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PHOX2B-mediated regulation of ALK expression: in vitro identification of a functional relationship between two genes
Tiziana Bachetti1, Daniela Di Paolo, Simona Di Lascio
1Laboratory of Molecular Genetics, G. Gaslini Children's Hospital, Genoa, Italy.
Background:
Neuroblastoma (NB) is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) and the transcription factor Paired-like Homeobox 2B (PHOX2B), in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis.
Methodology/Principal Findings:
Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target.
Conclusions/Significance:
These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.
Insights
Neuroblastoma (NB) pathogenesis involves Anaplastic Lymphoma Kinase (ALK) and Paired-like Homeobox 2B (PHOX2B). PHOX2B directly drives ALK transcription, revealing a key molecular interaction for NB development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Neuroblastoma (NB) is a significant pediatric cancer with complex genetic underpinnings.
- Mutations in Anaplastic Lymphoma Kinase (ALK) and Paired-like Homeobox 2B (PHOX2B) are found in some NB patients.
- Both ALK and PHOX2B are overexpressed in most NB samples, suggesting a functional link.
Purpose of the Study:
- To investigate the regulatory relationship between ALK and PHOX2B in neuroblastoma pathogenesis.
- To elucidate the molecular mechanisms underlying the co-involvement of ALK and PHOX2B in NB.
Main Methods:
- Correlation analysis of gene transcription levels in NB cell lines.
- Gene expression manipulation using siRNA knock-down and forced overexpression.
- Analysis of PHOX2B binding to the ALK promoter.
Main Results:
- A strong correlation was observed between ALK, PHOX2B, and PHOX2A transcription levels.
- PHOX2B and PHOX2A regulate each other's expression and also affect ALK levels.
- PHOX2B directly binds to the ALK promoter, driving its transcription.
Conclusions:
- PHOX2B directly regulates ALK transcription, explaining their concurrent involvement in NB.
- This study enhances understanding of molecular interactions in NB pathogenesis.
- Findings suggest potential for novel RNAi-based therapies targeting multiple genes in refractory NB.
