Tumor Biology

, Volume 37, Issue 9, pp 12731–12742 | Cite as

CYLD downregulates Livin and synergistically improves gemcitabine chemosensitivity and decreases migratory/invasive potential in bladder cancer: the effect is autophagy-associated

  • Lei Yin
  • Shuai Liu
  • Chensheng Li
  • Sentai Ding
  • Dongbin Bi
  • Zhihong Niu
  • Liping Han
  • Wenjia Li
  • Dexuan Gao
  • Zheng Liu
  • Jiaju LuEmail author
Original Article


Although GC (gemcitabine and cisplatin) chemotherapy remains an effective method for treating bladder cancer (BCa), chemoresistance is a major obstacle in chemotherapy. In this study, we determined whether gemcitabine resistance correlates with migratory/invasive potential in BCa and whether this relationship is regulated by the cylindromatosis (CYLD)-Livin module. First, we independently investigated the correlation of CYLD/Livin and gemcitabine resistance with the potential for tumor migration and invasiveness. Second, we found that co-transfected CYLD and Livin dramatically improved sensitivity to gemcitabine chemotherapy and decreased migration/invasion potential. Next, we determined that CYLD may regulate Livin by the NF-κB-dependent pathway. We also found that CYLD overexpression and Livin knockdown might improve gemcitabine chemosensitivity by decreasing autophagy and increasing apoptosis in BCa cells. Finally, the effects of CYLD-Livin on tumor growth in vivo were evaluated. Our study demonstrates that CYLD-Livin might represent a potential therapeutic for chemoresistant BCa.


Gemcitabine Cylindromatosis (CYLD) Livin Bladder cancer (BCa) Autophagy 



This work was supported by the Shandong Key Research and Development Project (No. 2015GSF118055 ), Medicine and Healthcare Technology Development Project of Shandong Province (No. 2014WS0341), and Natural Science Foundation of Shandong Province (No. 2014ZRB14513 and 2014ZRB14081).

Compliance with ethical standards

The study had obtained approval from the Committee on Animal Research of Shandong Provincial Hospital of Shandong University, and our care was in accordance with institutional guidelines.

Conflicts of interest



  1. 1.
    Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010;60(5):277–300.CrossRefPubMedGoogle Scholar
  2. 2.
    Kovalenko A, Chable-Bessia C, Cantarella G, Israel A, Wallach D, Courtois G. The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature. 2003;424(6950):801–5.CrossRefPubMedGoogle Scholar
  3. 3.
    Massoumi R. CYLD: a deubiquitination enzyme with multiple roles in cancer. Future Oncol. 2011;7(2):285–97.CrossRefPubMedGoogle Scholar
  4. 4.
    Wu W, Zhu H, Fu Y, Shen W, Xu J, Miao K, Hong M, Xu W, Liu P, Li J. Clinical significance of down-regulated cylindromatosis gene in chronic lymphocytic leukemia. Leuk Lymphoma. 2014;55(3):588–94.CrossRefPubMedGoogle Scholar
  5. 5.
    Ye H, Liu X, Lv M, Wu Y, Kuang S, Gong J, Yuan P, Zhong Z, Li Q, Jia H, Sun J, Chen Z, Guo AY. MicroRNA and transcription factor co-regulatory network analysis reveals miR-19 inhibits CYLD in T-cell acute lymphoblastic leukemia. Nucleic Acids Res. 2012;40(12):5201–14.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Urbanik T, Koehler BC, Wolpert L, Elssner C, Scherr AL, Longerich T, Kautz N, Welte S, Hovelmeyer N, Jager D, Waisman A, Schulze-Bergkamen H. CYLD deletion triggers nuclear factor-kappaB-signaling and increases cell death resistance in murine hepatocytes. World J Gastroenterol. 2014;20(45):17049–64.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pannem RR, Dorn C, Ahlqvist K, Bosserhoff AK, Hellerbrand C, Massoumi R. CYLD controls c-MYC expression through the JNK-dependent signaling pathway in hepatocellular carcinoma. Carcinogenesis. 2014;35(2):461–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Yan B. Research progress on Livin protein: an inhibitor of apoptosis. Mol Cell Biochem. 2011;357(1–2):39–45.CrossRefPubMedGoogle Scholar
  9. 9.
    Ma L, Huang Y, Song Z, Feng S, Tian X, Du W, Qiu X, Heese K, M W. Livin promotes Smac/DIABLO degradation by ubiquitin-proteasome pathway. Cell Death Differ. 2006;13(12):2079–88.CrossRefPubMedGoogle Scholar
  10. 10.
    Wang Y, Li Y, Zhou B, Zhang WY, Guan JT, Wang R, Yang L, Xia QJ, Zhou ZG, Sun XF. Expression of the apoptosis inhibitor livin in colorectal adenoma-carcinoma sequence: correlations with pathology and outcome. Tumour Biol. 2014;35(12):11791–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Zhao X, Yuan Y, Zhang Z, Feng X, Zhang J, Yuan X, Li J. Effects of shRNA-silenced livin and survivin on lung cancer cell proliferation and apoptosis. J BUON. 2014;19(3):757–62.PubMedGoogle Scholar
  12. 12.
    Ding ZY, Zhang H, Adell G, Olsson B, Sun XF. Livin expression is an independent factor in rectal cancer patients with or without preoperative radiotherapy. Radiat Oncol. 2013;8:281.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Liu Y, Guo Q, Zhang H, Li GH, Feng S, XZ Y, Kong LS, Zhao L, Jin F. Effect of siRNA-Livin on drug resistance to chemotherapy in glioma U251 cells and CD133 stem cells. EXP THER MED. 2015;10(4):1317–23.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Wang J, Zhang X, Wei P, Zhang J, Niu Y, Kang N, Zhang Y, Zhang W, Xing N. Livin, survivin and caspase 3 as early recurrence markers in non-muscle-invasive bladder cancer. World J Urol. 2014;32(6):1477–84.CrossRefPubMedGoogle Scholar
  15. 15.
    Zhang Y, Huang H, Zhou H, Du T, Zeng L, Cao Y, Chen J, Lai Y, Li J, Wang G, Guo Z. Activation of nuclear factor kappaB pathway and downstream targets survivin and livin by SHARPIN contributes to the progression and metastasis of prostate cancer. Cancer-Am Cancer Soc. 2014;120(20):3208–18.Google Scholar
  16. 16.
    Li M, Gao P, Zhang J. Crosstalk between autophagy and apoptosis: potential and emerging therapeutic targets for cardiac diseases. Int J Mol Sci. 2016;17(3).Google Scholar
  17. 17.
    Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E. Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy. 2005;1(2):84–91.CrossRefPubMedGoogle Scholar
  18. 18.
    Mizushima N, Yoshimori T, Levine B. Methods in mammalian autophagy research. Cell. 2010;140(3):313–26.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Choudhary GS, Al-Harbi S, Almasan A. Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis. Methods Mol Biol. 2015;1219:1–9.CrossRefPubMedGoogle Scholar
  20. 20.
    Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, Jin H, Xu H, Chen Q. Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein Cell. 2010;1(5):468–77.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Li F, Zhang J, Arfuso F, Chinnathambi A, Zayed ME, Alharbi SA, Kumar AP, Ahn KS, Sethi G. NF-kappaB in cancer therapy. Arch Toxicol. 2015;89(5):711–31.CrossRefPubMedGoogle Scholar
  22. 22.
    Liu S, Lv J, Han L, Ichikawa T, Wang W, Li S, Wang XL, Tang D, Cui T. A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells. Biochem Biophys Res Commun. 2012;420(1):78–83.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Chen N, Karantza-Wadsworth V. Role and regulation of autophagy in cancer. Biochim Biophys Acta. 2009;1793(9):1516–23.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Lee JG, Shin JH, Shim HS, Lee CY, Kim DJ, Kim YS, Chung KY. Autophagy contributes to the chemo-resistance of non-small cell lung cancer in hypoxic conditions. Respir Res. 2015;16:138.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Liu S, Xie F, Wang H, Liu Z, Liu X, Sun L, Niu Z. Ubenimex inhibits cell proliferation, migration and invasion in renal cell carcinoma: the effect is autophagy-associated. Oncol Rep. 2015;33(3):1372–80.PubMedGoogle Scholar
  26. 26.
    Hamacher-Brady A, Brady NR. Bax/Bak-dependent, Drp1-independent targeting of X-linked inhibitor of apoptosis protein (XIAP) into inner mitochondrial compartments counteracts Smac/DIABLO-dependent effector caspase activation. J Biol Chem. 2015;290(36):22005–18.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Sulaiman MK, Chu Z, Blanco VM, Vallabhapurapu SD, Franco RS, Qi X. SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas. Mol Cancer. 2015;14:78.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Yang JM, Xu Z, Wu H, Zhu H, Wu X, Hait WN. Overexpression of extracellular matrix metalloproteinase inducer in multidrug resistant cancer cells. Mol Cancer Res. 2003;1(6):420–7.PubMedGoogle Scholar
  29. 29.
    Gungor C, Zander H, Effenberger KE, Vashist YK, Kalinina T, Izbicki JR, Yekebas E, Bockhorn M. Notch signaling activated by replication stress-induced expression of midkine drives epithelial-mesenchymal transition and chemoresistance in pancreatic cancer. Cancer Res. 2011;71(14):5009–19.CrossRefPubMedGoogle Scholar
  30. 30.
    Vaidya KS, Sanchez JJ, Kim EL, Welch DR. Expression of the breast cancer metastasis suppressor 1 (BRMS1) maintains in vitro chemosensitivity of breast cancer cells. Cancer Lett. 2009;281(1):100–7.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Hoshino H, Miyoshi N, Nagai K, Tomimaru Y, Nagano H, Sekimoto M, Doki Y, Mori M, Ishii H. Epithelial-mesenchymal transition with expression of SNAI1-induced chemoresistance in colorectal cancer. Biochem Biophys Res Commun. 2009;390(3):1061–5.CrossRefPubMedGoogle Scholar
  32. 32.
    Zhu L, Hu Z, Liu J, Gao J, Lin B. Gene expression profile analysis identifies metastasis and chemoresistance-associated genes in epithelial ovarian carcinoma cells. Med Oncol. 2015;32(1):426.CrossRefPubMedGoogle Scholar
  33. 33.
    Volk-Draper L, Hall K, Griggs C, Rajput S, Kohio P, DeNardo D, Ran S. Paclitaxel therapy promotes breast cancer metastasis in a TLR4-dependent manner. Cancer Res. 2014;74(19):5421–34.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Maseki S, Ijichi K, Tanaka H, Fujii M, Hasegawa Y, Ogawa T, Murakami S, Kondo E, Nakanishi H. Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3beta/snail signalling pathway. Br J Cancer. 2012;106(6):1196–204.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Rosano L, Cianfrocca R, Spinella F, Di Castro V, Nicotra MR, Lucidi A, Ferrandina G, Natali PG, Bagnato A. Acquisition of chemoresistance and EMT phenotype is linked with activation of the endothelin a receptor pathway in ovarian carcinoma cells. Clin Cancer Res. 2011;17(8):2350–60.CrossRefPubMedGoogle Scholar
  36. 36.
    Augello C, Caruso L, Maggioni M, Donadon M, Montorsi M, Santambrogio R, Torzilli G, Vaira V, Pellegrini C, Roncalli M, Coggi G, Bosari S. Inhibitors of apoptosis proteins (IAPs) expression and their prognostic significance in hepatocellular carcinoma. BMC Cancer. 2009;9:125.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Wong JC, Fiscus RR. Resveratrol at anti-angiogenesis/anticancer concentrations suppresses protein kinase G signaling and decreases IAPs expression in HUVECs. Anticancer Res. 2015;35(1):273–81.PubMedGoogle Scholar
  38. 38.
    Chung CY, Park YL, Kim N, Park HC, Park HB, Myung DS, Kim JS, Cho SB, Lee WS, Joo YE. Expression and prognostic significance of livin in gastric cancer. Oncol Rep. 2013;30(5):2520–8.PubMedGoogle Scholar
  39. 39.
    Ding Z, Liu Y, Yao L, Wang D, Zhang J, Cui G, Yang X, Huang X, Liu F, Shen A. Spy1 induces de-ubiquitinating of RIP1 arrest and confers glioblastoma’s resistance to tumor necrosis factor (TNF-alpha)-induced apoptosis through suppressing the association of CLIPR-59 and CYLD. Cell Cycle. 2015;14(13):2149–59.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Hester CC, Moscato EE, Kazakov DV, Vanecek T, Moretto JC, Seiff SR. A new cylindromatosis (CYLD) gene mutation in a case of Brooke-Spiegler syndrome masquerading as basal cell carcinoma of the eyelids. Ophthal Plast Reconstr Surg. 2013;29(1):e10–1.CrossRefPubMedGoogle Scholar
  41. 41.
    Arora M, Kaul D, Varma N, Marwaha RK. Cellular proteolytic modification of tumor-suppressor CYLD is critical for the initiation of human T-cell acute lymphoblastic leukemia. Blood Cells Mol Dis. 2015;54(1):132–8.CrossRefPubMedGoogle Scholar
  42. 42.
    Iliopoulos D, Jaeger SA, Hirsch HA, Bulyk ML, Struhl K. STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer. Mol Cell. 2010;39(4):493–506.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Huang DH, Wang GY, Zhang JW, Li Y, Zeng XC, Jiang N. MiR-501-5p regulates CYLD expression and promotes cell proliferation in human hepatocellular carcinoma. Jpn J Clin Oncol. 2015;45(8):738–44.CrossRefPubMedGoogle Scholar
  44. 44.
    Cao S, Liu S, Wang F, Liu J, Li M, Wang C, Xi S. DMA(V) in drinking water activated NF-kappaB signal pathway and increased TGF-beta and IL-1beta expressions in bladder epithelial cells of rats. Mediat Inflamm. 2015;2015:790652.CrossRefGoogle Scholar
  45. 45.
    Jiang Y, Han Y, Sun C, Han C, Han N, Zhi W and Qiao Q. Rab23 is overexpressed in human bladder cancer and promotes cancer cell proliferation and invasion. Tumour Biol 2015.Google Scholar
  46. 46.
    Huang Z, Zhong Z, Zhang L, Wang X, Xu R, Zhu L, Wang Z, Hu S, Zhao X. Down-regulation of HMGB1 expression by shRNA constructs inhibits the bioactivity of urothelial carcinoma cell lines via the NF-kappaB pathway. Sci Rep. 2015;5:12807.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Sun Y, Guan Z, Liang L, Cheng Y, Zhou J, Li J, Xu Y. NF-kappaB signaling plays irreplaceable roles in cisplatin-induced bladder cancer chemoresistance and tumor progression. Int J Oncol. 2016;48(1):225–34.PubMedGoogle Scholar
  48. 48.
    Sun WL, Chen J, Wang YP, Zheng H. Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development. Autophagy. 2011;7(9):1035–44.CrossRefPubMedGoogle Scholar
  49. 49.
    Lv L, Liu HG, Dong SY, Yang F, Wang QX, Guo GL, Pan YF and Zhang XH. Upregulation of CD44v6 contributes to acquired chemoresistance via the modulation of autophagy in colon cancer SW480 cells. Tumour Biol 2016.Google Scholar
  50. 50.
    Xu R, Liu S, Chen H and Lao L. MicroRNA-30a downregulation contributes to chemoresistance of osteosarcoma cells through activating Beclin-1-mediated autophagy. Oncol Rep 2015.Google Scholar
  51. 51.
    Anbalagan S, Pires IM, Blick C, Hill MA, Ferguson DJ, Chan DA, Hammond EM. Radiosensitization of renal cell carcinoma in vitro through the induction of autophagy. Radiother Oncol. 2012;103(3):388–93.CrossRefPubMedGoogle Scholar
  52. 52.
    He Q, Zhou X, Li S, Jin Y, Chen Z, Chen D, Cai Y, Liu Z, Zhao T, Wang A. MicroRNA-181a suppresses salivary adenoid cystic carcinoma metastasis by targeting MAPK-Snai2 pathway. Biochim Biophys Acta. 2013;1830(11):5258–66.CrossRefPubMedGoogle Scholar
  53. 53.
    Chang B, Li S, He Q, Liu Z, Zhao L, Zhao T, Wang A. Deregulation of Bmi-1 is associated with enhanced migration, invasion and poor prognosis in salivary adenoid cystic carcinoma. Biochim Biophys Acta. 2014;1840(12):3285–91.CrossRefPubMedGoogle Scholar
  54. 54.
    Xie J, Liu J, Liu H, Liang S, Lin M, Gu Y, Liu T, Wang D, Ge H, Mo SL. The antitumor effect of tanshinone IIA on anti-proliferation and decreasing VEGF/VEGFR2 expression on the human non-small cell lung cancer A549 cell line. Acta Pharm Sin B. 2015;5(6):554–63.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Lei Yin
    • 1
  • Shuai Liu
    • 1
  • Chensheng Li
    • 2
  • Sentai Ding
    • 1
  • Dongbin Bi
    • 1
  • Zhihong Niu
    • 1
  • Liping Han
    • 3
  • Wenjia Li
    • 4
  • Dexuan Gao
    • 1
  • Zheng Liu
    • 1
  • Jiaju Lu
    • 1
    Email author
  1. 1.Department of UrologyShandong Provincial Hospital Affiliated to Shandong UniversityJinanChina
  2. 2.Department of Digestive DiseasesShandong Provincial Hospital Affiliated to Shandong UniversityJinanChina
  3. 3.Department of NeurologyShandong Provincial Qianfoshan Hospital, Shandong UniversityJinanChina
  4. 4.Shandong UniversityJinanChina

Personalised recommendations