TSPAN family of proteins are generally considered to assemble as multimeric complexes on the plasma membrane.Our previous work uncovered that TSPAN8 can translocate into the nucleus as a membrane-free form,a process that requires TSPAN8 palmitoylation and association with cholesterol to promote its extraction from the plasma membrane and subsequent binding with 14-3-3θ and importin-β.However,what upstream signal(s)regulate(s)the nuclear translocation of TSPAN8,the potential function of TSPAN8 in the nucleus,and the underlying molecular mechanisms all remain unclear.Here,we demonstrate that,epidermal growth factor receptor(EGFR)signaling induces TSPAN8 nuclear translocation by activating the kinase AKT,which in turn directly phosphorylates TSPAN8 at Ser129,an event essential for its binding with 14-3-3θ and importin B1.In the nucleus,phosphorylated TSPAN8 interacts with STAT3 to enhance its chromatin occupancy and therefore regulates transcription of downstream cancer-promoting genes,such as MYC,BCL2,MMP9,etc.The EGFR-AKT-TSPAN8-STAT3 axis was found to be hyperactivated in multiple human cancers,and associated with aggressive phenotype and dismal prognosis.We further developed a humanized monoclonal antibody hT8Ab4 that specifically recognizes the large extracellular loop of TSPAN8(TSPAN8-LEL),thus being able to block the extraction of TSPAN8 from the plasma membrane and consequently its nuclear localization.Importantly,both in vitro and in vivo studies demonstrated an antitumor effect of hT8Ab4.Collectively,we discovered an unconventional function of TSPAN8 and dissected the underlying molecular mechanisms,which not only showcase a new layer of biological complexity of traditional membrane proteins,but also shed light on TSPAN8 as a novel therapeutic target for refractory cancers.

State Key Laboratory of Oncogenes and Related Genes,Department of Oncology,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,China;Department of Breast Surgery,Shanxi Cancer Hospital,Chinese Academy of Medical Sciences,Taiyuan,Shanxi,China;Department of Medical Ultrasound,Shanghai Tenth People's Hospital,Tongji University Cancer Center,School of Medicine,Tongji University,Shanghai,China;Precision Research Center for Refractory Diseases,Institute for Clinical Research,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,China;Department of Pathology,Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai,China;State Key Laboratory of Membrane Biology,Tsinghua-Peking University Joint Center for Life Sciences,School of Life Science,Tsinghua University,Beijing,China;State Key Laboratory of Genetic Engineering,School of Life Sciences,Fudan University,Shanghai,China

[1]Xiaoqing Lu;Liwei An;Guangjian FanTi;Lijuan Zang;Weiyi Huang;Junjian Li;Jun Liu;Weiyu Ge;Yuwei Huang;Jingxuan Xu;Shaoqian Du;Yuan Cao;Tianhao Zhou;Huijing Yin;Li Yu;Shi Jiao;Hongxia Wang-.EGFR signaling promotes nuclear translocation of plasma membrane protein TSPAN8 to enhance tumor progression via STAT3-mediated transcription)[J].细胞研究（英文版）,2022(04):359-374

TSPAN8,TSPAN,multimeric,translocate,palmitoylation,importin,hT8Ab4

EGFR,signaling,promotes,nuclear,translocation,plasma,membrane,enhance,progression,via,STAT3,mediated,transcription,family,proteins,are,generally,considered,assemble,complexes,Our,previous,work,uncovered,that,into,nucleus,free,form,process,requires,association,cholesterol,its,extraction,from,subsequent,binding,However,what,upstream,potential,function,underlying,molecular,mechanisms,remain,unclear,Here,epidermal,growth,receptor,induces,by,activating,kinase,AKT,which,turn,directly,phosphorylates,Ser129,event,essential,B1,In,phosphorylated,interacts,chromatin,occupancy,therefore,regulates,downstream,promoting,genes,such,MYC,BCL2,MMP9,etc,axis,was,found,hyperactivated,multiple,cancers,associated,aggressive,phenotype,dismal,prognosis,We,further,developed,humanized,monoclonal,antibody,specifically,recognizes,large,extracellular,loop,LEL,thus,being,able,block,consequently,localization,Importantly,both,vitro,vivo,studies,demonstrated,antitumor,effect,Collectively,discovered,unconventional,dissected,only,showcase,new,layer,biological,complexity,traditional,but,also,shed,light,novel,therapeutic,target,refractory

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