Int. J. Cancer: 119, 2098–2104 (2006) ' 2006 Wiley-Liss, Inc.

The expression levels of the transcriptional regulators p300 and CtBP modulate the correlations between SNAIL, ZEB1, E-cadherin and vitamin D in human colon carcinomas Cristina Pena~ 1, Jose Miguel Garcıa1, Vanesa Garcıa1, Javier Silva1, Gemma Domınguez1, Rufo Rodrıguez2, Constanza Maximiano1, Antonio Garcıa de Herreros3, Alberto Munoz~ 4 and Felix Bonilla1* 1Department of Medical Oncology, Hospital Universitario Puerta de Hierro, Madrid, Spain 2Department of Pathology, Hospital Virgen de la Salud, Toledo, Spain 3Unitat de Biologıa Cellular i Molecular, Institut Municipal d’InvestigacioMe `dica-Universitat Pompeu-Fabra, Barcelona, Spain 4Instituto de Investigaciones Biomedicas ‘‘Alberto Sols,’’ Consejo Superior de Investigaciones Cientıficas-Universidad Autonoma de Madrid, Spain

ZEB1 and SNAIL repress CDH1 and induce epithelial–mesenchy- tion.20,21 However, overexpression of ZEB1 was not correlated mal transition (EMT). However, SNAIL and ZEB1 also activate with down-regulation of CDH1 in colorectal tumor tissues, or regulate other target in different ways. For instance, vita- CDH1 although a trend toward statistical association between ZEB1 min D receptor (VDR), which activates expression upon expression and CDH1 down-regulation was observed in patients ligand binding, is repressed by SNAIL but induced by ZEB1. We 21 examined whether the biological activity of SNAIL and ZEB1 in without SNAIL expression. In addition, transfection of ZEB1 into SW620 colon carcinoma cells up-regulates the expression of en- colon cancer is regulated by interacting cofactors. The mRNA 22 expression levels of SNAIL and ZEB1, and of transcriptional regu- dogenous VDR. lators p300 and CtBP, were measured by RT-PCR in tumor and The NAD1-dependent CtBP is involved in the con- normal tissue from 101 colon carcinoma patients. Overexpression trol of E-cadherin in several cell lines. Inactivation SNAIL CDH1 VDR of was associated with down-regulation of and of this factor in cell lines either by expression of the viral (p 5 0.004 and p < 0.001). CDH1 correlated with VDR (r 5 0.49; 9 23 p < 0.001). ZEB1 expression also correlated with VDR (r 5 0.23; E1A, which interacts directly with it, or by RNA interference p 5 0.019). However, when CtBP was strongly expressed, ZEB1 up-regulates CDH1 transcription. Moreover, analysis of CtBP- CDH1 r 52 p 5 knockout versus CtBP-rescued mouse embryo fibroblasts revealed was inversely correlated with ( 0.39; 0.053). Fur- 24 thermore, when there were elevated p300 expression levels, the that CDH1 is regulated by this cofactor. CtBP interacts with correlation between expression of ZEB1 and VDR was stronger ZEB1 and 2 through a PXDLS motif present in these mole- (r 5 0.38; p 5 0.070). Association between SNAIL expression and cules.25,26 Although this sequence is also present in the Drosoph- down-regulation of CDH1 and VDR was lost in tumors in which ila ortholog of SNAIL, where CtBP is involved in the repression p300 CtBP and were strongly expressed. These results indicate that of this factor in this organism,27,28 mammalian SNAIL lacks this the levels of expression of CtBP and p300 are critical for the action motif, which questions the involvement of CtBP in SNAIL in of SNAIL and ZEB1, which have a pivotal role in EMT, and show the importance of CtBP and p300 for tumor progression. human cells. Recently, the identification of a functional associa- ' 2006 Wiley-Liss, Inc. tion of mouse Snail and HDAC1/2 and the mSin3A that interact to repress E-cadherin expression through the SNAG motif of Snail has been described.29 Key words: human colorectal cancer; expression; p300; CtBP; Although SNAIL and ZEB have been classically epithelial-mesenchymal transition genes described as transcriptional repressors, they can also promote the activation of such mesenchymal markers as fibronectin and LEF1, in the case of SNAIL, or ovalbumin and VDR gene in the case of ZEB1.18,22,30 Moreover, ZEB1 and ZEB2 also have antagonistic A large number of biological processes involve regulation of 31 by transcriptional repression.1 ZEB1 and ZEB2 functions in the regulation of TGFb/BMP signaling. Such regu- are transcriptional repressors that contain zinc-fingers motifs in lation of the TGFb/BMP signaling is achieved through differential recruitment of coactivators and corepressors by ZEB1 or ZEB2.32 their DNA binding domain, and both recognize the same E-boxes 25,26 in their target genes.2,3 Despite the high homology and the identi- Repression by ZEB1 and ZEB2 requires CtBP. ZEB1 also has cal overall gene structure of ZEB1 and ZEB2, there are differen- an N-terminal region that binds the coactivators p300 and P/ 32 33–35 ces in their pattern of expression, the organization of their re- CAF, acetyltransferases that loosen chromatin structure. pressor domains and their specificity.3 ZEB1 and ZEB2 repress a Furthermore, binding of P/CAF to ZEB1 acetylates several lysine residues close to the CtBP interaction domain of ZEB1, displaces large number of regulators involved in differentiation and devel- 32 opmental events.2,4–12 One such regulator, E-cadherin ( by CtBP and switches ZEB1 from a repressor to an activator. In contrast, ZEB2 does not interact with p300 or P/CAF and thus CDH1 gene), is located in adherent junctions and contributes to 32 the maintenance of the adhesive and polarized phenotype of epi- only serves as a transcriptional repressor. In parallel, it has been 13 suggested that activation of ovalbumin and VDR genes by ZEB1 thelial cells. When E-cadherin is down-regulated, the epithelial 32 cells acquire a fibroblastoid morphotype accompanied by invasion depends on the recruitment of p300 and P/CAF by ZEB1. and migratory properties, which are associated with the epithelial– Cofactors p300 or CtBP vary in their regulation of specific cell mesenchymal transition (EMT).14–16 This transition also occurs lines.36,37 Thus, we hypothesize that variations in the concentra- after expression of SNAIL, another transcriptional repressor with zinc-finger motifs that represses transcription of CDH1 even more ~ efficiently than ZEB1 or ZEB2.17,18 Moreover, ZEB1 expression Grant sponsor: Fundacion Cientıfica de la Asociacion Espanola contra el Cancer; Grant sponsor: SAF; Grant number: SAF2004-01002; Grant spon- is induced by SNAIL and persists after SNAIL is down-regulated 18 sor: Fundacion Santander Central Hispano. in tumor cell lines. *Correspondence to: Department of Medical Oncology, Hospital Uni- Vitamin D (1a,25-dehydroxyvitamin D3) induces the expres- versitario Puerta de Hierro, C/ San Martın de Porres, 4, E-28035-Madrid, sion of CDH1 in cells expressing vitamin D receptors (VDRs).19 Spain. E-mail: [email protected] Received 26 January 2005; Accepted 28 March 2006 We have recently shown that SNAIL also represses VDR expres- DOI 10.1002/ijc.22083 sion in tumor cell lines and that its induction in human colorectal Published online 27 June 2006 in Wiley InterScience (www.interscience. carcinomas is associated with CDH1 and VDR down-regula- wiley.com).

Publication of the International Union Against Cancer CORRELATIONS BETWEEN EMT GENES REGARDING p300 AND CtBP 2099

TABLE I – SEQUENCE OF THE PRIMERS AND CONDITIONS FOR THE TABLE II – MEDIAN, MINIMUM AND MAXIMUM VALUES OF CDH1, VDR, AMPLIFICATION OF EACH GENE ZEB1, p300 AND CtBP EXPRESSION DATA

21 Primers Sequence T.A [Mg ] CDH1 VDR ZEB1 p300 CtBP (°C) (mM) Median 0.92 2.17 2.34 0.69 1.01 TBP F: TCTGGGATTGTACCGCAGC 59 3 Minimum 0.03 0.13 0.02 0.03 0.07 R: CGAAGTGCAATGGTCTTTAGG Maximum 209.97 66.20 417.19 8.4 157.74 SDHA F: TGGGAACAAGAGGGCATCTG 59 3 R: CCACCACTGCATCAAATTCATG F: ATTTGGGTCGCGGTTCTTG UBC 59 3 TABLE III – GEOMETRIC AVERAGE OF THE RATIOS (T/N) OF CDH1, VDR R: TGCCTTGACATTCTCGATGGT AND ZEB1 EXPRESSION REGARDING SNAIL PRESENCE IN TUMOR TISSUE SNAIL F: CACTATGCCGCGCTCTTTC 68 3 AND PEARSON CORRELATION COEFFICIENT (r) OF THE CORRELATION R: GGTCGTAGGGCTGCTGGAA BETWEEN THE EXPRESSION LEVELS OF CDH1, VDR AND ZEB1 CDH1 F: AGAACGCATTGCCACATACACTC 60 4 CDH1 VDR ZEB1 R: CATTCTGATCGGTTACCGTGATC VDR F: TTGCCATACTGCTGGACGC 60 2 Geometric average of the ratios (T/N) R: GGCTCCCTCCACCATCATT SNAIL ZEB1 F: GCCAATAAGCAAACGATTCTG 55 3 Yes 0.83 0.31 0.47 R: TTTGGCTGGATCACTTTCAAG No 1.93 0.91 0.43 CtBP F: ACTGTGGCCTTCTGCGACG 59 3 p 0.004 <0.001 0.832 R: GGTGTGGTACATCAGGGCCC Pearson correlation coefficient p300 F: GGCTGTATCAGAGCGTATTGTC 59 3 CDH1 _ r 5 0.49; p < 0.001 r 520.10; p 5 0.32 R: CCTCGAAATAAGGCAATTCC VDR ––r 5 0.23; p 5 0.019 tions of these cofactors could modulate the activity of ZEB1, and polation using a standard curve for each gene generated with a perhaps that of SNAIL, in the regulation of CDH1 and VDR. We serial dilution of a cDNA prepared from RNA extracted from examined this hypothesis in a large series of human colorectal car- SW480ADH cells. The expression level of a target gene in a cinomas. patient was calculated as the ratio: target in tumor tissue/target in normal tissue (T/N). The quantitative mRNA analysis was con- firmed by a duplicate analysis. The primers used are shown in Methods Table I. For the synthesis of the first strand of cDNA, 400 ng of Patients, tumor samples and RNA extraction total RNA was retro-transcribed using the Gold RNA PCR Core The present study, approved by the Research Ethics Board of Kit (PE Biosystems), following the manufacturer’s instructions. our hospital, (Hospital Universitario Puerta de Hierro), was based Random hexamers were used for cDNA synthesis. on a consecutive series of 101 patients undergoing surgery for colo- Real-time PCR was performed in a Light-Cycler apparatus rectal cancer between January 1998 and 2003. All the patients (Roche Diagnostics, Mannheim, Germany) using the LightCycler- were considered sporadic cases because no clinical antecedents of FastStart DNA Master SYBR Green I Kit (Roche Diagnostics, FAP were reported, and those with clinical criteria of hereditary Mannheim, Germany). Each reaction was performed in a final vol- non-polyposis colorectal cancer (Amsterdam criteria) were ume of 20 ll containing 2 ll of the cDNA product sample, a dif- excluded. Both normal and tumor tissue samples were obtained ferent MgCl2 concentration for each primer (Table I), 0.5 lMof sequentially immediately after surgery, immersed in RNA laterTM each primer as well as 13 reaction mix including FastStar DNA (Ambion, Austin, TX), snap-frozen in liquid nitrogen and stored at polymerase, reaction buffer, dNTPs and SYBR green. Thermal cy- 280°C until processing. cling for all genes was initiated with a denaturation step of 95°C ° All tumors were histologically examined by a pathologist to (i) for 10 min followed by 40 cycles (denaturation at 94 C for 0 sec, annealing at different temperature for each gene (Table I) for confirm the diagnosis of adenocarcinoma, (ii) verify the presence ° of tumor and select those samples with more than 75% neoplastic 5 sec, and elongation at 72 C for 5 sec, in which fluorescence was cells and (iii) establish the pathological stage. acquired). At the end of the PCR cycles, melting curve analyses as well as electrophoresis of the products on nondenaturing 8% poly- For 101 patients, RNAs were extracted from tumor and normal acrylamide gels, together with a molecular weight marker, fol- samples. RNA was obtained using RNeasy Mini Kit (Quiagen, lowed by sequencing, were performed to confirm the generation of Hilden, Germany) and quantified spectrophotometrically. the specific PCR product expected. Real time PCR Since SNAIL RNA was not detected in normal tissues, SNAIL expression was valued only as presence or absence in tumor tis- Data analysis sues. To specifically detect SNAIL gene, we used oligonucleotides The ratios (T/N) of gene expression were not normally distrib- that do not amplify the SNAIL retrogene. This retrogene, SNAIL- uted (Kolmogorov-Smirnov test, Lilliefors correction). For this like, is highly homologous to SNAIL, but considerably less active reason, we normalized the data distribution by using log10 for sta- than SNAIL in the repression of CDH1.38 Specific SNAIL primers tistical analysis. We also used the geometric (rather than the arith- were designed from a region with 16 bp differences between metic) average of the (T/N) to describe the expression gene data, SNAIL and SNAIL-like. We can thus distinguish between the 2 for the same reason. The variables analyzed (T/N), when com- genes when the amplification is carried out, and false results at- pared expression levels of CDH1, VDR, ZEB1, CtBP and p300 in tributable to SNAIL-like can be discarded. The primers are shown presence or absence of SNAIL expression in different tissue sam- in Table I. ples, were contrasted by ANOVA test. The correlation between CDH1, VDR, ZEB1, p300 and CtBP mRNA levels were calcu- genes expression levels (without SNAIL) was studied using the lated in the normal and tumor counterpart samples was calculated Pearson coefficient. by a relative quantification approach in which the amount of the The data were divided into quartiles (25, 50 and 75%), depend- target was expressed in relation to the geometric average of 3 ref- ing on the concentration of CtBP and p300, to examine the possi- erence housekeeping genes: TATA binding protein (TBP), succi- ble changes in the correlation between CDH1, VDR, ZEB1 and nate dehydrogenase complex subunit A (SDHA) and ubiquitin C SNAIL. Again, the correlations were studied using the Pearson (UBC), as it has been previously described.20 The relative concen- coefficient or the ANOVA test. The expression levels (T/N) defin- trations of target and reference genes were calculated by inter- ing the 4 groups for each gene were: CtBP, 0.60 (25%), 0.99 2100 PENA~ ET AL.

FIGURE 1 – Effect of CtBP (a)orp300 (b) expression on the correlation between VDR and ZEB1 in tumor tissues. r 5 Pearson correlation coefficient.

(50%) and 1.33 (75%); p300, 0.8 (25%), 1.45 (50%) and 2.33 down-regulation of CDH1 and VDR genes (p 5 0.004 and p < (75%). 0.001, respectively, ANOVA test) (Table III). Likewise, the In all statistical tests two-tailed p values 0.05 were considered expression levels of VDR and CDH1 were directly correlated, but statistically significant. levels of ZEB1 only correlated with VDR (Table III). Statistical analysis was performed using package SPSS version 11.0. Effect of CtBP and p300 on the correlation between ZEB1 and VDR expression Results We examined the effect of CtBP and p300 expression levels on Gene expression data the correlation between ZEB1 and VDR. Tumors were divided into The expression levels of CDH1, VDR, SNAIL, p300 and CtBP 4 groups on the basis of CtBP and p300 expression as described in mRNA in a series of 101 colorectal tumors and their normal mu- Material and Methods: CtBP-0 (T/N values lower than 0.60), cosa counterparts were measured by real time (RT)-PCR as CtBP-1 (from 0.60 to 0.99 T/N values), CtBP-2 (from 0.99 to 1.33 described in Material and Methods. The medians, the minimum T/N values) and CtBP-3 (T/N values higher than 1.33); p300-0 and the maximum values of gene expression are listed in Table II. (T/N values lower than 0.8), p300-1 (from 0.8 to 1.45 T/N values), SNAIL mRNA expression was detected in 62 of the 101 patients p300-2 (from 1.45 to 2.33 T/N values) and p300-3 (T/N values (61.4%). higher than 2.33). The correlation between ZEB1 and VDR was In a previous study, to validate these mRNA results and exam- not affected by the expression of CtBP (Fig. 1a). However, when ine whether E-cadherin protein expression in tumors cells was ho- VDR and ZEB1 were studied with regard to p300, we observed an mogeneous, we performed immunohistochemical analyses.21 We increase of the Pearson correlation coefficient between expression concluded that no significant changes in E-cadherin expression in levels of ZEB1 and VDR associated to p300 increase: 20.16 (p 5 different tumor regions were detected and that, there was a strict 0.938) for p300-0 group and 0.38 (p 5 0.070) for p300-3 group correlation between the CDH1 RNA and protein expression (Fig. 1b). results.21 Moreover, we also determined that deregulation of SNAIL and ZEB1 expression in tumor sample is due to a deregulation of these genes in the carcinoma cells and not in the mesenchymal cells.21 FIGURE 2 – Effect of CtBP (a)orp300 (b) expression on the corre- lation between E-cadherin and ZEB1 in tumor tissues. r 5 Pearson Correlations between ZEB1, SNAIL, CDH1 and VDR correlation coefficient. genes in colon tumors FIGURE 3 – Association between SNAIL and CDH1 expression 19,21 regarding CtBP (a)andp300 (b) expression. The graphs show the quar- As described in previous studies, the analysis of 101 pa- tiles 25, 50 and 75, values lower than 1.5 box lengths and the outliers tients showed an association between SNAIL expression and (O) of CDH1 expression. p values was calculated by ANOVA test. FIGURE 2.

FIGURE 3. 2102 PENA~ ET AL.

FIGURE 4 – Association between SNAIL and VDR expression regarding CtBP (a) and p300 (b) expression. The graphs show the quartiles 25, 50 and 75, values lower than 1.5 box lengths, the outliers (O) and the extremes (*)ofVDR expression. p values was calculated by ANOVA test.

Effect of CtBP and p300 on the inverse correlation high. Both correlations were near to statistical significance. How- between ZEB1 and CDH1 expression ever, the correlations between SNAIL and the down-regulation of Tumors with high CtBP expression present a better correlation CDH1 and VDR genes were lost when the levels of both cofactors between CDH1 and ZEB1 than the tumors with low CtBP expres- increase. sion. In this case, we observed an increase of the Pearson correla- It has been suggested that the mechanism by which ZEB1 acti- tion coefficient between ZEB1 and CDH1, but with negative sign vates VDR22 and ovalbumin30 involves recruitment of such coacti- due to the inverse correlation, related to CtBP increase. The Pear- vators as p300 or P/CAF and displacement of CtBP.32 Our results- son correlation coefficient between ZEB1 and CDH1 was 20.19 showing a higher correlation between ZEB1 and VDR in tumors in the CtBP-0 group (p 5 0.365) and 20.391 in the CtBP-3 group with p300 expression support this hypothesis (Pearson correlation (p 5 0.053) (Fig. 2a). coefficient in the p300-3 group was 0.38, regard to 0.23 in the In contrast, p300 expression did not affect the correlation complete series). Despite the statistical significance in the com- between ZEB1 and CDH1 (Fig. 2b). plete series between ZEB1 and VDR, the Pearson correlation coef- ficient is low (0.23), suggesting that it could be a random event. Effect of CtBP and p300 on the association between SNAIL The down-regulation of CDH1 by ZEB1 reported elsewhere9,18 expression and CDH1 or VDR down-regulation was not found in our study in the whole series. We previously With the exception of the CtBP-3 group, the association reported the loss of the association between overexpression of 21 between SNAIL expression in the tumor and down-regulation of SNAIL and down-regulation of CDH1 at high levels of ZEB. Fur- CDH1 and VDR was lost in tumor with high expression of CtBP thermore, a trend toward statistical significance was observed in or p300 (Figs. 3 and 4). the association between ZEB1 up-regulation and CDH1 down-reg- ulation in patients without SNAIL expression.21 It has been Discussion reported that SNAIL and ZEB1 recognize the same E-boxes in CDH1 and other target genes.2 Therefore, we proposed a model in In this study, we show that levels of coregulators CtBP or p300 which Either SNAIL or ZEB1 could alternatively bind to CDH1 affect the correlation between the expression of ZEB1, SNAIL, E-boxes and repress the expression of this gene. We also sug- CDH1 and VDR genes in human colon tumors. The direct correla- gested that variations in the levels of cofactors could modulate tion between ZEB1 and VDR was stronger when the levels of the ZEB1 activity.21 The results shown here support this model p300 were higher. Expression of ZEB1 correlated because overexpression of ZEB1 and down-regulation of CDH1 inversely with CDH1 only when the corepressor CtBP levels were correlated in tumors with high CtBP expression. CORRELATIONS BETWEEN EMT GENES REGARDING p300 AND CtBP 2103 It has recently been proposed that p300 could play a role in pro- of AIB1, a member of the SRC-1 family of nuclear receptors, may moting CDH1 expression in epithelial breast cells.39 Our data in contribute to the development of steroid-dependent cancers.40 colon cancer tumors did not reveal any association with p300 Again, the coactivator ARA70, an , enhances expression levels and the correlation between ZEB1 and CDH1, transcription of androgen-responsive genes in prostate cancer.41,42 since independently of p300 expression levels no correlation However, this is the first study to show that cofactor levels could between ZEB1 and CDH1 was observed. Perhaps the p300-medi- affect the progression of colorectal cancer. ated activation of CDH1 expression described by Liu et al. was in- Together, our results show that CtBP and p300 levels are impor- dependent of ZEB1 expression. tant factors in the control of the expression of genes crucial for the Our results also indicate that CtBP is not involved in the repres- EMT, and therefore, for tumor progression, at least in human co- sion of CDH1 by SNAIL, which is consistent with the lack of lon cancer. More studies should be performed in large series and binding sites for CtBP on the mammalian forms of this factor. in other types of cancer to verify this hypothesis. Therefore, the mechanism of action of SNAIL may have changed during evolution, and the function of CtBP-repression may now be performed by ZEB1 and 2. Acknowledgements Other authors have reported that variations in cofactors levels We thank R. Rycroft for help with the English manuscript and affect the development of cancer. For example, altered expression M.E. Gomez for selection of tumor samples.

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