E-Cadherin Regulates Human Nanos1, Which Interacts With

Research Article

E-Cadherin Regulates Human Nanos1, which Interacts with p120ctn and Induces Tumor Cell Migration and Invasion Kristin Strumane,1,3 Arnaud Bonnomet,4 Christophe Stove,1 Roosmarijn Vandenbroucke,1 Beatrice Nawrocki-Raby,4 Erik Bruyneel,2 Marc Mareel,2 Philippe Birembaut,4 Geert Berx,1 and Frans van Roy1

1Department for Molecular Biomedical Research, VIB and Ghent University, Ghent, Belgium; 2Laboratory of Experimental Cancerology, Ghent University Hospital, Ghent, Belgium; 3The Netherlands Cancer Institute, Amsterdam, the Netherlands; and 4Institut National de la Sante´et de la Recherche Medicale U514, Institut Fe´de´ratifde Recherche 53, and Laboratoire Pol Bouin, Centre Hospitalier Universitaire Maison Blanche, Reims, France

Abstract by direct binding to cytoplasmic Armadillo catenins (5). h-Catenin Down-regulation of the epithelial cell-cell adhesion molecule interacts tightly via its Armadillo repeats with the catenin binding domain of the COOH-terminal tail of E-cadherin and via its NH2- E-cadherin is frequently associated with tumor formation and a progression. Besides its role in physical cell-cell adhesion, terminal domain with -catenin. This is essential for the formation E-cadherin is also thought to be involved in intracellular of actin cytoskeleton-coupled cell junctions, although the detailed signaling in normal epithelial cells. In these cells, the mechanism is a matter of debate (6). In addition, p120 catenin Armadillo catenin p120ctn binds to the cytoplasmic domain (p120ctn) associates via its Armadillo repeat region with the juxtamembrane domain of the E-cadherin cytoplasmic tail. The of E-cadherin and stabilizes the adhesion complexes. On loss h of E-cadherin, cytoplasmic p120ctn might accumulate and interactions of E-cadherin with p120ctn and -catenin are necessary for dynamically regulating adequate cell-cell adhesion contribute to tumor malignancy. We used suppression subtractive hybridization to search for genes regulated by by modulating events such as cadherin clustering and the strength E-cadherin expression. We isolated human Nanos1 as a of the connection with the actin cytoskeleton (reviewed in ref. 5). transcript of which levels decrease on E-cadherin reexpression One major function of p120ctn is to stabilize cadherin complexes in a human breast cancer cell line. The hNanos1 protein bears at the cell membrane by controlling the entry of cadherins into the degradative endocytic pathway (reviewed in refs 7, 8). a COOH-terminal (CCHC)2 zinc finger domain and belongs to an evolutionarily conserved protein family sharing functions The molecular basis of the suppressive signals that are conferred in germ cell development in both vertebrates and inverte- by functional expression of E-cadherin on cancer cells has not been brates. We found an inverse correlation between E-cadherin fully elucidated. Loss of E-cadherin might induce signaling by h and hNanos1 expression in various cell lines and under catenins released from the cell membrane. Indeed, -catenin is also diverse conditions. Conditional expression of hNanos1 in a major component of the canonical Wnt signaling pathway human colorectal DLD1 cancer cells functionally abolished (reviewed in refs. 5, 9). When the Wnt signaling pathway is turned h cell-cell adhesion. It induced cytoplasmic translocation of on (e.g., by certain gene mutations in cancer), cytoplasmic - p120ctn, as well as strong migratory and invasive properties. catenin becomes protected from proteasomal degradation, trans- locates to the nucleus, binds LEF/Tcf, and acts as an oncogenic We also found that the NH2-terminal domain of hNanos1, which is conserved only among mammals, interacts with transcription cofactor. However, in the absence of such mutations h p120ctn. hNanos1 counteracted the stimulatory effect of or of a Wnt signal, the -catenin that is released from the p120ctn on cell protrusion formation. Together, these findings membrane on cadherin loss is rapidly degraded. Cadherins are describe a new function for hNanos1 as a downstream effector both necessary and sufficient for localization of p120ctn at the cell of E-cadherin loss contributing to tumor progression. Target- membrane (10). Cadherin deficiency does not significantly alter ing hNanos1 might be a promising strategy in the treatment of p120ctn levels but leaves it stranded in the cytoplasm and/or the E-cadherin–negative tumors in particular. (Cancer Res 2006; nucleus (10–12). In the cytoplasm, p120ctn interacts with Rho 66(20): 10007-15) GTPases (reviewed in refs. 7, 8, 13). It directly binds, via its Armadillo repeats, to RhoA and inhibits its activity by acting as a Introduction guanine nucleotide dissociation inhibitor. p120ctn also indirectly activates Rac1 and Cdc42 through guanine nucleotide exchange E-Cadherin is a major epithelial cell-cell adhesion molecule that factors such as Vav-2. The combined effects of cytoplasmic p120ctn functions as a tumor suppressor (1). Moreover, E-cadherin promote cell migration and, consequently, invasion and metastasis. deficiency plays a causative role in tumor cell invasion and In the nucleus, p120ctn can interact with the transcription factor metastasis (2–4). The extracellular domains of the transmembrane Kaiso, and it is likely that this also contributes to the malignant E-cadherin molecules form homophilic adhesion dimers stabilized phenotype (14, 15). Here, we describe the identification of human Nanos1 as a transcript that is down-regulated by E-cadherin. The human Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Nanos 1 gene encodes a (CCHC)2 zinc finger protein that is highly Requests for reprints: Frans van Roy, Department for Molecular Biomedical homologous to the Nanos orthologues in Drosophila and mouse, Research, VIB-Ghent University, Technologiepark 927, B-9052 Ghent-Zwijnaarde, which are known to have an evolutionarily conserved function in Belgium. Phone: 32-9-331-3604; Fax: 32-9-331-3500; E-mail: [email protected]. I2006 American Association for Cancer Research. embryonic patterning and germ line development (16–22). Our doi:10.1158/0008-5472.CAN-05-3096 finding that E-cadherin regulates the expression of hNanos1 in www.aacrjournals.org 10007 Cancer Res 2006; 66: (20). October 15, 2006

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Cancer Research carcinoma cells prompted us to seek a functional link between E- 2 mmol/L L-glutamine, 100 units/mLpenicillin, and 100 Ag/mLstrepto- cadherin and hNanos1 in epithelial cells. We generated cell systems mycin. The HEK293T cell line was obtained from the American Cell with inducible expression of hNanos1 and found that hNanos1 Type Culture Collection (Rockville, MD) and maintained in DMEM supple- impairs E-cadherin function in cell-cell adhesion and promotes cell mented with 5% FCS, 5% newborn bovine serum, 2 mmol/L L-glutamine, 0.4 mmol/Lsodium pyruvate, 100 units/mLpenicillin and 100 Ag/mL motility and invasion. Interestingly, we found that hNanos1 streptomycin. DLD1TR21-Teton cells, also called DLD1-Teton, were kindly interacts with the Armadillo protein p120ctn and counteracts the provided by Dr. H. Clevers (Hubrecht Laboratory, Utrecht, the Netherlands) effect of its overexpression on cell protrusion formation. and cultured in RPMI with 10% FCS. SW480 and transfected derivatives, kindly provided by Dr. Schwarte-Waldhoff (Ruhr-Universitat Bochum, Bochum, Germany) (24, 25), were maintained in DMEM supplemented Materials and Methods with 10% FCS and 100 units/mLpenicillin and 100 Ag/mLstreptomycin. Suppression subtractive hybridization and cloning of hNanos1. Generation of stable inducible cell systems. DLD1-Teton cells were Polyadenylated mRNA, obtained from subconfluent human E-cadherin– stably transfected with linearized pcDNA4/TO-Myc-hNanos1 by electro- negative MDA-MB-231 breast cancer cells and a derivative transfected cell poration. Clones were isolated after 2 weeks of selection in 500 Ag/mL line reexpressing E-cadherin, was isolated with the Fast Track 2.0 Kit zeocin and 10 Ag/mLblasticidin (Invitrogen). Expression was induced with (Invitrogen, Merelbeke, Belgium), in accordance with the recommendations doxycycline (1 Ag/mL; Sigma, St. Louis, MO) for 48 hours. of the manufacturer. cDNA subtraction was done using the PCR-select Immunofluorescence assays. Methanol fixation and immunofluores- cDNA subtraction kit (BD Biosciences Clontech, Erembodegem, Belgium). cence were done following standard procedures (11). The specific The subtracted cDNA libraries, generated by subcloning the PCR products antibodies used were mouse monoclonal antibody (mAb) 9E10 against the in the pGEM-T vector (Promega, Madison, WI), were screened for Myc-tag (dilution 1:500; Sigma), mouse mAb clone 36 against E-cadherin differentially expressed cDNAs using plus/minus colony hybridization. (1:100; Transduction Laboratories, Lexington, United Kingdom), mouse Finally, the differential expression of isolated clones was confirmed by mAb clone 14 against h-catenin (1:500; Transduction Laboratories), and Northern blotting. mouse mAb pp120ctn against p120ctn (1:500; Transduction Laboratories). The 538-bp hNanos1 cDNA fragment isolated in the suppression sub- The secondary antibody used was Alexa 594–coupled antimouse immuno- tractive hybridization (SSH) screen was used to probe a fetal kidney phage globulin (1:200; Molecular Probes, Eugene, OR). Nuclei were stained with cDNA library (Clontech). The full-length sequence of the hNanos1 cDNA was 4¶,6-diamidino-2-phenylindole (DAPI; Molecular Probes). Samples were obtained by sequencing the isolated specific cDNA clones and several EST examined with a Zeiss AxioImager microscope (Zeiss, Le Pecq, France). clones that were identified by BLAST analysis. To identify the 5¶ and 3¶ ends of Immunoblotting. Cells were lysed in buffer [50 mmol/LTris-HCl (pH the hNanos1-specific transcripts, rapid amplification of cDNA ends (RACE) 7.4), 150 mmol/LNaCl, 1% (v/v) Igepal, 1% (w/v) sodium deoxycholate, was done on RNA isolated from SW480 human colon cancer cells. Additional 5 mmol/Liodoacetamide, 0.1% (w/v) SDS] containing complete protease genomic sequence data were obtained by isolation and characterization of a inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). hNanos1-specific genomic PAC clone. A 16-kb HindIII fragment containing Proteins (0.5 Ag) were separated by 10% SDS-PAGE and transferred onto the single-exon hNanos1 gene was subcloned into the pGEM-11Zf(+) vector Immobilon-P membranes (Millipore Corp., Bedford, MA). After blocking (Promega) to produce pGEM11-hNanos1PAC74-HindIII. with 5% nonfat dry milk in TBS containing 0.1% Tween 20, the membranes Northern blot analysis. Total RNA was isolated from various human cell were incubated with primary antibody. The following mAbs were used: mAb lines with the RNeasy kit (Qiagen, Chatsworth, CA) according to the clone 36 against E-cadherin (1:2,500), mAb clone 14 against h-catenin instructions of the manufacturer. Total RNA (30 Ag) was glyoxylated, size (1:500), mAb pp120ctn against p120ctn (1:1,000), mAb clone 9E10 against fractionated in a 1% agarose gel, and transferred to a Hybond-N+ membrane the Myc tag (1:500), and mouse mAb against GAPDH (1:200; Chemicon (Amersham Pharmacia Biotech, Rainham, United Kingdom). Hybridization International, Temecula, CA). The secondary antibody used was horseradish was done following standard procedures. Probes were radioactively labeled peroxidase (HRP)–conjugated antimouse immunoglobulin antibody by random priming (RadPrime labeling kit, Gibco BRLLifeTechnologies, (1:1,000; DakoCytomation, Glostrup, Denmark). The proteins were visual- Paisley, United Kingdom). The hNanos1-specific probe used was the 538-bp ized with an enhanced chemiluminescence plus detection kit (Amersham insert of the pGEM-T clone obtained from the SSH analysis. The other probe Pharmacia Biotech, Buckinghamshire, United Kingdom). was an 800-bp fragment (exons 14-16) of the human E-cadherin cDNA. To Coimmunoprecipitation assays. Cells were rinsed with PBS and lysed control for the amounts of RNA loaded, a probe specific for glyceraldehyde- in buffer [0.5% NP40, 150 mmol/LTris-HCl (pH 7.4), 50 mmol/LNaCl, 3-phosphate dehydrogenase (GAPDH) was used in an additional hybrid- 1 Amol/Lleupeptin, 0.3 Amol/Laprotinin, 5 Amol/LPefablock]. Lysates ization on the same blot. Radioactive bands were quantified with a (400 Ag of protein) were incubated overnight at 4jCwith1Ag of the relevant PhosphorImager (Molecular Imager FX, Bio-Rad, Richmond, CA). antibody, and then for 2 hours with 50 ALof 50% protein G-Sepharose beads Plasmid constructs. The eukaryotic expression plasmid pCS3-hNanos1, or protein A-Sepharose beads (Amersham Pharmacia Biotech). Adsorbed encoding the complete hNanos1 open reading frame (ORF) fused to an immunoprecipitates were washed five times with lysis buffer and boiled for A NH2-terminal Myc-tag, was constructed by ligating an SgrAI(blunted)/SalI 5 minutes in sample buffer [60 mol/LTris-HCl (pH 6.8), 1.7% SDS, 6% fragment from pGEM11-hNanos1PAC74-HindIII into the BglII(blunted)/ glycerol, 0.1 mol/LDTT, 0.002% bromophenol blue]. Eluted proteins were

XhoI-cut pCS3 vector (23). Expression vectors encoding NH2-terminal or separated by 10% SDS-PAGE (for detection of p120ctn and a-catenin) or COOH-terminal fragments of hNanos1 were obtained by removing a PstI/ 12.5% SDS-PAGE (for detection of Myc-hNanos1 fusion proteins), followed XbaI fragment from pCS3-hNanos1 or by transferring a NotI(blunted)/MunI by immunoblotting as described above. An anti-Myc HRP-coupled antibody fragment from pCS3-hNanos1 to the XhoI(blunted)/MunI–cut pCS3 vector. (1:5,000; Invitrogen) was used for detection of Myc-tagged hNanos1. Other To construct the inducible vector pcDNA4/TO-Myc-hNanos1, a BamHI/ primary antibodies were mAb HECD-1 against E-cadherin (1:1,000; Takara, XbaI fragment from pCS3-hNanos1, encoding Myc-hNanos1, was cloned San Diego, CA), clone 14 against h-catenin (1:1,000), and pp120ctn against into pcDNA4/TO (Invitrogen). The expression constructs for human p120ctn (1:150). The secondary antibody was HRP-conjugated secondary p120ctn (isoform 3A) were pEFBOS-p120-3A (11) and those generated by antimouse antibody (1:3,000; Amersham Pharmacia Biotech). cloning the cDNA encoding p120-3A into pEGFP-C2 (Clontech), forming a Cell aggregation assay. Single-cell suspensions were prepared accord- fusion construct with NH2-terminal enhanced green fluorescent protein ing to an E-cadherin saving procedure (26). Cells were incubated for (EGFP). 30 minutes with continuous shaking in an isotonic buffer containing Cell culture and reagents. The human E-cadherin–negative breast 1.25 mmol/LCa 2+. Rat mAb DECMA-1 (Sigma) was used to functionally cancer cell line MDA-MB-231 (American Tissue Culture Collection, block E-cadherin, starting 30 minutes before aggregation at 4jC and Manassas, VA) and its E-cadherin–transfected derivative were maintained continuing throughout aggregation at 37jC. Particle diameters were in L-15 medium (Gibco BRL Life Technologies) supplemented with 10% FCS, measured with a Coulter particle size counter LS200 (Coulter Electronics

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Ltd., Miami, FL) at the start of the incubation at 37jC and after 30 minutes ring, phase-contrast images of outgrowth areas were recorded at a Â40 of incubation. The results were plotted against the volume distribution magnification every 15 minutes during 1 hour. Cell migration was (expressed as percent of the total cell volume). Statistical differences were characterized and quantified with a previously described software program determined by applying the Kolmogorov-Smirnov test to cumulative fast (29) that measures trajectories of peripheral cell nuclei and cell migration aggregation curves. speeds. Collagen type I invasion assay. Cells were seeded on top of a jellified collagen type I solution (0.09%; Upstate Biotechology, Lake Placid, NY). Invasion was scored on living cultures using a microscope with a computer- Results controlled stage as described (27). Cloning of hNanos1 mRNA as an E-cadherin–suppressed Cell dispersion assay. The spatial distribution of cells was characterized transcript. To identify genes the expression of which is modulated and quantified with an algorithmic program of cellular sociology based on by E-cadherin expression, we conducted comparative transcrip- three geometric models: Voronoi’s partition, Delaunay’s graph, and tome analysis of E-cadherin–negative and E-cadherin–positive minimum spanning tree (28). Details are described in Supplementary data. cancer cell lines. Through SSH analysis of the closely related In vitro cell migration assay. For each cell type, 105 cells were seeded in culture medium in a 22-mm dish with a 6-mm restriction ring placed inside. human breast cancer cell line MDA-MB-231 and its E-cadherin– Twenty-four hours after plating, the ring was removed, the cells were washed expressing counterpart, MDA2BE5.36 (11, 28), we isolated a 538-bp twice with PBS and then covered with culture medium with or without cDNA fragment the expression of which was lower in the doxycycline. In this model, the cells migrate as an outgrowth from the E-cadherin–expressing cells. Completion of the 4,035-nucleotide confluent area initially delineated by the ring. Five hours after removing the (nt) full-length cDNA sequence (GenBank accession no. AF458985),

Figure 1. Cloning and initial characterization of hNanos1 as an E-cadherin–suppressed transcript. A, schematic representation of the human Nanos1 cDNA. ALU, Alu repeat within the 3¶-UTR; ORF, open reading frame of 876 bp; ZFD, zinc finger domain. Arrows, two alternative poly(A) addition sites; bar, localization of the initially isolated SSH clone. B, Northern blot analysis of E-cadherin and hNanos1 mRNAs in MDA-MB-231 cells and in the E-cadherin reexpressing derivative cell line MDA2BE5.36. GAPDH hybridization was used as RNA loading control. The sizes of the alternative hNanos1 transcripts are indicated in nucleotide number. C, Clustal W alignments of the (CCHC)2 zinc finger domains of Nanos-related proteins 1 to 3 in three mammalian species: Hs, Homo sapiens (ref. 22; this report and our unpublished data); Mm, Mus musculus (20, 21); Rn, Rattus norvegicus (our unpublished data). Reverse and gray shadings depict residues that are identical or similar to the column consensus in at least 50% of the aligned sequences. Arrows, conserved cysteine and histidine residues of the (CCHC)2 motif. D, expression patterns of hNanos1 and E-cadherin mRNAs in various human cell lines. Strong expression of hNanos1 mRNA was detected by Northern blotting only in E-cadherin–deficient cell lines. GAPDH hybridization served as the RNA loading control.

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Restricted mRNA expression profile of hNanos1. Northern blot analysis of hNanos1 mRNA expression in a panel of E- cadherin–negative and E-cadherin–positive cell lines revealed that this transcript was not ubiquitously expressed. hNanos1 was expressed at high levels only in the E-cadherin–deficient cell lines SW480 (colon cancer), SK-BR-3 and MDA-MB-231 (breast cancer), SK-N-AS (neuroblastoma), and WERI-Rb-1 (retinoblastoma; Fig. 1D). Moderate expression levels were detected in the A431 (epidermoid), MDA2BE5.36 (breast cancer), and IMR32 (neuroblastoma) cell lines. hNanos1 transcripts were rare or absent in several other cell lines tested. In conclusion, high levels of hNanos1 mRNA were only seen in cell lines expressing low levels of E-cadherin mRNA. All hNanos1-positive cell lines analyzed expressed both the 1,810-nt and 4,035-nt transcripts. hNanos1 and E-cadherin expression are inversely related. E-Cadherin expression can be modulated in the hNanos1-positive human colon cancer cell line SW480 by treatment with phorbol ester, by transfection with Smad4 cDNA, or by growing the cells to confluency (24, 25). Treatment of SW480 cells with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to a gradual decrease in hNanos1 mRNA levels during the first 6 hours of treatment, after which hNanos1 mRNA levels were reestablished (Fig. 2A). At the end point of TPA treatment (24 hours), hNanos1 mRNA levels were even higher than in untreated cells. E-Cadherin mRNA levels had an inverse profile, as they increased during the first 6 hours of treatment and then Figure 2. Modulation of E-cadherin mRNA levels in SW480 human colorectal decreased to zero after 24 hours of TPA treatment. In Smad4- cancer cells induces inverse changes in hNanos1 transcripts. Transcripts were transfected SW480 clones D1 and D14, E-cadherin mRNA analyzed by Northern blot using GAPDH hybridization as RNA loading control. expression was induced and hNanos1 mRNA expression was Columns, mRNA expression levels obtained after quantification and normalization to GAPDH. hNanos1 and E-cadherin transcript levels were reduced (Fig. 2B) in comparison with E-cadherin–deficient mock- compared in (A) subconfluent SW480 cells treated with TPA (250 ng/mL) for the transfected SW480 clones K5 and K6. Finally, the inverse indicated durations; (B) mock-transfected SW480 clones (K5 and K6) and Smad4-transfected clones (D1 and D14; ref. 24); (C) control K5 clone and correlation between E-cadherin and hNanos1 mRNA expression Smad4-transfected D1 clone of SW480 cells that were either grown to was shown by culturing these cells at increasing cell densities, confluence (C) or as a dense multilayer (M). which resulted in induction of E-cadherin and reduction of hNanos1 with increasing cell density in both Smad4- and mock- transfected SW480 cells (Fig. 2C). which we originally called hEC-Rep1a for human E-cadherin hNanos1 disturbs E-cadherin–dependent cell-cell adhesion. repressed 1a, showed that the originally cloned cDNA fragment To determine the intracellular localization and function of was part of the 3¶ untranslated region (UTR) of the human hNanos1, we generated a human epithelial cancer cell system Nanos1 transcript (Fig. 1A), an orthologue of the Drosophila Nanos with inducible expression of Myc-tagged hNanos1. Addition of gene (16). According to the UCSC human genome browser (NCBI doxycycline induces tagged hNanos1 in DLD1-Teton-hNanos1 Build 35),5 hNanos1 is a single-exon gene located on chromosome clones, but not in DLD1-Teton-mock clones, as shown by 10q26.11. Northern blot analysis confirmed the differential immunoblotting (55-kDa band in Fig. 3A). This induction was expression of hNanos1 in the MDA-MB-231 cell line relative to its associated with an obvious morphotypic switch: cells lost the E-cadherin expressing derivative MDA2BE5.36, and also revealed ability to form compact colonies and showed more and longer hybridization to two transcripts, one of 1,810 nt and one of 4,035 nt cytoplasmic protrusions (Fig. 3B), although total levels of the cell h (Fig. 1B). These transcripts differ only in the size of their 3¶-UTR junctional proteins E-cadherin, p120ctn, and -catenin were not sequences due to the use of an alternative poly-adenylation signal decreased (Fig. 3C). (AAAAAA) at position 1,764 of the transcript (Fig. 1A), which was In a cell aggregation assay, noninduced DLD1-Teton-hNanos1 À also confirmed by 3¶ RACE experiments (data not shown). cells formed large aggregates within 30 minutes (Fig. 3D, dox Alignment of the predicted hNanos1 ORF of 292-amino-acid N30). Aggregation required functional E-cadherin, as it was residues with those of other Nanos-related proteins revealed a high prevented by addition of an antibody (DECMA-1) that functionally blocks E-cadherin. When hNanos1 was induced by addition of degree of conservation of the COOH-terminal (CCHC)2 zinc finger domain of 52 amino acids (Fig. 1C and Supplementary Fig. S1). In doxycycline, the cells failed to form aggregates and their particle size distribution curve resembled that of cells treated with contrast, the sequences and lengths of their NH2-terminal parts were poorly conserved (data not shown), as had been described for DECMA-1 (Fig. 3D,+dox N30). DLD1-Teton-mock cells showed several Nanos orthologues (30). aggregation after 30 minutes irrespective of doxycycline treatment (Fig. 3D). In summary, induction of hNanos1 in epithelial tumor cells interfered with their ability to form E-cadherin–dependent three-dimensional aggregates, although total levels of E-cadherin 5 http://genome.ucsc.edu. were not decreased.

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Immunofluorescence analysis revealed that induced Myc-tagged complex in human tumor cells by relocalizing the junctional hNanos1 in DLD1-Teton-hNanos1 cells was expressed as cytoplas- proteins without altering their expression levels. mic dots with perinuclear concentration (Fig. 3E, e). E-Cadherin, hNanos1 induces cell migration, cell dispersion, and h-catenin, and p120ctn were highly enriched at cell-cell contacts in invasion of epithelial cells. An in vitro cell migration model was DLD1-Teton-mock cells irrespective of doxycycline treatment (data used to determine whether induction of hNanos1 influences cell not shown), and a similar situation was seen in DLD1-Teton- migration. We monitored the migration of DLD1-Teton-mock and hNanos1 cells not treated with doxycycline (Fig. 3E, a-d). However, DLD1-Teton-hNanos1 cells (with or without doxycycline) growing expression of Myc-tagged hNanos1 induced less intense staining at out from a confluent monolayer into adjacent free space. Phase- the cell contacts for each of the three proteins investigated (Fig. 3E, contrast images showed that 6 hours after the start of the experi- f-h), and in the case of p120ctn, even prominent cytoplasmic ment, induced DLD1-Teton-hNanos1 cells (+Dox)clearlyhad staining was seen although nuclei remained always unstained migrated a longer distance than untreated DLD1-Teton-hNanos1 (Fig. 3E, h). These results indicate that strong expression of cells and DLD1-Teton-mock cells (with or without doxycycline; hNanos1 interferes with the function of the E-cadherin junctional Fig. 4A). Migration was characterized quantitatively in this assay.

Figure 3. Inducible expression of hNanos1 interferes with E-cadherin functionality in epithelial cells. A, immunoblotting using an anti-Myc antibody shows the presence of Myc-tagged hNanos1 in doxycycline-induced DLD1-Teton-hNanos1 cell clones, but not in doxycycline-treated DLD1-Teton-mock clones. B, expression of hNanos1 in doxycycline-treated DLD1-Teton-hNanos1 cells induces a clear morphotypic switch. C, immunoblotting analysis of protein levels expressed in DLD1-Teton-mock and DLD1-Teton- hNanos1 cells before (Àdox) or after (+dox) induction of Myc-tagged hNanos1 protein. The levels of GAPDH served as loading controls. D, aggregation assays of DLD1-Teton-mock and DLD1-Teton- hNanos1 cells. Particle size distribution curves are shown for cells that were either induced (+dox) or not (Àdox) for 48 hours before the assay, in the presence or absence of an E-cadherin–blocking antibody (+DECMA-1 or ÀDECMA-1) during the assay. The starting population is indicated as N0. Aggregation after a 30-minute incubation (N30) was always reversed by DECMA-1 (y and E). Whereas both induced and noninduced mock cells formed large aggregates (n and .; left), hNanos1-induction (+dox) abrogated aggregation of DLD1- Teton-hNanos1 cells (n vs .; right). E, immunofluorescence analysis of the cadherin/catenin complex in DLD1-Teton-hNanos1 cells before and after induction of Myc-tagged hNanos1 protein. Antigens detected are as indicated. Nuclei were counterstained with DAPI.

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Figure 4. hNanos1 induces directional migration of tumor cells on a two-dimensional substrate and invasion into three-dimensional collagen type I. A, phase-contrast micrographs of the outgrowth periphery of DLD1-Teton-mock and DLD1-Teton-hNanos1 cells, either left untreated (ÀDox) or treated with doxycycline (+Dox)att =0(a-d) and t = 1 hour (e-h). Bar,11Am. B, the migration trajectories of the cells were computed from images recorded every 15 minutes for 1 hour. Trajectories are shown for the noninduced (ÀDox) and induced (+Dox) DLD-1-Teton-mock and DLD1-Teton-hNanos1 cells. Bar,11Am. C, migration speeds of DLD1-Teton-mock and DLD1-Teton-hNanos1 cells, either left untreated or treated with doxycycline. Columns, mean of three different experiments; bars, SD. **, P < 0.01. D, induction of hNanos1 results in invasion of DLD1-Teton-hNanos1 cells into type I collagen gels (filled columns). White columns, data for DLD1- Teton-mock cells. Treatment with the E-cadherin–blocking antibody DECMA-1 was included as a positive control, resulting in induction of invasion in all conditions.

Migration trajectories of individual cells showed that the move- To further evaluate whether hNanos1 expression correlates with ments of DLD1-Teton-mock cells (untreated and treated) and invasion into a three-dimensional matrix, we did a collagen type-I untreated DLD1-Teton-hNanos1 cells were restricted and random, invasion assay. Expression of hNanos1 strongly induced invasive- in contrast to the strikingly directional migration of the induced ness in DLD1-Teton-hNanos1 cells, similar to that induced by the DLD1-Teton-hNanos1 cells (Fig. 4B). In addition, the migration E-cadherin–blocking antibody DECMA-1 (Fig. 4D). speed was significantly increased by 81% on induction of hNanos1 hNanos1 interacts with the Armadillo catenin p120ctn. In expression in the DLD1-Teton-hNanos1 cells (29.23 F 7.56 Am/h) as Drosophila, Nanos protein is recruited by Pumilio to the Nanos compared with the untreated DLD1-Teton-hNanos1 cells (16.14 F response elements in the 3¶-UTR of target mRNAs (31). The crystal 3.99 Am/h; Fig. 4C). No difference in cell migration speed between structure of the Pumilio Puf domain, consisting of eight so-called the untreated and treated DLD1-Teton-mock cells was observed. In PUM repeats forming a positively charged superhelix, resembles conclusion, we show that hNanos1 induction in DLD1-Teton- that of the Armadillo repeats (32). Based on the Nanos-Pumilio hNanos1 cells results in a rapid and directional cell migration. interaction and the topological similarities of the PUM and The changes in the migratory capacity of hNanos1-expressing Armadillo repeats, we investigated the possibility that hNanos1 cells were confirmed in a cell dispersion assay (ref. 28; Supplemen- interacts with human Armadillo proteins. Myc-tagged Nanos tary Fig. S2). Induction of hNanos1 expression in DLD1- indeed formed a complex with endogenous p120ctn in induced Teton-hNanos1 cells resulted in a more random spatial cell (+dox) DLD1-Teton-hNanos1 cells (Fig. 5A) but did not associate distribution of these cells compared with control cells, reflecting with the non-Armadillo repeat protein a-catenin (Fig. 5A) or with increased migratory and invasive properties of hNanos1-expressing E-cadherin (data not shown), whereas a-catenin associated with cells. E-cadherin as expected (not shown). These findings were

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. hNanos1 Induces Tumor Cell Migration and Invasion consolidated and extended in Madin-Darby canine kidney-Tetoff- line MDA-MB-231. We isolated a partial cDNA that was down- hNanos1 cells with inducible expression of hNanos1: reciprocal regulated in cells reexpressing E-cadherin. The completed full- coimmunoprecipitations showed that hNanos1 interacts with both length cDNA sequence was highly homologous to the evolutionarily endogenous h-catenin and endogenous p120ctn (data not shown). conserved gene Nanos. To identify which part of hNanos1 mediated this interaction with All the Nanos and Nanos-related proteins that have been p120ctn, we extended our analysis to HEK293T cells transiently characterized in several organisms contain a conserved COOH- cotransfected with constructs encoding full-length p120ctn (iso- terminal RNA-binding (CCHC)2 zinc finger domain (Fig. 1C and form 3A) and Myc-tagged hNanos1 (full-length protein, NH2- Supplementary Fig. S1; ref. 20, 21, 30, 35). In Drosophila, Nanos terminal or COOH-terminal fragment). Interestingly, we found that cooperates with Pumilio to repress translation by binding to Nanos the hNanos1-p120ctn interaction was mediated by the NH2- terminal part of hNanos1, which lacks the evolutionarily conserved zinc finger domain (Fig. 5B). hNanos1 abolishes arborization caused by p120ctn overexpression. The observed interactions between hNanos1 and Armadillo proteins indicate that hNanos1 might affect the functions of these interaction partners. Because p120ctn is known to function in cell migration by modulating Rho GTPases in the cytoplasm (reviewed in ref. 13), we analyzed the effect of hNanos1 expression on this function. Overexpression of p120ctn in epithelial cells increases lamellipodia formation. This effect is particularly striking in fibroblasts, in which overexpressed p120ctn induces dramatic arborization (33). Therefore, we analyzed the effect of hNanos1 expression on p120ctn functionality in transfected NIH 3T3 fibroblasts. Exogenous p120ctn and hNanos1 both resided in the cytoplasm in these cells. Coexpression of either full-length or an NH2-terminal fragment of hNanos1 in NIH 3T3 cells expressing high levels of ectopic p120ctn diminished formation of dendrite- like structures (Fig. 5C, b and c), whereas cotransfection of a backbone vector did not interfere with the phenotype induced by p120ctn (Fig. 5C, a). Notice in c and f that a cell that expresses EGFP-p120ctn but not the hNanos1 fragment still exhibits arborization, in contrast to a cell coexpressing both proteins.

Discussion E-Cadherin expression is frequently down-regulated in many different types of tumors (34). Because E-cadherin is a key cell-cell adhesion protein in epithelial cells, loss of its expression or function diminishes cell-cell contacts and contributes to tumor progression and invasion. However, as E-cadherin inactivation may also occur early in certain precursor lesions, its loss is thought to induce intracellular signaling that promotes tumor development and progression. Because little is known about such signaling pathways, we screened for transcripts that are differentially expressed on E-cadherin reexpression in the breast cancer cell

Figure 5. The NH2-terminal domain of hNanos1 associates with the p120ctn Armadillo protein, and hNanos1 expression abolishes p120ctn-induced arborization. A, coimmunoprecipitation experiments on lysates of the indicated DLD1-Teton cell lines, treated or not treated with doxycycline for 48 hours, show association of Myc-tagged hNanos1 with p120ctn, but not with a-catenin. B, reciprocal coimmunoprecipitation on lysates of HEK293T cells, transiently cotransfected with a human p120ctn (isoform 3A) construct and a hNanos1 construct, encoding either hNanos1 full-length (FL), its COOH-terminal zinc finger domain (ZnF), or its NH2-terminal domain (N-term). p120ctn associates with the full-length hNanos1 and with its NH2-terminal domain. C, transient cotransfection of NIH 3T3 fibroblast cells with 0.5 Ag of pCS3 backbone vector in combination with 0.25 Ag of a construct expressing EGFP-p120ctn fusion protein does not interfere with p120ctn-induced arborization (a; GFP fluorescence). In contrast, cotransfection with 0.5 Ag of a pCS3-hNanos1 construct, encoding either full-length hNanos1 or its NH2-terminal domain, abolishes formation of the dendrite-like extensions (b and c; GFP fluorescence). Cells expressing ectopic hNanos1 were visualized by immunostaining with anti-Myc-tag antibody (d-f). www.aacrjournals.org 10013 Cancer Res 2006; 66: (20). October 15, 2006

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Cancer Research response elements located in the 3¶-UTR of target transcripts (e.g., (13, 33). This extensive branching of cellular protrusions is known hunchback and cyclin B mRNAs; refs. 36, 37). Nanos regulates to result from p120ctn-induced reduction in Rho activity with several processes in Drosophila, including differentiation of the concomitant activation of Rac1 and Cdc42. This indicates a link anterior-posterior body axis (16), control of neuronal dendrite between hNanos1 and Rho GTPase signaling via cytoplasmic morphogenesis (38), establishment of germ cell identity (39), self- p120ctn. The underlying mechanisms deserve further investiga- renewal of germ line stem cells (40), migration and survival of tion. Unfortunately, several attempts to convincingly show an primordial germ cells (17, 18), oogenesis and spermatogenesis (19). effect of hNanos1 on the activity of small GTPases of the Rho Mice deficient in the individual Nanos genes are viable family were unsuccessful because the doxycycline treatment itself (20, 21). Nanos1 knockout mice are fertile and exhibit no obvious had a strong negative effect on the levels of GTP-bound Rac and phenotypic abnormalities. In contrast, Nanos2 is essential for germ Rho (data not shown). Nonetheless, it is worth mentioning that cell development in male mice, and Nanos3 is important in both cytoplasmic p120ctn has been observed in different types of male and female sexual differentiation. A human Nanos protein tumors (43–46). The cytoplasmic localization of hNanos1 induced was reported to interact with human Pumilio-2 and to be abundant in our epithelial tumor cell system supports the possibility that in germ line stem cells (22). hNanos1 has a function in the migratory effects mediated by In Drosophila, Nanos expression is regulated at the translational cytoplasmic p120ctn (7). It is tempting to speculate on a similar level (41). Whereas most Nanos mRNA is translationally repressed mechanism to explain the role of Nanos in particular normal cell in the Drosophila embryo, translational activation occurs at the types, such as in primordial germ cell migration (17) and embryonic posterior pole. This switch from translational repression neuronal dendrite morphogenesis (38), especially because we saw to activation is mediated by cis-acting signals in the 3¶-UTR of the an obvious increase in cytoplasmic p120ctn on hNanos1 induction Nanos mRNA (41, 42). Whether Nanos expression can be transla- in the tumor cell system we analyzed. tionally regulated also in mammals needs further investigation. Our The Armadillo protein p120ctn interacts with the NH2-terminal identification of two hNanos1 transcripts that differ only in the part of hNanos1, which is conserved only among mammals.6 The lengths of their 3¶-UTR might be relevant to this. In E-cadherin– more broadly conserved COOH-terminal part of Nanos and Nanos- deficient cancer cells, hNanos1 expression seems to be regulated at related proteins, containing the (CCHC)2 zinc finger domain, is the transcriptional level. We cloned the minimal hNanos1 promoter thought to represent the ancestral function of Nanos proteins in in a luciferase reporter construct and found that reporter gene germ line development and survival in various invertebrate and activity in MDA-MB-231 cells is higher than in the E-cadherin vertebrate species (47, 48). One can speculate that other specialized transfectant.6 This implies the presence of E-cadherin–sensitive functions of Nanos genes were generated later in evolution in regulatory sequences. weakly conserved domains (30, 35, 48). p120ctn is dispensable for We found that expression of the hNanos1 transcript was reduced cadherin stability and adherens junction assembly in Drosophila following E-cadherin reexpression in a human cancer cell line. and Caenorhabditis elegans (49, 50), but it is critically required in Analysis of a series of cell lines showed an inverse correlation mammals (reviewed in refs. 7, 8). The combination of these data between E-cadherin and hNanos1 expression. In the human supports the possibility that the p120ctn-hNanos1 interaction has colorectal cancer cell line SW480, hNanos1 expression was an important function in mammals but not in evolutionarily inversely regulated by various conditions modulating E-cadherin distinct organisms, such as flies and worms. expression. On TPA treatment, hNanos1 expression diminished In summary, our data show that hNanos1 is a downstream target during the first 6 hours, then increased during the next 18 hours; gene of E-cadherin capable of interfering with the proadhesive and this is in line with the attribution of different effects to TPA anti-invasive functions of E-cadherin and of interacting with depending on the length of treatment. Indeed, despite the strong Armadillo proteins including p120ctn. Further research should activation of protein kinase C by short-term TPA treatment, down- investigate the following: (i) how hNanos1 transcript levels are regulation of this important kinase by sustained TPA treatment is regulated; (ii) how it can interfere with E-cadherin functionality; well known. (iii) how it activates directional cell migration and invasion; (iv) We then sought to identify the function that hNanos1 might have what the molecular and biological consequences are of its in cancer cells. Using functional assays, we showed that hNanos1 interaction with Armadillo proteins; and (v) whether hNanos1 is expression impairs the function of E-cadherin in cell-cell adhesion. an appropriate molecular target for therapy of certain cancers in Levels and extracellular exposure of E-cadherin protein were not which the function of E-cadherin is defective. changed on hNanos1 induction in the epithelial cells analyzed, indicating that hNanos1 interferes with the functionality of E-cadherin rather than with its expression. Furthermore, hNanos1 Acknowledgments expression promoted cell migration and invasion of epithelial cells Received 8/30/2005; revised 7/10/2006; accepted 8/14/2006. in vitro. Together, these data indicate that hNanos1 has a function Grant support: Fund for Scientific Research (FWO)-Flanders, Geconcerteerde Onderzoeksacties of Ghent University, Interuniversity Attraction Poles Programme of in cancer cells deficient in E-cadherin. the Belgian Science Policy, Belgian Federation against Cancer, and Sixth Framework Intriguing in this respect is our discovery that hNanos1 binds Programme of the European Union (BRECOSM); Instituut voor de aanmoediging van the Armadillo protein p120ctn and triggers its translocation from Innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Flanders) and young scientist scholarship from the Centrum voor de studie en behandeling van cell-cell contacts to the cytoplasm. The hNanos1-p120ctn gezwelziekten, Ghent-Flanders (K. Strumane); and postdoctoral fellowships from association is correlated with abolishment of the arborization Fund for Scientific Research (FWO)-Flanders (G. Berx and C. Stove). The costs of publication of this article were defrayed in part by the payment of page phenotype induced in fibroblasts by overexpression of p120ctn charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank P. Vermassen and K. Staes for technical assistance; Drs. S. Bogaerts, J. Comijn, J. van Hengel, and G. Vanpoucke for stimulating discussions; Dr. A. Bredan for critical reading of the manuscript; and Dr. John G. Collard for providing PAK-CRIB 6 Our unpublished data. peptide for the Rac activity assay.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. E-Cadherin Regulates Human Nanos1, which Interacts with p120ctn and Induces Tumor Cell Migration and Invasion

Kristin Strumane, Arnaud Bonnomet, Christophe Stove, et al.

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