[Epigenetics 3:1, 28-37; January/February 2008]; ©2008 Landes Bioscience
Review
HDACs and HDAC inhibitors in colon cancer John M. Mariadason Department of Oncology; Montefiore Medical Center; Albert Einstein College of Medicine; Bronx, New York USA
Classification of HDACs
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HDACs derive their name from their ability to catalyze the deacetylation of lysine residues within DNA bound core histone proteins.5,6 These enzymes work in opposition to histone acetyltransferases (HATs) which catalyze lysine acetylation.7 However, it is now evident that histones are not the only substrate for HDACs, as multiple proteins subject to post-translational modification by deacetylation have been recently identified.8 The more specific term of lysine deacetylases to describe this family of enzymes has therefore been suggested.9 Eighteen mammalian HDACs have been identified to date (Fig. 1). These can be classified into one of four classes based upon their homology to a prototypical HDAC found in yeast. Class I HDACs (HDACs 1, 2, 3 and 8), are ubiquitously expressed, show homology to the yeast HDAC, Rpd3 and are approximately 50 kDa in size. Class I HDACs, with the possible exception of HDAC3,10 are also predominantly nuclear in localization.11 Class II HDACs (HDACs 4, 5, 6, 7, 9 and 10) have a high degree of homology to the yeast HDAC Hda-1, are larger in size (120–150 kDa) compared to class I HDACs,4,12 and are expressed in a tissue-specific manner. Class II HDACs exist in both the nucleus and cytoplasm, and the shuttling of class II HDACs out of the nucleus, which is regulated by 14-3-3 proteins, is a major mechanism by which their activity is regulated.12 Class II HDACs can be further separated into class IIa (HDACs 4, 5, 7 and 9) and IIb (HDACs 6 and 10) based upon the existence of tandem deacetylase domains in HDACs 6 and 10.13 Class I and II HDACs share significant homology at the deacetylase domain but differ in their N-terminal sequence. HDAC11, which shares some but not sufficient homology to both class I and II HDACs is assigned to its own class, class IV.14 The class III HDAC, or sir2 family named for their homology to the yeast Sir2 gene, are a highly conserved gene family which in humans presently comprises seven members, sirt1-7.15 Among these, sirt1, 2, 3 and 5 have a NAD-dependent deacetylase domain (distinct from the zinc-dependent deacetylase domains of class I and II HDACs), and catalyze the deacetylation of histone as well as a number of non-histone proteins. In contrast, Sirts 4 and 6 have an NAD+-dependent ADP ribosylation domain and catalyze protein ribosylation. Sirtuin family members have well-defined subcellular localizations, with sirt1, 6 and 7 localized to the nucleus, sirt2 to the cytoplasm and sirt3, 4 and 5 localized to the mitochondria. Class III HDACs share little homology to the first two classes, and are not inhibited by the widely used HDAC inhibitors butyrate, valproic acid, Trichostatin A or SAHA.9 As a result we will not focus extensively on class III HDACs in this review.
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The histone deacetylase (HDAC) family of transcriptional co-repressors have emerged as important regulators of colon cell maturation and transformation. Pharmacological inhibitors of class I and II HDAC activity (HDACi) are potent inducers of growth arrest, differentiation and apoptosis of colon cancer cells in vitro and in vivo, implicating a role for these HDACs in tumor promotion. Consistent with this role, expression of several HDACs are upregulated in colon tumors, while downregulation of specific HDACs inhibits growth of colon cancer cells in vitro and intestinal tumorigenesis in vivo. This review focuses on the function and transcriptional mechanisms by which class I and II HDACs regulate colon cell maturation and transformation, and on the mechanisms by which HDACi induce growth arrest, differentiation and apoptosis of colon cancer cells. The emerging role of the class III HDAC, Sirt1, in colon cancer progression is also discussed.
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Key words: class I HDAC, class II HDAC, Sirt1, HDAC inhibitors, colon cancer, transcription
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Introduction
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The ability of histone deacetylase inhibitors (HDACi) to induce growth arrest, differentiation and apoptosis in cell lines derived from a range of malignancies including colon cancer has been appreciated for several decades.1,2 Based upon these pre-clinical findings, several HDAC inhibitors are presently in clinical trial for the treatment of a variety of hematological and solid tumors including colon cancer, with one inhibitor, SAHA, recently approved for the treatment of cutaneous T-cell lymphoma. These HDACi inhibit the enzymatic activity of class I and II HDACs,3,4 implying a physiological role for these proteins in the maintenance of colon cell proliferation and survival, and inhibition of cell differentiation. The findings from several recent studies which have examined the expression pattern and function of class I and II HDACs in normal colon, and colon cancer cells, will be reviewed, as will the emerging role for the class III HDAC, Sirt1. The effects and mechanisms of action of HDACi on colon cancer cells in vitro and in vivo, their chemopreventive potential, and the status of HDACi in clinical trial for treatment of colon cancer will also be examined. Correspondence to: John M. Mariadason; Department of Oncology; Hofheimer 413; Montefiore Medical Center; 111 East 210th Street; Bronx, New York 10467 USA; Tel.: 718.920.2025; Fax: 718.882.4464; Email:
[email protected] Submitted: 12/28/07; Accepted: 02/15/08 Previously published online as an Epigenetics E-publication: http://www.landesbioscience.com/journals/epigenetics/article/5736 28
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Class I and II HDACs are Components of Large Transcriptional Co-Repressor Complexes
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Histone deacetylases do not directly bind DNA. Rather, they typically reside within high molecular weight (1–2 MDa), multisubunit transcriptional co-repressor complexes that are recruited by sequence-specific transcription factors to promoter regions.16 Distinct co-repressor complexes contain different HDACs. For example HDACs 1 and 2 are typically associated with the Sin3-SAP, NuRD and CoRest transcriptional co-repressor complexes. HDAC3 is a component of the closely related NCoR (nuclear receptor co-repressor) and SMRT (silencing-mediator of retinoic and thyroid receptors) co-repressor complexes.17 N-CoR and SMRT (also called N-CoR2) are paralogs encoded by distinct genes, which act as molecular platforms on Figure 1. Classes of mammalian HDACs. which the remainder of the co-repressor complex (including HDAC3, TBL1, TBLR1 and GPS2) assembles.18 NCoR and SMRT also serve as the principal contact between the co-repressor complex and its transcription factor partners, which among others, include nuclear hormone receptors (thyroid, retinoic acid and retinoid X receptors), NFκB, AP-1, SMAD proteins, c-Myb, PLZF, Bcl-6, MyoD and Sp1.19-21 The interaction of HDAC3 with the N-terminal DAD (deacetylase activating domain) domain of NCoR/SMRT, has also been shown to be essential for activation of HDAC3 deacetylase activity.22 Finally, the class II HDACs 4, 5 and 7 interact with the B-CoR,23 BCoRL124 and CtBP co-repressors.12 Class II HDACs have also been shown to interact with the N-CoR /SMRT-HDAC3 corepressor complex, although they have been shown to be inactive in this context and do not contribute to the enzymatic activity of the complex.25 While Figure 2. Non-histone substrates for class I and II HDACs. expression of HDACs 1, 2, 3 and 4 have now been described in colon cancer cells, the specific co-repressor complexes within which they Class I and II HDACs-Mechanisms of Action reside have not been defined for this particular cell type. Distinct co-repressor complexes are recruited to target promoters Functionally, HDACs regulate gene expression by at least two by sequence-specific transcription factors. For example, the HDAC1 mechanisms. First, upon recruitment to DNA by sequence-specific and 2 containing Sin3-SAP complex is recruited by E2F family transcription factors, they catalyze the deacetylation of specific members to repress target gene expression,26 while the class II lysine residues in DNA-bound core histone proteins. Lysine deacetHDAC containing B-CoR co-repressor complex is recruited by ylation of histones confers a positive charge on histone proteins, MEF2 to repress muscle cell specific genes.27 Consistent with the which among other effects, enhances their affinity for negatively differential recruitment of HDACs by sequence-specific transcrip- charged DNA. A consequence of this alteration in nucleosome tion factors, genomewide microarray analyses in yeast and drosophila conformation is reduced accessibility of the transcriptional regulahave demonstrated that different HDACs regulate distinct cellular tory machinery to the DNA template, resulting in transcriptional processes.28,29 For example, genes altered in expression following repression.31,32 Whether individual HDACs target specific lysine deletion of Rpd3 (homolog of mammalian class I HDACs) were residues within histone tails has not been extensively explored. enriched for genes involved in cell cycle progression. Conversely, However, preferential acetylation of H3K18 and H3K9 following deletion of Hda1 and Sir2 (homologs of mammalian class II and III knockdown of HDAC1 and HDAC3, respectively, has been reported HDACs respectively) resulted in enrichment of genes involved in in Hela cells, suggesting this may be a possibility.33 A second mechacarbohydrate utilization, and amino acid biosynthesis, respectively.30 nism by which HDACs regulate transcription is by catalyzing the Determining whether class I, II and III HDACs exert similar vari- deacetylation of sequence-specific, DNA binding, transcription ability in terms of genome-wide transcriptional targets in colon factors. Examples include p53,31 E2F,34 Sp1 and Sp3,32,35 TFIIEβ and TFIIF,36 GATA-1,37 TCF,38 HMG-1,39 Erα40 and c-Myb cancer cells remains to be determined. (Fig. 2).41 Acetylation and de-acetylation of sequence-specific transcription factors can either increase or decrease their DNA binding
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A number of recent studies have examined the expression profile of HDACs in normal colonic tissue.57-60 The class I HDACs, 1, 2, 3 and 8,52,57 and the class II HDAC, HDAC4 (Wilson and Mariadason, unpublished), are expressed in the normal colon and small intestine and primarily in the proliferating crypt compartment. The expression of HDACs 5, 6, 7, 9 and 10 in normal colon has not been reported. The expression of HDACs in the proliferative crypt compartment is consistent with a role for these proteins in the maintenance of cell proliferation and survival, and inhibition of differentiation. Indeed, direct evidence for such a role in vivo was elegantly demonstrated by the transient overexpression of HDAC1 and HDAC2 in fetal mouse intestinal explants, which resulted in abnormal intestinal development and differentiation.61 Likewise, intestinal cell number and thickness of the intestinal mucosa were reduced in HDAC2 mutant compared to wild type mice,52 consistent with a reduction in the proliferative capacity of the intestinal epithelium.
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Knockout mice for HDACs 1, 2, 4, 5, 6, 7 and 9 have now been generated. Knockout of HDAC1 results in embryonic lethality due to severe proliferation defects, and developmental retardation.49,50 Olson’s group also recently reported the generation of HDAC1 mice with conditional null alleles.50 Consistent with the findings of Lagger et al.,49 global knockout of HDAC1 resulted in embryonic lethality.50 Two independent strains of HDAC2 knockout mice have now been generated,50,51 with some differences in phenotype observed particularly in regards to the penetrance of the neonatal lethal phenotype, and with respect to the role of HDAC2 in mediating stress induced cardiac hypertrophy. Olson’s group generated HDAC2 mice with conditional null alleles in which global knockout of HDAC2 resulted in perinatal lethality due to a range of cardiac defects. Conversely, HDAC2 null animals generated using a gene trap LacZ insertion strategy are viable, although significant perinatal lethality is observed.51 Animals are smaller in size compared to WT littermates, and develop myocardial defects.51 Gottlicher’s group also recently described an intestinal phenotype in the LacZ-HDAC2 knockout mice in the form of shortened crypts and villi, and development of fewer adenomas when crossed to ApcMin mice.52 Coupled with the overexpression of HDAC2 in colon tumors and the in vitro observations described below, this observation supports a role for HDAC2 in intestinal tumor progression. However, given the phenotypic differences which exist between the two HDAC2 null strains, it would be of interest to determine whether the reduction in intestinal tumorigenesis observed following whole body HDAC2 deletion is phenocopyied by intestinal-specific HDAC2 deletion. Knockouts of the class II HDACs, HDAC4, HDAC5, HDAC7 and HDAC9 have each been generated by the Olson laboratory,53-55 and an HDAC6 knockout was recently generated and reported by Zhang et al.56 The primary phenotype observed in HDAC4 knockout mice is premature differentiation of chondrocytes, resulting in pronounced skeletal defects.53 HDAC5 and HDAC9 knockout mice are viable, fertile, and develop the similar phenotype of profoundly enlarged hearts in response to pressure overload.55 HDAC6 knockout mice are also viable and fertile and have a normal phenotype with the exception of a minor increase in cancellous bone mineral density, implicating a role for this deacetylase in bone biology. Tubulin is also hyperacetylated in multiple tissues in HDAC6 knockout mice, although the normal phenotype of these mice indicates this is not detrimental to normal development.56 Knockout of HDAC7 results
Class I and II HDAC Expression in Normal Colon
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in embryonic lethality due to a defect in endothelial cell-cell adhesion and subsequent dilation and rupture of blood vessels.54 While the intestinal phenotype of these animals was not described, the recent demonstration that specific class II HDACs (i.e., HDAC4, Wilson and Mariadason, unpublished) are expressed in the intestine and play a role in colon cell proliferation warrants investigation of the intestinal phenotype of these animals.
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activity, and subsequently may enhance or repress transcription.35,36 Some specificity of individual HDACs for different transcription factors has been described. This includes deacetylation of p53 by HDAC1,42 deacetylation of glucocorticoid receptor by HDAC2,43 and deacetylation of MEF2 by HDAC3.44 HDACs also elicit transcription-independent effects (Fig. 2). For example, a number of cytoplasmic proteins, including tubulin45 and HSP90,46 have now been shown to be acetylated by HDAC6. HDAC6-mediated deacetylation of HSP90 promotes its chaperone function,47 which includes the binding and stabilization of the oncogenic client proteins Akt, mutant p53, HIF1α, survivin and telomerase hTERT.48 The relative contribution of transcription-dependent versus independent effects in determining HDACi-induced effects on colon cancer cells is discussed below.
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Class I and II HDAC Expression in Colon Cancer
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Several studies have also examined HDAC expression in colon tumors.57-60,62 Most of these studies have reported increased expression of the class I HDACs, HDAC1,57,59,62,63 HDAC2,57,60,62,63 HDAC357 and HDAC8,57,58,60,62 in colon tumors relative to adjacent normal mucosa. Increased expression of HDACs 1, 2 and 8 in colon tumors has been demonstrated at both the protein and mRNA level, suggesting transcriptional activation may be a likely mechanism of overexpression. Consistent with this, expression of HDAC2 in colon cancer cell lines has been shown to be promoted by β-cateninTCF-myc signaling,60 the fundamental pathway deregulated in colon cancer. The mechanisms by which expression of other class I HDACs are upregulated in colon cancers, and the expression status of class II HDACs in colon tumors versus normal mucosa, are presently unknown. Overexpression of HDACs however, is not observed in all colon tumors. For example, a study by Ropero et al., identified the presence of a truncating mutation within an A9 repeat in exon 1 of the HDAC2 gene, in approximately 20% of MSI colon cancers (which equates to approximately 5% of all colon cancers).64 Homozygous inactivation of HDAC2 was demonstrated by loss of HDAC2 immunohistochemical staining in five of six patients.64 However, given related HDACs such as HDAC1, may compensate for HDAC2 loss,64 and given the basally high mutation rates of repetitive elements in MSI colon cancers, it remains unclear whether HDAC2 loss of function mutations actively contribute to colon cancer progression. Instead, as demonstrated by Ropero et al., HDAC2 mutations may have significant importance for determining sensitivity to HDACi, with HDAC2 mutant cell lines being refractory to HDACi-induced apoptosis.64
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HDACs has been shown to transactivate Sp3.57 At least two models by which HDACs may repress p21 in a Sp1-dependent manner can be envisioned. First, Sp1 and or Sp3 may recruit class I HDACs to the proximal p21 promoter where they would repress transcription by driving the deacetylation of surrounding histones.72 The resultant closed chromatin structure would repress gene expression by reducing access of the transcriptional machinery to the promoter. Second, binding of class I HDACs to Sp1 and or Sp3 may result in deacetylation of the transcription factors themselves, converting them from transcriptional activators into transcriptional repressors.32 Class II HDACs. While the role of the class II HDACs, 5, 6, 7 and 9 in colon cancer cells has not been reported, a recent study by our own group demonstrated that HDAC4 promotes growth of colon cancer cells in vitro and in vivo (Wilson et al., unpublished). As for class I HDACs, HDAC4 was found to repress p21 expression in HCT116 cells, an effect that is critical for the pro-proliferative effects of HDAC4, as demonstrated by the attenuated growth inhibitory response of p21 deficient HCT116 cells to HDAC4 knockdown. HDAC4 was also found to co-localize and directly interact with Sp1 in colon cancer cells. Furthermore, consistent with the model of recruitment of HDAC4 by Sp1 to the proximal p21 promoter, ChIP analyses demonstrated that downregulation of Sp1 in colon cancer cells resulted in loss of HDAC4 from the proximal p21 promoter. Collective interpretation of these findings indicates that multiple class I and II HDACs are simultaneously recruited to the p21 promoter to mediate its repression. As for class I HDACs, knockdown of HDAC4 also induced a modest degree of apoptosis in colon cancer cells involving cytochrome c release and PARP cleavage, demonstrating a pro-survival role for HDAC4 in this cell type. These findings however, are in contrast to studies in other cell types which demonstrate a role for HDAC4 in promoting apoptosis, such as in response to UV radiation in lung fibroblasts.73 HDAC4 undergoes caspasemediated cleavage in response to ionizing radiation, and translocation of the caspase-cleaved amino fragment of HDAC4 has been shown to induce mitochondrial cytochrome c release and to promote apoptosis in a caspase 9-dependent manner.73 HDAC4 therefore appears to be a complex protein with differing functions under basal and stressed conditions. Notably, the magnitude of induction of both p21 and apoptosis following knockdown of any individual HDAC, is markedly less that that induced by HDACi.57 Collectively, these findings suggest that the simultaneous inhibition of multiple HDACs, rather than inhibition of a specific HDAC family member, is responsible for the effects of HDACi. Class III HDACs. As recently reviewed elsewhere,15 class III HDACs (sirt1-7) have generated significant interest for their role in regulating lifespan in a wide range of organisms, particularly the increased longevity mediated by caloric-restriction. At the cellular level, sirtuins may facilitate this process by regulating energy metabolism, stress response, DNA repair, apoptosis and cell senescence.15 Given the importance of these processes for transformation, the likelihood that sirtuins play a role in cancer is also considerable, and is an area of intense investigation. At the molecular level, sirtuins may contribute to transformation through modulating gene transcription as a result of aberrant deacetylation of specific lysine residues within histones (H1K26,
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The expression profile of class II HDACs in colon cancer relative to normal colonic mucosa has been less extensively examined. In contrast to class I HDACs, reduced mRNA expression of HDAC465 and HDAC566 have been reported in colon tumors relative to normal mucosa. Although not observed in colon cancers, it is also notable that mutations in HDAC4 were identified in a genome-wide screening analysis of breast tumors.67 The significance of this mutation, which occurs within the HDAC domain, on HDAC4 activity and function remains to be determined. The expression of HDACs 6, 7, 9, 10 and 11 in colon tumors has presently not been reported. Examination of class II HDAC expression in colon tumors versus normal both at the protein level and in terms of their subcellular localization therefore remains an important question to address. Collectively, evidence to date indicates consistent overexpression of specific HDACs in colon cancer. HDAC overexpression may facilitate progression of colon tumors via at least two mechanisms. First, HDAC overexpression can contribute to the transcriptional repression of genes that normally function in growth arrest, differentiation and apoptosis, by inducing histone hypoacetylation in core promoter regions following their recruitment by sequence specific transcription factors. Second, HDAC overexpression could induce hypoacetylation and thus modulate the function of multiple non-histone proteins, including transcription factors and critical cytoplasmic proteins such as Hsp90. As discussed below, most of the data generated to-date in colon cancer cells has been in support of the transcriptional repression model. However, as more nonhistone targets of HDACs are identified and the consequences of their deacetylation determined, the complexity by which HDAC overexpression contributes to colon cancer progression is likely to be increasingly appreciated.
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Function of Class I and II HDACs in Colon Cancer Cells
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Class I HDACs. The effect of over and underexpression of multiple class I HDACs on the growth and survival of colon cancer cells has been investigated in vitro in a number of studies. The majority of these experiments have demonstrated a role for class I HDACs in promoting colon cell proliferation and survival.57,60 Knockdown of the class I HDACs 1, 2 and 3 reduces growth of several colon cancer cell lines including HCT116,57 HT2960 and SW480.68 Conversely, Ropero et al, observed reduced proliferation upon stable overexpression of HDAC2, in HDAC2 deficient RKO cells.64 The basis for this difference may be related to the different experimental strategies used (under versus overexpression) and the possibility that HDAC2 may have differing effects when overexpressed in cells adapted to proliferate in its absence. Mechanistically, the pro-proliferative effects of HDACs in colon cancer cells have been linked to transcriptional repression of the cdk-inhibitor, p21,57,68 while in non-colon cell lines, HDAC3mediated repression of p15INK4b has also been reported.69 Knockdown of HDACs 1, 2 and 3 induce p21 expression in colon cancer cell lines, while their overexpression represses basal as well as HDACi-mediated p21 induction.70 HDAC-mediated repression of p21 is likely regulated by Sp1/Sp3 transcription factors. Localization of class I HDACs to Sp1/Sp3 binding sites within the proximal p21 promoter has been demonstrated by ChIP analysis,71 physical interaction of class I HDACs with Sp1 and Sp3 has been demonstrated by co-immunoprecipitation,72 and knockdown of class I www.landesbioscience.com
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Lastly, the HDACi VPA has been shown to reduce adenoma formation in ApcMin mice.60 As discussed above, multiple HDACs are overexpressed in colon cancer, which in turn, may contribute to colon cancer progression by epigenomic repression of tumor suppressor genes, or by hypoacetylation and modification of function of non-histone substrates. HDACi’s therefore, likely elicit their anti-tumor effects by reversing these effects. For example, inhibition of HDAC activity can result in histone hyperacetylation and subsequently in transcriptional de-repression of tumor suppressor genes. HDACi may also hyperacetylate transcription factors, either increasing or decreasing their transcriptional activity and subsequently altering gene expression programs in favor of growth arrest and apoptosis. HDACi may also hyperacetylate cytoplasmic substrates potentially inhibiting their tumor promoting function via transcription-independent mechanisms. The majority of the evidence generated to-date has been in support of HDACi mediating their phenotypic effects through transcription dependent effects. However, while less evidence is presently available for HDACi working through modulation of function of cytoplasmic substrates, this is likely to become increasingly evident.
Growth Arrest
Treatment of colon cancer cells with HDACi typically induces G0/G1 growth arrest within 12–16 hours,85,86 although a G2/M arrest can also be induced in certain colon cancer cell lines87 when exposed to higher HDACi concentrations.88 HDACi induced growth arrest in colon cancer cells consistently involves induction of p21,70,89 which is induced rapidly (within 30 minutes) in a protein synthesis-independent manner, and independently of p53.90,91 Multiple studies utilizing p21 promoter deletion constructs have demonstrated that HDACi induce p21 in colon cancer cells in a Sp1/Sp3-dependent manner,90 a finding further supported by the attenuated HDACi-mediated induction of p21 following siRNAmediated Sp1 knockdown (Wilson and Mariadason, unpublished). Importantly, the critical role of p21 induction for mediating HDACi-induced growth arrest in colon cancer cells is demonstrated by the attenuated growth inhibitory effect of HDACi on p21 deficient HCT116 cells.70 In addition to p21, HDACi also induce expression of several other growth inhibitory proteins including p15INK4b,92 p1686 and GADD45α and β93 Notably, HDACi also downregulate expression of several pro-proliferative genes including c-myc,94,95 cyclin B196 and cyclin D197 in colon cancer cells. In contrast to p21 however, the relative importance of these effects in mediating HDACi-induced growth arrest, have not been directly tested. HDACi have also been shown to induce polyploidy in HCT116 colon cancer cell lines.88 These cells lose their ability to proliferate and commit to senescence, indicating a further mechanism by which HDACi exert growth arrest of colon cancer cells.88
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H3K9, H3K14 and H4K16),74 as well as through deacetylation and subsequent modulation of activity of a number of non-histone proteins implicated in cancer. These include p53, Ku70, FOXO, p300 and NFκB, each of which has been shown to be deacetylated by Sirt1.15 Sirt1 is the only class III HDAC studied to date in detail in colon cancer cells. Sirt1 is highly expressed in the cytoplasm of colon cancer cell lines and upregulated in colon cancers relative to normal colonic tissue.75 Sirt1 expression is induced in a p53-independent manner in SW620 colon cancer cells upon DNA damage, while Sirt1 knockdown leads to cell cycle arrest and apoptosis of colon cancer cells.75 One mechanism by which Sirt1 may promote transformation in colon cancer cells is by contributing to the epigenetic repression of tumor suppressor genes. For example, the Baylin laboratory recently demonstrated Sirt1 localization to the promoters of several aberrantly silenced tumor suppressor genes in colon cancer cells, in which CpG islands are hypermethylated, but not to these same promoters in cell lines in which the promoters are not hypermethylated and the genes are expressed.76 These included SFRP1, SFRP2, MLH1 and E-Cadherin. Pharmacological inhibition of SIRT1 using NAD or splitomicin, dominant negative expression of Sirt1, and siRNA mediated Sirt1 inhibition each caused increased H4-K16 and H3-K9 acetylation at endogenous promoters and re-expression of these genes. Notably, and somewhat unexpectedly, this de-repression occurred despite full retention of promoter DNA hypermethylation. Importantly, re-expression of SFRPs resulted in downregulation of β-catenin-TCF signaling in colon cancer cells,76 suggesting Sirt1mediated transcriptional repression may provide colon cancer cells with a growth advantage through further activation of this critical signaling pathway.
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Effects of Class I and II HDAC Inhibitors (HDACi) on Colon Cancer Cells
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Agents capable of inhibiting HDAC activity such as the short chain fatty acid, butyrate, have been recognized and utilized experimentally for several decades.77 Several other structurally unrelated classes of HDACi have since been described, including hydroxamic acids (TSA, SAHA), cyclic tetrapeptides (depsipeptide), benzamides (MS275) and electrophilic ketones (trifluoromethylketone).78 HDACi are potent inducers of histone acetylation, a consequence of inhibition of HDAC activity and altering the cellular balance between HATs and HDACs, in favor of HATs. The HDACi, butyrate has been shown to induce classical maturation and anti-tumor effects in colon cancer cell lines, including the inhibition of cell proliferation and the stimulation of differentiation and apoptosis.1,79,80 Similar effects on growth inhibition and induction of apoptosis in colon cancer cells have now been demonstrated for other, structurally similar (valproic acid),60 and structurally distinct HDAC-inhibitors. The latter include the hydroxamic acids Trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA)81,82 and the benzamide MS-275.83 Anti-tumor effects for HDACi have also been demonstrated in vivo, in animal models of intestinal cancer. First, HDACi including butyrate, TSA and A-423378.0 have been shown to efficiently reduce growth of colon cancer xenografts.64 Second, direct infusion of butyrate into the colon of rats using a surgical intubation model reduced aberrant crypt formation following AOM treatment.84 32
Apoptosis HDACi also potently induce apoptosis in colon cancer cells.79,80 While HDACi have been shown to induce apoptosis via both the intrinsic/mitochondrial and extrinsic/death receptor pathways in non colon cancer cells,98,99 most studies to date implicate the intrinsic pathway in HDACi-induced apoptosis in colon cancer cells.
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gelsolin in mutant K-Ras expressing cell lines has been implicated in the increased sensitivity to HDACi.113 Given Ras/BRAF mutations are observed in approximately 50% of colon cancers this observation has significant potential for stratification of patients likely to respond to HDACi in vivo. SW620 colon cancer cell sublines, selected based upon differences in basal mitochondrial membrane potential also demonstrate differences in response to HDACi, with lines with lower basal mitochondrial membrane potential more sensitive to HDACi-induced apoptosis.114 The basal proliferative and differentiation status of colon cancer cells also determines response, as illustrated by the markedly attenuated apoptotic response of differentiated Caco-2 and HT29 cl.19A cells compared to undifferentiated counterparts.115
Differentiation
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A unique feature of HDACi treatment of colon cancer cells, which contrasts with the effects of cytotoxic chemotherapeutic agents, is the induction of cell differentiation.1,80 Enhanced differentiation is evidenced morphologically by the formation of dome like structures, a characteristic of increased water absorption116 and improved tightjunction function,117 a feature of differentiated, polarized epithelial cells. Enhanced differentiation in response to HDACi treatment also manifests biochemically by the increased expression of the differentiation markers intestinal alkaline phosphatase1,80 which plays a role in lipid uptake, the sodium hydrogen exchanger, NHE3,118 the adherens junction protein E-cadherin119 and the cytoskeletal proteins villin120 and gelsolin.113 Notably, SCFAs appear to promote differentiation of colon cancer cells along the absorptive cell lineage, as the induction of absorptive cell markers such as iALP, is accompanied by a simultaneous inhibition of expression of the markers of goblet cell differentiation Muc2 and ITF.121,122 Induction of alkaline phosphatase and villin is also an indirect effect of butyrate, requiring de novo protein synthesis, and relatively late in onset,123 suggesting that HDACi-induced differentiation is the manisfestation of significant reprogramming of colon cancer cells to differentiate along the absorptive cell lineage. Mechanistically, the transcription factor, or factors, that drive this reprogramming remain to be determined. HDACi induce expression of the important intestinal-specific transcription factors KLF4,124 and cdx-2,125 however, the importance of their induction for HDACiinduced differentiation has not been directly demonstrated. Differentiation of colon cancer cells induced by HDACi, however, is limited. For instance markers such as sucrase-isomaltase and dipeptidylpeptidase IV, which are induced during spontaneous differentiation of Caco-2 colon cancer cells, are not induced following butyrate treatment of this cell line.116 This may in part be related to the simultaneous activation of apoptotic programs in butyrate treated cells, which may result in abrogation of the complete differentiation program. Butyrate also induces expression of the oncofetal CEA gene,126 and placental-like alkaline phosphatase.127 These genes are typically expressed during fetal development and pregnancy, respectively, and again in transformed cells. CEA in particular is extensively used as a colon tumor marker and as a readout of tumor burden. Induction of these genes indicates that the genetic reprogramming induced by HDACi is complex, and in some cases involves altered expression of markers not typically associated with differentiation of normal colonic epithelial cells.
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This involves a cascade of events including a decrease in mitochondrial membrane potential, cytochrome c release and caspase-9 and 3 activation.100 Importantly, inhibitors of caspase 3 and 9, but not 8, inhibit butyrate-induced apoptosis in Caco-2 cells,101 emphasizing the importance of the intrinsic apoptotic pathway. The initiating events in HDACi-induced apoptosis may involve transcriptional alteration of the balance between expression of pro and anti-apoptotic Bcl-2 family members, which regulate mitochondrial membrane integrity. For example, HDACi induce expression of the pro-apoptotic Bak protein,101,102 and downregulate expression of the anti-apoptotic protein, BclXL.101 HDACi also induce mitochondrial localization of Bax and Bak.103 HDACi-induced apoptosis has also been shown to involve nuclear to cytoplasmic shuttling of the orphan receptor Nurr77/TR3, which in turn is linked to localization of Bax to the mitochondria and cytochrome c release.103 HDACi also modulate expression of genes involved in the extrinsic apoptotic pathway, including upregulation of the pro-apoptotic DR5 gene,104 and downregulation of the anti-apoptotic caspase inhibitor, FLIP, in colon cancer cells.105 Consistent with these effects, HDACi sensitize colon cancer cells to TRAIL104,105 and FAS-induced apoptosis,106 in colon cancer cells typically resistant to apoptosis induced by these agents. These observations suggest that activation of both of these pathways may occur in parallel in colon cancer cells. HDACi induced apoptosis in colon cancer cells has also been linked to increased ROS production.107 However, while HDACiinduced apoptosis is inhibited by ROS scavengers in non colon cancer cell lines,108 this has not been demonstrated in colon cancer cells. Interestingly, both SAHA and butyrate induce the morphological features of autophagic cell death in Hela cells, suggesting promotion of autophagy may contribute to HDACi induced apoptosis in certain cell types.109 However, in CML cells, disruption of autophagy by chloroquine treatment augmented SAHA-induced apoptosis consistent with a role for autophagy in chemoresistance.110 Given these contrary findings, it would be of interest to determine the effect of both pharmacological and molecular disruption of autophagy on HDACi-induced cell killing in colon cancer cells, to assess the relative importance of this pathway in mediating the effects of HDACi.
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Molecular Determinants of HDACi-Induced Apoptosis
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With regards to biomarkers that may predict response to HDACiinduced apoptosis in colon cancer cells, the mutation status of Apc and K-Ras have been shown to be determinants of HDACi response. For example, re-introduction of Wild type Apc into HT29 cells results in increased sensitivity to HDACi.111 Ultimately, however, stratification of colon cancers according to Apc/β-catenin mutation status is unlikely to be of significant clinical use as the vast majority of colon tumors harbor mutations in one of these genes. Contrasting findings regarding the role of Apc/β-catenin/TCF signaling in HDACi-induced apoptosis have also been reported. For example, Bordonarro et al., reported that HDACi induce β-cateninTCF reporter activity,81 and that the magnitude of reporter activity induction correlated with HDACi-induced apoptosis.112 Experiments using isogenic colon cancer and nontransformed intestinal epithelial cell lines have also demonstrated that the presence of a k-ras mutation enhances sensitivity to HDACi-induced apoptosis.113 Mechanistically, reduced expression of STAT1 as well as www.landesbioscience.com
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HDAC Inhibitors in Clinical Trial for Colon Cancer Based upon the pre-clinical findings of HDACi in vitro and in animal models in vivo, a number of HDACi including phenylbutyrate, depsipeptide, SAHA, PXD101, MS-275 and valproic acid, have or are presently being evaluated in clinical trials for the treatment of hemopoietic and solid tumors.143 In October 2006, SAHA (zolinza, vorinostat, Merck) was the first HDACi approved for treatment of cutaneous T-cell lymphoma. Since its approval a number of trials have been initiated to determine the efficacy of the combination of SAHA with existing chemotherapeutics for treatment of patients with progressive metastatic or unresectable colorectal cancer (clinicaltrials.gov), including combination with 5FU/LV and FOLFOX (5FU-Leucovorin/oxaliplatin). Preclinical studies have demonstrated that combination of HDACi with 5FU can synergistically enhance cell killing in vitro, and inhibit tumor growth in xenograft models,144 establishing the potential of this approach. HDACi are also potent sensitizers to radiation therapy in multiple cell types, including colon cancer cells. Mechanistically, it has been proposed that HDACi elicit this effect either by inducing histone hyperacetylation thus providing increased access of radiation to DNA, or by inhibiting double stranded break repair, for example, by inducing the acetylation and subsequently inhibiting the activity of proteins involved in this process such as Ku70.145 Radiation therapy is commonly used in the treatment of rectal cancers, and it will be interest to monitor the success of clinical trials combining HDACi treatment with radiation therapy in rectal cancer. A mechanism by which HDACi may induce apoptosis is by re-induction of expression of transcriptionally downregulated genes. A common mechanism of transcriptional downregulation is promoter hypermethylation, and genome-wide epigenetic changes characterized by both global hypomethylation and locus specific hypermethylation are hallmarks of multiple cancers including colon cancer.146 Recently, the existence of subclasses of colon tumors with unique methylation profiles (CpG island methylator phenotype or CIMP) have been reported.147 While HDACi alone cannot induce re-expression of epigenetically silenced genes, HDACi in
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Consistent with the widespread role of HDACs in transcriptional regulation, HDACi treatment results in altered expression of large numbers of genes in colon cancer cells. The transcriptional changes induced by butyrate progressively increases with time from
