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Published OnlineFirst August 31, 2010; DOI: 10.1158/0008-5472.CAN-10-0860

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cular and Cellular Pathobiology

NP A2/B1 Modulates Epithelial-Mesenchymal Transition

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Tauler1, Enrique Zudaire2, Huaitian Liu3, Joanna Shih4, and James L. Mulshine1

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erogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been reported to be overexpressed inancer and in other cancers such as breast, pancreas, and liver. However, a mechanism linking hnRNP A2/rexpression and progression to cancer has not yet been definitively established. To elucidate this mech-, we have silenced hnRNPA2/B1 mRNA in non–small-cell lung cancer cell lines A549, H1703, and H358.cell lines present different levels of expression of epithelial-to-mesenchymal transition (EMT) markerss E-cadherin, fibronectin, and vimentin. Microarray expression analysis was performed to evaluate theof silencing hnRNP A2/B1 in A549 cells. We identified a list of target genes, affected by silencing of hnRNP, that are involved in regulation of migration, proliferation, survival, and apoptosis. Silencing hnRNP A2/uced formation of cell clusters and increased proliferation. In the anchorage-independent assay, silencingA2/B1 increased colony formation by 794% in A549 and 174% in H1703 compared with a 25% increase inration, in both cell lines, in a two-dimensional proliferation assay. Silencing hnRNP A2/B1 decreasedion in intermediate cell line A549 and mesenchymal cell line H1703; however, no changes in proliferationbserved in epithelial cell line H358. Silencing hnRNP A2/B1 in A549 and H1703 cells correlated with anse of E-cadherin expression and downregulation of the E-cadherin inhibitors Twist1 and Snai1. These
increa
data suggest that expression of hnRNP A2/B1 may play a role in EMT, in nonepithelial lung cancer cell linesA549 and H1703, through the regulation of E-cadherin expression. Cancer Res; 70(18); 7137–47. ©2010 AACR.

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eregeneous nuclear ribonucleoprotein A2/B1 (hnRNP) is a member of the hnRNP family of proteins. Thes are a complex group of RNA-binding proteins thatkey role in mRNA processing and telomere biogenesishnRNP A2/B1 overexpression has been described incancers, including breast, pancreas, liver, and gastroin-l cell lines (5–8). Recent reports have described a crit-le of hnRNP A2/B1 in the regulation of fundamentaly especially related to cancer, such as migration (9)robic glycolysis (10). We have previously reported thatA2/B1 is a marker for early lung cancer (11–15). Over-

RNP A2/B1 in exfoliated bronchial epitheliall sputum samples correlated with eventual

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ns: 1Laboratory of Lung Cancer Biology, Section of, Rush University Medical Center, Chicago, Illinois;is Core Facility, National Cancer Institute, NIH,ology Center, Gaithersburg, Maryland; 3Sciencernational Corporation, Rockville, Maryland; andrch Branch, Division on Cancer Treatment andl Cancer Institute, NIH, Bethesda, Maryland

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

uthor: Jordi Tauler, Rush University Medical Center,Building, 1750 West Harrison Street, Chicago, IL

-942-3595; Fax: 312-563-3377; E-mail: Jordi_Tauler@

5472.CAN-10-0860

ssociation for Cancer Research.

ls.org

Research. on November 16, 2cancerres.aacrjournals.org d from

pment of lung cancer (16). High levels of hnRNPexpression also colocalized with areas of genetic in-s assessed by microsatellite instability and loss ofzygosity (17). hnRNP A2/B1 has been consistently re-as an informative biomarker for lung cancer screen-

sing a variety of analytic approaches, includingssion studies and proteomic analysis (7, 8, 18, 19).ve also previously reported that hnRNP A2/B1 expres-s tightly controlled during lung development (20). At article has shown the impact of cell density onP A2/B1 expression, reporting that mouse lung cell9 and E10 both showed increase of hnRNP A2/B1 pro-elated to proliferation. At confluency, the nontumori-E10 cell line downregulated hnRNP A2/B1 mRNAction, whereas the tumorigenic E9 cell line showedrease of total hnRNP A2/B1 production (21).ever, the contribution of hnRNP A2/B1 to the carcino-process is still poorly understood. Some reports havesilencing tools in different cancer cell lines to studynction of hnRNP A2/B1 (22–24). Silencing hnRNP A2/combination with hnRNP A1 reduced cell growth byng cell death in cancer cell lines (22). Silencing hnRNPalone in squamous carcinoma cell line, Colo16, regu-

genes involved in cell cycle and proliferation; however,a modest effect in Colo16 proliferation (23, 24).ent evidence is showing a prominent role of hnRNPin fundamental biological functions such as regulation
l metabolism (10), migration and invasion (9, 25, 26),ration (22–24), and cellular response to mitochondrial
7137

020. © 2010 American Association for Cancer

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(26). Moreover, hnRNP A2/B1 activation of invasiontype involves different mechanisms including activa-f the CXCL-12/CXCR4 axis (25), alternative splicing53INP2 (9), and activation of invasive behavior, afterion of mitochondrial DNA, through hnRNP A2/B1–ted cooperative effect of NF-κB, NFAT, CREB,/EBPδ (26). Interestingly, it has been reported thation of mitochondrial DNA in cancer cells promoteslial-mesenchymal transition (EMT; ref. 27). Moreover,also been proposed that changes in morphology, in-, and metastasis potential could be the result of(28, 29).nvestigate how the overexpression of hnRNP A2/B1 af-his important functional biology in lung cancer, weied the expression of hnRNP A2/B1. Because hnRNPis playing a role in the regulation of migration activity,ed lung cancer cell lines with different levels of EMTtype markers such as mesenchymal H1703, intermedi-49, and epithelial H358 (30). We have, for the first time,significant changes in proliferation and migration inithelial H1703 and A549 lung cancer cell lines associat-h changes in cell morphology, suggesting that hnRNPmay be an important regulator of EMT and could pro-
mechanistic link between hnRNP A2/B1 overexpres- time P
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ultureslines used in this study include A549, H1703, andnon–small cell lung cancer (NSCLC) cell lines ob-from the American Type Culture Collection. Thesees present different EMT phenotypes as determinedexpression of EMT markers. H1703 has been classi-

s mesenchymal, whereas A549 and H358 display aepithelial phenotype (30). Cells were maintained in1640 supplemented with 5% fetal bovine serumHyclone), in 95% air and 5% CO2 at 37°C. Cells wered and seeded at 1 × 106 per flask in a new T75 flaskry passage.

expression vectorsonucleotides for the siRNA vectors were designed witheb-based software provided by Oligoengine. Sequencese three different oligonucleotides were as follows: siA,GCTGTAGCAAGAGAG; siD, CCAGGGGCTCATG-TG; siE, TTTTGGAGGTAGCCCCGGT. siRNA oligonu-des from Oligoengine were ligated and cloned inr.neo (Oligoengine) vector following the manufac-s protocol. Cells were transfected with Fugene 6e) following the manufacturer's protocol and selected00 μg/mL of G418 (Hyclone). After selection, trans-cells were maintained in medium supplemented withat 300 μg/mL (Hyclone). We have stably transfectedA549, H1703, and H358 cell lines with three different

ucts (siA, siD, and siE) and empty vector (EV) as al.

(PiercReady

r Res; 70(18) September 15, 2010

Research. on November 16, 2cancerres.aacrjournals.org ownloaded from

expression vectorsA expression vector for hnRNP A2/B1, based onRF cDNA clones system, was acquired from Origene.ine A549 was transfected with Fugene 6 (Roche),ansfected cells were selected using the protocol de-d above. Cell lines A549 EV-TC (Empty Vector True) and A549 A2B1rec (expressing hnRNP A2/B1) wereed.

array data generation9 EV and A549 siA cells were seeded at 1.5 × 106 in aask. At 60 hours postseeding, total RNA was extractedRNeasy mini-kit (Quiagen). First-strand cDNA, double-cDNA, and cRNA were synthesized and processeding to the manufacturer's protocol (Affymetrix). Mi-ay study was performed using Affymetrix HG U133.0. Protocol for data analysis can be found in Supple-ry Materials (Supplementary Microarray Data Analysisegends, and Supplementary Figs. S1–3).

ime PCRreal-time PCR of samples depicted in Figs. 1A,B, 4A,B,, cells were seeded at 2 × 106 in a T75 flask. For real-CR for samples in Fig. 5A to C, cells were seeded at106 in a T25 flask. Total RNA was extracted afterurs postseeding (Figs. 1A,B, 4A,B, and 5D) and at 0, 2,6 hours (Figs. 5A–C) with RNeasy Mini-kit (Qiagen).RNA (0.8–2 μg) for each analyzed sample was reverseribed in a final volume of 25 μL using SuperScripttrand Synthesis kit (Invitrogen). Quantitative real-timeeaction was performed in a final volume of 25 μL con-g 2 μL of cDNA (1:10 dilution) and 400 nmol/L of pri-and SybrGreen PCR master mix (Applied Biosystems),n in an Opticon Cycler (MJ Research) and in a 7300(Applied Biosystems). All samples were amplified inate using the following cycle scheme: 50°C for 2 min-5°C for 10 minutes; and 45 cycles of 95°C for 30 seconds,or 30 seconds. Final mRNA levels were normalized toNA levels. All results are plotted as columns (mean)ars (SD), calculated with 7300 Real-Time PCR Systemftware (Applied Biosystems). Primer sequences can bein Supplementary Table S1.

rn blottings were seeded at 3 × 105 per well in a six-well platehours. For each cell line, three wells were used. Forensity experiments, cells were seeded at describedies in T75 flask. To obtain whole-cell extracts, cellswashed with cold 1× PBS (Bio-Rad), scrapped from all plate or T75 flask, and disrupted with 150 μL of ly-ffer: 100 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl,iton X-100 (Bio-Rad) supplemented with 1× HALTse inhibitor cocktail (Pierce), and 1× HALT phospha-hibitor cocktail (Pierce). After 30 minutes of incubation, whole-cell extracts were cleared by centrifugation.n concentration was calculated with the BCA method
e), and 10 to 60 μg of total protein were loaded onGel 4% to 15% Tris-HCl from Bio-Rad. Western
Cancer Research



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g was performed following standard conditions withry antibody: diluted 1:1,000 for hnRNP A2/B1035; Santa Cruz), 1:1,000 for actin (sc1616; Santa
1:1,000 for E-cadherin (3195; Cell Signaling), 1:250ai1 (3879; Cell Signaling), 1:500 for fibronectin
was ucalcul

silencing to be used in microarray analysis. RNA from A549 EV and A549 siA wars, and confluent cells at 60 and 84 hours postseeding. Experiments were repea

acrjournals.org


68; Santa Cruz), and detected with ECL Plus Westernetection system (GE Healthcare). Images were ana-using a FluorChem SP system (Alphainnotech). Actin
sed as a loading control. Relative optical density wasated using ImageJ free software (31).
1. Validation of silenced hnRNP A2/B1. A, top, comparison of siRNA efficiency of siA and siD constructs in A549-transfected cells. Bottom,ion of hnRNP A2/B1 and hnRNP A1 in A549 after silencing hnRNP A2/B1. B, top, expression of hnRNP A2/B1 in transfected cells with a siA silencingct. Middle, hnRNP A2/B1 silencing in whole-cell extracts of A549 siA, H1703 siA, and H358 siA by Western blot. Bottom, relative opticalof hnRNP A2/B1 normalized to actin. C, top, effect of cell density on expression of hnRNP A2/B1 in A549 cells seeded at increasing density.cell extracts were obtained after 60 hours postseeding. Bottom, silencing efficiency is affected by cell density in A549 cells. A549 EV and A549s were seeded at increasing cell density. Whole-cell extracts were obtained after 72 hours postseeding. D, selection of optimal conditions for hnRNP

s extracted in cells growing in exponential phase at 18 andted three times.

Cancer Res; 70(18) September 15, 2010 7139



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th assay and anchorage-independent growth assaywth was determined using CellTiter 96 AQueous Oneon Cell Proliferation Assay (Promega) following thefacturer's protocol. Experiments were performed inll plates using RPMI 1640 containing 5% FBS. A549d A549 siA cells were seeded from 2,500 andper well. H1703 EV and H1703 siA cells were seeded00 and 30,000 per well. Assay reading was performed96-well plate reader (FLUOstar Optima, BMG Lab-The anchorage-independent growth of A549 EV,siA, H1703 EV, and H1703 siA cells was examinedt agar clonogenic assay. Briefly, 5,000 cells were re-ded in 1.5 mL of the culture medium containingBS and 0.3% agarose and plated in six-well plates.5 mL of presolidified culture media in 0.5% agarining 10% FBS. Plates were incubated at 37°C forks, and colonies larger than 0.1 mm in diameter wereed. All results are plotted as columns (mean) andSD).

tion assaymotaxis was assayed in 8-μm pore, 96-well ChempTx(Neuroprobe). Cells were resuspended at 100,000/mLeded at 35 μL/well (3,500 cells per well). Cells werein the upper chambers, and the lower chambers wereith RPMI–10% FBS. After a 4-hour migration period atonmigrating cells were wiped off the top surface of therane. Membranes were fixed and stained with Hema3emical Sciences), and the cells trapped in the pores ofembrane were counted.

nofluorescences were seeded in Lab-Tek II four-well glass chamber(Nunc) at 60,000 per chamber. Cells were incubatedhours at 37°C, fixed with formalin (Fisher Scientific)minutes at room temperature, washed with PBS andabilized with 100% ice-cold methanol at −20°C forutes, and washed with PBS and blocked with PBS con-g 5% of normal goat serum and 0.1% Triton X-100. Cellsncubated with primary antibody to E-cadherin (1:100)ght at 4°C, washed with PBS, and then incubated withary antibody conjugated with TRITC (1:100; Pierce) forurs. After washing, coverslips were attached to glasswith Vectashield mounting medium containing 4′,6-ino-2-phenylindole (Vector Laboratories). Cells wered using a fluorescence microscope (Olympus BX41,us America), and images were processed using Spotced v.4.6 software (Diagnostic Instruments).

lts

ing hnRNP A2/B1 in lung cancer cell linesough siA and siD sequences are separated by only 12tides, the silencing effects observed with these oligo-tides are very different. siD was able to show a mod-but not consistent silencing of hnRNP A2/B1. siE had
ct in silencing hnRNP A2/B1 (data not shown). siA wasly construct that showed consistent and significant si-
complsurfac



g of hnRNP A2/B1 compared with EV (Fig. 1A). Be-previous observations have suggested a cooperativection between hnRNP A2/B1 and hnRNP A1, we evalu-xpression of hnRNP A1 after silencing hnRNP A2/B1.d not observe changes in hnRNP A1 mRNA expressionA). Downregulation of hnRNP A2/B1 at the mRNA andn levels was confirmed in A549, H1703, and H358 cellFig. 1B).and others have reported that cell density affectsA2/B1 expression (11, 21) in several lung cancer cell

Cell density at seeding affects hnRNP A2/B1 expressionredictably determines the timing of hnRNP A2/B1 peakression in A549 cell line (Fig. 1C). We examined thence of cell density in silencing of hnRNP A2/B1 inAt 72 hours postseeding, A549 EV cells seeded at 1.25 ×r flask were 90% confluent, and cells seeded at 2.5 × 106

sk were at confluency. Maximal differences in hnRNPprotein levels between A549 EV and A549 siA cells wereed just as cells reached confluency (Fig. 1C).

s of hnRNP A2/B1 silencing on gene expression incell lineextracted RNA from A549 EV and siA cells at four dif-time points to evaluate conditions for optimal silenc-t 60 hours postseeding, expression of hnRNP A2/B1in A549 EV was still near maximum. At this timemRNA levels of hnRNP A2/B1 in A549 EV comparedA549 siA showed an efficiency of silencing aroundFig. 1D). Microarray analysis was performed underconditions. We obtained a list of 173 differentially ex-d genes between A549 EV and A549 siA cells (see list inmentary Materials; Supplementary Table S2). Networkation analysis resulted in these 173 genes being catego-into nine clusters according to their known functionalnship as described in the Ingenuity Pathway Knowl-ase (Table 1). Different significant biological functionsevealed by this method as reported in SupplementaryS3. The most relevant functions affected by silencingA2/B1 in A549 cell line were cellular movement, cel-evelopment, amino acid metabolism, and cell death.stingly, pathways central to embryonic development al-e found in the nine clusters. These results, in combina-ith 64 genes involved in cancer (see list Supplementaryials; Supplementary Table S4), are consistent with a roleRNP A2/B1 in cancer that could represent a modifica-developmental programs as we have reported previous-. Moreover, we have validated the microarray results byme PCR, using 27 of the 173 genes, and reported that 25show similar results after microarray and real-time PCRes (see table and figure in Supplementary Materials,mentary Fig. S4).

ing hnRNP A2/B1 affects morphology ofithelial cell linesr 72 hours postseeding, A549 EV cells consistently pre-a more homogeneous cell shape and size, and are

etely confluent. A549 siA cells did not cover the wholee of the flask and revealed a pattern of high-density

Cancer Research



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Molecules in network Score

CNB2, collagen(s), CP, ELOVL2, ENTPD1, ERK, Fascin,FHL1, FN1, FSCN1, GDF15, GJA1, GJC1, GSN, H19,G, IGFBP2, INHBB, LPAR1, Nfat, NPR3, NTS, PAPPA,

44

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hCPd

r, cell death, neurologic disease

gf, PDGF BB, PDGFC, PGM2L1, Pkc(s), PLC, PLCB4,Rac, S1PR3, SLC1A1, TGFB2, VCAN

BCB1, Akt, AKT3, Ap1, CD24, DNER, EDAR, ERBB3,IPK2, IFNβ, IL1, IL6ST, insulin, Jnk, LDL, LEPR, Mapk,FHAS1, NF-κB (complex), NR2F1, OAS1, P38 MAPK,

40

MPC

n, small-molecule biochemistry,behavior

SK9, PI3K, PPARGC1A, PRSS1, PRSS3, SERPINA1,TFPI2, TLE1, TNS3, Trypsin, UTRN, Vegf, WNT4

Actin, CA12, CACNA1A, caspase, Ck2, CPLX1, CUX1,DSG1, DSG3, F-actin, FSH, GPRC5B, HNRNPA2B1,HOXB8, JUP, MEIS2, MIRN346, NOVA1, NR3C2,

NRCAM, NRXN1, NTRK3, PBX1, Pka, PKN2, PSEN1,

24
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eath, cellular development, hairskin development and function

AB8B, S100A13, SLC14A2, SNAP25, Snare, SYT1,SYT3, VIL1, ZDHHC17

12, CASP3, CDC37, CIDEC, corticosterone, CPS1, CPVL,SP, HYAL1, hyaluronic acid, KCTD12, L-dopa, MAP3K3,MD, MSRB3, MYCN, MYO1A, PKN2, PPARA, PRKCZ,

22

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iochemistry, carbohydrate

N1, RAF1, SFI1, SLC1A2, SRPX, TGFA, TNF, TRAF6,UBXN10, VCP, VIP, WWOX, YWHAG, YWHAZ, ZFP36SL5, ALB, AMBP, ATG3, CD302, CEBPA, CLDN1, CRB1,IP1, ELOVL7, HIST2H4A, HNF1A, HNF4α dimer, HNF4A,T, HPX, LEF1, LEP, LGALS3, LUC7L2, MIRN31, MOCOS,

20
d metabolism, small-molecule
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1, PEG10, PPARA, PZP, SFRS1, SREBF1, SRI, SSFA2,TCF7L2, TRAF2, TRIM31, UBE2I, YWHAG

ADORA2B, aryl sulfotransferase, BDNF, β-estradiol,CEBPA, CHST7, cyclic AMP, DLG4, DRD1, GABAR-A,GABRA5, GABRB1, GABRB2, GABRB3, GABRG2,

ABRG3, HTR1A, MIRN196A1, MIRN196A2, MIRN196B,PDE1A, PKIB, potassium, pregnenolone, RAF1, SET,

20

metabolism

SE

expression, organ development,carbohydrate metabolism

TBP1, SLC6A14, STK32B, sulfotransferase, SULT1A1,SULT1A2, SULT1A3, TOX3

LX1, ANXA5, ATRX, calpain, CBX5, CDKN1A, CEBPA,CTNNBIP1, D-glucose, DCLK1, DSE, EP300, EYA4,HMGB3, KLF6, KRT4, LEF1, LEP, MBP, MIRN124,MIRN106B, NR3C1, PDX1, PPARA, PRKCZ, PSMD4,

20

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r cell cycle, cellular growth andproliferation

ASSF8, RIMKLB, SCN9A, ST8SIA4, STOM, TGFA,TM4SF18, TP53INP1, YY1

BCC6, ANXA2, AXIN1, BDNF, CADM1, CEBPA, CREB1,clic AMP, HRAS, ITGAE, KIAA1199, LEP, LETM2, MEST,3, NKX2-1, P4HA1, phosphatidylinositol, PITPNC1, PLOD1,

18

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17

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MYSMAD3, SUMF1, SVEP1, TCF7L2 (includes EG:6934), TGFA,
UBASH3B, USH1C, USH1G, VIP, ZFP36, ZFYVE9

E: After analysis through Ingenuity Pathways, we identified nine main gene networks. Genes affected by silencing of hnRNP

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red small cells together with foci of bigger cells (Fig. 2).96 hours postseeding, A549 siA cells were not fullyent but showed very compact regions of smaller cellscells were piling up. Similar results were observed inchymal H1703 cells. Confluent H1703 EV cells did notto each other and did not cluster. H1703 siA cells

d clusters of more compact cells with increased cell-ntact compared with H1703 EV cells (Fig. 2). In con-no changes were observed in the epithelial H358 cellter silencing hnRNP A2/B1 (data not shown).

ing hnRNP A2/B1 increases proliferation andy formation in nonepithelial cell lines
further investigate hnRNP A2/B1 function in lungcell lines, we evaluated proliferation by cell growth
compadid n



(MTS). After 4 days, A549 siA cells showed a signif-increase in proliferation compared with A549 EVA similar result was obtained with H1703 siA cellsred with H1703 EV cells (Fig. 3A). Moreover, silenc-RNP A2/B1 enhanced the ability to form colonies onnchorage-independent clonogenic assay. A549 siAed a 794% increase in the number of coloniesared with A549 EV. H1703 siA showed a 174% in-over H1703 EV in the number of colonies counted

eld (Fig. 3B). These changes in number of coloniesated with changes in the size and morphology ofolonies. A549 siA produced bigger colonies thanEV (Fig. 3B). H1703 siA formed bigger and more
ct colonies than H1703 EV (Fig. 3B). H358 siA cellsot show any differences in growth compared with
ureNPflasgnifμm.

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2. Morphologic changes after silencingA2/B1. Cells were seeded at 1 × 106

k in T75 flask. Images at 10× and 40×ication. Scale bar at 10×, 40 μm; at 40×,

Cancer Research

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EV, and both cell lines did not form colonies inenic assay (data not shown).

ing hnRNP A2/B1 inhibits migration in lungr cell linesddition, we evaluated the effect of hnRNP A2/B1 in mi-n. A549 EV and H1703 EV migration activity was sim-hereas H358 EV showed less migration (Fig. 3C). H1703owed less migration (4.45-fold) compared with H170349 siA showed 2.65-fold less migration compared withEV. H358 siA also exhibited less migration than H3586-fold decrease). Therefore, all cell lines showed a de-in migration after silencing hnRNP A2/B1, althoughfect was only significant in nonepithelial cell linesand in A549.

P A2/B1 affects expression of EMT markers inand H1703 cell linesroarray analysis showed downregulation of fibronectin,T marker, after silencing hnRNP A2/B1 in A549. We
onfirmed downregulation of fibronectin in A549 siAe compared with A549 EV (Fig. 4A and B). However,
field,E-cadh

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sion of fibronectin in mesenchymal H1703 was verynd we were not able to detect changes (Fig. 4A). Givenntral role of E-cadherin in EMT control, we explorednRNP A2/B1 silencing affected E-cadherin expression9). Although E-cadherin expression was not detectede microarray analysis, we evaluated changes inherin expression at the mRNA and protein levels innd H1703 cell lines. We have observed upregulation ofherin expression in A549 siA and H1703 siA cell linesared with their EV counterparts at the mRNA levelA). Also, we found a modest increase in E-cadherinn expression in A549 siA and an important increase insiA compared with H1703 EV (Fig. 4B). Through immu-rescence, we have shown that E-cadherin expression9 EV is present in the membrane. There was also astaining in the cytoplasm, whereas there was a moreant E-cadherin signal in the membrane of A549 siAlong areas of cell-cell contacts. H1703EV cells wereive for E-cadherin expression, but H1703 siA cellsd clusters of E-cadherin–positive cells. In the same

3. Silencing hnRNP A2/B1es proliferation andes migration. A, MTSation assay. *, P < 0.001,t's t test. B, anchorage-dent growth assay. Left,of colonies per field..001, Student's t test.0× magnification to showces in colonies afterg hnRNP A2/B1.ation assay. *, P < 0.001,t's t test. All experimentspeated at least three times.

nonclustered cells without cell-cell contacts wereerin negative (Fig. 4C).




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ssion of E-cadherin inhibitors Snai1 and Twist,ot ZEB-1, is affected by silencing hnRNP A2/B1 inithelial cell linesfurther study the effect of hnRNP A2/B1 in modulationT and regulation of E-cadherin expression, we have ex-the effect of hnRNP A2/B1 in E-cadherin inhibitors

, Snai1, and ZEB-1 (32, 33). We have observed thatiA cells showed a higher and constant level of expres-
f E-cadherin mRNA, whereas A549 EV had lower andegulated expression of E-cadherin (Fig. 5A). We exam-
gulateother



nai1 expression and found that at 2 hours postseeding,mRNA peaked in A549 EV cells. This peak was moreated in A549 siA cells. After 4 hours postseeding, Snai1was downregulated in both cell lines (Fig. 5B). Snai1

n expression followed the same pattern but was slightlyd in time (Fig. 5B). However, H1703 cell line did notthis peak of expression after 2 hours postseeding inmRNA expression (data not show). Twist was downre-

FigA2/A, randandanaextposimmExpthre

d in both A549 siA and H1703E-cadherin inhibitor, ZEB-1

020. © 2010 American Assoc

4. Silencing hnRNPeffect on EMT markers.time PCR for fibronectinadherin. B, fibronectinadherin Western blots from whole-cells after 24 hoursding. C, E-cadherin

ofluorescence staining.ents were repeated

siA cells (Fig. 5C). An-, was not affected by

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ng of hnRNP A2/B1 in A549 and H1703 cell lines (dataown).confirm the role of hnRNP A2/B1 in the regulation ofherin and E-cadherin inhibitors, we overexpressedA2/B1 in A549 by stable transfection. OverexpressionNP A2/B1 in A549 A2B1rec correlated with an increasei1 and Twist expression. Changes in E-cadherin expres-ere also observable (Fig. 5D).

ssion

and others have reported hnRNP A2/B1 as an earlyancer marker (11–15). However, despite increasingce of a prominent role for hnRNP A2/B1 in the regu-of mRNA processing (1, 2, 4), telomere maintenancell metabolism (10), migration and invasion (9, 25, 26),ration (22–24), and cellular response to mitochondrial
(26), there is no described mechanistic link for the con-on of hnRNP A2/B1 overexpression and the pathogen-
pholopholo

experiments.

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f lung cancer. To approach this question, we haveed hnRNP A2/B1 in a panel of lung cancer cell linesresent a different EMT phenotype: mesenchymal, intermediate A549, and epithelial H358 (30). EMT dif-iation has been reported to affect migration and prolif-n (28, 29), and EMT induction has been observed asf a response to mitochondrial stress (27).ults of silencing hnRNP A2/B1 have been reported in aer of human cancer cell lines but not in lung cancer cell22–24). In this study, we have analyzed, for the first time,ect of silencing hnRNP A2/B1 in lung cancer cell lines.sults lead to four main observations: (a) microarrayis expression in A549 reveals that silencing of hnRNPalone has a strong effect on genes that are fundamentaleral hallmarks of cancer (34). Several affected genes areto play a role during development and affect motility,

tion, and proliferation. (b) hnRNP A2/B1 regulates mor-
gy and cell growth in nonepithelial cell lines. Mor-gic changes correlate with a different growth pattern.
5. E-cadherin regulation.time PCR of E-cadherin,, top, real-time PCR of549. Bottom, Snai1 proteinion in A549. C, real-timer Twist. Left, A549. Right,D, real-time PCR of Snai1,nd E-cadherin afterression of hnRNP A2/B1 inesults are representative




Interesilencthan itake pwhichesis paffectewith cthat shmigrastudielines.silencA549 aAll

increaincrealationmesenposeEMT,procesnonepof E-cadownrpatterfact thSnai1Twist-Althouby sileationprolifemicroa(37) an(38–40We

cell dehave iand Vto thedensitnonepdensitditionDetachcells aThis

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stingly, a greater effect in proliferation is observed aftering hnRNP A2/B1 in an anchorage-independent assayn a two-dimensional assay. Changes in proliferationlace without inducing changes in hnRNP A1 expression,contradicts the cooperative hnRNP interaction hypoth-roposed by Patry and colleagues (22). (c) Migration isd by silencing of hnRNP A2/B1. These effects correlatehanges observed in the microarray expression analysisowed a number of genes involved in cell motility and

tion. The effect on migration is present in all cell linesd but is only statistically significant in nonepithelial cell(d) Proliferation and migration changes observed aftering hnRNP A2/B1 are more relevant in intermediatend in mesenchymal H1703 than in epithelial H358.these reported changes, including cell morphology,sed proliferation, decreased migration, together withse in E-cadherin, decrease of fibronectin, and downregu-of Snai1 and Twist are consistent with transition from achymal to an epithelial phenotype. Therefore, we pro-that silencing hnRNP A2/B1 expression may revertinducing a mesenchymal-to-epithelial transition (MET)s. Induction of MET after silencing hnRNP A2/B1 inithelial cell lines could be explained by the upregulationdherin expression as a consequence of Snai1 and Twistegulation. However, A549 and H1703 show a differentn of expression of E-cadherin inhibitors although theat Twist is downregulated in both cell lines but notmay indicate a predominant role for Twist in theSnai1 axis as it has been suggested previously (35).gh previous reports have linked suppression of EMTncing Snai1 and/or Twist with decrease in cell prolifer-(35, 36), in our study, reversion of EMT increases cellration. This could be explained by the fact that in ourrray, we detected upregulation of ErbB3 and versicand downregulation of TGF-β2, tumor suppressor HIPK2), and GDF15 (41).have also shown that there is a correlation betweennsity and hnRNP A2/B1 expression. Previous reportsndicated that VHL affects hnRNP A2/B1 expression,HL is also regulated by cell density (42, 43). Accordingreported observations, we suggest a link between celly, hnRNP A2/B1 expression, and EMT regulation inithelial cancer cells. Tumor cell growth increases celly and levels of hnRNP A2/B1 protein. Under these con-s, E-cadherin is downregulated, favoring EMT.ed cells, which may be in vitro analogues of metastatic
t low density, may show low levels of hnRNP A2/B1.
ion and upregulation ofRece

OnlineF

ran-Jones K, Wayman L, Kennedy DD, et al. hnRNP A2, a poten-ssDNA/RNA molecular adapter at the telomere. Nucleic Acids

s 2005;33:486–96.

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erin that would permit cells to attach to a new site.ver, epithelial-like cells with high expression of E-rin, such as H358, would not be expected to respondncing of hnRNP A2/B1 in a significant way. A conse-e of this hypothesis is that an increase of hnRNP A2/pression may be regulating EMT. Our results supportossibility because we have shown that overexpressionNP A2/B1 in A549 correlates with an upregulation ofand Twist expression.ur results, epithelial H358 cell line was not affected bying of hnRNP A2/B1. We suggest that cell lines fromus reports (22–24) may have a different status ofET differentiation that could explain these differentreported after hnRNP A2/B1 silencing. Alternatively,e–specific differences in regulation of hnRNP A2/B1to cell density may affect the functional effects ob-after silencing hnRNP A2/B1.fact that hnRNP A2/B1 has a role in the control of cellolism by regulating alternative splicing of M1/M2ate kynase isozymes (10) may explain the integrationdensity and regulation of hnRNP A2/1 expression. Thists a complex network of interaction between cell me-sm, cell density, cellular stress, and EMT differentiationch hnRNP A2/B1 may play a regulatory role. An impor-onsequence of the relationship between cell densitynRNP A2/B1 expression status is that to obtain consis-xperimental results while studying hnRNP A2/B1 func-ell handling, cell density at the time of splitting flasks,eeding density, all need to be carefully defined andrdized.ight of our preliminary description of the regulatory in-e of hnRNP A2/B1 and its effect on clonogenic growth,igration, and proliferation, coupled with other recents on hnRNP A2/B1 regulation of core biology, furthers are needed to understand the underlying mechanismulation of EMT/MET linked to hnRNP A2/B1 expres-nd cell density in lung cancer, relative to its contribu-lung carcinogenesis.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

Support

research was supported in part by intramural NCI funds, Rushity, and the Charles J. & Margaret Roberts Fund.

ived 03/10/2010; revised 06/30/2010; accepted 07/14/2010; publishedirst 08/31/2010.
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2010;70:7137-7147. Published OnlineFirst August 31, 2010.Cancer Res Jordi Tauler, Enrique Zudaire, Huaitian Liu, et al. Lung Cancer Cell LineshnRNP A2/B1 Modulates Epithelial-Mesenchymal Transition in

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