N-MYC Regulates Focal Adhesion Kinase Expression in Human Neuroblastoma

• Published in: The Journal of biological chemistry 2007-04, Vol.282 (17), p.12503-12516
• Main Authors: Beierle, Elizabeth A., Trujillo, Angelica, Nagaram, Abhilasha, Kurenova, Elena V., Finch, Richard, Ma, Xiaojie, Vella, Jennifer, Cance, William G., Golubovskaya, Vita M.
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; Focal Adhesion Kinase 1 - antagonists & inhibitors; Focal Adhesion Kinase 1 - biosynthesis; Focal Adhesion Kinase 1 - genetics; Gene Amplification; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Neoplastic - drug effects; Humans; Neuroblastoma - enzymology; Neuroblastoma - genetics; Protein Kinase Inhibitors - pharmacology; Proto-Oncogene Proteins c-myc - metabolism; Response Elements; RNA, Small Interfering - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M701450200

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.