MYCN, neuroblastoma and focal adhesion kinase (FAK)

Abstract

Neuroblastoma is the most common extracranial solid tumor of childhood. This tumor is characterized by poor survival, especially when it features amplification of the MYCN oncogene. The ability for human cancers to propagate is marked by their ability to invade and metastasize to distant sites. Focal adhesion kinase (FAK) is a key tyrosine kinase involved in the survival and metastasis of a number of human tumor types. We have shown that FAK is present in human neuroblastoma and that its expression in neuroblastoma is related to the MYCN oncogene. We have also demonstrated that inhibition of FAK in neuroblastoma leads to decreased tumor cell survival. The current review addresses the relationship between the MYCN oncogene, focal adhesion kinase and neuroblastoma.

Figure 1. FAK protein detection in formalin-fixed, paraffin embedded human neuroblastoma specimens

A. Antibody to p125^FAK was utilized to detect FAK staining in 70 human neuroblastoma specimens. This photomicrograph (40×) shows a MYCN amplified INSS stage 4 neuroblastoma specimen with significant staining for p125^FAK. B. This photomicrograph (40×) shows a MYCN non-amplified INSS stage 4 neuroblastoma specimen with no significant p125^FAK staining.

Figure 2. MYCN regulates the FAK promoter

A. Nucleotide sequence (GenBank Accession Number AY323812) of the 5’-flanking region of the FAK gene. The major transcription initiation site is marked as +1. The putative E-box binding site for MYCN is underlined. B. Dual luciferase assays are utilized to detect FAK promoter activity. Graph showing fold change in FAK promoter activity in MYCN− and MYCN+ isogenic neuroblastoma cell lines. There is a significant increase in FAK promoter activity with the P-280 construct in the MYCN+ cell line compared to the MYCN− cells (*P<0.01). When the E-box binding site is mutated (ΔP-280), the FAK promoter activity in the MYCN+ neuroblastoma cell lines significantly decreases compared to the wild type (P-280) promoter construct (†P<0.01).

Figure 3. FAK inhibition leads to decreased cell viability in MYCN amplified neuroblastoma cell lines

FAK inhibition is achieved with TAE226, a specific FAK kinase inhibitor. Neuroblastoma cells, SK-N-BE(2) (amplified MYCN) and SK-N-AS (non-amplified MYCN) are treated with TAE226 for 24 hours and viability is measured using Alamar Blue Assay. After 24 hours, cellular viability is significantly decreased in the MYCN amplified neuroblastoma cell line, SK-N-BE(2), compared to that in the non-amplified, SK-N-AS, cell line, demonstrating the biologic importance of FAK in MYCN amplified neuroblastoma cells.

Fig. 4. FAK inhibition has minimal effects upon normal ganglion cells

FAK is inhibited with a small molecule inhibitor, 1,2,4,5-benzenetetraamine tetrahydrochloride, for 24 and 48 hours in normal ganglion cells. Viability is measured using Alamar Blue Assay. The cellular viability in these normal cells is essentially unaffected, even after 48 hours of treatment, by FAK inhibition with 1,2,4,5-benzenetetraamine tetrahydrochloride.