Smoothened-independent activation of hedgehog signaling by rearranged during transfection promotes neuroblastoma cell proliferation and tumor growth.
Rearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) signaling, and high RET expression is closely related to the tumorigenesis and malignancy of neuroblastoma(NB).



We have investigated whether RET signals through hedgehog (HH) pathway in NB cell proliferation and tumor growth by in vitro cell culture and in vivo xenograft approaches.



The key members of both GDNF/RET and HH/GLI pathways are expressed in NB cell lines to different extents. Knockdown of RET in NB cells significantly attenuates the activity of HH signaling, whereas overexpression of RET robustly enhances the output of transcriptional activation by HH. Likewise, activation of RET by GDNF induces HH signaling, whereas knockdown of RET attenuates both basal and GDNF-induced activities of HH signaling. Moreover, protein kinase B lies on the downstream of GDNF/RET signaling module to inhibit the GSK3β, resulting in activation of HH signaling. Furthermore, either knockdown of RET by shRNA or inhibition of HH pathway by cyclopamine attenuates not only basal but also GDNF-induced proliferation of SH-SY5Y cells, and knockdown of either RET or smoothened in SH-SY5Y cell xenografts significantly attenuated the tumor growth. Finally, inhibition of HH signaling by GLI1 and GLI2 inhibitor, Gant61, reduces not only basal but also RET-induced proliferation of SH-SY5Y cells and outgrowth of xenografts.



GDNF/RET/AKT/GSK3β signaling module activates HH pathway to stimulate NB cells proliferation and tumor outgrowth.



Targeting HH pathway is a rational approach for therapeutic intervention of NB with high RET expression.
Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, China; Department of Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, China; Institute of Orthopaedics and Traumatology, Zhejiang Chinese Medical University, Hangzhou 310053, China.
Hongfeng
H
Ruan
Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, China; Department of Pharmacy, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
Huan
H
Luo
Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, China.
Jirong
J
Wang
Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, China.
Xing
X
Ji
Department of Pharmacy, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
Zhongmiao
Z
Zhang
Department of Emergence, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
Junsong
J
Wu
Department of Genetics, School of Medicine, Zhejiang University, Hangzhou 310058, China. Electronic address: zhangxianning@zju.edu.cn.
Xianning
X
Zhang
Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, China. Electronic address: xiwu@zju.edu.cn.
Ximei
X
Wu
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta / Q000378:metabolism; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000037:antagonists & inhibitors; D009447:Neuroblastoma / Q000378:metabolism*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors; D000090063:Proto-Oncogene Mas; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051096:Proto-Oncogene Proteins c-ret / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014162:Transfection / Q000379:methods; D014704:Veratrum Alkaloids / Q000494:pharmacology; D000071676:Zinc Finger Protein GLI1 / Q000037:antagonists & inhibitors; D000075746:Zinc Finger Protein Gli2
C551027:GANT 61; C082593:GLI2 protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D053823:Hedgehog Proteins; D051741:Kruppel-Like Transcription Factors; C000715470:MAS1 protein, human; D009687:Nuclear Proteins; D000090063:Proto-Oncogene Mas; D011725:Pyridines; D011743:Pyrimidines; D034741:RNA, Small Interfering; D014704:Veratrum Alkaloids; D000071676:Zinc Finger Protein GLI1; D000075746:Zinc Finger Protein Gli2; D051096:Proto-Oncogene Proteins c-ret; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; C000541:cyclopamine
GDNF; Hedgehog; Neuroblastoma; RET