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.

Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that regulates both cellular adhesion and apoptosis. FAK is overexpressed in a number of human tumors including neuroblastoma. Previously, we have shown that the MYCN oncogene, the primary adverse prognostic indicator in neuroblastoma, regulates the expression of FAK in neuroblastoma. In this study, we have examined the effects of FAK inhibition upon neuroblastoma using a small molecule [1,2,4,5-benzenetetraamine tetrahydrochloride (Y15)] to inhibit FAK expression and the phosphorylation of FAK at the Y397 site. Utilizing both non-isogenic and isogenic MYCN+/MYCN- neuroblastoma cell lines, we found that Y15 effectively diminished phosphorylation of the Y397 site of FAK. Treatment with Y15 resulted in increased detachment, decreased cell viability and increased apoptosis in the neuroblastoma cell lines. We also found that the cell lines with higher MYCN are more sensitive to Y15 tr...

One of the least-investigated areas of brain pathology research is glycosylation, which is a critical regulator of cell surface protein structure and function. β-Galactoside α2,6-sialyltransferase ( ST6GAL1 ) is the primary enzyme that α2,6 sialylates N-glycosylated proteins destined for the plasma membrane or secretion, thereby modulating cell signaling and behavior. We demonstrate a potentially novel, protumorigenic role for α2,6 sialylation and ST6GAL1 in the deadly brain tumor glioblastoma (GBM). GBM cells with high α2,6 sialylation exhibited increased in vitro growth and self-renewal capacity and decreased mouse survival when orthotopically injected. α2,6 Sialylation was regulated by ST6GAL1 in GBM, and ST6GAL1 was elevated in brain tumor-initiating cells (BTICs). Knockdown of ST6GAL1 in BTICs decreased in vitro growth, self-renewal capacity, and tumorigenic potential. ST6GAL1 regulates levels of the known BTIC regulators PDGF Receptor β ( PDGFRB ), Activated Leukocyte Cell Adhesion Molecule, and Neuropilin, which were confirmed to bind to a lectin-recognizing α2,6 sialic acid. Loss of ST6GAL1 was confirmed to decrease PDGFRB α2,6 sialylation, total protein levels, and the induction of phosphorylation by PDGF-BB. Thus, ST6GAL1 -mediated α2,6 sialylation of a select subset of cell surface receptors, including PDGFRB , increases GBM growth.