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¹ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
² ERATO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

	Abstract

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Numerous independent clinical and experimental studies indicate that estrogens confer a protective effect against development of intestinal tumors, however the molecular mechanisms involved remain unclear. Physiological effects of estrogens are predominantly mediated by the action of nuclear estrogen receptors (ERs). A multifunctional protein adenomatous polyposis coli (APC) is a tumor suppressor and thought to act as a gatekeeper in colon tumorigenesis, as loss of function APC mutations trigger the development of colorectal cancer. Here we report that APC physically associates with ERa in the ligand-dependent manner. We have shown in the endogenous setting that the ligand-activated ERa recruits APC to the promoters in ER target genes and that increased levels of ER-dependent recruitment of APC enhances the ER transactivation through stimulation of histone acetylation. Found in majority of human colon tumors APC truncation mutants lost the ability to interact with ER. Thus, here we present the first evidence of a functional interaction between APC and ER that may be accounted for a tumor protective action of estrogens.

	Introduction

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Estrogens control a wide range of vital physiological processes. Pleiotropic effects of estrogens are mediated mainly by the action of estrogen receptor (ER) and ERβ, two closely related members of the nuclear receptor superfamily of ligand-dependent transcription factors. Ligand-activated ERs directly bind to specific estrogen responsive element (ERE) present in estrogen-sensitive gene promoters. Recent studies of transcriptional complexes associated with EREs revealed high levels of complexity in the dynamics of ER-mediated transactivation. This involves recruitment to the target gene promoter of multiple co-factor complexes, composition of which determines an integral outcome of the receptor regulatory action (Yanagisawa et al. 2002; Ohtake et al. 2003; Kato et al. 2005; Metivier et al. 2006). ERs themselves can act as transcriptional co-factors via interaction with other DNA binding proteins (Kushner et al. 2000; Tzagarakis-Foster et al. 2002; Cvoro et al. 2006), and also mediate specific estrogen-induced physiological effects through non-genomic mechanisms of action (Bjornstrom & Sjoberg 2005; Vasudevan & Pfaff 2007).

The tumor suppressor adenomatous polyposis coli (APC) is a large multifunctional protein that involves in interaction with various protein complexes and structures in the cytoplasm, nucleus and cell membrane (Kawasaki et al. 2000, 2003, 2007; Bienz 2002; Jimbo et al. 2002; Faux et al. 2004). APC is considered to be a gatekeeper in colorectal tumorigenesis. Most colorectal tumors express truncated mutants of APC that represent about a half of the full-length wild-type APC protein. Mutational inactivation of only one of the two genomic APC alleles (APC^–/+ genotype) significantly compromises APC-dependent cell functions (a phenomenon of haploinsufficiency) and predisposes to development of colon cancer (Kinzler & Vogelstein 1996; Sieber et al. 2002; Venesio et al. 2003; Nathke 2004). Although the main function of APC as a tumor suppressor is thought to associate with its capacity to down-regulate intracellular β-catenin, a nuclear transducer of the canonical Wnt signaling (Bienz 2002; Nathke 2004), a complex network of APC protein interactions and high penetrance of APC mutations (Fodde et al. 2001; Gounari et al. 2005; Akiyama & Kawasaki 2006; Nathke 2006; Strom et al. 2007) suggests that APC is involved in a number of fundamental processes that maintain the normal cell physiology.

An increasing number of epidemiological, clinical and experimental evidence indicates that estrogens confer an overall protection against development of colon tumors. It has been reported that intake of estrogens during hormone replacement therapy lowered the risk of development of colorectal cancer to about 40%, and administration of estrogens reduced the reoccurrence of malignant polyps in patients recovering after surgical removal of colon tumors (Nanda et al. 1999; Slattery et al. 2001; Chlebowski et al. 2004). In studies on animals, male rats experimentally exposed to the carcinogen dimethylhydrazine had twice as high the risk of developing colon cancer and significantly shorter survival times than their female counterparts (Di Leo et al. 2001). Ovariectomy was shown to result in a sharp increase in intestinal adenoma number in the C57BL/6J-Min/+(Min/+) mouse, an animal model of APC-dependent colorectal cancer, while replacement of estradiol (E2) in ovariectomized Min/+ mice reduced tumor numbers to control baseline (Javid et al. 2005). Progressive loss of ER expression has been observed in human colorectal tumors (Foley et al. 2000). Experiments with ER gene knockout in APC-deficient mice have shown that both ER and ERβ act as inhibitory modifiers of APC-dependent colon tumorigenesis (Cho et al. 2007). Taken together, these data indicate that deficient ER signaling may contribute to development of colorectal cancer. However, mechanisms involved in the protective effects of estrogens remain unclear.

In this study we investigated a possibility of functional interaction between ERs and APC and showed that APC may physically associate with ER in the ligand-dependent manner and enhance the estrogen-dependent ERE transactivation.

	Results

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ER and APC associate in the ligand-dependent manner

Human breast cancer MCF7 and human colon cancer HCT116 cells express full-length APC and only MCF7 cells express endogenous ER. Therefore, HCT116 cells were transfected with human ER cDNA expression vector. Cell lysates were prepared from pretreated for 3 h with vehicle or 17β-E2 MCF7 cells and HCT116 cells ectopically expressing ER. Endogenous APC was precipitated with N-terminal APC-specific antibodies that recognize both the wild-type and truncated mutant proteins, and with anti-C-terminal APC antibodies, recognizing only full-length APC. Obtained immunocomplexes were subjected to Western blotting and analyzed by immunostaining with antibodies against ER.

High amounts of ER were detected in immunocomplexes from both MCF7 and HCT116 cells only after treatment with estrogen (Fig. 1A). Similar results were obtained in HeLa and HEK293 cells transfected with human ER cDNA expression vector (data not shown). A weak band of ectopic ER precipitated from HCT116 cells treated with vehicle (Fig. 1A) can be attributed to high levels of expression of the recombinant receptor. Deletion in the ligand-binding domain of ER abrogated its association with APC, and anti-APC antibodies failed to precipitate from HCT116 cells ectopically expressed FLAG-tagged ER(1–461) mutant protein (Fig. 1B).

View larger version (8K): [in this window] [in a new window]   Figure 1  ER associates with the full-length APC in the ligand-dependent manner. A and C, Immunostaining of endogenous (MCF7 cells) and ectopic (HCT116 and SW480 cells) ER in Western blots of APC immunocomplexes precipitated from cells expressing full-length (A) or truncated (C) endogenous APC proteins. B, Deletions in the ligand binding domain abolish interaction of ER with APC. FLAG-ER (1–461) mutant does not co-precipitate with APC from HCT116 cells.

View larger version (51K): [in this window] [in a new window]   Figure 2  Nuclear co-localization of APC and ER in the presence of estrogen. Confocal imaging micrographs of immunovisualized endogenous APC (green) and ectopic FLAG-ER (red) in HCT116 cells treated with vehicle or estradiol alone (E2) or together with leptomycin B (E2 + LMB), as indicated.

Colon cancer-specific truncation mutations abolish interaction of APC with ER

Next we analyzed interaction of ER with truncated APC mutants, which are characteristic to majority of colorectal cancers. Human colon carcinoma SW480 and CACO2 cells express only truncated APC (1–1338) and APC (1–1367) mutants, respectively. Since both colon cancer cell lines do not express detectable levels of endogenous ER, SW480 and CACO2 cells were transfected with human ER expression vector. In contrast to results from the wild-type APC expressing cells and using the same N-terminal APC-specific antibodies, no ER was precipitated with truncated APC from SW480 cells (Fig. 1C) and CACO2 cells (data not shown) even in the presence of ligand and despite high levels of ectopic ER expression.

ER recruits APC to the target gene promoter EREs

ERs exert their genomic regulatory action through binding to the promoter EREs and recruitment of various co-factor protein complexes to modulate the expression of estrogen-sensitive genes. To examine whether APC is recruited to the ERE, we performed ChIP assay of several well-characterized estrogen target genes in human breast cancer MCF7 cells. We found that in the presence of E2 both endogenous ER and APC associated with the ERE, but were undetectable at the exon 1 region, as shown here for the pS2 gene (Fig. 3A). Similar results were obtained for the c-fos and cathepsin D gene EREs (data not shown). Absence of signal for both ER and APC at genomic regions outside the ERE supports the specificity of estrogen-dependent APC recruitment to the ER target genes. This was further confirmed in the time course experiments shown that patterns of APC recruitment at the ERE followed the pattern of ER binding, as presented here for the c-fos gene ERE (Fig. 3B).

View larger version (10K): [in this window] [in a new window]   Figure 3  Estrogen-activated ER recruits APC to the ERE in the endogenous estrogen target gene promoters. ChIP with the indicated antibodies from MCF-7 cells was performed 3 h (A) or at indicated periods of time (B) after addition of E2.

Next, we investigated transcriptional effects of APC on the estrogen-dependent ER transactivation. ERE-dependent luciferase reporter plasmid was co-transfected into MCF7 cells together with either APC cDNA expression vector or empty vector. Increased levels of APC in MCF7 cells significantly enhanced luciferase reporter expression in the presence of ER agonist 17β-E2 and partial agonist tamoxifen. Predictably, over-expression of truncated APC (1–1309) produced no apparent effect on the reporter activity (Fig. 4A).

View larger version (13K): [in this window] [in a new window]   Figure 4  APC enhances the estrogen-dependent ER transactivation through stimulation of histone acetylation. (A) APC enhances ERE-dependent reporter expression. MCF-7 cells were co-transfected with ERE-tk-Luc reporter and either empty, or full-length APC, or truncated APC cDNA expression vector (light, black and grey bars, respectively). Luciferase activity was measured after overnight treatment with vehicle (VEH), estradiol (E2), tamoxifen (TOH), or ICI 182 780 (ICI), as indicated. The data represent the mean ± S.D. of three independent experiments. (B) Levels of the estrogen-dependent recruitment of APC directly correlate with the levels of histone acetylation at the target gene promoter ERE. ChIP with antibodies against ERa (ER), APC, acetylated histone H4 (AcH4), acetylated histone H3 (AcH3), K9-trimethylated (K9metH3) and K4-trimethylated (K4metH3) histone H3, as indicated, was performed from MCF-7 cells transfected with empty (light bars) or APC cDNA expression vector (dark bars) and treated with E2 for 3 h. Semiquantitative real time PCR amplification was performed at the cathepsin D gene ERE. (C) Tethering of transcriptionally active APC fragment at the reporter gene promoter enhances histone acetylation. 293F-pGL4.31 cells with stably incorporated GAL4RE-dependent luciferase gene were transfected with empty pM vector (Control) or pM-APC-C plasmid expressing GAL4DBD-APC (1441–2077) fusion protein (designated as APC-C). PCR at the reporter gene GAL4RE was performed after ChIP with anti K9 acetylated (Acet-K9) and K4-trimethylated (Met3-K4) histone H3 antibodies, as indicated.

Using ChIP assay with real time quantitative PCR amplification, we found that over-expression of APC in MCF7 cells did not affect the binding of ER, however, it resulted in a significant increase in the recruitment of APC, as shown here for the cathepsin D gene ERE (Fig. 4B). This finding further confirms that APC recruitment to the ERE is ER-dependent and indicates that amounts of the endogenous APC available for interaction with ER in these cells are limited and far bellow the saturation level. Significantly, increased recruitment of APC to the ERE resulted in a sharp increase in histone H3 and H4 acetylation and decrease in histone H3 methylation at K9, but we did not observe, however, a marked change in histone H3 methylation at K4, (Fig. 4B). Similar results were obtained for the c-fos and PS2 gene EREs (data not shown).

Estrogen-activated ER has been known to recruit histone acetyltransferases (HATs) at the ERE to activate the target gene expression. Therefore, it remained unclear whether the observed APC-associated increase in histone acetylation at the ERE was caused by a stabilization of ER-HAT complexes at the target promoters, or whether APC itself can independently recruit HATs. To address this question we used 293F-pGL4.31 cells containing stably incorporated GAL4-dependent luciferase reporter transgene (Yokoyama et al. 2008). 293F-pGL4.31 cells we transfected with expression constructs encoding the GAL4DBD (empty pM vector) or GAL4DBD-APC (1441–2077) fusion protein (pM-APC-C construct) that was previously shown to activate GAL4-dependent reporter gene expression (Kouzmenko et al. 2008). In ChIP assay experiments we found that binding of transcriptionally active APC fragment to the reporter gene promoter induced a marked increase in histone H3 acetylation at the GAL4RE (Fig. 4C). Taken together, these data suggest that APC enhances the estrogen-dependent ER transactivation through stimulation of histone acetylation at the EREs.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
Rising number of evidences from different lines of clinical (Nanda et al. 1999; Foley et al. 2000; Slattery et al. 2001; Chlebowski et al. 2004) and experimental (Di Leo et al. 2001; Javid et al. 2005; Cho et al. 2007) studies suggests that deficiency in estrogen signaling may contribute to the APC-dependent development of colorectal tumors. However, a possibility of functional interaction between APC and ER has not been investigated.

In this study we found that APC physically associated with ER in the ligand-dependent manner and that deletions in the receptor LBD abolished this interaction. Over-expression of APC enhanced activity of the ERE-dependent reporter. Significantly, APC truncation mutants from colon cancer cells did not interact with ER and had no apparent effect on the ERE transactivation.

In the endogenous setting, we have shown that the ligand-activated ER recruits APC to the promoters in ER target genes. Increased levels of the ER-dependent recruitment of APC associated with significant increase in the levels of histone H3 and H4 acetylation at the endogenous promoter EREs. This is consistent with our recent finding that nuclear protein complexes of APC contain HAT activity (Kouzmenko et al. 2008). Previously, APC was reported to negatively regulate β-catenin transactivation through tethering a repressor complex and reduction of histone H3 K4 methylation at the Wnt/β-catenin target gene promoters (Sierra et al. 2006). In our experiments, the estrogen-induced recruitment of APC did not affect the levels of histone H3 K4 methylation at the ER target gene promoters. This suggests that nuclear APC may be involved in functionally different transcriptional complexes.

It has been reported that estrogen-dependent transactivation of the pS2 gene requires a generation of DNA double strand break (DSB) followed by recruitment of the DNA-dependent protein kinase repair complex (DNAPK) at the gene promoter ERE, and that inhibition of the DSB DNA repair suppresses the ER transactivation (Ju et al. 2006). Recently we have found that nuclear APC associates with the DNAPK catalytic subunit (DNAPKcs) and that APC-DNAPKcs complex associates with chromatin after induction of DNA DSB and promotes DNA repair (Kouzmenko et al. 2008). These data suggest that APC may enhance the estrogen-dependent transactivation through stimulation of DSB DNA repair and/or histone acetylation at the target gene EREs.

Previously we reported that β-catenin physically associated with ER and enhanced the ER transactivation (Kouzmenko et al. 2004). Several lines of evidence indicate that ER interacts with APC independently of β-catenin: 1) β-catenin associates with ER regardless of the presence of ligand (Kouzmenko et al. 2004), in contrast, APC interacts with ER in the estrogen-dependent manner; 2) LBD deletion ER mutants retained the ability to interact with β-catenin (Kouzmenko et al. 2004), but not with APC; 3) while β-catenin binds to the full-length and truncated APC (Bienz 2002; Schneikert et al. 2007), colon cancer-associated APC mutants do not interact with ER.

Thus, in this study we obtained the first evidence of the ligand-dependent functional interaction between ER and APC. Our data provide a solid ground for further investigation on mechanisms by which estrogen signaling may contribute to or cooperate with the tumor suppression function of APC.

	Experimental procedures

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Immunoprecipitation and immunoblotting

Human colon carcinoma SW480 (APC truncated at aa1338, wild-type β-catenin), CACO2 (APC truncated at aa1367) and HCT 116 (wild-type APC, stabilized β-catenin mutant) cell lines and human breast cancer MCF7 cell line were purchased from the ATCC. Cells were grown in the presence of charcoal-stripped foetal calf serum (CSFCS). Colon cancer cells were transfected with FLAG-hER or FLAG-hER (1–461) expression vector and harvested 28–30 h post-transfection, after treatment for 3 h with vehicle (ethanol) or 10^–8 M 17β-E2 (Sigma). Anti-APC Ali 12–28 (Abcam) or C-20, or F-3 (Santa Cruz Biotechnologies) antibodies were used for immunoprecipitation. Preimmune mice or rabbit serum IgG were used as a control for nonspecific precipitation. Western blots were visualized with Anti-FLAG M2 (Sigma) or anti-ER HC-20 (Santa Cruz Biotechnology) antibodies.

Immunocytochemistry and microscopy

All techniques were performed as previously described (Ito et al. 2004). Confocal imaging micrographs were obtained using Zeiss Confocal Laser Scanning System 510.

Transfection and reporter assay

MCF7 cells grown in OPTI-MEM-5% CSFCS were transfected with 250 ng of ERE-tk-Luc or tk-Luc reporter and 1 ng of pRl (Promega) plasmid (control for transfection efficiency) together with 500 ng of empty (control), or full-length APC, or truncated APC (1–1309) cDNA expression vectors (Kawasaki et al. 2003). Cells were treated for 16–20 h with vehicle or 10^–8 M 17β-E2, or Tamoxifen (Sigma), or ICI 182 780 (Tocris), as indicated. To nullify nonspecific effects on the basal promoter, ERE-tk-Luc reporter activities were normalized against tk-Luc reporter activities from parallel experiments.

Chromatin immunoprecipitation (ChIP) assay

Association of the endogenous ER and APC with ERE of the estrogen target genes in MCF7 cells was analyzed using HC-20 (Santa Cruz Biotechnology) antibody for ER, and Ali 12–28 (Abcam) or F-3 (Santa Cruz Biotechnologies) antibodies for APC. PCR at the gene EREs was performed as previously described (Ohtake et al. 2003; Kouzmenko et al. 2004). As a control for nonspecific chromatin precipitation with these antibodies, several sets of primers were used to amplify gene DNA segments that do not contain ERE sequences. Effects of APC recruitment at the ERE on histone modification were analyzed in MCF7 cells transfected with either empty or APC cDNA expressing vector. Effect of promoter-bound APC on histone acetylation was investigated in 293F-pGL4.31 cells with stably incorporated GAL4-dependent luciferase reporter transgene (Yokoyama et al. 2008). 293F-pGL4.31 cells were transfected with empty pM vector (Clontech) or pM-APC-C plasmid expressing GAL4DBD-APC (1441–2077) fusion protein (Kouzmenko et al. 2008). ChIP was performed with antibodies against trimethyl-K4 histone H3, trimethyl-K9 histone H3, acetyl-K9 histone H3 (Abcam), acetyl-K14 histone H3, acetyl-K9/K18 histone H3, hyperacetylated histone H4 (Upstate). In addition, IgG from normal preimmune mice or rabbit serum were used as a negative control. Real time PCR was performed with SYBR Premix Ex Taq (Takara) and monitored using Smart Cycler II apparatus and software (Cepheid).

	Acknowledgements

 
We thank A. Yokoyama for providing 293F-pGL4.31 cells and members of the Department of Nuclear Signaling for constructive discussions, technical support and camaraderie. This work was funded in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and priority areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to S.K.), and by the Kato Nuclear Complex Project grant from the Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST).

	Footnotes

 
Communicated by: Kohei Miyazono

^* Correspondence: Email: uskato{at}mail.ecc.u-tokyo.ac.jp or alexk{at}iam.u-tokyo.ac.jp

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Received: 7 February 2008
Accepted: 7 April 2008

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