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Human Cancer Biology
Characterization of a Cleavage Stimulation Factor, 30 pre-RNA,Subunit 2, 64 kDa (CSTF2) as a Therapeutic Target for Lung Cancer
Masato Aragaki1,4, Koji Takahashi1, Hirohiko Akiyama5, Eiju Tsuchiya6, Satoshi Kondo4,Yusuke Nakamura1, and Yataro Daigo1,2,3
AbstractPurpose: This study aims to discover novel biomarkers and therapeutic targets for lung cancers.
Experimental Design: We screened for genes showing elevated expression in the majority of lung
cancers by genome-wide gene expression profile analysis of 120 lung cancers obtained by cDNAmicroarray
representing 27,648 genes or expressed sequence tags. In this process, we detected a gene encoding cleavage
stimulation factor, 30 pre-RNA, subunit 2, 64 kDa (CSTF2) as a candidate. Immunohistochemical stainingusing tissue microarray consisting of 327 lung cancers was applied to examine the expression of CSTF2
protein and its prognostic value. A role of CSTF2 in cancer cell growthwas examined by siRNA experiments.
Results: Northern blot and immunohistochemical analyses detected the expression of CSTF2 only in
testis among 16 normal tissues. Immunohistochemical analysis using tissue microarray showed an
association of strong CSTF2 expression with poor prognosis of patients with non–small cell lung cancer
(P ¼ 0.0079), and multivariate analysis showed that CSTF2 positivity is an independent prognostic factor.In addition, suppression of CSTF2 expression by siRNAs suppressed lung cancer cell growth, whereas
exogenous expression of CSTF2 promoted growth and invasion of mammalian cells.
Conclusions: CSTF2 is likely to play an important role in lung carcinogenesis and be a prognostic
biomarker in the clinic. Clin Cancer Res; 17(18); 5889–900. �2011 AACR.
Introduction
Primary lung cancer is the leading cause of cancer deathsin the world, and non–small cell lung cancer (NSCLC)accounts for approximately 80% of them (1). Detailedmolecular mechanism of lung carcinogenesis remainsunclear, although various genetic alterations in lung cancerwere reported (2). The patient with an advanced lungcancer often suffers from the fatal disease progression inspite of the improvement in surgical treatment and chemo-radiotherapy (1). Therefore, it is extremely important tounderstand the biology of lung cancer and to developmoreeffective treatments to improve the prognosis of patients
(2). Within the past 2 decades, some newly developedcytotoxic agents such as paclitaxel, docetaxel, gemcitabine,and vinorelbine have appeared to offer multiple choices fortreatment of patients with advanced NSCLC; however,those regimens could show only a modest survival benefitcompared with conventional platinum-based regimens(3). To date, a lot of molecular targeting agents have beenused in the clinical practice of lung cancer treatment,including tyrosine kinase inhibitors of epidermal growthfactor or VEGF as well as monoclonal antibodies againstthem (4, 5). However, only a small proportion of patientscan choose these treatment regimens due to the problem oftoxicity, and the ratio of patients who show good responseis limited even if all kinds of treatments are applied.
Systematic analysis of expression levels of thousands ofgenes by a cDNA microarray technology is an effectiveapproach to identify diagnostic and therapeutic targetmolecules involved in carcinogenic pathways (2). To iden-tify potential molecular targets for diagnosis and/or treat-ment of lung cancers, we previously analyzed genome-widegene expression profiles of 120 lung cancer tissue samplesby means of a cDNAmicroarray consisting of 27,648 genesor expressed sequence tags (EST) and tumor-cell popula-tions purified by laser microdissection (6–10). To verify thebiological and clinicopathologic significance of the respec-tive gene products, we have established a screening systemby combination of the tumor tissue microarray analysisof clinical lung cancer materials and RNA interference
Authors' Affiliations: 1Laboratory of Molecular Medicine, Human GenomeCenter, Institute of Medical Science, The University of Tokyo, Tokyo;2Department of Medical Oncology; 3Cancer Center, Shiga University ofMedical Science, Otsu; 4Department of Surgical Oncology, HokkaidoUniversity Graduate School of Medicine, Sapporo; 5Department of Tho-racic Surgery, Saitama Cancer Center, Saitama; and 6Kanagawa CancerCenter Research Institute, Yokohama, Japan
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Yataro Daigo, Institute of Medical Science, TheUniversity of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,Japan. Phone: 81-3-5449-5457; Fax: 81-3-5449-5406; E-mail:[email protected]
doi: 10.1158/1078-0432.CCR-11-0240
�2011 American Association for Cancer Research.
ClinicalCancer
Research
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technique (11–41). This systematic approach revealed thatcleavage stimulation factor, 30 pre-RNA, subunit 2, 64 kDa(CSTF2) gene was frequently transactivated in the majorityof primary lung cancers.
CSTF2 encodes a nuclear protein that contains a ribo-nucleoprotein (RNP)-type RNA binding domain in theN-terminal region. The protein is a member of cleavagestimulation factor complex (CSTF), which was reported toplay a role in polyadenylation of mRNA with 2 othermembers of cleavage stimulation factors in HeLa cells(42). In vitro binding assay revealed that human recombi-nant CstF2 can bind to synthesized GU-rich elementswithin the 30-untranslated region (30-UTR) of mRNAs (43).Elevated CSTF2 expression was reported in male germ cellsof mice and rats (44). In spite of the evidence of CSTF2function as a member of CSTF in vitro, the significance ofCSTF2 activation in carcinogenesis and its potential as abiomarker and a therapeutic target were not described.
We here report that overexpression of CSTF2 is critical forlung cancer growth and is a prognostic factor for NSCLCpatients.
Materials and Methods
Cell lines and tissue samplesFifteen human lung cancer cell lines used in this study
included 5 adenocarcinomas (NCI-H1781, NCI-H1373,LC319, A549, and PC-14), 5 squamous cell carcinomas(SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170, andLU61), 1 large-cell carcinoma (LX1), and 4 small-cell lungcancers (SBC-3, SBC-5, DMS114, and DMS273; details areshown in Supplementary Table S1). All cells were grown inmonolayer in appropriate media supplemented with 10%fetal calf serum (FCS) and were maintained at 37�C inhumidified air with 5% CO2. Human small airway epithe-lial cells were grown in optimized medium (CambrexBioscience, Inc.). Primary NSCLC tissue samples as wellas their corresponding normal tissues adjacent to resectionmargins had been obtained earlier with informed consentfrom patients having no anticancer treatment before tumorresection (6, 10). All tumors were staged on the basis ofthe pathologic tumor-node-metastasis classification of theInternational Union Against Cancer (45). Formalin-fixed
primary lung tumors and adjacent normal lung tissuesamples used for immunostaining on tissue microarrayshad been obtained from 327 patients (196 adenocarcino-mas, 98 squamous cell carcinomas, 23 large-cell carcino-mas, and 10 adenosquamous carcinomas; 99 female and228male patients; median age of 64.7 years with a range of29–85 years) undergoing surgery at Saitama Cancer Center.Those patients that received resection of their primarycancers did not receive any preoperative treatment, andamong them only patients with positive lymph nodemetastasis were treated with platinum-based adjuvantchemotherapies after their surgery. This study and theuse of all clinical materials mentioned were approved byindividual institutional ethics committees.
Semiquantitative reverse transcription-PCRA total of 3 mg of mRNA aliquot from each sample
was reversely transcribed to single-stranded cDNAs usingrandom primer (Roche Diagnostics) and Superscript II(Invitrogen). Semiquantitative reverse transcription-PCR(RT-PCR) experiments were carried out with the followingsets of synthesized primers specific for human CSTF2or with b-actin (ACTB)–specific primers as an internalcontrol: CSTF2, 50-GTCATGCAGGGAACAGGAAT-30 and50-TGAGTCATTCAAGGGTTAGGATG-30; ACTB, 50-GAGG-TGATAGCATTGCTTTCG-30 and 50-CAAGTCAGTGTACAG-GTAAGC-30. PCR reactions were optimized for the numberof cycles to ensure product intensity to be within the linearphase of amplification.
Northern blot analysisHuman multiple tissue blots covering 16 tissues (BD
Bioscience) were hybridized with a 32P-labeled 520-bp PCRproduct of CSTF2 that was prepared as a probe, usingprimers 50-CGAGGCTTGTTAGGAGATGC-30 and 50-CCCC-CATGTTAATTGGACTG-30. Prehybridization, hybridization,and washing were done following the manufacturer’sinstructions. The blots were autoradiographed with inten-sifying screens at �80�C for 14 days. The blots were alsohybridized with 32P-labeled PCR product of b-actin (ACTB)as a probe to verify the loading conditions.
Western blottingTumor cells were lysed in lysis buffer: 50 mmol/L Tris-
HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5%sodium deoxycholate, and Protease Inhibitor CocktailSet III (Calbiochem). The protein content of each lysatewas determined by a Bio-Rad protein assay kit with bovineserum albumin as a standard. Ten micrograms of eachlysate was resolved on 7.5% to 12% denaturing poly-acrylamide gels (with 3% polyacrylamide stacking gel)and transferred electrophoretically onto a nitrocellulosemembrane (GE Healthcare Biosciences). After blockingwith 5% nonfat dry milk in Tris-buffered saline and Tween20 (TBST), the membrane was incubated for 1 hour atroom temperature with a rabbit polyclonal anti-CSTF2antibody (ATLAS, Inc.; catalog no. HPA000427) that wasproved to be specific to human CSTF2, by Western blot
Translational Relevance
Because there is a significant correlation of cleav-age stimulation factor, 30 pre-RNA, subunit 2, 64 kDa(CSTF2) expression with poor prognosis for patientswith lung cancers, CSTF2 positivity in resected speci-mens could be an index that provides useful informa-tion to physicians in applying adjuvant therapy andintensive follow-up to the cancer patients who are likelyto relapse. Because CSTF2 is likely to play importantroles in cell proliferation and invasion, further func-tional analysis should contribute to the development ofa new therapeutic strategy targeting this pathway.
Aragaki et al.
Clin Cancer Res; 17(18) September 15, 2011 Clinical Cancer Research5890
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analysis using lysates of lung cancer cell lines. Immunore-active proteins were incubated with horseradish peroxidase(HRP)-conjugated secondary antibodies (GE HealthcareBio-sciences) for 1 hour at room temperature. After wash-ing with TBST, we used an enhanced chemiluminescenceWestern blotting analysis system (GE Healthcare Bio-sciences), as previously described (12).
Immunofluorescence analysisSBC-5, A549, and LC319 cells were seeded on cover-
slips. The cultured cells washed twice with cold PBS(–)were fixed in 4% paraformaldehyde solution for 60 min-utes at 4�C and rendered permeable by treatment for5 minutes with PBS(–) containing 0.1% Triton X-100.Cells were covered with CAS Block (Zymed) for 10 minutesto block nonspecific binding before the primary antibodyreaction. Then the cells were incubated with antibodyto human CSTF2 (ATLAS, Inc.; catalog no. HPA000427)for 1 hour at room temperature, followed by incubationwith Alexa488-conjugated goat anti-rabbit antibodies(Molecular Probes; 1:1,000 dilution) for 1 hour. Imageswere captured on a confocal microscope (TCS SP2-AOBS;Leica Microsystems).
Immunohistochemistry and tissue microarrayTo investigate the clinicopathologic significance of the
CSTF2 protein in clinical lung cancer samples that had beenformalin fixed and embedded in paraffin blocks, westained the sections by Envisionþ Kit/HRP (DakoCytoma-tion) in the following manner. For antigen retrieval, slideswere immersed in Target Retrieval Solution (pH 9;DakoCytomation) and boiled at 108�C for 15 minutesin an autoclave. A rabbit polyclonal anti-human CSTF2antibody (ATLAS, Inc.; catalog no. HPA000427) was addedto each slide after blocking of endogenous peroxidase andproteins, and the sections were incubated with HRP-labeled anti-rabbit IgG [Histofine Simple Stain MAX PO(G); Nichirei] as the secondary antibody. Substrate-chro-mogenwas added, and the specimens were counterstainedwith hematoxylin.Tumor tissue microarrays were constructed with 327
formalin-fixed primary NSCLCs which had been obtainedat Saitama Cancer Center (Saitama, Japan) with an iden-tical protocol to collect, fix, and preserve the tissues afterresection (5). Considering the histologic heterogeneity ofindividual tumors, tissue area for sampling was selected onthe basis of visual alignment with the corresponding he-matoxylin and eosin–stained section on a slide. Three, 4, or5 tissue cores (diameter, 0.6 mm; depth, 3–4 mm) takenfrom a donor tumor block were placed into a recipientparaffin block, using a tissue microarrayer (Beecher Instru-ments). A core of normal tissue was punched from eachcase, and 5-mm sections of the resulting microarray blockwere used for immunohistochemical analysis. Three inde-pendent investigators semiquantitatively assessed CSTF2positivity without prior knowledge of clinicopathologicdata. Because the intensity of staining within each tumortissue core was mostly homogeneous, the intensity of
CSTF2 staining was semiquantitatively evaluated by thefollowing criteria: strong positive (scored as 2þ), darkbrown staining in more than 50% of tumor cells complete-ly obscuring nucleus; weak positive (1þ), any lesser degreeof brown staining appreciable in tumor cell nucleus;absent (scored as 0), no appreciable staining in tumorcells. Cases were accepted as strongly positive if 2 or moreinvestigators independently defined them as such.
Statistical analysisStatistical analyses were carried out with the StatView
statistical program (SaS). Strong CSTF2 immunoreactivitywas assessed for association with clinicopathologic vari-ables such as gender, age, histologic type, smoking, andpathologic tumor-node-metastasis stage by using theFisher’s exact test. Survival curves were calculated fromthe date of surgery to the time of death related to NSCLCor to the last follow-up observation. Kaplan–Meier curveswere calculated for each relevant variable and for CSTF2expression; differences in survival times among patientsubgroups were analyzed by the log-rank test. Univariateand multivariate analyses were done with the Cox propor-tional hazard regression model to determine associationsbetween clinicopathologic variables and cancer-relatedmortality. First, we analyzed associations between deathand possible prognostic factors including gender, age,histology, smoking, pT classification, and pN classification,taking into consideration 1 factor at a time. Second, mul-tivariate analysis was applied on backward (stepwise) pro-cedures that always forced strong CSTF2 expression into themodel, along with any and all variables that satisfied anentry level of P < 0.05. As the model continued to addfactors, independent factors did not exceed an exit levelof P < 0.05.
RNA interference assayTo evaluate the biological functions of CSTF2 in
lung cancer cells, we used siRNA duplexes against CSTF2gene. The target sequences of the synthetic oligonucleo-tides for RNA interference were as follows: si-CSTF2-#1,50-GGCUUUAGUCCCGGGCAGA-30; si-CSTF2-#2, 50-CU-GAAUGGGCGCGAAUUCA-30; control 1 [EGFP: enhancedgreen fluorescence protein (GFP) gene, a mutant ofAequorea victoria GFP], 50-GAAGCAGCACGACUUCUUC-30; control 2 (LUC: luciferase gene from Photinus pyralis),50-CGUACGCGGAAUACUUCGA-30. Lung cancer celllines, A549 and LC319, were plated onto 10-cm dishes(8.0 � 105 per dish) and transfected with either of thesiRNA oligonucleotides (100 nmol/L), using 30 mL ofLipofectamine 2000 (Invitrogen) according to the manu-facturers’ instructions. After 7 days of incubation, cellviability was assessed by MTT assay.
Flow cytometryCells transfected with siRNA oligonucleotides were
plated at densities of 8 � 105 per 100-mm dish. Cellswere collected in PBS and fixed in 70% cold ethanol for30 minutes. After treatment with 100 mg/mL RNase
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(Sigma–Aldrich), the cells were stained with 50 mg/mLpropidium iodide (Sigma Aldrich) in PBS. Flow cytometrywas done on a Cell Lab Quanta SC (Beckman Coulter) andanalyzed by CXP Analysis software ver.2.2 (Beckman Coul-ter). The cells selected from at least 20,000 ungated cellswere analyzed for DNA content.
Cell growth assayCOS-7 and HEK293T cells that hardly expressed endog-
enous CSTF2 were plated at densities of 5.0 � 105 cellsper 100-mm dish, transfected with plasmids designed toexpress CSTF2 (pcDNA3.1/myc-His-CSTF2) or mock plas-mids (pcDNA3.1/myc-His). COS-7 and HEK293T cellswere cultured for 7 days in medium containing 0.4 and0.9 mg/mL of geneticin (Invitrogen), respectively. Cellviability was assessed by MTT assay. Colony numbers werealso counted with ImageJ software after the cells had beenfixed and stained by Giemsa solution.
Matrigel invasion assayCOS-7 and HEK293T cells, transfected with plasmids
designed to express CSTF2 (pcDNA3.1/myc-His-CSTF2)or mock plasmids (pcDNA3.1/myc-His), were grown tonear confluence in appropriate media containing 10%FCS. The cells were harvested by trypsinization, washedin the media without addition of serum or proteinaseinhibitor, and suspended in the media at a concentrationof 5.0 � 104/mL. Before preparing the cell suspension,the dried layer of Matrigel matrix (Becton DickinsonLabware) was rehydrated with the media for 2 hours atroom temperature. The media (0.75 mL) containing10% FCS was added to each lower chamber in 24-wellMatrigel invasion chambers, and 0.5 mL (2.5 � 104 cells)of cell suspension was added to each insert of the upperchamber. The plates of inserts were incubated for 24 hoursat 37�C. Then the chambers were processed, and cellsinvading through the Matrigel were fixed and stained byGiemsa as directed by the supplier (Becton DickinsonLabware).
Results
Expression of CSTF2 in lung cancers and normaltissues
To screen novel target molecules for development oftherapeutic agents and/or biomarkers for lung cancer, wefirst carried out genome-wide gene expression profileanalysis of 120 lung cancers by using a cDNA microarrayconsisting of 27,648 genes or ESTs (6–10). We identifiedthe CSTF2 transcript to be overexpressed (3-fold or higher)in the majority of lung cancer samples examined, whereasCSTF2 expression was scarcely detectable in any of 29normal tissues except testis. We confirmed CSTF2 over-expression by semiquantitative RT-PCR experiments in12 of 15 lung cancer tissues and in all of 15 lung cancercell lines examined (Fig. 1A). In addition, we detectedexpression of CSTF2 proteins in 9 lung cancer cell linesby Western blot analysis, using anti-CSTF2 antibody
(Fig. 1B). To determine the subcellular localization ofendogenous CSTF2 in lung cancer cells, we carried outimmunofluorescence analysis with anti-CSTF2 antibodyand found its staining in the nucleus of SBC-5, A549,and LC319 cells (Fig. 1C).
Northern blot analysis with a CSTF2 cDNA as a probeidentified a 2.6-kb transcript only in testis among 16normal human tissues examined (Fig. 2A). In addition,we examined expression of CSTF2 protein in 5 normaltissues (heart, lung, liver, kidney, and testis) as well aslung cancers, using anti-CSTF2 antibody. Positive stainingof CSTF2 was observed in the nucleus of testicular cellsand lung cancer cells, but not in other normal tissues(Fig. 2B).
Association of CSTF2 overexpression with poorprognosis for NSCLC patients
To verify the biological and clinicopathologic signifi-cance of CSTF2 in pulmonary carcinogenesis, we carriedout immunohistochemical staining on tissue microarrayscontaining primary NSCLC tissues from 327 patients whounderwent curative surgical resection at Saitama CancerCenter. CSTF2-positive staining with the anti-CSTF2 poly-clonal antibody was observed in the nucleus of lung cancercells, but staining was negative in any of their adjacentnormal lung cells or stromal cells surrounding tumor cells(Fig. 2C). We classified CSTF2 expression levels on thetissue array ranging from absent (scored as 0) to weak/strong positive (scored as 1þ or 2þ; Fig. 2D). Of the 327NSCLCs, CSTF2 was strongly stained in 77 cases (24%,score 2þ), weakly stained in 165 cases (50%, score 1þ),and not stained in 85 cases (26%: score 0; details are shownin Table 1). We then examined a correlation of CSTF2expression levels (strong positive vs. weak positive/absent)with various clinicopathologic variables and found thatstrong CSTF2 expression was associated with poor prog-nosis for patients withNSCLC after the resection of primarytumors (P ¼ 0.0079, log-rank test; Fig. 2E; SupplementaryTable S2), but not associated with any other clinicopath-ologic variables. In addition, we used univariate analysisto evaluate associations between patient prognosis andseveral clinicopathologic factors including gender (malevs. female), age (�65 vs.
(si-CSTF2-#1 and si-CSTF2-#2) or control siRNAs(si-EGFP and si-LUC) into A549 and LC319 cells in whichCSTF2 was endogenously overexpressed. The mRNAlevels of CSTF2 in cells transfected with si-CSTF2-#1and si-CSTF2-#2 were significantly decreased in compar-ison with those transfected with either control siRNAs(Fig. 3A). Cell viability and colony numbers investigatedby MTT assays were reduced significantly in the cellstransfected with si-CSTF2-#1 and si-CSTF2-#2 (Fig. 3B).To clarify the mechanism of tumor suppression by siRNAsagainst CSTF2, we carried out flow cytometric analysisof the tumor cells transfected with these siRNAs and
found a significant increase in the cells of sub-G1 fraction72 hours after the treatment (Fig. 3C and D).
Enhancement of mammalian cell proliferation andinvasion by CSTF2
To examine a potential role of CSTF2 in tumorigenesis,we constructed plasmids designed to express CSTF2(pcDNA3.1/myc-His-CSTF2) and transfected them intomammalian COS-7 and HEK293T cells. Exogenous CSTF2expression in the transfected cells was confirmed byWestern blot analysis (Fig. 4A). We carried out MTT andcolony formation assays and found that growth of the
Lung cancer cell linesAirway
epithelia
NC
I-H
1781
NC
I-H
1373
LC
319
A54
9
PC
14
SK
-ME
S-1
NC
I-H
520
NC
I-H
1703
NC
I-H
2170
LU
61
LX
1
SB
C-3
SB
C-5
DM
S11
4
DM
S27
3
SA
EC
CLCSCCSCDA
CLCSCCSCDA LCC
No
rmal
lu
ng
LC
1
LC
2
LC
3
LC
4
LC
5
LC
6
LC
7
LC
8
LC
9
LC
10
LC
11
LC
12
LC
13
LC
14
LC
15
A
B C
(kDa)
A54
9
LC
319
NC
I-H
1781
NC
I-H
520
NC
I-H
1703
NC
I-H
2170
DM
S11
4
SB
C-3
SB
C-5
SA
EC
Lung cancer cell linesAirway
epithelia
250-150-
100-75-
50-
37-
25-
IB: anti-ACTB
IB: anti-CSTF2
10 µm
10 µm
10 µm 20 µm
20 µm
20 µm
20 µm
20 µm
20 µm
Mer
ge
CS
TF
2D
AP
I
913CL945A5CBS
CSTF2
ACTB
CSTF2
ACTB
Figure 1. Expression of CSTF2 in lung tumors. A, expression of CSTF2 in 15 clinical lung cancers [lung adenocarcinoma (ADC), lung squamous cellcarcinoma (SCC), and small-cell lung cancer (SCLC)] and 15 lung cancer cell lines, examined by semiquantitative RT-PCR. Expression of b-actin (ACTB) geneserved as a quantity control. LCC, large-cell carcinoma. B, expression of CSTF2 protein in lung cancer cell lines, detected byWestern blot analysis. Expressionof ACTB protein served as a quantity control. IB, immunoblotting. C, subcellular localization of CSTF2 protein, examined by confocal microscopy. DAPI, 40,6-diamidino-2-phenylindole.
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COS-7 and HEK293T cells transfected with CSTF2 wassignificantly enhanced compared with those transfectedwith the mock vector (Fig. 4B; Supplementary Fig. S1).
Because strong CSTF2 expression was associated withpoor prognosis of NSCLC patients, we next examined byMatrigel assay the effect of CSTF2 expression on cellularinvasion. We transfected plasmids designed to expressCSTF2 into COS-7 and HEK293T cells and found thatoverexpression of CSTF2 significantly enhanced the inva-sive activity of cells through Matrigel (SupplementaryFig. S2). The result suggests that CSTF2 could contributeto the highly malignant phenotype of cancer cells.
Discussion
To develop molecular-targeting anticancer drugs thatare expected to be highly specific to malignant cells, withminimal risk of adverse reactions, we established an ef-fective screening system to identify proteins that wereactivated specifically in lung cancers. First, we analyzed agenome-wide expression profile of 120 lung cancer sam-ples through the genome-wide cDNA microarray systemcontaining 27,648 genes coupled with cancer cell puri-fication by laser microdissection (6–10). After verificationof very low or absent expression of such genes in normal
(kb)9.57.5
4.4
2.4
1.35
2.4
1.35
0.24
A
ACTB
CSTF2
reviLgnuLtraeH
CDA gnuLsitseTyendiK
B
CCL gnuLCCS gnuL SCLC
Figure 2. Expression of CSTF2 innormal tissues and association ofCSTF2 overexpression with poorprognosis of NSCLC patients.A, expression of CSTF2 in normalhuman tissues, detected byNorthern blot analysis.B, expression of CSTF2 in5 normal human tissues as well aslung cancer tissues [lungadenocarcinoma (ADC),squamous cell carcinoma(SCC), large-cell carcinoma(LCC), and small-cell lungcancer (SCLC), detected byimmunohistochemical staining,using a rabbit polyclonal anti-CSTF2 antibody. Counterstainingwith hematoxylin was done(�200).
Aragaki et al.
Clin Cancer Res; 17(18) September 15, 2011 Clinical Cancer Research5894
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organs by cDNA microarray analysis and multiple-tissueNorthern blot analysis, we analyzed the protein expres-sion of candidate targets among hundreds of clinical sam-ples on tissue microarrays, investigated loss of function
phenotypes using RNA interference systems, and furtherdefined biological functions of the proteins. Through theseanalyses, we identified candidate genes for the develop-ment of novel serum biomarkers (30–35), therapeutic
Figure 2. (Continued ) C,comparison of CSTF2 expressionlevels between normal lungs andtumors by immunohistochemistryin the same patient's tissues.Representative images of pairednormal lung and tumor tissuesfrom 8 patients (4 ADC, 2 SCC,1 LCC, and 1 SCLC) were shown.D, representative examples forstrong, weak, and absent CSTF2expression in lung ADC tissuesand a normal lung tissue (originalmagnification, �100). E, Kaplan–Meier analysis of survival ofpatients with NSCLC who hadundergone surgery (P ¼ 0.0079,log-rank test).
Case 1 (ADC) Case 2 (ADC) Case 3 (ADC) Case 4 (ADC)
Case 5 (SCC) Case 6 (SCC) Case 7 (LCC) Case 8 (SCLC)
Lung
tum
orN
orm
al lu
ngLu
ng tu
mor
Nor
mal
lung
C
CSTF2Strong positive
CSTF2Weak positive
CSTF2Negative
Lung cancer (ADC)D
Normal lung
Strong positive Weak positive Negative
CSTF2 negative or weak positive1.0
Lung cancer mortality: first study ECSTF2 negative or weak positive
ate
(× 1
00%
)
(N = 250)0.8
0.6
Surv
ival
ra
(N = 77)CSTF2 strong positive
0.4
0.2
P = 0.00790
Postoperarative days0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000
CSTF2 Activation in Lung Cancer
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drugs (11–29), and/or immunotherapy (35–41), whichwere upregulated in cancer cells but not expressed innormal organs, except testis, placenta, and/or fetus tissues.In this study, we report that CSTF2 encoding a member of
cleavage stimulation factors was frequently overexpressedin the great majority of clinical lung cancer samples andcell lines, and that its gene product plays an indispensablerole in the growth and invasion of lung cancer cells.
Table 1. Association between CSTF2 positivity in NSCLC tissues and patients’ characteristics
Total(N ¼ 327)
CSTF2 strongpositive (n ¼ 77)
CSTF2 weakpositive (n ¼ 165)
CSTF2 absent(n ¼ 85)
P (strong vs.weak/absent)
GenderMale 228 55 120 53 0.7775Female 99 22 45 32
Age, y
P < 0.0001
P < 0.0001
Anti-CSTF2siRNA
CSTF2
ACTB
CSTF2
ACTB
Anti-CSTF2siRNA
si -#1
LC319
ControlsiRNA
si-LUCsi -#2 si-EGFP
A549
ControlsiRNA
si -#1 si-LUCsi -#2 si-EGFP
si -#1 si-LUCsi -#2 si-EGFP
A
B2.5
2.0
1.5
1.0
0.5
0
1.6
1.2
0.8
0.4
0
P < 0.0001
P < 0.0001
si -#1 si-LUCsi -#2 si-EGFP
Rel
ativ
e ab
sorb
ance
(490
nm
/630
nm
)
Rel
ativ
e ab
sorb
ance
(490
nm
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Figure 3. Inhibition of growth of NSCLC cells by siRNAs against CSTF2. A, expression of CSTF2 in response to siRNA treatment for CSTF2 (si-CSTF2-#1or si-CSTF2-#2) or control siRNAs (si-EGFP or si-LUC) in A549 and LC319cells, analyzed by semiquantitative RT-PCR (top). B, MTT assays of thetumor cells transfected with si-CSTF2s or control siRNAs. C, flow cytometric analysis of the A549 cells and LC319 cells 72 hours after transfectionof the siRNAs for CSTF2 (si-CSTF2-#1) and control siRNAs (si-EGFP). D, left, percentage of cells at each phase; right, averaged percentage of cellsat sub-G1 phase of triplicate assays.
CSTF2 Activation in Lung Cancer
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CSTF2 encodes a 557–amino acid protein with anN-terminal RNP-type RNA-binding domain, a long Pro-and Gly-rich region, and a pentapeptide repeat regionthat forms an extended a-helix and comprises one of amultisubunit complex of CstF required for polyadenyla-tion and 30-end cleavage of mammalian pre-mRNAs (46).CstF cooperates with cleavage-polyadenylation specificityfactor to define the site of polyadenylation by recognizingthe highly conserved AAUAAA hexanucleotide and moredivergent GU-rich situated downstream of the actualcleavage site (47). The polyadenylatation and 30-endcleavage of pre-mRNAs are important modifications forproper transcription, splicing, transport, translation, andstability of mRNAs. This factor for polyadenylation isreported to change the length of the 30-UTR of mRNAand thus the binding sites of microRNAs (miRNA), whichtend to bind to these regions (48). Loss of miRNAcomplementary sites in 30-UTR of some oncogenes isone of the mechanisms of oncogene activation. In cancercells, 30-UTR of some oncogenes is likely to be shortenedby alternative cleavage and polyadenylation, resultingin avoidance of silencing by miRNA and activation ofoncogenes in cancer cells (49). It is still unknown whyCSTF2 was overexpressed in testis and cancer cells;however, CSTF2 may play important roles for activationof some oncogenes by changing the length of 30-UTR oftheir mRNAs in cancer cells.
To further elucidate the downstream pathways ofCSTF2 in lung cancer cells, we transfected siRNAs againstCSTF2 (si-CSTF2 #1) or EGFP (control siRNA) into lungcancer A549 cells that overexpressed CSTF2, and screenedby microarray analysis as described previously (16) genesthat were downregulated in accordance with the knock-down of CSTF2 expression at 24 and 48 hours after siRNAtransfection. The self-organizing map clustering analysisfor selecting genes showed the same expression pattern toCSTF2 in a time-dependent manner and subsequently thegene ontology database analysis identified 4 candidatedownstream pathways of CSTF2 including genes relatedto a member of interleukin, TGF-b, chemokine, andheterotrimeric G protein, some of which were reportedto have important roles in carcinogenesis (SupplementaryTables S3 and S4). Although further detailed analysesare necessary to determine the direct targets of CSTF2, theinformation implies the biological importance of CSTF2in human carcinogenesis.
In this study, we showed that CSTF2 gene was over-expressed in most lung cancers and likely to play animportant role in the growth of lung cancers. Moreover,clinicopathologic data through our tissue microarrayexperiments showed that patients with NSCLC, in whichCSTF2 was highly expressed, had shorter survival periodsthan those with CSTF2 weak positive/negative tumors.This is the first study that proves a potential of tissue
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Figure 4. Enhancement of cellulargrowth by CSTF2 introduction intomammalian cells. A, transientexpression of CSTF2 in COS-7and HEK293T cells, detected byWestern blot analysis. The cellswere introduced pcDNA3.1-myc-His-CSTF2 or mock vector. B, cellviability evaluated by the MTTassays. Assays were done 3 timesand in triplicate wells.
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CSTF2 expression as a prognostic biomarker for humancancers.Somatic epidermal growth factor receptor (EGFR)
mutation was reported to be biologically important forthe clinical response of NSCLCs to EGFR tyrosine kinaseinhibitors such as gefitinib or erlotinib (5, 50). To examinethe relevance of CSTF2 expression to this subgroup ofNSCLCs with EGFRmutation, we examined the correlationbetween CSTF2 overexpression and EGFR mutation statusin NSCLC tissues using specific antibodies to E746-A750deletion and those to a point mutation (L858R) in EGFR,and found that EGFR mutation was not associated withCSTF2 strong expression (P ¼ 0.2555, Fisher’s exact test;Supplementary Table S5; Supplementary Fig. S3). Thecombined results suggest that further functional analysisand screening to selectively inhibit CSTF2 function bysmall molecule compounds and/or nucleic acid drugscould be a potential therapeutic strategy that is expectedto have a powerful biological activity against broad clini-copathologic types of lung cancers.
In summary, CSTF2 might play an important role in thegrowth and progression of lung cancers. CSTF2 overexpres-sion in resected specimens may be a useful indicator ofadjuvant therapy to lung cancer patients who are likely tohave poor prognosis.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant Support
This work was supported in part by Grant-in-Aid for Scientific Research (B)and Grant-in-Aid for Scientific Research on Innovative Areas from The JapanSociety for the Promotion of Science. Y. Daigo is a member of Shiga CancerTreatment Project supported by Shiga Prefecture (Japan).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received January 26, 2011; revised June 22, 2011; accepted July 23, 2011;published OnlineFirst August 3, 2011.
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2011;17:5889-5900. Published OnlineFirst August 3, 2011.Clin Cancer Res Masato Aragaki, Koji Takahashi, Hirohiko Akiyama, et al. Subunit 2, 64 kDa (CSTF2) as a Therapeutic Target for Lung Cancer
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