PHOX2B-Mediated Regulation of ALK Expression: In Vitro Identification of a Functional Relationship between Two Genes Involved in Neuroblastoma

Tiziana Bachetti,Roberto Chiarle,Marta Bellotti,Ulrich Pfeffer,Valentina Mirisola,Irene Caffa,Daniela Di Paolo,Chiara Ferraris,Chiara Brignole,Patrizia Perri,Mirco Ponzoni,Diego Fornasari,Isabella Ceccherini,Michele Fiore,Silvia Borghini,Simona Di Lascio

doi:10.1371/journal.pone.0013108

Abstract

BackgroundNeuroblastoma (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 FindingsFollowing 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/SignificanceThese 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.

Highlights

Novel insights into the molecular pathogenesis of neuroblastoma (NB), a severe pediatric tumor originating from neural crest cells and accounting for 15% of childhood cancer mortality, have been gained after the identification of germline as well as somatically acquired mutations in the genes encoding the paired-like homeobox 2b (PHOX2B) transcription factor [1,2] and the Anaplastic Lymphoma Kinase (ALK) tyrosine kinase receptor [3]
ALK, Paired-like Homeobox 2B (PHOX2B) and PHOX2A resulted to be highly expressed in almost all analyzed NB cell lines (Figure 1) with respect to a pool of normal tissues and to HeLa cells, a cervix carcinoma cell line characterized by low level of ALK expression [31] and almost undetectable expression levels of the two PHOX2 genes [6]
ALK expression is regulated by PHOX2 genes ALK, PHOX2B and PHOX2A silencing was achieved through gene-directed siRNA in three NB cell lines, namely IMR-32, HTLA-230 and SH-SY5Y, this latter carrying a p.F1174L ALK mutation, showing a high expression of the three genes

Summary

Introduction

Novel insights into the molecular pathogenesis of neuroblastoma (NB), a severe pediatric tumor originating from neural crest cells and accounting for 15% of childhood cancer mortality, have been gained after the identification of germline as well as somatically acquired mutations in the genes encoding the paired-like homeobox 2b (PHOX2B) transcription factor [1,2] and the Anaplastic Lymphoma Kinase (ALK) tyrosine kinase receptor [3].The PHOX2B gene is involved in the specification of the noradrenergic phenotype during the development and differentiation of neural crest derivatives [4,5,6,7,8,9]. 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. 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

Methods

Results

Conclusion