ZNF217 Is a Marker of Poor Prognosis in Breast Cancer ThatDrives Epithelial–Mesenchymal Transition and Invasion
Julie A. Vendrell1,2, Aur�elie Thollet1,2, Nhan T. Nguyen1,2, Sandra E. Ghayad1,2, St�ephanie Vinot5, Ivan Bi�eche6,Evelyne Grisard1,2, V�eronique Josserand7,8, Jean-Luc Coll7,8, Pierre Roux5, Laura Corbo2, Isabelle Treilleux2,3,Ruth Rimokh2, and Pascale A. Cohen1,2,3,4
AbstractThe Kr€uppel-like zinc finger protein ZNF217 is a candidate oncogene in breast cancer. In this study, we
showed that high levels of expression of ZNF217 mRNA are associated with poor prognosis and thedevelopment of metastases in breast cancer. Overexpression of ZNF217 in breast cancer cells stimulatedmigration and invasion in vitro and promoted the development of spontaneous lung or node metastases inmice in vivo. ZNF217 also promoted epithelial–mesenchymal transition (EMT) in human mammary epithelialcells, and the TGF-b–activated Smad signaling pathway was identified as a major driver of ZNF217-inducedEMT. In addition, a TGF-b autocrine loop sustained activation of the TGF-b pathway in ZNF217-over-expressing mammary epithelial cells, most likely because of ZNF217-mediated direct upregulation of TGFB2or TGFB3. Inhibition of the TGF-b pathway led to the reversal of ZNF217-mediated EMT. Together, ourfindings indicate that ZNF217 mRNA expression may represent a novel prognostic biomarker in breastcancer. Therapeutic targeting of ZNF217 of the TGF-b signaling pathway may benefit the subset of patientswhose tumors express high levels of ZNF217. Cancer Res; 72(14); 3593–606. �2012 AACR.
IntroductionZNF217 is a candidate oncogene located on chromosome
20q13.2, a region that is frequently amplified in manytumors, including those of the breast (1). The first directevidence for a role for ZNF217 in oncogenesis was thatZNF217 could give rise to immortalized cells (2, 3). ZNF217is a Kr€uppel-like zinc finger protein that localizes to thenucleus (4) and interacts with corepressors and histone-modifying proteins, suggesting that it may be part of atranscriptional repressor complex (5–7). However, the roleof ZNF217 in transcriptional regulation is likely to be com-plex, as ZNF217 has also been shown to induce positivetranscriptional regulation of target genes (8, 9). Previousstudies have found that high levels of ZNF217 expression
have been associated with resistance to chemotherapy andwith deregulated apoptotic signals in breast cancer cells(10, 11). The precise molecular mechanisms involved inZNF217 prosurvival functions are currently unknown, butactivation of the Akt pathway (10), ErbB3 overexpression(12), Aurora-A overexpression (11), deregulated expressionof several members of the Bcl-2 family (11), eEF1A2 over-expression (13), and interaction with the HIF pathway (14)have been proposed.
High-level amplification of 20q13 is found in 6.8% (15), 8%(16), and 18% (17) of breast cancers. Although amplificationof 20q13 has been correlated with poor prognosis in breastcancer (15), contradictory data have emerged (16, 17).Although high levels of ZNF217 expression have been mainlyattributed to amplification of ZNF217 (18), other mechan-isms may be important in regulating ZNF217 expressionin cancer cells. Indeed, high levels of ZNF217 expressionhave been observed in breast tumors and breast cancer celllines in the absence of amplification (4, 18), and no statisticalcorrelation between ZNF217 mRNA expression levels andZNF217 amplification was found in breast tumors in whichonly the ZNF217 locus of the 20q13 region was amplified(16).
In this context, we thus sought to assess the potential valueof ZNF217 mRNA expression levels (independently of ZNF217amplification status) as a biomarker in breast cancer. Ourstudy reports for the first time the strong prognostic value ofZNF217mRNA expression levels in several independent breastcancer cohorts. We also show that ZNF217 overexpressionconfers invasive properties, both in vitro and in vivo, and
Authors' Affiliations: 1ISPB, Facult�e de Pharmacie, Universit�e Lyon 1,Lyon; 2INSERM U1052, CNRS UMR5286, Centre de Recherche enCanc�erologie de Lyon, Lyon; 3Centre L�eon B�erard, Lyon; 4ProfileXpert,SFR Lyon-Est, Lyon; 5Universit�e Montpellier 2, CRBM–CNRS, UMR5237,IFR 122, Montpellier; 6Laboratoire d'Oncog�en�etique, Institut Curie–HopitalRen�e Huguenin, St-Cloud; 7INSERM U823, Grenoble; and 8Universit�eJoseph Fourier, Institut Albert Bonniot, Grenoble, France
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
J.A. Vendrell and A. Thollet contributed equally to this work.
Corresponding Author: Pascale A. Cohen, ISPBL, Faculte de Pharmacie,8 Av Rockefeller, 69008 Lyon, France. Phone: 33-4-78-77-28-94; Fax: 33-4-78-77-72-49; E-mail: [email protected]
promotes epithelial–mesenchymal transition (EMT). Finally,biochemical and transcriptomic investigations allowed us toidentify themolecular pathways involved in the ZNF217-drivenphenotype.
Materials and MethodsBreast tumor cohorts
Women with primary breast tumors who had not receivedany therapy before surgery and who did (Metþ group) or didnot (Met� group) develop metastases while receiving che-motherapy and/or endocrine therapy were from the CentreL�eon B�erard (CLB, Lyon) or from the Centre Ren�e Huguenin(CRH, St Cloud; Supplementary Materials and Methods,Tables S1 and S2). Total RNA was extracted from frozentumor samples as previously described (19). Gene expressiondata from Ma and colleagues' study (ref. 20; GSE1379) orKaplan–Meier plotter (21) were also investigated.
were carried out as described previously (22). All statisticalanalyses assessing ZNF217 prognostic value were carried outusing SPSS Software. The data are divided at the medianvalue of ZNF217 mRNA expression into 2 groups with eitherhigh or low expression. P value less than 0.05 was consideredto be statistically significant.
ImmunohistochemistryExperiments were carried out as previously described
(ref. 22; see Supplementary Materials and Methods).
Cell cultureMDA-MB-231, MCF10A, and HEK-293T cells were pur-
chased from American Type Culture Collection (ATCC) andcultured as recommended. These cell lines have been routinelytested and authenticated by the ATCC. The identity of theMDA-MB-231 cells was also confirmed by sequencing KRASand TP53 genes. The D3H2LN cells were purchased fromCaliper and tested for invasive capacities by Boyden chamberand in vivo investigation.
ZNF217 stable transfectantsMDA-MB-231-pcDNA6, ZNF217-1, and ZNF217-2 cells have
been described previously (11). Populations of MCF10A-pcDNA6 and MCF10A-ZNF217 stable transfectants wereobtained using the same plasmid and selection process asdescribed in Thollet and colleagues (11).
Western blotsThe antibodies used were anti-ZNF217 (Abcam Ltd);
anti–phospho-FAKY397 and phospho-FAKY577 (Invitrogen);anti-FAK and anti-ErbB2 (Santa Cruz Biotechnology Inc.);anti–E-cadherin, anti–a-catenin, anti–b-catenin, and anti–N-cadherin (BD Biosciences); anti-occludin (Zymed Labora-tories); anti-vimentin (Dako); anti–a-tubulin (Sigma-Aldrich);and anti-ErbB3 (Cell Signalling).
Gene silencingCells were transfected with 5 nmol/L of Stealth siRNAs
targeting ZNF217 (Invitrogen; ref. 11), siRNAs targeting Smad4(Dharmacon; ref. 23) or scrambled control RNA (Invitrogen).
Soft-agar colony-formation assayExperiment was carried out as previously described (24).
Twenty days later, the cells were stained with 0.005% Cristalviolet (Sigma-Aldrich) for 1 hour.
Wound healing assayCells were plated, the medium was removed, and replaced
by FBS free medium 16 hours before wounding of theconfluent monolayer by scratching.
Matrigel invasion analysisA total of 106 cells were suspended in 1 mL Dulbecco's
Modified Eagle's Medium and seeded in the middle of aMatrigel layer (BD Biosciences).
Boyden chamber invasion assayA total of 5 � 104 cells were plated on top of a thick layer of
Matrigel in Transwell chambers (Fluroroblock; BD Bios-ciences). Cell invasion (24 hours) and quantification werecarried out as described in Vinot and colleagues (25).
In vivo experimentsPopulations of 2 � 106 stably transfected MDA-MB-231-
pcDNA6 and MDA-MB-231-ZNF217 cells were suspended inPBS/Matrigel v/v (BD Biosciences) and injected into the mam-mary fat pad of female Swiss nude mice (Charles River). Aftersacrifice, the presence of metastases in the lung and the lymphnodes was detected by histologic analysis of tissue sectionsstained with hematoxylin and eosin.
Luciferase stably transfected MDA-MB-231-ZNF217or MDA-MB-231-pcDNA6 cell populations were produced.Female athymic Swiss nude mice (Janvier) were eachinjected with 106 cells suspended in PBS. Before each bio-luminescence imaging (IVIS Kinetic; Caliper), anesthetizedmice received an intraperitoneal injection of Luciferin(Promega).
All animal studies were conducted in accordance withEuropean Union guidelines and approved by the regionalethics committee.
Transcriptomic analysisExperiments were carried out as previously described
(ref. 26; see Supplementary Materials and Methods).
Immunofluorescence assayFor E-cadherin staining (23), the E-cadherin antibody
and the Alexa Fluor 488 anti-mouse antibody were used.For actin staining (23), cells were incubated with tetra-methyl rhodamine isothiocyanate–conjugated phalloidin(Sigma-Aldrich). Images were acquired using an Axioplan2 microscope (Carl Zeiss). Recombinant human TGF-b1(5 ng/mL) was from Peprotech and SB431542 (10 nmol/L)from Sigma.
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Luciferase assayHEK-293T cells were transfected with 150 ng of CAGA-luc
plasmid (with repeated CAGA Smad-binding elements con-trolling firefly luciferase gene transcription; ref. 27) and 10 ngof pTK-RL (Renilla luciferase plasmid). Twenty-four hourslater, supernatants from MCF10A-pcDNA6 or MCF10A-ZNF217 cells were incubated for 24 hours with transfectedHEK-293T cells and luciferase activities were assessed.
Chromatin immunoprecipitationExperiments were carried out as previously described
using anti-ZNF217 antibody or anti-rabbit IgG (Abcam Ltd)as a negative control (ref. 23; see Supplementary Table S3).
Mammosphere assaySingle-cell suspensions were seeded in nonadherent mam-
mosphere culturing conditions (28). After 7 days, mammo-spheres were counted, collected, trypsinated, and replated innonadherent culturing conditions.
ResultsHigh levels of expression of ZNF217mRNA are associatedwith poor prognosis in breast cancerTo investigate the clinical relevance of ZNF217, we used
qRT-PCR to explore ZNF217 mRNA levels in a test set of 47samples of primary breast tumors (CLB1 cohort, Supplemen-tary Table S1) from women who had (Metþ group) or had not(Met� group) developed metastases while receiving therapy.High levels of ZNF217 mRNA in the primary tumor weresignificantly associated with the subsequent development ofmetastases (significant overexpression ofZNF217mRNA in theMetþ group, P ¼ 0.002, Supplementary Table S4) and shorterrelapse-free survival (RFS; P ¼ 0.003, Fig. 1A). These findingswere verified in an independent cohort from a different geo-graphic location, the CRH cohort (Supplementary Table S4and Fig. 1B).Retrospective analysis of gene expression array data from
Ma and colleagues (20) showed that ZNF217mRNA levels werestill significantly higher in the Metþ group than in the Met�
group (P ¼ 0.0007, Supplementary Table S4), and that highlevels of expression of ZNF217 were significantly associatedwith shorter RFS (P ¼ 0.01, Fig. 1C). Finally, the most remark-able results arose from our analysis of publicly availablemicroarray data for 2,414 breast cancer patients (21), in whichwe found that high levels of expression of ZNF217 wereassociated with poor prognosis, with a P value of 1 � 10�11
(Fig. 1D). Altogether, these data suggested that ZNF217 is anovel and powerful biomarker for poor prognosis in breastcancer.
Clinical relevance of ZNF217 mRNA levels for prognosisin an independent breast cancer cohortIn the independent CLB2 cohort (n ¼ 100, Supplementary
Table S2), ZNF217 mRNA was again significantly overex-pressed in the Metþ group compared with the Met� group(P ¼ 0.025, Supplementary Table S4), and high levels ofexpression of ZNF217 were significantly associated with
shorter RFS (P ¼ 0.02, Fig. 1E). No association was foundbetween expression of ZNF217 with other clinical para-meters (Supplementary Table S5). The prognostic value ofZNF217 mRNA was more informative than the ER, PR, orHER2 conventional biomarkers in this cohort (Log-ranktest, Table 1). However, lymph node status was associatedwith shorter RFS (P ¼ 0.013, Log-rank test, Table 1). Prog-nostic factors for RFS with a 0.05 significance level inunivariate analysis were then entered in a multivariate Coxmodel. Both ZNF217 mRNA levels and lymph node statuspersisted in the model (P < 0.05), revealing that these 2biomarkers are independent prognostic markers (Table 1).
We then constructed a signature based on ZNF217 mRNAexpression levels and lymph node status by dividing patientsinto 3 groups: patients who expressed low levels of ZNF217mRNA and had no or few (� 3) involved lymph nodes (groupA); patients who expressed high levels of ZNF217 mRNAexpression and no or 3 or less involved lymph nodes or thosewho presented with more than 3 involved lymph nodes andlow expression of ZNF217 mRNA (group B); and patientswho expressed high levels of expression of ZNF217 and hadmore than 3 involved lymph nodes (group C). The resultingKaplan–Meier curves for RFS are shown in Fig. 1F (P ¼0.004). Finally, we tested the ZNF217 mRNA levels/lymphnode status signature with respect to RFS and found that themodel associating ZNF217 mRNA levels and lymph nodestatus was a better fit (likelihood ¼ 199.83) than the modelwith ZNF217 mRNA levels only (likelihood ¼ 208.16, P ¼0.001) or the model with lymph node status only (likelihood¼ 207.74, P ¼ 0.001; ref. 29), showing that, in this cohort, theZNF217 mRNA level/lymph node status signature has thebest prognostic value.
We also investigated ZNF217 protein expression by immu-nohistochemistry in 10 ZNF217-mRNA–positive human breasttumors. ZNF217 expressionwas detected in all tumors andwasmainly located in the nucleus of the stained tumoral cells (nostaining was observed in the stromal cells). Moreover, themajority of the breast tumors investigated displayed a pro-portion of stained cells ranging from 50% to 80% and a positivebut variable level of ZNF217-protein expression (Fig. 1G).
Overexpression of ZNF217 stimulates migration andinvasiveness of breast carcinoma cells in vitro and in vivo
MDA-MB-231 cells, which have low endogenous levels ofZNF217, have been previously used by our group to establish2 stable cell lines (ZNF217-1 and ZNF217-2) that constitu-tively overexpress the ZNF217 protein (ref. 11; SupplementaryFig. S1A). Overexpression of ZNF217 strongly simulates cellsurvival and/or proliferation (Supplementary Fig. S1B). Tran-sient transfections with 2 ZNF217-targeted siRNAs resultedin a spectacular inhibition of cell proliferation/viability bothin control and in ZNF217-overexpressing cells (SupplementaryFig. S1C), supporting our previous results (11). Anchorage-independent growth is thought to be associated with aggres-siveness and metastasis in malignant cells. We found thatthe number of colonies formed when ZNF217 was overex-pressed was greater in ZNF217-1 and ZNF217-2 cells than incontrol cells (P < 0.001, Fig. 2A).
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A strong capacity for migration or invasion is anotherhallmark of tumor malignancy. We carried out wound healingexperiments at short time points (15 and 24 hours) and inserum-freemedium, to exclude interference attributable to the
proproliferative action of ZNF217. Overexpression of ZNF217in ZNF217-1 and ZNF217-2 cells stimulated cell migrationcompared with control cells (Fig. 2B), and this was entirelyreversed in the presence of siRNA-A ZNF217 or siRNA-B
Figure 1. ZNF217 mRNAexpression is associated withshorter RFS. Kaplan–Meieranalyses (log-rank test) for RFS areshown in theCLB1cohort (A); in theCRH cohort (B); in the Ma cohort(C); for all patients using Kaplan–Meier plotter (D); in the CLB2cohort (E). F, effect of the ZNF217mRNA level/lymph node statussignature on RFS among the CLB2cohort (n ¼ 100). G, ZNF217immunohistochemistry staining inexample cores of 3 ZNF217-mRNA–positive breast tumors.
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ZNF217 (Fig. 2C and Supplementary Fig. S2). Matrigel invasionassays showed that ZNF217-overexpressing cells are also moreinvasive than control cells (Fig. 2D). Supporting data camefrom the Boyden chamber assay in vitro (P < 0.001, Fig. 2E).Focal adhesion kinase (FAK) signaling is crucial in mediatingmigration and invasion, in particular, in those induced byErbB2 and ErbB3 receptor signaling in breast cancer cells(30, 31). We strikingly found increased levels of phospho-FAK/FAK total ratio associated with overexpression of ErbB2and ErbB3 proteins in both ZNF217-overexpressing clones(Fig. 2F), suggesting that this molecular pathway could beinvolved in the ZNF217-driven phenotype.We next explored whether deregulation of endogenous
ZNF217 expression could be detected in a breast cancer cellmodel of invasion. The D3H2LN cell line is a clonal subline ofMDA-MB-231 cells passaged twice in mice, and the resultingxenografts showed early and increased frequency of sponta-neous lymph nodemetastasis in vivo (32). Strikingly, comparedwith controls, D3H2LN cells showed significantly higherendogenous expression of ZNF217 protein (Fig. 3A) andincreased migratory properties (Fig. 3B). Moreover, the appli-cation of ZNF217-targeted siRNAs was sufficient to decreasethe migratory and invasive properties of D3H2LN cells (Fig. 3Band C).We then carried out in vivo investigations in nude mice.
Xenografts were established by injecting populations of MDA-MB-231-pcDNA6 control cells or MDA-MB-231-ZNF217 cellsinto the mammary fat pads of female nude mice. A significantincrease in the growth of MDA-MB-231-ZNF217 xenograftscompared with control xenografts was observed and 2 of 4mice injected with MDA-MB-231-ZNF217 cells (and 0 of 4control mice) spontaneously developed metastases, either inthe lung or in the lymph nodes (Figs. 3D–H). Supporting datacame from intravenous injection of luciferase stably trans-fected MDA-MB-231-ZNF217 or MDA-MB-231-pcDNA6 cellpopulations in the tails of the nude mice. Interestingly, 2 of5 MDA-MB-231-ZNF217–injected mice developed lung metas-
tases, although none (0 of 5) of the control mice developed anymetastases (Fig. 3I).
In summary, these data suggested that ZNF217 overexpres-sion is associated with aggressiveness and invasion, bothin vitro and in vivo.
Gene expression profiling of ZNF217-overexpressingcells
To provide new mechanistic insights into ZNF217-drivenmigration/invasion, we carried out global gene expressionprofiling on populations of MDA-MB-231-pcDNA6 andMDA-MB-231-ZNF217 cells. Focusing on significantly de-regulated genes, we found that the 10 top-ranked biologicfunctions (P values from 10�28 to 10�11) included responseto wounding, regulation of locomotion, locomotory behav-ior, cell motility, and anatomic structure morphogenesis,providing strong support at the molecular level for the cel-lular phenotype (migration/invasion) observed in ZNF217-overexpressing cells (Fig. 4A and Supplementary Table S6).We clearly identified deregulated expression of genes encod-ing adhesion proteins (Supplementary Fig. S3) and alsodiscovered a deregulation in the focal adhesion pathwayin ZNF217-overexpressing MDA-MB-231 cells (Fig. 4B), sup-porting the data presented in Fig. 2F.
ZNF217 promotes EMT in mammary epithelial cellsEMT is known to be involved in the invasive capacity of
transformed epithelial cells (33), and chromatin immunopre-cipitation (ChIP) assays have previously shown that theZNF217 complex is present on the E-cadherin promoter inbreast cancer cells, driving downregulation of luciferase activ-ity in a reporter gene assay (6). Compared with MCF10A-pcDNA6 control cells, stable transfection of ZNF217 in humanmammary epithelial MCF10A cells (34) was paired with inva-sion (Fig. 5A) and with significant decreased expression ofCDH1/E-cadherinmRNA (Fig. 5B) and was sufficient to triggersome features of EMT, including fibroblastic morphology
Table 1. Univariate and multivariate analyses of the prognostic value of ZNF217 gene expression andclinical parameters with regard to RFS in the CLB2 cohort (n ¼ 100)
(Fig. 5C), a significant reduction in expression of epithelialmarker proteins and an increase in levels of mesenchymalproteins (Fig. 5D). These data showed for the first time that inMCF10A cells, ZNF217 can induce the downregulation ofCDH1/E-cadherin expression at the endogenous level, both atthe mRNA and protein levels. Interestingly, ZNF217-inducedEMT was also associated with significant increased mRNAlevels of several transcriptional factors known to be majordrivers of EMT (Fig. 5E). A direct link between EMT and thegain of epithelial stem cell properties has been describedpreviously (35). We found that ZNF217 enhances mammo-sphere formation in MCF10A-ZNF217 cells (Fig 5F), indicatingthat ZNF217 overexpression stimulates the growth of cancerstem cell populations. Similar data were obtained usingZNF217-overexpressing MDA-MB-231 cells (SupplementaryFig. S4A and B).Nonpolar or cytoplasmic expression of E-cadherin protein
is a strong hallmark of EMT in cancer progression (36) andcan be induced with TGF-b1 (MCF10A-pcDNA6 cells, Fig. 6A,immunofluorescence assay). The immunofluorescence assayin MCF10A-ZNF217 cells showed that E-cadherin (validatingthe Western blot data presented in Fig. 5D) was poorlyexpressed, with cytoplasmic localization (Fig. 6A). Moreover,MCF10A-ZNF217 cells also displayed increased actin poly-merization, reorganization of stress fibers into large linearand parallel bundles, and fibroblast-like elongated morpho-logy (Fig. 6A), features also shown by MCF10A-pcDNA6cells treated with TGF-b1. Similar data were obtained in asecond model of human mammary epithelial cells (HMLEcells; ref. 37; Supplementary Fig. S5A–D). Promotion of EMTis thus another important feature of the deleterious role ofZNF217 in tumorigenesis.
The TGF-b pathway is a major driver of the ZNF217-induced features of EMTIn late stages of breast cancer tumorigenesis, TGF-b is
known to promote cancer progression and contribute to theacquisition of metastatic phenotypes, partly through itsability to stimulate EMT and cell migration (36, 38). TGF-b canonically elicits its cellular responses via TGF-b recep-tors and Smad proteins that regulate the transcription oftarget genes (39). We thus sought to determine whether theTGF-b pathway is involved in ZNF217-induced EMT usingSB431542, an ATP analog and inhibitor of the kinase activityof the TGF-b type I receptor, and a siRNA-based strategytargeting Smad4. Control experiments showed that exposureto SB431542 or Smad4 silencing of TGF-b1–treated MCF10A-pcDNA6 cells potently inhibited TGF-b1–induced featuresof EMT (Fig. 6A and B). Strikingly, treating with SB431542or silencing Smad4 was also sufficient to reverse the
ZNF217-induced EMT phenotype in MCF10A-ZNF217 cells(Fig. 6A and B). Similar data were obtained using HMLE-ZNF217 cells (Supplementary Fig. S6A and B). SB431542treatment also prevented ZNF217-promoted migration ofZNF217-overexpressing MCF10A cells (Fig. 6C). Overall,these data thus strongly suggested that, firstly, ZNF217-induced EMT and migration in mammary epithelial cells isreversible; and secondly, that ZNF217-induced EMT isTGF-b type I receptor dependent and Smad4 dependent.Supporting data showed that the expression of TGFB1,TGFB2, and TGFB3 mRNA was significantly higher inMCF10A-ZNF217 cells than in the control cells, and themost striking increase was observed for TGFB2 and TGFB3mRNA levels (Fig. 6D). Similar data were obtained usingHMLE-ZNF217 cells (Supplementary Fig. S7A), suggestingthat the upregulation of TGFB2 and TGFB3 mRNA levels isindependent of the cellular context in ZNF217-overexpres-sing mammary epithelial cells.
We then carried out ChIP experiments on several ZNF217-binding sites (6, 8) found in the �5 Kb to þ1 Kb promoterregion of the TGFB1, TGFB2, and TGFB3 genes (Supplemen-tary Table S3). PCR amplification of the TGFB2-P4 and theTGFB3-P1 regions was observed in the MCF10A-ZNF217cells and not in control cells (Fig. 6E), suggesting thatZNF217 can associate with TGFB2 or TGFB3 promoters andwould directly upregulate the transcription of these genes. Inthe TGFB1 gene promoter, only one ZNF217-binding siteexists (Supplementary Table S3), but no ZNF217 associationcould be detected in any cells (data not shown), suggestingthe involvement of other regulatory mechanisms. Similardata were obtained using HMLE-ZNF217 cells (Supplemen-tary Fig. S7B).
To investigate whether TGF-bs autocrine production waspaired with TGF-b pathway activation, supernatant fromMCF10A-ZNF217 cells was used to analyze the transcrip-tional activity of a CAGA (Smad binding elements)-luciferasereporter plasmid in HEK-293T cells. We found that thissupernatant induced a significant increase in CAGA-drivenluciferase activity (in comparison with supernatant fromMCF10A-pcDNA6 cells; Fig. 6F).
In conclusion, increased expression of TGF-bs may contrib-ute, via an autocrine mechanism, to the constitutive activationof the TGF-b pathway, and the TGF-b pathway is amajor driverof ZNF217-induced EMT features.
DiscussionThe key findings of our study were that high levels of
expression of ZNF217 mRNA are associated with poor prog-nosis and with the development of metastases in breast
Figure 2. ZNF217 promotes anchorage-independent growth, migration, and invasion. A, overexpression of ZNF217 in MDA-MB-231 cells increases theformation of colonies in soft agar (means � SD from 3 independent experiments). B, wound healing assay (representative of 3 independent experiments).C, wound healing assay in ZNF217-1 transfected or not (NT) with either scrambled RNA, siRNA-A ZNF217, or siRNA-B ZNF217 (representative of 3independent experiments). D, representative images of cell invasion Matrigel assay (3 independent experiments). E, Boyden chamber assay carried out intriplicate (mean�SD). F,Western blots of phospho-FAKY397, phospho-FAK Y577, FAK, ErbB2, and ErbB3 expression. Histograms represent quantificationof Western blot signals (mean � SD of 3 independent experiments). ���, P < 0.001 (Student t test).
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Figure 3. ZNF217 is involved in cell migration/invasion. A,Western blot analysis of ZNF217. Histograms represent quantification ofWestern blot signals (mean� SD of independent experiments). B, wound healing assay in D3H2LN cells transfected or not with either scrambled RNA, siRNA-A ZNF217, or siRNA-BZNF217 (representative of independent experiments). C, Boyden chamber assay carried out in triplicate (mean � SD). D, growth curves of MDA-MB-231-pcDNA6xenografts (n¼4) andMDA-MB-231-ZNF217 xenografts (n¼4;mean�SD). Histologic viewsof lungmetastasis (E) and thematchedmurine primarytumor (F); lymphnodemetastasis (G) and thematchedmurine primary tumor (H). N, normal tissue; T, tumoral tissue. I, representativebioluminescent imagesofmice intravenously injected with luciferase stably transfected MDA-MB-231-pcDNA6 or MDA-MB-231-ZNF217 cell populations. Red circled areas indicatelung metastases. �, P < 0.05; ��, P < 0.01; ���, P < 0.001 (Student t test).
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cancer patients. Our findings were powerfully validatedin 3 independent cohorts and in 2 retrospective analysesof publically available transcriptomic data. To our knowl-
edge, this is the first article that ZNF217 mRNA levelshave prognostic value in cancer patients, except for a veryrecent study that found elevated expression of ZNF217
Figure 4. Transcriptomic analysis of ZNF217-overexpressing MDA-MB-231 cell populations. A, enriched gene ontology biologic processes for genesdifferentially expressed in ZNF217-overexpressingMDA-MB-231 cells (comparedwith control cells). B, overview of the focal adhesion pathway. Genes in redrepresent deregulated genes in the MDA-MB-231-ZNF217 cells compared with control cells.
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Figure 5. ZNF217 induces invasion, EMT, and mammosphere formation in MCF10A cells. A, representative images of cell-invasion Matrigel assay (3independent experiments). B, gene expression of CDH1/E-Cadherinmeasured by qRT-PCR (means� SD of 3 independent experiments). C, representativeimages of MCF10A-pcDNA6 and MCF10A-ZNF217 cell morphology. D, Western blot of ZNF217, epithelial markers, and mesenchymal markers. Signalquantifications are presented in Supplementary Fig. S9. E, qRT-PCRmeasurement of transcription factors known to bemajor drivers of EMT (mean�SDof 3independent experiments). F, MCF10A-pcDNA6 and MCF10A-ZNF217 cells were grown in nonadherent culture conditions and mammospheres werecounted (mean � SD of 3 independent experiments). ��, P < 0.01; ���, P < 0.001 (Student t test).
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Figure 6. The TGF-b pathway iscrucial for ZNF217-induced EMTand migration. Immunofluo-rescence acquisition showing E-cadherin and actin subcellularlocalization in TGF-b1–treatedMCF10A-pcDNA6 and in MCF10A-ZNF217 cells under SB431542exposure (A) and after Smad4silencing (B; concomitant Westernblot of Smad4 expression ispresented in Supplementary Fig.S10A). C, wound healing assay inMCF10A-pcDNA6 and in MCF10A-ZNF217 treated or not withSB431542 (representative of 3independent experiments).Histograms representing thedistance between edges of thewound are presented inSupplementary Fig. S10B.D, qRT-PCR measurement ofexpression of TGF-b familymembers (mean � SD of 3independent experiments).E, ChIP assays using anti-ZNF217antibody or human IgG as a controlon TGFB2 and TGFB3 promoterregions. Immunoprecipitated ChIPDNA was analyzed by PCR usingZNF217 binding site–specificprimers (Supplementary Table S3).F, HEK-293T cells werecotransfected with the CAGA-Lucplasmid and the pTK-RL plasmid.HEK-293T cells were thenincubated with supernatant (1:1and 1:2 dilutions) from MCF10A-pcDNA6 and MCF10A-ZNF217cells. The luciferase activity resultsare means � SD of 3 independentexperiments. ��, P < 0.01;���, P < 0.001 (Student t test).G, model for ZNF217-driven EMT.
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to be associated with poor survival in patients with glioma(14).
We were thus able to classify breast tumors on the basis ofZNF217 mRNA expression as those with "good prognosis" orthose with "poor prognosis". This stratification could berefined in the CLB2 cohort using a signature based on bothZNF217mRNA expression levels and lymph node status, whichdefined 3 prognostic classes of breast cancer patients (good,intermediate, or poor). In the CRH cohort, although lymphnode status was associated with shorter RFS, we found thatZNF217 mRNA expression is an independent prognosticmarker that is more informative than lymph node status inthis cohort (P¼ 0.01, multivariate analysis, data not shown). Inthe Ma and colleagues cohort (20) and in the CLB1 cohort,lymph node status, ER, PR, and HER2 had no prognostic value(data not shown), showing that the prognostic value of ZNF217was again superior. Overall, ZNF217 mRNA expression is aprognostic marker that possesses an added value to currentbiomarkers. Assessing ZNF217mRNA expression levels (aloneor associated with lymph node status) would thus allow therestratification of patients with breast cancer into outcome-dependent subclasses.
Other major findings of our study were that overexpres-sion of ZNF217 protein strongly stimulates migration andinvasion in several in vitro independent breast cell modelsand is associated with the development of spontaneous lungor node metastases in mice. Interestingly, levels of ZNF217mRNA in the ZNF217-overexpressing cells used in this studywere in the same range as endogenous levels of ZNF217mRNA detected in ZNF217-positive breast tumor samples(data not shown). This supports the idea that similar levelsof ZNF217mRNA are associated with poor prognosis and thedevelopment of metastases in breast cancer patients andwith an aggressive and invasive phenotype in vitro or in miceexperiments. New immunohistochemical data also revealedheterogeneity in ZNF217 staining in human ZNF217-positivebreast tumors, and ZNF217 positively stained cells thusseem to represent a specific subpopulation of tumoral cellswithin the breast tumor. On the basis of the aggressiveZNF217-associated phenotype identified in vitro and in miceand the fact that ZNF217-positive breast cancer patients areprone to develop metastases, we suggest that high levels ofZNF217 expression in a specific subset of breast tumoralcells could provide these cells with a selective advantage fortumoral escape and generation of metastases. Remarkably,we also showed that ZNF217 enhances mammosphereformation, indicating that ZNF217 expression stimulatesthe growth of the cancer stem cell population in ZNF217-overexpressing cells.
Previous studies have shown that FAK signaling mediatesmigration and invasion, particularly in those induced byErbB2/ErbB3 receptor signaling in breast cancer (30, 31).Biochemical and transcriptomic analyses undertaken in ourstudy both indicate that the ErbB2/ErbB3/FAK signalingpathway is deregulated in ZNF217-overexpressing breastcancer cells. Our transcriptomic investigations also foundhigh ERBB3 mRNA expression levels in ZNF217-overexpres-sing MDA-MB-231 cells, supporting the recent finding that
ZNF217 regulates ErbB3 expression at the transcriptionallevel (12).
During oncogenesis, epithelial tumor cells undergo EMTand display enhanced migratory capacity and invasiveness(33, 40). Sustained TGF-bR signaling can be required for themaintenance of EMT for metastasis in mouse models (41).Another major finding of our study is that ZNF217 promotesEMT in independent models of human mammary epithelialcells. We thus propose a model for ZNF217-driven EMT thatincorporates the direct transcriptional downregulation ofE-cadherin expression and/or the constitutive activation ofthe TGF-b–activated Smad signaling pathway (Fig. 6G).Indeed, we showed that ZNF217 overexpression in MCF10Acells promotes downregulation of endogenous mRNA levelsof E-cadherin, supporting for the first time at the endoge-nous cellular level the observations made by Cowger andcolleagues (6). E-cadherin is known to be mainly functionallyinactivated by transcriptional repression at the promoterlevel via several transcription factors (known to be them-selves directly regulated at the transcriptional level by TGF-b signaling; refs. 40, 42, 43). ZNF217 could thus represent anew important transcription factor promoting EMT andbelonging to the family of zinc finger factors (such as Snailor Slug). Our results also show constitutive activation of theTGF-b pathway in MCF10A-ZNF217 or HMLE-ZNF217 cells.Supporting data showed overexpression in MCF10A-ZNF217cells of transcriptional factors known to be induced by TGF-b signaling (refs. 40, 42, 43; Fig. 5E). In addition, sustainedactivation of the TGF-b pathway was the consequence of aTGF-b autocrine loop (increased expression and secretion ofactive TGF-bs), and direct binding of ZNF217 to ZNF217-binding sites located within the TGFB2 or TGFB3 promotersmay contribute to the upregulated expression of these TGF-bRII ligands. Strikingly, inhibition of the TGF-b pathwayleads to the reversal of ZNF217-dependent features of EMT,supporting the finding that the TGF-b–activated Smadsignaling pathway is a major driver of ZNF217-induced EMT.Finally, the close cross-talk existing between ZNF217 and theTGF-b signaling pathway was also validated by our tran-scriptomic data in a third cellular model (MDA-MB-231 cells;Supplementary Fig. S8).
Overall, our new findings have important medical applica-tions: (i) theZNF217mRNAexpression level of a breast tumor isinformative and provides a novel and powerful biomarker ofpoor prognosis that could aid clinicians in therapeutic deci-sions; (ii) clinical strategies to counteract ZNF217-mediatedeffects, either by targeting ZNF217 directly and/or by targetingFAK or TGF-b signaling using clinically tested inhibitors (44,45), could be a potentially valuable approach to the manage-ment of breast cancer, particularly for the subpopulation ofbreast tumors we have identified that possesses high levels ofZNF217 mRNA expression and poor prognosis.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: R. Rimokh, P.A. CohenDevelopment of methodology: I. Bieche, V. Josserand, R. Rimokh, P.A. Cohen
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Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Thollet, N.T. Nguyen, S.E. Ghayad, S. Vinot, I.Bieche, E. Grisard, V. Josserand, J.-L. Coll, P. Roux, I. Treilleux, J.A. VendrellAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.A. Vendrell, A. Thollet, S. Vinot, I. Bieche, V.Josserand, J.-L. Coll, P. Roux, P.A. CohenWriting, review, and/or revision of themanuscript: J.A. Vendrell, A. Thollet,S.E. Ghayad, V. Josserand, J.-L. Coll, P. Roux, L. Corbo, I. Treilleux, P.A. Cohen, R.RimokhAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.E. Ghayad, P.A. Cohen, J.A. VendrellStudy supervision: P. Roux, P.A. Cohen, J.A. Vendrell
AcknowledgmentsThe authors thank the Centre de Ressources Biologiques of
the CLB and of the CRH and also thank Dr. Mattock for edition, S.
L�eon-Goddard for technical support, and Dr. Nguyen for fruitful dis-cussions.
Grant SupportThis work was supported by PRES/Lyon Science Transfert (LST607, Uni-
versit�e Lyon 1) and the Ligue Nationale Contre le Cancer (Grants 2009/2010,Comit�e 71) and the CLARA (Grant 2012/Oncostarter). Nhan T. Nguyen wassupported by the USTH PhD fellowships program.
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 indicate thisfact.
Received September 28, 2011; revised May 2, 2012; accepted May 3, 2012;published OnlineFirst May 16, 2012.
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