PHOX2B is a suppressor of neuroblastoma metastasis.

Osnat Naftali,Tsipi Meshel,Orit Sagi-Assif,Isaac P Witz,Shelly Maman,Ravit Ginat

doi:10.18632/oncotarget.7056

Abstract

Paired like homeobox 2B (PHOX2B) is a minimal residual disease (MRD) marker of neuroblastoma. The presence of MRD, also referred to as micro-metastases, is a powerful marker of poor prognosis in neuroblastoma.Lung metastasis is considered a terminal event in neuroblastoma. Lung micro-metastatic neuroblastoma (MicroNB) cells show high expression levels of PHOX2B and possess a less malignant and metastatic phenotype than lung macro metastatic neuroblastoma (MacroNB) cells, which hardly express PHOX2B.In vitro assays showed that PHOX2B knockdown in MicroNB cells did not affect cell viability; however it decreased the migratory capacity of the MicroNB-shPHOX2B cells. An orthotopic inoculation of MicroNB-shPHOX2B cells into the adrenal gland of nude mice resulted in significantly larger primary tumors and a heavier micro-metastatic load in the lungs and bone-marrow, than when control cells were inoculated.PHOX2B expression was found to be regulated by methylation. The PHOX2B promoter in MacroNB cells is significantly more methylated than in MicroNB cells. Demethylation assays using 5-azacytidine demonstrated that methylation can indeed inhibit PHOX2B transcription in MacroNB cells.These pre-clinical data strongly suggest that PHOX2B functions as a suppressor of neuroblastoma progression.

Highlights

Neuroblastoma is the most common extracranial solid tumor in children, and accounts for approximately 15% of all childhood cancer deaths [1, 2]
In a previous study we found that micrometastatic neuroblastoma (MicroNB) cells, but not MacroNB cells, express high mRNA levels of the minimal residual disease (MRD) marker Paired like homeobox 2B (PHOX2B) [18]
Control cells were infected with a non-silencing shRNA (MicroNBshControl). qRT-PCR assays showed that following infection with PHOX2B-specific shRNA, PHOX2B mRNA expression decreased almost four fold (p

Summary

Introduction

Neuroblastoma is the most common extracranial solid tumor in children, and accounts for approximately 15% of all childhood cancer deaths [1, 2]. Most children diagnosed with neuroblastoma over the age of one year present metastatic disease, when the presence of lung metastases is considered a rare and terminal event [3, 4]. Most patients with metastatic disease who achieve near complete remission typically relapse because of the presence of neuroblastoma micrometastasis, known as minimal residual disease (MRD) [5,6,7,8]. The current consensus is that micro-metastases remain in a dormant state, until “awakened” to progress towards overt metastases [10]. Understanding the mechanism regulating the progression of micro-metastases to overt metastasis is crucial for the development of efficient modalities to monitor and treat neuroblastoma metastasis

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