PHOX2B: a Regulator of Micro-Metastasis in Neuroblastoma

An orthotopic mouse model for human neuroblastoma metastasis, comprising local and metastatic variants originating from single tumors, was developed in our lab. Inoculation of these variants into a new set of nude mice generated two types of variants: lung macro and micro-metastatic cells, the latter not generating overt lung metastases. In previous work, these cells were characterized for tumorigenic and metastatic abilities, indicating a more malignant phenotype of the macro-metastatic cells.

PHOX2B is a transcription factor used as a minimal residual disease marker in neuroblastoma patients. Higher expression levels of PHOX2B were identified through qRT-PCR and western-blot analyses in micro as compared to macro-metastatic neuroblastoma cells.

Having a common genetic background, we hypothesized that an epigenetic event had led to the differential expression of PHOX2B in the micro and macro-metastatic neuroblastoma cells. Indeed, examination of the methylation pattern of the PHOX2B promoter revealed that 25 CpG dinucleotides in the promoter region are 31% more methylated in the macro than in the micro-metastatic cells. Further on, luciferase assay examining 1100bp region of the PHOX2B promoter, showed that in vitro methylation of that region lowered transcription by 93%. These results strongly imply that hyper-methylation could be the cause of PHOX2B lack of expression in the macro-metastatic cells.

Larger primary tumors were seen in mice inoculated with the micro-metastatic variant where PHOX2B was down-regulated, and 20 fold more human cells were detected in the lungs and bone marrow of these mice. These results strongly support the hypothesis postulating that PHOX2B can function as an inhibitor of tumor progression and metastasis.