Background. Neuroblastoma (NB) arises from embryonal neural crest secondary to a block in differentiation and long-term survival inversely correlates with the degree of differentiation. Inhibition of epigenetically controlled developmental programs is critical for NB de-differentiation and treatment with differentiation agents like retinoic acid (RA) has modestly improved survival. We have recently demonstrated a novel function for the histone chaperone CHAF1A (a subunit of the Chromatin Assembly Factor-1) in opposing NB differentiation.
Material and Methods. Loss-of-function studies were performed in NB RA-resistant cell lines upon CHAF1A knockdown. Conversely, gain-of-function studies were performed in NB RA-sensitive lines. Gene expression profiling (affymetrix u133+2 array) of knockdown and overexpressing lines was performed and quantitative-PCR assays used to validate most enriched metabolic gene sets. Inducible CHAF1A over-expressing cells were xenografted using a non-tumorigenic NB model.
Results. CHAF1A loss-of-function effectively drives neuronal differentiation in multiple NB RA-resistant lines. Conversely, CHAF1A overexpression in RA-sensitive lines induces colony formation and causes resistance to RA treatment. Moreover, GSEA and Q-PCR revealed that CHAF1A profoundly alters cell metabolism pathways (AA, glutamine, and glucose metabolism; p<0.01). Using conditional expression of CHAF1A (Tet-ON) in a NB line that has a poor engraftment rate, we demonstrate that CHAF1A overexpression significantly promotes tumor initiation in vivo with a much higher engraftment rate and tumor size (engraftment rate CHAF1A control: 2 out of 9, 22%; engraftment rate CHAF1A induced: 9 out of 12, 75%, p< 0.03).
Conclusion. These gain-of-function studies support our hypothesis that CHAF1A expression restricts neural crest differentiation and contributes to the resistance of NB tumors to RA therapy by altering glucose and glutamine metabolism. Importantly CHAF1A overexpression markedly enhances tumor initiation in vivo. Further understanding of the metabolic changes induced by CHAF1A will guide the development of novel differentiating therapies for high-risk neuroblastoma.