title: Nuclear Localization of DNAJB6 Is Associated With Survival of Patients With Esophageal Cancer and Reduces AKT Signaling and Proliferation of Cancer Cells.
abstract: OBJECTIVE
  The DnaJ (Hsp40) homolog, subfamily B, member 6 (DNAJB6) is part of a family of proteins that regulates chaperone activities. One of its isoforms, DNAJB6a, contains a nuclear localization signal and regulates β-catenin signaling during breast cancer development. We investigated the role of DNAJB6 in the pathogenesis of esophageal squamous cell carcinoma (ESCC).
  
  
  METHODS
  We performed immunohistochemical analyses of primary ESCC samples and lymph node metastases from a cohort of 160 patients who underwent esophagectomy with no preoperative chemoradiotherapy at Hong Kong Queen Mary Hospital. Data were collected on patient outcomes over a median time of 12.1 ± 2.9 months. Retrospective survival association analyses were performed. Wild-type and mutant forms of DNAJB6a were overexpressed in cancer cell lines (KYSE510, KYSE 30TSI, KYSE140, and KYSE70TS), which were analyzed in proliferation and immunoblot assays, or injected subcutaneously into nude mice. Levels of DNAJB6 were knocked down in ESCC cell lines (KYSE450 and T.Tn), immortalized normal esophageal epithelial cell lines (NE3 and NE083), and other cells with short hairpin RNAs, or by genome engineering. Bimolecular fluorescence complementation was used to study interactions between proteins in living cells.
  
  
  RESULTS
  In primary ESCC samples, patients whose tumors had high nuclear levels of DNAJB6 had longer overall survival times (19.2 ± 1.8 months; 95% confidence interval [CI], 15.6-22.8 mo) than patients whose tumors had low nuclear levels of DNAJB6 (12.6 ± 1.4 mo; 95% CI, 9.8-15.4 mo; P = .004, log-rank test). Based on Cox regression analysis, patients whose tumors had high nuclear levels of DNAJB6 had a lower risk of death than patients with low levels (hazard ratio, 0.562; 95% CI, 0.379-0.834; P = .004). Based on log-rank analysis and Cox regression analysis, the combination of the nuclear level of DNAJB6 and the presence of lymph node metastases at diagnosis could be used to stratify patients into groups with good or bad outcomes (P < .0005 for both analyses). There was a negative association between the nuclear level of DNAJB6 and the presence of lymph node metastases (P = .022; Pearson χ(2) test). Cancer cell lines that overexpressed DNAJB6a formed tumors more slowly in nude mice than control cells or cells that expressed a mutant form of DNAJB6a that did not localize to the nucleus. DNAJB6 knockdown in cancer cell lines promoted their growth as xenograft tumors in mice. A motif of histidine, proline, and aspartic acid in the J domain of DNAJB6a was required for its tumor-suppressive effects and signaling via AKT1. Loss of DNAJB6a resulted in up-regulation of AKT signaling in cancer cell lines and immortalized esophageal epithelial cells. Expression of a constitutively active form of AKT1 restored proliferation to tumor cells that overexpressed DNAJB6a, and DNAJB6a formed a complex with AKT1 in living cells. The expression of DNAJB6a reduced the sensitivity of ESCC to AKT inhibitors; the expression level of DNAJB6a affected AKT signaling in multiple cancer cell lines.
  
  
  CONCLUSIONS
  Nuclear localization of DNAJB6 is associated with longer survival times of patients with ESCC. DNAJB6a reduces AKT signaling, and DNAJB6 expression in cancer cells reduces their proliferation and growth of xenograft tumors in mice. DNAJB6a might be developed as a biomarker for progression of ESCC.
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Valen Zhuoyou
authors: VZ
authors: Yu
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Victor Chun-Lam
authors: VC
authors: Wong
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Wei
authors: W
authors: Dai
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Josephine Mun-Yee
authors: JM
authors: Ko
authors: Department of Cancer Molecular Pathology, Griffith Medical School and Menzies Health Institute Queensland, Griffith University, Gold Coast, Australia.
authors: Alfred King-Yin
authors: AK
authors: Lam
authors: Department of Pathology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region; Center for Cancer Research, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Kwok Wah
authors: KW
authors: Chan
authors: Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama.
authors: Rajeev S
authors: RS
authors: Samant
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region; Center for Cancer Research, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Hong Lok
authors: HL
authors: Lung
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Wai Ho
authors: WH
authors: Shuen
authors: Center for Cancer Research, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region; Department of Surgery, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Simon
authors: S
authors: Law
authors: Department of Pathology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Yuen Piu
authors: YP
authors: Chan
authors: Center for Cancer Research, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region; Department of Surgery, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Nikki Pui-Yue
authors: NP
authors: Lee
authors: Department of Surgery, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Daniel King Hung
authors: DK
authors: Tong
authors: Department of Surgery, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Tsz Ting
authors: TT
authors: Law
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region; Center for Cancer Research, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region.
authors: Victor Ho-Fun
authors: VH
authors: Lee
authors: Department of Clinical Oncology, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region; Center for Cancer Research, University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong, Special Administrative Region. Electronic address: mlilung@hku.hk.
authors: Maria Li
authors: ML
authors: Lung
mesh_terms: D021581:Active Transport, Cell Nucleus; D000368:Aged; D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000201:enzymology* / Q000235:genetics / Q000401:mortality / Q000473:pathology / Q000601:surgery; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000401:mortality / Q000473:pathology / Q000601:surgery; D000077277:Esophageal Squamous Cell Carcinoma; D016629:Esophagectomy; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D064593:Heterografts; D006801:Humans; D053208:Kaplan-Meier Estimate; D008207:Lymphatic Metastasis; D008297:Male; D008819:Mice, Nude; D008875:Middle Aged; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016016:Proportional Hazards Models; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012189:Retrospective Studies; D012307:Risk Factors; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D016896:Treatment Outcome
chemical_list: C485565:DNAJB6 protein, human; D050956:HSP40 Heat-Shock Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D047428:Protein Kinase Inhibitors; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt
keywords: Prognostic Marker; Signal Transduction; Tissue Microarray; Tumor Suppressor