Summary:
Title: Expression of Focal Adhesion Kinase (FAK)-Src Kinase-Paxillin (PAX)
system and its association with Disease Clinical Outcome in Advanced Stage
Neuroblastoma.
Background: Neuroblastoma (NB) is the most common extracranial tumor in
children, arising from the neural crest of the sympathetic ganglia and accounts
for 7-10% of all pediatric cancers. To date, the most important prognostic
marker for NB is the N-Myc gene. We already know from adult malignancies that
proteins such as Focal Adhesion Kinase, Src kinase and Paxillin are
overexpressed in adult malignancies and their degree of expression correlates
with tumor grading and patients’ survival. We have previously proposed that
those proteins interact with each other comprising the FAK-Src-Paxillin system.
Objective: The current study tests the hypothesis that the expression of the
FAK-Src-Paxillin system is increased in a. human NB cell lines and b. in biopsy
specimens of patients with advanced stage of NB and is associated with poor
clinical outcome.
Design/Methods: Initially, the cells of the following cell lines were cultured
in culture media: Κ562, ΝΙΗ/3T3, CCF-STTG1, SKNSH, IMR-32 and U-87-MG.
Subsequently, using western blot, the expression of FAK, Src kinase and
Paxillin was analyzed semi-quantitatively based on the intensity of the
staining using specific antibodies for these proteins. (Stark 1979).
Using immunohistochemistry with monoclonal antibody SC 1688 anti FAK, SC 5266
anti SRC and SC anti Paxillin, we determined the expression of FAK, Src kinase
and Paxillin in tissue specimens of 23 children with advanced (Stage IV) NB.
The biopsy material was obtained with Fine Needle Aspiration (FNA). The tissue
was maintained in the form of cell blocks (Orell and Sterrett's Fine Needle
Aspiration Cytology, Philadelphia 2005). The expression of FAK-Src-Paxillin for
each patient was correlated with the clinical outcome and with the presence of
N-Myc amplification.
Results: In human NB cell lines, the expression of FAK, Src kinase and Paxillin
was increased compared to the expression of b-actin.
The expression of FAK, Src kinase and Paxillin staining was present in 59%, 32%
and 33% respectively in biopsy specimens of children with neuroblastoma.
Simultaneous FAK, Src kinase and Paxillin staining was present in 30% of all
the patients studied. Out of the patients that did not survive, 70% showed
expression of FAK-Src-Paxillin proteins. Only 14% of the patients who died were
found to stain negative for all three proteins although they expressed positive
for N-myc amplification. The isolated increased expression of Paxillin was
associated to poorer survival and disease persistence. When the FAK and Src
kinase proteins were taken individually there was no statistically significant
association between its expression and the survival of the children. However,
when we analyzed the impact of simultaneous expression of the proteins to the
survival we found an additive effect. Children who were positive for all three
proteins and the N-Myc gene have had poor outcome when compared to children who
were negative for the proteins and the N-Myc gene.
Conclusions: The data provide evidence that FAK-Src-Paxillin system is
expressed by advanced-stage NB and provide a rationale for inhibition of these
proteins. We propose that the concurrent inhibition of the FAK-Src-Paxillin
system, with new-generation selective small molecules, may yield better results
in patients with NB.
