title: Mutations that disrupt PHOXB interaction with the neuronal calcium sensor HPCAL1 impede cellular differentiation in neuroblastoma.
abstract: Heterozygous germline mutations in PHOX2B, a transcriptional regulator of sympathetic neuronal differentiation, predispose to diseases of the sympathetic nervous system, including neuroblastoma and congenital central hypoventilation syndrome (CCHS). Although the PHOX2B variants in CCHS largely involve expansions of the second polyalanine repeat within the C-terminus of the protein, those associated with neuroblastic tumors are nearly always frameshift and truncation mutations. To test the hypothesis that the neuroblastoma-associated variants exert their effects through loss or gain of protein-protein interactions, we performed a large-scale yeast two-hybrid screen using both wild-type (WT) and six different mutant PHOX2B proteins against over 10 000 human genes. The neuronal calcium sensor protein HPCAL1 (VILIP-3) exhibited strong binding to WT PHOX2B and a CCHS-associated polyalanine expansion mutant but only weakly or not at all to neuroblastoma-associated frameshift and truncation variants. We demonstrate that both WT PHOX2B and the neuroblastoma-associated R100L missense and the CCHS-associated alanine expansion variants induce nuclear translocation of HPCAL1 in a Ca(2+)-independent manner, while the neuroblastoma-associated 676delG frameshift and K155X truncation mutants impair subcellular localization of HPCAL1, causing it to remain in the cytoplasm. HPCAL1 did not appreciably influence the ability of WT PHOX2B to transactivate the DBH promoter, nor did it alter the decreased transactivation potential of PHOX2B variants in 293T cells. Abrogation of the PHOX2B-HPCAL1 interaction by shRNA knockdown of HPCAL1 in neuroblastoma cells expressing PHOX2B led to impaired neurite outgrowth with transcriptional profiles indicative of inhibited sympathetic neuronal differentiation. Our results suggest that certain PHOX2B variants associated with neuroblastoma pathogenesis, because of their inability to bind to key interacting proteins such as HPCAL1, may predispose to this malignancy by impeding the differentiation of immature sympathetic neurons.
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.affiliation: Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Zhong
authors.affiliation: Chicago Center for Childhood Cancer and Blood Diseases, the University of Chicago, Chicago, IL, USA.
authors.forename: L
authors.initials: L
authors.lastname: Teng
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: N
authors.initials: N
authors.lastname: Bhatnagar
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: B
authors.initials: B
authors.lastname: Sharma
authors.affiliation: High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, People's Republic of China.
authors.forename: X
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Luther
authors.affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
authors.forename: L P
authors.initials: LP
authors.lastname: Haynes
authors.affiliation: Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK.
authors.forename: R D
authors.initials: RD
authors.lastname: Burgoyne
authors.affiliation: Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Vidal
authors.affiliation: Chicago Center for Childhood Cancer and Blood Diseases, the University of Chicago, Chicago, IL, USA.
authors.forename: S
authors.initials: S
authors.lastname: Volchenboum
authors.affiliation: Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Hill
authors.affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: George
mesh_terms: D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D051596:Neurocalcin / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D060066:Protein Interaction Maps; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: C520235:HPCAL1 protein, human; D018398:Homeodomain Proteins; C109220:NBPhox protein; D051596:Neurocalcin; D010455:Peptides; D014157:Transcription Factors; C019529:polyalanine; D002118:Calcium