Research Papers:

Aberrant epigenetic silencing of neuronatin is a frequent event in human osteosarcoma

Haleema Saeed, Sayantani Sinha, Christine Mella, Jeffrey S. Kuerbitz, Monica L. Cales, Mark A. Steele, Jennifer Stanke, Derek Damron, Fayez Safadi and Steven J. Kuerbitz _

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Oncotarget. 2020; 11:1876-1893. https://doi.org/10.18632/oncotarget.27583

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Haleema Saeed1,8, Sayantani Sinha2,9, Christine Mella1, Jeffrey S. Kuerbitz1,10, Monica L. Cales3,11, Mark A. Steele1, Jennifer Stanke1,12, Derek Damron2, Fayez Safadi4,5,6 and Steven J. Kuerbitz1,6,7

1 Division of Pediatric Hematology/Oncology, Akron Childrens Hospital, Akron, OH, USA

2 Department of Biological Sciences, Kent State University, Kent, OH, USA

3 College of Osteopathic Medicine, University of Pikeville, Pikeville, KY, USA

4 Department of Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH, USA

5 Musculoskeletal Research Group, Northeast Ohio Medical University, Rootstown, OH, USA

6 Rebecca D. Considine Research Institute, Akron Children’s Hospital, Akron, OH, USA

7 Department of Pediatrics, Northeast Ohio Medical University, Rootstown, OH, USA

8 Current affiliation: Shaukat Khanum Cancer Hospital and Research Centre, Lahore, Pakistan

9 Current affiliation: Department of Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

10 Current affiliation: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA

11 Current affiliation: Penn State Health St. Joseph, Reading, PA, USA

12 Current affiliation: Foundation Medicine, Boston, MA, USA

Correspondence to:

Steven J. Kuerbitz,email: [email protected]

Keywords: osteosarcoma; DNA methylation; neuronatin; tumor suppressor genes

Received: December 03, 2019     Accepted: April 03, 2020     Published: May 19, 2020


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.

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