Molecular Mechanism Of Perineural Invasion Research Articles

Molecular mechanisms of perineural invasion in pancreatic cancer

Molecular mechanisms of perineural invasion in pancreatic cancer

Cellular and molecular mechanisms of perineural invasion of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignant disease with a unique tumor microenvironment surrounded by an interlaced network of cancer and noncancerous cells. Recent works have revealed that the dynamic interaction between cancer cells and neuronal cells leads to perineural invasion (PNI), a clinical pathological feature of PDAC. The formation and function of PNI are dually regulated by molecular (e.g., involving neurotrophins, cytokines, chemokines, and neurotransmitters), metabolic (e.g., serine metabolism), and cellular mechanisms (e.g., involving Schwann cells, stromal cells, T cells, and macrophages). Such integrated mechanisms of PNI not only support tumor development, growth, invasion, and metastasis but also mediate the formation of pain, all of which are closely related to poor disease prognosis in PDAC. This review details the modulation, signaling pathways, detection, and clinical relevance of PNI and highlights the opportunities for further exploration that may benefit PDAC patients.

Identification of Genomic Alterations of Perineural Invasion in Patients with Stage II Colorectal Cancer.

PurposeThe molecular mechanism of perineural invasion (PNI) in stage II colorectal cancer (CRC) remains not to be defined clearly. This study aims to identify the genomic aberrations related to PNI in stage II CRC.Patients and MethodsUsing array-based comparative genomic hybridization (array-CGH), primary tumor tissues and paracancerous normal tissues of stage II CRC with PNI and without PNI were analyzed. We identified genomic aberrations by using Genomic Workbench and MD-SeeGH and validated the aberrations of selected genes by real-time polymerase chain reaction (PCR). Gene ontology (GO) and pathway analysis were performed to determine the most likely biological effects of these genes.ResultsThe most frequent gains in stage II CRC were at 7q11.21-q11.22, 8p11.21, 8p12-p11.23, 8q11.1-q11.22, 13q12.13-q12.2, and 20q11.21-q11.23 and the most frequent losses were at 17p13.1-p12, 8p23.2, and 118q11.2-q23. Four high-level amplifications at 8p11.23-p11.22, 18q21.1, 19q11-q12, and 20q11.21-q13.32 and homozygous deletions at 20p12.1 were discovered in Stage II CRC. Gains at 7q11.21-q22.1, 16p11.2, 17q23.3-q25.3, 19p13.3-p12, and 20p13-p11.1, and losses at 11q11-q12.1, 11p15.5-p15.1, 18p11.21, and 18q21.1-q23 were more commonly found in patients with PNI by frequency plot comparison together with detailed genomic analysis. It is also observed that gains at 8q11.1-q24.3, 9q13-q34.3, and 13q12.3-q13.1, and losses at 8p23.3-p12, 17p13.3-p11.2, and 21q22.12 occurred more frequently in patients without PNI. Further validation showed that the expression of FLT1, FBXW7, FGFR1, SLC20A2 and SERPINI1 was significantly up-regulated in the NPNI group compared to the PNI group. GO and pathway analysis revealed some genes enriched in specific pathways.ConclusionThese involved genomic changes in the PNI of stage II CRC may be useful to reveal the mechanisms underlying PNI and provide candidate biomarkers.

Establishment of a Convenient System for the Culture and Study of Perineurium Barrier In Vitro.

The perineurium serves as a selective, metabolically active diffusion barrier in the peripheral nervous system, which is composed of perineurial cells joined together by tight junctions (TJs). Not only are these junctions known to play an essential role in maintaining cellular polarity and tissue integrity, but also limit the paracellular diffusion of certain molecules and ions, whereas loss of TJs barrier function is imperative for tumour growth, invasion and metastasis. Hence, a detailed study on the barrier function of perineurial cells may provide insights into the molecular mechanism of perineural invasion (PNI). In this study, we aimed to develop an efficient procedure for the establishment of perineurial cell lines as a tool for investigating the physiology and pathophysiology of the peripheral nerve barriers. Herein, the isolation, expansion, characterization and maintenance of perineurial cell lines under favourable conditions are presented. Furthermore, the analysis of the phenotypic features of these perineurial cells as well as the barrier function for the study of PNI are described. Such techniques may provide a valuable means for the functional and molecular investigation of perineurial cells, and in particular may elucidate the pathogenesis and progression of PNI, and other peripheral nerve disorders.

Modulation of BDNF-TRKB Interactions on Schwann Cell-induced Oral Squamous Cell Carcinoma Dispersion In Vitro.

Perineural invasion (PNI) is a significant pathological feature in head and neck cancer. The molecular mechanisms of PNI are poorly understood. Contrary to the previous belief that cancer cells invade nerves, recent studies have shown that Schwann cells (SC) can dedifferentiate, intercalate between cancer cells, and promote cancer dispersion. Communication between cells through brain-derived neurotrophic factor (BDNF) activation of its receptor tropomyosin receptor kinase B (TRKB) may contribute to these cellular events. We aimed to determine the effect of TRKB inhibitor ANA-12 on the direction of cell migration and degree of SC-induced oral cancer cell dispersion. Cell migration and dispersion assays were performed in vitro using murine SC and oral carcinoma cell lines. Assays were performed with and without ANA-12. Although SCs preferentially migrated towards cancer cells in control medium, there was minimal SC-associated cancer cell dispersion. In contrast, treatment with ANA-12 reduced migration of SCs and cancer cells towards each other and initiated more SC-associated cancer cell dispersion. This pilot study shows that BDNF-TRKB signaling may have a role in regulating interactions between SC and oral cancer cells that affect cell migration, intercalation, and cancer cell dispersion. Further research into these interactions may provide important clues about the molecular and cellular mechanisms of PNI.

Pancreatic Stellate Cells Facilitate Perineural Invasion of Pancreatic Cancer via HGF/c-Met Pathway

Pancreatic cancer (PC) is a highly lethal cancer that has a strong ability for invasion and metastasis, poor prognosis, and a stubbornly high death rate due to late diagnosis and early metastasis. Therefore, a better understanding of the mechanisms of metastasis should provide novel opportunities for therapeutic purposes. As a route of metastasis in PC, perineural invasion (PNI) occurs frequently; however, the molecular mechanism of PNI is still poorly understood. In this study, we show that the hepatocyte growth factor (HGF)/c-Met pathway plays a vital role in the PNI of PC. We found that HGF promotes PC cell migration and invasion by activating the HGF/c-Met pathway, and enhances the expression of nerve growth factor (NGF) and matrix metalloproteinase-9 (MMP9) in vitro. Furthermore, HGF significantly increased PC cell invasion of the dorsal root ganglia (DRG) and promoted the outgrowth of DRG in cocultured models of PC cells and DRG. In contrast, the capacity for invasion and the phenomenon of PNI in PC cells were reduced when the HGF/c-Met pathway was blocked by siRNA. In conclusion, PSCs facilitate PC cell PNI via the HGF/c-Met pathway. Targeting the HGF/c-Met signaling pathway could be a promising therapeutic strategy for PC.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

Advances in the molecular mechanisms of perineural invasion in salivary adenoid cystic carcinoma

Salivary adenoid cystic carcinoma is a very common malignant neoplasm in the salivary glands. One of the most important features of salivary cystic carcinoma is perineural invasion, which renders its complete removal is very difficult through clinical surgery. Consequently, tumor recurrence occurs easily after clinical surgery. Therefore, the molecular mechanisms of perineural invasion in salivary adenoid cystic carcinoma are necessary to explore. These mechanisms are reviewed in this paper.

MMP1/PAR1/SP/NK1R paracrine loop modulates early perineural invasion of pancreatic cancer cells

The molecular mechanism of perineural invasion (PNI) is unclear, and insufficient detection during early-stage PNI in vivo hampers its investigation. We aimed to identify a cytokine paracrine loop between pancreatic ductal adenocarcinoma (PDAC) cells and nerves and established a noninvasive method to monitor PNI in vivo.Methods: A Matrigel/ dorsal root ganglia (DRG) system was used to observe PNI in vitro, and a murine sciatic nerve invasion model was established to examine PNI in vivo. PNI was assessed by MRI with iron oxide nanoparticle labeling. We searched publicly available datasets as well as obtained PDAC tissues from 30 patients to examine MMP1 expression in human tumor and non-tumor tissues.Results: Our results showed that matrix metalloproteinase-1 (MMP1) activated AKT and induced protease-activated receptor-1 (PAR1)-expressing DRG to release substance P (SP), which, in turn, activated neurokinin 1 receptor (NK1R)-expressing PDAC cells and enhanced cellular migration, invasion, and PNI via SP/NK1R/ERK. In animals, hind limb paralysis and a decreased hind paw width were observed approximately 20 days after inoculation of cancer cells in the perineurium. MMP1 silencing with shRNA or treatment with either a PAR1 or an NK1R antagonist inhibited PNI. MRI detected PNI as early as 10 days after implantation of PDAC cells. PNI also induced PDAC liver metastasis. Bioinformatic analyses and pathological studies on patient tissues corroborated the clinical relevance of these findings.Conclusion: In this study, we provided evidence that the MMP1/PAR1/SP/NK1R paracrine loop contributes to PNI during the early stage of primary tumor formation. Furthermore, we established a sensitive and non-invasive method to detect nerve invasion using iron oxide nanoparticles and MRI.

Cancer's got nerve: Schwann cells drive perineural invasion.

The invasion of cancer cells around and into nerves is associated with increased cancer aggression and poor patient outcome. As this perineural invasion increases disease severity, a better understanding of how the process is regulated may help in the development of therapeutics to target neuronal involvement in cancer. In this issue of the JCI, Deborde and colleagues show that direct contact between Schwann cells and cancer cells promotes cancer cell dissociation, migration, and invasion. Moreover, their data specifically suggest NCAM1 as an important molecular mediator of this Schwann cell-directed regulation of cancer cells in perineural invasion. The results of this study provide new insight into the cellular and molecular mechanisms of perineural invasion.

Nerve Growth Factor Expression Is Not Associated with Perineural Invasion in Extrahepatic Cholangiocarcinoma.

Although the presence of perineural invasion has been recognized as a poor prognostic factor in extrahepatic cholangiocarcinoma, the molecular mechanisms of perineural invasion in extrahepatic cholangiocarcinoma remain unclear. Nerve growth factor has been reported to be a candidate predictive biomarker of perineural invasion in some cancers. To investigate the impact of intratumoral nerve growth factor expression in resected extrahepatic cholangiocarcinoma on survival. Intratumoral nerve growth factor expression was investigated immunohistochemically in 112 patients with resected extrahepatic cholangiocarcinoma. Associations between nerve growth factor expression and clinicopathological factors were statistically evaluated, and risk factors for poor survival were analyzed using univariate and multivariate analyses. High and low nerve growth factor expression was observed in 62 (55%) and 50 (45%) patients, respectively. For all 112 patients, no significant correlation was found between nerve growth factor expression and presence of perineural invasion (P = 0.942). Moreover, nerve growth factor expression was not associated with recurrence-free survival (P = 0.861) and overall survival (P = 0.973). In multivariate analysis, lymph node metastasis (P = 0.004) was identified as an independent risk factor for early recurrence and the presence of perineural invasion (P = 0.002) and lymph node metastasis (P < 0.001) was identified as independent risk factors for poor survival. Intratumoral nerve growth factor expression is not associated with perineural invasion or recurrence-free and overall survival in patients with resected extrahepatic cholangiocarcinoma.

Schwann cells promote EMT and the Schwann-like differentiation of salivary adenoid cystic carcinoma cells via the BDNF/TrkB axis.

Perineural invasion (PNI) is a striking biological behavior observed in salivary adenoid cystic carcinoma (SACC). The present study was designed to establish a co-culture model of SACC cells with Schwann cells (SCs), and then study epithelial-mesenchymal transition (EMT) and the Schwann-like differentiation of SACC cells to investigate the likely molecular mechanism of PNI. The co-culture models of SCs with tumor cells (SACC-83, SACC-LM and MEC-1) were established using a Transwell system. An elevated concentration of brain-derived neurotrophic factor (BDNF) was detected by ELISA assay in the co-cultured medium of the SACC-83 group and SACC-LM group rather than the MEC-1 group. The EMT process and Schwann-like differentiation in SACC-83 cells were analyzed by RT-PCR, western blotting, immunofluorescence, photography, and migration and perineural invasion assays. The SACC-83 cells under the co-culture condition with SCs changed to a mesenchymal morphology and had higher migration and invasion capabilities compared with the solely cultured SACC-83 cells, accompanied by the downregulation of E-cadherin and upregulation of N-cadherin and vimentin. The co-cultured SACC-83 cells also developed Schwann-like differentiation with increased expression of SC markers, S100A4 and GFAP. However, inhibition of tropomyosin-related kinase B (TrkB) by K252a markedly blocked these effects. Additionally, the expression and correlation of TrkB, E-cadherin and S100A4 were analyzed by immunohistochemistry in 187 primary SACC cases. The levels of TrkB and S100A4 expression were both positively associated with PNI in the SACC cases, while E-cadherin expression was negatively associated with PNI. Elevated expression of TrkB was significantly correlated with the downregulated expression of E-cadherin and the upregulated expression of S100A4 in the SACC cases. Our results suggest that SCs play a pivotal role in the PNI process by inducing the EMT process and the Schwann-like differentiation of SACC cells via the BDNF/TrkB axis. Interruption of the interreaction between SACC cells and SCs by targeting the BDNF/TrkB axis may be a potential strategy for anti-PNI therapy in SACC.

Interaction Between Gastric Carcinoma Cells and Neural Cells Promotes Perineural Invasion by a Pathway Involving VCAM1.

Perineural invasion (PNI) is one of the important routes for local spread of gastric carcinoma associated with poor prognosis. However, the exact cellular characteristics and molecular mechanisms of PNI are still unclear. To identify the interaction between gastric carcinoma cells and neural cells, and whether vascular cell adhesion molecule-1 (VCAM1) is involved in this process. We adopted in vitro cell coculture assays to investigate the cellular and molecular interaction between gastric cancer cells and neural cells. We find upregulation of VCAM1 in clinical gastric cancer tissue samples. In in vitro tumor-neural cell coculture system, gastric cancer cells with high level of VCAM1 promote proliferation of neural progenitor cells and induce the process outgrowth and branching of neural cells. Reciprocally, neural cells enhance neurotropic migration and mobility of tumor cells. Repressing VCAM1 function through VCAM1 blocking antibody can attenuate these effects. Our study indicates that VCAM1 is significantly involved in tumor invasion via mediating nerve-tumor interaction, which is a mutually beneficial process. It is possible that interaction between neural cells and tumor cells might contribute to PNI of gastric carcinoma. Inhibiting the activity of VCAM1 could be a potential strategy targeting PNI in gastric carcinoma therapy.

Cellular and molecular mechanisms of perineural Invasion in gastric cancer

Perineural invasion(PNI) is one of the important routes for metastasis of gastric carcinoma and results in local recurrence and cancer pain after radical gastrectomy. The dismal prognosis of gastric cancer has been intimately associated with lymph node metastasis, hematogenous metastasis that partly caused by PNI. Nerve, vascular and lymphatic constitutes the tumor microenvironment which plays a decisive role in the development of cancer. Molecular interaction and morphological change contribute to reciprocal signaling interactions between tumor cell and nerve. However, the underlying mechanism of PNI in gastric cancer is still unclear and needs further study. Here, we present a brief review of literatures on the topic of PNI in gastric cancer.

Perineural invasion in pancreatic cancer: Advanced research in the neuro-cancer interactions

Pancreatic Cancer (PCa) is characterized by prominently local nerve alterations and perineural invasion (PNI), which frequently aff ects the extrapancreatic nerve plexus, causing severe pain and retropancreatic tumor extension. It precludes curative resection, promotes local recurrence, and at the last negatively influences the prognosis of patients. Recent research on PNI in PCa has revealed the critical involvement of numerous nerve- or cancer cell-derived molecules in vitro and in vivo. However, the mechanisms contributing to alteration and invasion of intrapancreatic nerves and the spread of cancer cells along extrapancreatic nerves in pancreatic cancer patients are still poorly understood. This review focuses on perineural invasion in pancreatic cancer and provides an outline of the characteristics and molecular mechanisms of perineural invasion in pancreatic cancer.

Molecular mechanisms of perineural invasion, a forgotten pathway of dissemination and metastasis

Invasion and metastasis are key components of cancer progression. Inflammatory mediators, including cytokines and chemokines, can facilitate tumor dissemination. A distinct and largely forgotten path is perineural invasion (PNI), defined as the presence of cancer cells in the perinerium space. PNI is frequently used by many human carcinomas, in particular by pancreas and prostate cancer, and is associated with tumor recurrence and pain in advanced patients. Neurotrophic factors have been identified as molecular determinants of PNI. A role for chemokines in this process has been proposed; the chemokine CX3CL1/Fractalkine attracts receptor positive pancreatic tumor cells to disseminate along peripheral nerves. Better understanding of the neurotropism of malignant cells and of the clinical significance of PNI would help the design of innovative strategies for the control of tumor dissemination and pain in cancer patients.

Synuclein-γ Is Closely Involved in Perineural Invasion and Distant Metastasis in Mouse Models and Is a Novel Prognostic Factor in Pancreatic Cancer

Perineural invasion is associated with the high incidence of local recurrence and a dismal prognosis in pancreatic cancer. We previously reported a novel perineural invasion model and distinguished high- and low-perineural invasion groups in pancreatic cancer cell lines. This study aimed to elucidate the molecular mechanism of perineural invasion. To identify key biological markers involved in perineural invasion, differentially expressed molecules were investigated by proteomics and transcriptomics. Synuclein-gamma emerged as the only up-regulated molecule in high-perineural invasion group by both analyses. The clinical significance and the biological property of synuclein-gamma were examined in 62 resected cases of pancreatic cancer and mouse models. Synuclein-gamma overexpression was observed in 38 (61%) cases and correlated with major invasive parameters, including perineural invasion and lymph node metastasis (P < 0.05). Multivariate analyses revealed synuclein-gamma overexpression as the only independent predictor of diminished overall survival [hazard ratio, 3.4 (95% confidence interval, 1.51-7.51)] and the strongest negative indicator of disease-free survival [2.8 (1.26-6.02)]. In mouse perineural invasion and orthotopic transplantation models, stable synuclein-gamma suppression by short hairpin RNA significantly reduced the incidence of perineural invasion (P = 0.009) and liver/lymph node metastasis (P = 0.019 and P = 0.020, respectively) compared with the control. This is the first study to provide in vivo evidence that synuclein-gamma is closely involved in perineural invasion/distant metastasis and is a significant prognostic factor in pancreatic cancer. Synuclein-gamma may serve as a promising molecular target of early diagnosis and anticancer therapy.

An Experimental Study of Salivary Tumor Models in Mice

Adenoid cystic carcinoma (ACC) is a relatively rare tumor that arises in glandular tissues of the head and neck region and sometimes has a protracted clinical course with perineural invasion and delayed occurrence of distant lung metastases. Treatment failure of salivary ACC is most often associated with perineural and hematogenous tumor spread. However, very little is known about the cellular and molecular mechanisms of perineural invasion and hematogenous distant metastasis. This study was designed to develop an orthotopic tumor model of parotid adenoid cystic carcinomas in athymic nude mice. A melanoma cell line was injected into the parotid gland of athymic mice to determine whether such implantation was technically feasible. The parotid ACC cell line was then injected into the parotid gland or the subcutaneous tissue of athymic mice at various concentrations of tumor cells, and the mice were thereafter followed for development of tumor nodules. The tumors were examined histopathologically for perineural invasion and regional or distant lung metastasis. We used an oral squamous cell carcinoma cell line as a control. Dr Jeffrey N. Myers (Department of Head and Neck surgery, University of Texas M.D. Anderson Cancer Center) kindly donated all cell lines. For this study, the exact logistic regression model was used to assess tumorigenicity and the effects of orthotopic (parotid) versus ectopic (subcutis) tumor development and metastases. The Ps for the location effect were evaluated against an a significance level of 0.05. n/a Implantation of tumor (melanoma) cell suspension into the parotid gland of nude mice was technically feasible and resulted in the formation of parotid tumors. A parotid ACC cell line, ACC3 did not demonstrate significantly higher tumorigenicity, but showed significantly higher lung metastatic potential in the parotid gland compared to the subcutis. By contrast, the mucosal squamous cell carcinoma cell line did not show significantly higher lung metastatic potential compared the subcutis. Perineural ACC tumor, from cells established in the parotid gland, needs further study. An orthotopic tumor model of salivary ACC in athymic nude mice was successfully developed that closely mimics the clinical findings in human salivary ACC. This model should facilitate improved understanding of the cellular and molecular mechanisms of tumorigenisis and metastasis of salivary ACC and aid in the development of targeted molecular therapies for salivary ACC.

Establishment of Perineural Invasion Models and Analysis of Gene Expression Revealed an Invariant Chain (CD74) as a Possible Molecule Involved in Perineural Invasion in Pancreatic Cancer

Perineural invasion causes frequent local recurrence even after resection and a poor prognosis for pancreatic cancer. We established perineural invasion models and analyzed the molecular mechanism of perineural invasion in pancreatic cancer. Seven pancreatic cancer cell lines with or without human peripheral nerves were s.c. implanted in nonobese diabetes/severe combined immunodeficient mice. We compared expression profiles among high and low perineural invasion cell lines by using an oligonucleotide microarray. We examined up-regulation of the invariant chain (CD74) in high perineural invasion cell lines in mRNA and protein levels and surgical cases immunohistochemically. Four of seven pancreatic cancer cell lines (CaPan1, CaPan2, CFPAC, and MPanc96) showed perineural invasion to s.c. transplanted human peripheral nerves. Moreover, CaPan1 and CaPan2 (high perineural invasion group) also resulted in a high frequency of perineural invasion to mouse s.c. peripheral nerves, whereas three pancreatic cancer cell lines HPAFII, AsPC1, and Panc1 (low perineural invasion group) did not show perineural invasion to either human or mouse nerves. We identified 37 up-regulated genes and 12 down-regulated genes in the high perineural invasion group compared with the low perineural invasion group. Among them, CD74 was up-regulated in the high perineural invasion group in mRNA and protein levels. Furthermore, immunohistochemical expression of CD74 in clinical cases revealed its significant overexpression in pancreatic cancer with perineural invasion (P < 0.008). This is the first report of perineural invasion models using human pancreatic cancer cell lines. In combination with gene expression profiling, it was indicated that CD74 could be a candidate molecule involved in perineural invasion. These models provide new approaches for study of perineural invasion in pancreatic cancer.

唾液腺腺様嚢胞癌の増殖・進展メカニズムに関する研究―ＮＣＡＭの発現とその機能について―

Neural cell adhesion molecule (NCAM) is one cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant glandular tumors, such as bile duct carcinoma or gallbladder carcinoma. However, it remains unclear whether NCAM is really associated with the development of salivary gland tumors. Previous studies have demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG in vitro. In this study, molecular mechanisms of perineural invasion were examined using HSG cells in which NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined immunohistochemically. The levels of NCAM mRNA and protein expression were found to decrease in a dose-dependent manner on treatment with anti-NCAM antibody. The MTT assay revealed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells undergo apoptosis in response to treatment with anti-NCAM antibody. The activation of caspases 3, 7, and 9 was observed in HSG cells after treatment with anti-NCAM antibody, thus confirming the induction of activated caspase-mediated apoptosis. The up-regulation of TGF-β1-mediated NCAM expression seemed to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. Immunohistochemical examinations revealed that NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC.These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism but also with tumorigenesis, including growth, development, and perineural invasion in human ACCs.