Cellular Oncology

, Volume 42, Issue 2, pp 143–155 | Cite as

Interfering with bromodomain epigenome readers as therapeutic option in mucoepidermoid carcinoma

  • Renata L. Markman
  • Liana P. Webber
  • Carlos H. V. Nascimento Filho
  • Leonardo A. Reis
  • Pablo A. Vargas
  • Marcio A. Lopes
  • Virgilio Zanella
  • Manoela D. Martins
  • Cristiane H. Squarize
  • Rogerio M. CastilhoEmail author
Original Article



Emerging evidence indicates that bromodomains comprise a conserved class of epigenome readers involved in cancer development and inflammation. Bromodomains are associated with epigenetic modifications of gene transcription through interactions with lysine residues of histone tails. Particularly, the bromodomain and extra-terminal domain (BET) family member BRD4 has been found to be involved in the control over oncogenes, including c-MYC, and in the maintenance of downstream inflammatory processes. The objective of this study was to evaluate the effect of pharmacologically displacing BRD4 in mucoepidermoid carcinoma (MEC) cells.


We assessed the presence of BRD4 levels in a panel of human MEC tissue samples in conjunction with histological grading and clinical information. In vitro studies were carried out using human MEC-derived cell lines. The BET inhibitor iBET762 was administered to MEC cells to assess the impact of disrupted BRD4 signaling on colony forming capacities and cell cycle status. The activation of cellular senescence induced by iBET762 was determined by immunohistochemical staining for p16ink4. Flow cytometry was used to identify populations of cancer stem cells in MEC-derived cell lines.


We found that primary human MECs and MEC-derived cell lines are endowed with high BRD4 expression levels compared to those in normal salivary glands. We also found that, by displacing BRD4 from chromatin using the BET inhibitor iBET762, MEC cells lose their colony forming capacities and undergo G1 cell cycle arrest and senescence. Finally, we found that targeted displacement of BRD4 from chromatin results in depletion of cancer stem cells from the overall MEC cell populations.


Our findings indicate that bromodomain-mediated gene regulation constitutes an epigenetic mechanism that is deregulated in MEC cells and that the use of BET inhibitors may serve as a feasible therapeutic strategy to manage MECs.


Mucoepidermoid carcinoma BRD4 Epigenetic Cancer stem cells iBET762 Epi-drug 



This work was conducted during a visiting scholar period at the University of Michigan, sponsored by the Capes Foundation within the Ministry of Education, Brazil (grant n. BEX / 88881.132606/2016-01 PDSE). This grant was funded by a University of Michigan School of Dentistry faculty grant, and a Cancer Center Support Grant (P30 CA046592). The authors declare no potential conflicts of interest with respect to authorships and/or publication of this article.

Author’s contributions

RLM performed most of the cell culture-based assays, IHC, IF, and participated in the organization of the figures. CHVNF performed the flow cytometry assays, LAR helped with the IHC and IF assays, and LPW helped with the immunofluorescence assays and its quantifications. VZ and MDM contributed to the collection of MEC tissue samples and their clinical data and helped with their re-evaluation. PAV, MAL, CHS and RMC contributed to the conception, design, data organization and writing of the manuscript. All authors gave their final approval and agreed to be accountable for all aspects of the work.

Compliance with ethical standards

Conflict of interest

None declared.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.


  1. 1.
    P.A. Vargas, R. Gerhard, V.J. Araujo Filho, I.V. de Castro, Salivary gland tumors in a Brazilian population: A retrospective study of 124 cases. Rev. Hosp. Clin. Fac. Med. Sao Paulo 57, 271–276 (2002)CrossRefGoogle Scholar
  2. 2.
    F.A. de Oliveira, E.C. Duarte, C.T. Taveira, A.A. Maximo, E.C. de Aquino, C. Alencar Rde, E.F. Vencio, Salivary gland tumor: a review of 599 cases in a Brazilian population. Head Neck Pathol. 3, 271–275 (2009)CrossRefGoogle Scholar
  3. 3.
    F.P. Fonseca, V. Carvalho Mde, O.P. de Almeida, A.L. Rangel, M.C. Takizawa, A.G. Bueno, P.A. Vargas, Clinicopathologic analysis of 493 cases of salivary gland tumors in a southern Brazilian population. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 114, 230–239 (2012)CrossRefGoogle Scholar
  4. 4.
    E.S. Choi, S. Oh, B. Jang, H.J. Yu, J.A. Shin, N.P. Cho, I.H. Yang, D.H. Won, H.J. Kwon, S.D. Hong, S.D. Cho, Silymarin and its active component silibinin act as novel therapeutic alternatives for salivary gland cancer by targeting the ERK1/2-Bim signaling cascade. Cell Oncol. 40, 235–246 (2017)Google Scholar
  5. 5.
    N. Saghravanian, N. Ghazi, M. Saba, Clinicopathologic evaluation of salivary gland neoplasms: a 38-year retrospective study in Iran. Ann. Diagn. Pathol. 17, 522–525 (2013)CrossRefGoogle Scholar
  6. 6.
    J.H. Mikesch, W. Hartmann, L. Angenendt, O. Huber, C. Schliemann, M.F. Arteaga, E. Wardelmann, C. Rudack, W.E. Berdel, M. Stenner and I. Grunewald, AAA+ ATPases Reptin and Pontin as potential diagnostic and prognostic biomarkers in salivary gland cancer - a short report. Cell Oncol. 41, 455–462 (2018)Google Scholar
  7. 7.
    L. Barnes, Universitäts-Spital Zurich. Dept. Pathologie., International Academy of Pathology., World Health Organization. and International Agency for Research on Cancer., Pathology and genetics of head and neck tumours, (IARC Press, Lyon, 2007)Google Scholar
  8. 8.
    M.R. Posner, T.J. Ervin, R.R. Weichselbaum, R.L. Fabian, D. Miller, Chemotherapy of advanced salivary gland neoplasms. Cancer 50, 2261–2264 (1982)CrossRefGoogle Scholar
  9. 9.
    S. Grisanti, V. Amoroso, M. Buglione, A. Rosati, R. Gatta, C. Pizzocaro, V.D. Ferrari, G. Marini, Cetuximab in the treatment of metastatic mucoepidermoid carcinoma of the salivary glands: a case report and review of literature. J. Med. Case Rep. 2, 320 (2008)CrossRefGoogle Scholar
  10. 10.
    T. Cerda, X.S. Sun, S. Vignot, P.Y. Marcy, B. Baujat, A.C. Baglin, A.M. Ali, S. Testelin, E. Reyt, F. Janot, J. Thariat, A rationale for chemoradiation (vs radiotherapy) in salivary gland cancers? On behalf of the REFCOR (French rare head and neck cancer network). Crit. Rev. Oncol. Hematol. 91, 142–158 (2014)CrossRefGoogle Scholar
  11. 11.
    A. Coca-Pelaz, J.P. Rodrigo, A. Triantafyllou, J.L. Hunt, A. Rinaldo, P. Strojan, M. Haigentz Jr., W.M. Mendenhall, R.P. Takes, V. Vander Poorten, A. Ferlito, Salivary mucoepidermoid carcinoma revisited. Eur. Arch. Otorhinolaryngol. 272, 799–819 (2015)CrossRefGoogle Scholar
  12. 12.
    R.M. Castilho, C.H. Squarize, L.O. Almeida, Epigenetic modifications and head and neck Cancer: Implications for tumor progression and resistance to therapy. Int J Mol Sci. 18 E15063 (2017)Google Scholar
  13. 13.
    M.S. Gilardini Montani, M. Granato, C. Santoni, P. Del Porto, N. Merendino, G. D'Orazi, A. Faggioni, M. Cirone, Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells. Cell Oncol. 40, 167–180 (2017)Google Scholar
  14. 14.
    M. Staberg, S.R. Michaelsen, R.D. Rasmussen, M. Villingshoj, H.S. Poulsen, P. Hamerlik, Inhibition of histone deacetylases sensitizes glioblastoma cells to lomustine. Cell Oncol. 40, 21–32 (2017)Google Scholar
  15. 15.
    M.F. Segura, B. Fontanals-Cirera, A. Gaziel-Sovran, M.V. Guijarro, D. Hanniford, G. Zhang, P. Gonzalez-Gomez, M. Morante, L. Jubierre, W. Zhang, F. Darvishian, M. Ohlmeyer, I. Osman, M.M. Zhou, E. Hernando, BRD4 sustains melanoma proliferation and represents a new target for epigenetic therapy. Cancer Res. 73, 6264–6276 (2013)CrossRefGoogle Scholar
  16. 16.
    M. Perez-Salvia, M. Esteller, Bromodomain inhibitors and cancer therapy: From structures to applications. Epigenetics 12, 323–339 (2017)CrossRefGoogle Scholar
  17. 17.
    C.A. French, I. Miyoshi, J.C. Aster, I. Kubonishi, T.G. Kroll, P. Dal Cin, S.O. Vargas, A.R. Perez-Atayde, J.A. Fletcher, BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19). Am. J. Pathol. 159, 1987–1992 (2001)CrossRefGoogle Scholar
  18. 18.
    D. Houzelstein, S.L. Bullock, D.E. Lynch, E.F. Grigorieva, V.A. Wilson, R.S. Beddington, Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4. Mol. Cell. Biol. 22, 3794–3802 (2002)CrossRefGoogle Scholar
  19. 19.
    K. Mochizuki, A. Nishiyama, M.K. Jang, A. Dey, A. Ghosh, T. Tamura, H. Natsume, H. Yao, K. Ozato, The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase. J. Biol. Chem. 283, 9040–9048 (2008)CrossRefGoogle Scholar
  20. 20.
    V.P. Wagner, M.A. Martins, M.D. Martins, K.A. Warner, L.P. Webber, C.H. Squarize, J.E. Nor, R.M. Castilho, Overcoming adaptive resistance in mucoepidermoid carcinoma through inhibition of the IKK-beta/IkappaBalpha/NFkappaB axis. Oncotarget 7, 73032–73044 (2016)CrossRefGoogle Scholar
  21. 21.
    N.A. Franken, H.M. Rodermond, J. Stap, J. Haveman, C. van Bree, Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006)CrossRefGoogle Scholar
  22. 22.
    M.D. Martins, Y. Jiao, L. Larsson, L.O. Almeida, C. Garaicoa-Pazmino, J.M. Le, C.H. Squarize, N. Inohara, W.V. Giannobile, R.M. Castilho, Epigenetic modifications of histones in periodontal disease. J. Dent. Res. 95, 215–222 (2016)CrossRefGoogle Scholar
  23. 23.
    E. Nicodeme, K.L. Jeffrey, U. Schaefer, S. Beinke, S. Dewell, C.W. Chung, R. Chandwani, I. Marazzi, P. Wilson, H. Coste, J. White, J. Kirilovsky, C.M. Rice, J.M. Lora, R.K. Prinjha, K. Lee, A. Tarakhovsky, Suppression of inflammation by a synthetic histone mimic. Nature 468, 1119–1123 (2010)CrossRefGoogle Scholar
  24. 24.
    O. Mirguet, R. Gosmini, J. Toum, C.A. Clement, M. Barnathan, J.M. Brusq, J.E. Mordaunt, R.M. Grimes, M. Crowe, O. Pineau, M. Ajakane, A. Daugan, P. Jeffrey, L. Cutler, A.C. Haynes, N.N. Smithers, C.W. Chung, P. Bamborough, I.J. Uings, A. Lewis, J. Witherington, N. Parr, R.K. Prinjha, E. Nicodeme, Discovery of epigenetic regulator I-BET762: Lead optimization to afford a clinical candidate inhibitor of the BET bromodomains. J. Med. Chem. 56, 7501–7515 (2013)CrossRefGoogle Scholar
  25. 25.
    S. Wang, A.M. Pike, S.S. Lee, M.A. Strong, C.J. Connelly, C.W. Greider, BRD4 inhibitors block telomere elongation. Nucleic Acids Res. 45, 8403–8410 (2017)CrossRefGoogle Scholar
  26. 26.
    L.O. Almeida, D.M. Guimaraes, M.D. Martins, M.A.T. Martins, K.A. Warner, J.E. Nor, R.M. Castilho, C.H. Squarize, Unlocking the chromatin of adenoid cystic carcinomas using HDAC inhibitors sensitize cancer stem cells to cisplatin and induces tumor senescence. Stem Cell Res. 21, 94–105 (2017)CrossRefGoogle Scholar
  27. 27.
    A. Newbold, K.J. Falkenberg, H.M. Prince, R.W. Johnstone, How do tumor cells respond to HDAC inhibition? FEBS J. 283, 4032–4046 (2016)CrossRefGoogle Scholar
  28. 28.
    D.M. Guimaraes, L.O. Almeida, M.D. Martins, K.A. Warner, A.R. Silva, P.A. Vargas, F.D. Nunes, C.H. Squarize, J.E. Nor, R.M. Castilho, Sensitizing mucoepidermoid carcinomas to chemotherapy by targeted disruption of cancer stem cells. Oncotarget 7, 42447–42460 (2016)Google Scholar
  29. 29.
    A. Adams, K. Warner, A.T. Pearson, Z. Zhang, H.S. Kim, D. Mochizuki, G. Basura, J. Helman, A. Mantesso, R.M. Castilho, M.S. Wicha, J.E. Nor, ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas. Oncotarget 6, 26633–26650 (2015)Google Scholar
  30. 30.
    V.P. Wagner, M.D. Martins, M.A.T. Martins, L.O. Almeida, K.A. Warner, J.E. Nor, C.H. Squarize, R.M. Castilho, Targeting histone deacetylase and NFkappaB signaling as a novel therapy for Mucoepidermoid carcinomas. Sci. Rep. 8, 2065 (2018)CrossRefGoogle Scholar
  31. 31.
    M. Granic, P. Suton, D. Mueller, I. Cvrljevic, I. Luksic, Prognostic factors in head and neck mucoepidermoid carcinoma: experience at a single institution based on 64 consecutive patients over a 28-year period. Int J Oral Maxillofac Surg. 47, 283-288 (2018)Google Scholar
  32. 32.
    A.C. Birkeland, S.K. Foltin, N.L. Michmerhuizen, R.C. Hoesli, A.J. Rosko, S. Byrd, M. Yanik, J.E. Nor, C.R. Bradford, M.E. Prince, T.E. Carey, J.B. McHugh, M.E. Spector, J.C. Brenner, Correlation of Crtc1/3-Maml2 fusion status, grade and survival in mucoepidermoid carcinoma. Oral Oncol. 68, 5–8 (2017)CrossRefGoogle Scholar
  33. 33.
    B.N. Devaiah, C. Case-Borden, A. Gegonne, C.H. Hsu, Q. Chen, D. Meerzaman, A. Dey, K. Ozato, D.S. Singer, BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin. Nat. Struct. Mol. Biol. 23, 540–548 (2016)CrossRefGoogle Scholar
  34. 34.
    X. Wu, D. Liu, X. Gao, F. Xie, D. Tao, X. Xiao, L. Wang, G. Jiang, F. Zeng, Inhibition of BRD4 suppresses cell proliferation and induces apoptosis in renal cell carcinoma. Cell. Physiol. Biochem. 41, 1947–1956 (2017)CrossRefGoogle Scholar
  35. 35.
    J. Zuber, J. Shi, E. Wang, A.R. Rappaport, H. Herrmann, E.A. Sison, D. Magoon, J. Qi, K. Blatt, M. Wunderlich, M.J. Taylor, C. Johns, A. Chicas, J.C. Mulloy, S.C. Kogan, P. Brown, P. Valent, J.E. Bradner, S.W. Lowe, C.R. Vakoc, RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478, 524–528 (2011)CrossRefGoogle Scholar
  36. 36.
    A. Chaidos, V. Caputo, K. Gouvedenou, B. Liu, I. Marigo, M.S. Chaudhry, A. Rotolo, D.F. Tough, N.N. Smithers, A.K. Bassil, T.D. Chapman, N.R. Harker, O. Barbash, P. Tummino, N. Al-Mahdi, A.C. Haynes, L. Cutler, B. Le, A. Rahemtulla, I. Roberts, M. Kleijnen, J.J. Witherington, N.J. Parr, R.K. Prinjha, A. Karadimitris, Potent antimyeloma activity of the novel bromodomain inhibitors I-BET151 and I-BET762. Blood 123, 697–705 (2014)CrossRefGoogle Scholar
  37. 37.
    J.E. Delmore, G.C. Issa, M.E. Lemieux, P.B. Rahl, J. Shi, H.M. Jacobs, E. Kastritis, T. Gilpatrick, R.M. Paranal, J. Qi, M. Chesi, A.C. Schinzel, M.R. McKeown, T.P. Heffernan, C.R. Vakoc, P.L. Bergsagel, I.M. Ghobrial, P.G. Richardson, R.A. Young, W.C. Hahn, K.C. Anderson, A.L. Kung, J.E. Bradner, C.S. Mitsiades, BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904–917 (2011)CrossRefGoogle Scholar
  38. 38.
    S.B. Baylin, P.A. Jones, Epigenetic determinants of Cancer. Cold Spring Harb Perspect Biol. 8(9) (2016).
  39. 39.
    Y. Yokoyama, H. Zhu, J.H. Lee, A.V. Kossenkov, S.Y. Wu, J.M. Wickramasinghe, X. Yin, K.C. Palozola, A. Gardini, L.C. Showe, K.S. Zaret, Q. Liu, D. Speicher, J.R. Conejo-Garcia, J.E. Bradner, Z. Zhang, A.K. Sood, T. Ordog, B.G. Bitler, R. Zhang, BET inhibitors suppress ALDH activity by targeting ALDH1A1 super-enhancer in ovarian Cancer. Cancer Res. 76, 6320–6330 (2016)CrossRefGoogle Scholar
  40. 40.
    N. Tasdemir, A. Banito, J.S. Roe, D. Alonso-Curbelo, M. Camiolo, D.F. Tschaharganeh, C.H. Huang, O. Aksoy, J.E. Bolden, C.C. Chen, M. Fennell, V. Thapar, A. Chicas, C.R. Vakoc, S.W. Lowe, BRD4 connects enhancer remodeling to senescence immune surveillance. Cancer Discov. 6, 612–629 (2016)CrossRefGoogle Scholar
  41. 41.
    M. Collado, M.A. Blasco, M. Serrano, Cellular senescence in cancer and aging. Cell 130, 223–233 (2007)CrossRefGoogle Scholar
  42. 42.
    R.M. Castilho, C.H. Squarize, L.A. Chodosh, B.O. Williams, J.S. Gutkind, mTOR mediates Wnt-induced epidermal stem cell exhaustion and aging. Cell Stem Cell 5, 279–289 (2009)CrossRefGoogle Scholar
  43. 43.
    H. Liu, M.M. Fergusson, R.M. Castilho, J. Liu, L. Cao, J. Chen, D. Malide, I.I. Rovira, D. Schimel, C.J. Kuo, J.S. Gutkind, P.M. Hwang, T. Finkel, Augmented Wnt signaling in a mammalian model of accelerated aging. Science 317, 803–806 (2007)CrossRefGoogle Scholar
  44. 44.
    J. Campisi, F. d’Adda di Fagagna, Cellular senescence: when bad things happen to good cells. Nat. Rev. Mol. Cell Biol. 8, 729–740 (2007)CrossRefGoogle Scholar
  45. 45.
    D. Hanahan, R.A. Weinberg, The hallmarks of cancer. Cell 100, 57–70 (2000)CrossRefGoogle Scholar

Copyright information

© International Society for Cellular Oncology 2018

Authors and Affiliations

  • Renata L. Markman
    • 1
    • 2
  • Liana P. Webber
    • 1
    • 3
  • Carlos H. V. Nascimento Filho
    • 1
    • 4
  • Leonardo A. Reis
    • 1
    • 2
  • Pablo A. Vargas
    • 2
  • Marcio A. Lopes
    • 2
  • Virgilio Zanella
    • 3
    • 5
  • Manoela D. Martins
    • 3
  • Cristiane H. Squarize
    • 1
    • 6
  • Rogerio M. Castilho
    • 1
    • 6
    Email author
  1. 1.Laboratory of Epithelial Biology, Department of Periodontics and Oral MedicineUniversity of Michigan School of DentistryAnn ArborUSA
  2. 2.Department of Oral Diagnosis, Piracicaba Dental SchoolState University of Campinas-UNICAMPPiracicabaBrazil
  3. 3.Department of Oral Pathology, School of DentistryFederal University of Rio Grande do Sul-UFRGSPorto AlegreBrazil
  4. 4.Genetics and Molecular Biology Research UnitSao Jose do Rio Preto Medical School-FAMERPSao Jose do Rio PretoBrazil
  5. 5.Head and Neck DepartmentSanta Rita Hospital - Santa Casa de Misericordia de Porto AlegrePorto AlegreBrazil
  6. 6.Comprehensive Cancer CenterUniversity of Michigan Ann ArborAnn ArborUSA

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