Background
The MYC family promotes neuroblastoma tumorigenesis at least in part through the induction of aerobic glycolysis by promoting the transcription of key glycolytic enzymes, such as LDHA. FX11 is a selective inhibitor of LDHA that has demonstrated preclinical efficacy in adult cancers. Herein, we hypothesized that FX11 would inhibit aerobic glycolysis and block growth of neuroblastoma cells.
Methods
We surveyed three MYCN-single copy and five MYCN-amplified neuroblastoma cell lines to correlate C-MYC/N-MYC protein levels with LDHA expression. Cell viability was measured with FX11 using a tetrazolium-based assay. Cell cycle analysis using propidium iodide with flow cytometry was performed to evaluate for growth arrest. Immunoblotting demonstrated PARP and Caspase 3 cleavage as evidence of apoptosis.
Results
LDHA is frequently expressed in both MYCN-amplified and MYCN-single copy cell lines. N-MYC and C-MYC protein levels did not correlate with LDHA protein expression. FX11 inhibits aerobic glycolysis and growth in three MYCN-amplified and one MYCN-single copy neuroblastoma cell lines. FX11 induces modest G1 cell cycle arrest with selective induction of apoptosis.
Conclusions
Small molecule LDHA inhibition is capable of blocking aerobic glycolysis and growth of neuroblastoma cell lines in vitro and merits further in vivo evaluation of its preclinical efficacy in neuroblastomas.
Neuroblastoma arises from the neural crest, the precursor cells of the
sympathoadrenal axis, and differentiation status is a key prognostic factor used
for clinical risk group stratification and treatment strategies. Neuroblastoma
tumor-initiating cells have been successfully isolated from patient tumor
samples and bone marrow using sphere culture, which is well established to
promote growth of neural crest stem cells. However, accurate quantification of
sphere-forming frequency of commonly used neuroblastoma cell lines has not been
reported. Here, we show that MYCN-amplified neuroblastoma cell
lines form spheres more frequently than non-MYCN-amplified cell
lines. We also show that sphere formation is directly sensitive to cellular
differentiation status. 13-cis-retinoic acid is a clinically
used differentiating agent that induces a neuronal phenotype in neuroblastoma
cells. Induced differentiation nearly completely blocked sphere formation.
Furthermore, sphere formation was specifically FGF-responsive and did not
respond to increasing doses of EGF. Taken together, these data suggest that
sphere formation is an accurate method of quantifying the stemness phenotype in
neuroblastoma.
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