Advertisement
Therapeutics, Targets, and Chemical Biology

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, Georgia A. Corner, Naseem Ahmed, Sanjay Goel, Do-Sun Byun, Shannon Nasser, Michele A. Houston, Minaxi Jhawer, Helena J.M. Smartt, Lucas B. Murray, Courtney Nicholas, Barbara G. Heerdt, Diego Arango, Leonard H. Augenlicht and John M. Mariadason
DOI: 10.1158/0008-5472.CAN-09-2327 Published January 2010

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

Keywords
  • HDACi
  • microarray
  • colon cancer cell lines

Introduction

Histone deacetylase inhibitors (HDACi) are a class of targeted therapeutics with promising antitumor efficacy in multiple malignancies. HDACi inhibit the HDAC family of transcriptional corepressors, which catalyze the deacetylation of lysine residues within target proteins (1). HDACi treatment likely regulates gene expression by at least two mechanisms. First, inhibition of HDAC activity results in hyperacetylation of DNA-bound histones, generating a chromatin environment more permissive to transcriptional activation (2, 3). Second, HDACi induce hyperacetylation of sequence-specific transcription factors (4–8), which can either increase or decrease transcriptional activity (8, 9).

Multiple structurally distinct HDACi have been described, including short-chain fatty acids (butyrate, valproic acid), hydroxamic acids (trichostatin A, SAHA), cyclic tetrapeptides, tetrapeptides, and benzamidines (2). Of these, SAHA is approved for the treatment of cutaneous T-cell lymphoma, whereas several others are in phase I and phase II clinical trials. These HDACi primarily inhibit the class I (HDACs 1, 2, 3 and 8) and class II HDACs (HDACs 4, 5, 6, 7, 9 and 10; ref. 2).

HDACi induce growth arrest, differentiation, and apoptosis in a variety of tumor cell lines, including colon cancer cells (2). Depending on the cell type, HDACi-induced apoptosis can be mediated by both the intrinsic/mitochondrial (10) or extrinsic/death receptor pathways (11, 12). In colon cancer cells, most studies implicate the intrinsic pathway, with a sequential series of events including loss of mitochondrial membrane potential, mitochondrial Bax localization, cytochrome c release, and caspase-9 activation observed upon HDACi treatment (12–14). However, HDACi have also been shown to sensitize colon cancer cells to TRAIL and Fas ligand–induced apoptosis (15, 16), suggesting activation of the two pathways may occur in parallel.

Whereas the downstream effectors of HDACi-induced apoptosis have been studied in detail (17, 18), the molecular events that initiate the process are less well defined. One postulated transcription-dependent mechanism is alteration in the balance in expression of proapoptotic and antiapoptotic genes in favor of apoptosis (2). For example, in colon cancer cells HDACi upregulate expression of the proapoptotic genes, Bax (19) and Bak (20), and downregulate expression of the antiapoptotic Bcl-XL gene (13).

Alternatively, HDACi can initiate apoptosis via transcription-independent mechanisms (21). For example, HDACi can promote apoptosis via hyperacetylation of HSP90 and subsequent depletion of prosurvival HSP90 client proteins (22, 23). HDACi-induced apoptosis has also been linked to aberrant mitosis (22, 24–27).

Therefore, whether HDACi-induced apoptosis is initiated primarily in a transcription-dependent or -independent manner is currently unclear. In the present study we identify a robust and reproducible transcriptional response selectively induced by HDACi in sensitive cell lines involving the induction and repression of 48 and 44 genes respectively. The induced genes were highly enriched for immediate-early (IE) and stress response (SR) genes. We show that this transcriptional response and subsequent apoptosis are coordinately induced by HDACi in a Sp1/Sp3-dependent manner. Notably, a significant percentage of the HDACi-induced genes are basally repressed in colon tumors, suggesting HDACi-induced apoptosis involves deregulation of a complex transcriptional network basally repressed in colon cancer cells.

Materials and Methods

Cell lines and cell culture

The 30 colon carcinoma cell lines used were Caco-2, Colo201, Colo205, Colo320, Dld-1, HCT116, HCT-15, HCT-8, HT29, LoVo, LS174T, RKO, SK-CO-1, SW1116, SW403, SW48, SW480, SW620, SW837, SW948, T84, WiDr, HT29-Cl.16E, HT29-Cl.19A, LIM1215, LIM2405, HCC2998, KM12, RW2982, and RW7213. The source and methods of maintenance have been previously described (28). RKO-EcR and RKO-EcR-KLF4 (also called RKO-RG24) were kindly provided to us by Dr. Vincent Yang and used as previously described (29).

Drug sensitivity assays

Methods used to determine apoptosis, clonogenic survival, and response of xenografts to HDACi treatment in vivo are described in the supplementary text.

Microarray experiments

Exponentially growing cells were treated with 5 mmol/L butyrate for 24 h, and total 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 preparation, hybridization conditions, and array scanning procedure were as previously described (5, 6). Probes were hybridized to 27,000 feature cDNA microarrays generated by the Albert Einstein College of Medicine microarray facility (30), and images were analyzed using Genepix Pro software (Axon Instruments, Union City, CA) and Lowess normalized. For each cell line, two independent butyrate treatment experiments were performed, and the mean fold change in expression following butyrate treatment relative to control was computed for each sequence. Microarray experiments using the Nimblegen platform are described in Supplementary Methods.

To identify genes differentially regulated (induced or repressed) in response to HDACi treatment in sensitive and resistant cell lines, the mean fold change in expression (butyrate relative to control) across the five sensitive and five resistant lines was computed for each gene. Differentially expressed genes were selected upon their ability to satisfy each of the following three criteria: (1) changed 2-fold or greater relative to control in either the sensitive or resistant panel; (2) differentially regulated by a factor of 1.5-fold or greater between sensitive and resistant cells; (3) significantly differentially regulated between sensitive and resistant cells by an unpaired t test, with a P value of <0.05 considered statistically significant.

To identify genes not changed in response to butyrate treatment, we first identified all genes for which the median fold induction in response to butyrate treatment was <10% (within the range of +1.1 to −1.1), for both the sensitive and resistant panel. From this list we then selected genes that were also not significantly differentially expressed between sensitive and resistant lines (P value resulting from a t test between sensitive and resistant is >0.05). Clones (2,751) that meet the above criteria were identified. From these, a random number generator was used to select 48 genes that were used for comparative analyses with the HDACi-induced gene list.

Promoter analysis

Promoter sequences for the 48 HDACi-induced genes as well as an equal number of control genes not changed by HDACi were obtained from the University of California Santa Cruz genome browser.4 The promoter region encompassing 1.5 kb upstream of the transcription start site (TSS) through 200 bp downstream was systematically searched for the presence of transcription factor binding sites using the MATCH tool (BIOBASE Biological Databases GmbH, Wolfenbüttel, Germany) on the TransFac public site. For more detailed guanine-cytosine (GC) content, Sp1, Sp3, and KLF4 binding site analyses, each promoter was subdivided into 1-kb windows beginning 10 kb downstream of the TSS and ending 10 kb upstream of the TSS. The GC content of each 1-kb window was computed using the freak tool in the EMBOSS suite accessed on the Washington State University Bioinformatics server5 (31). The frequency of Sp1, Sp3, and KLF4 binding sites in these windows were determined as above.

Quantitative reverse transcription–PCR, Western blotting, transient transfection, siRNA knockdown, chromatin immunoprecipitation analysis

Detailed methods are described in supplementary text. Primers used in quantitative reverse transcription–PCR (qRT-PCR) and chromatin immunoprecipitation (ChIP) analysis are described in Supplementary Tables S1 and S2, respectively.

Immunohistochemistry

We used the Protein Atlas online immunohistochemistry resource6 for images of metallothionein and activating transcription factor 3 (ATF3) staining in normal colon and colon tumor sections (32, 33).

Results

Identification of colon cancer cell lines sensitive and refractory to HDACi-induced apoptosis

To identify colon cancer cell lines sensitive and refractory to HDACi, a panel of 30 colon cancer cell lines was screened for HDACi-induced apoptosis by treatment with low (1 mmol/L), intermediate (5 mmol/L), or high (10 mmol/L) dose of the HDACi, sodium butyrate, for 72 hours. As shown in Fig. 1, a continuum response to butyrate-induced apoptosis was observed. The five cell lines with the highest apoptotic response to low-dose butyrate treatment were defined as butyrate “sensitive” (SW480, HCT116, HCT8, HCC2998, and SW948), and the five cell lines with the lowest apoptotic response to high-dose butyrate treatment defined as butyrate “resistant” (LIM2405, Colo320, RKO, HCT15, and HT29 cl.19A) for subsequent studies.

Figure 1.
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.

To validate these findings using an independent method, clonogenic survival assays were performed following 24 h butyrate treatment on the adherent-sensitive and -resistant cell lines. Consistent with the apoptosis data, colony formation was reduced to a significantly greater extent in sensitive compared with resistant lines (Fig. 1B and C; P = 0.01, unpaired t test). To confirm the differential response of colon cancer cells to HDACi in vivo, two sensitive (HCT116 and HCT8) and two resistant (LIM2405, HCT15) cell lines were grown as xenografts in severe combined immunodeficient mice and treated with the HDACi, VPA, for 2 weeks. VPA was used in these studies as it is structurally similar to butyrate but not metabolized as rapidly in vivo. Consistent with the in vitro findings, VPA treatment significantly inhibited growth of HCT116 and HCT8 xenografts but had minimal effects on growth of LIM2405 and HCT15 cells (Fig. 1D and E).

To confirm that the apoptotic effects of butyrate were due to the inhibition of HDAC activity, the cell lines were rescreened for apoptotic response to VPA, and to the structurally distinct hydroxamic acid–based HDAC inhibitors, Trichostatin A (TSA), and SAHA. As shown in Supplementary Fig. S1, cell lines sensitive and resistant to butyrate-induced apoptosis were likewise differentially sensitive to each of the other HDACi. Notably, the differential sensitivity of these cell lines to HDACi was specific to this class of agent and was not due to an inherent differential sensitivity of these cell lines to apoptosis in general, as a number of cell lines refractory to HDACi-induced apoptosis were sensitive to apoptosis induced by the mechanistically distinct chemotherapeutic agents, 5FU, CPT, and oxaliplatin (Supplementary Fig. S1B).

Differential sensitivity of cell lines to HDACi-induced apoptosis is not linked to the degree of HDAC inhibition or HDACi-induced growth arrest

Interestingly, HDACi treatment resulted in robust induction of histone H3 and histone H4 acetylation to a similar extent in both sensitive and resistant cell lines (P = 0.44 and P = 0.37 for Ac.H3 and Ac.H4, respectively; Supplementary Fig. S2), indicating differential sensitivity to HDACi was not due to differences in the degree of HDAC inhibition.

Consistent with this finding, HDACi treatment resulted in significant growth inhibition in all 10 cell lines (Supplementary Fig. S3). The percentage of cells in S phase was decreased by 73 ± 8.2% and 62 ± 1.8% in resistant and sensitive cell lines, respectively (P = 0.59), and the percentage of cells in G2-M was induced 359 ± 95% and 388 ± 112% in resistant and sensitive cell lines, respectively (P = 0.85) 24 h after HDACi treatment. Consistent with HDACi-induced growth inhibition, expression of p21WAF1/CIP1, a regulator of cell cycle progression and known HDACi target gene, was consistently induced by butyrate treatment in each of the 10 cell lines (Supplementary Fig. S4A and B).

HDACi-induced apoptosis is inhibited by actinomycin D

To determine the importance of transcription in HDACi-induced apoptosis, HCT116 cells were cotreated with HDACi and the RNA polymerase inhibitor, actinomycin D. Actinomycin D significantly attenuated HDACi-induced apoptosis, showing HDACi-induced apoptosis in colon cancer cells is dependent upon de novo transcription (Supplementary Fig. S5A and B).

IE and SR genes are preferentially induced by HDACi in sensitive colon cancer cell lines

Having identified sensitive and resistant cell lines and established the dependency of HDACi-induced apoptosis upon de novo transcription, the transcriptional basis for this differential sensitivity was determined. The five sensitive and five resistant cell lines were treated with 5 mmol/L butyrate for 24 hours and changes in gene expression profiled using 27,000-feature cDNA microarrays (entire database provided as Supplementary Table S3). Notably, the overall number of genes changed in response to HDACi treatment (P > 0.05, t test) and the range of transcriptional changes in terms of fold change was similar for sensitive and resistant cell lines.

Genes differentially induced by HDACi in sensitive versus resistant cell lines were identified using a stringent supervised analysis as described in Materials and Methods. Satisfying these criteria, 48 sequences were identified as significantly and preferentially induced by butyrate in sensitive cell lines (Fig. 2; Supplementary Table S4). Notably, 7 of these 48 genes, Fos, Jun, Atf3, Arc, Nr4a1 (Nur77), Egr1, and Egr3, are IE genes whereas an additional 7 genes have previously been classified as SR genes: Gadd45b, Ndrg4, Mt1B, Mt1E, Mt1F, Mt1H, and MtIX (Fig. 2A).

Figure 2.
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).

Forty-four genes preferentially repressed by HDACi in sensitive lines were also identified (Supplementary Table S4). These included several genes involved in organization of microtubules and the actin cytoskeleton (TRIP6, SRHML, PLXNB1, MAP7, LASP1, and LAD1), cell adhesion (OCLN, DSC2), transcriptional repression (NCoR2, SET), and apoptosis (FLIP, DAXX).

Validation of transcriptional changes

To validate the differential induction of HDACi-induced genes in sensitive and resistant lines, we performed qRT-PCR and Western blot analyses. As shown in Fig. 2B, Fos, EGR1, EGR3, and IGFBP3 mRNA induction by butyrate showed strong concordance with the microarray data, with robust and preferential induction in the sensitive cell lines. Preferential induction in sensitive cell lines of three additional genes, ATF3, Jun, and syntaxin, was confirmed at the protein level (Fig. 2C). To confirm that the transcriptional changes induced by butyrate were largely due to HDAC inhibition, gene expression changes induced in response to butyrate were compared with changes induced by VPA and SAHA in HCT116 cells by microarray analysis. Examination of the gene expression changes across the entire microarray revealed a marked similarity in the transcriptional changes induced by each of these agents (Supplementary Fig. S6A and B), whereas specific examination of the genes comprising the apoptosis signature revealed an even stronger correlation (r = 0.83 or greater), indicating the butyrate-induced changes are highly likely to be the consequence of HDAC inhibition.

Induction of IE genes is specific to HDACi-induced apoptosis

To confirm that the transcriptional response observed was specific to HDACi-induced apoptosis and not a nonspecific reflection of cells undergoing apoptosis, HCT116 cells were treated with HDACi or the mechanistically distinct chemotherapeutic agents 5FU and oxaliplatin for 24 hours. As shown in Supplementary Fig. S7, expression of Fos, EGR1, EGR3, and IGFBP3 mRNA and ATF3, Jun, and syntaxin protein expression were induced primarily in response to HDACi treatment. In contrast, p21, which is induced both by HDACi and in a p53-dependent manner in response to DNA damage, was induced by all agents tested (Supplementary Fig. S7).

Promoters of genes selectively induced by HDACi in sensitive cell lines are GC rich and are enriched for KLF4/Sp1/Sp3 binding sites

IE and SR gene expression has previously been shown to be coordinately induced in response to serum, 12-O-tetradecanoylphorbol-13-acetate, and growth factor treatment (34), suggesting that a common transcription factor or a set of transcription factors likely regulates their expression. To determine the identity of such a factor(s), a systematic bioinformatic search of the promoter regions of each of these genes was performed. As a control, the promoters of an equal number of genes not induced by HDACi were also analyzed. First, a nucleotide composition analysis of the region 10-kb upstream through 10-kb downstream of the TSS, measured in 1-kb windows, showed that the GC content of HDACi-induced gene promoters was significantly higher than that of the control set. As shown in Fig. 3A, promoter GC content increased significantly in both groups with increasing proximity to the TSS; however, the magnitude of this increase was significantly higher in HDACi-induced genes. To determine whether the promoters of HDACi-induced genes were enriched for specific transcription factor binding sites, the frequency of occurrence of all established transcription factor binding sites in the 1.5-kb promoter region upstream and 200-bp downstream of the TSS was computed using the MATCH database. As shown in Supplementary Table S5, the frequency of occurrence of multiple transcription factor binding sites was significantly different between HDACi-induced versus uninduced genes, with the vast majority of sites underrepresented in the promoters of genes induced by HDACi. The transcription factor binding sites most significantly enriched in HDACi-induced genes were the related GC-rich KLF4, Sp1, and Sp3 binding sites. To further confirm this finding and to examine the distribution of KLF4/Sp1/Sp3 binding sites within the promoters of these genes in more detail, we computed the frequency of occurrence of these sites in 1-kb increments beginning 10-kb upstream through 10-kb downstream of the TSS. As shown in Fig. 3B, the frequency of KLF4 and Sp1/Sp3 sites was significantly higher in HDACi-induced genes.

Figure 3.
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).

KLF4 is not a key regulator of HDACi induction of the IE/SR gene signature and apoptosis

To address the role of KLF4 in mediating the HDACi-induced transcriptional and apoptotic response, we first determined the effect of KLF4 overexpression on HDACi-induced apoptosis and IE gene expression in the HDACi-resistant RKO cell line. RKO cells have low levels of KLF4 due to hemizygous deletion of a portion of the KLF4 locus as well as promoter methylation (35). RKO-EcR-KLF4 cells are an isogenic derivative of RKO cells containing a stably integrated ponasterone-inducible KLF4 gene (29). Ponasterone treatment induced robust induction of KLF4 expression in RKO-EcR-KLF4 cells after 24 h; however, no change in the magnitude of HDACi-induced apoptosis or HDACi induction of IE gene expression was observed (Supplementary Fig. S8). Second, KLF4 mRNA was selectively downregulated in HDACi-sensitive HCT116 cells. Despite >70% knockdown of KLF4 mRNA expression, no change in HDACi-induced apoptosis was observed (Supplementary Fig. S8B), collectively indicating KLF4 does not play a central role in HDACi induction of IE/SR gene expression or in HDACi-induced apoptosis in colon cancer cells.

Activity of Sp1 and Sp3 is functionally implicated in HDACi induction of the IE/SR gene signature and apoptosis

Next, we determined the role played by Sp1 and Sp3 transcription factors in HDACi-induced gene expression and apoptosis using multiple independent approaches. First, to determine whether Sp1 and Sp3 were physically localized to the promoters of HDACi-responsive genes, we performed ChIP analyses in HCT116 cells interrogating the Sp1/Sp3 sites within the proximal promoter regions of the ATF3, EGR1, and JUN promoters. Pulldowns performed using anti-Sp1 or anti-Sp3 antibodies resulted in significant enrichment of the proximal promoter fragments of these genes, relative to the immunoprecipitations performed using No Ab or IgG controls (Fig. 3C). In comparison, for each of these genes, no enrichment of distal promoter regions devoid of Sp1/Sp3 sites was observed (Fig. 3D). To determine the functionality of these sites in terms of facilitating transcription in response to HDACi treatment, we also examined AcH3 levels at these sites before and after HDACi treatment by ChIP analysis. As shown in Fig. 3E, AcH3 levels were increased ∼2-fold 24 hours after HDACi treatment consistent with the transcriptional induction of these genes. Notably, enrichment of Sp1 or Sp3 or of the class I HDAC, HDAC3, at the Jun promoter was not significantly altered in response to HDACi treatment (data not shown), indicating these factors are retained at the promoter during transcription of the gene.

Second, HDACi-sensitive and -resistant colon cancer cell lines were transfected with wild-type or mutant Sp1/Sp3-luciferase reporter constructs and treated with HDACi for 24 hours. As shown in Fig. 3F, the magnitude of induction of Sp1/Sp3 reporter activity was 2- to 10-fold higher in HDACi-sensitive lines compared with HDACi-resistant lines, indicating preferential induction of Sp1/Sp3 driven transcription in sensitive cells. Minimal effects were observed on the mutant Sp1/Sp3-luciferase reporter construct in both sensitive and resistant lines (data not shown).

Third, whether pharmacologic inhibition of Sp1/Sp3 binding abrogated HDACi induction of IE genes and apoptosis was examined using the antibiotic mithramycin, which inhibits Sp1/Sp3 transcription by competitively binding to GC-rich elements (36). Cotreatment of HCT116 cells with mithramycin markedly attenuated butyrate and TSA-mediated induction of three of the four genes tested: FOS, EGR1, and EGR3 (Fig. 4A). In contrast, mithramycin treatment failed to inhibit HDACi induction of IGFBP3, instead potentiating the HDACi effect (Fig. 4A). Likewise, mithramycin markedly attenuated HDACi-induced apoptosis in both HCT116 and HCT8 cells at the 24-hour time point as assessed by cell monolayer morphology, the percentage of cells with a subdiploid DNA content or poly(ADP-ribose) polymerase (PARP) cleavage (Fig. 4B–D). However, this effect was lost at later time points (48 and 72 hours) when mithramycin alone induced significant apoptosis (data not shown).

Figure 4.
Figure 4.

The Sp1/Sp3 inhibitor, mithramycin (Mith, M, 1 μmol/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.

Finally, to directly determine the role of Sp1 and Sp3 in the HDACi-induced transcriptional and apoptotic response, Sp1 and Sp3 were downregulated in HCT116 cells using Sp1 and Sp3 targeting siRNAs. Effective downregulation of Sp1 and all three isoforms of Sp3 were confirmed by Western blot (Fig. 5A). As shown in Fig. 5B, downregulation of Sp1 and Sp3 resulted in a 17% and 46% inhibition of HDACi-induced apoptosis, respectively, and in the extent of HDACi-induced PARP cleavage (Fig. 5C).

Figure 5.
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.

Downregulation of Sp1 and Sp3, alone and in combination, resulted in a heterogeneous pattern of effects on HDACi-induced gene expression, which fell into three categories. First, for a subset of the genes (FOS, IGFBP3), Sp1 and Sp3 silencing had no effect on HDACi-induced gene expression (Fig. 5D). For a second subset (EGR3, JUN), silencing of Sp3 but not Sp1 partially attenuated HDACi-mediated transcriptional induction (Fig. 5E). For the final subset (GADD45, EGR1), individual silencing of both Sp1 and Sp3 inhibited HDACi-mediated transcriptional induction (Fig. 5F). Collectively, these results indicate that Sp1 and Sp3 regulate a significant, although not the entire component of the HDACi-induced transcriptional response associated with promotion of apoptosis in colon cancer cells.

Genes selectively induced by HDACi in sensitive cell lines are basally repressed in colon tumors

To independently assess the biological significance of the subset of genes preferentially induced by HDACi in sensitive cell lines, we compared the relative expression of this gene subset in colon tumors and normal colonic mucosa. For this, we generated a microarray database comparing gene expression differences 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 32 genes in the matched primary colon tumor/normal database. Notably, expression of 9 of these 32 genes (28%) was significantly repressed (>1.5-fold and P < 0.05) in colon tumors compared with normal colonic mucosa. In contrast, only 2 of 32 (6.3%) were upregulated in colon tumors by these same criteria (Fig. 6A). As a control, the expression pattern in colon tumor and normal tissue of an equal number of genes not induced by HDACi treatment was also examined. As shown in Fig. 6B, 5.7% of the genes on this control list were upregulated and 5.7% were downregulated (>1.5-fold and P < 0.05) in colon tumors relative to normal colonic mucosa (P = 0.004, χ2 test).

Figure 6.
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.

The HDACi-induced genes downregulated in colon tumors were ATF3, MT1B, MT1E, MT1F, MT1H, MT1X, NDRG4, PLEKHH2, and TGFB3 (Fig. 6C). As shown in Fig. 6D, the downregulation of MT1F and ATF3 in colon tumors was independently validated using the protein atlas immunohistochemistry database (33, 37). These data show that the subset of genes identified as selectively induced during HDACi-induced apoptosis was enriched for genes that are basally downregulated in colon tumors. These findings are also consistent with a previous report indicating downregulation of a number of IE genes in colon tumors (38).

Discussion

HDAC inhibitors induce growth arrest, differentiation, and apoptosis in tumor cells of diverse origins, and their efficacy for cancer treatment was recently underscored by the approval of SAHA for the treatment of cutaneous T-cell lymphoma. Whereas the downstream effectors of HDACi-induced apoptosis in tumor cells have been studied in detail (3, 17), the molecular events that initiate HDACi-induced apoptosis are unknown. Possibilities include transcription-dependent effects, such as altered expression of proapoptotic and antiapoptotic molecules, or transcription-independent effects, such as induction of aberrant mitosis, ROS production, or altered acetylation of prosurvival molecules, such as HSP90.

In the present study, the importance of de novo transcription in HDACi-induced apoptosis in colon cancer cell lines was established. Through gene expression profiling of multiple colon cancer cell lines sensitive and resistant to HDACi, we identified a reproducible transcriptional response preferentially induced in response to HDACi treatment in sensitive cell lines. Importantly, this transcriptional response was observed in response to multiple, structurally distinct HDACi and across a range of colon cancer cell lines with extensive molecular and mutational heterogeneity including differences in microsatellite instability status, ploidy, and the mutation and methylation status of multiple tumor suppressor genes and oncogenes (28).

A striking feature of the HDACi-induced transcriptional response is that a number of the induced genes, particularly the IE and SR genes, are coordinately induced in response to several other stimuli. This includes the coordinate induction of IE gene expression in response to serum stimulation, phorbol ester, and growth factor treatment (39, 40) and the coordinate induction of the metallothionein gene family, clustered within the q13 region of chromosome 16, in response to metal induction and oxidative stress (41). The coordinate induction of these genes in response to HDACi treatment suggested they likely reflected the modulation of a specific transcription factor or factors that coordinately regulate their expression.

Several findings collectively indicated a key role for Sp1/Sp3 transcription factors in driving this transcriptional response. First, the promoters of HDACi-induced genes had a high GC content and were significantly enriched for Sp1/Sp3 binding sites. Second, localization of Sp1 and Sp3 at the promoters of several of these genes was detectable by ChIP analysis. Third, induction of Sp1/Sp3 reporter activity was preferentially induced by HDACi in sensitive cell lines. Finally, disruption of Sp1/Sp3 DNA binding with mithramycin or siRNA-mediated downregulation of Sp1 or Sp3 attenuated induction of a number of HDACi targets. Importantly, both mithramycin and Sp1/Sp3 siRNA attenuated HDACi-induced apoptosis, establishing a direct link between Sp1/Sp3-mediated transcriptional response and initiation of HDACi-induced apoptosis. It is important to note, however, that expression of a subset of the genes induced by HDACi, including FOS and IGFBP3, were not modulated by Sp1 and/or Sp3 silencing, indicating additional transcription factors also likely participate in HDACi-induced transcriptional changes.

Whereas the precise mechanism by which initiation of this transcriptional response triggers apoptosis remains to be determined, it is noteworthy that several genes comprising the HDACi transcriptional response have previously been linked with HDACi-induced apoptosis through over and underexpression studies. These include IGFBP3 (42), EGR1 (43), NR4A1 (TR3/Nur77; ref. 14), and GADD45B (44). However, in each case, induction of these genes only partially accounts for the overall apoptotic response, suggesting that their collective induction, which may sufficiently alter the cellular transcriptional equilibrium in favor of apoptosis, may be the critical determinant of HDACi-induced apoptosis. Lending support to this concept is the finding that a number of these transcripts are basally repressed in colon tumors compared with normal colon tissue. In the context of tumorigenesis therefore, selective repression of these transcripts may alter the transcriptional balance in favor of cell survival.

Whereas HDACi treatment uniformly induces expression of antiproliferative genes, such as p21 in both sensitive and resistant cell lines, it is notable that several of the genes preferentially induced by HDACi in sensitive cells, including the IE genes FOS, JUN, EGR1, and EGR3, are typically linked to the promotion of cell proliferation, such as in response to serum or growth factor treatment (34). HDACi treatment therefore clearly induces a conflicting transcriptional response comprising both proproliferative and antiproliferative signals, which is magnified in HDACi sensitive cells. HDACi induction of IE gene expression is also sustained for over 24 hours, whereas growth factor induction is typically maximal at 30 minutes to 1 hour after stimulation and rapidly returns to baseline (38). Whether these conflicting transcriptional signals or the sustained induction of IE gene expression are perceived by the cells as a SR and whether this is ultimately the stimulus for apoptosis induction is worthy of further investigation. In support of this hypothesis, it is notable that cotreatment of colon cancer cells with agents such as phorbol 12-myristate 13-acetate, which independently induce IE gene expression, potentiates HDACi-induced apoptosis (45).

In conclusion, this study shows that HDACi-induced apoptosis in colon cancer cells is linked to a consistent transcriptional response involving induction of a transcriptional network enriched for IE and SR genes, which are regulated, to a large extent, by the Sp1/Sp3 transcription factors. Importantly, several components of this transcriptional response are basally repressed in colon tumors suggesting HDACi-induced apoptosis may be initiated through induction of a transcriptional network whose basal repression is linked to tumor cell survival.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Geoff Childs and Aldo Massimi at the AECOM cDNA microarray facility for generation of cDNA microarrays and assistance with array scanning and David Reynolds at the AECOM DNA sequencing facility.

Grant Support: NIH grants CA123316, CA100823, and CA88104.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Received June 24, 2009.
  • Revision received October 9, 2009.
  • Accepted November 6, 2009.

References

  1. ↵
    1. Xu WS,
    2. Parmigiani RB,
    3. Marks PA
    . Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene 2007;2637:5541–52.
    OpenUrl
  2. ↵
    1. Bolden JE,
    2. Peart MJ,
    3. Johnstone RW
    . Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 2006;59:769–84.
    OpenUrl
  3. ↵
    1. Mariadason JM
    . HDACs and HDAC inhibitors in colon cancer. Epigenetics 2008;31:28–37.
    OpenUrl
  4. ↵
    1. Gu W,
    2. Roeder RG
    . Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 1997;904:595–606.
    OpenUrl
  5. ↵
    1. Marzio G,
    2. Wagener C,
    3. Gutierrez MI,
    4. Cartwright P,
    5. Helin K,
    6. Giacca M
    . E2F family members are differentially regulated by reversible acetylation. J Biol Chem 2000;27515:10887–92.
    OpenUrl
  6. ↵
    1. Ammanamanchi S,
    2. Freeman JW,
    3. Brattain MG
    . Acetylated sp3 is a transcriptional activator. J Biol Chem 2003;27837:35775–80.
    OpenUrl
    1. Ferrante RJ,
    2. Kubilus JK,
    3. Lee J,
    4. et al
    . Histone deacetylase inhibition by sodium butyrate chemotherapy ameliorates the neurodegenerative phenotype in Huntington's disease mice. J Neurosci 2003;2328:9418–27.
    OpenUrl
  8. ↵
    1. Braun H,
    2. Koop R,
    3. Ertmer A,
    4. Nacht S,
    5. Suske G
    . Transcription factor Sp3 is regulated by acetylation. Nucleic Acids Res 2001;2924:4994–5000.
    OpenUrl
  9. ↵
    1. Imhof A,
    2. Yang XJ,
    3. Ogryzko VV,
    4. Nakatani Y,
    5. Wolffe AP,
    6. Ge H
    . Acetylation of general transcription factors by histone acetyltransferases. Curr Biol 1997;79:689–92.
    OpenUrl
  10. ↵
    1. Heerdt BG,
    2. Houston MA,
    3. Augenlicht LH
    . Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function. Cell Growth Differ 1997;85:523–32.
    OpenUrl
  11. ↵
    1. Insinga A,
    2. Monestiroli S,
    3. Ronzoni S,
    4. et al
    . Inhibitors of histone deacetylases induce tumor-selective apoptosis through activation of the death receptor pathway. Nat Med 2005;111:71–6.
    OpenUrl
  12. ↵
    1. Nebbioso A,
    2. Clarke N,
    3. Voltz E,
    4. et al
    . Tumor-selective action of HDAC inhibitors involves TRAIL induction in acute myeloid leukemia cells. Nat Med 2005;111:77–84.
    OpenUrl
  13. ↵
    1. Ruemmele FM,
    2. Schwartz S,
    3. Seidman EG,
    4. Dionne S,
    5. Levy E,
    6. Lentze MJ
    . Butyrate induced Caco-2 cell apoptosis is mediated via the mitochondrial pathway. Gut 2003;521:94–100.
    OpenUrl
  14. ↵
    1. Wilson AJ,
    2. Arango D,
    3. Mariadason JM,
    4. Heerdt BG,
    5. Augenlicht LH
    . TR3/Nur77 in colon cancer cell apoptosis. Cancer Res 2003;6317:5401–7.
    OpenUrl
  15. ↵
    1. Hernandez A,
    2. Thomas R,
    3. Smith F,
    4. et al
    . Butyrate sensitizes human colon cancer cells to TRAIL-mediated apoptosis. Surgery 2001;1302:265–72.
    OpenUrl
  16. ↵
    1. Bonnotte B,
    2. Favre N,
    3. Reveneau S,
    4. et al
    . Cancer cell sensitization to fas-mediated apoptosis by sodium butyrate. Cell Death Differ 1998;56:480–7.
    OpenUrl
  17. ↵
    1. Lindemann RK,
    2. Newbold A,
    3. Whitecross KF,
    4. et al
    . Analysis of the apoptotic and therapeutic activities of histone deacetylase inhibitors by using a mouse model of B cell lymphoma. Proc Natl Acad Sci U S A 2007;10419:8071–6.
    OpenUrl
  18. ↵
    1. Ruefli AA,
    2. Ausserlechner MJ,
    3. Bernhard D,
    4. et al
    . The histone deacetylase inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA) induces a cell-death pathway characterized by cleavage of Bid and production of reactive oxygen species. Proc Natl Acad Sci U S A 2001;9819:10833–8.
    OpenUrl
  19. ↵
    1. Bandyopadhyay D,
    2. Mishra A,
    3. Medrano EE
    . Overexpression of histone deacetylase 1 confers resistance to sodium butyrate-mediated apoptosis in melanoma cells through a p53-mediated pathway. Cancer Res 2004;6421:7706–10.
    OpenUrl
  20. ↵
    1. Chirakkal H,
    2. Leech SH,
    3. Brookes KE,
    4. Prais AL,
    5. Waby JS,
    6. Corfe BM
    . Upregulation of BAK by butyrate in the colon is associated with increased Sp3 binding. Oncogene 2006;2554:7192–200.
    OpenUrl
  21. ↵
    1. Johnstone RW,
    2. Licht JD
    . Histone deacetylase inhibitors in cancer therapy: is transcription the primary target? Cancer Cell 2003;41:13–8.
    OpenUrl
  22. ↵
    1. Park JH,
    2. Jong HS,
    3. Kim SG,
    4. et al
    . Inhibitors of histone deacetylases induce tumor-selective cytotoxicity through modulating Aurora-A kinase. J Mol Med (Berlin, Germany) 2008;861:117–28.
    OpenUrl
  23. ↵
    1. Bali P,
    2. Pranpat M,
    3. Bradner J,
    4. et al
    . Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. J Biol Chem 2005;28029:26729–34.
    OpenUrl
  24. ↵
    1. Xu WS,
    2. Perez G,
    3. Ngo L,
    4. Gui CY,
    5. Marks PA
    . Induction of polyploidy by histone deacetylase inhibitor: a pathway for antitumor effects. Cancer Res 2005;6517:7832–9.
    OpenUrl
    1. Taddei A,
    2. Maison C,
    3. Roche D,
    4. Almouzni G
    . Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases. Nat Cell Biol 2001;32:114–20.
    OpenUrl
    1. Dowling M,
    2. Voong KR,
    3. Kim M,
    4. Keutmann MK,
    5. Harris E,
    6. 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;42:197–206.
    OpenUrl
  27. ↵
    1. Blagosklonny MV,
    2. Robey R,
    3. Sackett DL,
    4. et al
    . Histone deacetylase inhibitors all induce p21 but differentially cause tubulin acetylation, mitotic arrest, and cytotoxicity. Mol Cancer Therap 2002;111:937–41.
    OpenUrl
  28. ↵
    1. Mariadason JM,
    2. Arango D,
    3. Shi Q,
    4. et al
    . Gene expression profiling-based prediction of response of colon carcinoma cells to 5-fluorouracil and camptothecin. Cancer Res 2003;6324:8791–812.
    OpenUrl
  29. ↵
    1. Dang DT,
    2. Chen X,
    3. Feng J,
    4. Torbenson M,
    5. Dang LH,
    6. Yang VW
    . Overexpression of Kruppel-like factor 4 in the human colon cancer cell line RKO leads to reduced tumorigenecity. Oncogene 2003;2222:3424–30.
    OpenUrl
  30. ↵
    1. Cheung VG,
    2. Morley M,
    3. Aguilar F,
    4. Massimi A,
    5. Kucherlapati R,
    6. Childs G
    . Making and reading microarrays. Nat Genet 1999;211 Suppl:15–9.
    OpenUrl
  31. ↵
    1. Rice P,
    2. Longden I,
    3. Bleasby A
    . EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet 2000;166:276–7.
    OpenUrl
  32. ↵
    1. Berglund L,
    2. Bjorling E,
    3. Oksvold P,
    4. et al
    . A genecentric human protein atlas for expression profiles based on antibodies. Mol Cell Proteomics 2008;710:2019–27.
    OpenUrl
  33. ↵
    1. Uhlen M,
    2. Bjorling E,
    3. Agaton C,
    4. et al
    . A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics 2005;412:1920–32.
    OpenUrl
  34. ↵
    1. Kovary K,
    2. Bravo R
    . The jun and fos protein families are both required for cell cycle progression in fibroblasts. Mol Cell Biol 1991;119:4466–72.
    OpenUrl
  35. ↵
    1. Zhao W,
    2. Hisamuddin IM,
    3. Nandan MO,
    4. Babbin BA,
    5. Lamb NE,
    6. Yang VW
    . Identification of Kruppel-like factor 4 as a potential tumor suppressor gene in colorectal cancer. Oncogene 2004;232:395–402.
    OpenUrl
  36. ↵
    1. Ray R,
    2. Snyder RC,
    3. Thomas S,
    4. Koller CA,
    5. Miller DM
    . Mithramycin blocks protein binding and function of the SV40 early promoter. J Clin Invest 1989;836:2003–7.
    OpenUrl
  37. ↵
    1. Bjorling E,
    2. Lindskog C,
    3. Oksvold P,
    4. et al
    . A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues. Mol Cell Proteomics 2008;75:825–44.
    OpenUrl
  38. ↵
    1. Tice DA,
    2. Soloviev I,
    3. Polakis P
    . Activation of the Wnt pathway interferes with serum response element-driven transcription of immediate early genes. J Biol Chem 2002;2778:6118–23.
    OpenUrl
  39. ↵
    1. Sassone-Corsi P
    . Goals for signal transduction pathways: linking up with transcriptional regulation. EMBO J 1994;1320:4717–28.
    OpenUrl
  40. ↵
    1. Thomson S,
    2. Clayton AL,
    3. Hazzalin CA,
    4. Rose S,
    5. Barratt MJ,
    6. Mahadevan LC
    . The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase. EMBO J 1999;1817:4779–93.
    OpenUrl
  41. ↵
    1. Haq F,
    2. Mahoney M,
    3. Koropatnick J
    . Signaling events for metallothionein induction. Mutat Res 2003;5331–2:211–26.
    OpenUrl
  42. ↵
    1. Collard TJ,
    2. Guy M,
    3. Butt AJ,
    4. et al
    . Transcriptional upregulation of the insulin-like growth factor binding protein IGFBP-3 by sodium butyrate increases IGF-independent apoptosis in human colonic adenoma-derived epithelial cells. Carcinogenesis 2003;243:393–401.
    OpenUrl
  43. ↵
    1. Pan L,
    2. Lu J,
    3. Wang X,
    4. et al
    . Histone deacetylase inhibitor trichostatin a potentiates doxorubicin-induced apoptosis by up-regulating PTEN expression. Cancer 2007;1098:1676–88.
    OpenUrl
  44. ↵
    1. Chen Z,
    2. Clark S,
    3. Birkeland M,
    4. et al
    . Induction and superinduction of growth arrest and DNA damage gene 45 (GADD45) α and β messenger RNAs by histone deacetylase inhibitors trichostatin A (TSA) and butyrate in SW620 human colon carcinoma cells. Cancer Lett 2002;1881–2:127–40.
    OpenUrl
  45. ↵
    1. Rahmani M,
    2. Dai Y,
    3. Grant S
    . The histone deacetylase inhibitor sodium butyrate interacts synergistically with phorbol myristate acetate (PMA) to induce mitochondrial damage and apoptosis in human myeloid leukemia cells through a tumor necrosis factor-α-mediated process. Exp Cell Res 2002;2771:31–47.
    OpenUrl
View Abstract
Back to top
Cancer Research: 70 (2)
January 2010
Volume 70, Issue 2

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Email Article
Citation Tools
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, Georgia A. Corner, Naseem Ahmed, Sanjay Goel, Do-Sun Byun, Shannon Nasser, Michele A. Houston, Minaxi Jhawer, Helena J.M. Smartt, Lucas B. Murray, Courtney Nicholas, Barbara G. Heerdt, Diego Arango, Leonard H. Augenlicht and John M. Mariadason
Cancer Res January 15 2010 (70) (2) 609-620; DOI: 10.1158/0008-5472.CAN-09-2327
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement

Keywords