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Annals of Surgical Oncology

, Volume 26, Issue 3, pp 907–917 | Cite as

Roles of Pin1 as a Key Molecule for EMT Induction by Activation of STAT3 and NF-κB in Human Gallbladder Cancer

  • Shinichiro Nakada
  • Satoshi KubokiEmail author
  • Hiroyuki Nojima
  • Hideyuki Yoshitomi
  • Katsunori Furukawa
  • Tsukasa Takayashiki
  • Shigetsugu Takano
  • Masaru Miyazaki
  • Masayuki Ohtsuka
Translational Research and Biomarkers

Abstract

Background

Despite developments in multidisciplinary treatment, the prognosis for advanced gallbladder cancer (GBC) still is poor because of its rapid progression. Epithelial–mesenchymal transition (EMT) plays a central role in promoting tumor invasion and metastasis in malignancies thorough signal transducer and activator of transcription-3 (STAT3) and nuclear factor κB (NF-κB) activation. Whereas Pin1 mediates STAT3 and NF-κB activation, the involvement of Pin1 in GBC progression is unclear.

Methods

Factors regulating Pin1-related STAT3 and NF-κB activation were evaluated using surgical specimens collected from 76 GBC patients, GBC cells, and orthotopic GBC xenograft mice.

Results

In the patients with GBC, high Pin1 expression in GBC was associated with aggressive tumor invasion and increased tumor metastasis, and was an independent factor for a poor prognosis. Pin1 expression was correlated with phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276), thereby activating STAT3 and NF-κB in GBC. Pin1-mediated STAT3 and NF-κB activation induced EMT in GBC. When Pin1 knockdown was performed in GBC cells, the phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276) was inhibited, and STAT3 and NF-κB activation was suppressed. Inactivation of STAT3 and NF-κB in Pin1-depleted cells decreased snail and zeb-2 expression, thereby reducing the rate of mesenchymal-like cells, suggesting that EMT was inhibited in GBC cells. PiB, a Pin1-specific inhibitor, inhibited EMT and reduced tumor cell invasion by inactivating STAT3 and NF-κB in vitro. Moreover, PiB treatment inhibited lymph node metastasis and intrahepatic metastasis in orthotopic GBC xenograft tumor in vivo.

Conclusions

Pin1 accelerates GBC invasion and metastasis by activating STAT3 and NF-κB. Therefore, Pin1 inhibition by PiB is an excellent therapy for GBC by safely inhibiting its metastasis.

Notes

Acknowledgment

This work was supported by JSPS KAKENHI (Grant No. 26462036) to Satoshi Kuboki.

Disclosure

There are no conflicts of interest.

Supplementary material

10434_2018_7132_MOESM1_ESM.doc (70 kb)
Supplementary material 1 (DOC 70 kb)
10434_2018_7132_MOESM2_ESM.tif (6.2 mb)
Receiver operating characteristic (ROC) curve analysis of the Pin1-labeling index in accordance with the 2-year survival rate (P < 0.001; AUC, 0.751). AUC, area under the curve
10434_2018_7132_MOESM3_ESM.tif (29.3 mb)
Effects of Pin1 knockdown on (A) STAT3 and (B) NF-κB activation assessed by EMSA in NOZ-1033 cells in vitro. The data are expressed as mean ± SEM with n = 3 for negative control and n = 4 for Pin1 siRNA per group. *P < 0.05 compared with negative control subjects. (C) Cell invasion assay in NOZ-1033 cells with Pin1 knockdown in vitro. The data are expressed as mean ± SEM with n = 3 for negative control and n = 4 for Pin1 siRNA per group. *P < 0.05 compared with negative control subjects. STAT3, signal transducer and activator of transcription-3; NF-κB, nuclear factor κB; EMSA, electrophoretic mobility shift assay; SEM, standard error of the mean
10434_2018_7132_MOESM4_ESM.tif (28.7 mb)
Effects of PiB treatment on (A) STAT3 and (B) NF-κB activation assessed by EMSA in NOZ-1033 cells in vitro. The data are expressed as mean ± SEM with four per group. *P < 0.05 compared with 0 μmol PiB. (C) Cell invasion assay in PiB-treated NOZ-1033 cells in vitro. The data are expressed as mean ± SEM with four per group. *P < 0.05 compared with 0 μmol PiB. STAT3, signal transducer and activator of transcription-3; NF-κB, nuclear factor κB; EMSA, electrophoretic mobility shift assay; SEM, standard error of the mean
10434_2018_7132_MOESM5_ESM.tif (26.5 mb)
Effects of juglone treatment on (A) STAT3 and (B) NF-κB activation assessed by EMSA in OCUG-1 cells in vitro. The data are expressed as mean ± SEM with four per group. *P < 0.05 compared with 0 μmol juglone. (C) Cell invasion assay in juglone-treated OCUG-1 cells in vitro. The data are expressed as mean ± SEM with four per group. *P < 0.05 compared with 0 μmol juglone. STAT3, signal transducer and activator of transcription-3; NF-κB, nuclear factor κB; EMSA, electrophoretic mobility shift assay; SEM, standard error of the mean

References

  1. 1.
    Ishihara S, Horiguchi A, Miyakawa S, Endo I, Miyazaki M, Takada T. Biliary tract cancer registry in Japan from 2008 to 2013. J Hepatobiliary Pancreat Sci. 2016;23:149–57.CrossRefGoogle Scholar
  2. 2.
    Higuchi R, Ota T, Araida T, et al. Surgical approaches to advanced gallbladder cancer: a 40-year single-institution study of prognostic factors and resectability. Ann Surg Oncol. 2014;21:4308–16.CrossRefGoogle Scholar
  3. 3.
    Valle J, Wasan H, Palmer DH, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010;362:1273–81.CrossRefGoogle Scholar
  4. 4.
    Kato A, Shimizu H, Ohtsuka M, et al. Downsizing chemotherapy for initially unresectable locally advanced biliary tract cancer patients treated with gemcitabine plus cisplatin combination therapy followed by radical surgery. Ann Surg Oncol. 2015;22:S1093–9.CrossRefGoogle Scholar
  5. 5.
    Dutta U. Gallbladder cancer: can newer insights improve the outcome? J Gastroenterol Hepatol. 2012;27:642–53.CrossRefGoogle Scholar
  6. 6.
    Thiery JP. Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002;2:442–54.CrossRefGoogle Scholar
  7. 7.
    Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer. 2003;3:362–74.CrossRefGoogle Scholar
  8. 8.
    Min C, Eddy SF, Dherr DH, Sonenshein GS. NF-kappaB and epithelial to mesenchymal transition of cancer. J Cell Biochem. 2008;104:733–44.CrossRefGoogle Scholar
  9. 9.
    Wendt MK, Balanis N, Carlin CR, Schiemann WP. STAT3 and epithelial–mesenchymal transitions in carcinomas. JAKSTAT. 2014;3:e28975.PubMedCentralGoogle Scholar
  10. 10.
    Yu H, Jove R. The STATs of cancer: new molecular targets come of age. Nat Rev Cancer. 2004;4:97–105.CrossRefGoogle Scholar
  11. 11.
    Yaffe MB, Schutkowski M, Shen M, et al. Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science. 1997;278:1957–60.CrossRefGoogle Scholar
  12. 12.
    Lu KP, Liou YC, Zhou XZ. Pinning down proline-directed phosphorylation signaling. Trends Cell Biol. 2002;12:164–72.CrossRefGoogle Scholar
  13. 13.
    Lu KP, Zhou XZ. The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease. Nat Rev Mol Cell Biol. 2007;8:904–16.CrossRefGoogle Scholar
  14. 14.
    Lufei C, Koh TH, Uchida T, Cao X. Pin1 is required for the Ser727 phosphorylation-dependent Stat3 activity. Oncogene. 2007;26:7656–64.CrossRefGoogle Scholar
  15. 15.
    Wakahara R, Kunimoto H, Tanino K, Kojima H, Shintaku H, Nakajima K. Phospho-Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho-Tyr705 largely through TC45. Genes Cells. 2012;17:132–45.CrossRefGoogle Scholar
  16. 16.
    Ryo A, Suizu F, Yoshida Y, et al. Regulation of NF-kappaB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell. 2003;12:1413–26.CrossRefGoogle Scholar
  17. 17.
    Shinoda K, Kuboki S, Shimizu H, et al. Pin1 facilitates NF-κB activation and promotes tumour progression in human hepatocellular carcinoma. Br J Cancer. 2015;113:1323–31.CrossRefPubMedCentralGoogle Scholar
  18. 18.
    Deryckere F, Gannon F. A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques. 1994;16:405.Google Scholar
  19. 19.
    Reichert M, Takano S, Heeg S, Bakir B, Botta GP, Rustgi AK. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells. Nat Protoc. 2013;8:1354–65.CrossRefPubMedCentralGoogle Scholar
  20. 20.
    Egberts JH, Schniewind B, Schafmayer C, et al. Establishment of a novel orthotopic xenograft model of human gallbladder carcinoma. Clin Exp Metastasis. 2007;24:141–8.CrossRefGoogle Scholar
  21. 21.
    Atkinson GP, Nozell SE, Harrison DK, Stonecypher MS, Chen D, Benveniste EN. The prolyl isomerase Pin1 regulates the NF-kappaB signaling pathway and interleukin-8 expression in glioblastoma. Oncogene. 2009;28:3735–45.CrossRefPubMedCentralGoogle Scholar
  22. 22.
    Henning L, Christner C, Kipping M, et al. Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone. Biochemistry. 1998;37:5953–60.CrossRefGoogle Scholar
  23. 23.
    Uchida T, Takamiya M, Takahashi M, et al. Pin1 and Par14 peptidyl prolyl isomerase inhibitors block cell proliferation. Chem Biol. 2003;10:15–24.CrossRefGoogle Scholar
  24. 24.
    Mantovani F, Tocco F, Girardini J, et al. The prolyl isomerase Pin1 orchestrates p53 acetylation and dissociation from the apoptosis inhibitor iASPP. Nat Struct Mol Biol. 2007;14:912–20.CrossRefGoogle Scholar
  25. 25.
    Lin W, Jiang L, Chen Y, et al. Vascular endotherial growth factor-D promotes growth, lymphangiogenesis, and lymphatic metastasis in gallbladder cancer. Cancer Lett. 2012;314:127–36.CrossRefGoogle Scholar
  26. 26.
    Mita Y, Ajiki T, Kamigaki T, et al. Antitumor effect of gemcitabine on orthotopically inoculated human gallbladder cancer cells in nude mice. Ann Surg Oncol. 2012;14:1374–80.CrossRefGoogle Scholar
  27. 27.
    Du Q, Jiang L, Wang XQ, Pan W, She FF, Chen YL. Establishment of and comparison between orthotopic xenograft and subcutaneous xenograft models of gallbladder carcinoma. Asian Pac J Cancer Prev. 2014;15:3747–52.CrossRefGoogle Scholar
  28. 28.
    Kuboki S, Sakai N, Clarke C, Schuster R, Blanchard J, Edwards MN, Lentsch AB. The peptidyl-prolyl isomerase, Pin1, facilitates NF-kappaB binding in hepatocytes and protects against hepatic ischemia/reperfusion injury. J Hepatol. 2009;51:296–306.CrossRefPubMedCentralGoogle Scholar
  29. 29.
    Lee TH, Tun-Kyi A, Shi R, et al. Essential role of Pin1 in the regulation of TRF1 stability and telomere maintenance. Nat Cell Biol. 2009;11:97–105.CrossRefGoogle Scholar
  30. 30.
    Liou YC, Ryo A, Huang HK, et al. Loss of Pin1 function in the mouse causes phenotypes resembling cyclin D1-null phenotypes. Proc Natl Acad Sci USA. 2002;99:1335–40.CrossRefPubMedCentralGoogle Scholar

Copyright information

© Society of Surgical Oncology 2019

Authors and Affiliations

  • Shinichiro Nakada
    • 1
  • Satoshi Kuboki
    • 1
    Email author
  • Hiroyuki Nojima
    • 1
  • Hideyuki Yoshitomi
    • 1
  • Katsunori Furukawa
    • 1
  • Tsukasa Takayashiki
    • 1
  • Shigetsugu Takano
    • 1
  • Masaru Miyazaki
    • 1
  • Masayuki Ohtsuka
    • 1
  1. 1.Department of General Surgery, Graduate School of MedicineChiba UniversityChibaJapan

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