Abstract
BackgroundNeuroblastoma is the most common pediatric extra-cranial nervous system tumor, originating from neural crest elements and giving rise to tumors in the adrenal medulla and sympathetic chain ganglia. Amplification of MYCN confers increased malignancy and poorer prognosis in high-risk neuroblastoma. Our SILAC proteomics analysis revealed over-expression of HSP90 in MYCN-amplified IMR-32 compared to the non-MYCN amplified SK-N-SH human neuroblastoma cells, rendering them highly resistant to therapeutic intervention.MethodsWe used cellular bio-functional (proliferation, migration/invasion, apoptosis, viability and stem-cell self-renewal) assays and Western blot analysis to elucidate the therapeutic efficacy of HSP90 inhibition with 17-AAG.Results17-AAG treatment significantly inhibited cellular proliferation, viability and migration/invasion and increased apoptosis in both cell lines. Moreover, drug treatment significantly abrogated stem-cell self-renewal potential in the MYCN-amplified IMR-32 cells. Differential tumorigenic protein expression revealed a novel mechanism of therapeutic efficacy after 17-AAG treatment with a significant downregulation of HMGA1, FABP5, Oct4, MYCN, prohibitin and p-L1CAM in SK-N-SH cells. However, we observed a significant up-regulation of p-L1CAM, MYCN and prohibitin, and significant down-regulation of Oct4, FABP5, HMGA1, p-ERK, cleaved/total caspase-3 and PARP1 in IMR-32 cells.ConclusionsHSP90 inhibition revealed a novel therapeutic mechanism of antitumor activity in MYCN-amplified neuroblastoma cells that may enhance therapeutic sensitivity.
Highlights
Heat Shock Proteins (HSPs) have multiple roles in eukaryotic cells, they mainly serve as molecular chaperones having a central role in cell survival and development [1]
Our SILAC proteomics analysis revealed over-expression of HSP90 in MYCN-amplified IMR-32 compared to the non-MYCN amplified SK-N-SH human neuroblastoma cells, rendering them highly resistant to therapeutic intervention
The results showed an important reduction in the stem cell capacity of sphere formation in the IMR-32 cells (Figure 10) whereby Sphere Formation Efficiency (SFE) was severely reduced from 20% and 10% (5% fetal bovine serum (FBS) and SC media, respectively) in the vehicletreated wells to less than 1% in the drug-treated wells with both 0.5 and 1 mM concentrations (p
Summary
Heat Shock Proteins (HSPs) have multiple roles in eukaryotic cells, they mainly serve as molecular chaperones having a central role in cell survival and development [1]. They facilitate native protein folding and stabilization, protein translocation, protein re-folding and renaturation, or degradation for proteins that are beyond rescuing [2], ensuring protein quality control and preventing protein aggregation that would otherwise lead to apoptosis or necrosis [3]. HSPs are classified based on the molecular mass, where the 90-kDa group consists of two major isoforms: the inducible HSP90a and the constitutively expressed HSP90b [3]. Our SILAC proteomics analysis revealed over-expression of HSP90 in MYCN-amplified IMR-32 compared to the non-MYCN amplified SK-N-SH human neuroblastoma cells, rendering them highly resistant to therapeutic intervention
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