Modulation of chemotherapeutic drug resistance in neuroblastoma SK-N-AS cells by the neural apoptosis inhibitory protein and miR-520f.

The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research.

MicroRNA-34a is a potent tumor suppressor molecule in vivo in neuroblastoma.

BACKGROUND: Neuroblastoma is a paediatric cancer which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis. METHODS: A synthetic miR-34a (or negative control) precursor molecule was transfected into NB1691luc and SK-N-ASluc neuroblastoma cells. Quantitative PCR was used to verify increased miR-34a levels in NB1691luc and SK-N-ASluc cell lines prior to in vitro and in vivo analysis. In vitro analysis of the effects of miR-34a over expression on cell growth, cell cycle and phosphoprotein activation in signal transduction pathways was performed. Neuroblastoma cells over expressing miR-34a were injected retroperitoneally into immunocompromised CB17-SCID mice and tumor burden was assessed over a 21 day period by measuring bioluminescence (photons/sec/cm²). RESULTS: Over expression of miR-34a in both NB1691luc and SK-N-ASluc neuroblastoma cell lines led to a significant decrease in cell number relative to premiR-negative control treated cells over a 72 hour period. Flow cytometry results indicated that miR-34a induced cell cycle arrest and subsequent apoptosis activation. Phosphoprotein analysis highlighted key elements involved in signal transduction, whose activation was dysregulated as a result of miR-34a introduction into cells. As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels. Although MAP3K9 is a predicted target of miR-34a, direct targeting could not be validated with luciferase reporter assays. Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved. Most notably, in vivo studies showed that tumor growth was significantly repressed after exogenous miR-34a administration in retroperitoneal neuroblastoma tumors. CONCLUSION: We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.