SMAD9-MYCN positive feedback loop represents a unique dependency for MYCN-amplified neuroblastoma.
SMAD9-MYCN positive feedback loop represents a unique dependency for MYCN-amplified neuroblastoma.
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DOI：
10.1186/s13046-022-02563-3
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发表时间：
2022-12-20
影响因子：
11.3
通讯作者：
Lv, Zhibao
中科院分区：
文献类型：
--
作者：
Tan, Kezhe;Mo, Jialin;Li, Meng;Dong, Yu;Han, Yujie;Sun, Xi;Ma, Yingxuan;Zhu, Kai;Wu, Wei;Lu, Li;Liu, Jiangbin;Zhao, Kewen;Zhang, Lei;Tang, Yujie;Lv, Zhibao
神经母细胞瘤是儿童时期最常见的颅外实体瘤，高危神经母细胞瘤患者预后较差。扩增的MYCN基因是NB高危的重要决定因素。我们利用公共的NB组织和细胞系数据进行了综合筛查，发现SMAD9在高危NB中发挥了重要作用。通过对超级增强子数据库(SEDB)和染色质免疫沉淀测序(CHIP-SEQ)数据库的研究，以及加入基因敲除和CRISPR干扰(CRISPRi)的生物学实验，以确定Smad9的上游调控机制。采用基因敲除与挽救、实时定量聚合酶链式反应(Q-RT-PCR)、细胞滴度Glo分析、克隆形成实验、异种皮下移植模型和免疫组织化学等方法研究Smad9在NB中的作用。对芯片序列数据进行综合分析，结合CRISPRi和双荧光素酶报告实验的验证，以及RNA序列(RNA-SEQ)数据和Q-RT-PCR验证的验证，分析SMAD9的下游调控机制。Smad9的高表达是由增强子区的转录因子MYCN、PHOX2B、GATA3和HAND2特异性诱导的。在体内外，Smad9的基因抑制抑制了MYCN扩增的NB细胞的增殖和致瘤性。进一步的研究表明，SMAD9与MYCN启动子结合并在转录上调节MYCN的表达，而MYCN与SMAD9增强子相互结合并反式激活SMAD9，从而形成与MYCN相关的癌细胞周期的正反馈环。这项研究表明，SMAD9与MYCN形成一个正转录反馈环，并代表着MYCN扩增的神经母细胞瘤独特的肿瘤依赖性。网上版载有补充材料，可在10.1186/s13046-022-02563-3查阅。
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. 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. 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. This study delineates that SMAD9 forms a positive transcriptional feedback loop with MYCN and represents a unique tumor-dependency for MYCN-amplified neuroblastoma. The online version contains supplementary material available at 10.1186/s13046-022-02563-3.
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影响因子：
25
作者：
Ito-Ishida A;Yamalanchili HK;Shao Y;Baker SA;Heckman LD;Lavery LA;Kim JY;Lombardi LM;Sun Y;Liu Z;Zoghbi HY
通讯作者：
Zoghbi HY
影响因子：
48
作者：
Langmead, Ben;Salzberg, Steven L.
通讯作者：
Salzberg, Steven L.
影响因子：
11.2
作者：
Izumi, Hideki;Kaneko, Yasuhiko
通讯作者：
Kaneko, Yasuhiko
影响因子：
4.7
作者：
Otte J;Dyberg C;Pepich A;Johnsen JI
通讯作者：
Johnsen JI
影响因子：
16
作者：
Heinz S;Benner C;Spann N;Bertolino E;Lin YC;Laslo P;Cheng JX;Murre C;Singh H;Glass CK
通讯作者：
Glass CK