TY - JOUR
T1 - Nuclear Localization of DNAJB6 Is Associated with Survival of Patients with Esophageal Cancer and Reduces AKT Signaling and Proliferation of Cancer Cells
AU - Yu, Valen Zhuoyou
AU - Wong, Victor Chun Lam
AU - Dai, Wei
AU - Ko, Josephine Mun Yee
AU - Lam, Alfred King Yin
AU - Chan, Kwok Wah
AU - Samant, Rajeev S.
AU - LUNG, Hong Lok
AU - Shuen, Wai Ho
AU - Law, Simon
AU - Chan, Yuen Piu
AU - Lee, Nikki Pui Yue
AU - Tong, Daniel King Hung
AU - Law, Tsz Ting
AU - Lee, Victor Ho Fun
AU - Lung, Maria Li
N1 - Funding Information:
The authors thank Professor Masaki Inagaki (Aichi Cancer Center Research Institute, Japan) for providing the pRK5-Myc-DNAJB6b plasmid. The authors acknowledge the German Collection of Microorganisms and Cell Culture for the KYSE cell lines. The authors thank Professor Gopesh Srivastava, Dr Annie Lai Man Cheung, and Professor George Tsao for cell lines. The authors acknowledge the University of Hong Kong Faculty of Medicine Core Facility for providing facilities for flow cytometry, confocal microscopy imaging, and live animal imaging; and the staff on the Cancer Molecular Pathology team at Griffith University for setting up one of the tissue microarrays. Supported by the General Research Fund from the Research Grants Council of the Hong Kong Special Administrative Region, People''s Republic of China (HKU 774411M to M.L.L.); and Griffith University funded one of the tissue microarrays.
PY - 2015/12/1
Y1 - 2015/12/1
N2 - Background & Aims 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 χ2 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.
AB - Background & Aims 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 χ2 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.
KW - Prognostic Marker
KW - Signal Transduction
KW - Tissue Microarray
KW - Tumor Suppressor
UR - http://www.scopus.com/inward/record.url?scp=84952983430&partnerID=8YFLogxK
U2 - 10.1053/j.gastro.2015.08.025
DO - 10.1053/j.gastro.2015.08.025
M3 - Journal article
C2 - 26302489
AN - SCOPUS:84952983430
SN - 0016-5085
VL - 149
SP - 1825-1836.e5
JO - Gastroenterology
JF - Gastroenterology
IS - 7
ER -