Functional Characterization of the Novel APC N1026S Variant Associated With Attenuated Familial Adenomatous Polyposis

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Functional Characterization of the Novel APC N1026S Variant Associated With Attenuated Familial Adenomatous Polyposis MIREIA MENÉNDEZ,* SARA GONZÁLEZ,* ANTÒNIA OBRADOR–HEVIA,* ANA DOMÍNGUEZ,‡ MARIA JESÚS PUJOL,‡ JOAN VALLS,* NÚRIA CANELA,‡ IGNACIO BLANCO,* ASUNCIÓN TORRES,§ ANTONIO PINEDA–LUCENA,储 VÍCTOR MORENO,* ORIOL BACHS,‡ and GABRIEL CAPELLÁ* *Laboratori de Recerca Translacional, Departament de Prevenció i Control del Càncer, Servei d’Epidemiologia i Registre del Càncer, Institut d’Investigació Biomèdica de Bellvitge—Institut Català d’Oncologia, L’Hospitalet de Llobregat, Barcelona, Spain; ‡Departament de Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Institut d’Investigacions Biomèdiques August Pi Sunyer, Universitat de Barcelona, Barcelona, Spain; §Unitat de Consell Genètic, Fundació Privada Lliga per a la Investigació i Prevenció del Càncer, Reus, Tarragona, Spain; and the 储Laboratorio de Biología Estructural, Departamento de Química Médica, Centro de Investigación Príncipe Felipe, Valencia, Spain

See Neklason DW et al on page 46 in CGH.

Background & Aims: We identified the APC N1026S variant of unknown malignant potential in the adenomatous polyposis coli (APC) gene in a Spanish attenuated familial adenomatous polyposis (AFAP) family. The variant was located in the first of the 4 highly conserved 15-amino acid (AA) repeats within the ␤-catenin union domain. Our aim was to determine its functional relevance to establish its pathogenicity. Methods: N1026S variant was analyzed in 22 members of the AFAP family studied, in 236 sporadic colorectal cancer cases, 203 matched controls, and 205 unrelated familial colorectal cancer cases. To assess its effects on ␤-catenin binding, ␤-catenin/Tcf-4 –mediated transcription and ␤-catenin subcellular distribution we performed affinity chromatography experiments, BIAcore 1000 (BIAcore AB, Uppsala, Sweden) assays, luciferase reporter assays, assessment of c-myc messenger RNA levels, and cell fractionation. Results: N1026S variant cosegregated with the disease in the AFAP family studied. None of the sporadic or familial cases as well as the controls analyzed was positive for the variant. N1026S variant completely precluded ␤-catenin binding to the first 15-AA repeat and diminished it when all four 15-AA repeats were present. Expression of APC N1026S in SW480 and DLD-1 cells did not diminish ␤-catenin/Tcf-4–mediated transcription as effectively as APC wild-type. N1026S did not decrease c-myc transcription in DLD1 cells and nuclear ␤-catenin in SW480 cells as effectively as WT. Conclusions: These findings strongly support a pathogenic role of the APC N1026S variant in the AFAP phenotype, reinforcing the importance of functional characterization of APC variants for genetic counseling.

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lassic familial adenomatous polyposis (FAP) is an autosomal-dominant inherited disease caused by germline mutations in the adenomatous polyposis coli (APC)

tumor-suppressor gene. FAP patients typically develop hundreds to thousands of adenomas in the colon and rectum by the third decade of life. If left untreated, one or more adenomas progress to carcinoma by 45 years of age.1 Truncating germline mutations in the APC gene are responsible for the majority of FAP cases,2 whereas in a minority of cases missense mutations are detected.3 Attenuated FAP (AFAP) patients generally present with a lower number (⬍100) of colorectal adenomas by their fourth decade and have a later age of onset of colorectal cancer (CRC). AFAP is associated mainly with germline mutations in the 5’ and 3’ ends of the APC gene.4 Several studies have shown that in some cases, mainly showing an autosomal-recessive pattern, AFAP is associated with biallelic germline mutations in the base-excision repair gene MYH.5 The APC protein plays an integral role in the Wnt signaling pathway because it binds and down-regulates ␤-catenin.6 The central region of the APC protein contains four 15-amino acid (AA) repeats that bind ␤-catenin.7,8 This region also contains seven 20-AA repeats that trigger ␤-catenin down-regulation through the formation of a protein complex with axin, conductin, and the glycogen synthase kinase-3␤.9 It also has been suggested that APC acts as a nuclear exporter of ␤-catenin,10 inducing its cytoplasmic degradation. Loss of functional APC, usually caused by truncating mutations that remove the ␤-catenin regulatory domain, leads to nuclear accumulation of ␤-catenin.6 There, it binds to members of the Lef/Tcf family of transcription factors, such as Tcf-4,11 activating transcription of target genes such as c-myc,12

Abbreviations used in this paper: AA, amino acid; AFAP, attenuated familial adenomatous polyposis; APC, adenomatous polyposis coli; CRC, colorectal cancer; FAP, familial adenomatous polyposis; GST, glutathione S-transferase; PCR, polymerase chain reaction; WB, western blotting; WT, wild-type. © 2008 by the AGA Institute 0016-5085/08/$34.00 doi:10.1053/j.gastro.2007.10.009

involved in early transformation of the colonic epithelium. In both classic FAP and AFAP, germline missense variants leading to single AA changes in the APC gene, such as I1307K, E1317Q, or D1822V, have been reported, although their contribution to FAP is controversial,3,13–17 limiting their use in genetic counseling.13,17 Cosegregation of the variant with the disease, absence of the variant in the control population, location within the protein, the nature of the AA substitution, and evolutionary conservation of the involved codon are used to estimate their pathogenicity. Unfortunately, these criteria frequently are insufficient to predict the significance of missense variants, reinforcing the need for functional assays. We identified the APC N1026S germline missense variant of unknown malignant potential in one Spanish AFAP family. This variant results in an asparagine to serine change in a highly conserved residue in the central third of the APC protein, a region that mediates ␤-catenin union and that has been shown to be sufficient for its tumor-suppressor activity.18 Also, the crystal structure of the first 15-AA repeat of the APC domain bound to the armadillo region of ␤-catenin8 revealed that the side chain of APC Asn1026 forms a hydrogen bond with ␤-catenin Asp390 and APC Ser1028. We hypothesized that the APC N1026S variant could affect the binding of APC with ␤-catenin, therefore having a dominant-negative effect on APC function. The aim of this study was to determine the biological and functional significance of the APC N1026S variant by investigating the effect of this mutation on the interaction between APC and ␤-catenin and its impact on ␤-catenin/Tcf-4 –mediated transcription and ␤-catenin subcellular localization.

Materials and Methods Subjects The APC N1026S variant was found in the index case of a single Spanish AFAP family. Twenty-two blood relatives of the AFAP family available were studied (Figure 1). In addition, 236 sporadic CRC cases and 203 ageand sex–matched controls were genotyped for the variant.19 A total of 205 familial colorectal patients also were included: 98 classic FAP, 40 AFAP, 13 hereditary nonpolyposis colorectal cancer patients defined by Amsterdam criteria I or II, and 54 hereditary nonpolyposis colorectal cancer–like patients (defined by 1 of these criteria: lack of only 1 Amsterdam criteria; diagnosis of CRC before age 45; or having 2 or more hereditary nonpolyposis colorectal cancer–related tumors).20 All subjects were informed and gave written consent to participate in the study, according to the Helsinki declaration. The local ethical committee cleared the study protocol.

Molecular Analysis Peripheral blood for DNA extraction was obtained from all individuals. The N1026S variant was assessed by

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Figure 1. Pedigree of the Spanish AFAP family in whom the APC N1026S missense variant was identified. The arrow indicates the index case. Ages, cancer type, age at diagnosis, and mutation status are shown. For more details see Supplementary File 1. White, unaffected; red, AFAP; green, CRC; purple, lung cancer (LC); blue, breast cancer (BC); yellow, thyroid cancer (ThC).

means of fluorescent hybridization probe melting curves using the LightCycler 2.0 System (Roche Diagnostics, Mannheim, Germany) (details are available on request). To discard the presence of other mutations in the index case of the AFAP family carrying the variant, we sequenced the whole coding region of APC and MYH genes and exon 3 of CTNNB1.

Plasmids Expression plasmids cytomegalovirus or cytomegalovirus immediate/early promoter (pCMV)-APC, containing full-length APC, and pCMV-APC1309⌬, which encodes a C-terminal–truncated APC mutant, were kindly provided by B. Vogelstein and K. Kinzler (Johns Hopkins University, Baltimore, MD). A vector for highlevel expression of native proteins from the CMV promoter (pcDNA3.1) plasmid (Invitrogen, Carlsbad, CA) was used as a negative control. Reporter plasmids pTOPFLASH and pFOPFLASH, which contain, respectively, 3 copies of the optimal or mutant ␤-catenin/Tcf-binding motif upstream of a firefly luciferase gene, were a generous gift of E. Sancho (Institut de Recerca Biomèdica de Barcelona, Barcelona, Spain). phRL-TK plasmid (Promega, Madison, WI), which contains a HSV thymidine kinase promoter driving a Renilla luciferase gene, was used as a control for transfection efficiency. pGEX-6P3–␤-catenin expression plasmid was kindly provided by M. Duñach (Universitat Autònoma de Barcelona, Barcelona, Spain). A DNA fragment corresponding to all 4 ␤-catenin– binding 15-AA repeats of APC (APC 4 ⫻ 15, AAs 1014 – 1197) was amplified by polymerase chain reaction (PCR) from the entire APC cDNA of plasmid pCMV-APC using primers containing EcoRI and XhoI sites at their 5’-ends, respectively. The 0.57-kilobase amplification fragment

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was digested with EcoRI and XhoI and cloned in the same sites of the pGEX-4T1 plasmid. The absence of mutations in this fragment was verified by DNA sequencing.

Site-Directed Mutagenesis The Ser1026 mutation was introduced into pCMVAPC and pGEX-APC-4 ⫻ 15 using the QuikChange SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA) following the manufacturer’s instructions (primer sequences available on request). After amplification, the nicked plasmid was transformed and sequenced to confirm the mutation.

Expression and Purification of Recombinant Proteins pGEX-6P3–␤-catenin, pGEX-4T1, and pGEX-APC-4 ⫻ 15 (wild-type [WT] and mutant) plasmids were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and purified by affinity chromatography on glutathione-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). GST domain was removed from GST–␤-catenin fusion protein by digestion with PreScission protease according to the manufacturer (Amersham Pharmacia Biotech).

␤-Catenin Binding Analysis by Affinity Chromatography Two chromatography columns were generated by coupling the same amount (3 mg) of WT (Asn1026 LDTPINYSLKYSDEQ) (bold face depicts the residue that is mutated) and mutant N1026S (Ser1026 LDTPISYSLKYSDEQ) peptides comprising the first ␤-catenin– binding 15-AA repeat of APC (AAs 1021–1035) to 3.5 mL of BrCN-activated Sepharose 4B (Amersham Pharmacia Biotech). Then, 20 ␮g of purified GST–␤-catenin or GST as a control were incubated with immobilized peptides in the binding buffer: 50 mmol/L KCl, 50 mmol/L HEPES pH 7.6, 1 mmol/L MgCl2, and 1 mmol/L ethylenediaminetetraacetic acid [EDTA] in a final volume of 1 mL for 30 minutes at 4°C. Beads then were washed extensively with binding buffer containing 300 mmol/L KCl. The unbound fraction was collected whereas bound proteins were eluted, first with buffer A (1 mol/L KCl pH 7.6) and then with buffer B (0.1 mol/L acetate and 0.5 mol/L NaCl pH 4). Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed by Western blotting (WB) with specific monoclonal antibodies against ␤-catenin (Transduction Laboratories, Lexington, KY) or GST (Upstate Biotechnology, Inc, Lake Placid, NY). Immunoblots were developed by a chemiluminescence detection system (Pierce, Rockford, IL).

␤-Catenin Binding Analysis by a Surface Plasmon Resonance Biosensor BIAcore 1000 (BIAcore AB, Uppsala, Sweden), a surface plasmon resonance biosensor, was used to co-

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valently immobilize the same amount (0.07 pmoles/mm2; 2800 resonance units) of WT (APC 4 ⫻ 15Asn) or mutant (APC 4 ⫻ 15Ser) GST fusion proteins on a CM5 sensor chip (BIAcore AB) at a flow rate of 10 ␮L/min using the amine coupling method as described by the manufacturer. Two ␮mol/L of purified ␤-catenin without GST in HBS-EP buffer (0.15 mol/L NaCl, 0.01 mol/L HEPES pH 7.4, 3 mmol/L EDTA, and 0.005% surfactant P20) were injected over the sensor surface at a flow rate of 30 ␮L/min. The sensor chip surface was regenerated with 0.05% sodium dodecyl sulfate between experiments. The association, dissociation, and regeneration phases were followed up in real-time. For each binding curve, the response obtained using control surfaces (no protein coupled) was subtracted. Sensograms were analyzed using BIAevaluation Software (v 4.1; BIAcore AB).

Structural Analysis of the Interaction Between the Mutant APC N1026S Peptide and ␤-Catenin The structural analysis of the APC/␤-catenin complex was performed based on the radiograph crystal structure8 deposited at the Protein Data Bank (ID code: 1JPP) for a 15-AA ␤-catenin– binding repeat from APC bound to the armadillo repeat region of ␤-catenin. By using that Protein Data Bank file as an input, the program PyMOL (De-Lano Scientific, San Carlos, CA; http:// www.pymol.org) was used for recreating absent atoms (side chains of residues Lys1030 and Asp1033) and for generating a mutant peptide containing a Ser residue at position 1026. Positional restrained energy minimization for both peptides was performed using the program GROMACS (Groningen Machine for Chemical Simulation, University of Groningen, Groningen, the Netherlands).21 This allowed the initiation of the docking process with energy-minimized structures, particularly for the mutant peptide whose structure was generated de novo. AutoDock (The Scripps Research Institute, La Jolla, CA)22 was used to analyze the structural consequences of this mutation in the interaction with ␤-catenin, generating a set of poses for both complexes. The docking protocol incorporated, during the definition of active torsion angles, the polar contact restraints obtained from the energy-minimization process.

Transient Transfection and Luciferase Reporter Assay SW480 and DLD-1 human colorectal carcinoma cell lines were transfected transiently by using the LipofectAMINE PLUS method (Invitrogen), according to the manufacturer. In luciferase reporter assays, cells were transfected with 0.5 ␮g of the TCF reporter construct (pTOPFLASH or pFOPFLASH), 50 ng of phRL-TK, and 3 ␮g of the expression plasmid pCMV-APC (APC WT, APC 1309⌬, or APC N1026S), or the empty vector pcDNA3.1. Firefly and Renilla luciferase activities were determined with a luminometer using the Dual-Luciferase Reporter

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Real-Time Quantitative Reverse-Transcription PCR Total RNA was isolated from SW480- and DLD1–transfected cells using Trizol reagent (Invitrogen), and APC and c-myc mRNA levels were analyzed 24 hours after transfection. A total of 500 ng of total RNA was reverse transcribed using pdN6 primers. Subsequent real-time qRT-PCR for APC and c-myc amplification was performed in the LightCycler 2.0 System (Roche). ␤-2-microglobulin was used as an internal control for normalization (primer sequences available on request).

Immunocytochemistry and Immunohistochemistry ␤-catenin immunocytochemistry was performed in SW480-transfected cells grown on coverslips. Cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) for 20 minutes, incubated with 3% hydrogen peroxide in water for 10 minutes, and permeabilized using 0.1% Triton X-100/PBS for 15 minutes. After blocking with goat serum for 1 hour, cells were incubated with primary (1:200, mouse anti–␤-catenin) and secondary (Envision anti-mouse; Dako, Hamburg, Germany) antibodies for 2 hours and 30 minutes, respectively. 3,3’Diaminobenzidine (Dako) was used as the chromogen. The protocol was adapted for paraffin-embedded tissues using the same monoclonal antibody. A paired normal adenoma sample of one affected N1026S carrier was analyzed. A sporadic colorectal carcinoma was used as a positive control.

Cell Fractionation SW480-transfected cells were resuspended in 100 ␮L of buffer C (250 mmol/L sucrose, 10 mmol/L Tris pH 7.5, 150 mmol/L NaCl, and 5 mmol/L EDTA) and lysed by passing 30 times through a 23-gauge needle. Cell lysates then were centrifuged at 5000 rpm for 5 minutes. After removal of the supernatant (cytoplasmic fraction), the pellet (nuclear fraction) first was washed 3 times with 100 ␮L of buffer C, then resuspended with 50 ␮L of buffer C and 0.1% Triton X-100, and finally sonicated 3 times for 10 seconds to shear DNA. Cytoplasmic and nuclear fractions were tested for purity by WB using lamin B and actin as nuclear and cytoplasmic markers,

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respectively. Then fractions were subjected to WB with specific monoclonal antibody against ␤-catenin.

Statistical Analysis To evaluate differences in ␤-catenin/Tcf-4 –mediated luciferase activity and c-myc mRNA levels between APC WT and APC N1026S we fitted a linear regression model adjusted by the effect of day in which the basal category was APC WT. Data were conveniently log-scaled (base 2) to improve normality. Box plots were used to visualize and interpret the observed effects with respect to pcDNA3.1. Significance was assessed by using the estimated parameters and their standard error from the model. To analyze the differences in ␤-catenin binding between WT and N1026S assessed by BIAcore, we focused on resonance unit maximum values. Because 3 curves were obtained for each condition, a Mann–Whitney test, which is a conservative and nonparametric test, was performed to assess significance. A significance level of .05 was set for P values.

Results N1026S Variant Cosegregated With the Disease in an AFAP Family We identified the novel APC N1026S germline missense variant in 1 of 40 Spanish AFAP families analyzed (Figure 1). The index case (III.10) developed 34 adenomas covering the entire colon during a 10-year follow-up period. His father (II.7) was diagnosed with CRC with 10 adenomas. One paternal uncle (II.3) developed more than 50 adenomas during the follow-up period and eventually underwent subtotal colectomy. The 2 sisters (III.9 and III.11) had accumulated 14 and 19 adenomas, respectively (Supplementary Table 1; supplementary material online at www.gastrojournal.org). No mutation was detected in the rest of the coding region of APC, the MYH gene, and exon 3 of CTNNB1. The N1026S variant cosegregated with the disease in the 9 affected members of the AFAP family studied and it was absent in 6 nonaffected members belonging to the second and third generations, 3 of whom were older than 55 years. In the youngest generation, 4 members are carriers that have been invited to undergo colonoscopy. None of the remaining CRC cases (236 sporadic and 204 familial) or controls (0 of 203) analyzed harbored the mutation.

N1026S Variant Completely Prevents ␤-Catenin Binding to the First 15-AA Repeat To evaluate APC N1026S effects on ␤-catenin binding, we performed affinity chromatography experiments using WT (Asn1026) and mutant (Ser1026) APC peptides (Figure 2). In the Asn1026 column incubated with GST–␤-catenin, we observed GST–␤-catenin in serial eluates (120-kilodalton band) after extensive washing (Figure 3A). However, in the Ser1026 column, all GST– ␤-catenin was collected in washes, indicating that

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Assay System (Promega) 24 hours after transfection. For the remaining analyses, cells transfected exclusively with pCMV and pcDNA3.1 plasmids were used. In SW480transfected cells, APC and ␤-catenin protein levels were assessed by WBs with specific monoclonal antibodies against APC (OP44; Oncogene Research Products, San Diego, CA) and ␤-catenin. As a loading control, actin was analyzed in all samples. For all transfectants, APC messenger RNA (mRNA) quantitative reverse transcriptionPCR was used to confirm that efficiency of transfections was comparable.

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Figure 2. Overview of the ␤-catenin– binding 15-AA repeats domain of the APC protein. Schematic representation and AA sequence of the peptide comprising first 15-AA repeat used in the binding assay and the cloned fragment (APC 4 ⫻ 15) used in the BIAcore assay are shown. APC 4 ⫻ 15 fragment includes the 4 ␤-catenin– binding 15-AA repeats (highlighted in boxes). N1026S variant is shown in bold.

N1026S variant completely precludes ␤-catenin binding in this experimental setting (Figure 3A). When columns were incubated with GST, all GST protein was collected in washes in both columns, indicating that binding of GST–␤-catenin to columns was specific (Figure 3A).

N1026S Variant Diminishes ␤-Catenin Binding to APC 4 ⴛ 15 Next, we performed surface plasmon resonance measurements with BIAcore to analyze differences in ␤-catenin binding to WT (APC 4 ⫻ 15Asn) or mutant (APC 4 ⫻ 15Ser) GST fusion proteins containing all 4 ␤-catenin– binding 15-AA repeats (Figure 2), to evaluate if the variant still prevents ␤-catenin binding when the other three 15-AA repeats are present. A difference of approximately 1490 resonance units (⌬resonance unit maximum) was observed in ␤-catenin binding between WT and mutant proteins, being higher for WT when compared with N1026S (P ⫽ .05) (Figure 3B).

Mutant N1026S Peptide Is Unable to Establish Stable Contacts With ␤-Catenin Once energy-minimized structures were obtained for both peptides, PyMOL was used for analyzing the number of polar contacts (ie, hydrogen bonds). For the WT peptide, the root mean square deviation between the coordinates available from the Protein Data Bank and the GROMACS minimized was only 0.083 Å. While the WT peptide has 4 polar contacts, the N1026S mutant has only 2. The change of Asn1026 for Ser abolishes tertiary contacts between Asn1026 and Ser1028, increasing the internal flexibility of the peptide. A docking strategy was pursued to evaluate the structural consequences of the mutation in the interaction with ␤-catenin as compared with the WT (Figure 4A). Figure 4B and C show an overlap of the clusters obtained during the docking process for the WT and the mutant peptide, respectively. An in-depth analysis of the different poses generated for the mutant peptide showed that the number of contacts with ␤-catenin was clearly lower than in the WT peptide.

Also, the mutation of Asn1026 to Ser precludes the formation of a hydrogen bond with Asp390 in ␤-catenin. Finally, it was observed that, on average, the number of internal polar contacts at the output of AutoDock was of 3.43 for the WT peptide, and of 1.40 for the mutant peptide. These data indicate that, from a structural point of view, the N1026S peptide is unable to establish stable contacts with ␤-catenin, mainly because of the flexibility of the peptide as a consequence of the lack of internal restraints.

N1026S Variant Does Not Diminish ␤-Catenin/Tcf-4 –Mediated Transcription as Effectively as APC WT The effects of APC N1026S expression on ␤-catenin/Tcf-4 –mediated transcription were assessed using a luciferase reporter assay. SW480 and DLD-1 cells were chosen for these studies because they exclusively express an endogenous mutant APC protein (truncated at AAs 1338 and 1427, respectively) that constitutively activates ␤-catenin/Tcf-4 –mediated transcription. Introduction of full-length APC into these cells substantially reduces ␤-catenin levels and down-regulates ␤-catenin/Tcf-4 –mediated transcriptional activity.6,23 ␤-catenin/Tcf-4 –mediated transcription was elevated in SW480 and DLD-1 cells transfected with APC 1309⌬ (Figure 5A). Expression of APC N1026S in SW480 and DLD-1 cells still reduced ␤-catenin/Tcf-4 –mediated transcription, but not as effectively as APC WT (SW480 cells: 7-fold vs 9-fold decrease when compared with pcDNA3.1; P ⫽ .00003; DLD-1 cells: 2.7-fold vs 5.7-fold decrease; P ⫽ .0107) (Figure 5A), in the presence of comparable APC protein and/or mRNA levels (Supplementary Figure 1; supplementary material online at www.gastrojournal.org; and data not shown).

N1026S Variant Moderately Activates c-myc Transcription Because c-myc has been broadly shown to be a target of the ␤-catenin/Tcf-4 pathway involved in deregulation of cell growth and proliferation,12 we evaluated by qRT-PCR whether c-myc up-regulation correlated to the expression of

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Figure 3. (A) Binding of GST–␤-catenin and GST to WT and mutant peptides. Peptides were incubated with 20 ␮g of GST–␤-catenin and GST. Bound (B) and not bound (NB) GST–␤-catenin and GST were affinity-purified and analyzed by WB. In the Asn1026 column we observed GST–␤catenin in eluates. In the Ser1026 column all GST–␤-catenin is collected with washes, indicating that ␤-catenin does not bind to the mutant peptide. The lack of GST in eluates in both columns indicates that binding of GST–␤-catenin to peptides was specific. All experiments were performed in triplicate. PC, pre-column; FT, flow through; W1, first wash; W2, second wash; Wn, last wash; EA, eluate buffer A; EB, eluate buffer B. (B) Sensogram showing the association and dissociation profiles of 2 ␮mol/L ␤-catenin injected over sensor surfaces immobilized with WT (thick line, APC 4 ⫻ 15Asn) or mutant (thin line, APC 4 ⫻ 15Ser) fusion proteins. The response in resonance units was recorded as a function of time. Experiments were performed in triplicate.

APC N1026S in SW480 and DLD-1 cells. In both cells, APC N1026S did not diminish c-myc transcription as effectively as APC WT (Figure 5B), but differences were significant only in DLD-1 cells (P ⫽ .001). These results suggest that the APC N1026S variant moderately activates transcription of one of the most described up-regulated targets of the ␤-catenin/Tcf-4 pathway: c-myc gene.

Effects of N1026S Variant on Nuclear and Cytoplasmic ␤-Catenin Distribution Next we explored whether the moderate up-regulation of ␤-catenin/Tcf-4 –mediated transcription induced by the N1026S variant was associated with changes in the total amount of endogenous cellular ␤-catenin and/or in its

subcellular distribution in transfected SW480 cells. No differences in the total amount of cellular ␤-catenin protein, as assessed by WB, were observed among the distinct transfectants (data not shown). Immunocytochemistry of ␤-catenin in parental SW480 cells revealed an intense cytoplasmic and nuclear staining in the overwhelming majority of cells that precluded meaningful comparisons with the remaining transfectants (data not shown). By using cell fractionation, N1026S resulted in a modest increase in nuclear ␤-catenin associated with a decrease in the cytoplasmic compartment in SW480 cells when compared with APC WT (Figure 6). Finally, an attempt was made to assess by immunohistochemistry whether N1026S was associated with changes in ␤-catenin localization in carrier tissues. No nuclear ␤-cate-

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Figure 5. ␤-catenin/Tcf-4 –mediated transcription and c-myc mRNA levels in SW480- and DLD-1–transfected cells. (A) Box plots representing the log-relative fold change of ␤-catenin/Tcf-4 –mediated transcription with respect to pcDNA3.1. TOPFLASH and FOPFLASH activities were normalized using the Renilla phRL-TK activity. ␤-catenin/Tcf-4 – mediated reporter activity was corrected for nonspecific transcription by the TOP/FOP ratio. Experiments were performed in duplicate or triplicate (11 and 7 replicas, respectively). (B) Box plots representing the log-relative fold change of c-myc mRNA levels with respect to pcDNA3.1. Results show the average of 3 independent experiments performed in duplicate or triplicate (8 and 6 replicas, respectively).

Figure 4. (A) Detail of the interaction between the most populated cluster generated by AutoDock for the WT peptide (green) and the radiograph-calculated structure (yellow) of the same peptide with ␤-catenin (surface representation). The root mean square deviation was of 0.42 Å between both structures, revealing that redocking was successful and that the parameters used were reasonable. (B) The high degree of overlapping of the 7 multiclusters obtained for the WT indicates the convergence of the model in its interaction with ␤-catenin. The close proximity of Asn1026 and Ser1028 explains why the side chains of these 2 residues form a hydrogen bond. (C) No consensus structure for the mutant N1026S peptide in its interaction with ␤-catenin is obtained, probably because of the conformational flexibility induced by the mutation.

Figure 6. Cell fractionation of SW480-transfected cells. Three micrograms of the expression plasmids containing APC WT, APC 1309⌬, and APC N1026S were transfected and 24 hours later, nuclear (N) and cytoplasmic (C) fractions were obtained. The amount of ␤-catenin in each fraction was analyzed by WB. ␤-catenin intensities were corrected with actin for the cytoplasmic fraction and lamin B for the nuclear one. Two independent experiments were performed, and means of intensities and SDs are shown.

nin was detected in the N1026S carrier normal colonic mucosa or adenoma analyzed (data not shown).

Discussion Although missense mutations represent a minority of FAP mutations,3 the difficulty in assessing the functional role of missense APC variants is a challenge in the diagnosis and counseling of FAP and AFAP families. Several missense variants in other genes, such as breast cancer genes (BRCA1) or mismatch repair genes, have been characterized functionally to determine their pathogenicity within familial breast/ovarian cancer or hereditary nonpolyposis colorectal cancer.24 –26 There is a lack of functional assays of APC missense variants, presumably because of the difficulty in designing and developing specific assays depending on the position of the variant along the different functional domains of the APC protein. We identified the novel APC N1026S germline missense variant of unknown malignant potential in a Spanish AFAP family. The variant cosegregated with the disease and was not present in any of the familial and sporadic CRC cases and controls studied. Its location within the critical area of the first 15-AA repeat of the APC gene in the highly conserved ␤-catenin union domain suggested a pathogenic role in the genesis of the observed phenotype. Because this information was not conclusive enough to be used in genetic counseling, we developed several assays to determine its functional significance. We first investigated the effect of the variant on the physical interaction between APC and ␤-catenin. A small mutant peptide encompassing the first ␤-catenin– binding 15-AA repeat of APC, for which the crystal structure was available, and a construct containing the four 15-AA repeats were used. Although N1026S completely precluded ␤-catenin binding to the first 15-AA repeat, we observed a partial decrease in APC 4 ⫻ 15 binding to ␤-catenin, likely to better reflect the whole protein situation. The diminished ␤-catenin binding was in concordance with the structural analysis of the N1026S APC peptide/ ␤-catenin complex. The Ser1026 peptide is predicted to be unable to establish stable contacts with ␤-catenin. The change for Ser precludes the formation of a hydrogen bond with ␤-catenin Asp390 and abolishes tertiary contacts between Asn1026 and Ser1028, increasing the internal flexibility of the peptide. In silico and in vitro observations were confirmed further in experiments using SW480 and DLD-1 cells, which show a constitutively active ␤-catenin/Tcf-4 –mediated transcription that can be reverted by inducing the whole APC WT protein expression.6,23 APC N1026S expression was associated with a lower decrease of ␤-catenin/Tcf-4 – mediated transcription when compared with APC WT in both cells. A modest increase in c-myc activation was

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observed only in DLD-1 cells. The magnitude of these changes is lower than that induced by APC 1309⌬, a truncating mutation usually associated with a severe FAP phenotype. Altogether, our results strongly suggest that the whole APC N1026S protein partially precludes ␤-catenin binding and activates its downstream effectors. APC has been described to regulate ␤-catenin activity by either facilitating its cytoplasmic degradation or promoting its translocation out of the nucleus, 2 distinct but not exclusive ways of modulation. In line with previous observations by Morin et al,27 we did not detect differences among SW480 transfectants in the total amount of endogenous cellular ␤-catenin. The low transfection efficiency obtained is likely to account for the lack of differences observed. Although the limited sensitivity of immunocytochemistry may preclude detection of modest changes, the increased amount of nuclear ␤-catenin, as assessed by cell fractionation, suggests that the APC N1026S protein, as a consequence of its decreased ability to interact with ␤-catenin, may impair nuclear ␤-catenin export. The consistency of binding analysis together with luciferase and c-myc expression results and cell fractionation assays strongly suggests that APC N1026S variant activates ␤-catenin/Tcf-4 –mediated transcription. The limitation associated to every individual assay further reinforces the need for a combined approach to elucidate the pathogenic significance of a given variant. The moderate activation of the ␤-catenin/Tcf-4 –mediated transcription is consistent with the attenuated phenotype observed within the family.28 Previous studies have described a correlation of the attenuated phenotype with the location of APC mutations,29 but not with its molecular nature. Although APC missense mutations are rare in classic FAP phenotype, they may be overrepresented in AFAP cases (González et al, unpublished observations). Thus, the approach used in this study may be performed to characterize those alterations present in AFAP families. The strategy used here to further characterize the significance of the APC N1026S variant also may be useful for other variants that are located within the ␤-catenin binding and/or down-regulation region, such as I1307K, E1317Q, or D1822V, associated to familial and sporadic colorectal tumors.13,15–17 Although for mutations outside the first 15-AA repeat chromatography, affinity experiments will have to be adapted; all mutations located within the APC 4 ⫻ 15 construct can be evaluated with our BIAcore design. In this regard, the same approach has allowed us to show that another APC variant, the L1129S, does not affect ␤-catenin binding, ruling out a putative pathogenic effect (data not shown). In vitro transfection studies could be useful for mutations predicted to affect the ␤-catenin transcription program. Finally, functional characterization of mutations in the oligomerization or

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microtubule-binding domains will need alternative strategies. In conclusion, our findings strongly support a pathogenic role of the APC N1026S variant in the AFAP phenotype. Therefore, the variant should be tested in all at-risk relatives so that they can be advised to adhere to a lifelong screening program,20 reinforcing the importance of functional characterization of APC variants for genetic counseling.

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