Oncogene (2011) 30, 3979–3984 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc SHORT COMMUNICATION E2F1 suppresses Wnt/b-catenin activity through transactivation of b-catenin interacting ICAT

ZWu1, S Zheng1,ZLi1, J Tan1 and Q Yu1,2

1Department of Cancer Biology and Pharmacology, Genome Institute of Singapore, A*Star (Agency for Science, Technology and Research), Biopolis, Singapore and 2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Deregulation of the pRb/ or Wnt/b-catenin pathway 2004; Clevers, 2006; Cadigan, 2008; Klaus and Birchmeier, occurs frequently in human cancers, which is often 2008). On the other hand, genetic inactivation of RB associated with inappropriate cell proliferation. Although pathway due to RB mutation or alterations in other the oncogenic roles of pRb/E2F1 and Wnt/b-catenin upstream regulators (such as CyclinD1, Cdk4 or ) pathways have been well studied, the functional interac- may lead to aberrant activation of another transcription tion between the two pathways has only recently been factor E2F1, leading to deregulation of Rb/E2F1 in a characterized. In particular, E2F1 has been recently variety of human cancers (Sherr and McCormick, 2002). reported to negatively regulate Wnt/b-catenin activity in Unlike Wnt/b-catenin signaling, E2F1 is also equipped human colorectal cancers, though the mechanism under- with an ability to induce , suggesting a potential lying this regulation is not fully understood. Here we tumor suppressor function of E2F1. Obviously, the para- provide evidence that b-catenin interacting protein 1 doxical behavior of E2F1 as an oncogene or as a tumor (CTNNBIP1), also known as ICAT (inhibitor of suppressor is operated in a context-dependent manner. b-catenin and TCF4), functions as a crucial node to Compared with other human cancers, a long-standing mediate the cross talk between E2F1 and b-catenin puzzle in most colorectal cancers is the observation that signaling. We show that ICAT is a direct transcriptional while Wnt/b-catenin signaling is activated, E2F1 activity target of E2F1, and that activation of ICAT by E2F1 is is kept at a low level as there are rare mutations in Rb, required for E2F1 to inhibit b-catenin activity. This study as opposed to some 30% inactivation or mutation rate provides a mechanistic insight into the antagonistic of Rb in other human cancers (Nevins, 2001). Moreover, interaction between E2F1 and b-catenin signaling. instead of acting as an oncogene, E2F1 in human Oncogene (2011) 30, 3979–3984; doi:10.1038/onc.2011.129; colorectal cancers may function as a tumor suppressor published online 2 May 2011 and the lower level of E2F1 expression is more correlated with a poor disease outcome (Bramis et al., Keywords: E2F1; Wnt/b-catenin; ICAT; cancer cells 2004). Recently, E2F1 activity in colorectal cancer was reported to be inhibited by CDK8 (Morris et al., 2008), an oncogene amplified in 50% of colorectal cancers (Firestein et al., 2008), leading to activation of b-catenin Introduction activity (Morris et al., 2008). These studies reveal a cross talk between the two critical signaling pathways in colo- Wnt/b-catenin signaling regulates the stability and rectal cancers and further support a tumor suppressor subcellular localization of b-catenin role of E2F1 in colorectal cancers by negatively regulat- and therefore b-catenin-dependent expression ing b-catenin pathway (Bernards, 2008). Mechanistically, (Logan and Nusse, 2004; Clevers, 2006; Polakis, 2007; several E2F1 targets such as Axin1/2 and SIAH1 have Klaus and Birchmeier, 2008). Deregulation of the Wnt/ been implicated in this process (Firestein et al., 2008; b-catenin pathway has been implicated in human Morris et al., 2008; Xie, 2009). In this study, we seek to malignance, owing to the genetic or epigenetic altera- further understand the mechanistic insights into E2F1 tions in negative or positive regulators of Wnt/b-catenin regulation of Wnt/b-catenin activity and identify b-catenin components, which leads to aberrant activation of its interacting protein 1 (CTNNBIP1), also known as ICAT target , such as c- and CyclinD1, promoting (inhibitor of b-catenin and TCF4), as a key E2F1 target cell growth, tissue invasion or metastasis (Moon et al., mediating this effect of E2F1.

Correspondence: Dr Q Yu, Department of Cancer Biology and Pharmacology, Genome Institute of Singapore, A*Star (Agency for Results and discussion Science, Technology and Research), Genome, #02-01, 60 Biopolis Street, 138672, Singapore. E-mail: [email protected] E2F1 regulates CTNNBIP1 (ICAT) expression Received 6 February 2011; revised 7 March 2011; accepted 15 March To identify the E2F1 targets that are responsible for 2011; published online 2 May 2011 E2F1-mediated inhibition of Wnt/b-catenin signaling, E2F1 suppresses Wnt/b-catenin activity through ICAT ZWuet al 3980 we used human osteosarcoma Saos-2 cells that express cells resulted in significant induction (X3 fold) of an inducible ER (estrogen )–E2F1 fusion protein. many well-known E2F1 target genes, including CCNA1, In this system, E2F1, which is fused to the hormone- CCNE1/2, BCL2L11, APAF-1, CASP3, CASP9 and binding domain of human , moves EZH2 (Bracken et al., 2003; Zhao et al., 2005; Iaquinta from the cytoplasm to the nucleus upon addition of and Lees, 2007; Polager and Ginsberg, 2008) in a time- estrogen receptor ligand 4-hydroxytamoxifen (hereafter dependent manner. Consistent with an inhibitory role referred to as OHT) and activates the expression of its of E2F1 on Wnt/b-catenin signaling, we observed the target genes (Vigo et al., 1999). To identify E2F1-regulated downregulation of a number of Wnt/b-catenin target genes, we used the Illumina Huamn Ref-8_V2 Sentrix genes, including CCND1, c-MYC, MMP7, BMP4, BeadChip (Illumina, San Diego, CA, USA) to compare ACCN1 and IL8 (Figure 1a). Among OHT-responsive the transcriptional profile between Saos-2 cells expres- genes, we noticed that CTNNBIP1, which encodes a sing ER-E2F1 and the empty vector (pBabe) that were b-catenin-interacting protein (ICAT), was markedly treated with OHT for 8, 24, or 48 h. As shown in a gene induced in an E2F1-dependent manner (Figure 1a). expression heatmap of representative E2F1-responsive ICAT has been previously reported to inhibit the genes (Figure 1a), OHT treatment of Saos-2 ER-E2F1 interaction of b-catenin with TCF4, leading to the

a b d pBabe ER-E2F1

OHT 0 8 24 48 0 8 24 48 h APAF-1 10.0 Saos-2 Saos-2 HCT116 7.5 CASP3 10.0 SIRT1 CCNE1 7.5 CCND3 ICAT 7.5 CCNE1 SIAH1 5.0 mRNA level GAB2 5.0 mRNA level 5.0 MAP3K5 ICAT ICAT EZH2 2.5 2.5 BCL2L11 2.5 CCNA1 Relative Relative Relative mRNA expression CCNE2 0.0 0.0 0.0 DIABLO OHT -+ -+ -+ -+ -+ -+ CHX - - - - + + - - CHX - - - - + + - - CASP9 OHT - + - + - + - + OHT - + - + - + - + AXIN1 pBabe ER-E2F1 WNT1 pBabe ER-E2F1 E132 pBabe ER-E2F1 E132 WNT8B U Up-regulated genes AXIN2 c e WNT4 1.5 ICAT Saos2 ER-E2F1 Saos-2 HCT116 WNT3A WNT8A SIAH1 1.0 WNT9B ACCN1 OHT - + - + - + OHT - + - + - + BMP4 ENC1 0.5 CyclinE1 CyclinE1 ETS2 c-MYC ICAT ICAT genes CCND1 Relativeexpressionm mRNA β-Actin β-Actin

MMP7 Down-regulated 0.0 -+ -+ IL8 OHT c-MYC CCND1 -3.0 0 3.0 Figure 1 Ectopic E2F1 upregulates ICAT expression. (a) heatmap showing the representative genes that are responsive to E2F1 activation in Saos-2 cells expressing pBabe (vector control) or ER-E2F1. Saos-2 cells infected with a retrovirus expressing ER-E2F1 or an empty vector (pBabe) were treated with OHT (300 nM) for the indicated time points. Total RNA was extracted using TRizol (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcription was performed using an RNA Amplification kit (Ambion, Austin, TX, USA). The microarray hybridization was performed using the Illumina Gene Expression Sentrix BeadChip HumanRef-8_V2 (Illumina, San Diego, CA, USA), and data analysis was performed and viewed using GeneSpring software from Agilent Technologies (Agilent, Santa Clara, CA, USA). Red, genes with higher expression levels; green, genes with lower expression levels. (b) ICAT, SIAH1 and CCNE1 mRNA expression levels in Saos-2 ER-E2F1 cells upon E2F1 activation. Saos-2 ER-E2F1 or pBabe control cells were treated with OHT (300 nM) for 24 h and the total RNA was isolated as in (a). Quantitative real-time PCR (qRT–PCR) was performed on a PRISM 7900 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA) using TaqMan probes of ICAT, SIAH1 and CCNE1 (Applied Biosystems). Samples were normalized to the levels of 18S ribosomal RNA. (c) Expression levels of CCND1 and c-MYC, two key Wnt/b-catenin target genes, were downregulated in Saos-2 ER-E2F1 cells upon E2F1 activation. Cell treatment and qRT–PCR were conducted as in (a). (d) ICAT expression levels were determined in pBabe, ER-E2F1 or in mutant ER-E2F1 (E132) systems in both Saos-2 (left) and HCT116 cells in the presence or absence of cycloheximide (10 mg/ml, 8 h). (e) Western blot analysis showing the induction of ICAT and CyclinE1 protein expression in ER-E2F1-expressing Saos-2 (left) or HCT116 (right) cells, but not in pBabe or E132 cells, upon OHT treatment using mouse anti-ICAT (Sigma-Aldrich, St Louis, MO, USA) or anti-CyclinE1 antibody (Santa Cruz, Santa Cruz, CA, USA). b-Actin was used as a loading control.

Oncogene E2F1 suppresses Wnt/b-catenin activity through ICAT ZWuet al 3981 ab 25 ICAT 1.2 CCNE1 E2F1 20 ssion ssion ICAT 0.8 15 IMR90/E1A

10 E2F1 siE2F1 -+ 0.4 ICAT E2F1 5 elative mRNA expre elative mRNAexpre CylinE1 ICAT Re R β-Actin 0 β-Actin 0.0 IMR90 IMR90/E1A NC siE2F1

c 20 d Saos-2- Saos2 15 Serum 0 16 24 h E2F1

10 ICAT cells

CylinE1 5 β-Actin Peercentage ofS phas

0 0 16 24 h Figure 2 Endogenous E2F1 upregulates ICAT expression. (a) Quantitative real-time PCR (qRT–PCR) and western blot showing the expression of ICAT and CCNE1 mRNA and protein levels in IMR90 and IMR90/E1A cells. (b) E2F1 knockdown downregulates ICAT mRNA and protein expressions. IMR90/E1A cells were transfected with negative control or E2F1 siRNA, which was described previously (Wu et al., 2010). Total mRNA and protein were extracted 48 h post-transfection, and the mRNA and protein levels of E2F1 and ICAT were detected by qRT–PCR and western blot, respectively. b-Actin was used as a loading control. (c) Saos-2 cells were serum starved for 48 h and then reentered into the after serum stimulation for the indicated times. The corresponding cell populations in S phase were determined by fluorescence activated cell sorting analysis. (d) Saos-2 cells were treated as in (c) and expression of E2F1, ICAT and CyclinE1 were determined by western blot analysis. b-Actin was used as a loading control. repression of b-catenin–TCF4-mediated transactivation activation of endogenous E2F1 (Nahle et al., 2002). (Tago et al., 2000). Quantitative reverse transcription– Indeed, IMR90/E1A cells express much more CCNE1 PCR validated the array data and showed that E2F1 and ICAT at both transcription and protein levels as activation by OHT induced the expression of ICAT in a compared with control IMR90 cells (Figure 2a). level similar to the bona fide E2F1 target CCNE1 Furthermore, siRNA-mediated knockdown of E2F1 in (Figure 1b), which was accompanied by the reduced IMR90/E1A cells resulted in marked decrease of ICAT expression of b-catenin targets c-MYC and CCND1 mRNA and protein expression (Figure 2b), confirming (Figure 1c). SIAH1, a previously reported E2F1 target the regulation of ICAT expression by the endogenous that can inhibit b-catenin activity as shown in lung E2F1. To further substantiate this conclusion, we cancer H1299 cells (Xie, 2009), was only modestly arrested Saos-2 in G0/G1 phase by serum starvation induced by E2F1 activation in our system (Figure 1b). and then released cells into the cell cycle by addition Furthermore, a DNA-binding defective mutant of E2F1 of serum (Figure 2c). Western blot in Figure 2d shows (E132) was unable to induce ICAT expression that while the serum-starved Saos-2 cells reentered (Figure 1d), and E2F1-induced expression of ICAT the cell cycle following the addition of serum, E2F1 mRNA was not affected by the protein synthesis expression was elevated over time, which was accom- inhibitor cycloheximide, as demonstrated in both Saos- panied by increased expression of cyclinE1 and ICAT 2 ER-E2F1 and HCT116 ER-E2F1 cells (Figure 1d). (Figure 2d). Taken together, these findings demonstrate These findings suggest that ICAT might be a direct that both the exogenous and endogenous E2F1 regulate target of E2F1. Finally, western blot analysis confirmed ICAT expression. the induction of ICAT protein expression by E2F1, but not by E132, in ER-E2F1-expressing Saos-2 and ICAT is a direct binding target of E2F1 HCT116 cells (Figure 1e). Sequence analysis identified three putative E2F1-bind- We next sought to investigate whether ICAT expres- ing sites (À1769/À1762, À402/À395 and À234/À226) in sion is regulated by endogenous E2F1. To this end, we the proximal region of the ICAT promoter (Figure 3a). first compared the expression levels of ICAT between This encourages a hypothesis that E2F1 may activate the human lung fibroblast IMR90 cells and IMR90 cells ICAT transcription by binding to its promoter. To test transformed by the oncoprotein E1A. E1A is known to this, three ICAT promoter fragments were isolated and inactivate Rb family members, which leads to the subcloned into a luciferase reporter plasmid and their

Oncogene E2F1 suppresses Wnt/b-catenin activity through ICAT ZWuet al 3982 response to ectopic E2F1 expression was examined. The transcription start site). The results show that upon results show that ICAT promoter fragment containing OHT treatment E2F1 binds strongly to the ICAT core two putative E2F1-binding sites within the genomic promoter region, which was not seen in pBabe or E132 region of À500/ þ 56 (fragment 1, F1) can be markedly cells (Figure 3c). By contrast, no enrichment of E2F1 activated by E2F1, but not by E132, in a dose- was found in the region containing the distal E2F1- dependent manner (Figure 3b). Of note, this activation binding site. In view of these results, we conclude that of ICAT promoter seemed to be more robust than the ICAT is a direct transcriptional target of E2F1. Bim promoter, which was used here as a positive control (Tan et al., 2006). Extension of the promoter region to include the distal E2F1-binding site (F2, À2000/ þ 56) ICAT induction is required for the E2F1-mediated did not seem to provide additional response to E2F1, inhibition of b-catenin activity indicating that the core promoter region between À500 To determine whether ICAT induction by E2F1 is func- and þ 56 is sufficient to give rise to a full E2F1 tionally required for E2F1-mediated inhibition of b-catenin response. The upstream promoter region between À2000 activity, we performed a series of experiments to examine and À1000 (F3) did not respond to E2F1 (Figure 3b). the effect of ICAT on the b-catenin–TCF4 reporter activity We next determined whether E2F1 can directly bind in the context of E2F1. It is well known that colon cancer to the ICAT gene promoter. We performed chromatin cells express constitutively activated b-catenin due to either immunoprecipitation assay using anti-E2F1 antibody in adenomatous polyposis coli (APC) or b-catenin mutations Saos-2 ER-E2F1, E132 mutant or pBabe cells, followed (Morris et al., 2008). In both colon cancer HCT116 and by quantitative PCR using primer sets flanking the SW480 cells, ectopic expression of E2F1 or ICAT results putative E2F1-binding sites in the core promoter region in marked inhibition of b-catenin/TCF4 activity, as (B500 bp upstream of the transcription start site) and determined by TCF4-dependent luciferase reporter the distal E2F1 site (B1.76 kb upstream of the activity (Figure 4a). A further dose–response analysis

a +1 Exon1

Luc ICAT/F1 (-500/+56)

Luc ICAT/F2 (-2000/+56) Luc ICAT/F3 (-2000/-1000)

E2F binding sites

b HCT116 c Saos-2 100 5 pGL-3 basic pGL3 basic pGL3-Bim pGL3-ICAT/F1 80 4 activity pGL3- ICAT/F1 IgG-ChIP E2F1-ChIP 60 pGL3-ICAT/F2 3 pGL3-ICAT/F3 40 2 Fold enrichment 20 1 Relative luciferase

0 0 0 50 100 0 50 100 (ng) OHT - + - + - + - + - + - + - + - +

pcDNA4-E2F1 pcDNA4-E132 pBabe ER-E2F1 E132 ER-E2F1 genomic location (-420/-200) (-1800/-1600) Figure 3 ICAT is a direct target of E2F1. (a) Schematic representation of the human ICAT promoter. Putative E2F-binding sites were predicted using the online TFSEACH program (www.cbrc.jp/research/db/TFSEARCH.html). Three promoter regions as indicated were cloned into pGL3 reporter plasmid. (b) Promoter luciferase reporter assay showing that E2F1 activates the ICAT gene promoter. HCT116 cells were transfected with empty vector pcDNA4, pcDNA4-E2F1 (50 or 100 ng) or a mutant E2F1 expression plasmid, pcDNA4-E132 (50 or 100 ng), together with pGL3 promoter luciferase construct pGL3 (100 ng, Promega, Madison, WI, USA) containing the indicated ICAT promoter regions as shown in (a). Relative luciferase activities were measured 48 h after transfection. Results were presented as fold induction after normalization to Renilla luciferase activity. Bim promoter (pGL3-Bim) that contains the predicted E2F-binding sites described previously was used as a positive control (Zhao et al., 2005). All experiments were performed at least twice in triplicate. (c) Chromatin immunoprecipitation analysis of the physical association between E2F1 and the ICAT promoter region in vivo. Saos-2 cells infected with an empty retrovirus (pBabe), or a retrovirus expressing either wild-type E2F1 (ER-E2F1) or a mutant ER-E2F1 (E132) were left untreated (À) or treated with OHT (300 nM) for 24 h. Crosslinked protein–chromatin complexes were immunoprecipitated by using antibodies against E2F1(c-20, Santa Cruz) or a non-specific IgG control (sc-2027, Santa Cruz) as described previously (Wu et al., 2010). The immunoprecipitated DNA was quantitated by real-time PCR using the PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems). The enrichment of specific genomic regions was assessed relative to the input DNA (mean±s.d. of triplicate measurements).

Oncogene E2F1 suppresses Wnt/b-catenin activity through ICAT ZWuet al 3983 of ectopic E2F1 or ICAT in HCT116 and SW480 cells cells. As shown in Figure 4c, treatment with GSK-3b showed that ICAT is a much more robust inhibitor of inhibitor LY2119301 induced a dramatic activation of b-catenin/TCF4 activity than E2F1 (Figure 4b), indicat- b-catenin activity in these cells, which was completely ing that ICAT might be a key E2F1 target mediating abolished by ectopic expression of E2F1 or ICAT this effect. Furthermore, as b-catenin is negatively (Figure 4c). regulated by GSK-3b, which can be relieved by applying In addition to ICAT, the array data shows that GSK-3b inhibitor LY2119301, we next tested whether E2F1 also induces the expression of AXIN1, AXIN2 and ectopic E2F1 or ICAT can also abolish GSK-3b SIAH1, which might also act as negative regulators of inhibitor-activated b-catenin in Saos-2 and HEK293 b-catenin activity (Morris et al., 2008; Xie et al., 2009).

a b 40 750 40 750

30 HCT116 500 SW480 30 HCT116 SW480 500 20 20 250 10 250 10 Relative luciferase activty 0 Relative luciferase activity 0 Relative luciferase activity

-++ - Realative luciferase activity pcDNA4-E2F1 pcDNA4-E2F1 -+ - 0 0 pcDNA4-ICAT -- + pcDNA4-ICAT --+ 0 50 200 400 10 50 (ng) 0 50 200 400 10 50 (ng)

E2F1 E2F1 pcDNA4-E2F1 pcDNA4-ICAT pcDNA4-E2F1 pcDNA4-ICAT ICAT ICAT β-Actin β-Actin d e

120 250 100 SW480 200 Saos-2 ER-E2F1 80 150 60 c 100 40 75 400 50 20 320 60 Relative luciferase activity 0 Saos-2 HEK293 Relative luciderase activity 0 45 240 GSKi + + + + pcDNA4-E2F1 - + + + + - + + + 30 160 OHT siAXIN2 --+ -- siAXIN2 - - + - 15 80 siICAT ---#1 #2 siICAT#1 ---+ 0 0 Relative luciferase activity Relative luciferase activity AXIN2 AXIN2 GSKi -+ -+ -+ GSKi -+ -+ -+ pcDNA4-ICATp - - + + - - pcDNA4-ICAT - - + + - - ICAT ICAT ----+ + pcDNA4-E2F1 pcDNA4-E2F1 ----+ + Active β-catenin Active β-catenin E2F1 E2F1 Total β-catenin Total β-catenin ICAT ICAT β-Actin β-Actin β-Actin β-Actin

Figure 4 ICAT induction is required for the E2F1-mediated inhibition of b-catenin activity. (a) Ectopic E2F1 or ICAT represses constitutively activated b-catenin activity in colorectal cancer cells. The human colorectal cancer cell lines HCT116 or SW480 were transfected with 100 ng of pTopFLASH luciferase reporter plasmid, along with 10 ng of Renilla luciferase plasmid as normalization control, in the presence or absence of 50 ng of E2F1 expression plasmid (pcDNA4-E2F1) or ICAT expression plasmid (pcDNA4- ICAT) using Fugene 6 (Roche Applied Science, Indianapolis, IN, USA). The relative luciferase activities were measured 48 h after transfection. Results were represented as fold induction after normalization to Renilla luciferase activity. Western blot (lower panel) was performed using antibodies against E2F1 (Santa Cruz) or ICAT (Sigma-Aldrich) to validate transfection efficiency, b-actin was used as a loading control. (b) Dose–response inhibition of constitutively activated b-catenin activity by ectopic E2F1 or ICAT in HCT116 or SW480 cells. Cells were transfected with 100 ng of pTopFLASH luciferase reporter plasmid, along with 10 ng of Renilla luciferase plasmid as a normalization control in the presence of increasing doses of E2F1 (pcDNA4-E2F1, 0, 50, 200 or 400 ng) or ICAT (pcDNA4-ICAT, 10 or 50 ng). The relative luciferase activities were measured 48 h after transfection as in (a). (c) Saos-2 or HEK293 cells treated with small-molecule GSK3-b inhibitor LY2119301 (GSKi, 1 mM) were transfected with or without 50 ng of E2F1 (pcDNA4-E2F1) or ICAT (pcDNA4-ICAT) together with the reporter plasmids for measuring luciferase activity as above. Western blot (lower panel) was performed using antibodies against E2F1 (Santa Cruz) or ICAT (Sigma-Aldrich) to validate transfection efficiency; b-actin was used as a loading control. (d) SW480 cells were transfected with 100 ng of E2F1 plasmid, with or without AXIN2 or ICAT siRNA (no. 1 or no. 2, respectively) using Lipofectamin RNAiMax (Invitrogen) followed by transfection with 100 ng of pTopFLASH luciferase reporter plasmid. Luciferase activity (upper panel) was measured 48 h after transfection as above. Western blot (lower panel) was performed to validate the AXIN2 or ICAT1 knockdown efficiency and the effect on the active b-catenin (anti-active b-catenin, Cell Signaling, Danvers, MA, USA) or the total b-catenin (anti-total b-catenin, Cell Signaling). b-Actin was used as a loading control. ICAT siRNA no. 1 was obtained from Sigma-Proligo targeting the following sequence: 50-GATATCAACAGCTTCCCTATT-30. ICAT siRNA no. 2 and the non-targeting control siRNA were purchased from Dharmacon (Lafayett, CO, USA). Specific AXIN2 siRNA was described previously (Kim et al., 2009). (e) Saos-2 ER-E2F1 cells transfected with negative control, AXIN2 or ICAT siRNA (no. 1) were treated with small-molecule GSK3-b inhibitor LY2119301 (GSKi, 1 mM), followed by transfection with 100 ng of luciferase reporter plasmids in the presence or absence of OHT (300 nM). pTopFLASH luciferase activity and western blot were conducted as in (d).

Oncogene E2F1 suppresses Wnt/b-catenin activity through ICAT ZWuet al 3984 It is worthwhile to note that the expression levels of indicating that in Saos-2 cells b-catenin activity is AXIN2 and ICAT were induced by OHT as early as 8 h, already repressed by ICAT, probably due to the Rb which was sustained for up to 48 h. This induction loss and thus a higher E2F1 activity in this cell line. kinetic was inversely correlated with the kinetics These findings demonstrate an active role of ICAT, but of CCND1 and c-MYC downregulation (Figure 1a). By not necessarily the AXIN2, in E2F1-mediated inhibition contrast, AXIN1 was not found to be induced by E2F1 in of b-catenin activity. We thus conclude that ICAT is a this system and SIAH1 was only modestly induced at 48 h key transcriptional target of E2F1 that is responsible for (Figures 1a and b). This suggests that the inhibition of the suppression of b-catenin activity. b-catenin signaling following E2F1 activation is more likely In summary, our data demonstrates a mechanistic link to be associated with the induction of AXIN2 or ICAT. between Rb/E2F1 pathway and Wnt/b-catenin through Furthermore, given that ectopic E2F1 can effectively ICAT, an inhibitor of Wnt/b-catenin activity. They abolish GSK-3b inhibitor-induced b-catenin activity, we further support a notion that E2F1 can have a tumor reasoned that the E2F1 target responsible for the suppressor role in colorectal cancer by antagonizing inhibition of b-catenin activity should act downstream Wnt/b-catenin activity. Given the relatively lower of GSK3b, which thus does not favor a role of AXIN2 activity of E2F1 in colorectal cancer (Nevins, 2001), it in this process. To further validate this hypothesis, we suggests that development of therapeutics that can depleted AXIN2 or ICAT and the corresponding effects activate the E2F1-ICAT function might be useful for on E2F1-mediated inhibition of b-catenin activity were colorectal cancer treatment. examined accordingly. In SW480 cells, the inhibitory effects of ectopic E2F1 on active b-catenin, as shown by Conflict of interest western blot (Figure 4d, lower panel), as well as on the TCF4-luciferase reporter activity (Figure 4d, upper The authors declare no conflict of interest. panel), were completely rescued by concomitant knock- down of ICAT, whereas AXIN2 knockdown had no Acknowledgements such effect (Figure 4d). Likewise, in Saos-2 ER-E2F1 cells, ICAT knockdown also abolished the inhibitory We thank Dr Kristian Helin (European Institute of Oncology, effect of E2F1 on GSK-3b inhibitor-induced b-catenin Milan) for the ER-E2F1 plasmids and Dr Claudio Brancolini activity, whereas AXIN2 knockdown only had a modest for providing the IMR90/E1A cells. This work was supported effect (Figure 4e). Intriguingly, ICAT knockdown over- by the Agency for Science, Technology and Research of shot the GSK inhibitor-induced activation of b-catenin, Singapore.

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Oncogene