flywch1, a novel suppressor of nuclear -catenin, regulates ...signal transduction flywch1, a novel...

Signal Transduction

FLYWCH1, a Novel Suppressor of Nuclearb-Catenin, Regulates Migration and Morphologyin Colorectal CancerBelal A. Muhammad1,2, Sheema Almozyan1, Roya Babaei-Jadidi1, Emenike K. Onyido1,Anas Saadeddin1,3, Seyed Hossein Kashfi1, Bradley Spencer-Dene4,5, Mohammad Ilyas6,Anbarasu Lourdusamy7, Axel Behrens8, and Abdolrahman S. Nateri1

Abstract

Wnt/b-catenin signaling plays a critical role during devel-opment of both normal and malignant colorectal cancertissues. Phosphorylation of b-catenin protein alters its traf-ficking and function. Such conventional allosteric regula-tion usually involves a highly specialized set of molecularinteractions, which may specifically turn on a particular cellphenotype. This study identifies a novel transcription mod-ulator with an FLYWCH/Zn-finger DNA-binding domain,called "FLYWCH1." Using a modified yeast-2-hybrid basedRas-Recruitment system, it is demonstrated that FLYWCH1directly binds to unphosphorylated (nuclear) b-cateninefficiently suppressing the transcriptional activity of Wnt/b-catenin signaling that cannot be rescued by TCF4.FLYWCH1 rearranges the transcriptional activity of b-cate-nin/TCF4 to selectively block the expression of specific

downstream genes associated with colorectal cancer cellmigration and morphology, including ZEB1, EPHA4, andE-cadherin. Accordingly, overexpression of FLYWCH1reduces cell motility and increases cell attachment. Theexpression of FLYWCH1 negatively correlates with theexpression level of ZEB1 and EPHA4 in normal versusprimary and metastatic colorectal cancer tissues in patients.Thus, FLYWCH1 antagonizes b-catenin/TCF4 signaling dur-ing cell polarity/migration in colorectal cancer.

Implications: This study uncovers a new molecular mecha-nism by which FLYWCH1 with a possible tumor suppressiverole represses b-catenin-induced ZEB1 and increases cadherin-mediated cell attachment preventing colorectal cancer metas-tasis. Mol Cancer Res; 16(12); 1977–90. �2018 AACR.

IntroductionActivated Wnt-signaling has significant implications both in

cellular homeostasis and carcinogenesis depending on thecontext of activation and the surrounding microenvironment.Accumulated evidence identified several proteins that interactwith nuclear-b-catenin and antagonize its transcriptional activity

leading to transcriptional repression of Wnt-target genes. Accord-ingly, any alterations of the Wnt/b-catenin pathway throughb-catenin corepressors is often involved in crucial biologicalprocesses ranging from normal development to cancer. Forinstance, Sox9 regulates the progression of the chondrocyte dif-ferentiation pathway during endochondral bone formationthrough inhibition of b-catenin (1), whereas the Wnt-signalingattenuator RUNX3 was proposed to function as a tumor suppres-sor during intestinal tumorigenesis (2). Moreover, expression ofChibby, a nuclear-b-catenin associated antagonist of the Wnt/Wingless pathway, was also linked to embryonic developmentand cancer (3). These findings indicate that b-catenin co-repres-sors function as potential gate-keepers for the activity of b-catenin,ensuring that a proper threshold of b-catenin is achieved before itsinteraction with members of T-cell factor (TCF) and lymphoidenhancer factor (LEF) transcription factors (TCF/LEF).

Furthermore, the paradox of b-catenin/TCF interaction, how-ever, is challenged by identification of new strategies for b-cate-nin-dependent but TCF-independent gene regulation. It has beenreported that b-catenin interacts with Prop-1 rather thanmembersof TCFs to regulate the cell-lineage determination during pituitarygland development (4). b-Catenin was also found to control thepluripotency of embryonic-stem cells through interaction andenhancement of Oct-4 activity independently of TCFs (5). Like-wise, the involvement of several other DNA-binding transcriptionfactors with b-catenin was reported in the literature. Some areenhancing the promoter binding ability of b-catenin and facilitatethe chromatin remodeling at the promoter site of Wnt-targetgenes such as p300/CBP and Brg-1 (6, 7), whereas others are

1Cancer Genetics and Stem Cell Group, Cancer Biology, Division of Cancer andStem Cells, School of Medicine, University of Nottingham, Nottingham, UnitedKingdom. 2Division of Experimental Haematology and Cancer Biology,Cincinnati Children's Hospital Medical Centre, Cincinnati, Ohio. 3Tres CantosMedicines Development Campus, GlaxoSmithKline, Cantos, Madrid, Spain.4Experimental Histopathology Laboratory, the Francis Crick Institute, London,United Kingdom. 5Advanced Cell Diagnostics, School of Medicine, Queen'sMedical Centre, University of Nottingham, Nottingham, United Kingdom.6Molecular Pathology Unit, School of Medicine, University of Nottingham,Nottingham, United Kingdom. 7Children's Brain Tumour Research Centre,School of Medicine, University of Nottingham, Nottingham, United Kingdom.8Adult Stem Cell Laboratory, the Francis Crick Institute, London, UnitedKingdom.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

B.A. Muhammad, S. Almozyan, and R. Babaei-Jadidi are co-first authors.

Corresponding Author: Abdolrahman S. Nateri, Cancer Genetics and Stem CellGroup, Cancer Biology, Division of Cancer and Stem Cells, School of Medicine,University of Nottingham, Nottingham NG7 2UH, UK. Phone: 44-115-8231306;Fax: 44-115-8231137; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-18-0262

�2018 American Association for Cancer Research.

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promoting nuclear localization of b-catenin such as Pygopus (8).Corepressors such as ICAT (inhibitor of b-catenin and TCF4) andp15RS antagonize Wnt-signaling through inhibiting the interac-tion and formation of b-catenin/TCF4 complex (9). Moreover,RUNX3 forms a ternary complex with b-catenin/TCF4 by whichattenuates Wnt-signaling activity (2). Similar to this mechanism,RAR, Chibby, Sox-9, and HIF1a compete with members ofTCF/LEF for binding to b-catenin (1, 3, 10, 11).

Notably, some of the b-catenin-binding transcription factorsincluding KLF4, Glis2/3, HIC1, and Osx belong to the Cys2-His2(C2H2) family of Zinc-finger proteins characterized by havingmultiple C2H2-type Zinc-finger DNA-binding domains (12–15).These proteins could potentially regulate the gene expressionprograms controlled by Wnt/b-catenin-signaling via their inter-action with b-catenin and/or TCFs, disrupting the formation ofb-catenin/TCF-complexes, and subsequently recruiting nuclear-b-catenin to thepromoter of specific target genes independently ofTCFs.However, this viewof nuclear-b-catenin recruitment has notyet been fully explored.

To further delineate the nuclear events of the Wnt-signalingpathway, we used a modified yeast two-hybrid Ras-recruitmentsystem (RRS) using mouse embryonic-cDNA library and identifiedseveral new proteins that bind b-catenin in a GSK-3b phosphory-lation-dependent and/or independent manner. One of the proteinswas FLYWCH1, a conserved nuclear protein containing multipleFLYWCH-type zinc-finger domains with unknown function.FLYWCH motif has first been described and annotated based onthe presence of FLYWCH consensus sequence (F/Y–Xn–L–Xn–F/Y–Xn–WXCX6-12CX17-22HXH; where X indicates any amino acid) inDrosophila (16,17). Inaddition,FLYWCHmotifswerealso identifiedand studied in two more proteins of C. elegans: PEB-1 (18) andFLYWCHtranscription factors; FLH-1,FLH-2, andFLH-3(19).Basedon Drosophila and C. elegans studies, FLYWCHmotifs may functionin protein–protein interactions and serve as DNA-binding domains(16–19). Accordingly, it can be predicted that human FLYWCH1may have similar biological activities. However, no evidence for thepresence of a transactivation-domain within the coding region ofthis protein was detected. To further characterize FLYWCH1, we setout to functionally describe its interaction with b-catenin andexplore its importance and role(s) in colorectal cancer.

Materials and MethodsHuman tissues

A total of 49 colorectal cancers tissues were arrayed on a tissuemicroarray (TMA) block in triplicates by theMolecular PathologyUnit from the consenting patients through the Nottingham tissuebank, as described previously (20).

AntibodiesAntibodies were purchased as follow: b-catenin (610154,

BD-Transduction and 9582; Cell Signaling Technology), ZEB2/SIP1 (H260; Santa Cruz Biotechnology), Vimentin (RV202; SantaCruz Biotechnology), ZEB1 (H-102 and E-20X; Santa CruzBiotechnology), E-cadherin (BD Biosciences), TCF4 (Upstate;05-511), FLYWCH1 (Sigma; HPA040753; Santa Cruz Biotech-nology; V18), GFP (3E6; Invitrogen), Flag-tag (F1804; Sigma),Myc-tag (9E10; Millipore), and b-actin (Abcam).

PlasmidsThe IMAGE-clone of human FLYWCH1-cDNA purchased from

Geneservice (ID: 4839062/AK34; GenBank: 84256). Human

FLYWCH1 gene is composed of 2,151 nucleotides encoding aprotein with 716aa residues called FLYWCH1. The IMAGE-cloneplasmid DNA then digested with several restriction-enzymesand cloned into different vectors, while fused with the MYC-tag,His6, and eGFP. The FLAG-b-cateninWT, FLAG-b-cateninS35A,FLAG-b-cateninS33A, S37A, T41A, S45A, TCF4, human c-jun-promot-er-Luc, TOP/FOP-FLASH-Luc reporters previously described(21, 22). The �1129/þ55 of ZEB1-promoter-Luc was fromDr. M. Saito (Tokyo, Japan), FLAG-GSK-3b-plasmid was fromProf B.W. O'Malley (Houston, TX), and the packaging plasmidswere kindly provided by Dr. D. Bonnet (London, UK). pEGFP-C2was purchased from Clontech, pBluescript-SK (Stratagene),pcDNA3.1 (Invitrogen), pET-His6, pGEX-KT (Addgene), thescrambled piLenti-scrambled control shRNA (scs)-GFP, andpiLenti-FLYWCH1-set-shRNA-GFP (set of 4) from ABM Inc.(https://www.abmgood.com/FLYWCH1-shRNA-Lentivectors-i008000.html).

Modified yeast-2-hybrid based RRSThe RRS uses the growth defective yeast strain cdc25-2, which is

deficient in Ras activity and cannot grow at 37�C (23). Briefly, theyeast cells stably transformed with pMET3-GSK-3b then cotrans-formed with a bait composed of the FLAG-tagged construct ofb-catenin lacking the 7-8 armadillo-domains (DARM) and fusedto oncogenic RasV12 (RasV12-FLAG-b-catenin/DARM). Further-more, the expression of GSK-3b and substrate-phosphorylationdependency is dependent on the presence (promoter-off) andabsence (promoter-on) of methionine in yeast. Thus, the activat-ed-GSK-3b (under the control of a methionine-regulated MET3-promoter) induces phosphorylation of FLAG-b-catenin�DARM.These cells then transformed with mouse embryonic-cDNAlibrary fused to the Src-myristylation signal (Stratagene). Glucoseplates were grown for 5 days at 25�C then plated onto minimalgalactose plates (þ/�methionine) at a restrictive temperature36 to 37�C. Yeast colonies exhibiting efficient methionine-dependent growth isolated and further analyzed.

ISH assayBoth sense and antisense FLYWCH1 RNA-probes generated by

cloning a short nucleotide sequence of human FLYWCH1-cDNAinto a pcDNA3 (details can be provided upon request). Bothprobes transcribed with T7-RNA polymerase using DIG-RNA-labeling mix (Roche) and ISH-assay carried out through the corefacility at the CRUK-LRI as described previously (24).

Immunofluorescence assayFor immunofluorescence (IF), colorectal cancer cell lines were

grown on coverslips, fixed for 30 minute s with 4% paraformal-dehyde and permeabilized with 0.5% Triton-X100 in PBS. Sam-ples were exposed to goat anti-rabbit antibodies conjugated toAlexa Fluor 594 (A11037; Invitrogen) and/or rabbit anti-mouseantibodies conjugated to Alexa Fluor 488 (A11059; Invitrogen).Tetramethylrhodamine-B isothiocyanate (TRITC) conjugatedPhalloidin (P1951; Sigma) used to label actinfilaments accordingto manufacturer's instruction. Finally, slides mounted in Vecta-shield-DAPI medium (Vector Labs) and analyzed by fluorescentmicroscopy.

Transient and stable cell transfectionsHuman colorectal cancers (HCT116, DLD-1, and SW480),

TIG119 fibroblasts, and HEK293T cells maintained in RPMIsupplemented with 10% FBS. Cells transiently transfected using

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Lipofectamine-2000 (Invitrogen). After 48 hours, cells processedfor transcriptional analysis, mRNA, or protein extraction. SW480cells stably knocked-down for FLYWCH1 by lentiviral transduc-tion of piLenti-FLYWCH1-set-shRNA-GFP and control piLenti-scsRNA- (scrambled control shRNA) GFP. Both cells selected in aPuromycin-containingmedium for 2weeks then the resistant cellstrypsinized, counted, and seeded into 96-well plates (1 cell/well).Finally, the resistant colonies derived from a single-seeded cellfurther amplified and expanded. The knocked-down status ofFLYWCH1 validated through RT-PCR and IF analysis. R-spondin1(293T-HA-Rspo1-Fc) and Wnt-3a (293T-HA-L) expressing celllines were a gift from Prof Hans Clevers (Hubrecht, the Nether-lands). R-spondin1/Wnt-3a conditioned media validated by aTOP/FOP-flash luciferase-reporter assay before using it.

Western blot, immunoprecipitation, and chromatin-immunoprecipitation assays

We performed Western blots (WB) and immunoprecipitations(IP) as previously described (25, 26). For in vitro binding assays,cDNAs encoding FLAG-b-catenin, andGFP-FLYWCH1were trans-lated by a T7-in vitro-coupled transcription/translation system(Promega). l-Phosphatase (BioLab) treatment was carried outwith 200 units for 60 minutes at 30�C. Immunocomplexes werecaptured by rotating for 2 to 3 hours with protein-A, Protein-G(Sigma), or secondary precoated Dynal-magnetic beads. Insome cases, conjugated Agarose beads were used. Immunopreci-pitates washed four times with lysis buffer, and SDS-PAGE sep-arated sample proteins then transferred onto PVDF-membranes(Amersham) following standard procedures. Chromatin-immu-noprecipitation (ChIP) analysis performed as described previ-ously (20, 21).

Competition assayRecombinant human b-catenin protein 1 mg/mL (Abcam) was

used to coat a 96-well ELISA plate overnight at 4�C. The plate waswashed three times with PBS-T and then blocked 2 hours at roomtemperature. For the TCF4-detection/calibration curve; after threewashes, TCF4 recombined protein (Autogen Bioclear) added tothe plate (range: 0.0–500 ng) and incubated at 37�C for 6 hours.The plate was washed three times PBS-T and incubated with2 mg/mL of TCF4-FITC (Autogen Bioclear) for 1 hour at roomtemperature. Following washing with PBS-T (three times) 50 mLPBS was added. The level of fluorescent was estimated in afluorescence microplate reader green setting excitation 494 nmand emission 518 nm. For the FLYWCH1-detection/calibrationcurve; after 3� washes, FLWYCH1 recombined protein (Sigma)added to the plate (range: 0.0–500 ng). Plate incubated at 37�Cfor 6 hours, and after three washes with PBS-T, 2 mg/mL ofFLYWCH1-HRP (Sigma) added to the wells and incubated 1 hourat room temperature. The plate washed with PBS-T (3�). Tetra-methylbenzidine (TMB) reagent A and B mixed and 100 mL ofmixed solution transferred to each well and incubated at 37�C for20 minutes. Fifty microliters of TMB stop solution was added toeach well and absorbance read at OD (450 nm) in the microplatereader. The experiment for secondary antibodies interaction havebeen carried out, and therewas no significant difference observed.Next, based on the calibration curve, TCF-4 100 ng/mL andFLWYCH1 80 ng/mL and mixer of both proteins added to wellsalready coated with human b-catenin protein (each triplicate).The plate incubated at 37�C for 6 hours and after three washeswith PBS-T incubated with 2 mg/mL of TCF4-FITC. The plate

incubated for 1 hour at room temperature and washed withPBS-T and added 50 mL PBS and read in fluorescence microplatereader green setting excitation 494 nm and emission 518 nm.

Electrophoretic mobility shift assaysFor studying DNA–protein interactions, we used the LightShift

chemiluminescent electrophoretic mobility shift assays (EMSA)Kit (Thermo Scientific) that uses a non-isotopic method to detectDNA–protein interactions. Biotin end-labeled DNA contains3� of Tcf-DNA-binding site incubated with SW480 nuclear pro-tein extract in the absent and/or present of recombinant TCF4 andFLYWCH1 proteins as outlined above. This reaction is thensubjected to gel electrophoresis on a native polyacrylamide gel.The biotin end-labeled DNA is detected using the streptavidin–horseradish peroxidase conjugate and the chemiluminescentsubstrate according to the manufacturer's protocol (ThermoScientific).

Luciferase assayA firefly reporter gene (TOP-FLASH) driven by three copies

of Tcf-binding elements (ATCAAAG) upstream to TK-minimal-promoter (which is specifically regulated by Wnt/b-catenin sig-naling) was used. Luciferase-activity measured using the Dual-Luciferase system (Promega) as described (20). For statisticalanalysis, the ratio of Firefly-Luc activity was normalized toRenilla-Luc. Data were expressed as fold-induction from threeindependent experiments.

Cell spreading, migration, and wound healing assaysColorectal cancer cells synchronized in G0-phase of the cell

cycle by serum-free starvation for 18 hours before cell motilityassays. Electric cell-substrate impedance-sensing (ECIS) systemwas used for cell attachment and spreading assays in a conflu-ent layer, according to manufacturer's guidelines. The systemwas run for 24 hours after which the quantification data ofcell attachment and spreading was collected. Cell migrationand wound healing assays assay were performed as previouslydescribed (24). All assays were performed at least three times intriplicate.

RT-PCR and quantitative RT-PCR assaysWe extracted total RNA from homogenized primary tumors

and cultured cells using Trizol (Sigma) or RNeasy Purification Kit(Qiagen). Superscript-III first-strand synthesis (Invitrogen) andoligo(dT) primers were used to convert the RNA to cDNA accord-ing to the manufacturer's instructions. qPCR was accomplishedwith SYBR-green (Roche), and the data were analyzed usingLightCycler-480 software (Roche) and primers below.

50-AGGTGACAGCATTGCTTCTG (forward: b-actin)50-AGGGAGACCAAAGCCTTCAT (reverse: b-actin)50-GTCTGTAGGAAGGCACAGCCTGTCG (forward: CDH1)50-AGGACCAGGACTTTGACTTGAGC (reverse: CDH1)50-GCTGGAGGTCTGCGAGGA (forward: CCND1)50-CATCTTAGAGGCCACGAACA (reverse: CCND1)50-AGAAGGTGTGGGCAGAAGAA (forward: CD44)50-AAATGCACCATTTCCTGAGA (reverse: CD44)50-ACCCCAAGATCCTGAAACAG (forward: c-Jun)50-ATCAGGCGCTCCAGCTCG (reverse: c-Jun)50-AAAACCAGCAGCCTCCCGC (forward: c-Myc)50-GGCTGCAGCTCGCTCTGC (reverse: c-Myc)50-GGCAAGCATGAGACTGTGAA (forward: ENFB1)

FLYWCH1/b-Catenin Axis Regulates Cell Migration

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50-ACTCCAAGGTGGCATTGTTC (reverse: ENFB1)50-CTGCTGGATCAACCAGGAAT (forward: ENFB2)50-TCTAGCACAGACGGCAACAG (reverse: ENFB2)50-AGGATTACCCTGTGGTGGTC (forward: EPHB2)50-TACAACGCCACAGCCATAAA (reverse: EPHB2)50-TCGTGGTCATCGCTATCGTCT (forward: EPHB3)50-AAACTCCCGAACAGCCTCATT (reverse: EPHB3)50-CGACAAAGAGCGTTTCATCA (forward: EPHA4)50-GCTTCACCCAAGTGGACATT (reverse: EPHA4)50-GCAAAGGTCGAAGACCAGGA (forward: FLYWCH1)50-TTCCTGGTGTACGAGTCCTT (reverse: FLYWCH1)50-GGAGACCGACCAGGAGAC (forward: FSCN1)50-CATTGGACGCCCTCAGTG (reverse: FSCN1)50-GACAACAGCAGTATGGACG (forward: LGR5)50-GCATTACAAGTAAGTGCCAG (reverse: LGR5)50-GGCATACACCTACTCAACTACGG (forward: ZEB1)50-TGGGCGGTGTAGAATCAGAGTC (reverse: ZEB1)50-AATGCACAGAGTGTGGCAAGGC (forward: ZEB2)50-CTGCTGATGTGCGAACTGTAGG (reverse: ZEB2)

Statistical analysisThe significance of differences between mean and median was

determined using the Student t test and theMann–WhitneyU test,as appropriate. Significance testing was performed using SPSSversion 15. Mean � SD (�, P < 0.05; ��, P < 0.01; ��, P < 0.001)values are shown.

ResultsFLYWCH1 and its physical interaction with b-catenin

FLYWCH1 is an uncharacterized protein product of the humanFLYWCH1 gene. The sequence of this gene is publicly available(http://www.ncbi.nlm.nih.gov/gene/84256), yet its functionremained undefined. The first evidence of FLYWCH1/b-catenininteraction comes fromour large-scale screening using amodifiedyeast-2-hybrid based RRS (Fig. 1A–C; ref. 23). As the bait, we usedthe FLAG-tagged construct of b-catenin lacking the seven to eightarmadillo-domains (DARM; FLAG-b-cateninDARM) to avoid anyinteraction with TCF/LEF and other known nuclear proteins(Fig. 1B). The expression of GSK-3b and substrate-phosphoryla-tion dependency is dependent on the presence (promoter-off) andabsence (promoter-on) of methionine in yeast (Fig. 1C). This RRSutilizes an embryonic cDNA library (pMyr-cDNA library). Thus,cdc25-2 mutant yeasts can grow at 37�C, when a phosphoryla-tion-dependent and -independent interaction between a proteintarget and RasV12-b-cateninDARM takes place. Therefore, todetermine GSK-3b phosphorylation-independent interactionsbetweenhumanb-catenin andmyristoylated target proteins, yeastcolonies exhibiting efficient methionine-dependent growth iso-lated and further analyzed. The b-cateninDARM(bait)-dependentgrowth of these clones was further analyzed on galactose-containingmedium at 37�C. This screening allowed us to identifyseveral proteins that specifically bound to unphosphorylated-b-catenin (Supplementary Table S1). The FLYWCH1 proteincontains a tandem array of five FLYWCH-type Zinc-finger motifs(Fig. 1D and E) and a putative nuclear localization signal (NLS)motif (KRAK; Supplementary Fig. S1A) closely resembling theclassical NLS motif consensus sequences (K-R/K-X-K/R). We test-ed multiple antibodies from various commercial sources, unfor-tunately despite a lot of efforts no commercial antibody can

detect the endogenous FLYWCH1 protein in WB and IP, whileworking on IF assays. Therefore, the full-length cDNA of humanFLYWCH1 tagged with MYC-epitope (MYC-FLYWCH1) or eGFP(GFP-FLYWCH1) were used for biochemical/molecular analyses(WB, IP, Co-IP, etc.) in this study.

To confirm our initial screening data regarding FLYWCH1/b-catenin interaction, we first carried out a coupled in vitro tran-scription/translation (IVT) assay of both FLYWCH1 and b-cateninin the presence and absence of constitutively active GSK-3b. Treat-ment of the IVT product with l-phosphatase resulted in a singleunphosphorylated, faster-migrating form of FLAG-b-cateninWT

resembling FLAG-b-cateninS33A (Supplementary Fig. S1B, bottom,lanes 2 and 3). Interestingly, our IP assay (using a-GFP antibody)confirmed the interaction of GFP-FLYWCH1 with both FLAG-b-cateninWT (treated with l-phosphatase) and FLAG-b-cateninS33A

(Supplementary Fig. S1B, top, lanes 2 and 3).Next, to further address the association of unphosphorylated

b-catenin with FLYWCH1, we expressed and purified GST-b-cateninWT, GST-b-catenin4A, andHis-FLYWCH1proteins frombacteria for an in vitro binding assay. Co-IP with His-antibodyand WB with b-catenin-antibody revealed that FLYWCH1 coulddirectly bind the unphosphorylated b-catenin4A (amutant clonethat lacks the phosphorylation sites S33, S37, and S45, whichneeded to prime b-catenin for subsequent phosphorylation atS41, S33, and S37 by GSK-3; Fig. 1F). We also confirmed theinteraction of GFP-FLYWCH1 with ectopically expressed b-cate-nin (FLAG-b-cateninS33A and FLAG-b-catenin4A) in HEK293cells using both a-GFP and a-FLAG antibodies, respectively(Fig. 1G; Supplementary Fig. S1C). Further Co-IP analysesshowed that b-catenin4A interacts with FLYWCH1 regardlessof GSK status (Supplementary Fig. S1D, lane 3 vs. lane 4). Asingle mutation of b-catenin (b-cateninS33A) showed strongerinteraction with FLYWCH1 than the wild-type (b-cateninWT;Supplementary Fig. S1D, lane 5 vs. lane 1), whereas the GSK3inhibitor-BIO (5micromolar) enhanced the interaction of bothb-cateninS33A and b-cateninWT with FLYWCH1 (SupplementaryFig. S1D, lanes 6 and 2). Overall, these data indicate thatphosphorylation of any of these sites may dampen theFLYWCH1 interaction with b-catenin.

The b-catenin has three main domains, the N-terminus,C-terminus, and the armadillo repeats, to which most of itsnuclear partners bind (27–29). Thus, we generated a series ofb-catenin internal deletions to investigate which region isinvolved in the FLYWCH1/b-catenin interaction (SupplementaryFig. S2A). Two b-catenin mutants (MD3 and DC) were weaklyexpressed, presumably due to their degradation in the cyto-plasmic b-catenin-destruction complexes (SupplementaryFig. S2A, lanes 7 and 8). These two deletions were thereforeexcluded from further Co-IP analyses. Co-IP analysis showed thatthe N-terminal deletion of b-catenin (DN) was sufficient to abortFLYWCH1/b-catenin interaction (Fig. 1H). The b-catenin proteinhas no classical nuclear localization or nuclear export signals (30).Therefore, b-catenin deletions may not affect the subcellulardistribution of the mutant proteins. Intriguingly, the armadillorepeats region of b-catenin (represented by deletions MD1and MD2 in Supplementary Fig. S2A) containing the TCF/LEFbinding domain was not essential for this interaction (Fig. 1H).Furthermore, we generated several deletion mutant clones ofFLYWCH1 to map the interaction of FLYWCH1 with b-catenin(Supplementary Figs. S2B–S2D). Co-IP revealed that b-cateninlost its interaction with the C-terminal deletion mutant of

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FLYWCH1 (DC350) in which the last two FLYWCH motifsare removed (Supplementary Fig. S2E). Full-length FLYWCH1is expressed in the nucleus in a punctate format (SupplementaryFig. S3A) presumably due to the presence of an NLS within the C-terminus domain (Supplementary Fig. S1A). However, theprotein product of GFP-FLYWCH1-DC350 is diffused into boththe cytoplasm and nucleus (Supplementary Fig. S3A), and asignificant fraction of its protein remained in the nucleus(Supplementary Fig. S3B). Therefore, the cellular distributionof FLYWCH1-DC350 protein may not result in a complete lossof FLYWCH1/b-catenin interaction. Next, we investigated theinteraction of FLYWCH1 with endogenous b-catenin in SW480cells, which contain a high level of nuclear b-catenin due to

mutations in the APC gene. Our Co-IP assay showed a directinteraction between endogenous b-catenin and FLYWCH1,whereas the C-terminal deletion of FLYWCH1 (DC350) haslost the ability to interact (Fig. 1I). Taken together, these datasuggest that the FLYWCH1-C-terminus domain is essential forefficient b-catenin interaction and complete nuclear localiza-tion of FLYWCH1.

FLYWCH1 represses the transcriptional activity of b-cateninby preventing the association of TCF4/b-catenin to theTcf-DNA-binding elements

To elucidate the consequences of FLYWCH1/b-catenin inter-action on the transcriptional activity of b-catenin/TCF4, we first

Figure 1.

Identification of FLYWCH1 as an interacting protein with unphosphorylated-b-catenin in yeast cells, in vitro and in vivo. A, Schematic representation ofthe modified yeast two-hybrid Ras Recruitment System (RRS) shows candidates interact with phosphorylated (left) and/or unphosphorylated (right)b-catenin (see detailed description in the text). GDP, guanosine diphosphate; MP, Met3 promoter; pA, polyA signal; P, phosphorylated amino acid. B and C,Ras, GSK-3b, FLAG, and phospho-b-catenin protein expression in yeast. Phospho-b-catenin is induced by withdrawal of methionine (C). D, Tandem repeats offive FLYWCH encoding domains across human FLYWCH1 protein. E, Alignment of FLYWCH domains sequence of human FLYWCH1 showing the core aaresidues (F, L, Y, W, C, and H) of FLYWCH consensus sequence highlighted in yellow. The starting and ending residue numbers given for each domain indicatethe actual aa numbers within the FLYWCH1 protein sequence. Identical residues are indicated by asterisks (�) at the bottom line and the non-identical residuesby colons (:). F, His-tag pull-down using purified GST-b-cateninWT, GST-b-catenin4A and 6xHis-FLYWCH1, followed by immunoblotting with anti-b-cateninand anti-His antibodies in the present of GSK-3b. G, Co-IP and immunoblotting of HEK293T cells cotransfected with the indicated constructs usinganti-FLAG-conjugated agarose beads for Co-IP and anti-GFP and anti-FLAG antibodies for immunoblotting as indicated. H, Co-IP and immunoblotting ofHEK293T cells cotransfected with the indicated constructs using anti-FLAG-conjugated agarose beads for Co-IP and, anti-GFP and anti-FLAG antibodies forimmunoblotting. In top panels, arrowhead indicates protein bands of GFP-FLYWCH1 detected by the anti-GFP antibody on both Co-IP and input membranes.In bottom panels, arrowheads indicate protein bands of FLAG-b-catenin, and mutants detected by anti-FLAG antibody on both IP and input membranes.Asterisks (�) indicate unspecific bands. I, Endogenous interaction between b-catenin and FLYWCH1 in the lysate of SW480 cells transfected with theindicated constructs. Co-IP was performed using anti-b-catenin-conjugated agarose beads. Normal IgG (IgG) was used as a negative control. Theimmunoprecipitated complexes were analyzed by WB with anti-b-catenin and anti-MYC antibodies. Asterisks (�) indicate unspecific bands.






performed a dual-luciferase TOP/FOP-FLASH reporter assayas described previously (20, 21). Intriguingly, FLYWCH1 effec-tively suppressed the reporter activity induced by b-catenin indifferent cell lines (Figs 2A; Supplementary Fig. S3C). Theluciferase-activity of all deletion mutants of b-catenin exceptthe N-terminal deletion (b-catenin�DN) was significantlyinhibited by FLYWCH1 (Supplementary Fig. S3D, lane 3).Similarly, the C-terminus removal of FLYWCH1 (DC350) haslost its suppressive activity against b-catenin signaling (Fig. 2B,lane 6). Thus, in line with our Co-IP results, the luciferase dataalso confirmed that the b-catenin-N-terminus and theFLYWCH1-C-terminus are required for both their physical andfunctional interaction.

To explore the downstream effect of the functional interactionof FLYWCH1/b-catenin, we show that the c-jun-promoter-Luc (aputative b-catenin-target gene) was effectively suppressed byFLYWCH1 (Fig. 2C). This suppression, however, was impairedby FLYWCH1-siRNA, whereas the TCF4 and b-catenin proteinlevels were unaffected (Fig. 2C). Intriguingly, our Co-IP assaysshowed that the interaction between b-catenin and TCF4 ismitigated by the ectopic expression of FLYWCH1 (Fig. 2D).Moreover, the luciferase assay showed that overexpression ofTCF4 only slightly rescued the suppression effect of FLYWCH1on b-catenin (Fig. 2E) giving the notion that FLYWCH1 maycompete with TCF4 for binding to b-catenin. To further confirmthis notion, we performed a competitive binding assay of human

Figure 2.

FLYWCH1 competes with TCF4 for binding to b-catenin. A, Effect of overexpressed FLYWCH1 on the firefly luciferase reporter (TOP-FLASH) induced by theendogenous level of b-catenin in colorectal cancer cell-lines (SW480, HCT116, and SW620). Data are mean � SD (n ¼ 6; �� , P < 0.01; �� , P < 0.001).B, FLYWCH1 C-terminal deletion (DC350) lost the ability to repress b-cateninS33A activity (lane 6). Data are mean � SD (n ¼ 3; �� , P < 0.001).C, Suppression of the b-cateninS33A-induced c-jun promoter activity by FLYWCH1. Data are mean � SD (n ¼ 6; �� , P < 0.01; �� , P < 0.001). D, Co-IP ofHEK293T lysate cotransfected with the indicated constructs using anti-FLAG-conjugated agarose beads. Both the Input and Co-IP samples were immunoblottedwith anti-HA and anti-GFP antibodies. Asterisks (�) indicate unspecific bands. E, Effect of overexpressed FLYWCH1 on the firefly luciferase reporter (TOP-FLASH)induced by the overexpressed TCF4/b-catenin in HEK293T cells as indicated. Data are mean � SD (n ¼ 6; �� , P < 0.001). F, Schematic diagram of abiotinylated-oligonucleotide-mediated ChIP method shows the streptavidin/biotinylated Tcf/Lef-protein bound complex (top) and immunoblots for theindicated constructs using the anti-HA antibody for TCF4 (lower panels). The streptavidin/biotinylated DNA/protein-bound complexes were immunoprecipitatedusing streptavidin-coupled Dynabeads. Biotinylated oligos containing x3 Tcf-DNA consensus sites (TOP), could not equally pull-down the HA-TCF4 fromstreptavidin/biotinylated beads in the lysate of SW480 cells transfected with the MYC-FLYWCH1 or empty vector. The experiment was repeated on threeindependent occasions. G, Quantitative PCR analysis of Tcf-DNA-binding sites in the c-jun promoter to monitor chromatin occupancy of TCF4 by ChIP onsorted for GFPþ cells by flow-cytometry using HCT116 cells transfected with GFP-FLYWCH1, GFP, and HA-TCF4, respectively. Data aremean� SD (n¼ 3; �, P <0.05;�� , P < 0.01). H, Effect of shRNA-mediated knockdown of b-catenin, compared with the control scrambled control shRNA (scs) on the transcriptionalrepression of firefly luciferase reporter (TOP-FLASH) mediated by FLYWCH1 in SW480 cells. Data are mean � SD (n ¼ 6; �� , P < 0.001).

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b-catenin protein to determine if the b-catenin and TCF4 inter-action values influenced by FLYWCH1 using competition andan ELISA based binding assays in vitro (Supplementary Fig. S4A–S4C). The result shows that FLYWCH1 protein, indeed, competeswith TCF4 protein for binding to b-catenin.

FLYWCH1 may compete with TCF4 via its binding to theTcf-DNA binding sites. To explore this possibility, we first usedthe LightShift Chemiluminescent EMSAassayusing SW480nucle-ar protein extract incubated with a biotin end-labeled DNAcontains 3� of Tcf-DNA-binding site in the absent and/or presentof recombinant TCF4 and FLYWCH1 proteins. Interestingly, theamount of DNA–TCF4–protein interaction is substantiallyreduced by FLYWCH1 protein (Supplementary Fig. S4D). Ourbiotinylated-oligonucleotide-mediated ChIP assay (31) alsoshowed that the biotinylated oligos (containing 3� Tcf-DNAconsensus sites, TOP) could not equally pull-down the HA-TCF4from streptavidin/biotinylated beads in the presence ofMYC-FLYWCH1 (Fig. 2F). Furthermore, our endogenousTCF4-ChIP assay in HCT116 cells (contains high endogenouslevel of TCF4) show that binding of the TCF4 to the Tcf-sites of thec-jun-promoter was reduced by both endogenous and exogenouslevels of FLYWCH1 (Fig. 2G). These data strongly indicate thecompetition of FLYWCH1 with TCF4 to bind to the Tcf-DNAbinding sites.

Next, we assessed the role of b-catenin in this competition. Ourdata show that knockdown of b-catenin (20) remarkably rescuedthe suppression effect of FLYWCH1 (Fig. 2H), indicating thenecessity of b-catenin for the FLYWCH1-mediated transcriptionalrepression. To further support this notion, we performed a

b-catenin-ChIP assay based on standard and qPCR analysis(Fig. 3A–B) to the Tcf-DNA-binding sites within c-jun promoter.b-Catenin-ChIP experiments were performed in normal fibro-blasts (TIG119) that lack nuclear-b-catenin (Supplementary Fig.S4E; ref. 21) and colorectal cancer cells (DLD-1 and SW480) thatcontain nuclear b-catenin. Notably, in contrast to the TIG119cells, immunofluorescent assay demonstrated that the b-catenindifferentially expressed in the normal colonic epithelial cell lineCCD-CoN-841, including some nuclear expression (Supplemen-tary Fig. S4E). Expectedly, the ChIP assays showed an efficientbinding of both b-catenin/TCF4 and b-catenin/FLYWCH1 (toa different extent) to the Tcf-DNA-binding elements of thec-jun-promoter in colorectal cancer cells, but not fibroblasts(Fig. 3A). These data further confirm the b-catenin dependencyof the FLYWCH1-mediated transcriptional repression. Moreover,FLYWCH1 overexpression also reduced the interaction ofb-catenin/TCF4 to the c-jun-Tcf-sites (Fig. 3B). We also show thatthe competition of FLYWCH1 and TCF4 to the Tcf-DNA-bindingelements is not through the direct interaction of TCF4 andFLYWCH1 proteins as no FLYWCH1 was co-IPed with TCF4 inTIG119-fibroblasts that lack nuclear b-catenin (SupplementaryFig. S4F).

To elucidate the physiologic relevance of FLYWCH1/b-catenininteraction and activation of Wnt-signaling, we examined theFLYWCH1 expression under the influence of canonical Wnt3a-ligand and R-spondin-1 that bind to and activate Wnt-receptors(32, 33). TheWB assays showed thatWnt3a/R-Spondin treatmentremarkably increased the accumulation of nuclear b-catenin andWnt-target genes such as cyclin-D1 and c-Jun (Fig. 3C), whereas

Figure 3.

Competition between FLYWCH1/b-catenin and TCF4/b-catenin complexes for their interaction to Tcf-DNA-binding sites. A, b-Catenin-ChIP assay showsbinding of FLYWCH1/b-catenin and TCF4/b-catenin complexes to the human c-jun promoter in SW480 and DLD-1 cells (right), as well as TIG119 fibroblasts (left).SW480 and DLD-1 cells were transfected to express FLYWCH1 and TCF4. B, b-Catenin-ChIP coupled with qPCRwas used to quantify binding of FLYWCH1/b-cateninversus TCF4/b-catenin to the c-jun promoter, as outlined in (A), using real-time PCR. Data are mean � SD (n ¼ 3; � , P < 0.05; �� , P < 0.01; �� , P < 0.001). C,Western-blotting analysis of mock vs. treatedWnt3aþR-spondin 1 in HCT116 cells after 24 hours using the indicated antibodies. D and E, SW480 and DLD-1 cell-lineswere treated with Wnt3aþR-spondin1 conditional media or mock after 24 hours. RNA was extracted and FLYWCH1-mRNA expression was assessed by real-timeqRT-PCR (D) and standard RT-PCR (N ¼ 3, 100bp marker was used; E). Data are mean � SD (n ¼ 6; �� , P < 0.01). F, Levels of cyclin D1/D2 gene expressionassociated with the activation of Wnt signaling altered FLYWCH1 gene expression levels in SW480 cells using WB assays. SW480 cells were transfected toexpress FLYWCH1 (lanes 3 and 4) and control GFP (lanes 1 and 2).






downregulated the FLYWCH1 gene expression (Fig. 3D and E).Further analyses showed that overexpression of FLYWCH1 in theWnt3a/R-Spondin-treated SW480 cells leads to decreased expres-sion of both cyclin-D1 and cyclin-D2 (Fig. 3F, lane 3 vs. lane 4),indicating that FLYWCH1 acts as a negative transcriptional mod-ulator for the Wnt-target genes.

Altogether, our findings suggest that FLYWCH1 represses thetranscriptional activity of b-catenin by preventing the associationof TCF4/b-catenin to the Tcf-DNA-binding elements via compet-ing with TCF4. Our data also indicate that FLYWCH1may act as anegative regulator of Wnt/b-catenin signaling pathway throughtranscriptional repression of Wnt-target genes.

FLYWCH1 modulates cell morphologyTo explore the biological activity of FLYWCH1, we first exam-

ined FLYWCH1 transfected cells under the microscope andobserved changes in cell shape and morphology. It is noteworthyto mention that stable overexpression of full-length FLYWCH1has been attempted in SW480 and HCT116 cells usingGFP-FLYWCH1 lentiviral vector (pLVX-GFP-FLYWCH1-PuroR)based on Puro-selection with no success (SupplementaryFig. S5A). GFP-FLYWCH1 showed different cellular localizationpattern (Supplementary Fig. S5A) anddifferent truncated formsofthe protein (Supplementary Fig. S5B), indicating possible spon-taneous mutation(s) of FLYWCH1 in these cell lines. To explorethis possibility, the N-terminal coding region of the genome-integrated FLYWCH1 flanked by eGFP was amplified by PCRand sequenced. Expectedly, the sequencing analysis revealed apointmutation, resulted in a stop-codon at aa277 (stable„1), anda relatively large internal in-frame deletion (537nt) mapped tonucleotides 1405–1942 within the C-terminal domain (D468-648aa, stable „2) of FLYWCH1 (Supplementary Fig. S5C andS5D). These deletions were corresponding to the truncated pro-tein bands detected on WB (Supplementary Fig. S5B). Indeed,consistent with our previous data, the stably expressed truncatedform of FLYWCH1 (D468-648aa) lost its physical interactionand suppression activity against TCF4/b-catenin (SupplementaryFig. S5E–S5F). Data from the The catalogue of Somatic Mutationsin Cancer (COSMIC) database revealed that somatic mutation inFLYWCH1 is rare in human cancers. Out of 32,801 primaryhuman cancer samples, only 154 samples (a mutation rate of0.47%) have somatic mutations. Interestingly, over 23% of thesemutations found in the large intestine and 3.9% of these wereframeshift deletions (Supplementary Fig. S6A). Therefore, colo-rectal cancer cells transiently expressingGFP-FLYWCH1were usedfor biological analysis.

With the possible frameshift mutation and loss of actionassociated with FLYWCH1 overexpressing cells, we first tested theeffect of FLYWCH1-knockdown on cell-cycle progression inSW480 and HCT116 cells stained with propidium iodide (PI)using flow cytometric analyses. We observed that FLYWCH1-knockdown increased cell number at G2–M transition in bothSW40 and HCT116 cells (Supplementary Fig. S6B). Next, wetested the morphologic changes associated to transient overex-pression of FLYWCH1. Our microscopic study for Phalloidinstained SW480 cells showed that untransfected cells displayedbranched, flat, and elongated shape with prominent actin fibers.Although FLYWCH1-expressing cells showed a different pheno-type, characterized by shorter, circular epithelial-likemorphologywith actin fibers concentrated at the edges of the cells (Fig 4A,middle horizontal panel). Consistent with this observation,

FLYWCH1-knockdown cells exhibited irregular branched shapeswith numerous elongated projections (filopodia) in all directionscompared with controls (Fig. 4A, lower; Supplementary Fig. S7C,S7D and S7F vs. S7G), suggesting that FLYWCH1 may modulatecellular biological activities through changes in cell morphology.

Previous studies have showed that b-catenin protein complexesredistribute from the membrane to the nucleus upon Wnt, irra-diation, TGFb1, and Rac1 activation or fibrosis and the nuclearsubstructures formed were readily identifiable (34–38). Toexplore this, we tested TIG119 cells, a normal embryonic fibro-blast lacking nuclear b-catenin (Supplementary Fig. S4E), whentreated with Wnt3A and stained for anti-b-catenin (Supplemen-tary Fig. S7I). Thus, activation of b-catenin signaling may show anuclear substructures foci-like pattern similar to the subnuclearlocalization of FLYWCH1.

Changes in cell morphology through actin cytoskeleton reor-ganization and filopodia formation have been involved in cellproliferation, migration, and invasion (39, 40). Therefore, weinvestigated whether alteration of cell morphology caused byFLYWCH1 correlates to cell motility. Interestingly, our in vitroscratch assay showed that thewound-closure of FLYWCH1expres-sing cells versus FLYWCH1-knockdown and control cells issignificantly reduced in SW480 cells (Fig. 4B; Supplementary Fig.S7E), indicating negative effect of FLYWCH1on cellmigration. Tofurther delineate the effect of FLYWCH1 on cell attachment andmotility, we evaluated the attachment and spreading capacityof FACS-sorted SW480-expressing GFP, GFP-FLYWCH1, andGFP-FLYWCH1shRNA using electric cell-substrate impedance sens-ing (ECIS; ref. 41). The ECIS analyses showed that cells over-expressing GFP-FLYWCH1 established a significantly higher resis-tance value comparing to FLYWCH1shRNA and control cells(�6,000 ohms vs. 2,000–�3,000 ohm). But the capacitancedecreases in cells overexpressing GFP-FLYWCH1 versusFLYWCH1-knockdown and control cells (�1.5F vs. �2.5–�4 mF; Fig. 4C, right). These data indicate migration/invasiondefects caused by FLYWCH1 possibly due to increased cell–cellattachment. Indeed, the ECIS analyses showed that cells-expressing GFP-FLYWCH1 were quickly and tightly attachedwithin 1 to 2 hours period compared with cells expressing GFP(10–12 hours), whereas cells expressing FLYWCH1shRNA weredelayed for over 24 hours (Fig. 4C, left). Also, cells expressingGFP-FLYWCH1 have restricted the current flow by slow spreadingover the electrode leading linearly to decreased capacitance. Thisindicates increased cell attachment of GFP-FLYWCH1overexpres-sing cells. To investigate if such activity may also modulatemetastatic colorectal cancer cells, we tested the effects ofFLYWCH1 on cell morphology and migration using a metastaticcolorectal cancer cell line, SW620 (Supplementary Fig. S8A–S8C).These data indicate that FLYWCH1 inhibits cell migration andinvasion in SW620 cells. Thus, these results suggest thatFLYWCH1 may negatively regulate cell migration through altera-tions in cell–cell adhesion.

Molecular mechanisms of FLYWCH1-regulated cell migrationTo explore the mechanisms underlying the effect of FLYWCH1

on cell migration, we first used a humanWNT-signaling pathwaycDNA-array to determine the expression profiles of 84 genesrelated to WNT-mediated signal transduction according tothe manufacturer's instructions; http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-243Z.html (Supplementary TableS2). These data further highlighted differences in transcript

Muhammad et al.




http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-243Z.html

http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-243Z.html


expression between FACS-sorted cells overexpressing GFP-FLYWCH1 and GFP proteins. Notably, many of these changesassociated with cell polarity and invasion (e.g., MMP7, DVL, FZD,JUN, and several WNT-target genes; Fig. 5A; SupplementaryTable S2). Interestingly, our further qRT-PCR analysis showedthat expression of a limited subset of genes (CD44, LGR5, c-JUN,EPHA4, CCND1, EFNB2, and FSCN1) were suppressed by theectopic expression of FLYWCH1, whereas expression of others(c-MYC, EFNB1, EPHB2, and EPHB3) were unaffected in bothSW480 andHCT116 cells (Fig. 5B, left, blue columns). Strikingly,the transcriptional repression of CD44, LGR5, c-JUN, EPHA4,CCND1, EFNB2, FSCN1, ZEB1, and c-MYC was significantlyreversed by the FLYWCH1-knockdown, whereas the expression

of others such as EFNB1 and EPHB3were unchanged (Fig. 5B, left,red columns). These data suggest selective repression of Wnt-target genes via FLYWCH1/b-catenin interaction. Moreover, ourWB analysis showed a sufficient correlation between transcriptand protein levels of some of these target genes in FLYWCH1-knockdown cells but less correlation in FLYWCH1-overexpressingcells (Fig. 5B, right). Not surprising that mRNA levels cannotalways correspond to protein levels due to posttranslationalmodifications (42). Furthermore, in a normal embryonicfibroblast lacking nuclear b-catenin and stably expressing theMYC-FLYWCH1, the cyclin-D1 expression is downregulatedwhereas FSCN1, SFRP1, c-MYC, and the c-JUN mRNA levelsremained unchanged (Supplementary Fig. S8D and S8E) further

Figure 4.

FLYWCH1 modulates cell adhesion and morphology. A, SW480 cells stably expressing GFP (top, control), transiently transfected with GFP-FLYWCH1 (middle),or stably expressing GFP-FLYWCH1-shRNA (lower). These cells were stained with Phalloidin and visualized under fluorescent microscope. Selected GFPexpressing cells are boxed and magnified by the right far panel for each condition. Scale bars, 50 mm. B, Percentage of wound closure of GFP-FACS-sortedSW480 cells expressingGFP (control, blue), GFP-FLYWCH1 (green), or GFP-FLYWCH1-shRNA (red), following a scratchwound assay.Wound closurewasmeasuredat time zero (T0), 24 hours (T24) and 48 hours (T48) as indicated. Data are mean � SD (n ¼ 6; � , P < 0.05; �� , P < 0.01; �� , P < 0.001). Representative images areshown in Supplementary Fig. S3. C, The impedance, reflecting cell adhesion and spreading, was measured for GFP-FACS-sorted SW480 cells expressing GFP(control, blue), GFP-FLYWCH1 (green), or GFP-FLYWCH1-shRNA (orange) using electric cell-substrate impedance sensing (ECIS), over 30 hours. Data showmean of duplicate wells of representative ECIS plates). The majority of GFP-FLYWCH1O/E cells were attached in 2 to 3 hours and cell spreading was readilydetected by an impedance increase (c-left graph), compared with GFP (control) and FLYWCH1shRNA cells.






supporting the notion of selective negative regulation of Wnt-target genes by FLYWCH1/b-catenin interaction. However, effectsof FLYWCH1 on cyclin-D1 gene expression via b-catenin-inde-pendent TCF/LEF activity cannot be excluded.

Notably, the expression of the cell–cell adhesion receptor,E-cadherin (CDH1) was upregulated by FLYWCH1 overexpres-sion in contrast to FLYWCH1-knockdown. Some of the premen-tioned responded genes such asZEB1,CDH1, and EPHA4 are wellknown for their involvement in epithelial–mesenchymal transi-tion (EMT) and cell migration. Thus, next, we asked if FLYWCH1could regulate cell migration via EMT/MET process. Our WBshowed no significant differences in the levels of a mesenchymalmarker, Vimentin, or an EMT transcription factor, ZEB2 (Fig. 5C).However, significant differences between E-cadherin and ZEB1mRNA and protein expression were detected in response to thepresence and/or absence of FLYWCH1 (see above and Fig. 5B andC). Moreover, the level of Snail protein is accumulated inFLYWCH-knocked down cells (similar to ZEB1), whereas thelevel of Twist protein is downregulated (SupplementaryFig. S9A). Overall, these data suggest a synergistic action of theseEMT-activating transcription factors may ultimately lead to apartial or steady state level of EMT process transition in colorectalcancer cells with altered FLYWCH1 expression. However, furtherinvestigation is required to explore the possible mechanism inmore details.

Previous studies found that b-catenin/TCF4 activity modulatesexpression of ZEB1 in SW480 cells (43). Accordingly, it isexpected that FLYWCH1 interaction with b-catenin may alsoregulate b-catenin–mediated transcription of ZEB1. Indeed,

overexpression of FLYWCH1 massively decreased the transcrip-tion of human ZEB1-promoter in SW480 cells (Fig. 5D and E).This effect, however, was not found when a mutant form ofFLYWCH1 (FLYWCH1DC), which lacks the b-catenin–bindingdomain was used (Fig. 5D). Notably, overexpression of ZEB1rescued the inhibitory effect of FLYWCH1 overexpression on cellmigration (Supplementary Fig. S9B and S9C). These data supportthe notion that FLYWCH1-mediated suppression of ZEB1 maydepend on the formation of a transcriptional activation complexwith nuclear b-catenin.

ZEB1 and EPHA4, which were among the most affected genesby the FLYWCH1-knockdown (Fig. 5B), are important deter-minants of tumor progression serving as an inducer of invasionand metastasis in colorectal cancers (44, 45). Therefore, wesought to examine whether the expression of FLYWCH1 corre-lates with the expressions of these genes in human pathologicsamples. First, to examine the correlation between FLYWCH1and ZEB1, CDH1 and EPHA4 gene expression, we obtainedthe expression data of colon cancer from the TCGA. Level 3Illumina RNA-Seq data for colon adenocarcinoma (COAD)were downloaded from cBioPortal, which contained geneexpression data for 469 primary tumor samples of COAD(Supplementary Fig. S9D–S9G). The data indicated a signifi-cant negative correlation between FLYWCH1 and ZEB1and EPHA4 (Supplementary Fig. S9D and S9E), whereas thecorrelation between FLYWCH1 and CDH1 or ZEB1 and CDH1is very weak and is not significant (Supplementary Fig. S9F andS9G). These data suggest that FLYWCH1 may possess someantimetastatic activity.

Figure 5.

FLYWCH1 regulates certain b-catenin/TCF4 target genes involved in the invasion. A, Bivariate scatter plot showing the distribution of up- anddownregulated 84 WNT-associated genes from SW480 cells expressing GFP-FLYWCH1 (experimental) vs. GFP (control) using the human WNT- RT2 profilerPCR array. Expression was determined by qRT-PCR. Downregulated genes (blue), and upregulated genes (yellow). B, The mRNA expression (left) andWestern-blotting analysis of several tumorigenic and proinavsive b-catenin/TCF4 target genes in SW480 cells expressing GFP-FLYWCH1 (blue) andGFP-FLYWCH1-shRNA (red) relatively to the control cells expressing GFP (blank). Data are mean � SD (n ¼ 3; � , P < 0.05; ��, P < 0.01; ��, P < 0.001).C, Western blot analysis of EMT markers and/or regulatory factors in SW480 cell lysate using the indicated constructs and antibodies. D, Effect of FLYWCH1on ZEB1 transcription activity mediated by b-catenin/TCF4. Data are mean � SD (n ¼ 6; � or o, P < 0.05; �� or oo, P < 0.01; �� or ooo, P < 0.001). E, Effectof FLYWCH1 on ZEB1-promoter activity and transcription.

Muhammad et al.





Next, the expression of FLYWCH1 in a series of early andadvanced/metastatic patient's colorectal cancers was assessedusing ISH assay on a TMA. Our analysis showed a negativecorrelationbetween FLYWCH1expression and tumor progression(70.8% vs. 25.7%, P � 0.017; Fig. 6A). Furthermore, theexpression pattern of ZEB1 and EPHA4 versus FLYWCH1 in acohort of 33 (normal, primary, andmetastatic) tumor tissueswerealso investigated. Our data showed that ZEB1 expression is highlyincreased in advanced tumors, whereas FLYWCH1 expression issignificantly repressed (Fig. 6B). Interestingly, a significantlinear correlation between downregulation of FLYWCH1 and

upregulation of ZEB1 and EPHA4 in normal versus advancedstages, but not in the primary tumors, was observed (Fig. 6B).

DiscussionDespite the fact that substantial progress toward identification

of full human proteome, not all the annotated protein-codinggenes have been characterized. One such example is the proteinproduct of FLYWCH1 gene, which contains five conservedFLYWCH-type Zinc-finger domains (Fig. 1). This work demon-strates identification, characterization, and functional analysis of

Figure 6.

Low FLYWCH1-mRNA expression significantly associated with advanced tumors. A, Top panel shows photomicrographs of colorectal cancer-TMA specimensanalyzed by ISH using a specific FLYWCH1 probe with strong (left), moderate (middle), and weak (right) expression. The lower panel shows a correlationbetween FLYWCH1 expression and tumor staging. Weak expression of FLYWCH1 is associated with more advanced stage of a tumor. N/A: samples were notavailable on slides after staining and perhaps wiped off through the ISH process. Scale bars: 50 mm. B, qRT-PCR analysis of FLYWCH1, EPHA4, and ZEB1mRNA expression in a cohort of thirty-three; normal, primary, and metastatic tumor tissues from patients in Nottingham, UK, normalized to Hypoxanthine-guaninephosphoribosyltransferase (HPRT). Data are mean � SEM (n ¼ 3; �P � 0.05; �� , P � 0.01). Experiments were performed in triplicate for each sample andrepeated on two independent occasions. C, Schematic representation shows the adverse effect of FLYWCH1 on cancer cell migratory potentials throughinhibition of b-catenin signaling pathway.






FLYWCH1 protein for the first time. Based on bioinformaticsanalysis, FLYWCH1 may have potential DNA-binding abilitiesdue to the presence of highly conserved NLS and FLYWCH-typeZinc-finger motifs, which appears to be evolutionarily conservedin mammals (Fig. 1; Supplementary Fig. S1). It should be notedthat, although the endogenous FLYWCH1 can be detected by IF,despite lot of efforts no commercial antibody can detect theendogenous FLYWCH1 by WB or IHC analysis.

We demonstrated that FLYWCH1 inhibits b-catenin–mediatedtranscriptional activation by competing with TCF4 to bind tob-catenin. We also show direct evidence of cell migration andinvasion defects caused by overexpression of FLYWCH1 thatmimic a canonical Wnt loss-of-function phenotype. Moreover,our analyses revealed repression of a surprisingly limitedsubset of b-catenin-target genes by FLYWCH1 including thoseinvolved in regulation of cell migration and morphology.Alterations in cell motility and morphology are initial stepstoward invasion and metastasis, one of the critical hallmark ofcancer. Wnt/b-catenin signaling pathway, through differentmechanisms, is often linked to regulation of various biologicaltraits such as modulation of collective cell migration and cellbranching (46–48). Indeed, signaling through this pathwaymodulates the expression of multiple genes that control filo-podia formation and consequently, cell morphology, migra-tion, and invasion. Examples of these genes include CDH1,Fascin1, c-Jun, and Eph/ephrin molecules (49–52). One of themost widely studied critical protein for the establishment offilopodia is the actin-binding protein, Fascin. Notably, ourdata show that Fascin1 gene expression is regulated byFLYWCH1 (Fig. 5B). During development, Fascin promotescell migration through filopodia formation. High Fascinexpression is correlated with invasion and metastasis of var-ious tumors (53, 54).

In line with these findings, the reduced cell migration andmorphologic changes observed in this study might be due tomodulation of the genetic program downstream of b-catenin/TCF4 by FLYWCH1. Especially, as FLYWCH1 negatively regulatesthe b-catenin/TCF transactivation with no effect on the level ofeither b-catenin or TCF4 proteins (Fig. 2). The possible mecha-nism by which continuous Wnt3a/R-spondin1 treatment down-regulates FLYWCH1 is currently unclear. It is possible that theWnt/b-catenin-TCF4 via a negative feedback loop regulates theFLYWCH1 transcription product. In turn, FLYWCH1, togetherwith Wnt via the b-catenin/TCF4, activates "on" or "off" decisionto control the downstream genes fundamental to cell migration.Although this genetic modulation is likely to represent a tran-scriptional hierarchy, nevertheless, it is essential to understandwhy and how FLYWCH1 selectively represses specific b-catenin/TCF-target genes and uncover the molecular mechanisms behindthis. This study, so far, focused on the assessment of the con-sequences of FLYWCH1 aberrations on theWnt/b-catenin canon-ical pathway. However, a possible role of FLYWCH1 in combi-nation with different ligands and receptors in regulating cellmorphology and migration via alternative noncanonical Wnt/signaling responses (e.g., mediated by small GTPases, Rac1, andRhoA; ref. 55), remains unknown.

Defects in cell migration and actin cytoskeleton rearrange-ment could be related to EMT (56). However, the decreased cellmigration found in our study is likely due to mechanisms otherthan EMT. No significant change in Vimentin expression indi-cates that its expression might not be regulated by FLYWCH1

and, thereby, ruling out the involvement of FLYWCH1 duringEMT. Thus, it appears that FLYWCH1may control cell migrationvia b-catenin-dependent E-cadherin upregulation and ZEB1downregulation leading to increased cell–cell and cell–matrixadhesion.

The role of E-cadherin and the adherens junctions inmediatingepithelial differentiation, by the establishment of cellular adhe-sion and polarity, is well recognized. Consistent with our obser-vations, upregulation of E-cadherin has been widely associatedwith reduced cell motility and invasion in addition to morpho-logic changes from mesenchymal-like to epithelial-like shape invarious types of cancer (57, 58). Another interesting observationof our study is the negative correlation between FLYWCH1expression and tumor progression (Fig. 6A and B). One possiblehypothesis is that tumor cells have developed mechanisms tosuppress FLYWCH1 expression (Fig. 6C), facilitating changes incell morphology and migration which promotes metastasis.FLYWCH1 is a newly characterized gene. Our findings demon-strate that FLYWCH1 could have potential tumor suppressoractivity. In advanced colorectal cancer, FLYWCH1 similar to mosttumor suppressor genes is significantly inhibited. A likely hypoth-esis is that tumors with high degrees of methylation are morelikely to inactivate genes critical for tumor progression andmetastasis. However, the underlyingmechanisms associated withthe possible epigenetic and posttranscriptional regulation ofFLYWCH1 gene expression remained unknown. Furthermore,FLYWCH1 functions as a transcription modulator that can bindto DNA and/or RNA and in b-catenin-dependent or independentmanners. Therefore, outside the scope of the current studies, acomprehensive genomic analysis to identify the direct targetgenes regulated by FLYWCH1, using both ChIP-sequencing andRNA-sequencing or RIP-sequencing analyses, will further clarifyother functions of this protein.

In conclusion, our findings revealed a new molecular mech-anism underlying negative signal cross-talk between FLYWCH1and b-catenin. In this manner, the FLYWCH1/b-catenin com-plex represses selective Wnt-target genes, including Eph/ephrinand ZEB1, which mainly associated with cell polarityand migration during colorectal cancer development andmetastasis. Prospectively, FLYWCH1 has potential to representa biomarker and therapeutic target against metastatic colorectalcancer in the future.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B.A. Muhammad, A. Saadeddin, A.S. NateriDevelopment of methodology: B.A. Muhammad, R. Babaei-Jadidi,A. Saadeddin, M. IlyasAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B.A. Muhammad, S. Almozyan, R. Babaei-Jadidi,E.K. Onyido, S. Hossein Kashfi, M. IlyasAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B.A. Muhammad, S. Almozyan, R. Babaei-Jadidi,E.K. Onyido, S. Hossein Kashfi, M. Ilyas, A. Lourdusamy, A.S. NateriWriting, review, and/or revision of the manuscript: B.A. Muhammad,R. Babaei-Jadidi, E.K. Onyido, S. Hossein Kashfi, B. Spencer-Dene, M. Ilyas,A. Lourdusamy, A.S. NateriAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): B.A. Muhammad, B. Spencer-Dene,A. Lourdusamy, A. Behrens, A.S. NateriStudy supervision: A. Saadeddin, A.S. Nateri

Muhammad et al.





AcknowledgmentsWe are grateful to B.W. O'Malley, M. Saito, and A. McIntyre for providing

essential reagents. We thank R. Muraleedharan and W. Fadhil for technical helpand advice on histology. We also thank the fantastic fundraising efforts of AlisonSimsandher family inmemoryofDaz Sims to support thework inour laboratory.This work was supported by the Medical Research Council (grant numberG0700763) to A.S. Nateri and University of Nottingham, Nottingham, UK.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 15, 2018; revised June 29, 2018; accepted August 1, 2018;published first August 10, 2018.

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Muhammad et al.




2018;16:1977-1990. Published OnlineFirst August 10, 2018.Mol Cancer Res Belal A. Muhammad, Sheema Almozyan, Roya Babaei-Jadidi, et al. Migration and Morphology in Colorectal Cancer

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