increased pea3/e1af and decreased net/elk-3 - carcinogenesis

Carcinogenesis vol.30 no.8 pp.1433–1442, 2009doi:10.1093/carcin/bgp129Advance Access publication May 29, 2009

Increased PEA3/E1AF and decreased Net/Elk-3, both ETS proteins, characterizehuman NSCLC progression and regulate caveolin-1 transcription in Calu-1 andNCI-H23 NSCLC cell lines

Karin A.Sloan�, Hector A.Marquez1, Jun Li, Yuxia Cao,Anne Hinds, Carl J.O’Hara2, Satinder Kathuria3, MariaI.Ramirez, Mary C.Williams and Hasmeena Kathuria

Pulmonary Center, 1Department of Medicine, and 2Department of Pathology,Boston University School of Medicine, 72 East Concord Street, R304, Boston,MA 02118, USA and 3Department of Pathology, Edward Hines, Jr. VAHospital, 5000 South 5th Avenue, Hines, IL 60141, USA

�To whom correspondence should be addressed. Tel: þ1 617 638 4860;Fax: þ1 617 536 8093;Email: [email protected]

Caveolin-1 protein has been called a ‘conditional tumor suppres-sor’ because it can either suppress or enhance tumor progressiondepending on cellular context. Caveolin-1 levels are dynamic innon-small-cell lung cancer, with increased levels in metastatictumor cells. We have shown previously that transactivationof an erythroblastosis virus-transforming sequence (ETS) cis-element enhances caveolin-1 expression in a murine lung epi-thelial cell line. Based on high sequence homology between themurine and human caveolin-1 promoters, we proposed that ETSproteins might regulate caveolin-1 expression in human lung tu-morigenesis. We confirm that caveolin-1 is not detected in well-differentiated primary lung tumors. Polyoma virus enhanceractivator 3 (PEA3), a pro-metastatic ETS protein in breast cancer,is expressed at low levels in well-differentiated tumors and highlevels in poorly differentiated tumors. Conversely, Net, a knownETS repressor, is expressed at high levels in the nucleus of well-differentiated primary tumor cells. In tumor cells in metastaticlymph node sites, caveolin-1 and PEA3 are highly expressed,whereas Net is now expressed in the cytoplasm. We studied tran-scriptional regulation of caveolin-1 in two human lung cancer celllines, Calu-1 (high caveolin-1 expressing) and NCI-H23 (lowcaveolin-1 expressing). Chromatin immunoprecipitation-bindingassays and small interfering RNA experiments show that PEA3 isa transcriptional activator in Calu-1 cells and that Net is a tran-scriptional repressor in NCI-H23 cells. These results suggestthat Net may suppress caveolin-1 transcription in primary lungtumors and that PEA3 may activate caveolin-1 transcription inmetastatic lymph nodes.

Introduction

Caveolin-1 protein is essential for the formation of caveolae, whichare plasma membrane invaginations that sequester proteins, enzymesand signaling molecules such as epidermal growth factor receptor andSrc family kinases, and generally maintain them in an inactive form(1). Caveolin-1 levels are dynamic in tumorigenesis, and it is probablythat changes in caveolin-1 protein levels affect tumor progression byinfluencing cell signaling. In lung adenocarcinomas, virtually all met-astatic lymph nodes have increased caveolin-1 expression in tumorcells by immunohistochemistry. Conversely, in most primary lungtumors, caveolin-1 expression is exceedingly low (2–5). Little isknown about the regulation of caveolin-1 transcription in lung tumor-

igenesis, whether regulation is dependent on cellular context andwhich environmental factors influence expression.

Evidence suggests that caveolin-1 has a causal role in tumorigen-esis and acts as a ‘conditional tumor suppressor’ that can preventtumor development and promote metastasis depending on cellularcontext (6). The principle of a protein being either tumor suppressingor promoting depending on context has been demonstrated for otherproteins including transforming growth factor beta (1). In support ofthe tumor-suppressing function of caveolin-1, overexpressing caveo-lin-1 in murine fibroblast cell lines blocks cell cycle progression inG0/G1 (7). Caveolin-1�/� mice are more susceptible to skin tumorsupon carcinogenic exposure and caveolin-1�/� mice crossed withinducible breast cancer mice exhibit early-onset tumor formationand increased metastases (8,9). Caveolin-1 has been mapped to a re-gion of chromosome 7q31.1 often deleted in cancers (10).

Known mechanisms for loss of tumor suppressor function ofcaveolin-1 are tyrosine phosphorylation at the N-terminus, whichcauses recruitment of proteins involved in anchorage-independentgrowth; serine phosphorylation at Ser80, which converts caveolin-1to a secreted protein and a dominant-negative P132L mutation inbreast cancer, which leads to a misfolded caveolin-1 protein (reviewedin ref. 1). In addition, E-cadherin expression is lost as tumors prog-ress, and caveolin-1 requires E-cadherin to function as a tumorsuppressor (11).

In several malignancies, caveolin-1 promotes the malignant pheno-type. Increased caveolin-1 expression has been correlated with me-tastasis in breast, prostate and colon carcinomas (1). In lung, there isno caveolin-1 expression in low-invasive lung adenocarcinoma celllines, but there is abundant expression in highly invasive cell lines.Induced caveolin-1 gene expression in a low-invasive lung adenocar-cinoma cell line increases cell migration and invasiveness (4). Inprostate cancer, crossing caveolin-1�/� mice with TRAMP micethat develop spontaneous prostate tumors has shown that loss ofcaveolin-1 impedes progression to highly invasive and metastaticdisease (12).

We previously identified a cis-element, containing the erythro-blastosis virus-transforming sequence (ETS) DNA-binding sequence5#-GGAA/T3-#, in the proximal mouse caveolin-1 promoter. Activa-tion of this element by the ETS protein ERM (ETS-related moleculePEA3) strongly enhances caveolin-1 gene expression in a mouse lungepithelial cell line (13,14). In Ewing’s sarcoma, caveolin-1 is a majortarget of the ETS fusion protein EWS-FLI-1 (Ewing sarcoma gene-friend leukemia 1). Knockdown of caveolin-1 expression downregu-lates the malignant phenotype of Ewing’s sarcoma cell lines. Loss ofcaveolin-1 expression reduces the growth of Ewing sarcoma gene cell-derived tumors in nude mice xenografts (15).

We hypothesized that the caveolin-1 promoter is differentially reg-ulated, probably by ETS proteins, in primary lung tumors and in met-astatic lymph node sites, given that caveolin-1 expression levels differmarkedly in these cell populations. We identified Net as a putative ETStranscriptional repressor responsible for suppressing caveolin-1 in pri-mary lung tumors and Polyoma virus enhancer activator 3 (PEA3) asa candidate transcriptional activator. Net expression in normal adulthuman lung is not well characterized, but immunohistochemistry innormal rat lung shows Net to have an inverse correlation with caveolin-1,being highly expressed in type II cells and minimally expressed intype I cells (data not shown). In cervical cancer, loss of Net as a tran-scriptional repressor has been implicated in cellular transformation (16).

PEA3 has little to no expression in normal lung but is expressed indistal lung epithelium during lung development and in human lungtumor cells (17–19). PEA3 is a member of the ETS subfamily com-posed of PEA3, ER81, and ERM. These members can transactivate

Abbreviations: ChIP, chromatin immunoprecipitation; EDTA, ethylenediami-netetraacetic acid; ETS, erythroblastosis virus-transforming sequence; mRNA,messenger RNA; NSCLC, non-small-cell lung cancer; PCR, polymerase chainreaction; PEA3, Polyoma virus enhancer activator 3; QRT, quantitative real-time; RT, reverse transcription; SDS, sodium dodecyl sulfate; siRNA, smallinterfering RNA.

� The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 1433

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target genes interchangeably via the PEA3 consensus element (20).PEA3 factors mediate pro-metastatic signatures and have been impli-cated in proliferation, differentiation and tumorigenesis (21,22).Known target genes of PEA3 include osteopontin, matrilysin, uroki-nase plasminogen activator (uPA), matrix metalloproteinase-1 and-9 and cyclooxygenase-2 (21,23). It has recently been reported thatPEA3 regulates a cigarette smoke-responsive region in the matrixmetalloproteinase-1 promoter (24).

Herein, we correlate expression patterns of caveolin-1 and the ETStranscription factors Net and PEA3 in human lung cancer specimensby immunohistochemistry. Our findings comparing primary humanlung cancer specimens with metastatic lymph node specimens showincreased PEA3 and caveolin-1 expression in metastatic cells. Theexpression pattern of Net, a known ETS repressor, on the other hand,changes from nuclear staining in primary tumors to cytoplasmic stain-ing in advanced and metastatic sites. These findings suggest that inmetastatic cells, Net resides in the cytoplasm in an inactive form.Chromatin immunoprecipitation (ChIP) assays, mutation analysisand small interfering RNA (siRNA) experiments in NCI-H23 (low-caveolin-1 expressing) and Calu-1 (high caveolin-1 expressing) lungcancer cell lines show that Net is a transcriptional repressor of cav-eolin-1 in NCI-H23 cells and PEA3 is a transcriptional activator inCalu-1 cells. Subsequent ChIP assays and siRNA studies in A549 andNCI-H1299 cells confirm the role of ETS proteins in the transcrip-tional regulation of caveolin-1. Since caveolin-1 is thought to promotecell migration and invasiveness, understanding the molecular mecha-nisms regulating its dynamic expression levels has important impli-cations for preventing and treating metastatic non-small-cell lungcancer (NSCLC).

Materials and methods

Immunohistochemistry

Archived paraformaldehyde-fixed, paraffin-embedded human lung adenocar-cinomas were studied. A pathologist reviewed hematoxylin and eosin-stainedsections and assigned a tumor grade of well, moderate or poorly differentiated(25). Thyroid transcription factor immunohistochemistry confirmed lung ori-gin of specimens (26). Antigen retrieval for caveolin-1 is 0.05% citraconicanhydride (pH 7.4, 98�C, 45 min) and for PEA3 and Net is Vector unmaskingbuffer (microwave low power, 15 min) (27,28). Sections were incubated withprimary antibody [mouse anti-caveolin-1 antibody (610406, BD Biosciences,San Jose, CA), mouse monoclonal anti-PEA3 antibody (sc-113, Santa CruzBiotechnology, Santa Cruz, CA) or goat polyclonal anti-Net antibody (sc-17860, Santa Cruz Biotechnology)] in phosphate-buffered saline (4�C, 16 h).Antibody binding was detected using Vectastain Elite ABC kit with diamino-benzidine as chromagenic substrate. Control slides lacking primary antibodywere included in all procedures. Sections were counterstained with methylgreen, hematoxylin or left unstained and photographed in a Leitz Aristopanmicroscope using ImagePro software. Photographs shown are representativefrom n 5 5 samples.

Characterization and culture of the cell lines

Human NSCLC cell lines were studied based on messenger RNA (mRNA) andprotein analyses showing that Calu-1 and NCI-H1299 cells express highcaveolin-1 levels, A549 cells express moderate caveolin-1 levels and NCI-H23 cells express low caveolin-1 levels (29,30). Calu-1 cells are derivedfrom a pleural lung squamous carcinoma metastatic site. NCI-H1299 cellsare derived from a metastatic lymph node. NCI-H23 and A549 cells are derivedfrom primary lung carcinomas.

Calu-1 cells were maintained in McCoy’s 5A (modified) medium (Invitro-gen, Carlsbad, CA); NCI-H1299 and NCI-H23 cells were maintained in RPMI1640 medium (Invitrogen) with 10 mM N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid and A549 cells were maintained in Dulbecco’s modifiedEagle’s medium (Invitrogen). All cells were maintained in media containing10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin G and 100 lg/ml streptomycin sulfate, were incubated at 37�C in 5% CO2 and harvested at80% confluence for experiments.

DNA constructs

We amplified the proximal 1.0 kb of the published human caveolin-1 promotersequence (GenBank accession number AJ133269) using primers designed withadapters for KpnI and SacI. We characterized two ETS sites, the �190 ETS site(described previously) (15) and the �966 ETS site (identified by Transcription

Element Search System). Two-step polymerase chain reaction (PCR) was per-formed with Advantage-HF 2 PCR kit (Clontech, Mountain View, CA) [94�C,30 s (94�C, 30 s; 68�C, 4 min; five cycles) and (94�C, 15 s; 65�C, 4 min; 30cycles) 68�C, 10 min] using the CTB-11K1 BAC clone (chromosome 7) astemplate (Invitrogen). DNA was purified using QIAquick PCR purification kit(Qiagen) and digested with KpnI and SacI. Fragments were run on a 1%agarose gel, purified with QIAquick Gel Extraction kit (Qiagen), ligated withpGL3-basic vector (Promega, Madison, WI) and sequenced. In the �477 to�557 region, which is highly guanine rich, all clones contained at least onebase pair difference from the published sequence. All constructs contain an Asubstituted for a G at position �490.

Similarly, constructs containing wild-type (�190WT and �966WT) andmutated ETS sites (�190MUT and �966MUT) were created by PCR. Eachmutated construct contained a one base pair substitution in the core ETS site.The same mutation at the �190 ETS site decreased caveolin-1 transcription inEwing’s sarcoma cell lines (15). After sequence verification, MatInspectorconfirmed that no other known cis-elements were created in the mutatedconstructs.

Oligonucleotides are as follows: �190WT forward—5#-CAGGGTACCGCG-CAGCACACGTCCGGGCCAA-3#, �190MUT forward—5#-CAGGGTACC-GCGCAGCACACGTACGGGCCAA-3#, �966WT forward—5#-CAGGGTAC-CGTCAAATCTTTCCTCACAGCC-3#, �966MUT forward—5#-CAGGGTAC-CGTCAAATCTTCCCTCACAGCC-3# and reverse—5#-GACTGAGCTCGGG-CTGTGCTTTAAGGGAAC-3# (nucleotide differences in bold underlinedalphabets).

RNA purification

Total RNA was isolated from cell lines with TRIzol (Invitrogen) and treated forDNA contamination using DNA free (Ambion, Austin, TX). A total of 500 ngRNA was reverse transcribed using TaqMan reagents (Applied Biosystems,Foster City, CA) (25�C, 10 min; 37�C, 60 min and 95�C, 5 min).

Real-time reverse transcription–PCR

Caveolin-1, PEA3, Net and Ets1 mRNA expression were analyzed in cell lines byquantitative real-time (QRT) reverse transcription (RT)–PCR using the ABI Prism7000 sequence detector (Applied Biosystems). RTs were diluted 1:25. Primersand probe sequences are as follows: Caveolin-1: forward—5#-CTAATCCAAG-CATCCCTTTGCC-3#, reverse—5#-TTTATTACTGCCTCCTCCCCCA-3#;PEA3: forward—5#-CCCTACCAACACCAGCTGTC-3#, reverse—5#-GA-GAAGCCCTCTGTGTGGAG-3# (18); Net: forward—5#-TCCACTGCTCTC-CAGCATAC-3#, reverse—5#-AATTGTGGCCAGACGTCATC-3#(18) andb-actin: forward—5#-CCCTGAAGTACCCCATCGAG-3#, reverse—5#-CAGATTTTCTCCATGTCGTCCC-3#, probe 5#-ACGGCATCGTCACCA-3#.For Ets1, TaqMan gene expression assay Hs00901425_m1 (Applied Biosystems)was used. Reactions were performed in 50 ll and amplified (95�C, 10 min;40 cycles: 95�C � 15 s and 60�C � 1 min) using SYBR Green or TaqMan PCRMaster Mix (Applied Biosystems). The relative amounts of mRNA for caveolin-1,PEA3, Net and Ets1 were determined using calibration curves from human adulttotal lung (caveolin-1, Net and Ets1) and Calu-1 cells (PEA3) and normalized tob-actin. All experiments were three different samples performed in duplicate. Datawere analyzed by Student’s t-test with P , 0.05 considered significant.

Purification of protein, western blots and protein densitometry

Cell monolayers were trypsinized, washed in phosphate-buffered saline, centri-fuged, resuspended in lysis buffer with protease inhibitors and incubated withrotation (4�C, 60 min) as described (14). Lysate was centrifuged (10 min,13 000 r.p.m., 4�C). Supernatant (20–50 lg protein) was electrophoresed ona 12% polyacrylamide gel and transferred to polyvinylidene difluoride mem-branes. For caveolin-1, polyvinylidene difluoride membranes were blocked in1X Tris-buffered saline Tween-20 containing 5% dry milk (1 h, RT), exposedovernight at 4�C to mouse anti-human caveolin-1 antibody (1:1000) followedby anti-mouse secondary antibody (1:10 000, 1 h, RT). For PEA3, mouse anti-PEA3 antibody (1:1000) and anti-mouse secondary antibody (1:20 000) wereused. For Ets1, rabbit polyclonal anti-Ets1 antibody (1:1000) (sc-350, SantaCruz Biotechnology) and anti-rabbit secondary antibody (1:20 000) were used.Immunoblots were probed for b-actin to control for equal loading. Binding oflabeled horseradish peroxidase-secondary antibodies was detected with Super-Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). All ex-periments were performed in triplicate. Densitometry was performed using theLuminescent Image Analyzer (LAS-4000, Fujifilm, Valhalla, NY). Data wereanalyzed using Student’s t-test with P values ,0.05 considered significant.

Chromatin immunoprecipitation assays

Cells were fixed with 1% formaldehyde, incubated (37�C, 15 min), washedwith phosphate-buffered saline, resuspended in lysis buffer [1% sodium do-decyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 50 mMTris pH 8, 1 mM phenylmethylsulfonyl fluoride, 1 mM pepstatin A and 1 mM

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aprotinin] and sonicated on ice to 500–1000 base pair fragments with a FisherScientific sonicator (Power 5, five cycles of 5 min, 25 s on, 5 s off). Lysate wascentrifuged (RT, 4000 r.p.m., 5 min). Supernatant was divided into aliquots.One aliquot was stored as input DNA. Three micrograms of anti-PEA3, anti-Net, anti-Ets1 or non-specific IgG (mouse IgG for PEA3, goat IgG for Net andrabbit IgG for Ets1) antibodies were added to each of the other aliquots.Dilution buffer (0.01% SDS, 1% Triton X-100, 2 mM EDTA pH 8, 20 mMTris–HCl pH 8 and 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride and1 mM Aprotinin) was added and samples were incubated (4�C, overnight) withrotation.

AG beads (Santa Cruz Biotechnology) pretreated with 9:1 dilution: lysisbuffer, bovine serum albumin 100 lg/ml and salmon sperm 500 lg/ml wereadded to samples. Samples were rotated (4�C, 2 h) and centrifuged (4000r.p.m., 2 min). Pellets were washed in wash buffer (1% Triton X-100, 0.1%SDS, 150 mM NaCl, 2 mM EDTA pH 8, 20 mM Tris–HCl pH 8, 1 mMphenylmethylsulfonyl fluoride, 1 mM aprotinin) and then with final washbuffer (1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA pH 8,20 mM Tris–HCl pH 8). Immune complexes were eluted with elution buffer(1% SDS, 100 mM NaHCO3), incubated with rotation (RT, 15 min) and centri-fuged (4000 r.p.m., 2 min). Proteinase K (500 lg/ml) and RNase A (500 lg/ml)were added to supernatants and input DNA and then incubated (37�C, 30 min).Cross-links were reversed (65�C, overnight) and DNA was purified.

DNA fragments were analyzed by PCR for caveolin-1 fragments spanningthe �190 ETS site (234 bp amplicon), �966 ETS site (175 bp amplicon) and forb-actin (171 bp amplicon, control for non-specific binding). Primers for cav-eolin-1 �190 ETS site were previously published (15). Primers for caveolin-1�966 ETS site: 5#-CAGGAACAGACAAAATACTTTAATCG-3# and5#-CCATATTTGCAAAATACACAAAATGT-3#; primers for b-actin:5#-CCAAAACTCTCCCTCCTCCT-3# and 5#-CTCGAGCCATAAAAGG-CAAC-3#.

Transfection and reporter assay activity

Caveolin-1 promoter-luciferase [wild-type (�190WT; �966WT) or mutatedETS sites (�190MUT; �966MUT)] and Renilla luciferase control plasmidswere transiently cotransfected into cell lines using GeneJammer transfectionreagent (Stratagene, La Jolla, CA) as described (13). Optimal conditions forNCI-H23 cells were 2 � 105 cells/35 mm dish, 1 lg DNA constructs and 6 lltransfection reagent. Optimal conditions for Calu-1 cells were 3 � 105 cells/35mm dish, 2 lg DNA constructs and 12 ll transfection reagent.

Cells incubated in standard growth conditions for 48 h were harvested andanalyzed for firefly and Renilla luciferase activity with the Dual-LuciferaseReporter Assay System (Promega). Luminescence was detected in a BertholdLumat LB 9501 luminometer (Berthold, Nashua, NH). Four experiments wereperformed, each in duplicate. Firefly luciferase activity was normalized toRenilla luciferase activity. For each experiment, fold difference in normalizedluciferase activity between mutated and wild-type constructs (with wild-typedesignated as 1) was calculated. Data are expressed as the mean of fourexperiments ±SE and analyzed by Student’s t-test with P ,0.05 consideredsignificant.

siRNA

ON-TARGETplus SMARTpool siRNA mixtures targeting PEA3 (L-004207-00), Net (L-010320-00), ETS1 (L-003887-00) and non-targeting controlsiRNA (D-001810-10) were obtained (Dharmacon, Lafayette, CO). Cells (2–3 � 105/35 mm dish) were treated with 50 nM siRNA mixture targeting PEA3(Calu-1), Net (NCI-H23) or Ets-1 (A549, NCI-H1299) using DharmaFECTGeneral Transcription Protocol. Controls were cells transfected with non-targeting control siRNA. siRNA-treated cells were cultured in standard con-ditions for 48–72 h. RNA was isolated, DNase treated, reverse transcribed andanalyzed by QRT RT–PCR. Protein was isolated, electrophoresed and ana-lyzed by western blot. Data (n 5 3) are expressed as the average fold differ-ence between targeting siRNA compared with non-targeting control siRNA(with non-targeting control siRNA designated as 1). Herein, Calu-1 is referredas Calu-1 (cav-high) and NCI-H23 as NCI-H23 (cav-low).

Results

Similar to caveolin-1, PEA3 has minimal expression in well-differentiated lung adenocarcinomas but is expressed at high levelsin tumor cells metastatic to lymph nodes by immunohistochemistry

Immunohistochemistry for caveolin-1 was performed on primary tu-mor specimens (n 5 15; 5 well-, 5 moderate- and 5 poorly differen-tiated adenocarcinomas). Consistent with the known expressionpattern in normal adult lung, areas of adjacent normal lung expresscaveolin-1 in blood vessels and alveolar type I (but not type II) cells,

serving as technical controls within each section. Well- and moder-ately differentiated primary lung adenocarcinomas have little or nocaveolin-1 expression in tumor cells (Figure 1a). Poorly differentiatedprimary tumors have little or no caveolin-1 expression in 4/5 speci-mens (Figure 1a) and 1/5 specimen shows high expression in tumorcells. All metastatic lymph nodes (n 5 5) express caveolin-1 in epi-thelial tumor cells (Figure 1a–b). Adjacent lymphocytes do not ex-press detectable levels of caveolin-1. As expected, blood vesselswithin lymph nodes express high levels of caveolin-1 (Figure 1b).

Our findings are consistent with previous immunohistochemicalstudies of primary and metastatic lung adenocarcinomas (2,4,5). Inan immunohistochemical study on 95 human lung adenocarcinomas,in just 4% of cases did the percentage of caveolin-1-positive tumor cellsin the primary tumor exceed 30%, and all four of these patients hadtumors that were metastatic to lymph nodes. A total of 34/35 metastaticlymph nodes had high caveolin-1 expression in this study (4).

We characterized the expression pattern of PEA3 in primary tumorsand metastatic lymph nodes. We found that caveolin-1 and PEA3protein expression positively correlate. PEA3 expression is minimalin well-differentiated tumors and is increasingly positive in the nucleiof tumor cells in moderate- and poorly differentiated tumors (Figure1a–b). PEA3 is expressed in the nucleus of metastatic lymph nodetumor cells.

Immunohistochemistry shows that Net is expressed in the nucleus ofprimary lung adenocarcinoma cells but in the cytoplasm of tumorcells metastatic to lymph node

Net, on the other hand, is highly expressed in the nucleus of primarytumors (Figure 1a). While Net staining is mostly nuclear in well-differentiated and moderately differentiated primary tumors, it ismostly cytoplasmic in poorly differentiated tumors. High-power viewof metastatic lymph nodes shows positive Net staining in cytoplasmand not in nuclei of tumor cells (Figure 1b).

PEA3 binds to the caveolin-1 promoter and is a transcriptionalactivator of caveolin-1. ChIP assays. QRT–PCR and western blotsconfirm that Calu-1 (cav-high) and NCI-H23 (cav-low) cells differmarkedly in caveolin-1 mRNA and protein levels (Figure 2). In Calu-1 (cav-high) cells, PEA3, but not Net, binds at the both �190 and�966 ETS sites (Figure 3).

Mutation of ETS cis-elements. Transient transfections of wild-typeand mutated constructs into Calu-1 (cav-high) cells show no differ-ence in luciferase activity between wild-type and mutated �966 ETSsite constructs (Figure 3). Mutation of the �190 ETS site, however,results in a 50% decrease in luciferase activity, indicating that thisETS site transactivates the caveolin-1 promoter.

siRNA PEA3. We transfected either an siRNA mixture targetingPEA3 or a non-targeting siRNA control into Calu-1 (cav-high) cells.We confirm effective knockdown of PEA3 mRNA and protein byQRT–PCR and western blot (Figure 4). Silencing of PEA3 resultsin a statistically significant decrease in caveolin-1 mRNA and proteinlevels, further supporting the role of PEA3 as a direct transcriptionalactivator of caveolin-1 in lung cancer cells.

Net binds to the human caveolin-1 promoter and is a transcriptionalrepressor of caveolin-1. ChIP assays. In NCI-H23 (cav-low) cells,neither PEA3 nor Net bind at the �190 ETS site, whereas both Netand PEA3 bind at the �966 ETS site (Figure 5). Net does not bind thecaveolin-1 promoter at these sites in Calu-1 (cav-high) cells. Thispreferential binding of Net is unlikely due to differences in transcrip-tion factor abundance since both cell lines express Net and PEA3mRNA at similar levels by QRT–PCR (data not shown). The cellulardistribution (cytoplasmic versus nuclear) of Net and PEA3 in NCI-H23 (cav-low) and Calu-1 (cav-high) cells is not known and may becontributing to the differential binding patterns to the caveolin-1 pro-moter in these cell lines.

PEA3 and Net regulate caveolin-1 in NSCLC cell lines

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Fig. 1. (a) Immunohistochemical localization of caveolin-1, PEA3 and Net in human lung adenocarcinomas and metastatic lymph node sites. (A) Well-differentiated tumor [(i) hematoxylin and eosin (H&E) staining, �40]. (ii) Ttf-1 staining is localized to tumor cells. (iii) Tumor cells express little or no caveolin-1.Surrounding connective tissue cells stain positive for caveolin-1. (iv) Most tumor cells do not express PEA3. (v) Net staining in tumor cells is positive, in a nuclearpattern. (B) Moderately differentiated tumor [(i) H&E staining, �4 and (ii) ttf-1 staining]. The pattern of expression is similar to panel A, with (iii) little or nocaveolin-1 staining in tumor cells, (iv) PEA3 staining mostly negative in tumor cells and (v) Net staining positive in tumor cells. (C) Poorly differentiated tumor

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Mutation of ETS cis-elements. Transient transfections of wild-typeand mutated constructs into NCI-H23 (cav-low) cells show no differ-ence in luciferase activity between wild-type and mutated �190 and�966 ETS sites constructs (Figure 5). Since neither Net nor PEA3bind the �190 ETS site, mutation of this site did not result in a differ-ence in luciferase activity, as expected. Both Net and PEA3 bind the �966 ETS site, and it is possible that mutation of this site allowedneither the repressor nor the activator to bind, thus resulting in nodifference in luciferase activity.

siRNA Net. We transfected either an siRNA mixture targeting Net ora non-targeting siRNA control into NCI-H23 (cav-low) cells. We con-firm effective knockdown of Net mRNA by QRT–PCR (Figure 6).Silencing of Net results in a statistically significant increase incaveolin-1 mRNA and protein levels. Taken together, the ChIP assaysand siRNA data confirm that Net is a transcriptional repressor ofcaveolin-1 in NCI-H23 (cav-low) cells.

Ets1 binds to the human caveolin-1 promoter and is a minimaltranscriptional activator of caveolin-1

ChIP assays. ChIP assays were performed in A549 and NCI-H1299cells to confirm ETS transcription factor binding to the caveolin-1promoter. In both cell lines, neither PEA3 nor Net binds at the �190

ETS site or the �966 ETS site. Since Ets1 has been shown to be a pro-metastatic ETS transcription factor, we performed ChIP assays todetermine whether Ets1 binds to the caveolin-1 promoter. Ets1 bindsat the �190 ETS site, but not the �966 ETS site, in both A549 andNCI-H1299 cells, thus confirming the �190 site as an important ETS-transactivating site. Supplementary Figure 1 (available atCarcinogenesisOnline) is a representative gel from NCI-H1299 cells.

siRNA Ets1. We transfected either an siRNA mixture targeting Ets1or a non-targeting siRNA control into A549 and NCI-H1299 cells. Weconfirm effective knockdown of Ets1 mRNA by QRT–PCR. SilencingEts1 results in a minimal decrease in caveolin-1 mRNA and proteinlevels in both A549 and NCI-H1299 cells. Representative graphs andimmunoblots are from NCI-H1299 cells (Supplementary Figure 1 isavailable at Carcinogenesis Online).

Discussion

In lung tumorigenesis, there is clear evidence that caveolin-1 expres-sion is altered. In normal adult lung, caveolin-1 is expressed in alve-olar type I epithelial cells, endothelial cells and fibroblasts. Consistentwith the published literature demonstrating dynamic changes in cav-eolin-1 expression, we confirm by immunohistochemistry that most

Fig. 2. Calu-1 (cav-high) cells express higher levels of caveolin-1 mRNA and protein compared with NCI-H23 (cav-low) cells. (A) QRT-PCR analysis shows thatcaveolin-1 mRNA is significantly higher in Calu-1 (cav-high) cells compared with NCI-H23 (cav-low) cells. The relative amounts of caveolin-1 were calculatedusing calibration curves obtained with mRNA from normal human adult lung and are expressed as fold difference between Calu-1 and NCI-H23 cells (with NCI-H23mRNA levels designated as 1). Data are expressed as the mean of three assays ±SE; �indicates P,0.05. (B) Representative western blot analysis for caveolin-1 totalprotein in Calu-1 (cav-high) and NCI-H23 (cav-low) cell lines. Analysis of 20 lg of protein per lane using a monoclonal mouse anti-caveolin-1 antibody showshigher protein abundance in Calu-1 (cav-high) cells. The two immunoreactive bands detected at �21 kDa represent the a- and b-isoforms of caveolin-1 (50).Immunoblot for b-actin shows equal protein loading. (C) Schematic representation of the ETS-binding sites in the proximal 1.0 kb of the human caveolin-1 promoter.

[(i) H&E staining, �40 and (ii) ttf-1 staining]. (iii) Tumor cells continue to be negative for caveolin-1, but (iv) PEA3 expression in tumor cells is now mostlypositive. (v) Tumor cells continue to stain positive for Net, but localization of Net is nor mostly cytoplasmic. (D) Metastatic lymph node from the primary tumorshown in panel C [(i) H&E staining, �40 and (ii) ttf-1 staining]. Tumor cells in metastatic lymph node sites exhibit positive staining for (iii) caveolin-1, (iv) PEA3and (v) Net, but Net expression is now cytoplasmic. (yellow arrows, negative cells, purple nuclei; red arrows, positive cells). (b) Immunohistochemical localizationof Caveolin-1, Net and PEA3 in metastatic lymph node. (A) (i and ii) Epithelial tumor cells express high-caveolin-1 levels in the cytoplasm, whereas adjacentlymphocytes do not express detectable levels of caveolin-1. (iii) As expected, blood vessels within the lymph nodes express high levels of caveolin-1. (B) (i and ii)High-power view of metastatic LN shows positive Net staining in cytoplasm and not in nuclei of tumor cells. Surrounding lymphocytes are negative. Consistentwith the known expression pattern of Net, blood vessels express Net. (C) Conversely, immunostaining for PEA3 is positive in nuclei of metastatic tumor cells. (redarrows, cytoplasm of tumor cells; black arrows, nuclei of tumor cells; yellow arrows, surrounding stromal cells and lymphocyte and yellow arrowheads, bloodvessels).


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Supplementary Figure 1


primary lung adenocarcinomas do not express detectable caveolin-1protein but that virtually all metastatic lymph nodes have highcaveolin-1 expression in tumor cells.

Based on our previous findings that the ETS protein ERM stronglyenhances caveolin-1 gene expression in a mouse lung alveolar epithe-

lial cell line, we hypothesized that caveolin-1 is an ETS target genein lung tumorigenesis. Identifying the ETS transcription factors reg-ulating caveolin-1 expression is complex. In humans, there are 27ETS family members with multiple members expressed in any in-dividual cell (18). Many different ETS proteins can bind the same

Fig. 3. PEA3,but notNet, binds to the caveolin-1promoter (�190and�966ETS sites) in Calu-1 (cav-high) cells. Mutating the�190ETS site in Calu-1 (cav-high) cellsdecreases luciferase activity. (A and B) Representative chromatin immunoprecipitation assay of�190 and �966ETS sites of the human caveolin-1 promoter using IgGcontrol or antibodies against Net or PEA3 in Calu-1 (cav-high) cells. After immunoprecipitation, samples were analyzed by PCR for caveolin-1 fragments spanningeach ETS site. (A) There is binding of PEA3 at the �190 ETS site (234 bp amplicon). (B) There is also binding of PEA3 at the �966 ETS site (175 bp amplicon). (C)Immunoprecipitated samples were analyzed by PCR for b-actin as a control (171 bp amplicon) and there is no binding at the b-actin promoter (I, input DNA; NT, notemplate). (D) The indicated luciferase reporter constructs were transiently transfected into Calu-1 (cav-high) cells. Fold differences in normalized luciferase activitybetween mutated and wild-type constructs (with wild-type designated as 1) are shown [ETS site is underlined with nucleotide differences capitalized]. There is noa 50% decrease in luciferase activity for the mutated �190 construct compared with wild-type. There is no statistical difference in luciferase activity between themutated and wild-type �966 construct. Data are expressed as a mean of three transfections with duplicate assays ±SE; �indicates P , 0.05.

Fig. 4. Silencing of PEA3 results in significantly decreased caveolin-1 mRNA and protein expression in Calu-1 (cav-high) cells. (A–D) Calu-1 (cav-high) cellswere transfected with siRNA solution targeted to PEA3 (PEA3 siRNA) or to non-targeting siRNA control (control) for 48–96 h. Total RNA and protein fromtransfected cells were purified for analysis by QRT-PCR and western blot. (A) QRT-PCR shows PEA3 mRNA is effectively decreased in cells transfected withPEA3 siRNA compared with control siRNA. (B) Western blot analysis shows PEA3 protein levels are decreased in cells transfected with PEA3 siRNA comparedwith control siRNA. (C) Caveolin-1 mRNA expression is decreased in cells transfected with PEA3 siRNA compared with control siRNA. (D) Representativewestern blot and densitometry show that silencing PEA3 decreases caveolin-1 protein expression. Equal amounts of cell lysates were blotted for caveolin-1 andb-actin. Data are expressed as fold difference between PEA3 siRNA compared with control siRNA (with control siRNA designated as 1). All data are expressed asthe mean of three assays ±SE; �indicates P , 0.05.

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ETS cis-element, and ETS factors are able to repress or activatetranscription depending on promoter context and cell type (20).Genes such as uPA, upregulation of which confers a pro-metastaticphenotype in breast cancer, can be regulated by more than one spe-cific ETS factor. Binding of ETS proteins is facilitated by binding of

other transactivating factors to cis-elements in proximity to ETSsites (22).

For our studies, we selected PEA3 as a candidate activator of caveo-lin-1 transcription in metastastic lung tumors because it has been linkedto oncogenesis in many carcinomas, including lung (31). Introducing

Fig. 5. Both PEA3 and Net bind to the caveolin-1 promoter (�966 ETS site) in NCI-H23 (cav-low) cells. (A and B) Representative chromatinimmunoprecipitation assay of �190 and �966 ETS sites of the human caveolin-1 promoter using IgG control or antibodies against Net or PEA3 in NCI-H23 (cav-low) cells. After immunoprecipitation, samples were analyzed by PCR for caveolin-1 fragments spanning each ETS site. (A) Neither Net nor PEA3 are bound atthe �190 ETS site. (B) At the �966 ETS site, there is binding of both PEA3 and Net (175 bp amplicon). (C) Immunoprecipitated samples analyzed by PCR forb-actin as a control (171 bp amplicon) show there is no binding at the b-actin promoter (I, input DNA and NT, no template). (D) The indicated luciferase reporterconstructs were transiently transfected into NCI-H23 (cav-low) cells. Fold differences in normalized luciferase activity between mutated and wild-type constructs(wild-type designated as 1) are shown [ETS site is underlined with nucleotide differences capitalized]. There is no statistical difference in luciferase activitybetween the mutated and wild-type �190 or �966 constructs. Data are expressed as a mean of three transfections with duplicate assays ±SE.

Fig. 6. Silencing of Net results in significantly increased caveolin-1 mRNA and protein expression in NCI-H23 (cav-low) cells. (A–C) NCI-H23 (cav-low) cellswere transfected with siRNA solution targeted to Net (Net siRNA) or to non-targeting siRNA control (control). Total RNA and protein were purified 48 hposttransfection for QRT-PCR and western blot analysis. (A) QRT-PCR analysis shows that Net mRNA is effectively decreased in cells transfected with NetsiRNA compared with control siRNA. (B) Caveolin-1 mRNA expression is significantly higher in cells transfected with Net siRNA compared with control siRNA.(C) Representative western blot and densitometry show that silencing Net increases caveolin-1 protein expression. Equal amounts of cell lysates were blotted forcaveolin-1 and b-actin. Data are expressed as fold difference between PEA3 siRNA compared with control siRNA (with control siRNA designated as 1). All dataare expressed as the mean of three assays ±SE; � indicates P , 0.05.


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the PEA3 gene into NSCLC cell lines that lack exogenous PEA3expression causes increased motility and invasiveness by activatingthe Rho/ROCK pathway (18,19,32). Induced expression of PEA3 in-duces epithelial/mesenchymal transition of lung epithelial cells (33).

In primary lung tumors where caveolin-1 expression is low, wehypothesized that the potent transcriptional inhibitor Net repressescaveolin-1 transcription by binding at an ETS cis-element. Althoughpromoter methylation is known to play a role in the silencing ofcaveolin-1 in breast, ovarian, prostate and colorectal cancer (34–37)and a dominant-negative P132L mutation is known to occur in breastcancer (38), these gene silencing mechanisms are not known to playa role in lung cancer.

Little is known about Net in normal lung or in lung tumorigenesis.During development, Net is expressed at sites of vasculogenesis andangiogenesis. It is expressed in pulmonary vasculature and to a lowerlevel throughout the lung parenchyma during mouse development(39). Net is expressed in adult human lung by QRT–PCR (18), butthe specific expressing cells are not known. Interestingly, Net canswitch to a transcriptional activator upon RAS (rat sarcoma)/ERK(extracellular signal-regulated kinase) activation and phosphorylationby mitogen-activated protein kinases (40,41). Phosphorylated Net hasbeen shown to positively regulate vascular endothelial growth factorexpression and promoter activity in NIH-3T3 cells (42). Studies ofhuman tumors (Kaposi’s sarcoma, prostate cancer and head and neckcancer) show phosphorylated Net to be highly expressed in tumorcells but not in normal surrounding tissue (42).

We correlated the expression pattern of caveolin-1 with PEA3 andNet by immunohistochemistry in human lung specimens. We foundthat caveolin-1 protein expression positively correlates with PEA3expression. Similar to caveolin-1, PEA3 staining is negative inwell-differentiated tumors and highly expressed in metastatic tumorcells. Net, a known transcriptional repressor, is highly expressed inprimary tumor cells, where caveolin-1 is not expressed. While Netstaining is mostly nuclear in well-differentiated primary tumors, it ismostly cytoplasmic, and therefore likely inactive, in poorly differen-tiated tumors and metastatic tumor cells.

We chose two representative human lung cancer [Calu-1 (cav-high)and NCI-H23 (cav-low)] cell lines as models to investigate the tran-scriptional regulation of caveolin-1 by PEA3 and Net. ChIP assayswere performed to characterize Net and PEA3 binding to two ETS cis-elements in the human proximal caveolin-1 promoter in both celllines. We show that PEA3 binds the caveolin-1 promoter at bothETS sites (�190 and �966) in Calu-1 (cav-high) cells. Transienttransfections show a 50% decrease in luciferase activity in the mu-tated �190 ETS site construct compared with the wild-type constructin Calu-1 (cav-high) cells. There is no difference in luciferase activitybetween wild-type and mutated �966 ETS site constructs. Interest-ingly, the same mutation of the �190 ETS site decreases caveolin-1transcription in Ewing’s sarcoma cells. Cotransfection of the Ewingsarcoma gene-friend leukemia 1 fusion protein with either wild-typeor mutated �190 ETS site constructs shows that the fusion proteinonly transactivates the wild-type construct (15). Supporting data fromour siRNA experiments show that silencing PEA3 in Calu-1 (cav-high) cells causes a decrease in caveolin-1 mRNA and protein expres-sion. Thus, PEA3 binds the caveolin-1 promoter at an important ETS-transactivating site and activates caveolin-1 transcription in Calu-1(cav-high) cells.

In NCI-H23 (cav-low) cells, both Net and PEA3 bind at the �966ETS site. The mechanism by which ETS family members compete foroccupancy of the same promoter is not known (22). Although ETSproteins generally bind as monomers, co-operative interactions in-cluding homo-dimerization occur (20,43). Silencing Net in NCI-H23 (cav-low) cells increases caveolin-1 mRNA and protein expres-sion. We show by ChIP assays and siRNA experiments that Net bindsto the promoter and represses caveolin-1 in NCI-H23 (cav-low) cells.

It is probably that other ETS proteins, cis-elements and transcriptionfactors are involved in caveolin-1 regulation in lung tumorigenesis.The caveolin-1 gene is regulated by Sp1-, E2F/DP-1-, p53- and steroidresponse element-specific enhancers in skin fibroblasts (44,45). The

tumor suppressor gene adenomatous polyposis coli, which is lost in85% of sporadic colon cancers, induces caveolin-1 transcription viaFOXO1a (46).

Observations suggest that multiple ETS factors act as an ‘ETS reg-ulatory network’ to regulate the pathways involved in tumorigenesis. Inbreast cancer, ETS proteins such as ETS-1, ETS-2 and PEA3 are pro-metastatic, whereas the ETS proteins PDEF (prostate-derived ETS fac-tor), Ese(epithelial specific ETS3)-2, and Ese-3 are antimetastatic (22).Ets-1 regulates hypoxia-inducible genes that facilitate tumor cell sur-vival (47). Ets-1 appears to be a predictor of poor prognosis aftersurgical resection in lung adenocarcinoma patients (48).

While we show that silencing PEA3 and Net in human lung cancercell lines statistically alter caveolin-1 mRNA and protein levels, theseeffects are not complete. We believe that similar to breast cancer,there are ETS regulatory networks, that transcriptionally regulatecaveolin-1 in lung tumorigenesis. To explore whether redundantETS proteins regulate caveolin-1 expression, we performed ChIP as-says and siRNA experiments in A549 and NCI-H1299 NSCLC cells.We show that Ets1, but not PEA3 or Net, bind to the caveolin-1 pro-moter in these cells. Silencing Ets1 in these cells has minimal effecton endogenous caveolin-1 expression. Differences in cofactor abun-dance probably account for these differential binding patterns.

In addition, we raise the possibility that an ETS fusion proteinregulates caveolin-1 expression, with the first fusion protein inNSCLC, a rare fusion tyrosine kinase EML4-ALK (echinoderm mi-crotubule associate protein like 4-anaplastic lymphoma kinase), re-cently reported (49). Gene fusions involving four different ETSfactors, including all three PEA3 family members, are present in.50% of all human prostate cancers, and ETS fusion proteins havebeen described in breast cancer and acute myelogenous leukemia (50–52).

It is hypothesized that loss of caveolin-1 expression is important intransformation of normal epithelial cells to cancer cells, with caveo-lin-1 functioning as a tumor suppressor gene in this context. Theinverse correlation of Net and caveolin-1 by immunohistochemistryin primary lung tumors and our results from ChIP and siRNA experi-ments in NCI-H23 (cav-low) cells suggest that Net represses caveolin-1transcription in primary lung tumors. When tumors become metastatic,caveolin-1 levels are increased, and it has been suggested that thefunction of caveolin-1 changes to that of a metastasis promoter. Webelieve that, based on our results, there are several potential mecha-nisms for increased caveolin-1 in metastasis. One possibility is that,since there is increased PEA3 expression as tumors progress, PEA3 ismore readily able to bind, perhaps by competing with Net binding, tothe caveolin-1 promoter.

Alternatively, since Net immunohistochemical staining is nuclearin the primary tumor cells and cytoplasmic in the metastatic tumorcells, perhaps a change in cellular location leads to inability of Net tobind and repress the caveolin-1 promoter in metastasis. Nuclear loca-tion has been shown to be important for the transcription factor nu-clear factor-jB to be active and tumor promoting (53). Anotherpossibility is that Net is non-phosphorylated in primary tumors butis phosphorylated in metastatic tumors, causing Net to change froma repressor to an activator. Future studies will include ChIP assays infresh human lung tumor samples to determine if the promoter-bindingpatterns seen in our cell line model are representative of primarytumors and metastatic sites. We will also characterize the phosphor-ylation status of Net in primary tumors versus metastatic sites.

We have shown by ChIP assays, promoter analysis and siRNAexperiments that PEA3 is a transcriptional activator and that Net isa transcriptional repressor of caveolin-1 in lung cancer cell lines.Based on these results and the patterns of expression of PEA3 andNet we observed in human lung cancer specimens, it is probably thatNet is suppressing caveolin-1 transcription in primary lung tumorsand that PEA3 is activating caveolin-1 transcription in metastaticsites. Understanding the transcriptional regulation of caveolin-1 inlung tumorigenesis may provide insight into how to prevent caveolin-1upregulation, probably an important step in metastasis. If overexpressionof caveolin-1 promotes cancer cell migration and seeding, blocking

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caveolin-1 expression in metastatic tumor cells is a potential therapeutictarget in advanced NSCLC.

Supplementary material

Supplementary Figure 1 can be found at http://carcin.oxfordjournals.org/

Funding

American Cancer Society (IRG-72-001-34-IRG) to H.K.; AmericanLung Association Lungevity to H.K.; National Institute of Health/National Heart, Lung, and Blood Institute Training Grant to K.A.S.

Acknowledgements

Conflict of Interest Statement: None declared.

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Supplementary material


http://carcin.oxfordjournals.org/

http://carcin.oxfordjournals.org/

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Received November 14, 2008; revised May 15, 2009; accepted May 16, 2009

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increased pea3/e1af and decreased net/elk-3 - carcinogenesis

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