Abstract
The paternally imprinted neuronatin (NNAT) gene has been identified as a target of aberrant epigenetic silencing in diverse cancers, but no association with pediatric bone cancers has been reported to date. In screening childhood cancers, we identified aberrant CpG island hypermethylation in a majority of osteosarcoma (OS) samples and in 5 of 6 human OS cell lines studied but not in normal bone-derived tissue samples. CpG island hypermethylation was associated with transcriptional silencing in human OS cells, and silencing was reversible upon treatment with 5-aza-2’-deoxycytidine. Expression of NNAT was detectable in osteoblasts and chondrocytes of human bone, supporting a potential role in bone homeostasis. Enforced expression of NNAT in human OS cells lacking endogenous expression resulted in significant reduction in colony formation and in vitro migration compared to nonexpressor control cells. We next analyzed the effect of NNAT expression on intracellular calcium homeostasis and found that was associated with an attenuated decay of calcium levels to baseline following ATP-induced release of calcium from endoplasmic reticulum (ER) stores. Furthermore, NNAT expression was associated with increased cytotoxicity in OS cells from thapsigargin, an inhibitor of calcium reuptake into ER and an inducer of the ER stress response. These results suggest a possible tumor suppressor role for NNAT in human osteosarcoma. Additional study is needed ascertain sensitization to ER stress-associated apoptosis as a mechanism of NNAT-dependent cytotoxicity. In that case, epigenetic modification therapy to effect NNAT transcriptional derepression may represent a therapeutic strategy potentially of benefit to a majority of osteosarcoma patients.
Highlights
Osteosarcoma (OS) is the most common primary bone tumor in children, adolescents, and young adults
We have previously shown that hypermethylation of the NNAT 5′ CpG island is a frequent event in acute leukemias of childhood [14]
We first reported a potential role for epigenetic dysregulation of the NNAT gene in human oncogenesis, identifying aberrant 5′CpG island hypermethylation associated with transcriptional silencing in pediatric acute leukemias [14]
Summary
Osteosarcoma (OS) is the most common primary bone tumor in children, adolescents, and young adults. Molecular and cytogenetic analyses have indicated a substantial prevalence of structural variation in OS [2,3,4,5]. Despite this genomic complexity, and excepting the well-established roles for aberrations in the RB transcriptional corepressor 1 (RB1) [6, 7], and tumor protein p53 (TP53) [8,9,10] tumor suppressor pathways in hereditary OS predisposition as well as sporadic tumors, few pathognomonic cytogenetic abnormalities or consistent genetic mutations have emerged either as clear and predominant drivers of tumorigenesis or as biomarkers for histologic features or clinical behavior. Few studies, have explored the potential role of epigenetically-mediated gene dysregulation in the pathogenesis of OS
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