Finally, how histone deacetylase inhibitors disrupt mitosis!

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Cell Cycle 10:16, 2658-2661; August 15, 2011; © 2011 Landes Bioscience

Finally, how histone deacetylase inhibitors disrupt mitosis! Brian Gabrielli,* KeeMing Chia and Robyn Warrener The University of Queensland Diamantina Institute; Princess Alexandra Hospital; Brisbane, QLD Australia

T

he disruption of normal mitosis by histone deacetylase inhibitors is a significant contributor to the anticancer effects of these drugs. However, the mechanism by which these drugs affect mitosis is poorly understood. A number of recent papers have now thrown considerable light onto how these drugs elicit this very distinctive cell cycle disruption.

Histone deacetylase inhibitors (HDACi) have a range of cellular effects, from transcriptional changes, a consequence of changes in histone and transcription factor acetylation, to decreasing protein stability through acetylation of HSP90 chaperones and other non histone acetylation targets, such as α-tubulin.1,2 A common feature of all HDACi-treated tumor cells is defects in mitosis. It is likely that these mitotic defects contribute significantly to the cytotoxic activity of these drugs, as cells that have been induced to arrest in G2 phase are insensitive to killing by these drugs.3-7 However, the mechanism by which HDACi cause these mitotic defects has, until very recently, remained elusive. Several recent papers have now suggested a likely mechanism. The mitotic defects produced by HDACi treatment are characterized by a prolonged delay in prometaphase, due to an inability to establish a correctly aligned metaphase plate, and failure to form a distinct mid-spindle structure and to undergo anaphase.3,4,8-10 These failures result in the cells delaying in mitosis for a few hours prior to exiting without establishing a normal metaphase plate or undergoing anaphase partitioning of their replicated genome. The delay in mitosis and subsequent premature mitotic exit suggest a partial failure of spindle assembly checkpoint (SAC). During the initial

delay in prometaphase after HDACi treatment, this checkpoint appears functional, and many of the kinetochore components (Bub1, BubR1, Mad2) localize normally.9 However, the arrest cannot be sustained, and exit from mitosis occurs with the normal loss of localization of these SAC components from the kinetochore.3,8,9 HDACi treatment effectively disrupted SAC function, demonstrated by the lack of stable mitotic arrest with either taxol or nocodazole treatment,3,4,6 although the cells did delay in mitosis longer than untreated controls, similar in duration to HDACi treatment alone.9 A key finding was that HDACi treatment resulted in a loss of localization of the chromosomal passenger complex (CPC) from the centromeres of mitotic cells. The CPC is comprised of four proteins, Aurora B, survivin, INCENP and Borealin, which essentially act as activating and targeting subunits for Aurora B kinase activity. HDACi treatment has little effect on CPC complex formation or Aurora B activity as measured by the phosphorylation of its primary mitotic substrate histone H3 Ser10.9-11 However, the normal accumulation of this complex at the centromere was lost with HDACi treatment.9 The loss of CPC centromeric localization correlated with the absence of BubR1 phosphorylation, which is required for SAC activity.12 Aurora B activity and its localization at the centromere are essential for BubR1 activity and SAC function,13,14 indicating that the loss of CPC localization was responsible for the instability of SAC function. One possible mechanism by which CPC localizes to the centromere was by binding to centromere-targeted protein heterochromatin protein 1(HP1). HP1 localizes to the centromeric regions through binding

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Key words: HDACi, Haspin, chromosomal passenger complex Abbreviations: HDACi, histone deacetylase inhibitor; CPC, chromosomal passenger complex; HP1, heterochromatin protein 1; SAC, spindle assembly checkpoint Submitted: 06/15/11 Revised: 07/01/11 Accepted: 07/08/11 DOI: 10.4161/cc.10.16.16953 *Correspondence to: Brian Gabrielli; Email: [email protected]

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to trimethylated histone H3 Lys915 and has been reported to associate with the CPC component INCENP.16 As HDACi treatment causes hyperacetylation of H3 Lys9, it was possible that HDACi treatment might inhibit H3 Lys9 methylation, thereby blocking HP1 binding at the centromere and CPC localization. However, short-term treatment of cells with HDACi that was sufficient to produce the defective mitosis had little effect on either the levels of trimethylated H3 Lys9 or HP1 binding. In addition, complete demethylation of trimethylated H3 Lys9 or depletion of HP1 had little effect on mitosis,17 and it has recently been reported that HP1α is localized to the inner centromere by direct binding to INCENP in mitosis.18 This provides conclusive evidence for a lack of H3 Lys9 involvement in CPC localization. The actual mechanism controlling CPC centromeric localization has now been defined in three papers published in Science in October last year that suggested a potential molecular basis for the mitotic defects observed with HDACi. These papers demonstrate that the centromeric localization of the CPC is through survivin binding to phosphorylated histone H3 Thr3 via its conserved BIR domain.19-21 Blocking H3 Thr3 phosphorylation by depleting the responsible kinase, Haspin, or expressing a BIR mutant of survivin resulted in the same loss of centromeric CPC localization and accompanying accumulation of CPC along the chromosome arms as observed with HDACi treatment. It also resulted in the loss of SAC function in response to taxol but not nocodazole treatment, whereas HDACi treatment overcame both nocodazole- and taxol-induced mitotic arrest.9,20,22 HDACi treatment appears to mimic the loss of H3 Thr3 phosphorylation by means of the increased trimethylation of H3 Lys4.23 This was a rapid effect, and elevated levels of trimethylation of H3 Lys4 were detected within 10 h, as cells progressed into mitosis.17 Trimethylated H3 Lys4 inhibits survivin binding to phosphorylated H3 Thr3 and inhibits Haspin phosphorylation of H3 Thr3 in vitro.20,24 Although the effect of HDACi treatment on H3 Thr3 phosphorylation has not been demonstrated in vivo, the loss of CPC binding strongly suggests

that the increased H3 Lys4 trimethylation is blocking binding rather than affecting Thr3 phosphorylation directly. The increased H3 Lys4 trimethylation is a consequence of increased localization of an H3 Lys4 methyltransferase, possibly MLL4, that is dependent on the increased H3 Lys9 acetylation induced by HDACi treatment, and decreased expression of H3 Lys4 demethylases, such as RBP2, in response to HDACi treatment.25 Haspin expression has not been reported to be affected by HDACi treatment. Blocking Haspin activity and the resultant loss of both H3 Thr3 phosphorylation and CPC centromeric localization can account for many of the mitotic features of HDACi treatment, but there are several that are not readily explained. One is the ability of HDACi to overcome SAC activity in both nocodazole- and taxol-arrested cells,3,4,6,9 whereas Haspin depletion only bypasses taxol arrest.20 The basis for this difference is not clear, but it suggests that HDACi affects additional critical mitotic regulators. The other major difference is that HDACi treatment causes the sister chromatid arms to remain closely associated, rather than attached only at the primary constriction, through centromeric cohesin attachments, resulting in failure of sister chromatid separation in mitosis,9,10,17 whereas Haspin inhibition/ depletion cause loss of sister chromatid cohesion.26 This suggests that HDACi treatment does not directly affect Haspin activity but rather only the H3Thr3 phosphorylation and/or survivin binding, since cohesin association is maintained along the chromosome arms. Indeed, the effect of HDACi is very similar to the phenotype produced with overexpression of Haspin, where Haspin localizes with the cohesin complex along the arms of the chromosomes, thus maintaining a “closed arm” appearance. Interestingly, selective HDAC3 depletion produces the same increased H3 Thr3 phosphorylation across the chromosome arms as Haspin overexpression in short-term depletion experiments, although over 5 d of depletions, there was a complete loss of H3 Thr3 phosphorylation and sister chromatid cohesion.11 The authors also showed a loss of Sgo1 with HDAC3 depletion, and siRNA depletion of Sgo1 had an identical

effect on H3 Thr3 phosphorylation as HDAC3 depletion. HDAC3 binds the chromosome associated AKAP95 in mitosis,27 and selective HDAC3 depletion has been demonstrated to produce many of the same effect as HDACi on mitosis, including premature mitotic exit through inactivation of SAC activity.6,17,28 Class I selective HDACi produce the full spectrum of mitotic defects observed with pan-isoform inhibitors,7,29 indicating that it is inhibition of HDAC3 that is responsible for the majority of the mitotic effects observed with HDACi. HDACi treatment may also affect other mitotic pathways in addition to H3 Thr3 that are likely to contribute to the spectrum of mitotic defects observed. Plk1 is a potential candidate due to its role in phosphorylating the cohesin SA2 subunit and, thereby, regulating the unloading of the cohesin complex from the chromosome arms in early mitosis.30 HDACi treatment may regulate Plk1 activity or function directed at the cohesin complex, thereby blocking sister chromatid separation and contributing to the “closed arm” appearance in HDACi-treated mitotic cells. A possible mechanism of action of HDACi treatment in this respect is stabilizing the acetylation of a cohesin component to block sister chromatid separation, possibly by blocking Plk1-dependent cohesin unloading. In budding yeast, Eco1 acetylates the cohesin Smc3 subunit, which is required for sister chromatid cohesion, and Hos1-dependent deacetylation is required for separation.31,32 Two Eco1 orthologs has been discovered in mammalian cells ESCO1 and ESCO2 that have roles in sister chromatid cohesion. In addition, San, an N-terminal acetyl transferase, is required for sister chromatid cohesion,33 and it may be the acetylation catalyzed by San that is the target of HDACi. Confirmation of San as a target of HDACi would require further investigation. A demonstrated HDACi target that does produce mitotic defects is HSP90. HDACi treatment stabilizes acetylation of HSP90, leading to inhibition of its chaperone function.34 Directly inhibiting HSP90 function using small molecule inhibitors such as 17-AAG causes mitotic defects, which are, in part, a consequence


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of destabilization of HSP90 clients Aurora A and Plk1.35,36 We and others have observed that the level of the HSP90 client Aurora A is reduced in HDACitreated cells, although this appears to be a selective destabilization of the cytoplasmic and not the centrosome localized pool (reviewed in ref. 37; unpublished observations). The lack of effect on the centrosomal Aurora A is likely to account for the lack of monopolar spindle phenotype normally associated with Aurora A inhibition.8,9,38 A similar selective destabilization of the chromosome-associated pool of Plk1 that is responsible for cohesin phosphorylation and unloading could also explain maintenance of sister chromatid cohesion observed with HDACi treatment. In conclusion, HDACi affect a number of components of the mitotic machinery, which combine to disrupt mitosis in tumor cells. These appear to include inhibiting survivin binding to phosphorylated H3 Thr3, which results in the mislocalization of both the CPC and destabilization of Aurora A and probably Plk1, possibly disrupting Plk1-dependent cohesin unloading. Together, the effects of HDACi treatment on these proteins can account for the mitotic defects reported with HDACi treatment.

6. Magnaghi-Jaulin L, Eot-Houllier G, Fulcrand G, Jaulin C. Histone deacetylase inhibitors induce premature sister chromatid separation and override the mitotic spindle assembly checkpoint. Cancer Res 2007; 67:6360-7; PMID:17616695; DOI:10.1158/0008-5472.CAN-06-3012. 7. Park JH, Jung Y, Kim TY, Kim SG, Jong HS, Lee JW, et al. Class I histone deacetylase-selective novel synthetic inhibitors potently inhibit human tumor proliferation. Clin Cancer Res 2004; 10:5271-81; PMID:15297431; DOI:10.1158/1078-0432.CCR03-0709. 8. Robbins AR, Jablonski SA, Yen TJ, Yoda K, Robey R, Bates SE, et al. Inhibitors of histone deacetylases alter kinetochore assembly by disrupting pericentromeric heterochromatin. Cell Cycle 2005; 4:717-26; PMID:15846093; DOI:10.4161/cc.4.5.1690. 9. Stevens FE, Beamish H, Warrener R, Gabrielli B. Histone deacetylase inhibitors induce mitotic slippage. Oncogene 2008; 27:1345-54; PMID:17828304; DOI:10.1038/sj.onc.1210779. 10. Cimini D, Mattiuzzo M, Torosantucci L, Degrassi F. Histone hyperacetylation in mitosis prevents sister chromatid separation and produces chromosome segregation defects. Mol Biol Cell 2003; 14:3821-33; PMID:12972566; DOI:10.1091/mbc.E03-01-0860. 11. Eot-Houllier G, Fulcrand G, Watanabe Y, MagnaghiJaulin L, Jaulin C. Histone deacetylase 3 is required for centromeric H3K4 deacetylation and sister chromatid cohesion. Genes Dev 2008; 22:2639-44; PMID:18832068; DOI:10.1101/gad.484108. 12. Huang H, Hittle J, Zappacosta F, Annan RS, Hershko A, Yen TJ. Phosphorylation sites in BubR1 that regulate kinetochore attachment, tension and mitotic exit. J Cell Biol 2008; 183:667-80; PMID:19015317; DOI:10.1083/jcb.200805163. 13. Becker M, Stolz A, Ertych N, Bastians H. Centromere localization of INCENP-Aurora B is sufficient to support spindle checkpoint function. Cell Cycle 2010; 9:1360-72; PMID:20372054; DOI:10.4161/ cc.9.7.11177. 14. Morrow CJ, Tighe A, Johnson VL, Scott MI, Ditchfield C, Taylor SS. Bub1 and aurora B cooperate to maintain BubR1-mediated inhibition of APC/CCdc20. J Cell Sci 2005; 118:3639-52; PMID:16046481; DOI:10.1242/jcs.02487. 15. Bannister AJ, Zegerman P, Partridge JF, Miska EA, Thomas JO, Allshire RC, et al. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 2001; 410:120-4; PMID:11242054; DOI:10.1038/35065138. 16. Ainsztein AM, Kandels-Lewis SE, Mackay AM, Earnshaw WC. INCENP centromere and spindle targeting: identification of essential conserved motifs and involvement of heterochromatin protein HP1. J Cell Biol 1998; 143:1763-74; PMID:9864353; DOI:10.1083/jcb.143.7.1763. 17. Warrener R, Chia K, Warren WD, Brooks K, Gabrielli B. Inhibition of histone deacetylase 3 produces mitotic defects independent of alterations in histone h3 lysine 9 acetylation and methylation. Mol Pharmacol 2010; 78:384-93; PMID:20562223; DOI:10.1124/mol.109.062976. 18. Kang J, Chaudhary J, Dong H, Kim S, Brautigam CA, Yu H. Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sisterchromatid cohesion in human cells. Mol Biol Cell 2011; 22:1181-90; PMID:21346195; DOI:10.1091/ mbc.E11-01-0009. 19. Kelly AE, Ghenoiu C, Xue JZ, Zierhut C, Kimura H, Funabiki H. Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B. Science 2010; 330:235-9; PMID:20705815; DOI:10.1126/science.1189505. 20. Wang F, Dai J, Daum JR, Niedzialkowska E, Banerjee B, Stukenberg PT, et al. Histone H3 Thr-3 phosphorylation by Haspin positions Aurora B at centromeres in mitosis. Science 2010; 330:231-5; PMID:20705812; DOI:10.1126/science.1189435.

21. Yamagishi Y, Honda T, Tanno Y, Watanabe Y. Two histone marks establish the inner centromere and chromosome bi-orientation. Science 2010; 330:239-43; PMID:20929775; DOI:10.1126/science.1194498. 22. Shin HJ, Baek KH, Jeon AH, Kim SJ, Jang KL, Sung YC, et al. Inhibition of histone deacetylase activity increases chromosomal instability by the aberrant regulation of mitotic checkpoint activation. Oncogene 2003; 22:3853-8; PMID:12813458; DOI:10.1038/sj.onc.1206502. 23. Nightingale KP, Gendreizig S, White DA, Bradbury C, Hollfelder F, Turner BM. Cross-talk between histone modifications in response to histone deacetylase inhibitors: MLL4 links histone H3 acetylation and histone H3K4 methylation. J Biol Chem 2007; 282:4408-16; PMID:17166833; DOI:10.1074/jbc. M606773200. 24. Eswaran J, Patnaik D, Filippakopoulos P, Wang F, Stein RL, Murray JW, et al. Structure and functional characterization of the atypical human kinase haspin. Proc Natl Acad Sci USA 2009; 106:20198-203; PMID:19918057; DOI:10.1073/pnas.0901989106. 25. Huang PH, Chen CH, Chou CC, Sargeant AM, Kulp SK, Teng CM, et al. Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases. Mol Pharmacol 2011; 79:197-206; PMID:20959362; DOI:10.1124/mol.110.067702. 26. Dai J, Sullivan BA, Higgins JM. Regulation of mitotic chromosome cohesion by Haspin and Aurora B. Dev Cell 2006; 11:741-50; PMID:17084365; DOI:10.1016/j.devcel.2006.09.018. 27. Li Y, Kao GD, Garcia BA, Shabanowitz J, Hunt DF, Qin J, et al. A novel histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity. Genes Dev 2006; 20:2566-79; PMID:16980585; DOI:10.1101/gad.1455006. 28. Ishii S, Kurasawa Y, Wong J, Yu-Lee LY. Histone deacetylase 3 localizes to the mitotic spindle and is required for kinetochore-microtubule attachment. Proc Natl Acad Sci USA 2008; 105:4179-84; PMID:18326024; DOI:10.1073/pnas.0710140105. 29. Chia K, Beamish H, Jafferi K, Gabrielli B. The histone deacetylase inhibitor MGCD0103 has both deacetylase and microtubule inhibitory activity. Mol Pharmacol 2010; 78:436-43; PMID:20538840; DOI:10.1124/mol.110.065169. 30. Hauf S, Roitinger E, Koch B, Dittrich CM, Mechtler K, Peters JM. Dissociation of cohesin from chromosome arms and loss of arm cohesion during early mitosis depends on phosphorylation of SA2. PLoS Biol 2005; 3:69; PMID:15737063; DOI:10.1371/ journal.pbio.0030069. 31. Unal E, Heidinger-Pauli JM, Kim W, Guacci V, Onn I, Gygi SP, et al. A molecular determinant for the establishment of sister chromatid cohesion. Science 2008; 321:566-9; PMID:18653894; DOI:10.1126/ science.1157880. 32. Beckouët F, Hu B, Roig MB, Sutani T, Komata M, Uluocak P, et al. An Smc3 acetylation cycle is essential for establishment of sister chromatid cohesion. Mol Cell 2010; 39:689-99; PMID:20832721; DOI:10.1016/j.molcel.2010.08.008. 33. Hou F, Chu CW, Kong X, Yokomori K, Zou H. The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshinindependent manner. J Cell Biol 2007; 177:587-97; PMID:17502424; DOI:10.1083/jcb.200701043. 34. Drysdale MJ, Brough PA, Massey A, Jensen MR, Schoepfer J. Targeting Hsp90 for the treatment of cancer. Curr Opin Drug Discov Devel 2006; 9:48395; PMID:16889231. 35. de Cárcer G. Heat shock protein 90 regulates the metaphase-anaphase transition in a polo-like kinasedependent manner. Cancer Res 2004; 64:5106-12; PMID:15289312; DOI:10.1158/0008-5472.CAN03-2214.


Acknowledgments

This work was supported by funding from Cancer Council Queensland. B.G. is a National Health and Medical Research Council Senior Research Fellow. References 1. Blagosklonny MV, Robey R, Sackett DL, Du L, Traganos F, Darzynkiewicz Z, et al. Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest and cytotoxicity. Mol Cancer Ther 2002; 1:937-41; PMID:12481415. 2. Lindemann RK, Gabrielli B, Johnstone RW. HistoneDeacetylase Inhibitors for the Treatment of Cancer. Cell Cycle 2004; 3:779-88; PMID:15153801; DOI:10.4161/cc.3.6.927. 3. Dowling M, Voong KR, Kim M, Keutmann MK, Harris E, Kao GD. Mitotic spindle checkpoint inactivation by trichostatin a defines a mechanism for increasing cancer cell killing by microtubule-disrupting agents. Cancer Biol Ther 2005; 4:197-206; PMID:15753652; DOI:10.4161/cbt.4.2.1441. 4. Warrener R, Beamish H, Burgess A, Waterhouse NJ, Giles N, Fairlie D, et al. Tumor cell-selective cytotoxicity by targeting cell cycle checkpoints. FASEB J 2003; 17:1550-2; PMID:12824307. 5. Krauer KG, Burgess A, Buck M, Flanagan J, Sculley TB, Gabrielli B. The EBNA-3 gene family proteins disrupt the G2 /M checkpoint. Oncogene 2004; 23:1342-53; PMID:14716295; DOI:10.1038/ sj.onc.1207253.

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36. Lyman SK, Crawley SC, Gong R, Adamkewicz JI, McGrath G, Chew JY, et al. High-content, high-throughput analysis of cell cycle perturbations induced by the HSP90 inhibitor XL888. PLoS ONE 2011; 6:17692; PMID:21408192; DOI:10.1371/ journal.pone.0017692. 37. Park JH, Jong HS, Kim SG, Jung Y, Lee KW, Lee JH, et al. Inhibitors of histone deacetylases induce tumor-selective cytotoxicity through modulating Aurora-A kinase. J Mol Med 2008; 86:117-28; PMID:17851643; DOI:10.1007/s00109-007-0260-8. 38. Andrews PD, Knatko E, Moore WJ, Swedlow JR. Mitotic mechanics: the auroras come into view. Curr Opin Cell Biol 2003; 15:672-83; PMID:14644191; DOI:10.1016/j.ceb.2003.10.013.


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