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Tumor and Stem Cell Biology

ATIP3, a Novel Prognostic Marker of Breast Cancer PatientSurvival, Limits Cancer Cell Migration and Slows MetastaticProgression by Regulating Microtubule Dynamics

Angie Molina1,2,3, Lauriane Velot1,2,3, Lydia Ghouinem1,2,3, Mohamed Abdelkarim4,7,Benjamin Pierre Bouchet10, Anny-Claude Luissint1,2,3, Im�ene Bouhlel1,2,3, Marina Morel1,2,3,El�ene Sapharikas1,2,3, Anne Di Tommaso1,2,3, St�ephane Honor�e8, Diane Braguer8, Nad�ege Gruel5,Anne Vincent-Salomon5, Olivier Delattre5, Brigitte Sigal-Zafrani5, Fabrice Andr�e9, Benoit Terris1,2,3,6,Anna Akhmanova10, M�elanie Di Benedetto4,7, Clara Nahmias1,2,3, and Sylvie Rodrigues-Ferreira1,2,3

AbstractMetastasis, a fatal complication of breast cancer, does not fully benefit from available therapies. In this

study, we investigated whether ATIP3, the major product of 8p22MTUS1 gene, may be a novel biomarker andtherapeutic target for metastatic breast tumors. We show that ATIP3 is a prognostic marker for overallsurvival among patients with breast cancer. Notably, among metastatic tumors, low ATIP3 levels associatewith decreased survival of the patients. By using a well-defined experimental mouse model of cancermetastasis, we show that ATIP3 expression delays the time-course of metastatic progression and limits thenumber and size of metastases in vivo. In functional studies, ATIP3 silencing increases breast cancer cellmigration, whereas ATIP3 expression significantly reduces cell motility and directionality. We report herethat ATIP3 is a potent microtubule-stabilizing protein whose depletion increases microtubule dynamics. Ourdata support the notion that by decreasing microtubule dynamics, ATIP3 controls the ability of microtubuletips to reach the cell cortex during migration, a mechanism that may account for reduced cancer cell motilityand metastasis. Of interest, we identify a functional ATIP3 domain that associates with microtubules andrecapitulates the effects of ATIP3 on microtubule dynamics, cell proliferation, and migration. Our study is amajor step toward the development of new personalized treatments against metastatic breast tumors thathave lost ATIP3 expression. Cancer Res; 73(9); 2905–15. �2013 AACR.

IntroductionThe occurrence of distant metastasis is a dreadful compli-

cation of breast cancer and a leading cause of death bymalignancy in women worldwide. Metastasis is a multistepprocess that involves cancer cell migration and invasion across

the extracellular matrix to reach the blood flow, followed byextravasation and colonization of secondary organs (1). Amongmillions of invasive cancer cells that reach the blood circula-tion, only few will establish at distant sites and grow asmetastases (2–5). Breast cancer metastases can remain latentfor several years following primary tumor removal, and theidentification ofmolecularmarkers thatmay predict the risk ofmetastasis occurrence, and/or progression is of invaluable helpfor the follow-up of the patients and choice of therapeuticoptions (5, 6). Over the past decade, extensive studies have ledto the classification of breast tumors into distinct molecularsubtypes, allowing subsequent development of efficient tar-geted treatments for a majority of primary tumors (7–9).However, available therapies have limited effect on cancermetastasis and new genetic determinants contributing toessential steps of the metastatic process need to becharacterized.

Microtubule-targeting drugs such as taxanes are used forstandard first-line treatment of breast cancer metastasis, andnew microtubule-targeting agents, such as epothilones anderibulin, are under clinical evaluation (10). Microtubules arepolarized and highly dynamic structures that rapidly switchbetween periods of polymerization (growth) and depolymer-ization (shrinkage) at the plus ends, a process termed dynamic

Authors' Affiliations: 1Institut National de la Sant�e et de la RechercheMedicale (Inserm), U1016, Institut Cochin; 2CNRS, UMR8104; 3Universit�eParis Descartes, Sorbonne Paris Cit�e; 4CNRS7033, UMRS940, IGM; 5Insti-tut National de la Sant�e et de la Recherche Medicale (Inserm), U830,Translational ResearchDepartment, InstitutCurie; 6PathologyDepartment,Hopital Cochin, Paris; 7Universit�e Paris 13, Bobigny; 8Aix-Marseille Uni-versity, CRO2, Institut National de la Sant�e et de la Recherche Medicale(Inserm) UMR911, Marseille; 9Department of Medicine, Institut GustaveRoussy, Institut National de la Sant�e et de la Recherche Medicale (Inserm),U981, Villejuif, France, and 10Cell Biology, Faculty of Science, UtrechtUniversity, Utrecht, the Netherlands

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

C. Nahmias and S. Rodrigues-Ferreira contributed equally to this work.

Corresponding Author:Clara Nahmias, Institut Cochin, Dept EMC, 22 rueM�echain 75014 Paris, France. Phone: 33-1405-16410; Fax: 33-1405-16430; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-3565

�2013 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 2905

on July 8, 2020. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 8, 2013; DOI: 10.1158/0008-5472.CAN-12-3565

http://cancerres.aacrjournals.org/

instability (11–13). The extent and rate of microtubule growth,as well as transitions between growth and shrinkage, areparameters of dynamic instability that can be measured bytracking end-binding proteins at the microtubule plus ends(13–15). Dynamic instability is essential for the microtubuleplus ends to explore the cytosol and ensure cytoskeletonreorganization during cell division and migration. Targetingthe expression or activity of metastasis genes that regulatemicrotubule dynamics represents a promising option for can-cer therapy.

ATIP3 is a microtubule-associated protein encoded by 8p22candidate tumor suppressor gene MTUS1 (16–18). We havepreviously shown that ATIP3/MTUS1 levels are significantlydownregulated in 47.7% of invasive breast carcinomas and62.4% ofmetastatic tumors (19). Restoring ATIP3 expression atnormal levels in breast cancer cells significantly reduces cancercell proliferation in vitro and tumor growth in vivo (19).However, effects of ATIP3 on breast cancer metastasis havenot yet been evaluated.

In this study, we investigated whether ATIP3 may representa new biomarker and therapeutic target for breast cancermetastasis. We present evidence that low ATIP3 levels corre-late with the decreased probability of survival among patientswith breast cancer metastasis, and that ATIP3 expression intoATIP3-deficient cancer cells markedly impairs the establish-ment of metastatic foci in vivo. Loss of ATIP3 increases breastcancer cell migration and alters microtubule dynamics. Weshow that ATIP3 associates with microtubules through acentral basic domain that retains the functional properties ofthe full-length protein. Our study thus identifiesATIP3 as a newpromising therapeutic target against metastatic breast tumorsof poor prognosis.

Materials and MethodsBreast tumor samples and gene arrays

Microarray data for a series of 150 infiltrating ductal primarybreast carcinomas and 11 normal breast tissues from theInstitut Curie (Paris, France) and clinical data for the patientswere described elsewhere (19, 20). Gene expression profilesfrom an independent cohort of 162 invasive breast carcinomaswere obtained from patients included in the prospectivedatabase of the Institut Gustave Roussy (IGR; Villejuif, France)between 1984 and 1994. This study was approved by theInstitutional Review Boards of the IGR. Data have been sub-mitted to the Array Express data repository at the EuropeanBioinformatics Institute (Saffron Walden, United Kingdon;http://www.ebi.ac.uk/arrayexpress/) under accession numberE-MTAB-1389. MTUS1 gene expression in a meta-analysis of2,898 patients with breast cancer with known clinical outcomewas retrieved from Kaplan–Meier plotter database (21, 22).

Cell lines, plasmid constructs, and transfectionsHuman breast cancer cell linesMDA-MB-468 andMCF7 and

stable clones were described previously (19). MDA-MB-231-Luc-D3H2LN breast cancer cells (designated D3H2LN)obtained from Caliper Life Science (Xenogen) were derivedfrom an in vivo-selected metastatic subclone of MDA-MB-231cells expressing luciferase and grown as described (23). HeLa

cells were provided by Dr. Mounira Amor-Gueret (InstitutCurie, Orsay, France). RPE-1 [human telomerase reverse tran-scriptase (h-TERT)-immortalized, retinal pigment epithelial]cells were from Dr. Franck Perez (Institut Curie, Paris). MRC5-SV lung fibroblasts were grown in Dr. A. Akhmanova's labo-ratory as described (24). All cells were used at passages 2 to 20after thawing and grown as described by the provider. Cellswere routinely authenticated by morphologic observation andtested for absence of mycoplasma contamination usingMycoAlert Assay detection kit (Lonza).

Plasmid constructs are described in the SupplementaryMethods. Transfections using ATIP3-specific siRNAs (si#1 andsi#2) were conducted as described (19) and verified by immu-noblotting using anti-MTUS1 polyclonal antibodies (ARP-44419, Aviva Systems).

Intracardiac experimental mouse model of metastasisExperimental metastasis was conducted as described

(23, 25, 26) following intracardiac injection of stableATIP3-negative [WT (wild-type), GFP] or positive (Cl3, Cl6)D3H2LN cell clones. All injected cells showed similar via-bility as measured by Annexin V apoptosis kit (BeckmanCoulter). The experiment was carried out with the approvalof the D�epartement d'Exp�erimentation Animale, Institutd'H�ematologie, Hopital St-Louis ethical committee, and wasconducted twice (9 mice per group).

Clonogenicity, cell migration, and adhesion assaysAnalyses of colony formation, Boyden chambers chemo-

taxis, transendothelial migration, wound healing, and celladhesion were conducted as described (23). Time-lapsevideomicroscopy analyses of cell motility are described inthe Supplementary Methods. For cell polarity measure-ments, transiently transfected D3H2LN were allowed tomigrate for 90 minutes and analyzed using bright fieldmicroscopy. Polarized cells were identified on the basis ofnucleus position and cytoplasm extension at the leadingedge.

Immunostaining, fluorescence microscopy, analysis ofmicrotubule dynamics

Cells were plated on glass coverslips and transfected for 24hours (plasmids) or 72 hours (siRNA), fixed in ice-coldmethanol for 5 minutes, and incubated for 1 hour at roomtemperature with anti-a-tubulin clone F2C (27), monoclonalanti-g-tubulin (Sigma), anti-end-binding 1 (EB1; clone 5; BDBiosciences), or anti-acetylated tubulin (clone 6-11B-1; Sigma).Secondary antibodies and fluorescence images capture aredescribed in the Supplementary Methods.

Linescan analyses of a-tubulin and EB1 fluorescence inten-sity were done (ImageJ) on a 6 mm line along the length ofmicrotubule tip. At least 10 microtubules per cell in 4 separatecells were measured. EB1-comet maximal intensity wasobtained by subtracting the intensity value of the EB1-dot(100 a.u.) to the maximal staining intensity.

Analyses of microtubule stability, regrowth, and microtu-bule dynamic instability are described in the SupplementaryMethods.

Molina et al.

Cancer Res; 73(9) May 1, 2013 Cancer Research2906




Statistical analysisStatistical analyses were done using JMP-7 and GraphPad

Prism softwares. Overall survival (OS) curves were plottedaccording to the method of Kaplan–Meier and compared bythe log-rank test. Data in bar graphs (mean þ/� SD) wereanalyzed using 2-tail unpaired Student t test. Dot plot analyseswere done using Mann–Whitney test. P < 0.05 was consideredstatistically significant.

ResultsATIP3 is a prognostic marker of poor outcome inmetastatic breast cancerThe prognostic value of ATIP3 as a marker for metastatic

progression and OS was evaluated in 3 independent cohorts ofpatients with breast cancer. Comparison ofMTUS1 Affymetrixprobeset intensities with clinicopathologic data of the patientsin a panel of 150 invasive breast carcinomas (SupplementaryTable S1) showed that the overall probability of survival isstrongly reduced in patients with tumors expressing low ascompared with normal ATIP3 transcript levels (Fig. 1A andSupplementary Fig. S1A). Relapse-free survival (RFS) of thepatients was also significantly reduced in low ATIP3-expres-sing tumors (Supplementary Fig. S1B). Similar results wereobtained by analyzing MTUS1 levels in an independent cohortof 162 patients with breast cancer (Fig. 1B and SupplementaryTable S2) and in a meta-analysis of 2,898 patients with breastcancer (Fig. 1C and Supplementary Fig. S1C and S1D). Of note,correlation between ATIP3 expression and OS of the patientswas independent of the estrogen receptor (ER) status of thetumor (Fig. 1D).Tumors were then classified according to their metastatic

properties and MTUS1 probeset intensities were comparedwith the probability of patient survival. As shown in Fig. 1E, thepercentage of patients withmetastatic disease surviving after 5years was markedly reduced when tumors expressed lowATIP3 (6.25%) compared with normal ATIP3 levels (31.6%),whereas in patients with nonmetastatic tumors, 5-year sur-vival (Fig. 1E and Supplementary Fig. S1E) and OS rates (Fig. 1Fand H) were independent of the levels of ATIP3. Withinpatients with metastatic disease, OS rates (Fig. 1F and H andSupplementary Fig. S1E) and survival time (Fig. 1G and I) werealso reducedwhen tumors expressed low levels of ATIP3. Thus,ATIP3 expression is an important indicator of clinical outcomefor patients with metastatic breast tumors. Correlationbetween low ATIP3 levels and reduced survival rates amongpatients with advanced breast cancer suggests major effects ofATIP3 on metastatic progression.

ATIP3 limits breast cancer metastatic colonization invivoIn vivo effects of ATIP3 on the metastatic potential of breast

cancer cells were evaluated using a well-defined experimentalmouse model of metastasis monitored by intravital biolumi-nescence imaging (23, 25, 26). Highly metastatic, ATIP3-neg-ative, D3H2LN breast cancer cells were transfected with eitherGFPorGFP-ATIP3, and independent stable cell clones (Cl3 andCl6) expressing moderate levels of ATIP3 were selected (Fig.2A, left). All cell clones exhibited similar levels of luciferase

activity (Fig. 2A, right). Metastatic cancer cells were injectedintracardiacally into the bloodstream of nude mice to reca-pitulate the late, rate-limiting, steps of the metastatic process,and examinemetastatic dissemination to various organs whileavoiding any effect of ATIP3 on primary tumor growth. Fourgroups of 18 mice were analyzed in two independent experi-ments. For each animal, the total number of metastatic fociand the number of photons/s were quantified every 2 days for24 days (Supplementary Table S3). As shown in Fig. 2B, thetime-course of metastasis formation was markedly delayed inmice injected with ATIP3-positive as compared with ATIP3-negative cell clones. The number of cancer cells growing atsecondary sites increased exponentially from day 17 afterinjection of ATIP3-positive clones, as compared with day 10for mice injected with control cells (Fig. 2B). As shown in Fig.2C, the number of mice developing metastasis was stronglydiminished upon ATIP3 expression. Importantly, the numberof detectable metastases per mouse was also significantlyreduced at all times in the presence of ATIP3 (Fig. 2D). At day24, the number of mice invaded with large metastases reached13 of 18 (72.2%) in the control group as compared with 2 of 18(11.1%) following injection of ATIP3-positive cells (Fig. 2E andF), indicating a prominent effect of ATIP3 on cancer cellgrowth and colonization at secondary sites. Accordingly, onday 24, the total number of photons/s per mouse was 50- and25-fold lower following injection with Cl3 and Cl6 clones,respectively, compared with WT (Supplementary Fig. S2A).For ethical reasons, mice had to be sacrificed at day 24,therefore OS of the two groups of mice could not be quantified.Furthermore, ex-vivo and histologic analysis of metastaticnodules (Supplementary Fig. S2B) confirmed that biolumines-cent signals indeed correspond to metastases of human tumorcells having infiltrated mouse tissues. Metastases were mainlydetected in the bones, the lungs, and the brain, which are themost frequent sites of metastatic dissemination of humanbreast tumors. No preferential location of metastatic nodulesin ATIP3-positive versus ATIP3-negative cell types could beobserved. Altogether, these results identify ATIP3 as a potentantimetastatic molecule, and support a role for ATIP3 inmetastatic growth and colonization in vivo.

ATIP3 impairs breast cancer cell proliferation andmigration

Metastatic colonization involves cancer cell migration, inva-sion through the extracellular matrix, and proliferation at thesecondary site. As expected from our previous studies (19), cellproliferation was significantly reduced in ATIP3-positiveclones Cl3 and Cl6 as compared with control (SupplementaryFig. S3A). In addition, Boyden chambers assays of chemotaxisand invasion revealed more than 90% reduction in the promi-gratory properties of Cl3 compared with GFP (Fig. 3A). Similareffects were observed using stably transfected MDA-MB-231cells (Supplementary Fig. S3B). Conversely, ATIP3 silencing inmetastaticMDA-MB-468 breast cancer cells expressing endog-enous ATIP3 induced a 2- to 2.5-fold increased chemotacticmigration (Fig. 3B), suggesting that cancer cells having lostATIP3 may acquire a promigratory phenotype and may bemore prone to develop distant metastasis.

Antimetastatic Effects of ATIP3 in Breast Cancer

www.aacrjournals.org Cancer Res; 73(9) May 1, 2013 2907




The ability to migrate through a monolayer of endothelialcells (transendothelialmigration)was significantly reduced (58� 16%) in Cl3 compared with control (Fig. 3C). Adhesion ofclones Cl3 and Cl6 to endothelial cells was significantly ele-vated (3-fold and 2.8-fold, respectively) compared with WT(Fig. 3D), suggesting that increased tumor-endothelial celladhesionmay account for reduced transendothelial migration.

Cell adhesion to collagen I was also increased in Cl3 (1.85-fold)and Cl6 (1.93-fold) compared with WT (Fig. 3E). Altogether,these data indicate that ATIP3 concomitantly increases celladhesion and limits cell migration.

The consequences of ATIP3-silencing on cancer cell motilitywere analyzed in HeLa cells that express endogenous ATIP3and are well suited for analyses of wound closure. As shown

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Figure 1. Low levels of ATIP3 predict poor outcome amongmetastatic tumors. A, OS curves for patients from the Institut Curie cohort, with tumors expressingnormal (> 0.5) or low (< 0.3) ATIP3 levels, relative to the median value of MTUS1 probeset (212096_s_at) in normal tissues. B, OS curves for patientsfrom the IGRcohort,with tumors expressingnormalor lowATIP3 levels [inferior or superior to themedian valueofMTUS1probeset (A_23_P347169) intensitiesin the 162 tumors analyzed]. C, OS curves for patients with tumors expressing normal to high (gray) or low (black) MTUS1 (212096_s_at) usingKaplan–Meier plotter. The best performing threshold was used as a cutoff. D, OS curves from patients expressing normal or low ATIP3 as in A,among ER negative (ER�, left) and positive (ERþ, right) tumors. E, percentage of patients remaining alive after 5 years with nonmetastatic (�) andmetastastic (þ) tumors expressing ATIP3 levels as in A. F, OS curves for patients with nonmetastatic (left) or metastatic (right) tumors expressingATIP3 levels as in A. G, OS time (in months) for patients with metastatic tumors expressing ATIP3 levels as in A. Median values are on the right. �, P¼ 0.0119.H, OS curves as in F for patients from the IGR cohort. Nonmetastatic (left) and metastatic (right) tumors were classified according to ATIP3 levels as in B.I, OS time (in months) for patients as in G with metastatic tumors classified as in H. Number of patients is under brackets. �, P ¼ 0.0172.

Molina et al.





in Fig. 3F, ATIP3 silencing in HeLa cells increased (1.84- to 2.6-fold) directional migration. Conversely, stable ATIP3 expres-sion into D3H2LN (Cl3 and Cl6, Fig. 3G) and MCF7 cells(Supplementary Fig. S3C) significantly reduced wound closure.Time-lapsemicroscopy (SupplementaryMovies S1 and S2) andtracking of D3H2LN-migrating cells further indicated thatstable ATIP3 expression impairs both cancer cell velocity(0.34 mm/s and 0.55 mm/s for Cl3 and GFP clones, respectively;ref. Fig. 3H) and directionality (Fig. 3I). Similar results wereobtained (Supplementary Fig. S3D and S3E) by analyzing celltracking following transient transfection of GFP or GFP-ATIP3

into D3H2LN cells (Supplementary Movies S3 and S4). Ofnote, the number of GFP-ATIP3–positive cells reaching thewound edge was reduced compared with GFP-expressingcells. GFP-ATIP3 expressing cells were overtaken by nontrans-fected cells reaching the border of the wound (SupplementaryFig. S3F), further confirming the inhibitory effect of ATIP3 oncancer cell migration.

ATIP3 alters microtubule dynamicsWe hypothesized that ATIP3, being closely associated with

microtubules (19),may limit cell proliferation andmigration by

Figure 2. ATIP3 expression slowsmetastatic progression in vivo. A,characterization of stablytransfected D3H2LN cell clones.Left, immunoblots of nontransfected(WT) and GFP-ATIP3-expressingD3H2LN clones (Cl3, Cl6) using anti-GFP antibodies, reprobed with anti-a-tubulin (a-tub) antibodies. Right,measurement of luciferase activityper cell (n ¼ 3). B, number ofphotons/s per mouse (n ¼ 9) at days6 to 24 following tumor cellinoculation. C, left, representativepictures of bioluminescence (5/9mice) at day 17 following intracardiacinjection. Scale is on the right. Right,number of mice with at least onedetectable metastasis at day 17.D, total number of metastastic sitesper mouse at indicated days afterinoculation of control (Ctrl, WT andGFP, n ¼ 18) and ATIP3 positive(ATIP3, Cl3 and Cl6, n ¼ 18) cells.��,P < 0.01, ��,P < 0.001. E, numberof mice with large metastases atdifferent times after inoculation as inD. F, representative pictures (day 24)as in C.

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regulating microtubule dynamics. We first analyzed the con-sequences of ATIP3 depletion on the sensitivity of microtu-bules to nocodazole that prevents repolymerization of dynam-ic microtubules. Stable microtubules that are not affected bynocodazole treatment are typically stained by anti-acetylatedtubulin. As shown in Fig. 4A, ATIP3-silenced HeLa cells werehighly sensitive to nocodazole. The number of cells retainingstable microtubules was decreased by 51%� 10 and 53%� 14following transfection of siRNA#1 and siRNA#2 compared withcontrol. Conversely, stable transfection of GFP-ATIP3 intoMCF7 cells significantly increased thenumber of cells retainingstable, nocodazole-resistant, microtubules as assessed by anti-

acetylated tubulin labeling (Fig. 4B) and immunoblotting (Fig.4C). ATIP3 expression also significantly delayed microtubuleregrowth following nocodazole washout (Fig. 4D). At 5 min-utes, microtubule length around the centrosome was reducedby 57 � 20% in GFP-ATIP3 compared with GFP-transfectedclones, supporting the notion that ATIP3 may impair micro-tubule dynamics.

The effects of ATIP3 on microtubule dynamic instabilityparameters were further analyzed by measuring EB1 proteinaccumulation at the microtubule plus tips (13–15, 28, 29) inRPE-1 epithelial cells and lung fibroblasts (MRC5-SV), whichhave a sparse microtubule array and are well suited for

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Figure 3. ATIP3 reduces breastcancer cell migration. A, Boydenchamber migration of stableD3H2LN cell clones across filterscoated (coll) or not (no coll) withcollagen I. Results (percent) aremean � SEM (n ¼ 3). Right,representative picture of cellsmigrating to the bottom of the well.B, Boyden chamber assay usingATIP3-positive (WT and siCtrl) andATIP3-negative (si#1 and si#2)MDA-MB-468 cells. Results areshown as in A. Top, immunoblotsof MDA-MB-468 cells after siRNAsilencing [anti-MTUS1, reprobedwith anti-a-tubulin (a-tub)antibodies]. Right, representativepictures of the lower face of thefilter. C, transendothelial migration,mean � SEM (n ¼ 3). D and E,cancer cell adhesion (mean�SEM,n ¼ 3) to endothelial cells (D) andcollagen (E). F, migration of ATIP3-positive (siCtrl) and ATIP3-silenced(si#1, si#2) HeLa cells (n ¼ 3). Left,representativepictures ofwoundattimes T0 and T22. Right,quantification (percent) of woundclosure at T22. Results are mean�SEM (n ¼ 2). Top, immunoblots ofsiRNA-transfected HeLa cells as inB. G, directional migration ofstably transfected D3H2LN clones(n ¼ 4). Left, representativepictures of wound at times T0 andT7. Right, quantification of woundclosure at T4 and T7. Results aremean � SD (n ¼ 3). H and I, celltracking of D3H2LN stable clonesduring wound closure. H, cellvelocity scattered dot plot. I,diagrams of migration trajectories(12 hours). Number of cells is underbrackets. Directionality coefficient(Dir) is inside the graph. A, B, F, andG,magnification,�100. �,P < 0.05;��, P < 0.001; ��, P < 0.0001.

Molina et al.





distinguishing individualmicrotubule tips. As shown in Fig. 5A,ATIP3 expression in RPE-1 cells led to a significant reduction inthe number and size of EB1 comets that rather appeared asdots. Decreased accumulation of EB1 at microtubule plus endswas not associated with decreased EB1 expression (Supple-mentary Fig. S4A). In ATIP3-depleted HeLa cells, significantlymore EB1 comets of increased length and intensity weredetected compared with control cells (Fig. 5B), suggesting thatATIP3 silencing increases microtubule dynamics. Time-lapsetotal internal reflection fluorescence (TIRF) videomicroscopyanalysis of EB1-GFP comets (Supplementary Movie S5) andsubsequent microtubule-tips tracking indicated that microtu-bule growth episodes were significantly longer in ATIP3-silenced HeLa cells compared with control (Fig. 5C). ATIP3depletion increased microtubule growth rate and decreased

the time spent in pause aswell as the frequency of catastrophes(Fig. 5C), accounting for increased microtubule dynamics.Conversely, videomicroscopy of EB3-GFP comets followingexpression of mCherry-ATIP3 in MRC5-SV cells (Supplemen-tary Movie S6), and corresponding kymographs (Supplemen-tary Fig. S4B), indicated that ATIP3 expression decreasesmicrotubule dynamics and reduces the rate of microtubulegrowth.

Microtubule stabilization and decreased growth rate atthe cell periphery should be responsible for an inhibition ofmicrotubule targeting and capture at the cell cortex (30). Asshown in Fig. 5D, in migrating D3H2LN cells, microtubulesprojected radially toward the cell periphery and microtubuleplus ends were close to the cell edge (mean distance 1.43 �0.7 mm), whereas in the presence of ATIP3, microtubules

Figure 4. ATIP3 reducesnocodazole sensitivity andmicrotubule outgrowth. A,immunostaining (anti-a-tubulin(a-tub) and anti-acetylated tubulin[Ac-tub antibodies] of ATIP3-positive(siCtrl) and -negative (si#1) HeLacells incubated without (0) or with1 mmol/L nocodazole (Nz). Right,immunoblots of HeLa cells aftersiRNA silencing (anti-MTUS1,reprobed with antitubulinantibodies). Bottom, quantification(%) of cells retaining stablemicrotubules, mean � SEM (n ¼ 3).B, immunostaining of stable MCF7clones incubated with or without 10mmol/L nocodazole, as in A. Right,quantification as in A,mean�SEM (n¼ 3). C, immunoblot analysis ofacetylated-tubulin (Ac-tub) and ezrincontent in stably transfected MCF7clones, either nontreated (�) ortreated with DMSO (D) or increasingconcentrations of nocodazole. Right,quantification of the ratio betweenAc-tub and ezrin intensity. D,microtubule regrowth in transientlytransfected RPE-1 cells (n ¼ 4).Shown is a-tubulin staining atindicated times after nocodazole(10 mmol/L) washout. Right,quantification of microtubule densityat 4 mm around the centrosome,mean � SD (n ¼ 4 to 10 cells).�, P < 0.05; ��, P < 0.001. Scale bar,10 mm.

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were bended and more than 50% of microtubule tips did notreach the cell margin (mean distance 2.31� 1.2 mm). Of note,reduced ability of microtubules to reach the cell cortex inmigrating ATIP3-positive cells was accompanied by a 34%decrease in cell polarity (Fig. 5E). Taken together, theseresults suggest that ATIP3-dependent regulation of micro-tubule dynamics results in decreased ability of microtubules

to reach the cell cortex, which contributes to reduced cellpolarity and migration.

Microtubule-binding domain D2 recapitulates thefunctional effects of ATIP3

The ATIP3 polypeptide was cleaved into 3 fragments des-ignated D1, D2, and D3 (Fig. 6A), which were fused to GFP and

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Figure 5. ATIP3 regulatesmicrotubule dynamics. A,immunostaining (anti-EB1, anti-mCherry antibodies) of RPE-1 cellstransiently transfected withmCherry-ATIP3 (Ch-ATIP3). Insets,EB1 comet-like structures inATIP3-negative (1) and positive (2)cells. Distribution of EB1 (black),a-tubulin (dashed) andATIP3 (gray)at the microtubule tip (linescans)and quantification of cometsintensity (scattered dot plot).Number of comets analyzed isunder brackets. Shown is 1experiment out of 5. B, EB1localization in siRNA-silencedHeLa cells. Insets, EB1 comet-likestructures in ATIP3-positive (1) andATIP3-negative (2) cells.Distribution of EB1 (black) anda-tubulin (dashed) at themicrotubule tip (linescans).Quantification of comets intensityas in A. Shown is 1 experiment outof 3. C, time-lapse images ofsiRNA-silenced HeLa cellsexpressing EB1-GFP. Arrowheadindicates theposition of EB1cometover time (in seconds). Parametersof microtubule dynamics (EB1-GFP comets) in siRNA-transfectedHeLa cells (n ¼ 100 comets) areshown in scattered dot plot andhistograms. D, immunostaining[anti-a-tubulin (a-tub) and anti-GFP] of transfected D3H2LN inmigration. Arrows indicate thedirection of migration. Cell margin(black line) is visualized by brightfield microscopy. Insets showmicrotubule array at the cell borderof ATIP3-negative (1, 2) andGFP-ATIP3-positive (3, 4) cells.Right, immunoblots (anti-GFP,antitubulin) of transientlytransfected D3H2LN cells. Bottomleft, quantification of microtubulesreaching given distance from thecell cortex in GFP- and GFP-ATIP3- expressing cells. Bottomright, mean distance betweenmicrotubules and cell cortex.Number of microtubules analyzedis under brackets. E, quantification(percent) of polarizedD3H2LNcellsduring migration. Number ofcells is under brackets. �, P < 0.05;��, P < 0.001, ��, P < 0.0001.A, B, C, and D, scale bar, 10 mm.

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expressed in RPE-1 cells (Fig. 6B). As shown in Fig. 6C, the GFP-D1 fusion protein did not associate withmicrotubules and wasrather diffuse in the cytosol. Accordingly, GFP-D1 expressionhadno significant effect on the number, size, or intensity of EB1comets (Fig. 6D). In contrast, GFP-D2 clearly colocalized withthe microtubule cytoskeleton and centrosome in living cells(Fig. 6C). As for GFP-ATIP3, GFP-D2 was entirely retrieved inthe pellet fraction in microtubule cosedimentation assays(Supplementary Fig. S5A). Of interest, upon expression ofGFP-D2, accumulation of EB1 as comet-like structures at themicrotubule plus ends was strongly impaired (Fig. 6E), indi-cating that expression of theD2domain is sufficient to stabilizemicrotubules. Expression of GFP-D3 (Fig. 6C) led to theformation of large aggregates containing tubulin, probablydue to oligomerization of coiled-coil motifs present in theC-terminal region of ATIP3 (31). Because of these aggregates,functional properties of GFP-D3 could not be evaluatedfurther.Altogether our results identify D2 as the ATIP3 domain able

to associate with microtubules and suppress their dynamics.

Of importance, the D2 domain also retained the ability ofATIP3 to inhibit cell proliferation (91.6% inhibition for GFP-D2and GFP-ATIP3 compared with GFP; ref. Fig. 7A). In woundhealing assays, cells expressing GFP-D2 showed reduced cellmigration and directionality (Fig. 7B). Cell tracking of transienttransfectants (Supplementary Movie S7) indicated that similarto GFP-ATIP3, GFP-D2–positive cells mostly remained at theback of the wound and were overtaken by untransfected cells(Fig. 7C and Supplementary Fig. S5B). Thus, the microtubule-binding domain D2 is sufficient to recapitulate the functionalfeatures of ATIP3.

DiscussionWe report here that ATIP3 is an important prognostic

marker for survival of the patients with breast cancer, inde-pendently of the ER status of the tumor. Using 3 differentpatient cohorts, we show that among metastatic breasttumors, low ATIP3 levels correlate with reduced probabilityfor overall survival of the patients, suggesting that ATIP3 maybe an important indicator of metastatic progression.

Figure 6. The D2 region of ATIP3decorates and stabilizesmicrotubules. A, scheme of ATIP3regions D1, D2, and D3. Amino acidnumbering is according to accessionnumber NP_001001924. B,immunoblots (anti-GFP, antitubulin)of RPE-1 cells transfected (24 hours)with GFP-D1, GFP-D2, and GFP-D3.C, immunostaining (anti-GFP,antitubulin) of RPE-1 cells transientlytransfected as in A. D, anti-EB1immunostaining of GFP-D1–transfected RPE-1 cells. Insets showEB1 comets in GFP-D1-negative (1)and GFP-D1-positive (2) cells.Distribution of EB1 (blue), a-tubulin(pink), and GFP-D1 (green) at themicrotubule tip (linescans) andquantification of comets intensity(scattered dot plot). Number ofcomets is under brackets. E, GFP-D2-transfected RPE-1 cells stainedwith anti-EB1 antibodies andanalyzed as in D. ��, P < 0.0001.B, C, and D, scale bar, 10 mm.

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Examination of ATIP3 levels in breast tumors may contributeto identify a population of patients at high risk of fatalcomplication, who should be the subject of careful medicalfollow-up.

Using a bioluminescence-based experimental mouse modelfor cancer metastasis (23, 25, 26), we showed that restoringATIP3 expression into highly metastatic ATIP3-deficientD3H2LNbreast tumor cells significantly delays the time-courseofmetastasis and reduces the number of detectablemetastasesper mouse at all times examined. ATIP3 expression in cancercells also strongly reduces the size of metastatic foci as well asthe number of mice fully invaded with large metastases. Theseobservations, together with above mentioned results onhuman patients, suggest that ATIP3 may have a prominenteffect on metastatic colonization.

Essential steps of metastatic progression include the abilityof cancer cells to reach a secondary organ and grow in the newmicroenvironmental context (3–5). This requires active cellmigration and proliferation, two important biologic processesthat are significantly increased in breast cancer cells followingATIP3 silencing. By promoting dual effects on cancer cellproliferation and migration, ATIP3 likely regulates both early(tumorigenic) and late (metastatic) stages of cancer develop-

ment. Beneficial actions of ATIP3 on a wide range of cancer-related processes, including invasion, transendothelial migra-tion, cell migration, and proliferation may explain its potentanti-metastatic effects in preclinical studies.

Other studies have shown that the MTUS1 gene encodingATIP3 is significantly downregulated in various types of can-cers including from the pancreas (32), ovary (33), head-and-neck (34, 35), colon (36), and bladder (37). Low MTUS1 levelswere also correlated with reduced overall survival of thepatients with bladder cancer (37) and oral tongue squamouscell carcinomas (35), highlighting the potential importanceof ATIP3 as a new prognostic marker in a variety of solidtumors.

At themolecular level, we show that ATIP3 is amicrotubule-associated protein with potent microtubule-stabilizing effects.We propose that by stabilizing microtubules, ATIP3 decreasestheir dynamics therefore leading to impaired ability of micro-tubule tips to reach the cell cortex during migration. Micro-tubule dynamics at the cell cortex is essential for generating apolarized microtubule array, required for cell polarity andmigration (30). Reduced microtubule dynamics may thusrepresent a major mechanism accounting for anti-migratoryand anti-metastatic effects of ATIP3 in breast cancer. Accord-ingly, loss of ATIP3 leads to increasedmicrotubule growth rate,less time spent in pause, and decreased frequency of cata-strophes. Alteration of microtubule dynamics parameters inATIP3-depleted cells may explain uncontrolled cancer cellmotility that is associated with increased metastasis and poorprognosis in patients with ATIP3-negative breast cancer. Theassociation of ATIP3 with the microtubule lattice involves aninternal basic region designated D2 whose expression is suf-ficient to recapitulate all effects of the full-length protein onmicrotubule stabilization, as well as cell proliferation, motility,and directional migration. The microtubule-binding D2 regionthus represents the functional domain of ATIP3. Furthercharacterization of this domain and identification of intracel-lular interacting partners may help deciphering the molecularmechanisms by which ATIP3 limits breast cancer cell migra-tion and hence, metastasis. Our study paves the way to thedesign of peptides or small molecules able to mimic the effectsof ATIP3, which is a prerequisite for the development oftargeted therapy. These may be particularly beneficial to thesubset of breast tumors that have lost ATIP3 expression andare prone to metastasize.

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

Authors' ContributionsConception and design: S. Honor�e, M. Di Benedetto, C. Nahmias, S. Rodrigues-FerreiraDevelopment of methodology: A. Molina, L. Ghouinem, M. Abdelkarim, M. DiBenedettoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Velot, B.P. Bouchet, A.-C. Luissint, I. Bouhlel,A. Vincent-Salomon, O. Delattre, B. Sigal-Zafrani, F. Andr�e, B. Terris, M. DiBenedetto, S. Rodrigues-FerreiraAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Molina, L. Velot, S. Honor�e, D. Braguer, N. Gruel,O. Delattre, F. Andr�e, B. Terris, A. Akhmanova, M. Di Benedetto, C. Nahmias,S. Rodrigues-Ferreira

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Figure 7. Microtubule-binding domain D2 is the functional domain ofATIP3. A, colony formation of GFP-, GFP-ATIP3-, GFP-D2- transfectedMCF7 cells, and quantification (mean � SD, n ¼ 4). Shown is 1representative experiment out of 3. B, migration trajectories (17 hours)covered by GFP- (n ¼ 30), GFP-ATIP3- (n ¼ 17), and GFP-D2- (n ¼ 24)expressing D3H2LN cells. Directionality coefficient (Dir) is inside thegraph. C, aligned dot plots show Euclidean distance covered byuntransfected (�) and transfected cells (þ) cells as indicated. �, P < 0.05;��, P < 0.001; ��, P < 0.0001.

Molina et al.





Writing, review, and/or revisionof themanuscript:A.Molina, L. Velot, A.-C.Luissint, A. Di Tommaso, S. Honor�e, B. Sigal-Zafrani, F. Andr�e, A. Akhmanova, C.Nahmias, S. Rodrigues-FerreiraAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. Morel, E. SapharikasStudy supervision: C. Nahmias, S. Rodrigues-Ferreira

AcknowledgmentsThe authors thank the Cochin Imaging facility, the Hospital St Louis Animal

facility, Nicolas Cagnard (Genomics facility of the University Paris Descartes),Muriel Andrieu, Karine Labroqu�ere (Cybio platform), Maryline Favier, andCorinne Lesaffre (Histology facility) of the Cochin Institute.

Grant SupportThiswork was supported by the University Paris Descartes, Inserm, CNRS, the

Ligue Contre le Cancer-IDF, Inserm-Transfert, the association "Le Cancer duSein, Parlons-en!", Roche laboratories, the fondation RAJA-Dani�ele-Marcovici,Odyssea, and Prolific.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 10, 2012; revised January 29, 2013; accepted February 4,2013; published OnlineFirst February 8, 2013.

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2013;73:2905-2915. Published OnlineFirst February 8, 2013.Cancer Res Angie Molina, Lauriane Velot, Lydia Ghouinem, et al. Progression by Regulating Microtubule DynamicsSurvival, Limits Cancer Cell Migration and Slows Metastatic ATIP3, a Novel Prognostic Marker of Breast Cancer Patient

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