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Molecular and Cellular Biochemistry

, Volume 396, Issue 1–2, pp 257–268 | Cite as

Activated hedgehog pathway is a potential target for pharmacological intervention in biliary tract cancer

  • Tobias Kiesslich
  • Christian Mayr
  • Julia Wachter
  • Doris Bach
  • Julia Fuereder
  • Andrej Wagner
  • Beate Alinger
  • Martin Pichler
  • Pietro Di Fazio
  • Matthias Ocker
  • Frieder Berr
  • Daniel NeureiterEmail author
Article

Abstract

Hedgehog (Hh) signalling contributes to carcinogenesis and represents a valid druggable target in human cancers, possibly also in biliary tract cancer (BTC). We analysed the expression of Hh components in BTC using eight heterogeneously differentiated cell lines, xenograft tumours and a human tissue microarray. The dose-, time- and cell line-dependent effects of two Hh inhibitors (cyclopamine and Gant-61) were analysed in vitro for survival, apoptosis, cell cycle distribution and possible synergism with conventional chemotherapeutic agents. In human BTC samples, the sonic Hh ligand and the Gli1 transcription factor showed increased expression in tumours compared to normal adjacent tissue and were significantly associated with high tumour grade and positive lymph node status. In BTC cell lines, we could confirm the Hh component expression at varying extent within the employed cell lines in vitro and in vivo indicating non-canonical signalling. Both Hh inhibitors showed dose-dependent cytotoxicity above 5 µM with a stronger effect for Gant-61 inducing apoptosis whereas cyclopamine rather inhibited proliferation. Cytotoxicity was associated with low cytokeratin expression and higher mesenchymal marker expression such as vimentin. Additionally, drug combinations of Gant-61 with conventional chemotherapy (cisplatin) exerted synergistic effects. In conclusion, Hh pathway is significantly activated in human BTC tissue compared to normal adjacent tissue. The current data demonstrate for the first time an effective anticancer activity of especially Gant-61 in BTC and suggest second generation Hh pathway inhibitors as a potential novel treatment strategy in BTC.

Keywords

Biliary tract cancer Hedgehog Oncogenic signalling Pharmacological inhibition Cyclopamine Gant-61 

Notes

Acknowledgments

This study was supported by funds of the Oesterreichische Nationalbank (Anniversary fund, project number: 14842), the research fund of the Paracelsus Medical University Salzburg (Grant No. A-12/02/006-KIE) and the ‘Wissenschaftlicher Verein‘of the Institute of Pathology Salzburg. The authors are grateful to the pharmacy at Salzburger Landeskliniken for providing cisplatin.

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

11010_2014_2161_MOESM1_ESM.pdf (58 kb)
Supplementary material 1 (PDF 57 kb)

References

  1. 1.
    Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15(6):801–812. doi: 10.1016/j.devcel.2008.11.010 PubMedCrossRefGoogle Scholar
  2. 2.
    Lum L, Beachy PA (2004) The hedgehog response network: sensors, switches, and routers. Science 304(5678):1755–1759. doi: 10.1126/science.1098020 PubMedCrossRefGoogle Scholar
  3. 3.
    Weiss GJ, Von Hoff DD (2010) Hunting the hedgehog pathway. Clin Pharmacol Ther 87(6):743–747. doi: 10.1038/clpt.2010.34 PubMedCrossRefGoogle Scholar
  4. 4.
    Kiesslich T, Neureiter D (2012) Advances in targeting the hedgehog signaling pathway in cancer therapy. Expert Opin Ther Targets 16(2):151–156. doi: 10.1517/14728222.2012.652948 PubMedCrossRefGoogle Scholar
  5. 5.
    Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, Tibes R, Weiss GJ, Borad MJ, Hann CL, Brahmer JR, Mackey HM, Lum BL, Darbonne WC, Marsters JC Jr, de Sauvage FJ, Low JA (2009) Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Engl J Med 361(12):1164–1172. doi: 10.1056/NEJMoa0905360 CrossRefGoogle Scholar
  6. 6.
    Patel T (2011) Cholangiocarcinoma–controversies and challenges. Nat Rev Gastroenterol Hepatol 8(4):189–200. doi: 10.1038/nrgastro.2011.20 PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, Madhusudan S, Iveson T, Hughes S, Pereira SP, Roughton M, Bridgewater J, Investigators ABCT (2010) Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med 362(14):1273–1281. doi: 10.1056/NEJMoa0908721 PubMedCrossRefGoogle Scholar
  8. 8.
    Kiesslich T, Neureiter D, Wolkersdorfer GW, Plaetzer K, Berr F (2010) Advances in photodynamic therapy for the treatment of hilar biliary tract cancer. Future Oncol 6(12):1925–1936. doi: 10.2217/fon.10.147 PubMedCrossRefGoogle Scholar
  9. 9.
    Wagner A, Kiesslich T, Neureiter D, Friesenbichler P, Puespoek A, Denzer UW, Wolkersdorfer GW, Emmanuel K, Lohse AW, Berr F (2013) Photodynamic therapy for hilar bile duct cancer: clinical evidence for improved tumoricidal tissue penetration by temoporfin. Photochem Photobiol Sci 12(6):1065–1073. doi: 10.1039/c3pp25425a PubMedCrossRefGoogle Scholar
  10. 10.
    Hopfner M, Schuppan D, Scherubl H (2008) Targeted medical therapy of biliary tract cancer: recent advances and future perspectives. World J Gastroenterol 14(46):7021–7032PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70PubMedCrossRefGoogle Scholar
  12. 12.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. doi: 10.1016/j.cell.2011.02.013 PubMedCrossRefGoogle Scholar
  13. 13.
    Kiesslich T, Berr F, Alinger B, Kemmerling R, Pichler M, Ocker M, Neureiter D (2012) Current status of therapeutic targeting of developmental signalling pathways in oncology. Curr Pharm Biotechnol 13(11):2184–2220PubMedCrossRefGoogle Scholar
  14. 14.
    Kasper M, Regl G, Frischauf AM, Aberger F (2006) GLI transcription factors: mediators of oncogenic hedgehog signalling. Eur J Cancer 42(4):437–445. doi: 10.1016/j.ejca.2005.08.039 PubMedCrossRefGoogle Scholar
  15. 15.
    Varjosalo M, Taipale J (2007) Hedgehog signaling. J Cell Sci 120(Pt 1):3–6. doi: 10.1242/jcs.03309 PubMedGoogle Scholar
  16. 16.
    Fingas CD, Bronk SF, Werneburg NW, Mott JL, Guicciardi ME, Cazanave SC, Mertens JC, Sirica AE, Gores GJ (2011) Myofibroblast-derived PDGF-BB promotes hedgehog survival signaling in cholangiocarcinoma cells. Hepatology 54(6):2076–2088. doi: 10.1002/hep.24588 PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Fingas CD, Mertens JC, Razumilava N, Sydor S, Bronk SF, Christensen JD, Rizvi SH, Canbay A, Treckmann JW, Paul A, Sirica AE, Gores GJ (2013) Polo-like kinase 2 is a mediator of hedgehog survival signaling in cholangiocarcinoma. Hepatology 58(4):1362–1374. doi: 10.1002/hep.26484 PubMedCrossRefGoogle Scholar
  18. 18.
    Jinawath A, Akiyama Y, Sripa B, Yuasa Y (2007) Dual blockade of the hedgehog and ERK1/2 pathways coordinately decreases proliferation and survival of cholangiocarcinoma cells. J Cancer Res Clin Oncol 133(4):271–278. doi: 10.1007/s00432-006-0166-9 PubMedCrossRefGoogle Scholar
  19. 19.
    Kurita S, Mott JL, Almada LL, Bronk SF, Werneburg NW, Sun SY, Roberts LR, Fernandez-Zapico ME, Gores GJ (2010) GLI3-dependent repression of DR4 mediates hedgehog antagonism of TRAIL-induced apoptosis. Oncogene 29(34):4848–4858. doi: 10.1038/onc.2010.235 PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Razumilava N, Gradilone SA, Smoot RL, Mertens JC, Bronk SF, Sirica AE, Gores GJ (2014) Non-canonical hedgehog signaling contributes to chemotaxis in cholangiocarcinoma. J Hepatol 60(3):599–605. doi: 10.1016/j.jhep.2013.11.005 PubMedCrossRefGoogle Scholar
  21. 21.
    Kiesslich T, Alinger B, Wolkersdorfer GW, Ocker M, Neureiter D, Berr F (2010) Active Wnt signalling is associated with low differentiation and high proliferation in human biliary tract cancer in vitro and in vivo and is sensitive to pharmacological inhibition. Int J Oncol 36(1):49–58PubMedGoogle Scholar
  22. 22.
    Wachter J, Neureiter D, Alinger B, Pichler M, Fuereder J, Oberdanner C, Di Fazio P, Ocker M, Berr F, Kiesslich T (2012) Influence of five potential anticancer drugs on wnt pathway and cell survival in human biliary tract cancer cells. Int J Biol Sci 8(1):15–29PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    de Groen PC, Gores GJ, LaRusso NF, Gunderson LL, Nagorney DM (1999) Biliary tract cancers. N Engl J Med 341(18):1368–1378. doi: 10.1056/NEJM199910283411807 PubMedCrossRefGoogle Scholar
  24. 24.
    Detre S, Saclani Jotti G, Dowsett M (1995) A “quickscore” method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. J Clin Pathol 48(9):876–878PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Kiesslich T, Neureiter D, Alinger B, Jansky GL, Berlanda J, Mkrtchyan V, Ocker M, Plaetzer K, Berr F (2010) Uptake and phototoxicity of meso-tetrahydroxyphenyl chlorine are highly variable in human biliary tract cancer cell lines and correlate with markers of differentiation and proliferation. Photochem Photobiol Sci 9(5):734–743. doi: 10.1039/b9pp00201d PubMedCrossRefGoogle Scholar
  26. 26.
    ComboSyn Incorporated: CompuSyn for drug combinations and for general dose-effect analysis. Available via http://www.combosyn.com. Accessed 25 June 2014
  27. 27.
    Chou TC (2006) Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 58(3):621–681. doi: 10.1124/pr.58.3.10 PubMedCrossRefGoogle Scholar
  28. 28.
    Heretsch P, Tzagkaroulaki L, Giannis A (2010) Cyclopamine and hedgehog signaling: chemistry, biology, medical perspectives. Angew Chem Int Ed Engl 49(20):3418–3427. doi: 10.1002/anie.200906967 PubMedCrossRefGoogle Scholar
  29. 29.
    Lauth M, Bergstrom A, Shimokawa T, Toftgard R (2007) Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci USA 104(20):8455–8460. doi: 10.1073/pnas.0609699104 PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Cai C, Rodepeter FR, Rossmann A, Teymoortash A, Lee JS, Quint K, Di Fazio P, Ocker M, Werner JA, Mandic R (2011) Nef from SIV(mac239) decreases proliferation and migration of adenoid-cystic carcinoma cells and inhibits angiogenesis. Oral Oncol 47(9):847–854. doi: 10.1016/j.oraloncology.2011.06.502 PubMedCrossRefGoogle Scholar
  31. 31.
    Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, Holcomb T, Stinson J, Gould SE, Coleman B, LoRusso PM, Von Hoff DD, de Sauvage FJ, Low JA (2009) Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 361(12):1173–1178. doi: 10.1056/NEJMoa0902903 PubMedCrossRefGoogle Scholar
  32. 32.
    Metcalfe C, de Sauvage FJ (2011) Hedgehog fights back: mechanisms of acquired resistance against Smoothened antagonists. Cancer Res 71(15):5057–5061. doi: 10.1158/0008-5472.CAN-11-0923 PubMedCrossRefGoogle Scholar
  33. 33.
    Kim YJ, Park SB, Park JY, Park SW, Chung JB, Song SY, Bang S (2012) The sonic hedgehog pathway as a treatment target for extrahepatic biliary tract cancer. Mol Med Rep 5(1):12–16. doi: 10.3892/mmr.2011.598 PubMedGoogle Scholar
  34. 34.
    El Khatib M, Kalnytska A, Palagani V, Kossatz U, Manns MP, Malek NP, Wilkens L, Plentz RR (2013) Inhibition of hedgehog signaling attenuates carcinogenesis in vitro and increases necrosis of cholangiocellular carcinoma. Hepatology 57(3):1035–1045. doi: 10.1002/hep.26147 PubMedCrossRefGoogle Scholar
  35. 35.
    Szkandera J, Pichler M, Absenger G, Stotz M, Weissmueller M, Samonigg H, Asslaber M, Lax S, Leitner G, Winder T, Renner W, Gerger A (2014) A functional germline variant in GLI1 implicates hedgehog signaling in clinical outcome of stage II and III colon carcinoma patients. Clin Cancer Res 20(6):1687–1697. doi: 10.1158/1078-0432.CCR-13-1517 PubMedCrossRefGoogle Scholar
  36. 36.
    Cooper MK, Porter JA, Young KE, Beachy PA (1998) Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280(5369):1603–1607PubMedCrossRefGoogle Scholar
  37. 37.
    Berman DM, Karhadkar SS, Maitra A, Montes De Oca R, Gerstenblith MR, Briggs K, Parker AR, Shimada Y, Eshleman JR, Watkins DN, Beachy PA (2003) Widespread requirement for hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425(6960):846–851. doi: 10.1038/nature01972 PubMedCrossRefGoogle Scholar
  38. 38.
    Yauch RL, Dijkgraaf GJ, Alicke B, Januario T, Ahn CP, Holcomb T, Pujara K, Stinson J, Callahan CA, Tang T, Bazan JF, Kan Z, Seshagiri S, Hann CL, Gould SE, Low JA, Rudin CM, de Sauvage FJ (2009) Smoothened mutation confers resistance to a hedgehog pathway inhibitor in medulloblastoma. Science 326(5952):572–574. doi: 10.1126/science.1179386 PubMedCrossRefGoogle Scholar
  39. 39.
    Omenetti A, Porrello A, Jung Y, Yang L, Popov Y, Choi SS, Witek RP, Alpini G, Venter J, Vandongen HM, Syn WK, Baroni GS, Benedetti A, Schuppan D, Diehl AM (2008) Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans. J Clin Invest 118(10):3331–3342. doi: 10.1172/JCI35875 PubMedPubMedCentralGoogle Scholar
  40. 40.
    Nakanuma Y, Harada K, Ishikawa A, Zen Y, Sasaki M (2003) Anatomic and molecular pathology of intrahepatic cholangiocarcinoma. J Hepatobiliary Pancreat Surg 10(4):265–281. doi: 10.1007/s00534-002-0729-3 PubMedCrossRefGoogle Scholar
  41. 41.
    Oishi N, Wang XW (2011) Novel therapeutic strategies for targeting liver cancer stem cells. Int J Biol Sci 7(5):517–535PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Ishimura N, Isomoto H, Bronk SF, Gores GJ (2006) Trail induces cell migration and invasion in apoptosis-resistant cholangiocarcinoma cells. Am J Physiol Gastrointest Liver Physiol 290(1):G129–G136. doi: 10.1152/ajpgi.00242.2005 PubMedCrossRefGoogle Scholar
  43. 43.
    Mott JL, Kurita S, Cazanave SC, Bronk SF, Werneburg NW, Fernandez-Zapico ME (2010) Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NF-kappaB. J Cell Biochem 110(5):1155–1164. doi: 10.1002/jcb.22630 PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Omenetti A, Diehl AM (2011) Hedgehog signaling in cholangiocytes. Curr Opin Gastroenterol 27(3):268–275. doi: 10.1097/MOG.0b013e32834550b4 PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    ClinicalTrials.gov, U.S. National Institutes of Health. Available via http://www.clinicaltrials.gov. Accessed 25 June 2014

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Tobias Kiesslich
    • 1
    • 2
  • Christian Mayr
    • 1
  • Julia Wachter
    • 1
    • 3
  • Doris Bach
    • 1
  • Julia Fuereder
    • 1
  • Andrej Wagner
    • 1
  • Beate Alinger
    • 4
  • Martin Pichler
    • 5
  • Pietro Di Fazio
    • 6
  • Matthias Ocker
    • 7
    • 8
  • Frieder Berr
    • 1
  • Daniel Neureiter
    • 4
    Email author
  1. 1.Department of Internal Medicine IParacelsus Medical University/Salzburger Landeskliniken (SALK)SalzburgAustria
  2. 2.Institute of Physiology and PathophysiologyParacelsus Medical UniversitySalzburgAustria
  3. 3.Department of Internal Medicine IILandesklinikum Gänserndorf-MistelbachMistelbachAustria
  4. 4.Institute of PathologyParacelsus Medical University/Salzburger Landeskliniken (SALK)SalzburgAustria
  5. 5.Department of Experimental TherapeuticsThe University of Texas MD Anderson Cancer CenterHoustonUSA
  6. 6.Department of Visceral, Thoracic and Vascular SurgeryPhilipps-University MarburgMarburgGermany
  7. 7.Institute for Surgical ResearchPhilipps-University MarburgMarburgGermany
  8. 8.Experimental Medicine OncologyBayer Pharma AGBerlinGermany

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