RUNX3 Destabilize MYCN In Neuroblastoma

Objective:Neuroblastoma is the most common malignant extracranial solid tumor of childhood. This neuronal cancer accounts for 15% of cancer deaths in children. In neuroblastoma, amplification of oncogene MYCN is the best characterized genetic factor for high-risk tumor and therapy resistance. RUNX3, which encodes the Runt-related transcription factor, was identified as a tumor suppressor gene in solid tumors of diverse origins, such as gastric cancer, lung cancer, colon cancer, but neuroblastoma. We previously found that high expression of RUNX3 is correlated with better prognosis in neuroblastoma patients in which MYCN is highly expressed. We therefore concluded that RUNX3 is a novel tumor suppressor in neuroblastma, related to suppressing the oncogenic activity of MYCN. The objective of this research is to elucidate the potential mechanism of RUNX3 to inhibit oncogene product MYCN.Methods:In this research, We chose MYCN amplified neuroblastoma cell lines SK-N-BE, NLF and MYCN non-amplified neuroblastoma cell lines SY5Y, SK-N-AS as well as human embryonic kidney cell line HEK293. Transfection was used to enhance RUNX3 and MYCN expression levels in above cell lines. Colony formation assays were used to check the effect of RUNX3 overexpression in different neuroblastoma cell lines. PCR and Western blot were used to research the mRNA and protein levels of MYCN with and without RUNX3 transfection. MYCN ubiquitination assays were performed to determine MYCN degradation process with and without RUNX3 transfection. Immunoprecipition were used to identify the physical interactions between RUNX3 and MYCN. Results:1. Ectopic expression of RUNX3 led to a strong reduction in colony formation in both MYCN amplified and non-amplified neuroblastoma cell lines, confirming the tumor suppressor activity of RUNX3 in neuroblastoma.2. The effect of overexpressing RUNX3 in neuroblastoma cell lines.2.1 The protein levels of MYCN were decreased by overexpressed RUNX3 in a dose dependent manner in MYCN amplified neuroblastoma cell lines such as SK-N-BE and NLF. But mRNA levels of MYCN were not changed by ectopic RUNX3 expression.2.2 Time Course experiment indicated that MYCN protein levels decreased in response to RUNX3 overexpression.2.3 Overexpression of RUNX3 shortened the half-life of MYCN protein.2.4 Immunofluorescence microscopy showed that RUNX3 and MYCN are mutually exclusive in the nucleus.These results suggest that RUNX3 regulates MYCN through post-translational mechanism.3. RUNX3-mediated MYCN degradation was inhibited by a proteasome inhibitor MG132, indicating that RUNX3 destabilizes MYCN through the proteasome function.4. Physical interaction between RUNX3 and MYCN.4.1 Immunoprecipitation assay demonstrates that RUNX3 interacts with MYCN in vivo.4.2 The GST pulldown assay showed that the Runt domain of RUNX3 is responsible for the binding of RUNX3 and MYCN.4.3 RUNX3 and MYCN co-localized in nucleus detected by immunofluorescence microscopy. These results suggest that RUNX3 and MYCN physically interact in vitro and in vivo.5. Overexpression of RUNX3 yielded accumulation of ubiquitin-conjugated MYCN protein in the presence of proteasome inhibitor MG132 treatment, suggesting that RUNX3 promotes ubiquitination of MYCN, results in MYCN degradation.Conclusion:RUNX3 interacts with MYCN in neuroblastoma, And facilitates MYCN degradation. This molecular mechanism may explain tumor suppressor activity of RUNX3 in neurblastoma.Concluding remark:Amplification of oncogene MYCN is an important indicator to predict risk, response to therapy, life-time survival, and overall mortality in neuroblastoma patients. In this study, I reported that RUNX3 invalidates oncogenic MYCN function by promoting degradation of MYCN protein. This key feature of RUNX3 may help to develop a novel therapeutic approach to block MYCN signaling by increasing RUNX3 activity in neuroblastoma patients.