Tumor Biology

, Volume 36, Issue 2, pp 1155–1162 | Cite as

Evaluation of transforming growth factor-β1 suppress Pokemon/epithelial–mesenchymal transition expression in human bladder cancer cells

  • Wei Li
  • Amritha Kidiyoor
  • Yangyang Hu
  • Changcheng Guo
  • Min Liu
  • Xudong Yao
  • Yuanyuan Zhang
  • Bo Peng
  • Junhua Zheng
Research Article


Transforming growth factor-β1 (TGF-β1) plays a dual role in apoptosis and in proapoptotic responses in the support of survival in a variety of cells. The aim of this study was to determine the function of TGF-β1 in bladder cancer cells and the relationship with POK erythroid myeloid ontogenic factor (Pokemon). TGF-β1 and its receptors mediate several tumorigenic cascades that regulate cell proliferation, migration, and survival of bladder cancer cells. Bladder cancer cells T24 were treated with different levels of TGF-β1. Levels of Pokemon, E-cadherin, Snail, MMP2, MMP9, Twist, VEGF, and β-catenin messenger RNA (mRNA) and protein were examined by real-time quantitative fluorescent PCR and Western blot analysis, respectively. The effects of TGF-β1 on epithelial–mesenchymal transition of T24 cells were evaluated with wound-healing assay, proliferation of T24 was evaluated with reference to growth curves with MTT assay, and cell invasive ability was investigated by Transwell assay. Data show that Pokemon was inhibited by TGF-β1 treatment; the gene and protein of E-cadherin and β-catenin expression level showed decreased markedly after TGF-β1 treatment (P < 0.05). While the bladder cancer cell after TGF-β1 treatment showed a significantly reduced wound-closing efficiency at 6, 12, and 24 h, mechanistic analyses demonstrated that different levels of TGF-β1 promotes tumor cell growth, migration, and invasion in bladder cancer cells (P < 0.01, P < 0.05, respectively). In summary, our findings suggest that TGF-β1 may inhibit the expression of Pokemon, β-catenin, and E-cadherin. The high expression of TGF-β1 leads to an increase in the phenotype and apical-base polarity of epithelial cells. These changes of cells may result in the recurrence and progression of bladder cancer at last. Related mechanism is worthy of further investigation.


Bladder cancer Transforming growth factor-β1 Pokemon Epithelial–mesenchymal transition T24 cells 


Conflicts of interest

This work was supported in part or in whole by the National Natural Science Foundation of China (Grant No. 31100702/ C100307) and Specialized Research Fund for the Doctoral Program of Higher Education in China (Grant No. 20110072120054). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.


  1. 1.
    Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin. 2014;64:9–29.CrossRefPubMedGoogle Scholar
  2. 2.
    Witjes JA, Comperat E, Cowan NC, De Santis M, Gakis G, Lebret T, et al. EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. Eur Urol. 2014;65:778–92.CrossRefPubMedGoogle Scholar
  3. 3.
    Rye PD, Nustad K, Stigbrand T. Tumor marker workshops. Tumour Biol. 2003;24:165–71.CrossRefPubMedGoogle Scholar
  4. 4.
    Lampropoulos P, Zizi-Sermpetzoglou A, Rizos S, Kostakis A, Nikiteas N, Papavassiliou AG. Prognostic significance of transforming growth factor beta (TGF-beta) signaling axis molecules and E-cadherin in colorectal cancer. Tumour Biol. 2012;33:1005–14.CrossRefPubMedGoogle Scholar
  5. 5.
    Zhou L, Lopes JE, Chong MM, Ivanov II, Min R, Victora GD, et al. TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function. Nature. 2008;453:236–40.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Luo S, Kleemann GA, Ashraf JM, Shaw WM, Murphy CT. TGF-beta and insulin signaling regulate reproductive aging via oocyte and germ line quality maintenance. Cell. 2010;143:299–312.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sanchez-Capelo A. Dual role for TGF-beta1 in apoptosis. Cytokine Growth Factor Rev. 2005;16:15–34.CrossRefPubMedGoogle Scholar
  8. 8.
    Duangkumpha K, Techasen A, Loilome W, Namwat N, Thanan R, Khuntikeo N and Yongvanit P. BMP-7 blocks the effects of TGF-beta-induced EMT in cholangiocarcinoma. Tumour Biol 2014;Google Scholar
  9. 9.
    Smith AL, Iwanaga R, Drasin DJ, Micalizzi DS, Vartuli RL, Tan AC, et al. The miR-106b-25 cluster targets Smad7, activates TGF-beta signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer. Oncogene. 2012;31:5162–71.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Waerner T, Alacakaptan M, Tamir I, Oberauer R, Gal A, Brabletz T, et al. ILEI: a cytokine essential for EMT, tumor formation, and late events in metastasis in epithelial cells. Cancer Cell. 2006;10:227–39.CrossRefPubMedGoogle Scholar
  11. 11.
    Jeon BN, Yoo JY, Choi WI, Lee CE, Yoon HG, Hur MW. Proto-oncogene FBI-1 (Pokemon/ZBTB7A) represses transcription of the tumor suppressor Rb gene via binding competition with Sp1 and recruitment of co-repressors. J Biol Chem. 2008;283:33199–210.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Canesin G, Cuevas EP, Santos V, Lopez-Menendez C, Moreno-Bueno G, Huang Y, Csiszar K, Portillo F, Peinado H, Lyden D and Cano A. Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization. Oncogene 2014; 0.Google Scholar
  13. 13.
    Thievessen I, Seifert HH, Swiatkowski S, Florl AR, Schulz WA. E-cadherin involved in inactivation of WNT/beta-catenin signalling in urothelial carcinoma and normal urothelial cells. Br J Cancer. 2003;88:1932–8.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wu Y, Ginther C, Kim J, Mosher N, Chung S, Slamon D, et al. Expression of Wnt3 activates Wnt/beta-catenin pathway and promotes EMT-like phenotype in trastuzumab-resistant HER2-overexpressing breast cancer cells. Mol Cancer Res. 2012;10:1597–606.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Saeb-Parsy K, Wilson A, Scarpini C, Corcoran M, Chilcott S, McKean M, et al. Diagnosis of bladder cancer by immunocytochemical detection of minichromosome maintenance protein-2 in cells retrieved from urine. Br J Cancer. 2012;107:1384–91.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Miles FL, Tung NS, Aguiar AA, Kurtoglu S, Sikes RA. Increased TGF-beta1-mediated suppression of growth and motility in castrate-resistant prostate cancer cells is consistent with Smad2/3 signaling. Prostate. 2012;72:1339–50.CrossRefPubMedGoogle Scholar
  17. 17.
    Pasche B. Role of transforming growth factor beta in cancer. J Cell Physiol. 2001;186:153–68.CrossRefPubMedGoogle Scholar
  18. 18.
    Smith AL, Robin TP, Ford HL. Molecular pathways: targeting the TGF-beta pathway for cancer therapy. Clin Cancer Res. 2012;18:4514–21.CrossRefPubMedGoogle Scholar
  19. 19.
    Eder IE, Stenzl A, Hobisch A, Cronauer MV, Bartsch G, Klocker H. Transforming growth factors-beta 1 and beta 2 in serum and urine from patients with bladder carcinoma. J Urol. 1996;156:953–7.CrossRefPubMedGoogle Scholar
  20. 20.
    Champelovier P, El Atifi M, Mantel F, Rostaing B, Simon A, Berger F, et al. In vitro tumoral progression of human bladder carcinoma: role for TGFbeta. Eur Urol. 2005;48:846–51.CrossRefPubMedGoogle Scholar
  21. 21.
    Park BJ, Park JI, Byun DS, Park JH, Chi SG. Mitogenic conversion of transforming growth factor-beta1 effect by oncogenic Ha-Ras-induced activation of the mitogen-activated protein kinase signaling pathway in human prostate cancer. Cancer Res. 2000;60:3031–8.PubMedGoogle Scholar
  22. 22.
    Iles RK. Ectopic hCGbeta expression by epithelial cancer: malignant behaviour, metastasis and inhibition of tumor cell apoptosis. Mol Cell Endocrinol. 2007;260–262:264–70.CrossRefPubMedGoogle Scholar
  23. 23.
    Eissa S, Salem AM, Zohny SF, Hegazy MG. The diagnostic efficacy of urinary TGF-beta1 and VEGF in bladder cancer: comparison with voided urine cytology. Cancer Biomark. 2007;3:275–85.CrossRefPubMedGoogle Scholar
  24. 24.
    Peinado H, Olmeda D, Cano A. Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer. 2007;7:415–28.CrossRefPubMedGoogle Scholar
  25. 25.
    Nollet F, Kools P, van Roy F. Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members. J Mol Biol. 2000;299:551–72.CrossRefPubMedGoogle Scholar
  26. 26.
    Chaw SY, Majeed AA, Dalley AJ, Chan A, Stein S, Farah CS. Epithelial to mesenchymal transition (EMT) biomarkers—E-cadherin, beta-catenin, APC and Vimentin—in oral squamous cell carcinogenesis and transformation. Oral Oncol. 2012;48:997–1006.CrossRefPubMedGoogle Scholar
  27. 27.
    Froeling FE, Mirza TA, Feakins RM, Seedhar A, Elia G, Hart IR, et al. Organotypic culture model of pancreatic cancer demonstrates that stromal cells modulate E-cadherin, beta-catenin, and Ezrin expression in tumor cells. Am J Pathol. 2009;175:636–48.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    De Wever O, Pauwels P, De Craene B, Sabbah M, Emami S, Redeuilh G, et al. Molecular and pathological signatures of epithelial–mesenchymal transitions at the cancer invasion front. Histochem Cell Biol. 2008;130:481–94.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Yilmaz M, Christofori G. EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev. 2009;28:15–33.CrossRefPubMedGoogle Scholar
  30. 30.
    van der Horst G, Bos L, van der Pluijm G. Epithelial plasticity, cancer stem cells, and the tumor-supportive stroma in bladder carcinoma. Mol Cancer Res. 2012;10:995–1009.CrossRefPubMedGoogle Scholar
  31. 31.
    Shinozaki S, Mashima H, Ohnishi H, Sugano K. IL-13 promotes the proliferation of rat pancreatic stellate cells through the suppression of NF-kappaB/TGF-beta1 pathway. Biochem Biophys Res Commun. 2010;393:61–5.CrossRefPubMedGoogle Scholar
  32. 32.
    Voloshenyuk TG, Landesman ES, Khoutorova E, Hart AD, Gardner JD. Induction of cardiac fibroblast lysyl oxidase by TGF-beta1 requires PI3K/Akt, Smad3, and MAPK signaling. Cytokine. 2011;55:90–7.CrossRefPubMedGoogle Scholar
  33. 33.
    Diaz-Benitez CE, Navarro-Fuentes KR, Flores-Sosa JA, Juarez-Diaz J, Uribe-Salas FJ, Roman-Basaure E, et al. CD3zeta expression and T cell proliferation are inhibited by TGF-beta1 and IL-10 in cervical cancer patients. J Clin Immunol. 2009;29:532–44.CrossRefPubMedGoogle Scholar
  34. 34.
    Tas F, Karabulut S, Serilmez M, Ciftci R, Duranyildiz D. Clinical significance of serum transforming growth factor-beta 1 (TGF-beta1) levels in patients with epithelial ovarian cancer. Tumour Biol. 2014;35:3611–6.CrossRefPubMedGoogle Scholar
  35. 35.
    Salvo E, Garasa S, Dotor J, Morales X, Pelaez R, Altevogt P, et al. Combined targeting of TGF-beta1 and integrin beta3 impairs lymph node metastasis in a mouse model of non-small-cell lung cancer. Mol Cancer. 2014;13:112.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Wei Li
    • 1
    • 2
  • Amritha Kidiyoor
    • 2
  • Yangyang Hu
    • 1
  • Changcheng Guo
    • 1
  • Min Liu
    • 1
  • Xudong Yao
    • 1
  • Yuanyuan Zhang
    • 2
  • Bo Peng
    • 1
  • Junhua Zheng
    • 1
  1. 1.Department of Urology, Shanghai Tenth People’s HospitalTongji University School of MedicineShanghaiChina
  2. 2.Institute for Regenerative MedicineWake Forest UniversityWinston-SalemUSA

Personalised recommendations