Asparaginyl Β-hydroxylase Research Articles

Safety/efficacy of a phase I/II study of SNS-301 added to pembrolizumab in patients with ASPH+ locally advanced unresectable or metastatic/recurrent squamous cell carcinoma of the head and neck (SCCHN).

e18510 Background: SNS-301 is a first-in-class immune activating agent targeting human aspartyl (asparaginyl) β-hydroxylase (ASPH). ASPH is overexpressed in +20 tumor types. SNS-301 is an inactivated λ-bacteriophage, engineered to express a highly immunogenic fragment of ASPH fused to the phage gpD coat protein, previously shown to safely and effectively generate CD4, CD8 and B cell responses in a Phase I study (NCT04034225). Anti-PD-1 antibodies such as pembrolizumab and nivolumab can induce durable clinical responses in 13-16% SCCHN patients. While ASPH is frequently expressed in SCCHN, effective de novo responses to this tumor-specific antigen may be limited due to immune tolerance. Our hypothesis is that SNS-301 will break tolerance and trigger a significant anti-ASPH T-cell immune response, which in combination with anti-PD-1 blockade, will result in improved anti-tumor responses in this difficult-to-treat patient population. Methods: This is a Phase I/II study of SNS-301 delivered intradermally in combination with pembrolizumab. The population consists of 30 patients with ASPH+ locally advanced unresectable or metastatic/recurrent SCCHN who are actively receiving pembrolizumab or nivolumab for ≥12 weeks and have a best response of stable disease (SD) or first evidence of progressive disease (PD). A safety run-in will be performed for safety and any dose limiting toxicities (DLT) using a modified rolling six design prior to proceeding to full enrollment. Patients will provide a pre-SNS-301 tumor sample, an on-treatment tumor biopsy and an optional biopsy at PD to characterize the tumor microenvironment and correlate with clinical outcomes. SNS-301 in combination with pembrolizumab is given until confirmed PD or unacceptable toxicity. Study Objectives: Safety, tolerability and anti-tumor activity such as objective response rate, duration of response, progression-free and overall survival, immune responses, tumor/immune biomarkers and their associated treatment outcome. Results: Three patients have been enrolled. There have been no DLTs. Emerging safety data includes G1 adverse events with one G1 injection site pain. Patient status includes 1 PD and 2 patients with SD of at least six weeks duration. Immunological analyses will be presented. Conclusions: Early safety/efficacy warrant further evaluation of this combination therapy. Trial will be proceeding to full enrollment after the safety run-in is completed. Clinical trial information: NCT04034225.

Early diagnosis of prostate cancer, AABH (Aspartyl Asparaginyl β-hydroxylase), a predictive biomarker: A serum immunoassay and personalized medicine.

353 Background: Prostate Cancer (PC) is the most common form of cancer in men in the United States. In their lifetime, approximately 1 out of every 6 American men will develop PC. The chances of developing PC increase dramatically after age 50 with more than 70% of cases occurring in individuals over 65 years of age. Methods: We have investigated the use of aspartyl (asparaginyl) β-hydroxylase (AABH) as a cancer biomarker. We have developed a sandwich ELISA for its detection. Here this assay was utilized to screen sera from patients with prostate cancer and PSA values in various ranges. Results: AABH levels were assessed in sera from 263 individuals with PC and 73 men over 50 years of age not known to have cancer. The levels of AABH in the two groups were >3.00 ng/ml and <3.00 ng/ml, respectively. The overall sensitivity and specificity of the test were 97.5% and 96%. AABH and PSA levels were also compared. The AABH values and sensitivities in individuals with PC and PSA values in the ranges <2 ng/ml, 2-4 ng/ml and >4 ng/ml were >3.00 ng/ml, 99% (n=115); > 3.00 ng/ml, 98% (n=64) and >3.00 ng/ml, 95% (n=114). Conclusions: The present study of AABH indicates that cancer group can be clearly distinguished from normal healthy controls. Moreover, the study indicates that AABH might be used to identify disease in individuals with questionable PSA values. These results support to the establishment of routine screening of serum AABH as a means to detect those who need referral for prostate biopsy. The use of AABH testing may enhance current prostate cancer screening practices by both identifying individuals with low but suspicious PSA values who should be recommended for biopsy and reducing the number of false positive biopsies indicated by positive PSA tests. Serum AABH levels are significantly elevated in individuals with prostate cancer. Using a cutoff value of 3ng/ml, overall sensitivity of the assay for prostate cancer is 97%. The test has a specificity of 96% in healthy men over 50 years of age. AABH is elevated in prostate cancer tissue but NOT in normal prostate or in Benign Prostatic Hyperplasia (BPH). Serum AABH levels are NOT correlate with PSA levels.

Research progress of aspartyl-(asparaginyl) β-hydroxylase in tumors

Aspartyl-(asparaginyl) β-hydroxylase (AAH) is a type 2 transmembrane protein and a member of the α-ketoglutarate-dependent dioxygenase family of proteins. AAH catalyzes hydroxylation of specific aspartyl and asparaginyl residues in epidermal growth factor-like domains of proteins. Overexpression of AAH is found in many types of cancer tissues, however, AAH expression is found only in normal placenta and paranephros tissues. AAH can translocate to cell membrane from endoplasmic reticulum membrane when it is overexpressed in cancer cell, which hydroxylate proteins such as Notch, Jagged, and cellular adhesion molecules that regulate cell migration and invasion. AAH is a potential biomarker for cancer diagnosis. Key words: Aspartyl-(asparaginyl) β-hydroxylase; Neoplasms; Diagnosis

Potential Role of Phosphorylation as a Regulator of Aspartyl-(asparaginyl)-β-hydroxylase: Relevance to Infiltrative Spread of Human Hepatocellular Carcinoma

Abundant expression of aspartyl-(asparaginyl)-β-hydroxylase (AAH) correlates with infiltrative growth of hepatocellular carcinoma (HCC). Herein, we examine the role of phosphorylation in relation to AAH's protein expression, hydroxylase activity, promotion of cell motility, and activation of Notch signaling in human Huh7 hepatoma cells. Predicted glycogen synthase kinase-3β (GSK-3β) , protein kinase A (PKA), protein kinase C (PKC), and casein kinase 2 (CK2) phosphorylation sites encoded by human AAH cDNA were ablated by S/T→A site-directed mutagenesis using N-Myc-tagged constructs in which gene expression was controlled by a cytomegalovirus promoter. Functional consequences were assessed in transiently transfected Huh7 cells. Cells transfected with wildtype AAH had significantly increased AAH expression, catalytic activity, HES-1 expression, and directional motility relative to controls. Single phosphorylation site mutations in the C-terminus largely abrogated these effects and further inhibited catalytic activity relative to that in cells transfected with empty vector, whereas the effects of single point mutations within the N-terminus were more varied. In contrast, AAH cDNAs carrying multiple phosphorylation site mutations exhibited wildtype levels of AAH catalytic activity suggesting that the effects of AAH phosphorylation are complex and non-uniform. AAH expression and function can be modulated by direct phosphorylation of the protein. These findings suggest additional strategies for inhibiting infiltrative growth of HCC.

The aspartyl (asparaginyl) beta-hydroxylase in carcinomas.

Aspartyl-(asparaginyl)-β-hydroxylase (AAH) is a member of the α-ketoglutarate-dependent dioxygenase family that catalyzes the hydroxylation of aspartyl and asparaginyl residues epidermal growth factor (EGF)-like domains of protein. In human tumorous cell lines from main systems of body, including tumor cells of kidney, throat, breast, liver, bladder, cervical and ovary, the AAH can be detected at both the transcriptional level and the translational level, and moreover, the AAH expression is usually increased, which is associated with the development and progression of carcinomas. Thus, AAH may play an important role in different carcinomas and may be a potential hub in carcinogenesis. In this review, we will discuss the role of AAH in carcinomas, focusing on liver cancers and other digestive tumors, lung cancers, and tumors of nervous system.

A cell-surface β-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a poor prognosis as a result of widespread intra- and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(asparaginyl)-β-hydroxylase (ASPH) is a cell-surface enzyme that generates enhanced cell motility, migration, invasion, and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer-assisted drug design. Candidate compounds were tested for inhibition of β-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion, and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biological effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor, MO-I-1100, was selected because it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion, and anchorage-independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. These studies suggest that the enzymatic activity of ASPH is important for hepatic oncogenesis. Reduced β-hydroxylase activity generated by the SMI MO-I-1100 leads to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease.

Role of aspartyl-(asparaginyl)-β-hydroxylase mediated notch signaling in cerebellar development and function

BackgroundAspartyl-(Asparaginyl)-β-Hydroxylase (AAH) is a hydroxylating enzyme that promotes cell motility by enhancing Notch-Jagged-HES-1 signaling. Ethanol impaired cerebellar neuron migration during development is associated with reduced expression of AAH.MethodsTo further characterize the role of AAH in relation to cerebellar development, structure, and function, we utilized an in vivo model of early postnatal (P2) intracerebro-ventricular gene delivery to silence AAH with small interfering RNA (siAAH), or over-express it with recombinant plasmid DNA (pAAH). On P20, we assessed cerebellar motor function by rotarod testing. Cerebella harvested on P21 were used to measure AAH, genes/proteins that mediate AAH's downstream signaling, i.e. Notch-1, Jagged-1, and HES-1, and immunoreactivity corresponding to neuronal and glial elements.ResultsThe findings demonstrated that: 1) siAAH transfection impaired motor performance and blunted cerebellar foliation, and decreased expression of neuronal and glial specific genes; 2) pAAH transfection enhanced motor performance and increased expression of neuronal and glial cytoskeletal proteins; and 3) alterations in AAH expression produced similar shifts in Notch-1, Jagged-1, and HES-1 protein or gene expression.ConclusionsThe results support our hypothesis that AAH is an important mediator of cerebellar development and function, and link AAH expression to Notch signaling pathways in the developing brain.

Glial Vascular Degeneration in CADASIL

CADASIL is a genetic vascular dementia caused by mutations in the Notch 3 gene on Chromosome 19. However, little is known about the mechanisms of vascular degeneration. We characterized upstream components of Notch signaling pathways that may be disrupted in CADASIL, by measuring expression of insulin, IGF-1, and IGF-2 receptors, Notch 1, Notch 3, and aspartyl-(asparaginyl)-β-hydroxylase (AAH) in cortex and white matter from 3 CADASIL and 6 control brains. We assessed CADASIL-associated cell loss by measuring mRNA corresponding to neurons, oligodendroglia, and astrocytes, and indices of vascular degeneration by measuring smooth muscle actin (SMA) and endothelin-1 expression in isolated vessels. Immunohistochemical staining was used to assess SMA degeneration. Significant abnormalities, including reduced cerebral white matter mRNA levels of Notch 1, Notch 3, AAH, SMA, IGF receptors, myelin-associated glycoproteins, and glial fibrillary acidic protein, and reduced vascular expression of SMA, IGF receptors, Notch 1, and Notch 3 were detected in CADASIL-lesioned brains. In addition, we found CADASIL-associated reductions in SMA, and increases in ubiquitin immunoreactivity in the media of white matter and meningeal vessels. No abnormalities in gene expression or immunoreactivity were observed in CADASIL cerebral cortex. In conclusion, molecular abnormalities in CADASIL are largely restricted to white matter and white matter vessels, corresponding to the distribution of neuropathological lesions. These preliminary findings suggest that CADASIL is mediated by both glial and vascular degeneration with reduced expression of IGF receptors and AAH, which regulate Notch expression and function.

Aspartyl‐(asparaginyl) β‐Hydroxylase, Hypoxia‐Inducible Factor‐1α and Notch Cross‐Talk in Regulating Neuronal Motility

Aspartyl-(Asparaginyl)-β-Hydroxylase (AAH ) promotes cell motility by hydroxylating Notch. Insulin and insulin-like growth factor, type 1 (IGF-I) stimulate AAH through Erk MAP K and phosphoinositol-3-kinase-Akt (PI3K-Akt). However, hypoxia/oxidative stress may also regulate AAH . Hypoxia-inducible factor-1alpha (HIF-1α) regulates cell migration, signals through Notch, and is regulated by hypoxia/oxidative stress, insulin/IGF signaling and factor inhibiting HIF-1α (FIH) hydroxylation. To examine cross-talk between HIF-1α and AAH , we measured AAH , Notch-1, Jagged-1, FIH, HIF-1α, HIF-1β and the hairy and enhancer of split 1 (HE S-1) transcription factor expression and directional motility in primitive neuroectodermal tumor 2 (PNET2) human neuronal cells that were exposed to H2O2 or transfected with short interfering RNA duplexes (siRNA) targeting AAH , Notch-1 or HIF-1α. We found that: (1) AAH , HIF-1α and neuronal migration were stimulated by H2O2; (2) si-HIF-1α reduced AAH expression and cell motility; (3) si-AAH inhibited Notch and cell migration, but not HIF-1α and (4) si-Notch-1 increased FIH and inhibited HIF-1α. These findings suggest that AAH and HIF-1α crosstalk within a hydroxylation-regulated signaling pathway that may be transiently driven by oxidative stress and chronically regulated by insulin/IGF signaling.

Ethanol inhibition of aspartyl-asparaginyl-β-hydroxylase in fetal alcohol spectrum disorder: Potential link to the impairments in central nervous system neuronal migration

Fetal alcohol spectrum disorder (FASD) is caused by prenatal exposure to alcohol and associated with hypoplasia and impaired neuronal migration in the cerebellum. Neuronal survival and motility are stimulated by insulin and insulin-like growth factor (IGF), whose signaling pathways are major targets of ethanol neurotoxicity. To better understand the mechanisms of ethanol-impaired neuronal migration during development, we examined the effects of chronic gestational exposure to ethanol on aspartyl (asparaginyl)-β-hydroxylase (AAH) expression, because AAH is regulated by insulin/IGF and mediates neuronal motility. Pregnant Long–Evans rats were pair-fed isocaloric liquid diets containing 0, 8, 18, 26, or 37% ethanol by caloric content from gestation day 6 through delivery. Cerebella harvested from postnatal day 1 pups were used to examine AAH expression in tissue, and neuronal motility in Boyden chamber assays. We also used cerebellar neuron cultures to examine the effects of ethanol on insulin/IGF–stimulated AAH expression, and assess the role of GSK-3β–mediated phosphorylation on AAH protein levels. Chronic gestational exposure to ethanol caused dose-dependent impairments in neuronal migration and corresponding reductions in AAH protein expression in developing cerebella. In addition, prenatal ethanol exposure inhibited insulin and IGF-I–stimulated directional motility in isolated cerebellar granule neurons. Ethanol-treated neuronal cultures (50 mM × 96 h) also had reduced levels of AAH protein. Mechanistically, we showed that AAH protein could be phosphorylated on Ser residues by GSK-3β, and that chemical inhibition of GSK-3β and/or global Caspases increases AAH protein in both control- and ethanol-exposed cells. Ethanol-impaired neuronal migration in FASD is associated with reduced AAH expression. Because ethanol increases the activities of both GSK-3β and Caspases, the inhibitory effect of ethanol on neuronal migration could be mediated by increased GSK-3β phosphorylation and Caspase degradation of AAH protein.

Impaired Placentation in Fetal Alcohol Syndrome

Intrauterine growth restriction (IUGR) is one of the key features of fetal alcohol syndrome (FAS), and IUGR can be mediated by impaired placentation. Insulin-like growth factors (IGF) regulate placentation due to stimulatory effects on extravillous trophoblasts, which are highly motile and invasive. Previous studies demonstrated that extravillous trophoblasts express high levels of aspartyl-(asparaginyl) β-hydroxylase (AAH), a gene that is regulated by IGF and has a critical role in cell motility and invasion. The present study examines the hypothesis that ethanol impaired placentation is associated with inhibition of AAH expression in trophoblasts. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0% or 37% ethanol by caloric content. Placentas harvested on gestation day 16 were used for histopathological, mRNA, and protein studies to examine AAH expression in relation to the integrity of placentation and ethanol exposure. Chronic ethanol feeding prevented or impaired the physiological conversion of uterine vessels required for expansion of maternal circulation into placenta, a crucial process for adequate placentation. Real-time quantitative RT-PCR analysis demonstrated significant reductions in IRS-1, IRS-2, and significant increases in IGF-II and IGF-II receptor mRNA levels in ethanol-exposed placentas. These abnormalities were associated with significantly reduced levels of AAH expression in trophoblastic cells, particularly within the mesometrial triangle (deep placental bed) as demonstrated by real time quantitative RT-PCR, Western blot analysis, ELISA, and immunohistochemical staining. Ethanol-impaired placentation is associated with inhibition of AAH expression in trophoblasts. This effect of chronic gestational exposure to ethanol may contribute to IUGR in FAS.

Differential growth factor regulation of aspartyl-(asparaginyl)-β-hydroxylase family genes in SH-Sy5y human neuroblastoma cells

BackgroundAspartyl (asparaginyl)-β-hydroxylase (AAH) hydroxylates Asp and Asn residues within EGF-like domains of Notch and Jagged, which mediate cell motility and differentiation. This study examines the expression, regulation and function of AAH, and its related transcripts, Humbug and Junctin, which lack catalytic domains, using SH-Sy5y neuroblastoma cells.ResultsReal time quantitative RT-PCR demonstrated 8- or 9-fold higher levels of Humbug than AAH and Junctin, and lower levels of all 3 transcripts in normal human brains compared with neuroblastic tumor cells. AAH and Humbug expression were significantly increased in response to insulin and IGF-I stimulation, and these effects were associated with increased directional motility. However, over-expression of AAH and not Humbug significantly increased motility. Treatment with chemical inhibitors of Akt, Erk MAPK, or cyclin-dependent kinase 5 (Cdk-5) significantly reduced IGF-I stimulated AAH and Humbug expression and motility relative to vehicle-treated control cells. In addition, significantly increased AAH and Humbug expression and directional motility were observed in cells co-transfected with Cdk-5 plus its p35 or p25 regulatory partner. Further studies demonstrated that activated Cdk-5 mediated its stimulatory effects on AAH through Erk MAPK and PI3 kinase.ConclusionAAH and Humbug are over-expressed in SH-Sy5y neuroblastoma cells, and their mRNAs are regulated by insulin/IGF-1 signaling through Erk MAPK, PI3 kinase-Akt, and Cdk-5, which are known mediators of cell migration. Although AAH and Humbug share regulatory signaling pathways, AAH and not Humbug mediates directional motility in SH-Sy5y neuroblastoma cells.

Ethanol Reduces Aspartyl‐(asparaginyl)‐β‐hydroxylase Protein Stability Through Increased GSK‐3β Activity: Relevance to Impaired Neuronal Migration in the Fetal Alcohol Syndrome

Gestational exposure to ethanol causes fetal alcohol syndrome, which is associated with impaired neuronal migration in the central nervous system. Previous studies linked expression of aspartyl-(asparaginyl)-β-hydroxylase (AAH) to cell motility and showed that AAH was regulated by insulin and IGF-I stimulation. The present work examines the effects of ethanol on neuronal migration in the context of AAH expression. In an in vivo rat model of gestational ethanol exposure, and an in vitro culture model using human SH-Sy5y cells, impaired neuronal migration was associated with reduced levels of AAH protein expression by Western blot analysis and confocal microscopy. However, there were no associated reductions in AAH mRNA abundance, suggesting the effect was mediated at a post-transcriptional level. Since AAH protein has multiple GSK3β, CK2, and PKA phosphorylation sites, and ethanol increases both GSK3β and PKA activities, we examined the effects of ethanol on AAH protein phosphorylation. Immunoprecipitation/Western blot analysis demonstrated increased AAH phosphorylation in ethanol-treated cells, and increased AAH expression and motility in cells treated with chemical inhibitors of CK2, PKA, or GSK3β. These results suggest that ethanol impaired neuronal migration during development is mediated in part by GSK3β, and possibly PKA and CK2 phosphorylation followed by accelerated degradation of AAH protein in the brain. NIH # T32-DK060415

Clinicopathological correlates of aspartyl (asparaginyl) β-hydroxylase over-expression in cholangiocarcinoma

Aspartyl (asparaginyl) β-hydroxylase (AAH) expression in surgically resected intrahepatic cholangiocarcinoma significantly correlated with tumor size, growth type, differentiation, vascular invasion, and prognosis after surgery. AAH may have a role in regulating invasive or metastatic tumor cell growth of human intrahepatic cholangiocarcinoma. Background: Recent studies demonstrated increased expression of the AAH gene in the majority of cholangiocarcinomas. The present study was undertaken to determine the relationship between high or low levels of AAH expression and the clinical course of intrahepatic cholangiocarcinoma (ICC). Methods: AAH expression was examined in 50 surgically resected primary ICCs, 12 samples of normal liver, and 12 cases of primary sclerosing cholangitis (PSC). The sections were evaluated by immunohistochemical staining with the FB-50 monoclonal antibody to human AAH protein. The sections were examined under code and graded for relative levels of AAH immunoreactivity. The results were analyzed with respect to multiple clinical and histopathological variables to determine correlates of AAH expression in ICCs. Results: Forty-six of the 50 (92%) ICCs had AAH immunoreactivity, whereas the 12 normal liver and 12 PSC specimens were AAH negative. In the ICC specimens, the highest levels of AAH immunoreactivity were detected at the infiltrating margins that interfaced with uninvolved liver tissue, and the lowest levels occurred in the central portions of the tumors. Multivariate analysis demonstrated that high levels of AAH expression were correlated with tumor size ( P < 0.05), infiltrative growth pattern ( P < 0.01), aggressive histological grade ( P < 0.01), vascular invasion (P < 0.05), and poor prognosis (P < 0.05). Conclusions: These findings suggest that AAH has an important role in regulating invasive or metastatic tumor cell growth of human ICC, and that high levels of AAH expression correlate with poor prognosis.

Antisense oligodeoxynucleotides directed against aspartyl (asparaginyl) β-hydroxylase suppress migration of cholangiocarcinoma cells

Background: Aspartyl (asparaginyl) β-hydroxylase (AAH) is an α-ketoglutarate-dependent dioxygenase that hydroxylates aspartate and asparagine residues in EGF-like domains of proteins. The consensus sequence for AAH β-hydroxylation occurs in signaling molecules such as Notch and Notch homologs, which have roles in cell migration. Aim: This study evaluated the potential role of AAH in cell migration using cholangiocarcinoma cell lines as models due to their tendency to widely infiltrate the liver. Methods: Five human cholangiocarcinoma cell lines established from human tumors were examined for AAH expression and motility. The effect of antisense oligodeoxynucleotide inhibition of AAH on cholangiocarcinoma cell migration was investigated. Results: Western blot analysis detected the ∼86 kDa AAH protein in all five cholangiocarcinoma cell lines, and higher levels of AAH in cell lines derived from moderately or poorly differentiated compared with well-differentiated tumors. Immunocytochemical staining and fluorescence activated cell sorting analysis revealed both surface and intracellular AAH immunoreactivity. Using the phagokinetic non-directional migration assay and a novel ATPLite luminescence-based directional migration assay, we correlated AAH expression with motility. Correspondingly, antisense and not sense or mutated antisense AAH oligodeoxynucleotides significantly inhibited AAH expression and motility in cholangiocarcinoma cells. Conclusions: AAH over-expression may contribute to the infiltrative growth pattern of cholangiocarcinoma cells by promoting motility.

Role of the Aspartyl-Asparaginyl-β-Hydroxylase Gene in Neuroblastoma Cell Motility

Aspartyl (asparaginyl) β-hydroxylase (AAH) is overexpressed in various malignant neoplasms, and high levels of immunoreactivity mainly occur in infiltrating or metastasized tumors. In addition, AAH is abundantly expressed in normally invasive placental trophoblastic cells. These observations led to the hypothesis that AAH may have a role in motility and aggressive behavior of tumor cells. The present study demonstrates that AAH is overexpressed in primary human malignant neuroectodermal tumors, including medulloblastomas and neuroblastomas, and that AAH expression is at a low level or undetectable in the normal mature brain. In the Sy5y neuroblastoma cell line, endogenous expression of the ∼86-kd AAH protein was demonstrated by Western blot analysis, and immunoreactivity predominantly localized to the cell surface by immunocytochemical staining and FACS analysis. Sy5y cells that were stably transfected with the human AAH cDNA had increased levels of proliferating cell nuclear antigen and Bcl-2, and reduced levels of p21/Waf1 and p16. In addition, increased AAH expression enhanced Sy5y cell motility, whereas antisense oligodeoxynucleotide inhibition of AAH significantly reduced Sy5y cell motility and increased the levels of p21/Waf1 and p16. The findings suggest that AAH overexpression contributes to the malignant phenotype of neuroectodermal tumor cells by increasing motility and enhancing proliferation, survival, and cell cycle progression. Because AAH expression is at a low level or undetectable in normal brain, the AAH gene may be a target for treating primitive neuroectodermal tumors.