title: N-MYC regulates focal adhesion kinase expression in human neuroblastoma.
abstract: N-MYC is a transcription factor that plays an important role in cellular survival in neuroblastoma, and amplification of the N-MYC oncogene is the primary adverse prognostic indicator for neuroblastoma. Focal adhesion kinase (FAK) is a survival factor that has been shown to be overexpressed in many types of human cancers. In this study, we investigated the role of N-MYC regulation of FAK expression in neuroblastoma. We first found a correlation between N-MYC and FAK expression in neuroblastoma. Real time quantitative PCR demonstrated an increase in FAK mRNA abundance in the N-MYC-amplified IMR-32 compared with the nonamplified SK-N-AS neuroblastoma cell lines. FAK protein expression also correlated positively with N-MYC expression in the N-MYC-amplified IMR-32 versus nonamplified SK-N-AS neuroblastoma cell lines. The same results were seen with the isogenic N-MYC(+) (Tet(-)) and N-MYC(-) (Tet(+)) neuroblastoma cell lines. Promoter-reporter assays showed that activity of the FAK promoter was increased in the N-MYC-amplified IMR-32 cell line, in the N-MYC-transfected SK-N-AS nonamplified cell line, and in the isogenic N-MYC(+) (Tet(-)) neuroblastoma cell lines compared with the nonamplified and N-MYC-nonexpressing cell lines. We also identified two N-MYC binding sites in the FAK promoter sequence and showed binding of N-MYC transcription factor to the FAK promoter through electrophoretic mobility shift, chromatin immunoprecipitation, and dual luciferase assays. Finally down-regulation of FAK expression in N-MYC-inducible neuroblastoma cell lines with FAK small interfering RNA or a dominant-negative FAK inhibitor (AdFAK-CD) significantly decreased viability and increased apoptosis in the N-MYC(+) (Tet(-)) cells compared with the isogenic N-MYC(-) (Tet(+)) cells, demonstrating the biological significance of FAK overexpression in the N-MYC-expressing cell lines. This is the first report linking N-MYC and FAK in neuroblastoma, and it clearly demonstrates that N-MYC induces FAK expression. The results indicate that N-MYC regulation of FAK expression can control cellular functions in isogenic N-MYC(-/+) (Tet(+/-)) neuroblastoma cell lines.
authors.affiliation: Department of Surgery, University of Florida, Gainesville, Florida 32610, USA. beierea@surgery.ufl.edu
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Beierle
authors.forename: Angelica
authors.initials: A
authors.lastname: Trujillo
authors.forename: Abhilasha
authors.initials: A
authors.lastname: Nagaram
authors.forename: Elena V
authors.initials: EV
authors.lastname: Kurenova
authors.forename: Richard
authors.initials: R
authors.lastname: Finch
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Ma
authors.forename: Jennifer
authors.initials: J
authors.lastname: Vella
authors.forename: William G
authors.initials: WG
authors.lastname: Cance
authors.forename: Vita M
authors.initials: VM
authors.lastname: Golubovskaya
mesh_terms: D045744:Cell Line, Tumor; D051417:Focal Adhesion Kinase 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D005784:Gene Amplification; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D009447:Neuroblastoma / Q000201:enzymology* / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D020218:Response Elements
chemical_list: D047428:Protein Kinase Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human