various stiffness of PDMS, the expressions of matrix metalloproteinases 2 (MMP2), MMP9, MMP14, RhoA, Rac1, Rho-associated protein kinase 1 (ROCK1) and ROCK2 were up-regulated with increasing substrate stiffness, whereas that of tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2 and TIMP4 were down-regulated with increasing substrate stiffness. Our results showed that substrate stiffness regulated the activities of MMPs and TIMPs and then modulate migratory and invasive ability of ACC2 cells via RhoA/ROCK pathway. This work indicate that matrix stiffness played an important role in progression of SACC, which not only can help understand the strong invasive ability of SACC, but also suggested that therapeutically targeting matrix stiffness may help reduce migration and invasion of SACC and improve effective therapies.

Involvement of non-B cell-derived immunoglobulin G in the metastasis and prognosis of salivary adenoid cystic carcinoma.

Cancer cell-derived immunoglobulin G (cancer-IgG) has been implicated in the pathogenesis and progression of various types of cancer. However, its role in salivary adenoid cystic carcinoma (SACC) remains unclear. The present study aimed to investigate the effects of cancer-IgG on metastasis and prognosis in 96 patients with SACC. Immunohistochemical staining showed that cancer-IgG expression was present in all 96 individual SACC tissues. Additionally, high cancer-IgG expression was significantly correlated with metastasis, nerve invasion and recurrence in SACC (P<0.05). Moreover, cancer-IgG expression was significantly correlated with the survival duration of patients with SACC (P<0.05). Proliferation, cell motility and invasion all decreased significantly following knockdown of cancer-IgG in SACC cells (P<0.05) through population-doubling time, wound healing and transwell invasion assays. Additionally, cancer-IgG-knockdown in SACC cells induced the increased expression of E-cadherin and matrix metalloproteinase 9, and promoted the epithelial-mesenchymal transition, but decreased the expression of F-actin filaments. Taken together, these results showed that the high expression of cancer-IgG was strongly associated with metastasis, recurrence and invasion in SACC, suggesting that cancer-IgG expression could serve as a useful biomarker to predict the prognosis of the disease.

MiR-582-5p inhibits invasion and migration of salivary adenoid cystic carcinoma cells by targeting FOXC1.

Neurotropism of salivary adenoid cystic carcinoma (SACC) and pulmonary metastasis may lead to in treatment failure. miR-582-5p plays important roles in tumorigenesis, invasion and migration. Here, we aim to determine the effect of miR-582-5p and its role in SACC invasion and metastasis. Six primary human SACC samples and matching adjacent normal tissues were analyzed by microarray analysis. Next, quantitative real-time PCR was carried out to evaluate miR-582-5p expression in 16 primary human SACC samples and matching adjacent normal tissues. Cell invasion and migration were also analyzed, and a luciferase reporter assay and western analysis were conducted. Cell growth and apoptosis assay were performed to confirm the effect of miR-582-5p and Forkhead box C1 (FOXC1) siRNA in cell proliferation and apoptosis. SACC tumorigenesis and metastasis were investigated in vivo experiment. Clinical samples from 110 patients were analyzed using in situ hybridization and immunohistochemistry. Microarray analysis revealed that miR-582-5p was significantly downregulated in the SACC samples compared with the matching adjacent normal tissues. Regulation of miR-582-5p expression significantly influenced the migration, invasion and proliferation ability of SACC cells by targeting FOXC1. E-cadherin was increased, while vimentin and snail were decreased with downregulation of FOXC1, suggesting that FOXC1 may regulate the epithelial-to-mesenchymal transition (EMT) of SACC cells by transactivating snail. In vivo, miR-582-5p overexpression suppressed the tumorigenesis and pulmonary metastasis of SACC. Lower expression of miR-582-5p expression predicts unfavorable prognoses and high rates of metastasis. miR-582-5p could suppress effect on the process of invasion and migration in SACC cell lines, and this could occur through its target gene FOXC1.

BRD4 inhibition suppresses cell growth, migration and invasion of salivary adenoid cystic carcinoma

BackgroundBromodomain-containing protein 4 (BRD4) inhibition is a new therapeutic strategy for many malignancies. In this study, we aimed to explore the effect of BRD4 inhibition by JQ1 on in vitro cell growth, migration and invasion of salivary adenoid cystic carcinoma (SACC).MethodsThe human normal epithelial cells and SACC cells (ACC-LM and ACC-83) were treated with JQ1 at concentrations of 0, 0.1, 0.5 or 1 μM. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Cell apoptosis and cell cycle distribution was evaluated by Flow cytometry. Immunofluorescence staining was used to examine the expression of BRD4 in SACC cells. The quantitative real-time polymerase chain reaction (qRT-PCR) assay and western blot assay were performed to examine messenger RNA (mRNA) and protein levels in SACC cells. Wound-healing assay and transwell assay were used to evaluate the activities of migration and invasion of SACC cells.ResultsJQ1 exhibits no adverse effects on proliferation, cell cycle and cell apoptosis of the normal human epithelial cells, while suppressed proliferation and cell cycle, and induced apoptosis of SACC cells, down-regulated the mRNA and protein levels of BRD4 in SACC cells, meanwhile reduced protein expressions of c-myc and BCL-2, two known target genes of BRD4. Moreover, JQ1 inhibited SACC cell migration and invasion by regulating key epithelial–mesenchymal transition (EMT) characteristics including E-cadherin, Vimentin and Twist.ConclusionsBRD4 is an important transcription factor in SACC and BRD4 inhibition by JQ1 may be a new strategy for SACC treatment.

Cytokeratin-14 contributes to collective invasion of salivary adenoid cystic carcinoma.

Collective invasion of cells plays a fundamental role in tissue growth, wound healing, immune response and cancer metastasis. This paper aimed to investigate cytokeratin-14 (CK14) expression and analyze its association with collective invasion in the invasive front of salivary adenoid cystic carcinoma (SACC) to uncover the role of collective invasion in SACC. Here, in the clinical data of 121 patients with SACC, the positive expression of CK14 was observed in 35/121(28.93%) of the invasive front of SACC. CK14 expression in the invasive front, local regional recurrence and distant metastasis were independent and significant prognostic factors in SACC patients. Then, we found that in an ex vivo 3D culture assay, CK14 siRNA receded the collective invasion, and in 2D monolayer culture, CK14 overexpression induced a collective SACC cell migration. These data indicated that the presence of characterized CK14+ cells in the invasive front of SACC promoted collective cell invasion of SACC and may be a biomarker of SACC with a worse prognosis.

PRL-3 promotes migration and invasion and is associated with poor prognosis in salivary adenoid cystic carcinoma.

Background PRL‐3 had been found to be involved in tumorigenesis in various malignancies. In this study, we investigated the role of PRL‐3 in the development, migration, and invasion of salivary adenoid cystic carcinoma (SACC).MethodsImmunohistochemistry (IHC) was used to analyze the role of PRL‐3 in the development and prognosis of SACC. Then, we overexpressed or inhibited the expression of PRL‐3 in paired SACC cells to analyze the role of PRL‐3 in the migration and invasion of SACC. In vitro migration and invasion assays were used. Western blotting was used to detect metastasis‐related protein levels.Results IHC results confirmed that the deregulation of PRL‐3 was a frequent event in SACC; the upregulation of PRL‐3 was related to clinical stages, vital status, and distant metastasis, which was associated with reduced overall survival and disease‐free survival. SACC‐LM cells with higher migratory and invasive abilities had more robust PRL‐3 protein expression than SACC‐83 cells with lower migratory and invasive abilities. PRL‐3 overexpression promoted cell migration, invasion, and proliferation, led to simultaneous upregulation of phosphorylated PRL‐3, pERK1/2, Slug, vimentin, and downregulation of E‐cadherin in SACC‐83 cells. However, the inhibition of PRL‐3 by PRL‐3 inhibitor or PRL‐3 siRNA in SACC‐LM cells inhibited cell migration, invasion, and proliferation, resulted in simultaneous downregulation of phosphorylated PRL‐3, pERK1/2, Slug, vimentin, and upregulation of E‐cadherin.ConclusionsOur results confirm that PRL‐3 plays an important role in the development of SACC and contributes to the migratory and invasive abilities of SACC.

MTUS1/ATIP3a down-regulation is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma

BackgroundMicrotubule-associated tumor suppressor gene (MTUS1) has been identified as tumor suppressor gene in many malignant tumors. In this study, we investigated the role of MTUS1 in the development of salivary adenoid cystic carcinoma (SACC) and its functional effect on the migration and invasion of SACC.MethodsArchival clinical samples including 49 primary SACC were examined for MTUS1 expression by immunohistochemistry. Statistical analyses were performed to evaluate the correlation between MTUS1 with histopathological features and survival. The expression of MTUS1/ATIP (AT2 receptor-interacting protein) isoforms was determined in SACC tissue samples and cell lines using quantitative RT-PCR assays. Then we investigated whether the migration and invasion of SACC were mediated by MTUS1/ATIP3a using in vitro cell migration and invasion assay.ResultsWe confirmed that the down-regulation of MTUS1 was a frequent event in SACC, and was correlated with distant metastasis and associated with reduced overall survival and disease free survival. Isoform specific quantitative RT-PCR assays revealed that ATIP1, ATIP3a and ATIP3b were the major isoforms of the MTUS1 gene products in SACC, and were significant down-regulation in SACC as compared to matching normal tissues. For functional analyses, we found that SACC-LM cells (SACC cell line with higher migration and invasion ability) possessed a lower expression level of ATIP3a compared to SACC-83 cells (lower migration and invasion ability). Restoration of ATIP3a expression in SACC-LM cells induced anti-proliferative activity and inhibited the migration and invasion ability. Knockdown of ATIP3a promoted the proliferation, migration and invasion ability of SACC-83 cells. Restoration of ATIP3a inhibited the phosphorylation of ERK (extracellular-regulated kinase) 1/2, the expression of Slug and Vimentin in SACC-LM cells, while knockdown of ATIP3a increased the phosphorylation of ERK1/2, the expression of Slug and Vimentin in SACC-83 cells.ConclusionsOur studies confirm that MTUS1 plays an important role in the progression of SACC, and may serve as a biomarker or therapeutic target for patients with SACC. MTUS1/ATIP3a down-regulation contributes to the proliferation, migration and the invasion abilities of SACC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1209-x) contains supplementary material, which is available to authorized users.

Abstract 5328: MicroRNA-181a suppresses salivary adenoid cystic carcinoma metastasis by targeting the ERK/Slug pathway.

Abstract MicroRNA deregulation plays a major role in the initiation and progression of many cancers. The objective of this study is to evaluate the role of miR-181a in the metastasis of salivary adenoid cystic carcinoma (SACC). We identified a panel of differentially expressed microRNAs in two pairs SACC cell lines with different metastatic potential using microarrays. Among these differentially expressed microRNAs, the down-regulation of miR-181a was further validated by quantitative RT-PCR. Functional analysis indicated that miR-181a inhibits SACC cell migration, invasion and proliferation in vitro and in vivo. Ectopic transfection of miR-181a inhibited the expression of NGF, VEGF, TGFβ2, ERK1/2, pERK1/2 and Slug in SACC-LM cells which had higher expression of these genes compared to SACC-83 cells; while inverse results were found in miR-181a LNA transfected SACC-83 cells. The direct targeting of miR-181a to the Slug and ERK2 mRNAs were confirmed by luciferase reporter gene assays. SiRNA-mediated knockdown of Slug, ERK2 led to reduced cell migration and invasion of SACC, and knockdown of ERK2 suppresses the expression of Slug. Taken together, these results suggest that microRNA deregulation (i.e., down-regulation of miR-181a) contributes to the metastasis of SACC. MiR-181a-mediated suppression of SACC metastasis may be achieved in part by directly targeting Slug and ERK2 and regulating ERK/Slug pathway. Citation Format: Qianting He, Xiaofeng Zhou, Su Li, Zhujian Chen, Dan Chen, Yuchen Cai, Zhonghua Liu, Tingting Zhao, Anxun Wang. MicroRNA-181a suppresses salivary adenoid cystic carcinoma metastasis by targeting the ERK/Slug pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5328. doi:10.1158/1538-7445.AM2013-5328

EMMPRIN contributes to the in vitro invasion of human salivary adenoid cystic carcinoma cells

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein that is involved in tumor invasion by stimulating matrix metalloproteinase (MMP) expression. Our previous immunohistochemical study found that the expression of EMMPRIN in salivary adenoid cystic carcinoma (SACC) was positively correlated with tumor perineural and perivascular invasion. The present study was designed to further investigate the role of EMMPRIN in the invasion of SACC. Western blot results showed that EMMPRIN was upregulated in the highly metastatic SACC cell line SACC-LM, compared to SACC-83, a SACC cell line with low metastatic ability. Blocking of EMMPRIN by its antibody significantly decreased the adhesion, secretion of MMP-2 and MMP-9, and invasion activity of SACC-LM cells in vitro (P<0.01). Co-cultures of SACC-LM cells with fibroblasts significantly produced elevated levels of MMP-2 and MMP-9, and promoted the in vitro invasion activity of SACC-LM cells, compared with cultures of SACC-LM cells alone (P<0.01). These results indicate that EMMPRIN may play an important role in the invasion of SACC by stimulating the expression of MMP-2 and MMP-9 in tumor and stromal cells.

Coexpression of Schwann cell marker GFAP and myoepithelial cell marker alpha-SMA in salivary adenoid cystic carcinoma

To observe the expression of Schwann cell marker GFAP and myoepithelial cell marker alpha-SMA in salivary adenoid cystic carcinoma (ACC), and to evaluate the relationship of GFAP, alpha-SMA and perineural invasion in ACC. Immunohistochemical SABC method, double-label immunofluorescence and CLSM were used to detect the expression of GFAP and alpha-SMA proteins in salivary ACC tissue samples. In salivary ACC tissue samples, both GFAP and alpha-SMA proteins were positive, which were coexpressed in cytoplasm of the same onco-myoepithelial cells. There may be Schwann cell differentiation in onco-myoepithelial cell of salivary ACC, and it may be the pathological base of perineural invasion in salivary ACC.

Nerve Growth Factor and Tyrosine Kinase A in Human Salivary Adenoid Cystic Carcinoma: Expression Patterns and Effects on In Vitro Invasive Behavior

Perineural invasion is a frequent occurrence in salivary adenoid cystic carcinoma (ACC) and may prevent complete surgical resection. Studies have indicated that nerve growth factor (NGF) and its high-affinity receptor tyrosine kinase A (TrkA) may play a role in perineural invasion in several malignancies in which perineural invasion is observed. The present study was conducted to investigate the expression of NGF and TrkA in salivary ACC and to examine the effects of NGF on adhesion, migration and invasion capacities of a salivary ACC cell line (SACC-83) in vitro. Expression of NGF and TrkA was explored using immunohistochemistry in paraffin-embedded tissues of 32 cases of salivary ACC. The effects of NGF on in vitro adhesion, migration, and invasion capacities of the SACC-83 cell line were examined using an MTT assay and a modified Boyden chamber assay respectively. In ACC specimens, 31 (96.9%) and 32 (100%) tumors showed immunoreactivity for NGF and TrkA respectively. Significant correlations were found between NGF/TrkA expression levels and perineural invasion (P < .05). In cell adhesion assay, the percent adherences of SACC-83 cells co-cultured with 25 ng/ml NGF at 1.5 hours and 5, 25 ng/ml NGF at 6 hours were significantly higher than that co-cultured with 0 ng/ml NGF (P < .05). However, high concentration of NGF (500 ng/ml) resulted in a significant inhibition of invasion (P < .05). Overexpression of NGF and TrkA in human salivary ACC tissues may constitute a reason for perineural invasion in salivary ACC.