Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase

Published OnlineFirst January 12, 2010; DOI: 10.1158/0008-5472.CAN-09-2327

Therapeutics, Targets, and Chemical Biology Cancer Research Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Andrew J. Wilson1, Anderly C. Chueh2, Lars Tögel2, Georgia A. Corner1, Naseem Ahmed1, Sanjay Goel1, Do-Sun Byun1, Shannon Nasser1, Michele A. Houston1, Minaxi Jhawer1, Helena J.M. Smartt1, Lucas B. Murray1, Courtney Nicholas1, Barbara G. Heerdt1, Diego Arango3, Leonard H. Augenlicht1, and John M. Mariadason1,2

Abstract Histone deacetylase inhibitors (HDACi) induce growth arrest and apoptosis in colon cancer cells and are being considered for colon cancer therapy. The underlying mechanism of action of these effects is poorly defined with both transcription-dependent and -independent mechanisms implicated. We screened a panel of 30 colon cancer cell lines for sensitivity to HDACi-induced apoptosis and correlated the differences with gene expression patterns induced by HDACi in the five most sensitive and resistant lines. A robust and reproducible transcriptional response involving coordinate induction of multiple immediate-early (fos, jun, egr1, egr3, atf3, arc, nr4a1) and stress response genes (Ndrg4, Mt1B, Mt1E, Mt1F, Mt1H) was selectively induced in HDACi sensitive cells. Notably, a significant percentage of these genes were basally repressed in colon tumors. Bioinformatics analysis revealed that the promoter regions of the HDACi-induced genes were enriched for KLF4/Sp1/Sp3 transcription factor binding sites. Altering KLF4 levels failed to modulate apoptosis or transcriptional responses to HDACi treatment. In contrast, HDACi preferentially stimulated the activity of Spl/Sp3 and blocking their action attenuated both the transcriptional and apoptotic responses to HDACi treatment. Our findings link HDACi-induced apoptosis to activation of a Spl/Sp3-mediated response that involves derepression of a transcriptional network basally repressed in colon cancer. Cancer Res; 70(2); 609–20. ©2010 AACR.

Introduction mechanisms. First, inhibition of HDAC activity results in hyperacetylation of DNA-bound histones, generating a chroma- Histone deacetylase inhibitors (HDACi) are a class of tar- tin environment more permissive to transcriptional geted therapeutics with promising antitumor efficacy in mul- activation (2, 3). Second, HDACi induce hyperacetylation of sequence-specific transcription factors (4–8), which can ei- tiple malignancies. HDACi inhibit the HDAC family of ther increase or decrease transcriptional activity (8, 9). transcriptional corepressors, which catalyze the deacetyla- Multiple structurally distinct HDACi have been described, tion of lysine residues within target proteins (1). HDACi including short-chain fatty acids (butyrate, valproic acid), hy- treatment likely regulates gene expression by at least two droxamic acids (trichostatin A, SAHA), cyclic tetrapeptides, tetrapeptides, and benzamidines (2). Of these, SAHA is ap- proved for the treatment of cutaneous T-cell lymphoma, Authors' Affiliations: 1Department of Oncology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York; 2Ludwig whereas several others are in phase I and phase II clinical Institute for Cancer Research, Melbourne, Australia; and 3Molecular trials. These HDACi primarily inhibit the class I (HDACs 1, Biology and Biochemistry Research Center, Barcelona, Spain 2, 3 and 8) and class II HDACs (HDACs 4, 5, 6, 7, 9 and 10; Note: Supplementary data for this article are available at Cancer Re- ref. 2). search Online (http://cancerres.aacrjournals.org/). HDACi induce growth arrest, differentiation, and apoptosis Current address for J.M. Mariadason: Ludwig Institute for Cancer Research, Melbourne Centre for Clinical Sciences Austin Health, Level in a variety of tumor cell lines, including colon cancer cells 7, Harold Stokes Building, 145-163 Studley Road, Heidelberg, Victoria (2). Depending on the cell type, HDACi-induced apoptosis 3084, Australia. can be mediated by both the intrinsic/mitochondrial (10) Corresponding Author: John M. Mariadason, Ludwig Institute for Can- or extrinsic/death receptor pathways (11, 12). In colon cancer cer Research, Melbourne Centre for Clinical Sciences Austin Health, Level 7, Harold Stokes Building, 145-163 Studley Road, Heidelberg, cells, most studies implicate the intrinsic pathway, with a se- Victoria 3084, Australia. Phone: 613-9496-3068; Fax: 613-9496-5334; quential series of events including loss of mitochondrial E-mail: [email protected]. membrane potential, mitochondrial Bax localization, cyto- doi: 10.1158/0008-5472.CAN-09-2327 chrome c release, and caspase-9 activation observed upon ©2010 American Association for Cancer Research. HDACi treatment (12–14). However, HDACi have also been

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shown to sensitize colon cancer cells to TRAIL and Fas ligand– hybridized to 27,000 feature cDNA microarrays generated induced apoptosis (15, 16), suggesting activation of the by the Albert Einstein College of Medicine microarray facility two pathways may occur in parallel. (30), and images were analyzed using Genepix Pro software Whereas the downstream effectors of HDACi-induced ap- (Axon Instruments, Union City, CA) and Lowess normalized. optosis have been studied in detail (17, 18), the molecular For each cell line, two independent butyrate treatment ex- events that initiate the process are less well defined. One pos- periments were performed, and the mean fold change in ex- tulated transcription-dependent mechanism is alteration in pression following butyrate treatment relative to control was the balance in expression of proapoptotic and antiapoptotic computed for each sequence. Microarray experiments using genes in favor of apoptosis (2). For example, in colon cancer the Nimblegen platform are described in Supplementary cells HDACi upregulate expression of the proapoptotic genes, Methods. Bax (19) and Bak (20), and downregulate expression of the To identify genes differentially regulated (induced or re- antiapoptotic Bcl-XL gene (13). pressed) in response to HDACi treatment in sensitive and re- Alternatively, HDACi can initiate apoptosis via transcrip- sistant cell lines, the mean fold change in expression tion-independent mechanisms (21). For example, HDACi (butyrate relative to control) across the five sensitive and five can promote apoptosis via hyperacetylation of HSP90 and resistant lines was computed for each gene. Differentially ex- subsequent depletion of prosurvival HSP90 client proteins pressed genes were selected upon their ability to satisfy each (22, 23). HDACi-induced apoptosis has also been linked to of the following three criteria: (1) changed 2-fold or greater aberrant mitosis (22, 24–27). relative to control in either the sensitive or resistant panel; Therefore, whether HDACi-induced apoptosis is initiated (2) differentially regulated by a factor of 1.5-fold or greater primarily in a transcription-dependent or -independent between sensitive and resistant cells; (3) significantly differ- manner is currently unclear. In the present study we iden- entially regulated between sensitive and resistant cells by an tify a robust and reproducible transcriptional response se- unpaired t test, with a P value of <0.05 considered statistically lectively induced by HDACi in sensitive cell lines involving significant. the induction and repression of 48 and 44 genes respective- To identify genes not changed in response to butyrate ly. The induced genes were highly enriched for immediate- treatment, we first identified all genes for which the median early (IE) and stress response (SR) genes. We show that fold induction in response to butyrate treatment was <10% this transcriptional response and subsequent apoptosis (within the range of +1.1 to −1.1), for both the sensitive are coordinately induced by HDACi in a Sp1/Sp3-depen- and resistant panel. From this list we then selected genes dent manner. Notably, a significant percentage of the HDA- that were also not significantly differentially expressed be- Ci-induced genes are basally repressed in colon tumors, tween sensitive and resistant lines (P value resulting from a suggesting HDACi-induced apoptosis involves deregulation t test between sensitive and resistant is >0.05). Clones (2,751) of a complex transcriptional network basally repressed in that meet the above criteria were identified. From these, a colon cancer cells. random number generator was used to select 48 genes that were used for comparative analyses with the HDACi-induced gene list. Materials and Methods Promoter analysis. Promoter sequences for the 48 HDA- Ci-inducedgenesaswellasanequalnumberofcontrol Cell lines and cell culture. The 30 colon carcinoma cell genes not changed by HDACi were obtained from the Uni- lines used were Caco-2, Colo201, Colo205, Colo320, Dld-1, versity of California Santa Cruz genome browser.4 The pro- HCT116, HCT-15, HCT-8, HT29, LoVo, LS174T, RKO, SK- moter region encompassing 1.5 kb upstream of the CO-1, SW1116, SW403, SW48, SW480, SW620, SW837, transcription start site (TSS) through 200 bp downstream SW948, T84, WiDr, HT29-Cl.16E, HT29-Cl.19A, LIM1215, was systematically searched for the presence of transcrip- LIM2405, HCC2998, KM12, RW2982, and RW7213. The source tion factor binding sites using the MATCH tool (BIOBASE and methods of maintenance have been previously described Biological Databases GmbH, Wolfenbüttel, Germany) on the (28). RKO-EcR and RKO-EcR-KLF4 (also called RKO-RG24) TransFac public site. For more detailed guanine-cytosine were kindly provided to us by Dr. Vincent Yang and used (GC) content, Sp1, Sp3, and KLF4 binding site analyses, as previously described (29). each promoter was subdivided into 1-kb windows beginning Drug sensitivity assays. Methods used to determine apo- 10 kb downstream of the TSS and ending 10 kb upstream of ptosis, clonogenic survival, and response of xenografts to the TSS. The GC content of each 1-kb window was comput- HDACi treatment in vivo are described in the supplementary ed using the freak tool in the EMBOSS suite accessed on text. the Washington State University Bioinformatics server5 Microarray experiments. Exponentially growing cells (31). The frequency of Sp1, Sp3, and KLF4 binding sites in were treated with 5 mmol/L butyrate for 24 h, and total these windows were determined as above. RNA was extracted using the RNeasy kit (Qiagen). For cDNA microarray analysis, 50 μg of RNA was labeled with Cy3 dUTP (control) or Cy5 dUTP (butyrate-treated). Probe prep- aration, hybridization conditions, and array scanning proce- 4 http://genome.ucsc.edu/ dure were as previously described (5, 6). Probes were 5 http://www.bioinformatics2.wsu.edu/emboss/

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Quantitative reverse transcription–PCR, Western blot- HDACi-induced apoptosis were sensitive to apoptosis in- ting, transient transfection, siRNA knockdown, chromatin duced by the mechanistically distinct chemotherapeutic immunoprecipitation analysis. Detailed methods are de- agents, 5FU, CPT, and oxaliplatin (Supplementary Fig. S1B). scribed in supplementary text. Primers used in quantitative Differential sensitivity of cell lines to HDACi-induced ap- reverse transcription–PCR (qRT-PCR) and chromatin immu- optosis is not linked to the degree of HDAC inhibition or noprecipitation (ChIP) analysis are described in Supplemen- HDACi-induced growth arrest. Interestingly, HDACi treat- tary Tables S1 and S2, respectively. ment resulted in robust induction of histone H3 and histone Immunohistochemistry. We used the Protein Atlas online H4 acetylation to a similar extent in both sensitive and resis- immunohistochemistry resource6 for images of metallothio- tant cell lines (P =0.44andP = 0.37 for Ac.H3 and Ac.H4, nein and activating transcription factor 3 (ATF3) staining in respectively; Supplementary Fig. S2), indicating differential normal colon and colon tumor sections (32, 33). sensitivity to HDACi was not due to differences in the degree of HDAC inhibition. Results Consistent with this finding, HDACi treatment resulted in significant growth inhibition in all 10 cell lines (Supplemen- Identification of colon cancer cell lines sensitive and re- tary Fig. S3). The percentage of cells in S phase was de- fractory to HDACi-induced apoptosis. To identify colon creased by 73 ± 8.2% and 62 ± 1.8% in resistant and cancer cell lines sensitive and refractory to HDACi, a panel sensitive cell lines, respectively (P = 0.59), and the percentage of 30 colon cancer cell lines was screened for HDACi-induced of cells in G2-M was induced 359 ± 95% and 388 ± 112% in apoptosis by treatment with low (1 mmol/L), intermediate resistant and sensitive cell lines, respectively (P = 0.85) 24 h (5 mmol/L), or high (10 mmol/L) dose of the HDACi, sodium after HDACi treatment. Consistent with HDACi-induced butyrate, for 72 hours. As shown in Fig. 1, a continuum re- growth inhibition, expression of p21WAF1/CIP1, a regulator of sponse to butyrate-induced apoptosis was observed. The five cell cycle progression and known HDACi target gene, was cell lines with the highest apoptotic response to low-dose bu- consistently induced by butyrate treatment in each of the tyrate treatment were defined as butyrate “sensitive” (SW480, 10 cell lines (Supplementary Fig. S4A and B). HCT116, HCT8, HCC2998, and SW948), and the five cell lines HDACi-induced apoptosis is inhibited by actinomycin D. with the lowest apoptotic response to high-dose butyrate To determine the importance of transcription in HDACi- treatment defined as butyrate “resistant” (LIM2405, Colo320, induced apoptosis, HCT116 cells were cotreated with HDACi RKO, HCT15, and HT29 cl.19A) for subsequent studies. and the RNA polymerase inhibitor, actinomycin D. Actinomy- To validate these findings using an independent method, cin D significantly attenuated HDACi-induced apoptosis, clonogenic survival assays were performed following 24 h bu- showing HDACi-induced apoptosis in colon cancer cells is tyrate treatment on the adherent-sensitive and -resistant cell dependent upon de novo transcription (Supplementary lines. Consistent with the apoptosis data, colony formation Fig. S5A and B). was reduced to a significantly greater extent in sensitive IE and SR genes are preferentially induced by HDACi in compared with resistant lines (Fig. 1B and C; P = 0.01, un- sensitive colon cancer cell lines. Having identified sensitive paired t test). To confirm the differential response of colon and resistant cell lines and established the dependency of cancer cells to HDACi in vivo, two sensitive (HCT116 and HDACi-induced apoptosis upon de novo transcription, the HCT8) and two resistant (LIM2405, HCT15) cell lines were transcriptional basis for this differential sensitivity was deter- grown as xenografts in severe combined immunodeficient mined. The five sensitive and five resistant cell lines were mice and treated with the HDACi, VPA, for 2 weeks. VPA treated with 5 mmol/L butyrate for 24 hours and changes was used in these studies as it is structurally similar to bu- in gene expression profiled using 27,000-feature cDNA micro- tyrate but not metabolized as rapidly in vivo. Consistent with arrays (entire database provided as Supplementary Table S3). the in vitro findings, VPA treatment significantly inhibited Notably, the overall number of genes changed in response to growth of HCT116 and HCT8 xenografts but had minimal ef- HDACi treatment (P > 0.05, t test) and the range of transcrip- fects on growth of LIM2405 and HCT15 cells (Fig. 1D and E). tional changes in terms of fold change was similar for sensi- To confirm that the apoptotic effects of butyrate were due tive and resistant cell lines. to the inhibition of HDAC activity, the cell lines were re- Genes differentially induced by HDACi in sensitive versus screened for apoptotic response to VPA, and to the structur- resistant cell lines were identified using a stringent super- ally distinct hydroxamic acid–based HDAC inhibitors, vised analysis as described in Materials and Methods. Satis- Trichostatin A (TSA), and SAHA. As shown in Supplementary fying these criteria, 48 sequences were identified as Fig. S1, cell lines sensitive and resistant to butyrate-induced significantly and preferentially induced by butyrate in sensi- apoptosis were likewise differentially sensitive to each of the tive cell lines (Fig. 2; Supplementary Table S4). Notably, 7 of other HDACi. Notably, the differential sensitivity of these cell these 48 genes, Fos, Jun, Atf3, Arc, Nr4a1 (Nur77), Egr1, and lines to HDACi was specific to this class of agent and was not Egr3, are IE genes whereas an additional 7 genes have previ- due to an inherent differential sensitivity of these cell lines to ously been classified as SR genes: Gadd45b, Ndrg4, Mt1B, apoptosis in general, as a number of cell lines refractory to Mt1E, Mt1F, Mt1H, and MtIX (Fig. 2A). Forty-four genes preferentially repressed by HDACi in sensitive lines were also identified (Supplementary Table 6 http://www.proteinatlas.org S4). These included several genes involved in organization

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Figure 1. A, relative response of 30 colon carcinoma cell lines to HDACi-induced apoptosis. Cell lines were treated with 1, 5, or 10 mmol/L butyrate for 72 h. Columns, mean; bars, SEM; n =3.B, clonogenic survival of sensitive and resistant lines following 24 h of HDACi treatment (n =3–4). C, response of two sensitive and two resistant cell lines to VPA treatment in vivo. D, quantification of colony formation in B. Columns, mean percentage colony formation following 5 mmol/L butyrate treatment relative to control for the sensitive and resistant cell lines; bars, SEM. E, quantification of tumor size following HDACi treatment in vivo (n =4–5). *, P < 0.05.

of microtubules and the actin cytoskeleton (TRIP6, SRHML, ilarity in the transcriptional changes induced by each of PLXNB1, MAP7, LASP1,andLAD1), cell adhesion (OCLN, these agents (Supplementary Fig. S6A and B), whereas specif- DSC2), transcriptional repression (NCoR2, SET), and apo- ic examination of the genes comprising the apoptosis signa- ptosis (FLIP, DAXX). ture revealed an even stronger correlation (r =0.83or Validation of transcriptional changes. To validate the greater), indicating the butyrate-induced changes are highly differential induction of HDACi-induced genes in sensitive likely to be the consequence of HDAC inhibition. and resistant lines, we performed qRT-PCR and Western blot Induction of IE genes is specific to HDACi-induced apo- analyses. As shown in Fig. 2B, Fos, EGR1, EGR3, and IGFBP3 ptosis. To confirm that the transcriptional response ob- mRNA induction by butyrate showed strong concordance served was specific to HDACi-induced apoptosis and not a with the microarray data, with robust and preferential induc- nonspecific reflection of cells undergoing apoptosis, tion in the sensitive cell lines. Preferential induction in sen- HCT116 cells were treated with HDACi or the mechanistical- sitive cell lines of three additional genes, ATF3, Jun,and ly distinct chemotherapeutic agents 5FU and oxaliplatin for syntaxin, was confirmed at the protein level (Fig. 2C). To con- 24 hours. As shown in Supplementary Fig. S7, expression firm that the transcriptional changes induced by butyrate of Fos, EGR1, EGR3, and IGFBP3 mRNA and ATF3, Jun, and were largely due to HDAC inhibition, gene expression syntaxin protein expression were induced primarily in re- changes induced in response to butyrate were compared sponse to HDACi treatment. In contrast, p21, which is in- with changes induced by VPA and SAHA in HCT116 cells duced both by HDACi and in a p53-dependent manner in by microarray analysis. Examination of the gene expression response to DNA damage, was induced by all agents tested changes across the entire microarray revealed a marked sim- (Supplementary Fig. S7).

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Promoters of genes selectively induced by HDACi in sen- 12-O-tetradecanoylphorbol-13-acetate, and growth factor sitive cell lines are GC rich and are enriched for KLF4/Sp1/ treatment (34), suggesting that a common transcription fac- Sp3 binding sites. IE and SR gene expression has previously tor or a set of transcription factors likely regulates their ex- been shown to be coordinately induced in response to serum, pression. To determine the identity of such a factor(s), a

Figure 2. A, heat map of the 48 genes selectively induced by butyrate in sensitive cell lines following 24 h of treatment. B, QPCR validation of select gene expression changes following 24 h of treatment with 5 mmol/L butyrate (But 5). C, Western blot validation of differential induction of select proteins in sensitive (HCT8 and HCT116) and resistant cell lines (LIM2405).

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systematic bioinformatic search of the promoter regions of to the promoters of HDACi-responsive genes, we performed each of these genes was performed. As a control, the promo- ChIP analyses in HCT116 cells interrogating the Sp1/Sp3 ters of an equal number of genes not induced by HDACi were sites within the proximal promoter regions of the ATF3, also analyzed. First, a nucleotide composition analysis of the EGR1, and JUN promoters. Pulldowns performed using anti- region 10-kb upstream through 10-kb downstream of the Sp1 or anti-Sp3 antibodies resulted in significant enrichment TSS, measured in 1-kb windows, showed that the GC content of the proximal promoter fragments of these genes, relative of HDACi-induced gene promoters was significantly higher to the immunoprecipitations performed using No Ab or IgG than that of the control set. As shown in Fig. 3A, promoter controls (Fig. 3C). In comparison, for each of these genes, no GC content increased significantly in both groups with in- enrichment of distal promoter regions devoid of Sp1/Sp3 creasing proximity to the TSS; however, the magnitude of sites was observed (Fig. 3D). To determine the functionality this increase was significantly higher in HDACi-induced of these sites in terms of facilitating transcription in response genes. To determine whether the promoters of HDACi- to HDACi treatment, we also examined AcH3 levels at these induced genes were enriched for specific transcription factor sites before and after HDACi treatment by ChIP analysis. As binding sites, the frequency of occurrence of all established shown in Fig. 3E, AcH3 levels were increased ∼2-fold 24 transcription factor binding sites in the 1.5-kb promoter re- hours after HDACi treatment consistent with the transcrip- gion upstream and 200-bp downstream of the TSS was tional induction of these genes. Notably, enrichment of Sp1 computed using the MATCH database. As shown in Supple- or Sp3 or of the class I HDAC, HDAC3, at the Jun promoter mentary Table S5, the frequency of occurrence of multiple was not significantly altered in response to HDACi treatment transcription factor binding sites was significantly different (data not shown), indicating these factors are retained at the between HDACi-induced versus uninduced genes, with the promoter during transcription of the gene. vast majority of sites underrepresented in the promoters of Second, HDACi-sensitive and -resistant colon cancer cell genes induced by HDACi. The transcription factor binding lines were transfected with wild-type or mutant Sp1/Sp3-lu- sites most significantly enriched in HDACi-induced genes ciferase reporter constructs and treated with HDACi for 24 were the related GC-rich KLF4, Sp1, and Sp3 binding sites. hours. As shown in Fig. 3F, the magnitude of induction of To further confirm this finding and to examine the distri- Sp1/Sp3 reporter activity was 2- to 10-fold higher in HDA- bution of KLF4/Sp1/Sp3 binding sites within the promoters Ci-sensitive lines compared with HDACi-resistant lines, indi- of these genes in more detail, we computed the frequency cating preferential induction of Sp1/Sp3 driven transcription of occurrence of these sites in 1-kb increments beginning in sensitive cells. Minimal effects were observed on the mu- 10-kb upstream through 10-kb downstream of the TSS. As tant Sp1/Sp3-luciferase reporter construct in both sensitive shown in Fig. 3B, the frequency of KLF4 and Sp1/Sp3 sites and resistant lines (data not shown). was significantly higher in HDACi-induced genes. Third, whether pharmacologic inhibition of Sp1/Sp3 bind- KLF4 is not a key regulator of HDACi induction of the ing abrogated HDACi induction of IE genes and apoptosis IE/SR gene signature and apoptosis. To address the role was examined using the antibiotic mithramycin, which in- of KLF4 in mediating the HDACi-induced transcriptional hibits Sp1/Sp3 transcription by competitively binding to and apoptotic response, we first determined the effect of GC-rich elements (36). Cotreatment of HCT116 cells with mi- KLF4 overexpression on HDACi-induced apoptosis and IE thramycin markedly attenuated butyrate and TSA-mediated gene expression in the HDACi-resistant RKO cell line. RKO induction of three of the four genes tested: FOS, EGR1, and cells have low levels of KLF4 due to hemizygous deletion of EGR3 (Fig. 4A). In contrast, mithramycin treatment failed to a portion of the KLF4 locus as well as promoter methylation inhibit HDACi induction of IGFBP3, instead potentiating the (35). RKO-EcR-KLF4 cells are an isogenic derivative of RKO HDACi effect (Fig. 4A). Likewise, mithramycin markedly at- cells containing a stably integrated ponasterone-inducible tenuated HDACi-induced apoptosis in both HCT116 and KLF4 gene (29). Ponasterone treatment induced robust in- HCT8 cells at the 24-hour time point as assessed by cell duction of KLF4 expression in RKO-EcR-KLF4 cells after monolayer morphology, the percentage of cells with a subdi- 24 h; however, no change in the magnitude of HDACi- ploid DNA content or poly(ADP-ribose) polymerase (PARP) induced apoptosis or HDACi induction of IE gene expression cleavage (Fig. 4B–D). However, this effect was lost at later was observed (Supplementary Fig. S8). Second, KLF4 mRNA time points (48 and 72 hours) when mithramycin alone in- was selectively downregulated in HDACi-sensitive HCT116 duced significant apoptosis (data not shown). cells. Despite >70% knockdown of KLF4 mRNA expression, Finally, to directly determine the role of Sp1 and Sp3 in the no change in HDACi-induced apoptosis was observed (Sup- HDACi-induced transcriptional and apoptotic response, Sp1 plementary Fig. S8B), collectively indicating KLF4 does not and Sp3 were downregulated in HCT116 cells using Sp1 and play a central role in HDACi induction of IE/SR gene expres- Sp3 targeting siRNAs. Effective downregulation of Sp1 and sion or in HDACi-induced apoptosis in colon cancer cells. all three isoforms of Sp3 were confirmed by Western blot Activity of Sp1 and Sp3 is functionally implicated in (Fig. 5A). As shown in Fig. 5B, downregulation of Sp1 and HDACi induction of the IE/SR gene signature and apopto- Sp3 resulted in a 17% and 46% inhibition of HDACi-induced sis. Next, we determined the role played by Sp1 and Sp3 tran- apoptosis, respectively, and in the extent of HDACi-induced scription factors in HDACi-induced gene expression and PARP cleavage (Fig. 5C). apoptosis using multiple independent approaches. First, to Downregulation of Sp1 and Sp3, alone and in combina- determine whether Sp1 and Sp3 were physically localized tion, resulted in a heterogeneous pattern of effects on

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HDACi-induced gene expression, which fell into three catego- ly 2 of 32 (6.3%) were upregulated in colon tumors by these ries. First, for a subset of the genes (FOS, IGFBP3), Sp1 and same criteria (Fig. 6A). As a control, the expression pattern in Sp3 silencing had no effect on HDACi-induced gene expres- colon tumor and normal tissue of an equal number of genes sion (Fig. 5D). For a second subset (EGR3, JUN), silencing of not induced by HDACi treatment was also examined. As Sp3 but not Sp1 partially attenuated HDACi-mediated tran- shown in Fig. 6B, 5.7% of the genes on this control list were scriptional induction (Fig. 5E). For the final subset (GADD45, upregulated and 5.7% were downregulated (>1.5-fold and P < EGR1), individual silencing of both Sp1 and Sp3 inhibited 0.05) in colon tumors relative to normal colonic mucosa (P = HDACi-mediated transcriptional induction (Fig. 5F). Col- 0.004, χ2 test). lectively, these results indicate that Sp1 and Sp3 regulate The HDACi-induced genes downregulated in colon tumors a significant, although not the entire component of the were ATF3, MT1B, MT1E, MT1F, MT1H, MT1X, NDRG4, HDACi-induced transcriptional response associated with PLEKHH2,andTGFB3 (Fig. 6C).AsshowninFig.6D,the promotion of apoptosis in colon cancer cells. downregulation of MT1F and ATF3 in colon tumors was in- Genes selectively induced by HDACi in sensitive cell lines dependently validated using the protein atlas immunohisto- are basally repressed in colon tumors. To independently as- chemistry database (33, 37). These data show that the subset sess the biological significance of the subset of genes prefer- of genes identified as selectively induced during HDACi- entially induced by HDACi in sensitive cell lines, we induced apoptosis was enriched for genes that are basally compared the relative expression of this gene subset in colon downregulated in colon tumors. These findings are also con- tumors and normal colonic mucosa. For this, we generated a sistent with a previous report indicating downregulation of a microarray database comparing gene expression differences number of IE genes in colon tumors (38). between 12 matched normal colon and colon tumor pairs. Of the 48 genes preferentially induced by HDACi in sensitive cells, corresponding gene expression data were available for Discussion 32 genes in the matched primary colon tumor/normal database. Notably, expression of 9 of these 32 genes (28%) was HDAC inhibitors induce growth arrest, differentiation, and significantly repressed (>1.5-fold and P < 0.05) in colon tu- apoptosis in tumor cells of diverse origins, and their efficacy mors compared with normal colonic mucosa. In contrast, on- for cancer treatment was recently underscored by the

Figure 3. A, mean GC content of 48 genes preferentially induced by HDACi in sensitive cell lines and an equal number of control genes. B, frequency of KLF4/Sp1/Sp3 binding sites in the promoters of HDACi-induced genes. *, P < 0.05. C and D, quantitative ChIP analysis in untreated HCT116 cells showing Sp1 and Sp3 localization to proximal promoters of HDACi-induced genes (C) but not distal promoter regions (D). E, ChIP analysis showing induction of histone H3 hyperacetylation in the proximal promoter regions of three genes following 24 h of treatment of HCT116 cells with 5 mmol/L butyrate (B5). F, preferential induction of Sp1/Sp3 reporter activity in HDACi-sensitive cell lines treated with butyrate, SAHA, or VPA for 24 h (n = 2).

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Figure 4. The Sp1/Sp3 inhibitor, mithramycin (Mith, M,1μmol/L), attenuates HDACi induction of (A) target gene expression (HCT116), (B) cell viability (HCT116), (C) subdiploid DNA content (HCT116 and HCT8), or (D) PARP cleavage (HCT116). Cells were treated with HDACi (5 mmol/L butyrate, 1 μmol/L TSA) for 24 h.

approval of SAHA for the treatment of cutaneous T-cell lym- cell lines. Importantly, this transcriptional response was ob- phoma. Whereas the downstream effectors of HDACi- served in response to multiple, structurally distinct HDACi induced apoptosis in tumor cells have been studied in detail and across a range of colon cancer cell lines with extensive (3, 17), the molecular events that initiate HDACi-induced ap- molecular and mutational heterogeneity including differ- optosis are unknown. Possibilities include transcription- ences in microsatellite instability status, ploidy, and the mu- dependent effects, such as altered expression of proapoptotic tation and methylation status of multiple tumor suppressor and antiapoptotic molecules, or transcription-independent genes and oncogenes (28). effects, such as induction of aberrant mitosis, ROS produc- A striking feature of the HDACi-induced transcriptional re- tion, or altered acetylation of prosurvival molecules, such sponse is that a number of the induced genes, particularly as HSP90. the IE and SR genes, are coordinately induced in response In the present study, the importance of de novo transcrip- to several other stimuli. This includes the coordinate induction in HDACi-induced apoptosis in colon cancer cell lines tion of IE gene expression in response to serum stimulation, was established. Through gene expression profiling of multi- phorbol ester, and growth factor treatment (39, 40) and the ple colon cancer cell lines sensitive and resistant to HDACi, coordinate induction of the metallothionein gene family, we identified a reproducible transcriptional response prefer- clustered within the q13 region of chromosome 16, in re- entially induced in response to HDACi treatment in sensitive sponse to metal induction and oxidative stress (41). The

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coordinate induction of these genes in response to HDACi Several findings collectively indicated a key role for Sp1/ treatment suggested they likely reflected the modulation of Sp3 transcription factors in driving this transcriptional re- a specific transcription factor or factors that coordinately sponse. First, the promoters of HDACi-induced genes had a regulate their expression. high GC content and were significantly enriched for Sp1/Sp3

Figure 5. A, silencing efficiency of Sp1 and Sp3 protein expression. B and C, silencing of Sp1 and Sp3 in HCT116 cell partially attenuates HDACi-induced apoptosis at 24 h as assessed by (B) subdiploid DNA content and (C) PARP cleavage and (D–F) HDACi-induced transcriptional response.

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Figure 6. Expression of multiple HDACi-induced genes is reduced in primary colon tumors relative to adjacent normal mucosa. The percentage of (A) HDACi-induced and (B) uninduced (control) sequences significantly overexpressed or underexpressed in colon tumors. C, identity of the nine genes induced by HDACi and significantly repressed in colon tumors relative to normal colonic epithelium (*, P < 0.05). D, immunohistochemical validation of reduced expression of ATF3 and metallothionein in normal colon and colon tumors. Images downloaded from the publicly available protein atlas database (32, 33) as per the image use policy of protein atlas.

binding sites. Second, localization of Sp1 and Sp3 at the pro- NR4A1 (TR3/Nur77; ref. 14), and GADD45B (44). However, moters of several of these genes was detectable by ChIP anal- in each case, induction of these genes only partially accounts ysis. Third, induction of Sp1/Sp3 reporter activity was for the overall apoptotic response, suggesting that their col- preferentially induced by HDACi in sensitive cell lines. Final- lective induction, which may sufficiently alter the cellular transcriptional equilibrium in favor of apoptosis, may be ly, disruption of Sp1/Sp3 DNA binding with mithramycin or the critical determinant of HDACi-induced apoptosis. Lend- siRNA-mediated downregulation of Sp1 or Sp3 attenuated in- ing support to this concept is the finding that a number of duction of a number of HDACi targets. Importantly, both mi- these transcripts are basally repressed in colon tumors com- thramycin and Sp1/Sp3 siRNA attenuated HDACi-induced pared with normal colon tissue. In the context of tumorigen- apoptosis, establishing a direct link between Sp1/Sp3-medi- esis therefore, selective repression of these transcripts may ated transcriptional response and initiation of HDACi- alter the transcriptional balance in favor of cell survival. induced apoptosis. It is important to note, however, that Whereas HDACi treatment uniformly induces expression expression of a subset of the genes induced by HDACi, in- of antiproliferative genes, such as p21 in both sensitive and cluding FOS and IGFBP3, were not modulated by Sp1 and/ resistant cell lines, it is notable that several of the genes pref- or Sp3 silencing, indicating additional transcription factors erentially induced by HDACi in sensitive cells, including the also likely participate in HDACi-induced transcriptional changes. IE genes FOS, JUN, EGR1, and EGR3, are typically linked to Whereas the precise mechanism by which initiation of this the promotion of cell proliferation, such as in response to se- transcriptional response triggers apoptosis remains to be de- rum or growth factor treatment (34). HDACi treatment termined, it is noteworthy that several genes comprising the therefore clearly induces a conflicting transcriptional re- HDACi transcriptional response have previously been linked sponse comprising both proproliferative and antiproliferative with HDACi-induced apoptosis through over and underex- signals, which is magnified in HDACi sensitive cells. HDACi pression studies. These include IGFBP3 (42), EGR1 (43), induction of IE gene expression is also sustained for over

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Transcriptional Basis of HDACi-Induced Apoptosis

24 hours, whereas growth factor induction is typically maxi- Disclosure of Potential Conflicts of Interest mal at 30 minutes to 1 hour after stimulation and rapidly returns to baseline (38). Whether these conflicting tran- No potential conflicts of interest were disclosed. scriptional signals or the sustained induction of IE gene expression are perceived by the cells as a SR and whether this Acknowledgments is ultimately the stimulus for apoptosis induction is worthy of further investigation. In support of this hypothesis, it is We thank Dr. Geoff Childs and Aldo Massimi at the notable that cotreatment of colon cancer cells with agents AECOM cDNA microarray facility for generation of cDNA such as phorbol 12-myristate 13-acetate, which indepen- microarrays and assistance with array scanning and David dently induce IE gene expression, potentiates HDACi- Reynolds at the AECOM DNA sequencing facility. induced apoptosis (45). In conclusion, this study shows that HDACi-induced apoptosis in colon cancer cells is linked to a consistent tran- Grant Support scriptional response involving induction of a transcriptional network enriched for IE and SR genes, which are regulated, NIH grants CA123316, CA100823, and CA88104. to a large extent, by the Sp1/Sp3 transcription factors. Impor- The costs of publication of this article were defrayed in tantly, several components of this transcriptional response part by the payment of page charges. This article must are basally repressed in colon tumors suggesting HDACi- therefore be hereby marked advertisement in accordance induced apoptosis may be initiated through induction of a with 18 U.S.C. Section 1734 solely to indicate this fact. transcriptional network whose basal repression is linked to Received 6/24/09; revised 10/9/09; accepted 11/6/09; tumor cell survival. published OnlineFirst 1/12/10.

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Apoptotic Sensitivity of Colon Cancer Cells to Histone Deacetylase Inhibitors Is Mediated by an Sp1/Sp3-Activated Transcriptional Program Involving Immediate-Early Gene Induction

Andrew J. Wilson, Anderly C. Chueh, Lars Tögel, et al.

Cancer Res 2010;70:609-620. Published OnlineFirst January 12, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-2327

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