Functional Analysis of HDACs in Tumorigenesis

  • Melissa Hadley
  • Satish Noonepalle
  • Debarati Banik
  • Alejandro VillagraEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1983)


HDACs, originally described as histone modifiers, have recently been demonstrated to modify a variety of other proteins that are involved in diverse cellular processes unrelated to the chromatin environment. This includes deacetylation of nonhistone targets involved in multiple signaling pathways. In this regard, a considerable number of reports have analyzed the role of nonspecific inhibition of HDACs through pan-HDACi in cancer as well as processes of immune regulation. However, with pan-HDACi there is a lack of understanding about the exact contribution of inhibition of each individual HDAC, which makes the rational design of improved drug candidates extremely difficult. Additionally, current approaches using nonselective HDACi in the clinic have critical limitations, including pan-HDACi which elicit poor activity in solid tumors and cardiac toxicity, class I HDACi which activate multiple apoptotic pathways, limiting its use for longer periods of time, and class I-HDAC6i that evidenced a number of adverse effects in initial clinical trials. Therefore, there is a growing interest in the identification of more selective HDACi, and the subsequent development of accurate functional tests to identify the effectiveness and selectivity of these inhibitors. In this chapter, we are describing some selected methodologies to identify the individual activities of HDACs. In addition, we present specific methods to identify enzymatic and nonenzymatic molecular targets of HDACs.


HDAC inhibitors Acetylation Deacetylation Tumor digestion Immunoprecipitation Chromatin immunoprecipitation HDAC activity assay 



This research was supported entirely by Team Award, Melanoma Research Foundation. We thank Kimberlyn Acklin from the flow cytometry core laboratory and Erica Palmer for assistance with animal experiments.


  1. 1.
    Scholz C et al (2015) Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat Biotechnol 33(4):415–423CrossRefGoogle Scholar
  2. 2.
    Choudhary C et al (2009) Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325(5942):834–840CrossRefGoogle Scholar
  3. 3.
    Kim SC et al (2006) Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23(4):607–618PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Ozdag H et al (2006) Differential expression of selected histone modifier genes in human solid cancers. BMC Genomics 7:90PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Fraga MF et al (2005) Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 37(4):391–400PubMedCrossRefGoogle Scholar
  6. 6.
    Haberland M, Montgomery RL, Olson EN (2009) The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet 10(1):32–42PubMedPubMedCentralCrossRefGoogle Scholar
  7. 7.
    Seto E, Yoshida M (2014) Erasers of histone acetylation: the histone deacetylase enzymes. Cold Spring Harb Perspect Biol 6(4):a018713PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Leipe DD, Landsman D (1997) Histone deacetylases, acetoin utilization proteins and acetylpolyamine amidohydrolases are members of an ancient protein superfamily. Nucleic Acids Res 25(18):3693–3697PubMedPubMedCentralCrossRefGoogle Scholar
  9. 9.
    Glozak MA, Seto E (2007) Histone deacetylases and cancer. Oncogene 26(37):5420–5432PubMedCrossRefGoogle Scholar
  10. 10.
    Gregoretti IV, Lee YM, Goodson HV (2004) Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis. J Mol Biol 338(1):17–31PubMedCrossRefGoogle Scholar
  11. 11.
    Grunstein M, Gasser SM (2013) Epigenetics in Saccharomyces cerevisiae. Cold Spring Harb Perspect Biol 5(7):a017491PubMedPubMedCentralCrossRefGoogle Scholar
  12. 12.
    Gao L, Cueto MA, Asselbergs F, Atadja P (2002) Cloning and functional characterization of HDAC11, a novel member of the human histone deacetylase family. J Biol Chem 277(28):25748–25755PubMedCrossRefGoogle Scholar
  13. 13.
    Reichert N, Choukrallah MA, Matthias P (2012) Multiple roles of class I HDACs in proliferation, differentiation, and development. Cell Mol Life Sci 69(13):2173–2187PubMedCrossRefGoogle Scholar
  14. 14.
    Yang XJ, Seto E (2008) The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men. Nat Rev Mol Cell Biol 9(3):206–218PubMedPubMedCentralCrossRefGoogle Scholar
  15. 15.
    Bosch-Presegue L, Vaquero A (2011) The dual role of sirtuins in cancer. Genes Cancer 2(6):648–662PubMedPubMedCentralCrossRefGoogle Scholar
  16. 16.
    Villagra A et al (2009) The histone deacetylase HDAC11 regulates the expression of interleukin 10 and immune tolerance. Nat Immunol 10(1):92–100PubMedCrossRefGoogle Scholar
  17. 17.
    Suliman BA, Xu D, Williams BRG (2012) HDACi: molecular mechanisms and therapeutic implications in the innate immune system. Immunol Cell Biol 90(1):23–32PubMedCrossRefGoogle Scholar
  18. 18.
    Zhang H, Xiao Y, Zhu Z, Li B, Greene MI (2012) Immune regulation by histone deacetylases: a focus on the alteration of FOXP3 activity. Immunol Cell Biol 90(1):95–100PubMedCrossRefGoogle Scholar
  19. 19.
    Das Gupta K, Shakespear MR, Iyer A, Fairlie DP, Sweet MJ (2016) Histone deacetylases in monocyte/macrophage development, activation and metabolism: refining HDAC targets for inflammatory and infectious diseases. Clin Transl Immunol 5(1):e62CrossRefGoogle Scholar
  20. 20.
    Mullican SE et al (2011) Histone deacetylase 3 is an epigenomic brake in macrophage alternative activation. Genes Dev 25(23):2480–2488PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    True O, Matthias P (2012) Interplay between histone deacetylases and autophagy—from cancer therapy to neurodegeneration. Immunol Cell Biol 90(1):78–84PubMedCrossRefGoogle Scholar
  22. 22.
    Bolden JE et al (2013) HDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses. Cell Death Dis 4:e519PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Hancock WW (2011) Rationale for HDAC inhibitor therapy in autoimmunity and transplantation. Handb Exp Pharmacol 206:103–123PubMedCrossRefGoogle Scholar
  24. 24.
    Iyer A, Fairlie DP, Brown L (2012) Lysine acetylation in obesity, diabetes and metabolic disease. Immunol Cell Biol 90(1):39–46PubMedCrossRefGoogle Scholar
  25. 25.
    Rao R, Fiskus W, Ganguly S, Kambhampati S, Bhalla KN (2012) HDAC inhibitors and chaperone function. Adv Cancer Res 116:239–262PubMedCrossRefGoogle Scholar
  26. 26.
    Kovacs JJ et al (2005) HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol Cell 18(5):601–607PubMedCrossRefGoogle Scholar
  27. 27.
    Kamemura K et al (2012) Depression of mitochondrial metabolism by downregulation of cytoplasmic deacetylase, HDAC6. FEBS Lett 586(9):1379–1383PubMedCrossRefGoogle Scholar
  28. 28.
    Koya RC et al (2012) BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy. Cancer Res 72(16):3928–3937PubMedPubMedCentralCrossRefGoogle Scholar
  29. 29.
    Bannister AJ, Miska EA (2000) Regulation of gene expression by transcription factor acetylation. Cell Mol Life Sci 57(8–9):1184–1192PubMedCrossRefGoogle Scholar
  30. 30.
    Rikiishi H (2011) Autophagic and apoptotic effects of HDAC inhibitors on cancer cells. J Biomed Biotechnol 2011:830260PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Zhang J, Zhong Q (2014) Histone deacetylase inhibitors and cell death. Cell Mol Life Sci 71(20):3885–3901PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Gryder BE, Sodji QH, Oyelere AK (2012) Targeted cancer therapy: giving histone deacetylase inhibitors all they need to succeed. Future Med Chem 4(4):505–524PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Dickinson M, Johnstone RW, Prince HM (2010) Histone deacetylase inhibitors: potential targets responsible for their anti-cancer effect. Investig New Drugs 28(Suppl 1):S3–S20CrossRefGoogle Scholar
  34. 34.
    Walkinshaw DR, Tahmasebi S, Bertos NR, Yang XJ (2008) Histone deacetylases as transducers and targets of nuclear signaling. J Cell Biochem 104:1541–1552PubMedCrossRefGoogle Scholar
  35. 35.
    Li X, Yang H, Huang S, Qiu Y (2014) Histone deacetylase 1 and p300 can directly associate with chromatin and compete for binding in a mutually exclusive manner. PLoS One 9(4):e94523PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Doetzlhofer A et al (1999) Histone deacetylase 1 can repress transcription by binding to Sp1. Mol Cell Biol 19(8):5504–5511PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Wu Y, Zhang X, Salmon M, Zehner ZE (2007) The zinc finger repressor, ZBP-89, recruits histone deacetylase 1 to repress vimentin gene expression. Genes Cells 12(8):905–918PubMedCrossRefGoogle Scholar
  38. 38.
    Lee TI, Johnstone SE, Young RA (2006) Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat Protoc. 1(2):729–48PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Nelson JD, Denisenko O, Bomsztyk K (2006) Protocol for the fast chromatin immunoprecipitation (ChIP) method. Nat Protoc. 1(1):179–85PubMedCrossRefGoogle Scholar
  40. 40.
    Cheng F et al (2014) Divergent roles of histone deacetylase 6 (HDAC6) and histone deacetylase 11 (HDAC11) on the transcriptional regulation of IL10 in antigen presenting cells. Mol Immunol 60(1):44–53PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Melissa Hadley
    • 1
  • Satish Noonepalle
    • 1
  • Debarati Banik
    • 1
  • Alejandro Villagra
    • 1
    Email author
  1. 1.The George Washington University Cancer CenterN.W. George Washington UniversityWashingtonUSA

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