The FASEB Journal • Research Communication

AP-2� and AP-2� regulate tumor progression viaspecific genetic programs

Francesca Orso,*,†,‡ Elisa Penna,*,† Daniela Cimino,*,† Elena Astanina,*Federica Maione,* Donatella Valdembri,* Enrico Giraudo,*,‡ Guido Serini,*,‡

Piero Sismondi,* Michele De Bortoli,*,‡ and Daniela Taverna*,†,‡,1

*Institute for Cancer Research and Treatment, †Molecular Biotechnology Center, Department ofOncological Sciences, and ‡Center for Complex Systems in Molecular Biology and Medicine,University of Torino, Torino, Italy

ABSTRACT The events occurring during tumor for-mation and progression display similarities to some ofthe steps in embryonic morphogenesis. The family ofAP-2 proteins consists of five different transcriptionfactors (�, �, �, �, and �) that play relevant roles inembryonic development, as demonstrated by the phe-notypes of the corresponding knockout mice. Here, weshow that AP-2� and AP-2� proteins play an essentialrole in tumorigenesis. Down-modulation of AP-2 ex-pression in tumor cells by RNA interference (RNAi) ledto enhanced tumor growth and reduced chemotherapy-induced cell death, as well as migration and invasion.Most of these biological modulations were rescued byAP-2 overexpression. We observed that increased xeno-transplant growth was mostly due to highly enhancedproliferation of the tumor cells together with reducedinnate immune cell recruitment. Moreover, we showedthat migration impairment was mediated, at least inpart, by secreted factors. To identify the genetic pro-grams involved in tumorigenesis, we performed wholegenome microarray analysis of AP-2� knockdown cellsand observed that AP-2� regulates specific genes in-volved in cell cycle, cell death, adhesion, and migration.In particular, we showed that ESDN, EREG, and CXCL2play a major role in AP-2 controlled migration, asablation of any of these genes severely altered migra-tion.—Orso, F., Penna, E., Cimino, D., Astanina, E.,Maione, F., Valdembri, D., Giraudo, E., Serini, G.,Sismondi, P., De Bortoli, M., Taverna, D. AP-2� andAP-2� regulate tumor progression via specific geneticprograms. FASEB J. 22, 2702–2714 (2008)

Key Words: transcription factor � migration � microarray

The events occurring during embryonic develop-ment resemble tumor progression, and the geneticprograms activated during these multistep processeshave many genes in common. In particular, transcrip-tion factors that are master regulators of embryonicmorphogenesis, such as Twist (1), FOXC2 (2), andGoosecoid (3), play essential roles in malignancy.Other transcription factors, such as the various AP-2family members, regulate embryogenesis and have thepotential to modulate tumor progression.

AP-2 transcription factors are a family of five closelyrelated and developmentally regulated proteins, AP-2�,�, �, �, and ε, encoded by five different genes (4–6).Structurally, AP-2 proteins contain a unique highlyconserved helix-span-helix dimerization motif at theC-terminal half of the protein, a DNA-binding basicregion, and a less conserved proline and glutamine-richactivation domain at the amino terminus. AP-2 proteinscan form homodimers or heterodimers and directlytransactivate their target genes by binding to GC-richconsensus sequences in their regulatory regions (7).They can also indirectly modulate genes by functionalprotein-protein interactions with other transcriptionfactors such as c-myc, pRB, and p53 (8–11).

AP-2 transcription factors are involved in physiologi-cal processes, such as morphogenesis, but also in patho-logical processes such as tumorigenesis and geneticdisease. In fact, AP-2 proteins orchestrate a variety ofcell processes, including cell growth, cell adhesion, andtissue differentiation (4). They play specific and non-redundant roles during embryogenesis, as demon-strated by the different phenotypes of mice deficientfor AP-2�, AP-2�, or AP-2� (12–15). AP-2 proteins playa relevant role in tumorigenesis by regulating key genesinvolved in neoplasia, such as p21Waf1/Cip1 (16), trans-forming growth factor-� (17), estrogen receptor � (18),BCL-2 (19), c-KIT (20), type IV collagenase/gelati-nase/MMP-2, E-cadherin, vascular endothelial growthfactor (VEGF) (21), integrin �5, �7, �3, and �5 (22),c-myc (23), and ERBB-2 (4).

Several investigations suggest that members of theAP-2 family act as tumor suppressor genes. Studies ofprimary breast tumors showed a progressive loss of AP-2expression during conversion to malignancy (24, 25).Similar findings have been reported for cutaneousmelanomas (26) and colon (27) and prostate cancers(28). In melanomas, the role of AP-2� as a tumorsuppressor is directly linked to its ability to activate aspecific genetic program involving the regulation of the

1 Correspondence: Molecular Biotechnology Center, Depart-ment of Oncological Sciences, University of Torino, Via Nizza,52, 10126 Torino, Italy. E-mail: daniela.taverna@unito.it

doi: 10.1096/fj.08-106492

2702 0892-6638/08/0022-2702 © FASEB

tyrosine kinase receptor c-KIT, the adhesion moleculeMCAM/MUC18, E-cadherin, the thrombin receptorPAR-1, the metalloprotease MMP-2 and the angiogenicfactor VEGF (4). However, there are examples oftumors in which AP-2 act like oncogenes (29, 30). Forexample AP-2 up-regulate gene expression of the proto-oncogene ERBB-2, known to be overexpressed in manymalignant tumors, including 25–30% of primary hu-man breast cancers (31). High levels of AP-2 wereobserved in ERBB-2-overexpressing breast tumors (32).Moreover, estrogens are known to modulate ERBB-2expression via AP-2 (33), and a significant correlationexists between expression of AP-2 and estrogen recep-tors (ERs) (34). Most probably, the involvement of AP-2in tumorigenesis depends on which AP-2 isoforms andin which ratio with one another are present in cells incombination with AP-2-modulating factors.

The aim of our work was to dissect the roles of AP-2�and AP-2� in tumor formation and progression. Usingmicroarray analysis of AP-2� down-modulated cells, weshow that these members of the AP-2 family regulatetumor growth and chemotherapy-induced-cell death, aswell as migration and invasion via specific geneticprograms. We also present data showing that migrationis mediated, at least in part, by secreted factors, and weshow that ESDN, EREG, and CXCL2 played a major rolein the control of tumor cell movement.

MATERIALS AND METHODS

See also Supplemental Data.

Cell culture

The following human cell lines were used; their origins andgeneral properties, as illustrated by American Type CultureCollection (ATCC, Manassas, VA, USA), are as follows: HeLa:cervix adenocarcinoma (AC), HPV-18 positive; MDA-MB-231:breast AC, pleural effusion; MDA-MB-435: breast ductal car-cinoma, pleural effusion (microarrays cluster these cells withcell lines of melanoma origin and away from lines of breastorigin, possibly due to neuroendocrine expression patternsseen in some breast ACs or to the fact that this cell line haslost lineage fidelity; alternatively, this cell line was establishedfrom cells of a coexisting occult melanoma); 293T: fetus,kidney, SV-40T-antigen positive, highly transfectable; Phoe-nix: virus-packaging cell line. All of the cells were grown assuggested by ATCC.

Reagents, antibodies, and DNA constructs

Transient knockdown of various genes was performed usingspecific or mutated or nonsilencing (NS) annealed double-stranded custom or commercial siRNAoligos (Qiagen, Stanford,CA, USA). Target sequences were as follows: oligo2�, 5�-AAG-CAGTAGCTGAATTTCTCA-3�; oligo4�, 5�-AACATCCCAGAT-CAAACTGTA-3�; oligo1�, 5�AAGGTCCCATTTCCATGACCA-3�; oligo2�, 5�-GAGGTGTTCTCAGAAGAGCCA-3�; oligo2�mut,5�-AAGCAGTAGCTCAATTTGTGA-3�; oligo4�mut, 5�-AA-CATCCCAGATCAAAGTCTA-3�; oligo1�mut, 5�-AAGGTC-CCATTTCCATCAGGA-3�; oligo2�mut, 5�-GAGGTGTTCTCA-GAACACGGA-3�; NS, 5�-AATTCTCCGAACGTGTCACGT-3�;

ESDN, catalog no. SI02663304; EREG, catalog no. SI03019387;CXCL2, catalog no. SI02627758. The oligo2�, oligo4�, andoligo4�mut sequences were used to obtain stable knockdown aswell. BglII and HindIII sites were added at the 5� and 3� ends,respectively, of the oligo sequences. Oligos were phosphory-lated, annealed, and cloned into pSUPER.retro.puro vector(OligoEngine, Seattle, WA, USA) at the BglII and HindIII sites.The following pSUPER.retro.puro-shRNA vectors were ob-tained: AP-2�shRNA2, AP-2�shRNA4, and AP-2�shRNA4mut.Lentivirus shRNA expression vectors for hAP-2� or hAP-2� werepurchased from Open Biosystems (Huntsville, AL, USA): AP-2�,shRNA4922 (catalog no. RHS3979–4922) and shRNA4923 (cat-alog no. RHS3979–4923); AP-2�, shRNA19744 (catalog no.RHS3979–19744), and shRNA19745 (catalog no. RHS3979–19745) and used for stable shRNA expression. pSP(RSV)AP-2�and pSP(RSV)AP-2� expression vectors, a gift from Dr. H. Hurst(29, 31), were used to overexpress AP-2� and AP-2� in cells.Primary antibodies used were anti-AP-2� mAb 3B5, AP-2� pAbC-18, AP-2� mAb 6E4/4, GAPDH pAb V-18, and Ki67 pAb C-20(Santa Cruz Biotechnology, Santa Cruz, CA, USA); F4/80 mAb(Serotec, Oxford, UK); VEGF� pAb (R&D, McKinley Place, MN,USA); NG-2 pAb (Chemicon, Billerica, MA, USA); Ly6G mAb(Abcam, Cambridge, UK); and MECA-32 mAb (BD, FranklinLakes, NJ, USA), used at the producer’s suggested concentra-tions. Secondary antibodies used were goat anti-mouse or anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-conjugated, donkey anti-goat IgG HRP-conjugated (Santa CruzBiotechnology); anti-rabbit Alexa-Fluor-488 or -555, anti-rat Al-exa-Fluor-488, and anti-goat Alexa Fluor-488 (Molecular Probes,Invitrogen Life Technologies, Carlsbad, CA, USA), all used atthe producer’s suggested concentrations.

Transient and stable transfections of siRNAoligos andplasmids

To obtain transient siRNAoligo-mediated gene silencing, cellswere transfected using RNAiFect (Qiagen) reagents, and toobtain transient expression of pSUPER.retro.puro-shRNAand cDNA-overexpression vectors, Lipofectamine2000 (In-vitrogen Life Technologies) was used, in both cases accordingto the manufacturer’s instructions. In some cases, a secondtransfection was carried out 24 h after the first transfection.Cells were tested for overexpression or knockdown 48 to 72 hlater. For stable cDNA overexpression, pSUPER.retro.purowas used as selection vector, and complete medium contain-ing 3 �g/ml puromycin was added to the cells 72 h aftertransfection. Clones were picked and expanded 1 wk later.

Retroviral or lentiviral infections and gene silencing withshRNAs

Retroviral (42) or lentiviral (43) infections were performed asdescribed previously.

Microarrays and data analysis

Total RNA was isolated 48 h after transient transfections usingphenolic TRIzol Reagent (Invitrogen Life Technologies),according to the manufacturer’s instructions. Equal amountsof labeled cRNA were synthesized for each sample and usedto hybridize 44K Whole Human Genome Oligo MicroarrayArrays (Agilent Technologies, Palo Alto, CA, USA) followingAgilent’s protocol. Then, slides were scanned, and imageswere analyzed using Feature Extraction software ver. 7.6(Agilent Technologies). Raw data files were processed asdescribed by Chiorino et al. (44). Microarrays were performedat the Fondo Edo Tempia (Biella, Italy).

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Quantitative real-time polymerase chain reaction (qRT-PCR)

RNA was purified as for microarray analysis and qRT-PCR,and calculations were carried out as described by Bookoutand Mangelsdorf (45). Primer sequences are available onrequest.

Protein preparation and immunoblotting

Total protein extracts were prepared and used for Westernblot analysis as described by Orso et al. (34).

Proliferation assay

The proliferation assays were performed according to theprotocol described by Kueng et al. (46).

Soft agar and xenotransplant assays

Soft agar and xenotransplant assays were performed as de-scribed by Hynes et al. (47).

Immunohistochemistry

Tissue samples were processed and immunofluorescenceperformed on sections as described by Giraudo et al. (48).

Cell death analysis

Cell death was analyzed in cells kept in growth medium withor without staurosporine (STS; Sigma Aldrich, St. Louis, MO,USA) or paclitaxel (PTX; Bristol-Myers Squibb, Rocky Hill,NJ, USA), and data were acquired and analyzed as describedby Rasola et al. (49). Xenotransplant cell death was analyzedusing the Dead End fluorimetric terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL) assay (Pro-mega, Madison, WI, USA).

In vitro migration and invasion assays

105 cells were resuspended in 200 �l serum-free Dulbeccomodified Eagle medium (DMEM) and seeded in the upperchambers of 24-well Falcon cell culture inserts (BD Bio-sciences) over a porous membrane (8.0 �m pore size)precoated or not with 2 �g/well Matrigel (BD Biosciences).The lower chamber was filled with either serum-free (negativecontrol) or 10% FBS-DMEM. After 18 or 24 h, migrated cells,which had stuck to the lower side of the membrane were fixedin 2% glutaraldehyde, stained with 0.1% crystal violet, andphotographed using a Leica DM IRB microscope equippedwith a CoolSNAP-Pro charge-coupled device (CCD) camera(Media Cybernetics, Silver Spring, MD, USA). Migration andinvasion were evaluated either by counting the number ofcells that reached the lower side of the membrane or bymeasuring the area occupied by these cells by using the ScionImage 1.62 software (http://www.scioncorp.com/ or http://rsb.info.nih.gov/nih-image/). For conditioned medium(CM) experiments, cells were kept in serum-free mediumovernight; medium was then collected and used as stimulusfor migration in the lower chambers.

Bioinformatic analysis of the promoters

Retrieval of regulative elements (RRE) software for retrievalof noncoding regulative elements from annotated genomedatabases was used to identify noncoding or regulatory re-

gions associated to annotated genes and to localize putativebinding sites for the AP-2 transcription factors as described byLazzarato et al. (50).

Chromatin immunoprecipitation (ChiP) assays

ChIP was performed using the ChIP-IT Kit (Active Motif,Carlsbad, CA, USA) reagents and protocols. Primer pairswere designed using RRE software (see above) and sequencesare available on request. In some cases, qRT-PCR was per-formed to evaluate the ChIP.

Statistical analyses

Statistical significance of the various experiments was testedby performing an F test followed by a 2-tailed t test. P indicatesthe probability of identity of the distributions. Xenograftdistributions are represented by box and whisker plots, wherethe box represents the interquartile range (central 50% ofthe data points), horizontal lines in the boxes represent themedian, and vertical bars represent a spread of 1.5� inter-quartile range.

RESULTS

Down-regulation and overexpression of AP-2� andAP-2� in cells

To perform AP-2 gene silencing, we first analyzed AP-2�and AP-2� protein (Fig. 1A) and mRNA (Fig. 1B) levels inHeLa, MDA-MB-435, and MDA-MB-231 cells by Westernblot and qRT-PCR analyses. While MDA-MB-231 cellsexpressed very low AP-2� and AP-2� levels, MDA-MB-435contained high amounts of both isoforms, and HeLa cellsexpressed only AP-2�. siRNAs or shRNAs were used todown-modulate AP-2� or AP-2� expression in the threecell lines in a transient or stable manner. Silencing wasverified by Western blot (Fig. 1A, C) and qRT-PCR (Fig.1B). Strong (60–90% reduction) and specific AP-2� pro-tein (Fig. 1A) and mRNA (Fig. 1B) down-modulationwere obtained in HeLa and MDA-MB-435 cells transientlytransfected with oligo2� or oligo4� siRNAoligos whencompared with generic NS or mutated (oligo4�mut)siRNA sequences. Similarly strong reduction (60–90%)and specific AP-2� silencing were obtained in MDA-MB-435 transiently transfected with oligo1� or oligo2� siR-NAoligos compared with control sequences (NS oroligo2�mut). Silencing of both AP-2� and AP-2� wasobtained in MDA-MB-435 cells following transient trans-fection of oligo2� and oligo2�. In all cases, silencing wasverified 48 or 72 h post-transfection. Alternatively, HeLa(Fig. 1C) or MDA-MB-435 (Fig. 1C) cells were trans-duced with the following retrovirus or lentivirus vec-tors: empty pSUPER.retro.puro (retro-empty), AP-2�shRNA2, AP-2�shRNA4, or AP-2�shRNA4mut (Fig.1C); empty pLKO.1 (lenti-empty), AP-2�shRNA4922,AP-2�shRNA4923, AP-2�shRNA19744, or AP-2�shRNA-19745 (Fig. 1C); and AP-2� or AP-2� shRNA stableexpressing cell lines were obtained showing high andspecific AP-2� or AP-2� gene silencing. It is importantto observe that in MDA-MB-435 cells, transient or stable

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silencing of AP-2� leads sometimes to a slight reductionof AP-2� protein levels (Fig. 1B, C). This could be dueto an miRNA-like effect on AP-2� translation or to atranscriptional regulation of AP-2� by AP-2�. AP-2� orAP-2� transient or stable overexpression was obtainedin HeLa or MDA-MB-231 cells following transfection ofpSP(RSV)NN (empty) or pSP(RSV)AP-2� or pSP(RS-V)AP-2� expression vectors (Fig. 1C). pSUPER.retro.

puro vector was used as a selection plasmid to obtainstable AP-2-overexpressing MDA-MB-231 clones.

AP-2� and AP-2� modulate cell proliferation anddrug-induced cell death

Analysis of cell proliferation (Fig. 2A) and cell death(Fig. 2B) was performed on HeLa, MDA-MB-435, or

Figure 1. AP-2� and AP-2� knockdown and overexpression in tumor cells. Proteins (A, C) or mRNA (B) were extracted fromparental or modified tumor cells (HeLa, MDA-MB-435, MDA-MB-231) and analyzed by Western blot (A, C) or qRT-PCR (B) forAP-2� or AP-2�. HeLa (A, B) and MDA-MB-435 (A, B) cells were transiently transfected with generic NS, specific (oligo2�,oligo4�, oligo1�, oligo2�), or mutated (olig4�mut, oligo1�mut, oligo2�mut) AP-2� or AP-2� siRNAoligos, one by one or incombination. Alternatively, HeLa cells (C) were infected with retroviruses for the expression of one or more of the followingvectors: pSUPER.retro.puro (retro-empty), AP-2�shRNA2, AP-2�shRNA4, and AP-2�shRNA4mut. MDA-MB-435 cells (C) wereinfected with lentiviruses for the expression of one or more of the following vectors: pLKO.1 (lenti-empty), AP-2�shRNA4922,AP-2�shRNA4923, AP-2�shRNA19744, or AP-2�shRNA19745. Transient or stable AP-2� or AP-2� overexpression was obtainedin HeLa (C) and MDA-MB-231 (C) cells, respectively, by transfection of either pSP(RSV)AP-2� or pSP(RSV)AP-2� expressionvectors. For stable overexpression, the selection vector retro-empty was cotransfected with the expression vectors. Threerepresentative MDA-MB-231 clones were used: pSP(RSV)NN#2 (empty vector), pSP(RSV)AP-2�#1, and pSP(RSV)AP-2�#28.mAb 3B5 and 6E4/4 mAbs were used to detect AP-2� and AP-2� protein expression, respectively (A, C). Glyceraldheyde-3-phosphate-dehydrogenase (GAPDH) expression was used for protein loading controls. Each qRT-PCR was performed intriplicate and repeated 3 times, and 18S was used as reference gene. One representative experiment is shown in each panel. Leftpanel in B shows AP-2� and AP-2� expression levels normalized over MDA-MB-231 cells.

2705ROLE OF AP-2 IN TUMOR PROGRESSION

MDA-MB-231 cells permanently expressing AP-2�shRNAs, AP-2�shRNAs, AP-2�cDNA, or AP-2�cDNA.No difference in growth was observed for retrovirus-infected HeLa cells expressing retro-empty, AP-2�shRNA2, or AP-2�shRNA4 (data not shown) vectorsfollowing serum stimulation for 6 days (Fig. 2A). How-ever, when the same experiment was performed inlentivirus-infected MDA-MB-435 cells expressing lenti-empty, AP-2�shRNA4923, or AP-2�shRNA19745 vec-tors, a slight retardation in cell growth at early, but notlate, time points was observed (Fig. 2A), suggesting thatAP-2� or AP-2� is important for cell proliferation ofhighly sparse but not confluent MDA-MB-435 cells.When the same HeLa, MDA-MB-435, or MDA-MB-231cells were used to analyze STS-induced (Fig. 2B, C) orPTX-induced (data not shown) cell death by flowcytometry, an important difference in cell survival wasobserved if AP-2� but not AP-2� was down-modulatedor when either isoform was overexpressed. In fact,increased cell survival (62.5 or 66.4 vs. 34.7%) was

observed in HeLa cells expressing either AP-2�shRNA2or AP-2�shRNA4 vectors vs. control retro-empty-vector-expressing cells grown in complete medium for 24 h inthe presence or absence of STS. Similarly, MDA-MB-435 cells survived better if expressing AP-2�shRNA4923(58.6%) compared with lenti-empty-expressing controlcells (43.9%), while a slight but statistically significant(P�0.05; 2-tailed Student’s t test) increase in cell deathwas observed in AP-2�19745 shRNA-expressing cells. In-terestingly, in STS-stimulated low-AP-2�-expressing cells,we observed an accumulation of cells in the initial stagesof cell death, suggesting that AP-2� may have a role inregulating the transition between early to more advancedphases of programmed cell death. When the stable MDA-MB-231 clones (Fig. 2B, C) expressing pSP(RSV)NN(clone 2), pSP(RSV)AP-2� (clone 1), or pSP(RSV)AP-2�(clone 28) vectors were treated with STS for 24 h, arelevant increase in cell death was observed in both AP-2�-and AP-2�-overexpressing clones (19.4 and 13.9%, respec-tively) compared with control cells (36.9%).

Figure 2. AP-2� and AP-2� modulate cell proliferation and drug-induced cell death. A) Analysis of proliferation in HeLa orMDA-MB-435 cells expressing the indicated vectors. Cells were plated and starved for 24 h in serum-free medium, then 10% FBSwas added onto cells. Cells were fixed and stained at the indicated days, and optical density was measured. The experiments wereperformed in triplicate and repeated at least twice. B, C) Investigation of cell death in retrovirus- or lentivirus-infected HeLa orMDA-MB-435 cells expressing the indicated vectors, as well as in AP-2�- or AP-2�-overexpressing MDA-MB-231 clonesp(SP)RSVNN#2, p(SP)RSVAP-2�#1, and p(SP)RSVAP-2�#28. Cells were grown in complete medium with or without 25 or 50ng/ml STS for 24 h (HeLa and MDA-MB-231) or 72 h (MDA-MB-435). Results are displayed in bidimensional plots showingTMRM and AnnexinV-FITC staining intensities. Propidium iodide (PI) was used to evaluate the percentage of PI-permeabledead cells. H, percentage of healthy cells, delimited by the gate (R1). The experiment was repeated 4 times. Results in B are fromrepresentative experiments; results in C are means sd for the 4 experiments.

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AP-2� or AP-2� down-regulation enhances growth inxenotransplants via increased proliferation

The tumorigenic potential of HeLa or MDA-MB-435cells expressing empty, AP-2�shRNA, or AP-2�shRNAvectors was analyzed in vitro (soft agar, Fig. 3A) and invivo (xenotransplants, Fig. 3B). A 3- to 6-fold increase inthe number of colonies grown in soft agar was observedfollowing AP-2� or AP-2� knockdown in MDA-MB-435(AP-2�shRNA4923 or AP-2�shRNA19745) and forAP-2� ablation in HeLa (AP-2�shRNA2 or AP-2�shRNA4) cells compared with controls. In vivo xeno-transplant growth was increased in the same way (Fig.3B). HeLa or MDA-MB-435 cells expressing empty,AP-2�shRNA, or AP-2�shRNA vectors were injectedsubcutaneously in the flanks of 10 immunodeficientnude mice, and tumor diameters were measured twicea week. Xenotransplants were harvested 21 days (MDA-MB-435) or 35 days (HeLa) postinjection. Tumor vol-ume (data not shown) and weight plots (Fig. 3B)showed that AP-2�shRNA- or AP-2�shRNA-expressingcells grew faster (data not shown) and gave rise tobigger tumors compared with controls; in the case ofHeLa cells, control xenografts stopped growing by day15 (data not shown). The same experiment was re-peated for Hela cells, except that the tumors wereharvested at day 14 postinjection, and a similar growthcurve was observed (data not shown). In this case, aswell as at day 21 postinjection for MDA-MB-435, 3control, AP-2�shRNA-expressing, or AP-2�shRNA-ex-pressing xenografts were fixed and used for immuno-histochemical analysis. Increased proliferation, as mea-sured by analysis of nuclear antigen Ki67 expression(Supplemental Fig. S1), was observed in absence ofAP-2� or AP-2�, explaining the observed increase intumor growth. TUNEL analysis of cell death showed nodifference between the various tumor groups (data notshown). No striking differences were noticed in tumorangiogenesis, as studied by staining endothelial cellsand pericytes for MECA-32 and NG2, respectively (datanot shown). Reduced infiltration of macrophages and

neutrophils was observed in AP-2�shRNA-expressingHeLa xenografts compared with controls (data notshown), but not in AP-2�shRNA- or AP-2�shRNA-ex-pressing MDA-MB-435 tumors (data not shown), prob-ably because of the later time points used for thisanalysis. All these data suggest that AP-2� and AP-2�regulate tumor growth mostly during tumor establish-ment and early growth.

AP-2� and AP-2� coordinate cell migration, motility,and adhesion

So far we have demonstrated the importance of AP-2�and AP-2� in tumor establishment and growth. Tounderstand the role of AP-2� and AP-2� in tumorprogression, we used transwell assays to analyze migra-tion through a porous membrane toward serum (che-motaxis) for AP-2� or AP-2� knockdown HeLa orMDA-MB-435 cells, as well as for HeLa or MDA-MB-231cells overexpressing AP-2� or AP-2� (Fig. 4A). Reducedmigration (40–80%) was observed when cells weresilenced for AP-2� or AP-2� in a transient or stablemanner, while cells overexpressing at least one AP-2isoform showed increased migration. Motility of HeLacells expressing either retro-empty or AP-2�shRNA2vectors was monitored by a wound-healing assay. Afterwounding, the movement of migrating cells was fol-lowed by time-lapse microscopy for 18 h as the woundrecovered. A 40% reduction of motility was observedfor AP-2�shRNA2-expressing cells compared with con-trol cells (Supplemental Fig. S2A). Adhesion propertieswere also investigated in this cell line. In particular, weperformed adhesion assays to plastic, laminin 1 (datanot shown), collagen IV, or fibronectin (SupplementalFig. S2B). AP-2�shRNA2 cells showed increased adhe-sion to collagen IV and fibronectin compared withcontrol cells, whereas no statistically significant differ-ences were observed in adhesion to plastic or laminin 1.Finally, to simulate tumor invasion in vivo, we investi-gated invasion through matrigel of MDA-MB-435 AP-2�and AP-2� knockdown cells and of MDA-MB-231 cells

Figure 3. AP-2� and AP-2� down-modulation enhances tumor growth. Retrovirus- or lentivirus-infected HeLa or MDA-MB-435cells expressing the indicated vectors were plated in 0.45% soft agar (A) and the number of grown colonies was evaluated 25days later. The experiment was performed in triplicate and repeated 3 times. Alternatively, the same cells were injectedsubcutaneously in nude mice, and tumor weight was measured at the end of the experiment (B). The experiments wererepeated twice. The increased tumorigenicity both in vitro (A) and in vivo (B) is statistically significant. ***P 0.05; 2-tailedStudent’s t test; n � number of animals per group. Representative experiments are shown.

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overexpressing AP-2� or AP-2� compared with controls.We observed a clear reduction in the invasion capabilityof AP-2�shRNA4923- and AP-2�shRNA19745-express-ing MDA-MB-435 cells (40 and 80% decrease, respec-tively) compared with control cells and an obviousincrease in invasion for pSP(RSV)AP-2�#1 (3-fold in-crease) and pSP(RSV)AP-2�#28 (6-fold increase) MDA-MB-231 clones compared with controls (Fig. 4B). Alltogether, these data suggest that AP-2� and AP-2� leadtumor progression toward malignancy.

Migration defects are at least in part due to secretedfactors

We then investigated whether migration impairmentwas cell autonomous or due to secreted factorsregulated by AP-2�. We analyzed the effect of serum-

free CM produced by HeLa cells, expressing retro-empty, AP-2�shRNA2, or AP-2�shRNA4 vectors oncontrol HeLa cells (Fig. 4C) in a transwell assay(chemotaxis). Migration toward serum was used ascontrol. Reduced chemotaxis of control HeLa cellswas observed only when CM from AP-2�shRNA2 orAP-2�shRNA4 cells was used as stimulus (lower cham-ber) but not when control CM (produced by retro-empty-expressing Hela cells) was added, suggestingthat migration impairment was at least in part due tosecreted molecules. The effect of AP-2�-null murineembryo fibroblast (MEF) CM was tested on MDA-MB-435 tumor cell migration, and also in this case, weobserved reduced migration compared with controlAP-2�-positive MEF CM (data not shown). These datasuggest that AP-2� regulates secreted factors that areinvolved in tumor cell migration.

Figure 4. AP-2� and AP-2� modulatetumor cell migration and invasion.A) Analysis of migration in transwelldishes for retrovirus-infected HeLacells expressing either retro-emptyor AP-2�shRNA2 vectors or forMDA-MB-435 transiently transfectedwith generic NS, specific (oligo2�,oligo4�, oligo1�, oligo2�), or mu-tated (oligo4�mut, oligo2�mut)AP-2� or AP-2� siRNAoligos one byone or in combination. Transientlyand stably AP-2� or AP-2� overex-pressing HeLa and MDA-MB-231cells were used as well to investigatemigration. Cells were plated in se-rum-free medium in the upperchamber and allowed to migrate

over medium containing 10% FBS for 18 h. B) Evaluation of invasion through matrigel in lentivirus-infectedMDA-MB-435 cells expressing lenti-empty, AP-2�shRNA4923, or AP-2�shRNA19745 vectors and in AP-2�- andAP-2�-overexpressing MDA-MB-231 clones. Cells were plated in serum-free medium in the upper chamber of matrigelprecoated transwells and let migrate over 10% FBS medium for 24 h. C) Investigation to test whether migration is dueto secreted factors regulated by AP-2�. Panel shows analysis of transwell migration of control retro-empty-expressingHeLa cells over serum-free CM derived from overnight cultures of HeLa cells expressing retro-empty, AP-2�shRNA2,or AP-2�shRNA4 vectors present in the lower chamber (chemotaxis). Migration over medium containing 10% FBS wasused as a control. Each experiment was performed in triplicate and repeated twice. The area occupied by migratingor invading cells or the total number of cells was measured. ***P 0.05; 2-tailed Student’s t test. Representativeexperiments are shown.

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Identification of potential AP-2�-regulated genes bymicroarray analysis

To identify which AP-2�-regulated genes were involvedin tumor formation (growth and cell death) and pro-gression, we performed microarray analysis on HeLacells transiently transfected either with the NS or theoligo2� siRNAoligos. RNA samples were extracted from3 independent transfections, labeled, and hybridized toAgilent Whole Human Genome Oligo Microarrays 44K.Pronounced AP-2� knockdown (90% reduction) wasobtained (similar to Fig. 1A, B) for all transfections. Inall, 721 differentially expressed genes [fold change(FC)�1.5; pv0.01] were identified as either signifi-cantly up-regulated (309) or down-regulated (412) inthe absence of AP-2�. For a complete list of newlyidentified genes, see Supplemental Table S1; a selectgroup of the most highly ranked differentially ex-pressed genes (FC�2) is shown in Table 1. Some ofthese genes had been previously demonstrated to beAP-2 regulated (i.e., CDKN1A, PPARG, and KLF2), andthey represent our internal controls. By performing aGene Ontology (GO) analysis, we observed genes in-volved in cell cycle (i.e., CDKN1A, CCNDBP1, SESN3,SESN1), cell death (i.e., HIPK3, BIRC3) and cell adhe-sion or motility (i.e., ITGBL1, WISP2, PODXL, CXCL1,CXCL2, EREG, ESDN); see Table 1. We validated ourmicroarray data by performing qRT-PCR analyses for 11randomly chosen new modulated genes (BIRC3, CCNDBP1,ESDN, EREG, FASTK, HRASL3, INSL4, KRT17, IL11,CXCL1, and CXCL2) and 3 known AP-2-regulated genes(CDKN1A, KLF2, and PPARG) using RNA samples from3 independent transfections (Fig. 5A, top panel). Theexpression levels of ESDN, EREG, and CXCL2 were alsotested by qRT-PCR in lentivirus-infected MDA-MB-435cells expressing lenti-empty, AP-2�shRNA4923, or AP-2�shRNA19745. EREG and CXCL2 were not expressedin these cells, whereas ESDN was expressed at highlevels in control cells, and its expression increasedfollowing AP-2� and AP-2� down-modulation (data notshown). As a further demonstration that these geneswere, in fact, AP-2� regulated, we performed the oppo-site experiment. HeLa cells were transiently transfectedwith either empty pSP(RSV)NN or pSP(RSV)AP-2�expression vectors to obtain transient AP-2� overex-pression. At 72 h after AP-2� transfection, we extractedRNA, verified AP-2� levels, and analyzed the expressionof ESDN, EREG, KLF2, CXCL1, CXCL2, and PPARG byqRT-PCR. Reduced expression of these genes was ob-served, as expected (Fig. 5A, bottom panel). To furtherinvestigate whether the regulation of these genes viaAP-2� was direct, we used a bioinformatic approach(RRE software available at http://www.bioinformatica.unito.it/bioinformatics/rre/rre.html) to identify po-tential AP-2� binding sites in the regulatory regions[�2000 to 500 relative to transcription start site(TSS)] of several genes. Binding sites with differentscores were identified for ESDN, EREG, CXCL2, KRT16,TPM3, and AIM1 and AP-2� binding were assayed

experimentally by ChIP analysis for some of the highestscore sites. As shown in Fig. 5B, we confirmed AP-2�binding to the promoter of ESDN, but we were not ableto show specific AP-2� binding to EREG or CXCL2promoters. Further investigations for AP-2� binding tothe lower score binding sites will be necessary for theselast two genes. However, we cannot exclude an indirectAP-2� regulation for EREG and CXCL2 genes. CDKN1Apromoter was used as a positive control for AP-2�binding. The specificity of the assay was tested onAP-2�-silenced 293T cells using a qRT-PCR analysis anda decrease in AP-2� binding was observed when com-pared with control cells (data not shown).

AP-2� regulates tumor cell migration via ESDN,EREG, and CXCL2

Our microarray analysis showed that a subset of genespotentially involved in migration were highly modu-lated. We selected three of these for functional valida-tion: ESDN (FC�5.7), EREG (FC�2.5), and CXCL2(FC�3.0). We transiently transfected HeLa cells withspecific single siRNAoligos against ESDN (oligoESDN),EREG (oligoEREG), or CXCL2 (oligoCXCL2) or with acombination of the three oligos (siRNA MIX) andanalyzed migration. At 48 h after transfection, genesilencing was observed (Fig. 5C, top panel), and migra-tion in response to serum was analyzed (Fig. 5C, bottompanel). An increased number of migrating cells (4-foldincrease) was observed following ESDN, EREG, andCXCL2 knockdown one by one or in combination(siRNA MIX), suggesting a direct involvement of thesegenes in cell migration. Moreover, transient ESDNknockdown in AP-2�-silenced cells reverted the AP-2�shRNA effect in HeLa cell migration, suggesting adirect link between ESDN and AP-2� (data not shown).All of these data taken together suggest that AP-2�regulates tumor cell migration via the modulation ofsome specific genes, in particular ESDN, EREG, andCXCL2.

DISCUSSION

AP-2 transcription factors are known to participate intumorigenesis. However, it is not understood whetherand how the various family members contribute toneoplasia or whether they act by promoting or sup-pressing tumorigenesis; contrasting results, even for thesame kind of tumors, can be found in the literature. Weexplored the function of AP-2� and AP-2� duringcervix, breast, and melanoma/breast tumor formationand progression and demonstrated a major role forthese two isoforms. In fact, knockdown of AP-2� orAP-2� induced increased tumor growth both in vitroand in vivo. Increased proliferation but no change incell death was observed in xenotransplants togetherwith increased innate immune cell infiltration (macro-phages and neutrophils) in the early stages of tumorgrowth. However, we found that chemotherapy-in-

2709ROLE OF AP-2 IN TUMOR PROGRESSION

TABLE 1. Differentially expressed genes in AP-2� siRNA-expressing HeLa cells

Accession number Name Description FC Function

NM_012142 CCNDBP1 Cyclin d-type binding-protein 1 �3.7 Cell cycleNM_004566 PFKFB3 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 �3.6 Cellular metabolismNM_004078 CSRP1 Cysteine and glycine-rich protein 1 �3.4 Intracellular organelleNM_001956 EDN2 Endothelin 2 �3.2 Protein bindingNM_000366 TPM1 Tropomyosin 1 (alpha) �3.0 Cell motilityBC036402 MAD MAX dimerization protein 1 �2.9 Cell proliferationNM_000422 KRT17 Keratin 17 �2.7 DevelopmentNM_004791 ITGBL1 Integrin, beta-like 1 (with EGF-like repeat domains) �2.6 Cell adhesionAK093577 MRTF-B Myocardin-related transcription factor B �2.6 Cell differentiationNM_000526 KRT14 Keratin 14 (epidermolysis bullosa simplex, Dowling-

Meara, Koebner)�2.6 Development

AK091132 SESN3 Sestrin 3 �2.6 Cell cycleAY004867 TPM3 Tropomyosin 3 �2.5 Cell motilityNM_005557 KRT16 Keratin 16 (focal non-epidermolytic palmoplantar

keratoderma)�2.5 Development

BC030112 HIPK3 Homeodomain interacting protein kinase 3 �2.4 ApoptosisNM_002670 PLS1 Plastin 1 (I isoform) �2.3 Actin bindingNM_002195 INSL4 Insulin-like 4 (placenta) �2.3NM_019006 AWP1 Protein associated with PRK1 �2.3 DNA bindingNM_005302 GPR37 G protein-coupled receptor 37 (endothelin receptor

type B-like)�2.3 Cell communication

NM_001630 ANXA8 Annexin A8 �2.2 Blood coagulationNM_033015 FASTK FAST kinase �2.2 ApoptosisNM_001613 ACTA2 Actin, alpha 2, smooth muscle, aorta �2.1 Muscle contractionNM_014454 SESN1 Sestrin 1 �2.1 Cell cycleNM_004508 IDI1 Isopentenyl-diphosphate delta isomerase �2.1 Alcohol metabolismNM_001165 BIRC3 Baculoviral IAP repeat-containing 3 �2.1 ApoptosisNM_024949 BOMB BH3-only member B protein �2.1NM_018440 PAG Phosphoprotein associated with glycosphingolipid-

enriched microdomains�2.1 Cell communication

NM_000641 IL11 Interleukin 11 �2.1 Wound healingNM_000389 CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) �2.1 Cell cycleNM_019001 SEP1 Strand-exchange protein 1 �2.1 Cell cycleNM_007069 HRASLS3 HRAS-like suppressor 3 �2.1 Cell cycleNM_006708 GLO1 Glyoxalase I �2.0 ApoptosisNM_004535 MYT1 Myelin transcription factor 1 �2.0 Cell differentiationNM_003881 WISP2 WNT1 inducible signaling pathway protein 2 �2.0 Cell adhesionNM_139241 FGD4 Actin-filament binding protein frabin �2.0 Actin cytoskeletonNM_021995 UTS2 Urotensin 2 2.0 Cell communicationNM_012388 PLDN Pallidin homolog (mouse) 2.0 Cell communicationNM_005397 PODXL Podocalyxin-like 2.0 Cell adhesionAK127078 FMN Homo sapiens similar to formin (LOC342184), mRNA 2.1 Actin cytoskeletonNM_012297 G3BP2 Ras-GTPase activating protein SH3 domain-binding

protein 22.1 Cell communication

NM_005261 GEM GTP binding protein overexpressed in skeletalmuscle

2.1 Cell communication

NM_004163 RAB27B RAB27B, member RAS oncogene family 2.2 Cell communicationNM_004734 DCAMKL1 Doublecortin and CaM kinase-like 1 2.2 Cell communicationNM_057161 KLHDC3 Kelch domain containing 3 2.3 Cell cycleAL050206 PPP2R2A Protein phosphatase 2 (formerly 2A), regulatory

subunit B (PR 52), alpha isoform2.3 Cell communication

BF733908 TPM3 Tropomyosin 4 2.3 Cell motilityU83115 AIM1 Absent in melanoma 1 2.4 UnknownNM_001511 CXCL1 Chemokine (C-X-C motif) ligand 1 (melanoma

growth stimulating activity, alpha)2.4 Cell communication

NM_001432 EREG Epiregulin 2.5 Cell communicationNM_015869 PPARG Peroxisome proliferative activated receptor, gamma 2.6 Cell communicationNM_002089 CXCL2 Chemokine (C-X-C motif) ligand 2 3.0 Cell communicationNM_012395 PFTK1 PFTAIRE protein kinase 1 3.3 Biopolymer metabolismNM_016270 KLF2 Kruppel-like factor 2 (lung) 3.4D42039 MESDC2 Mesoderm development candidate 2 3.9 DevelopmentNM_080927 ESDN Endothelial and smooth muscle cell-derived

neuropilin-like protein5.7 Cell adhesion

Microarray analysis (Whole Human Genome Agilent 44K) was performed on HeLa cells transiently transfected with either generic NS orspecific AP-2� (oligo2�) siRNAoligos; 721 modulated genes were found. Cutoff values: pv 0.01; FC � 1.5. A selection of the most highlymodulated genes (FC�2) is shown.

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duced cell death was AP-2� or AP-2� dependent. Forthe first time, we observed that altered AP-2� or AP-2�expression affected migration or invasion partially bysecreted growth factors; this strongly suggests thatAP-2� and AP-2� have important roles in cervix andbreast cancer progression. By performing whole-ge-nome analysis of gene expression, we identified theAP-2�-dependent genetic programs that orchestratetumor cell growth, cell death, and movement. In par-ticular, we showed that the AP-2-regulated genes ESDN,EREG, and CXCL2 have a major function in epithelialcell motility, because their ablation altered cell migra-tion.

Alteration of AP-2 expression patterns have beendescribed during malignant transformation of manytissues. Loss of AP-2 correlates with the acquisition of amalignant phenotype in melanomas (26) and breast(24, 25), prostate (28), and colorectal (27) cancers inwhich expression of AP-2� and/or AP-2� is oftenpronounced in normal tissues or benign tumors,whereas it decreases or disappears when the tumorsbecome invasive. Induced AP-2� overexpression invarious cell lines led to reduced tumorigenicity in vitroand in vivo (21, 35). Similarly, AP-2� overexpression in

the mammary gland of mice also negatively influencedErbB-2-induced tumor incidence (36). We observedthat tumor cell growth in xenotransplants and soft agarassays increased following AP-2� or AP-2� knockdown,which is in agreement with the above-mentioned re-sults; these findings reinforce the role of tumor sup-pressors for AP-2� and AP-2�. The fact that a similarincrease in proliferation was not observed in cell cul-ture plates (Fig. 2A) could indicate that either thesurrounding tissue or growth without contact inhibi-tion or even anoikis is playing an essential role here. Astrong increase in proliferation but no change in celldeath, angiogenesis, or VEGF production was observedin the xenotransplants, which suggests that cell cyclealteration is the first cause of tumor growth. Further-more, many genes involved in the cell cycle turned outto be regulated by AP-2� in our microarray analysis.Among them, we observed down-modulation of the cellcycle inhibitor p21Waf1/Cip1, which could be one of themajor factors responsible for increased cell growth(16). However, AP-2-regulated genes such as cyclinD-type binding protein 1, cyclin G2, CDK6, ERK1,p38MAPK, and WISP2 (see Table 1 and Supplemental

Figure 5. ESDN, EREG, and CXCL2 play a direct role in migration. A) Top panel: qRT-PCR validation of microarray data (Table1 and Supplemental Table 1S) for 14 genes on RNA extracted from Hela cells transiently transfected with generic NS or specificAP-2� (oligo2�) siRNAoligos. qRT-PCR analyses were performed in triplicate, and 3 independent transfections were performed.Microarray analysis and qRT-PCR-fold changes are shown for each validated gene as average values. Bottom panel: Evaluationof mRNA expression levels of genes potentially involved in cell migration, such as ESDN, EREG, CXCL1, and CXCL2, in HeLacells transiently overexpressing AP-2�-pSP(RSV)AP-2� vs. control pSP(RSV)NN cells. qRT-PCR analyses were performed intriplicate for two independent transfections; average values are shown. B) ChIP verification of AP-2 binding sites identified inthe regulatory regions (�2000 to 500 relative to TSS) of ESDN, EREG, and CXCL2 genes by using the RRE software. ChIP onCDKN1A promoter was used as a positive control. Chromatin from HeLa cells was cross-linked to proteins, extracted, andimmunoprecipitated with either AP-2� Abs (C-18 or 3B5) or nonspecific IgG (negative control) or RNA polymerase II Ab(positive control). DNA was analyzed by PCR, using primers flanking the AP-2� putative binding sites in CDKN1A, ESDN, EREG,and CXCL2 promoters. Input: not immunoprecipitated DNA. The experiment was repeated 3 times; one representativeexperiment is shown. C) Assessment of migration for HeLa cells transiently transfected with generic NS or specific siRNAoligos(oligoESDN, oligoEREG, or oligoCXCL2) or a mixture of the 3 siRNAoligos (siRNA MIX). Top panel: qRT-PCR evaluation ofESDN, EREG, and CXCL2 down-modulation. Bottom panel: Analysis of migration by transwell assays. Cells were plated inserum-free medium in the upper chamber of a transwell and let to migrate over 10% FBS medium for 18 h. The area occupiedby migrating cells was measured. The experiments were performed in triplicate and repeated twice. Representative experimentsare shown. ***P 0.05; 2-tailed Student’s t test.

2711ROLE OF AP-2 IN TUMOR PROGRESSION

Table S1) are certainly playing an important role in thiscontext as well.

Even if physiological cell death was not altered in cellcultures or xenotransplants, we found that drug-in-duced cell death was dependent on AP-2� or AP-2�when only one isoform was present in the cell. If bothisoforms were present, AP-2� had a major effect, againillustrating the importance of the ratio of the differentAP-2 isoforms. It has been previously observed thatendogenous AP-2� was induced by chemotherapeuticdrugs in various cancer cells (37). Moreover, using atetracycline-inducible system, the same investigatorsshowed that the response to chemotherapy dependedon the levels of AP-2� expression in an AP-2-empty cellsystem (MDA-MB-231 cells). Our AP-2� results agreewith those findings. Aggressive tumors that do notexpress AP-2� and AP-2� anymore most probably re-spond poorly to chemotherapy and have a higherchance of progressing on to metastatic cancers. It istherefore essential to discover why cells become che-moresistant and how we can revert chemoresistance inhuman cancer, analyzing changes in gene expressionassociated with malignant transformation.

The devastating phenotype of AP-2� and AP-2�knockout mice (12, 14) shows that these two genes playcrucial roles during embryogenesis. For the AP-2�-nullmouse, the cranial portion of the neural tube fails toclose, and the two hemispheres of the brain developopen. Medial nasal and mandibolar prominences fail toundergo midline fusion, and the same happens for theventral body wall (12). One can postulate that alteredcell migration or adhesion occur in AP-2�-null miceduring those important phases of embryogenesis. Inthe same way, we can postulate a role for AP-2 in tumorsduring epithelial-mesenchymal transition. AP-2� is cru-cial for melanoma progression due to the regulation ofgenes involved in cell movement (21). In the presentwork, we present data showing for the first time thatAP-2� and AP-2� regulate tumor cell migration andinvasion directly. In fact, removal of AP-2� or AP-2�impairs migration and invasion, whereas overexpres-sion increases movement. Our results are similar tothose observed by Jaeger et al. (36) in their in vivomouse tumorigenesis study. In that work, the investiga-tors analyzed tumor progression of mammary carcino-mas overexpressing ERBB-2 and AP-2� and observed anincreased proportion of advanced invasive stage carci-nomas, suggesting that these tumors are more prone toescape from their primary organs. Jaeger and col-leagues also suggested a dual role for AP-2. Expressionof AP-2 acted to prevent tumor formation, as men-tioned previously, but once the tumor was formed,invasion occurred. The fact that migration and invasionwere reduced in cells with AP-2�- or AP-2�-silencedexpression suggests that RNAi could be used in therapyfor those advanced tumors that remain AP-2 positive.Obviously, for those cases, an appropriate antigrowththerapy will be necessary to control the positive effect ofAP-2�-silencing on tumor growth. An appropriatemouse model will be necessary to test this approach.

Analysis of gene expression in HeLa cells lackingAP-2� revealed the modulation of several genes in-volved in cell motility. Extracellular products, such asthe HER/ErbB receptor ligand EREG; chemokines,such as CXCL1, CXCL2, and IL11; and surface mole-cules that can also be cleaved, such as ESDN, wereobserved to be major players in cell migration in oursystem. It is very important to recall that various studies(38, 39) have shown that EREG, chemokines, and ESDNare among the genes that characterize invasive breastcancer signatures. ESDN is a novel transmembraneprotein, which shows structural similarities to neuropi-lins, cell surface receptors for VEGF, and semaphorins(40). It contains a long N-terminal secretory signal thatseems essential to guide the protein to the cell surfaceand to induce cleavage, which indicates that ESDNcould be secreted like neuropilin-1 (40). Recently, ithas been shown that ESDN coordinates motility in lungcancer via interaction with SEMA4B (41). We are nowattempting the identification of these molecules in theculture medium of HeLa cells. The same moleculescould modulate not only tumor cell motility but alsothe recruitment of innate immune cells to the tumor.An increased production and secretion of these mole-cules by the tumor cells could be the cause of reducedmacrophage and neutrophil infiltration during theearly steps of tumorigenesis. It is intriguing to note thatalthough ESDN, EREG, and CXCL2 correlate negativelywith migration in our system, in other systems theirexpression has a positive influence on invasion, and infact highly aggressive breast cancers show increasedlevels of these molecules compared with controls (39).The discrepancy could be due to the fact that we arespecifically looking at AP-2-modulated migration,whereas in other systems the interplay of other tran-scription factors and motility molecules could be re-sponsible for cell movement.

In conclusion, our study is the first to analyze the roleof AP-2 in the various steps of tumorigenesis from aphenotypic and global transcription point of view. Wehave identified novel mediators for AP-2 functions, andwe want to characterize the importance of some ofthem, starting with ESDN, EREG, and CXCL2 duringtumor progression in terms of signal transduction andformation of metastasis in vivo. We believe that ourwork sets up the basis to identify a signature of AP-2-regulated genes involved in tumor progression to use astargets for gene therapy.

This work was supported by grants from the University ofTorino (Local Research Founding 2004, 2005, 2006), Re-gione Piemonte (CIPE2004), Ministero dell’Universita eRicerca (FIRB RBNE0157EH), Ministero della Sanita (PF2003/2005), Association for the Application of Biotechnolo-gies in Oncology (ABO grant TO47), Associazione ItalianaRicerca sul Cancro, and Fondazione Guido Berlucchi. F.O.and E.P. are fellows of the Regione Piemonte. We thankMichela Fassetta for performing some experiments; Dr. F.Girolami for helping us with the animal injections; B. Marti-noglio for technical assistance; Dr. H. Hurst for providing thepSP(RSV)NN, pSP(RSV)AP-2�, and pSP(RSV)AP-2� vectors;and Prof. B. White for helping us with the manuscript.

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Received for publication January 22, 2008.Accepted for publication April 2, 2008.

2714 Vol. 22 August 2008 ORSO ET AL.The FASEB Journal