title: Functional role of asparaginyl endopeptidase ubiquitination by TRAF6 in tumor invasion and metastasis.
abstract: BACKGROUND
  Asparaginyl endopeptidase (AEP) has been implicated in human cancer development. However, the molecular mechanisms underlying AEP regulation, including the role of pro-AEP activation, remain elusive.
  
  
  METHODS
  We investigated the regulation of AEP by TRAF6 and its effects on tumor progression and metastasis in cancer cell lines, murine models, and specimens from patients using biochemical analyses, confocal microscopy, immunoelectron microscopy, and migration-invasion assays. The sera of healthy donors and breast cancer patients were examined by enzyme-linked immunosorbent assay, and a tissue array of 314 breast cancer specimens was assessed for AEP and TRAF6 by immunohistochemistry. Furthermore, the effects of AEP inhibitors or monoclonal antibodies on pulmonary metastasis were evaluated in murine models. The statistical significance between groups was determined using two-tailed Student t tests.
  
  
  RESULTS
  We demonstrate that TRAF6 ubiquitinates the proform of AEP through K63-linked polyubiquitin, reversible by USP17, and forms a complex with HSP90α to subsequently promote pro-AEP intracellular stability as well as secretion. Disrupting the interaction between pro-AEP and TRAF6 or inhibiting HSP90α reduced pro-AEP secretion and consequently reduced tumor metastasis. Higher circulating AEP levels were detected in the sera of breast cancer patients, and AEP inhibitors or neutralizing antibodies remarkably decreased tumor metastasis in murine models. Notably, TRAF6 and AEP were overexpressed in human breast neoplasms and correlated with poor prognosis. Patients with low AEP/TRAF6 expression survived for a mean of 111 months (95% confidence interval [CI] = 108 to 115 months), whereas those with high AEP/TRAF6 expression survived for a mean of only 61 months (95% CI = 42 to 79 months; P < .001).
  
  
  CONCLUSIONS
  Our study elucidates a novel mechanism of AEP regulation and an alternative oncogenic pathway for TRAF6 in breast cancer, which suggests that AEP and TRAF6 protein levels may have prognostic implications in breast cancer patients. Thus, AEP may serve as a biomarker as well as new therapeutic target.
authors.affiliation: Affiliations of authors: Laboratory of Targeted Tumor Therapy, Key Laboratory of Systems Biology, Shanghai Advanced Research Institute (QL, BP, CW, YZ, FG) Institute of Health Sciences (YL, XC, BL, FG) Chinese Academy of Sciences, Shanghai, China; Department of Neurosurgery, Renji Hospital (YQ,YL) Shanghai First People's Hospital (YG) and Department of Radiation Oncology, Ruijin Hospital (CX), School of Medicine, Shanghai Jiao-Tong University, Shanghai, China; Scripps Research Institute, La Jolla, CA (GFK, TSE); Sorrento Thearpeutics, Inc, San Diego, CA (GFK); Department of Neuro-Oncology, University of Texas, MD Anderson Cancer Center, Houston, TX (ZL); Cancer Research Center, Shenzhen University, Shenzhen, China (DL); Shanghai Institute for Advanced Immunochemical Studies, Shanghai Tech University, Shanghai, China (BJ).
authors.forename: Yingying
authors.initials: Y
authors.lastname: Lin
authors.forename: Yongming
authors.initials: Y
authors.lastname: Qiu
authors.forename: Cheng
authors.initials: C
authors.lastname: Xu
authors.forename: Qiaoling
authors.initials: Q
authors.lastname: Liu
authors.forename: Baowei
authors.initials: B
authors.lastname: Peng
authors.forename: Gunnar F
authors.initials: GF
authors.lastname: Kaufmann
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Bin
authors.initials: B
authors.lastname: Lan
authors.forename: Chongyang
authors.initials: C
authors.lastname: Wei
authors.forename: Desheng
authors.initials: D
authors.lastname: Lu
authors.forename: Yueshan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Guo
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Lu
authors.forename: Biao
authors.initials: B
authors.lastname: Jiang
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Edgington
authors.forename: Fang
authors.initials: F
authors.lastname: Guo
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D018450:Disease Progression; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009374:Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D011237:Predictive Value of Tests; D011379:Prognosis; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism*; D054875:Ubiquitination; D000099240:Asparaginyl Endopeptidase
chemical_list: D014408:Biomarkers, Tumor; D003546:Cysteine Endopeptidases; D018841:HSP90 Heat-Shock Proteins; D048029:TNF Receptor-Associated Factor 6; D000099240:Asparaginyl Endopeptidase; C533585:HSP90AA2P protein, human