PHOX2B-mediated regulation of ALK expression: in vitro identification of a functional relationship between two genes involved in neuroblastoma.
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.


RESULTS
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
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.
Laboratory of Molecular Genetics, G. Gaslini Children's Hospital, Genoa, Italy.
Tiziana
T
Bachetti
Daniela
D
Di Paolo
Simona
S
Di Lascio
Valentina
V
Mirisola
Chiara
C
Brignole
Marta
M
Bellotti
Irene
I
Caffa
Chiara
C
Ferraris
Michele
M
Fiore
Diego
D
Fornasari
Roberto
R
Chiarle
Silvia
S
Borghini
Ulrich
U
Pfeffer
Mirco
M
Ponzoni
Isabella
I
Ceccherini
Patrizia
P
Perri
D000077548:Anaplastic Lymphoma Kinase; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D020868:Gene Silencing; D018398:Homeodomain Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D034741:RNA, Small Interfering; D020794:Receptor Protein-Tyrosine Kinases; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation
D018398:Homeodomain Proteins; C109220:NBPhox protein; D034741:RNA, Small Interfering; D014157:Transcription Factors; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases