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Reduced proliferation and colony formation of head and neck squamous cell carcinoma (HNSCC) after dual targeting of EGFR and hedgehog pathways

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Abstract

Purpose

The hedgehog signalling pathway (Hh) is frequently active in head and neck squamous cell carcinoma (HNSCC). Overexpressed Hh associates with poor prognosis. The Hh inhibitor vismodegib targets smoothened, and based on molecular data, may prevent resistance to EGFR targeting.

Methods

To elucidate potential roles of vismodegib in HNSCC therapy, its sole effects and those combined with cisplatin, docetaxel, and cetuximab on HNSCC cell lines were assessed by MTT metabolisation and BrdU incorporation. Colony formation (CF) of primary HNSCC cells was studied utilizing the FLAVINO-protocol. Combinatory effects were analysed regarding antagonism, additivity or synergism. Associations between the ex vivo detected mode of action of vismodegib with other treatments related to patient characteristics were assessed and progression-free survival (PFS) in patient groups compared using Kaplan–Meier curves.

Results

Vismodegib suppressed BrdU incorporation significantly stronger than MTT turnover; CF was significantly inhibited at ≥20 µM vismodegib while concentrations <20 µM acted hormetic. Combining 20 µM vismodegib plus docetaxel (T), cisplatin (P), and cetuximab (E), additively enhanced anti-tumoral activity in HNSCC samples from patients with superior PFS highlighting a potential role for ex-vivo testing of this combination for use as a prognostic classifier.

Conclusion

We provide ex-vivo evidence for vismodegib’s potential in HNSCC therapies, especially if combined with cetuximab, cisplatin and docetaxel.

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References

  1. Zigon G, Berrino F, Gatta G et al (2011) Prognoses for head and neck cancers in Europe diagnosed in 1995–1999: a population-based study. Ann Oncol 22:165–174. doi:10.1093/annonc/mdq306

    Article  CAS  PubMed  Google Scholar 

  2. Ferlay J, Steliarova-Foucher E, Lortet-Tieulent J et al (2013) Cancer incidence and mortality patterns in Europe: estimates for 40 countries in 2012. Eur J Cancer 49:1374–1403. doi:10.1016/j.ejca.2012.12.027

    Article  CAS  PubMed  Google Scholar 

  3. Macfarlane GJ, Zheng T, Marshall JR et al (1995) Alcohol, tobacco, diet and the risk of oral cancer. A pooled analysis of three case–control studies. Eur J Cancer Part B Oral Oncol 31:181–187. doi:10.1016/0964-1955(95)00005-3

    Article  Google Scholar 

  4. Andl T, Kahn T, Pfuhl A et al (1998) Etiological involvement of oncogenic human papillomavirus in tonsillar squamous cell carcinomas lacking retinoblastoma cell cycle control. Cancer Res 58:5–13

    CAS  PubMed  Google Scholar 

  5. Gillison ML (2000) Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J Ntnl Cancer Inst 92:709–720. doi:10.1093/jnci/92.9.709

    Article  CAS  Google Scholar 

  6. Seshacharyulu P, Ponnusamy MP, Haridas D, Jain M, Ganti AK, Batra SK (2012) Targeting the EGFR signaling pathway in cancer therapy. Expert Opin Ther Targets 16:15–31. doi:10.1517/14728222.2011.648617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Grandis JR, Tweardy DJ (1993) Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer. Cancer Res 53:3579–3584

    CAS  PubMed  Google Scholar 

  8. Bonner JA, Harari PM, Giralt J et al (2006) Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 354:567–578. doi:10.1056/NEJMoa053422

    Article  CAS  PubMed  Google Scholar 

  9. Rivera F, García-Castaño A, Vega N, Vega-Villegas ME, Gutiérrez-Sanz L (2009) Cetuximab in metastatic or recurrent head and neck cancer: the EXTREME trial. Expert Rev Anticancer Ther 9:1421–1428. doi:10.1586/era.09.113

    Article  CAS  PubMed  Google Scholar 

  10. Dietz A, Boehm A, Mozet C, Wichmann G, Giannis A (2008) Current aspects of targeted therapy in head and neck tumors. Eur Arch Otorhinolaryngol 265(Suppl 1):S3–S12. doi:10.1007/s00405-008-0697-6

    Article  PubMed  Google Scholar 

  11. Scales SJ, Sauvage FJ de (2009) Mechanisms of hedgehog pathway activation in cancer and implications for therapy. Trends Pharmacol Sci 30:303–312. doi:10.1016/j.tips.2009.03.007

    Article  CAS  PubMed  Google Scholar 

  12. Fujii K, Miyashita T (2014) Gorlin syndrome (nevoid basal cell carcinoma syndrome): update and literature review. Pediatr Int 56:667–674. doi:10.1111/ped.12461

    Article  CAS  PubMed  Google Scholar 

  13. Romer J, Curran T (2005) Targeting medulloblastoma: small-molecule inhibitors of the sonic hedgehog pathway as potential cancer therapeutics. Cancer Res 65:4975–4978. doi:10.1158/0008-5472.CAN-05-0481

    Article  CAS  PubMed  Google Scholar 

  14. Varnat F, Duquet A, Malerba M et al (2009) Human colon cancer epithelial cells harbour active HEDGEHOG-GLI signalling that is essential for tumour growth, recurrence, metastasis and stem cell survival and expansion. EMBO Mol Med 1:338–351. doi:10.1002/emmm.200900039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Berman DM, Karhadkar SS, Maitra A et al (2003) Widespread requirement for hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425:846–851. doi:10.1038/nature01972

    Article  CAS  PubMed  Google Scholar 

  16. Watkins DN, Berman DM, Burkholder SG, Wang B, Beachy PA, Baylin SB (2003) Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 422:313–317. doi:10.1038/nature01493

    Article  CAS  PubMed  Google Scholar 

  17. Karhadkar SS, Bova GS, Abdallah N et al (2004) Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 431:707–712. doi:10.1038/nature02962

    Article  CAS  PubMed  Google Scholar 

  18. Chung CH, Dignam JJ, Hammond ME et al (2011) Glioma-associated oncogene family zinc finger 1 expression and metastasis in patients with head and neck squamous cell carcinoma treated with radiation therapy (RTOG 9003). J Clin Oncol 29:1326–1334. doi:10.1200/JCO.2010.32.3295

    Article  PubMed  PubMed Central  Google Scholar 

  19. Dimitrova K, Stoehr M, Dehghani F et al (2013) Overexpression of the hedgehog signalling pathway in head and neck squamous cell carcinoma. Onkologie 36:279–286. doi:10.1159/000350322

    Article  CAS  PubMed  Google Scholar 

  20. Fertig EJ, Markovic A, Danilova LV et al (2013) Preferential activation of the hedgehog pathway by epigenetic modulations in HPV negative HNSCC identified with meta-pathway analysis. PLoS One 8:e78127. doi:10.1371/journal.pone.0078127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Keysar SB, Le PN, Anderson RT et al (2013) Hedgehog signaling alters reliance on EGF receptor signaling and mediates anti-EGFR therapeutic resistance in head and neck cancer. Cancer Res 73:3381–3392. doi:10.1158/0008-5472.CAN-12-4047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gan GN, Eagles J, Keysar SB et al (2014) Hedgehog signaling drives radioresistance and stroma-driven tumor repopulation in head and neck squamous cancers. Cancer Res 74:7024–7036. doi:10.1158/0008-5472.CAN-14-1346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mozet C, Stoehr M, Dimitrova K, Dietz A, Wichmann G (2013) Hedgehog targeting by cyclopamine suppresses head and neck squamous cell carcinoma and enhances chemotherapeutic effects. Anticancer Res 33:2415–2424

    CAS  PubMed  Google Scholar 

  24. Sekulic A, Migden MR, Oro AE et al (2012) Efficacy and safety of vismodegib in advanced basal-cell carcinoma. N Engl J Med 366:2171–2179. doi:10.1056/NEJMoa1113713

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Dietz A, Tschöp K, Wichmann G, Granzow C (2015) Method and kit for the ex vivo evaluation of the response of a tumor to conditions to be tested 09.04.2009. Accessed 10 Sep 2015

  26. Dietz A, Boehm A, Horn I et al (2010) Assay-based response evaluation in head and neck oncology: requirements for better decision making. Eur Arch Otorhinolaryngol 267:483–494. doi:10.1007/s00405-009-1191-5

    Article  PubMed  Google Scholar 

  27. Horn I, Wichmann G, Mozet C et al (2010) Heterogeneity of epithelial and stromal cells of head and neck squamous cell carcinomas in ex vivo chemoresponse. Cancer Chemother Pharmacol 65:1153–1163. doi:10.1007/s00280-009-1124-4

    Article  PubMed  Google Scholar 

  28. Stoehr M, Mozet C, Boehm A, Aigner A, Dietz A, Wichmann G (2014) Simvastatin suppresses head and neck squamous cell carcinoma ex vivo and enhances the cytostatic effects of chemotherapeutics. Cancer Chemother Pharmacol 73:827–837. doi:10.1007/s00280-014-2412-1

    Article  CAS  PubMed  Google Scholar 

  29. Schrader C, Boehm A, Reiche A, Dietz A, Mozet C, Wichmann G (2012) Combined effects of lapatinib and cisplatin on colony formation of head and neck squamous cell carcinoma. Anticancer Res 32:3191–3199

    CAS  PubMed  Google Scholar 

  30. Zhao M, Sano D, Pickering CR et al (2011) Assembly and initial characterization of a panel of 85 genomically validated cell lines from diverse head and neck tumor sites. Clin Cancer Res 17:7248–7264. doi:10.1158/1078-0432.CCR-11-0690

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Sobin LH, Gospodarowicz MK, Wittekind C (2010) TNM classification of malignant tumours. Wiley, Oxford

    Google Scholar 

  32. Qualtrough D, Rees P, Speight B, Williams AC, Paraskeva C (2015) The hedgehog inhibitor cyclopamine reduces β-catenin-Tcf transcriptional activity, induces E-cadherin expression, and reduces invasion in colorectal cancer cells. Cancers 7:1885–1899. doi:10.3390/cancers7030867

    Article  PubMed  PubMed Central  Google Scholar 

  33. Yoo YA, Kang MH, Lee HJ et al (2011) Sonic hedgehog pathway promotes metastasis and lymphangiogenesis via activation of Akt, EMT, and MMP-9 pathway in gastric cancer. Cancer Res 71:7061–7070. doi:10.1158/0008-5472.CAN-11-1338

    Article  CAS  PubMed  Google Scholar 

  34. Bora-Singhal N, Perumal D, Nguyen J, Chellappan S (2015) Gli1-mediated regulation of Sox2 facilitates self-renewal of stem-like cells and confers resistance to EGFR inhibitors in non-small cell lung cancer. Neoplasia (New York, N.Y.) 17:538–551. doi:10.1016/j.neo.2015.07.001

    Article  CAS  Google Scholar 

  35. Zahreddine HA, Culjkovic-Kraljacic B, Assouline S et al (2014) The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation. Nature 511:90–93. doi:10.1038/nature13283

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Della Corte CM, Bellevicine C, Vicidomini G et al (2015) SMO gene amplification and activation of the hedgehog pathway as novel mechanisms of resistance to anti-epidermal growth factor receptor drugs in human lung cancer. Clin Cancer Res 21:4686–4697. doi:10.1158/1078-0432.CCR-14-3319

    Article  CAS  PubMed  Google Scholar 

  37. Bowles DW, Keysar SB, Glogowska MJ et al (2014) A pilot study of cetuximab and the hedgehog inhibitor IPI-926 in recurrent/metastatic (R/M) head-and-neck squamous cell carcinoma. Int J Radiat Oncol Biol Phys 88:511. doi:10.1016/j.ijrobp.2013.11.152

    Article  Google Scholar 

  38. Singh S, Wang Z, Liang Fei D et al (2011) Hedgehog-producing cancer cells respond to and require autocrine hedgehog activity. Cancer Res 71:4454–4463. doi:10.1158/0008-5472.CAN-10-2313

    Article  CAS  PubMed  Google Scholar 

  39. Yauch RL, Gould SE, Scales SJ et al (2008) A paracrine requirement for hedgehog signalling in cancer. Nature 455:406–410. doi:10.1038/nature07275

    Article  CAS  PubMed  Google Scholar 

  40. Rhim AD, Oberstein PE, Thomas DH et al (2014) Stromal elements act to restrain, rather than support, pancreatic ductal adenocarcinoma. Cancer cell 25:735–747. doi:10.1016/j.ccr.2014.04.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Chun SG, Park H, Pandita RK et al (2015) Targeted inhibition of histone deacetylases and hedgehog signaling suppress tumor growth and homologous recombination in aerodigestive cancers. Am J Cancer Res 5:1337–1352

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Brechbiel J, Miller-Moslin K, Adjei AA (2014) Crosstalk between hedgehog and other signaling pathways as a basis for combination therapies in cancer. Cancer Treat Rev 40:750–759. doi:10.1016/j.ctrv.2014.02.003

    Article  CAS  PubMed  Google Scholar 

  43. Calabrese EJ, Nascarella MA (2010) Tumor resistance explained by hormesis. Dose Response 8:80–82. doi:10.2203/dose-response.09-063.Calabrese

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Graham RA, Lum BL, Cheeti S et al (2011) Pharmacokinetics of hedgehog pathway inhibitor vismodegib (GDC-0449) in patients with locally advanced or metastatic solid tumors: the role of alpha-1-acid glycoprotein binding. Clin Cancer Res 17:2512–2520. doi:10.1158/1078-0432.CCR-10-2736

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gajjar AJ, Gururangan S, Qaddoumi IA et al (2013) A prospective phase II study to determine the efficacy of GDC 0449 (vismodegib) in adults with recurrent medulloblastoma (MB): a Pediatric Brain Tumor Consortium study (PBTC 25B). J Clin Oncol 31:suppl; abstr 2035

    Google Scholar 

  46. Berlin J, Bendell JC, Hart LL et al (2013) A randomized phase II trial of vismodegib versus placebo with FOLFOX or FOLFIRI and bevacizumab in patients with previously untreated metastatic colorectal cancer. Clin Cancer Res 19:258–267. doi:10.1158/1078-0432.CCR-12-1800

    Article  CAS  PubMed  Google Scholar 

  47. Kaye SB, Fehrenbacher L, Holloway R et al (2012) A phase II, randomized, placebo-controlled study of vismodegib as maintenance therapy in patients with ovarian cancer in second or third complete remission. Clin Cancer Res 18:6509–6518. doi:10.1158/1078-0432.CCR-12-1796

    Article  CAS  PubMed  Google Scholar 

  48. Kim EJ, Sahai V, Abel EV et al (2014) Pilot clinical trial of hedgehog pathway inhibitor GDC-0449 (vismodegib) in combination with gemcitabine in patients with metastatic pancreatic adenocarcinoma. Clin Cancer Res 20:5937–5945. doi:10.1158/1078-0432.CCR-14-1269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Anett Reiche for excellent technical assistance and the patients and their families for participation in the study.

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    Authors and Affiliations

    1. ENT-Research Laboratory, Clinic for Otorhinolaryngology, Head and Neck Surgery, University Hospital Leipzig, Liebigstraße 21, Liebigstr. 10-14, 04103, Leipzig, Germany

      Hannes Liebig, Georg Günther, Marlen Kolb, Christian Mozet, Andreas Boehm, Andreas Dietz & Gunnar Wichmann

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    1. Hannes Liebig
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    2. Georg Günther
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    4. Christian Mozet
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    Correspondence to Gunnar Wichmann.

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    Funding

    The study was supported by the Grant EPGZ268406-G from Roche Pharma AG.

    Conflict of interest

    Gunnar Wichmann received funding for a translational research project on “Efficacy of vismodegib (GDC-0449) on HNSCC ex vivo, EPGZ268406-G” from Roche Pharma AG. All other authors declare no conflict of interest.

    Ethical approval

    The study was performed in accordance with the ethical standards as laid down in the 1964 declaration of Helsinki and its later amendments (vote 201-10-12072010 of the local ethics committee at the Medical Faculty of the University Leipzig).

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    H. Liebig and G. Günther equally contributed to the paper.

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    Liebig, H., Günther, G., Kolb, M. et al. Reduced proliferation and colony formation of head and neck squamous cell carcinoma (HNSCC) after dual targeting of EGFR and hedgehog pathways. Cancer Chemother Pharmacol 79, 411–420 (2017). https://doi.org/10.1007/s00280-017-3239-3

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    • DOI: https://doi.org/10.1007/s00280-017-3239-3

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