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

Abstract 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. Introduction Malignant (primitive) neuroectodermal tumors are often highly fatal as a result of extensive tissue infiltration and metastatic spread. Research conducted within the last decade demonstrated that specific adhesion molecules or receptors such as CD44 (Merzak et al, 1994; Radotra and McCormick, 1997; Radotra et al, 1994) and integrins (Goldbrunner et al, 1996, 1998; Paulus et al, 1996; Tysnes et al, 1996), as well as matrix degrading/modifying enzymes such as hyaluronidase (Liu et al, 1996; Pilkington, 1996) and metalloproteinases (MMPs) (Apodaca et al, 1990; Giese and Westphal, 1996; Nakano et al, 1995; Pilkington, 1996; Sawaya et al, 1996), are critical regulators of infiltrative and metastatic tumor cell growth. In addition, a third important factor is that the recipient tissues express extracellular matrix molecules such as laminin, fibronectin, and collagen type IV that enable tumor cell attachment and infiltration (Giese et al, 1998; Goldbrunner et al, 1996; Merzak et al, 1995). Similar adhesion molecule- or receptor-mediated interactions with extracellular matrix occur during embryonic cell migration (Romanic and Madri, 1994), trophoblast implantation (Canete-Soler et al, 1995), and axonal regeneration (Fitch and Silver, 1997; Frisen, 1997; Patterson, 1985). Infiltrative growth of malignant neoplasms may be controlled by inhibiting the expression of genes that encode matrix-degrading or matrix-modifying enzymes. For example, glioblastoma cell invasiveness has been linked to down-regulation or inactivation of the p16 tumor suppressor gene (Ueki et al, 1996) and increased MMP-2 expression (Rooprai and McCormick, 1997), whereas experimental restoration of p16 expression and down-regulation of MMP-2 inhibit glioma cell invasiveness (Mohanam et al, 1995). Moreover, tissue inhibitors of MMP (Mohanam et al, 1995; Nakano et al, 1995) can modulate the integrity of extracellular matrix and tumor cell invasion. In this regard, the human aspartyl (asparaginyl) β-hydroxylase (AAH) gene is of interest because of its potential role in regulating infiltrative or metastatic growth of malignant neoplasms through β-hydroxylation of extracellular matrix molecules or receptors that contain the required epidermal growth factor-like domain consensus sequence (Ince et al, 1997, 2000; Lavaissiere et al, 1996). The human AAH cDNA encodes a 757-amino acid protein with a predicted Mr of ∼86 kd (Lavaissiere et al, 1996). AAH is a member of the α-ketoglutarate–dependent dioxygenase family, which includes prolyl-3, prolyl-4, and lysyl hydroxylases (Jia et al, 1992; Wang et al, 1991). AAH catalyzes posttranslational hydroxylation of β carbons of specific aspartate and asparagine residues in certain epidermal growth factor-like domains present in a number of proteins, including receptors and receptor ligands involved in cell growth and differentiation, and extracellular matrix molecules, such as tenascin, laminin, and thrombospondin (Lavaissiere et al, 1996). Importantly, the consensus sequence for AAH hydroxylation is also present in the epidermal growth domains of Notch and oncogenic Notch homologs; Notch has a demonstrated role in neuronal migration during development. AAH protein contains a relatively compact and highly charged carboxyl region that can be proteolytically cleaved from the amino terminal region to generate an approximately 52-kd or 56-kd catalytically active fragment (Jia et al, 1992, 1994; Wang et al, 1991). Site-directed mutagenesis studies demonstrated that the 675His residue present in the C-terminal fragment is essential for catalytic activity (Dinchuk et al, 2000; Jia et al, 1992) and that the 675His → Ala mutated human AAH cDNA exerts a dominant-negative phenotype with respect to AAH activity in transfected cells (Dinchuk et al, 2000; Ince et al, 2000). We previously demonstrated AAH overexpression in hepatocellular carcinomas, cholangiocarcinomas, and adenocarcinomas of breast or lung origin and low to undetectable AAH expression in normal tissue counterparts (Lavaissiere et al, 1996). Further studies revealed that AAH overexpression was linked to malignant transformation rather than regeneration or benign proliferation of intrahepatic bile ducts (Ince et al, 2000). Clues regarding the potential role of AAH in cellular transformation stemmed from our investigations of a broad range of immunohistochemically stained normal and neoplastic tissues that disclosed high levels of AAH mainly in infiltrated and metastasized tumors and in normally invasive trophoblastic cells (Lavaissiere et al, 1996). These observations suggested a link between AAH overexpression and cellular invasiveness. The present study characterizes AAH expression in primary human primitive (malignant) neuroectodermal tumors of central nervous system (CNS) or peripheral origin, because these neoplasms cause death mainly by tissue infiltration and metastatic spread. In addition, we conducted experiments to examine the potential role of AAH in motility and aggressive growth of neuroblastoma cells. Results AAH Immunoreactivity in Surgical Biopsy Specimens of Human Malignant Neuroectodermal Tumors AAH immunoreactivity was examined in human primitive neuroectodermal tumor (PNET) biopsy specimens subcategorized as supratentorial neuroblastoma (n = 8), peripheral (sympathetic and non-CNS) ne (...truncated)