SMAD9-MYCN positive feedback loop represents a unique dependency for MYCN-amplified neuroblastoma

Abstract

Abstract Background Neuroblastoma (NB) is the most common extracranial solid tumor occurring during childhood and high-risk NB patients have a poor prognosis. The amplified MYCN gene serves as an important determinant of a high risk of NB. Methods We performed an integrative screen using public NB tissue and cell line data, and identified that SMAD9 played an important role in high-risk NB. An investigation of the super-enhancers database (SEdb) and chromatin immunoprecipitation sequencing (ChIP-seq) dataset along with biological experiments of incorporating gene knockdown and CRISPR interference (CRISPRi) were performed to identify upstream regulatory mechanism of SMAD9. Gene knockdown and rescue, quantitative real-time PCR (Q-RT-PCR), cell titer Glo assays, colony formation assays, a subcutaneous xenograft model and immunohistochemistry were used to determine the functional role of SMAD9 in NB. An integrative analysis of ChIP-seq data with the validation of CRISPRi and dual-luciferase reporter assays and RNA sequencing (RNA-seq) data with Q-RT-PCR validation was conducted to analyze the downstream regulatory mechanism of SMAD9. Results High expression of SMAD9 was specifically induced by the transcription factors including MYCN, PHOX2B, GATA3 and HAND2 at the enhancer region. Genetic suppression of SMAD9 inhibited MYCN-amplified NB cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that SMAD9 bound to the MYCN promoter and transcriptionally regulate MYCN expression, with MYCN reciprocally binding to the SMAD9 enhancer and transactivating SMAD9, thus forming a positive feedback loop along with the MYCN-associated cancer cell cycle. Conclusion This study delineates that SMAD9 forms a positive transcriptional feedback loop with MYCN and represents a unique tumor-dependency for MYCN-amplified neuroblastoma.