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).
J Natl Cancer Inst. 2014 Apr;106(4):dju012. doi: 10.1093/jnci/dju012. Epub 2014 Mar 7.
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.
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.
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).
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.
天冬酰胺内肽酶（AEP）参与了人类癌症的发展。然而，AEP 调控的分子机制，包括前体-AEP 激活的作用，仍然难以捉摸。
我们使用生化分析、共聚焦显微镜、免疫电子显微镜和迁移侵袭试验，研究了 TRAF6 对 AEP 的调控作用及其在癌细胞系、小鼠模型和患者标本中的肿瘤进展和转移的影响。通过酶联免疫吸附试验检测了健康供体和乳腺癌患者的血清，并通过免疫组织化学检测了 314 例乳腺癌标本的 AEP 和 TRAF6 的组织阵列。此外，还在小鼠模型中评估了 AEP 抑制剂或单克隆抗体对肺转移的影响。使用双尾学生 t 检验确定组间的统计学意义。
我们证明 TRAF6 通过 K63 连接的多泛素化使 AEP 的前体发生泛素化，这种泛素化可被 USP17 逆转，并与 HSP90α 形成复合物，从而促进前体-AEP 的细胞内稳定性和分泌。破坏前体-AEP 与 TRAF6 的相互作用或抑制 HSP90α 可减少前体-AEP 的分泌，从而减少肿瘤转移。在乳腺癌患者的血清中检测到较高的循环 AEP 水平，AEP 抑制剂或中和抗体可显著减少小鼠模型中的肿瘤转移。值得注意的是，TRAF6 和 AEP 在人类乳腺癌肿瘤中过度表达，与不良预后相关。AEP/TRAF6 低表达的患者平均存活 111 个月（95%置信区间[CI] = 108 至 115 个月），而 AEP/TRAF6 高表达的患者平均存活 61 个月（95%CI = 42 至 79 个月；P<.001）。
我们的研究阐明了 AEP 调控的新机制和 TRAF6 在乳腺癌中的另一种致癌途径，这表明 AEP 和 TRAF6 蛋白水平可能对乳腺癌患者具有预后意义。因此，AEP 可能既是一种生物标志物，也是一个新的治疗靶点。
