Differential aminoacylase expression in neuroblastoma

Neuroblastoma, a cancer of the sympathetic nervous system, is the most common extracranial solid tumor in children.1 Tumor cells likely emerge from the sympatho-adrenal lineage of the autonomic nervous system, with the majority of tumors forming in the adrenal medulla and the paraspinal sympathetic ganglia. Complete tumor regression occurs in a subset of infants with seemingly advanced cases.2 Unfortunately, most advanced stage neuroblastomas are fatal because they are widely metastatic or refractory to therapy. High-risk tumors frequently exhibit amplification of the proto-oncogene MYCN3 and expression of the TrkB neurotrophin receptor and its conjugate ligand, brain-derived neurotrophic factor (BDNF).4, 5 However, despite advances in tumor characterization, only a fraction of malignancies display such features. Identifying novel molecular markers is essential toward advancing our understanding of the etiology of neuroblastoma as well as developing appropriate clinical interventions. N-acetylation protects proteins from proteolytic degradation by aminopeptidases present in eukaryotic cells, thus increasing their half-life. Subsequent to cleavage by intracellular proteases (e.g., cathepsins, calpains, proteasomes, metalloproteinases), the released NH2-blocked dipeptide is catabolized by acylpeptide hydrolase to release an NH2-α-amino acid which undergoes deacetylation by an aminoacylase.6 Aminoacylases are cytosolic, zinc-dependent metalloenzymes responsible for catalyzing the deacetylation of N-acyl-L-amino acids to generate acetate and a free amino acid, which then is available for protein synthesis.7 Aminoacylase 1 (ACY1), the most widely expressed aminoacylase (e.g., kidney, liver, pancreas, spleen, thymus, brain, lung, and heart and skeletal muscle), is involved in the hydrolysis of a wide range of N-acetylated amino acids.8 ACY1 serves as a putative tumor suppressor in renal cell carcinoma9 and small cell lung cancer.10 Aspartoacylase (ASPA, aminoacylase 2), a central nervous system (CNS) glial protein, is best known for its role in myelin lipid synthesis via deacetylation of neuronally derived N-acetyl-L-aspartate (NAA).11 Missense mutations in ASPA result in Canavan disease, a fatal childhood leukodystrophy characterized by dysmyelination and spongi-form degeneration of white matter.12 Aminoacylase 3 (ACY3), which is primarily expressed in liver and kidney, is the least understood family member because it deacetylates N-acetylated aromatic amino acids, which are present at relatively low frequencies in eukaryotic proteins, and mercapturic acids.13, 14 Given the putative tumor suppressor role of ACY1, we sought to characterize aminoacylase expression in neuroblastoma. We report, for the first time, aminoacylase expression in six well-characterized neuroblastoma cell lines (i.e., TrkB-positive, MYCN-amplified SMS-KCNR and SK-N-BE and TrkB-negative, non-MYCN-amplified SK-N-AS, SK-N-SH, SH-SY5Y, and SH-EP). In addition to differential aminoacylase expression in these cell lines under basal conditions and in response to neural differentiation agents, we characterized the expression pattern in normal human adrenal gland and neuroblastoma tumors. Each aminoacylase displayed discrete expression patterns suggesting each subserves distinct functions.