Characterization of a Cleavage Stimulation Factor, 3 Pre-RNA

Published OnlineFirst August 3, 2011; DOI: 10.1158/1078-0432.CCR-11-0240

Clinical Cancer Human Cancer Biology Research

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

Abstract Purpose: 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 cDNA microarray 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 staining using 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 growth was 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 (2). Within the past 2 decades, some newly developed cytotoxic agents such as paclitaxel, docetaxel, gemcitabine, Primary lung cancer is the leading cause of cancer deaths and vinorelbine have appeared to offer multiple choices for in the world, and non–small cell lung cancer (NSCLC) treatment of patients with advanced NSCLC; however, accounts for approximately 80% of them (1). Detailed those regimens could show only a modest survival benefit molecular mechanism of lung carcinogenesis remains compared with conventional platinum-based regimens unclear, although various genetic alterations in lung cancer (3). To date, a lot of molecular targeting agents have been were reported (2). The patient with an advanced lung used in the clinical practice of lung cancer treatment, cancer often suffers from the fatal disease progression in including tyrosine kinase inhibitors of epidermal growth spite of the improvement in surgical treatment and chemo- factor or VEGF as well as monoclonal antibodies against radiotherapy (1). Therefore, it is extremely important to them (4, 5). However, only a small proportion of patients understand the biology of lung cancer and to develop more can choose these treatment regimens due to the problem of effective treatments to improve the prognosis of patients toxicity, and the ratio of patients who show good response is limited even if all kinds of treatments are applied. Systematic analysis of expression levels of thousands of Authors' Affiliations: 1Laboratory of Molecular Medicine, Human Genome genes by a cDNA microarray technology is an effective Center, Institute of Medical Science, The University of Tokyo, Tokyo; approach to identify diagnostic and therapeutic target 2Department of Medical Oncology; 3Cancer Center, Shiga University of 4 molecules involved in carcinogenic pathways (2). To iden- Medical Science, Otsu; Department of Surgical Oncology, Hokkaido University Graduate School of Medicine, Sapporo; 5Department of Tho- tify potential molecular targets for diagnosis and/or treat- racic Surgery, Saitama Cancer Center, Saitama; and 6Kanagawa Cancer ment of lung cancers, we previously analyzed genome-wide Center Research Institute, Yokohama, Japan gene expression profiles of 120 lung cancer tissue samples Note: Supplementary data for this article are available at Clinical Cancer by means of a cDNA microarray consisting of 27,648 genes Research Online (http://clincancerres.aacrjournals.org/). or expressed sequence tags (EST) and tumor-cell popula- Corresponding Author: Yataro Daigo, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, tions purified by laser microdissection (6–10). To verify the Japan. Phone: 81-3-5449-5457; Fax: 81-3-5449-5406; E-mail: biological and clinicopathologic significance of the respec- [email protected] tive gene products, we have established a screening system doi: 10.1158/1078-0432.CCR-11-0240 by combination of the tumor tissue microarray analysis 2011 American Association for Cancer Research. of clinical lung cancer materials and RNA interference

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Translational Relevance primary lung tumors and adjacent normal lung tissue samples used for immunostaining on tissue microarrays Because there is a significant correlation of cleav- had been obtained from 327 patients (196 adenocarcino- age stimulation factor, 30 pre-RNA, subunit 2, 64 kDa mas, 98 squamous cell carcinomas, 23 large-cell carcino- (CSTF2) expression with poor prognosis for patients mas, and 10 adenosquamous carcinomas; 99 female and with lung cancers, CSTF2 positivity in resected speci- 228 male patients; median age of 64.7 years with a range of mens could be an index that provides useful informa- 29–85 years) undergoing surgery at Saitama Cancer Center. tion to physicians in applying adjuvant therapy and Those patients that received resection of their primary intensive follow-up to the cancer patients who are likely cancers did not receive any preoperative treatment, and to relapse. Because CSTF2 is likely to play important among them only patients with positive lymph node roles in cell proliferation and invasion, further func- metastasis were treated with platinum-based adjuvant tional analysis should contribute to the development of chemotherapies after their surgery. This study and the a new therapeutic strategy targeting this pathway. use of all clinical materials mentioned were approved by individual institutional ethics committees.

technique (11–41). This systematic approach revealed that Semiquantitative reverse transcription-PCR cleavage stimulation factor, 30 pre-RNA, subunit 2, 64 kDa A total of 3 mg of mRNA aliquot from each sample (CSTF2) gene was frequently transactivated in the majority was reversely transcribed to single-stranded cDNAs using of primary lung cancers. random primer (Roche Diagnostics) and Superscript II CSTF2 encodes a nuclear protein that contains a ribo- (Invitrogen). Semiquantitative reverse transcription-PCR nucleoprotein (RNP)-type RNA binding domain in the (RT-PCR) experiments were carried out with the following N-terminal region. The protein is a member of cleavage sets of synthesized primers specific for human CSTF2 stimulation factor complex (CSTF), which was reported to or with b-actin (ACTB)–specific primers as an internal play a role in polyadenylation of mRNA with 2 other control: CSTF2,50-GTCATGCAGGGAACAGGAAT-30 and members of cleavage stimulation factors in HeLa cells 50-TGAGTCATTCAAGGGTTAGGATG-30; ACTB,50-GAGG- (42). In vitro binding assay revealed that human recombi- TGATAGCATTGCTTTCG-30 and 50-CAAGTCAGTGTACAG- nant CstF2 can bind to synthesized GU-rich elements GTAAGC-30. PCR reactions were optimized for the number within the 30-untranslated region (30-UTR) of mRNAs (43). of cycles to ensure product intensity to be within the linear Elevated CSTF2 expression was reported in male germ cells phase of amplification. of mice and rats (44). In spite of the evidence of CSTF2 function as a member of CSTF in vitro, the significance of Northern blot analysis CSTF2 activation in carcinogenesis and its potential as a Human multiple tissue blots covering 16 tissues (BD biomarker and a therapeutic target were not described. Bioscience) were hybridized with a 32P-labeled 520-bp PCR We here report that overexpression of CSTF2 is critical for product of CSTF2 that was prepared as a probe, using lung cancer growth and is a prognostic factor for NSCLC primers 50-CGAGGCTTGTTAGGAGATGC-30 and 50-CCCC- patients. CATGTTAATTGGACTG-30. Prehybridization, hybridization, and washing were done following the manufacturer’s Materials and Methods instructions. The blots were autoradiographed with inten- sifying screens at 80C for 14 days. The blots were also Cell lines and tissue samples hybridized with 32P-labeled PCR product of b-actin (ACTB) Fifteen human lung cancer cell lines used in this study as a probe to verify the loading conditions. included 5 adenocarcinomas (NCI-H1781, NCI-H1373, LC319, A549, and PC-14), 5 squamous cell carcinomas Western blotting (SK-MES-1, NCI-H520, NCI-H1703, NCI-H2170, and Tumor cells were lysed in lysis buffer: 50 mmol/L Tris- LU61), 1 large-cell carcinoma (LX1), and 4 small-cell lung HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% cancers (SBC-3, SBC-5, DMS114, and DMS273; details are sodium deoxycholate, and Protease Inhibitor Cocktail shown in Supplementary Table S1). All cells were grown in Set III (Calbiochem). The protein content of each lysate monolayer in appropriate media supplemented with 10% was determined by a Bio-Rad protein assay kit with bovine fetal calf serum (FCS) and were maintained at 37Cin serum albumin as a standard. Ten micrograms of each humidified air with 5% CO2. Human small airway epithe- lysate was resolved on 7.5% to 12% denaturing poly- lial cells were grown in optimized medium (Cambrex acrylamide gels (with 3% polyacrylamide stacking gel) Bioscience, Inc.). Primary NSCLC tissue samples as well and transferred electrophoretically onto a nitrocellulose as their corresponding normal tissues adjacent to resection membrane (GE Healthcare Biosciences). After blocking margins had been obtained earlier with informed consent with 5% nonfat dry milk in Tris-buffered saline and Tween from patients having no anticancer treatment before tumor 20 (TBST), the membrane was incubated for 1 hour at resection (6, 10). All tumors were staged on the basis of room temperature with a rabbit polyclonal anti-CSTF2 the pathologic tumor-node-metastasis classification of the antibody (ATLAS, Inc.; catalog no. HPA000427) that was International Union Against Cancer (45). Formalin-fixed proved to be specific to human CSTF2, by Western blot

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analysis using lysates of lung cancer cell lines. Immunore- CSTF2 staining was semiquantitatively evaluated by the active proteins were incubated with horseradish peroxidase following criteria: strong positive (scored as 2þ), dark (HRP)-conjugated secondary antibodies (GE Healthcare brown staining in more than 50% of tumor cells complete- Bio-sciences) for 1 hour at room temperature. After wash- ly obscuring nucleus; weak positive (1þ), any lesser degree ing with TBST, we used an enhanced chemiluminescence of brown staining appreciable in tumor cell nucleus; Western blotting analysis system (GE Healthcare Bio- absent (scored as 0), no appreciable staining in tumor sciences), as previously described (12). cells. Cases were accepted as strongly positive if 2 or more investigators independently defined them as such. Immunofluorescence analysis SBC-5, A549, and LC319 cells were seeded on cover- Statistical analysis slips. The cultured cells washed twice with cold PBS(–) Statistical analyses were carried out with the StatView were fixed in 4% paraformaldehyde solution for 60 min- statistical program (SaS). Strong CSTF2 immunoreactivity utes at 4C and rendered permeable by treatment for was assessed for association with clinicopathologic vari- 5 minutes with PBS(–) containing 0.1% Triton X-100. ables such as gender, age, histologic type, smoking, and Cells were covered with CAS Block (Zymed) for 10 minutes pathologic tumor-node-metastasis stage by using the to block nonspecific binding before the primary antibody Fisher’s exact test. Survival curves were calculated from reaction. Then the cells were incubated with antibody the date of surgery to the time of death related to NSCLC to human CSTF2 (ATLAS, Inc.; catalog no. HPA000427) or to the last follow-up observation. Kaplan–Meier curves for 1 hour at room temperature, followed by incubation were calculated for each relevant variable and for CSTF2 with Alexa488-conjugated goat anti-rabbit antibodies expression; differences in survival times among patient (Molecular Probes; 1:1,000 dilution) for 1 hour. Images subgroups were analyzed by the log-rank test. Univariate were captured on a confocal microscope (TCS SP2-AOBS; and multivariate analyses were done with the Cox propor- Leica Microsystems). tional hazard regression model to determine associations between clinicopathologic variables and cancer-related Immunohistochemistry and tissue microarray mortality. First, we analyzed associations between death To investigate the clinicopathologic significance of the and possible prognostic factors including gender, age, CSTF2 protein in clinical lung cancer samples that had been histology, smoking, pT classification, and pN classification, formalin fixed and embedded in paraffin blocks, we taking into consideration 1 factor at a time. Second, mul- stained the sections by Envisionþ Kit/HRP (DakoCytoma- tivariate analysis was applied on backward (stepwise) pro- tion) in the following manner. For antigen retrieval, slides cedures that always forced strong CSTF2 expression into the were immersed in Target Retrieval Solution (pH 9; model, along with any and all variables that satisfied an DakoCytomation) and boiled at 108C for 15 minutes entry level of P < 0.05. As the model continued to add in an autoclave. A rabbit polyclonal anti-human CSTF2 factors, independent factors did not exceed an exit level antibody (ATLAS, Inc.; catalog no. HPA000427) was added of P < 0.05. to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with HRP- RNA interference assay labeled anti-rabbit IgG [Histofine Simple Stain MAX PO To evaluate the biological functions of CSTF2 in (G); Nichirei] as the secondary antibody. Substrate-chro- lung cancer cells, we used siRNA duplexes against CSTF2 mogenwas added, and the specimens were counterstained gene. The target sequences of the synthetic oligonucleo- with hematoxylin. tides for RNA interference were as follows: si-CSTF2-#1, Tumor tissue microarrays were constructed with 327 50-GGCUUUAGUCCCGGGCAGA-30; si-CSTF2-#2, 50-CU- formalin-fixed primary NSCLCs which had been obtained GAAUGGGCGCGAAUUCA-30; control 1 [EGFP: enhanced at Saitama Cancer Center (Saitama, Japan) with an iden- green fluorescence protein (GFP) gene, a mutant of tical protocol to collect, fix, and preserve the tissues after Aequorea victoria GFP], 50-GAAGCAGCACGACUUCUUC- resection (5). Considering the histologic heterogeneity of 30; control 2 (LUC: luciferase gene from Photinus pyralis), individual tumors, tissue area for sampling was selected on 50-CGUACGCGGAAUACUUCGA-30. Lung cancer cell the basis of visual alignment with the corresponding he- lines, A549 and LC319, were plated onto 10-cm dishes matoxylin and eosin–stained section on a slide. Three, 4, or (8.0 105 per dish) and transfected with either of the 5 tissue cores (diameter, 0.6 mm; depth, 3–4 mm) taken siRNA oligonucleotides (100 nmol/L), using 30 mLof from a donor tumor block were placed into a recipient Lipofectamine 2000 (Invitrogen) according to the manu- paraffin block, using a tissue microarrayer (Beecher Instru- facturers’ instructions. After 7 days of incubation, cell ments). A core of normal tissue was punched from each viability was assessed by MTT assay. case, and 5-mm sections of the resulting microarray block were used for immunohistochemical analysis. Three inde- Flow cytometry pendent investigators semiquantitatively assessed CSTF2 Cells transfected with siRNA oligonucleotides were positivity without prior knowledge of clinicopathologic plated at densities of 8 105 per 100-mm dish. Cells data. Because the intensity of staining within each tumor were collected in PBS and fixed in 70% cold ethanol for tissue core was mostly homogeneous, the intensity of 30 minutes. After treatment with 100 mg/mL RNase

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(Sigma–Aldrich), the cells were stained with 50 mg/mL (Fig. 1B). To determine the subcellular localization of propidium iodide (Sigma Aldrich) in PBS. Flow cytometry endogenous CSTF2 in lung cancer cells, we carried out was done on a Cell Lab Quanta SC (Beckman Coulter) and immunofluorescence analysis with anti-CSTF2 antibody analyzed by CXP Analysis software ver.2.2 (Beckman Coul- and found its staining in the nucleus of SBC-5, A549, ter). The cells selected from at least 20,000 ungated cells and LC319 cells (Fig. 1C). were analyzed for DNA content. Northern blot analysis with a CSTF2 cDNA as a probe identified a 2.6-kb transcript only in testis among 16 Cell growth assay normal human tissues examined (Fig. 2A). In addition, COS-7 and HEK293T cells that hardly expressed endog- we examined expression of CSTF2 protein in 5 normal enous CSTF2 were plated at densities of 5.0 105 cells tissues (heart, lung, liver, kidney, and testis) as well as per 100-mm dish, transfected with plasmids designed to lung cancers, using anti-CSTF2 antibody. Positive staining express CSTF2 (pcDNA3.1/myc-His-CSTF2) or mock plas- of CSTF2 was observed in the nucleus of testicular cells mids (pcDNA3.1/myc-His). COS-7 and HEK293T cells and lung cancer cells, but not in other normal tissues were cultured for 7 days in medium containing 0.4 and (Fig. 2B). 0.9 mg/mL of geneticin (Invitrogen), respectively. Cell viability was assessed by MTT assay. Colony numbers were Association of CSTF2 overexpression with poor also counted with ImageJ software after the cells had been prognosis for NSCLC patients fixed and stained by Giemsa solution. To verify the biological and clinicopathologic significance of CSTF2 in pulmonary carcinogenesis, we carried Matrigel invasion assay out immunohistochemical staining on tissue microarrays COS-7 and HEK293T cells, transfected with plasmids containing primary NSCLC tissues from 327 patients who designed to express CSTF2 (pcDNA3.1/myc-His-CSTF2) underwent curative surgical resection at Saitama Cancer or mock plasmids (pcDNA3.1/myc-His), were grown to Center. CSTF2-positive staining with the anti-CSTF2 poly- near confluence in appropriate media containing 10% clonal antibody was observed in the nucleus of lung cancer FCS. The cells were harvested by trypsinization, washed cells, but staining was negative in any of their adjacent in the media without addition of serum or proteinase normal lung cells or stromal cells surrounding tumor cells inhibitor, and suspended in the media at a concentration (Fig. 2C). We classified CSTF2 expression levels on the of 5.0 104/mL. Before preparing the cell suspension, tissue array ranging from absent (scored as 0) to weak/ the dried layer of Matrigel matrix (Becton Dickinson strong positive (scored as 1þ or 2þ; Fig. 2D). Of the 327 Labware) was rehydrated with the media for 2 hours at NSCLCs, CSTF2 was strongly stained in 77 cases (24%, room temperature. The media (0.75 mL) containing score 2þ), weakly stained in 165 cases (50%, score 1þ), 10% FCS was added to each lower chamber in 24-well and not stained in 85 cases (26%: score 0; details are shown Matrigel invasion chambers, and 0.5 mL (2.5 104 cells) in Table 1). We then examined a correlation of CSTF2 of cell suspension was added to each insert of the upper expression levels (strong positive vs. weak positive/absent) chamber. The plates of inserts were incubated for 24 hours with various clinicopathologic variables and found that at 37C. Then the chambers were processed, and cells strong CSTF2 expression was associated with poor prog- invading through the Matrigel were fixed and stained by nosis for patients with NSCLC after the resection of primary Giemsa as directed by the supplier (Becton Dickinson tumors (P ¼ 0.0079, log-rank test; Fig. 2E; Supplementary Labware). Table S2), but not associated with any other clinicopathologic variables. In addition, we used univariate analysis Results to evaluate associations between patient prognosis and several clinicopathologic factors including gender (male Expression of CSTF2 in lung cancers and normal vs. female), age (65 vs. <65 years), histology (nonade- tissues nocarcinoma vs. adenocarcinoma), smoking history To screen novel target molecules for development of (smoker vs. nonsmoker), pT stage (tumor size; T2–T3 vs. therapeutic agents and/or biomarkers for lung cancer, we T1), pN stage (lymph node metastasis; N1–N2 vs. N0), and first carried out genome-wide gene expression profile CSTF2 expression (score 2þ vs. 0, 1þ). All of these para- analysis of 120 lung cancers by using a cDNA microarray meters except smoking history were significantly associated consisting of 27,648 genes or ESTs (6–10). We identified with poor prognosis (Table 2). Multivariate analysis by the the CSTF2 transcript to be overexpressed (3-fold or higher) Cox proportional hazard model indicated that pT stage, pN in the majority of lung cancer samples examined, whereas stage, age, and strong CSTF2 positivity were independent CSTF2 expression was scarcely detectable in any of 29 prognostic factors for NSCLC (Table 2). normal tissues except testis. We confirmed CSTF2 overexpression by semiquantitative RT-PCR experiments in Inhibition of growth of lung cancer cells by siRNA for 12 of 15 lung cancer tissues and in all of 15 lung cancer CSTF2 cell lines examined (Fig. 1A). In addition, we detected To assess whether upregulation of CSTF2 plays a role expression of CSTF2 proteins in 9 lung cancer cell lines in growth or survival of lung cancer cells, we trans- by Western blot analysis, using anti-CSTF2 antibody fected synthetic oligonucleotide siRNAs against CSTF2

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CSTF2 Activation in Lung Cancer

A CDA CCS CLCS Normal lung 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 CSTF2

ACTB

Lung cancer cell lines Airway ALCD CCS CCCS LC epithelia NCI-H1781 NCI-H1373 LC319 A549 PC14 SK-MES-1 NCI-H520 NCI-H1703 NCI-H2170 LU61 LX1 SBC-3 SBC-5 DMS114 DMS273 SAEC CSTF2

ACTB BCS 5CB 945A L 913C

Airway Lung cancer cell lines epithelia DAPI

A549 LC319 NCI-H1781 NCI-H520 NCI-H1703 NCI-H2170 DMS114 SBC-3 SBC-5 SAEC 10 µm 20 µm 20 µm (kDa) 250- 150-

100- 75-

IB: anti-CSTF2 CSTF2 50-

37- 10 µm 20 µm 20 µm

25-

IB: anti-ACTB Merge

10 µm 20 µm 20 µm

Figure 1. Expression of CSTF2 in lung tumors. A, expression of CSTF2 in 15 clinical lung cancers [lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC), and small-cell lung cancer (SCLC)] and 15 lung cancer cell lines, examined by semiquantitative RT-PCR. Expression of b-actin (ACTB) gene served as a quantity control. LCC, large-cell carcinoma. B, expression of CSTF2 protein in lung cancer cell lines, detected by Western blot analysis. Expression of 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.

(si-CSTF2-#1 and si-CSTF2-#2) or control siRNAs found a significant increase in the cells of sub-G1 fraction (si-EGFP and si-LUC) into A549 and LC319 cells in which 72 hours after the treatment (Fig. 3C and D). CSTF2 was endogenously overexpressed. The mRNA levels of CSTF2 in cells transfected with si-CSTF2-#1 Enhancement of mammalian cell proliferation and and si-CSTF2-#2 were significantly decreased in compar- invasion by CSTF2 ison with those transfected with either control siRNAs To examine a potential role of CSTF2 in tumorigenesis, (Fig. 3A). Cell viability and colony numbers investigated we constructed plasmids designed to express CSTF2 by MTT assays were reduced significantly in the cells (pcDNA3.1/myc-His-CSTF2) and transfected them into transfected with si-CSTF2-#1 and si-CSTF2-#2 (Fig. 3B). mammalian COS-7 and HEK293T cells. Exogenous CSTF2 To clarify the mechanism of tumor suppression by siRNAs expression in the transfected cells was confirmed by against CSTF2, we carried out flow cytometric analysis Western blot analysis (Fig. 4A). We carried out MTT and of the tumor cells transfected with these siRNAs and colony formation assays and found that growth of the

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(kb) 9.5 7.5

4.4 CSTF2

2.4 1.35

0.24 Figure 2. Expression of CSTF2 in normal tissues and association of 2.4 ACTB CSTF2 overexpression with poor 1.35 prognosis of NSCLC patients. A, expression of CSTF2 in normal B human tissues, detected by Northern blot analysis. eH art gnuL reviL B, expression of CSTF2 in 5 normal human tissues as well as lung cancer tissues [lung adenocarcinoma (ADC), squamous cell carcinoma (SCC), large-cell carcinoma (LCC), and small-cell lung cancer (SCLC), detected by immunohistochemical staining, using a rabbit polyclonal anti- yendiK Test si gnuL CDA CSTF2 antibody. Counterstaining with hematoxylin was done (200).

CCS gnuL CCS gnuL CCL SCLC

COS-7 and HEK293T cells transfected with CSTF2 was Discussion significantly enhanced compared with those transfected with the mock vector (Fig. 4B; Supplementary Fig. S1). To develop molecular-targeting anticancer drugs that Because strong CSTF2 expression was associated with are expected to be highly specific to malignant cells, with poor prognosis of NSCLC patients, we next examined by minimal risk of adverse reactions, we established an ef- Matrigel assay the effect of CSTF2 expression on cellular fective screening system to identify proteins that were invasion. We transfected plasmids designed to express activated specifically in lung cancers. First, we analyzed a CSTF2 into COS-7 and HEK293T cells and found that genome-wide expression profile of 120 lung cancer sam- overexpression of CSTF2 significantly enhanced the inva- ples through the genome-wide cDNA microarray system sive activity of cells through Matrigel (Supplementary containing 27,648 genes coupled with cancer cell puri- Fig. S2). The result suggests that CSTF2 could contribute fication by laser microdissection (6–10). After verification to the highly malignant phenotype of cancer cells. of very low or absent expression of such genes in normal

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C Case 1 (ADC) Case 2 (ADC) Case 3 (ADC) Case 4 (ADC) Lung tumor Normal lung

Case 5 (SCC) Case 6 (SCC) Case 7 (LCC) Case 8 (SCLC)

Continued Figure 2. ( )C, Lung tumor comparison of CSTF2 expression levels between normal lungs and tumors by immunohistochemistry in the same patient's tissues. Representative images of paired normal lung and tumor tissues from 8 patients (4 ADC, 2 SCC, Normal lung 1 LCC, and 1 SCLC) were shown. D, representative examples for strong, weak, and absent CSTF2 D Lung cancer (ADC) Normal lung expression in lung ADC tissues and a normal lung tissue (original CSTF2 CSTF2 CSTF2 magnification, 100). E, Kaplan– Strong positive Weak positive Negative Meier analysis of survival of patients with NSCLC who had undergone surgery (P ¼ 0.0079, log-rank test).

E Lung cancer mortality: first study

1.0 CSTF2 negative or weak positive

0.8 (N = 250)

0.6 ate (× 100%)

0.4

0.2 CSTF2 strong positive Survival ra (N = 77) 0 P = 0.0079

0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 Postoperarative days

organs by cDNA microarray analysis and multiple-tissue phenotypes using RNA interference systems, and further Northern blot analysis, we analyzed the protein expres- defined biological functions of the proteins. Through these sion of candidate targets among hundreds of clinical sam- analyses, we identified candidate genes for the develop- ples on tissue microarrays, investigated loss of function ment of novel serum biomarkers (30–35), therapeutic

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Table 1. Association between CSTF2 positivity in NSCLC tissues and patients’ characteristics

Total CSTF2 strong CSTF2 weak CSTF2 absent P (strong vs. (N ¼ 327) positive (n ¼ 77) positive (n ¼ 165) (n ¼ 85) weak/absent)

Gender Male 228 55 120 53 0.7775 Female 99 22 45 32 Age, y <65 150 33 77 40 0.6016 65 177 44 88 45 Histology ADC 196 45 97 54 0.7910 Non-ADCa 131 32 68 31 Smoking Smoker 234 51 124 59 0.2497 Nonsmoker 93 26 41 26 pT factor T1 135 26 68 41 0.1458 T2 þ T3 192 51 97 44 pN factor N0 208 45 107 56 0.2824 N1 þ N2 119 32 58 29

Abbreviation: ADC, adenocarcinoma. aThe category "non-ADC" comprises squamous cell carcinoma plus large-cell carcinoma and adenocarcinoma.

drugs (11–29), and/or immunotherapy (35–41), which cleavage stimulation factors was frequently overexpressed were upregulated in cancer cells but not expressed in in the great majority of clinical lung cancer samples and normal organs, except testis, placenta, and/or fetus tissues. cell lines, and that its gene product plays an indispensable In this study, we report that CSTF2 encoding a member of role in the growth and invasion of lung cancer cells.

Table 2. Cox proportional hazards model analysis of prognostic factors in patients with NSCLCs

HR 95% CI Unfavorable/favorable P

Univariate analysis CSTF2 1.617 1.130–2.314 Strong(þ)/weak(þ)or(–) 0.0085a Gender 1.512 1.037–2.206 Male/female 0.0317a Age, y 1.672 1.188–2.353 65/<65 0.0032a Histology 1.434 1.033–1.989 Non-ADC/ADC 0.0311a Smoking 1.252 0.858–1.826 Smoker/nonsmoker 0.2437 pT factor 2.176 1.505–3.146 T2 þ T3/T1 <0.0001a pN factor 1.965 1.416–2.728 N1 þ N2/N0 <0.0001a Multivariate analysis CSTF2 1.545 1.078–2.214 Strong(þ)/weak(þ)or(–) 0.0177a Gender 1.293 0.850–1.969 Male/female 0.2301 Age, y 1.858 1.306–2.643 65/<65 0.0006a Histology 1.009 0.696–1.464 Non-ADC/ADC 0.9607 pT factor 1.695 1.144–2.510 T2 þ T3/T1 0.0085a pN factor 1.990 1.413–2.802 N1 þ N2/N0 <0.0001a

NOTE: The category "non-ADC" comprises squamous cell carcinoma plus large-cell carcinoma and adenocarcinoma. Abbreviations: ADC, adenocarcinoma; CI, confidence interval. aP < 0.05.

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A A549 LC319 Anti-CSTF2 Control Anti-CSTF2 Control siRNA siRNA siRNA siRNA

si -#1 si -#2 si-EGFP si-LUC si -#1 si -#2 si-EGFP si-LUC

CSTF2 CSTF2

ACTB ACTB

B P < 0.0001 P < 0.0001 1.6 2.5 P < 0.0001 P < 0.0001

2.0 1.2

1.5 0.8

1.0 (490 nm/630 nm) (490 nm/630 nm) Relative absorbance Relative absorbance 0.4 0.5

0 0 si -#1 si -#2 si-EGFP si-LUC si -#1 si -#2 si-EGFP si-LUC C D 72 h AllAll phasephases SubG1Sub-G1 phasephase

-is GE PF is C- TS F2 100% 35% P = 0.0028

30% 150 150 80% G2−M 25% S 60% 20% 100 100 G 15% 1

A549 40% Counts Sub-G 50 50 10% 1 20% 5% 0 0 0 200 400 600 800 0 200 400 600 800 0% 0% si-EGFP si-CSTF2 si-EGFP si-CSTF2 A-2LF A-2LF

100% 25% P = 0.0044

80% 150 150 20% G2−M

60% 15% S 100 100 G 40% 10% 1 LC319 Counts

50 50 Sub-G1 20% 5%

0 0 0% 0 200 400 600 800 0 200 400 600 800 0% si-EGFP si-CSTF2 si-EGFP si-CSTF2 FL2-A FL2-A

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-#1 or 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 the tumor cells transfected with si-CSTF2s or control siRNAs. C, flow cytometric analysis of the A549 cells and LC319 cells 72 hours after transfection of the siRNAs for CSTF2 (si-CSTF2-#1) and control siRNAs (si-EGFP). D, left, percentage of cells at each phase; right, averaged percentage of cells at sub-G1 phase of triplicate assays. www.aacrjournals.org Clin Cancer Res; 17(18) September 15, 2011 5897

Aragaki et al.

A COS-7 HEK293T

(kDa) CSTF2 Mock (kDa) CSTF2 Mock 75- 75-

IB: anti-Myc IB: anti-Myc (CSTF2) (CSTF2) 50- 50-

37- 37-

IB: anti-ACTB IB: anti-ACTB Figure 4. Enhancement of cellular growth by CSTF2 introduction into mammalian cells. A, transient B expression of CSTF2 in COS-7 and HEK293T cells, detected by 1.6 P 1.2 P < 0.0001 < 0.0001 Western blot analysis. The cells were introduced pcDNA3.1-myc- His-CSTF2 or mock vector. B, cell 1.2 0.9 viability evaluated by the MTT assays. Assays were done 3 times and in triplicate wells.

0.8 0.6 (490 nm/630 nm) (490 nm/630 nm) Relative absorbance 0.4 Relative absorbance 0.3

0 0 CSTF2 Mock CSTF2 Mock

CSTF2 encodes a 557–amino acid protein with an To further elucidate the downstream pathways of N-terminal RNP-type RNA-binding domain, a long Pro- CSTF2 in lung cancer cells, we transfected siRNAs against and Gly-rich region, and a pentapeptide repeat region CSTF2 (si-CSTF2 #1) or EGFP (control siRNA) into lung that forms an extended a-helix and comprises one of a cancer A549 cells that overexpressed CSTF2, and screened multisubunit complex of CstF required for polyadenyla- by microarray analysis as described previously (16) genes tion and 30-end cleavage of mammalian pre-mRNAs (46). that were downregulated in accordance with the knock- CstF cooperates with cleavage-polyadenylation specificity down of CSTF2 expression at 24 and 48 hours after siRNA factor to define the site of polyadenylation by recognizing transfection. The self-organizing map clustering analysis the highly conserved AAUAAA hexanucleotide and more for selecting genes showed the same expression pattern to divergent GU-rich situated downstream of the actual CSTF2 in a time-dependent manner and subsequently the cleavage site (47). The polyadenylatation and 30-end gene ontology database analysis identified 4 candidate cleavage of pre-mRNAs are important modifications for downstream pathways of CSTF2 including genes related proper transcription, splicing, transport, translation, and to a member of interleukin, TGF-b, chemokine, and stability of mRNAs. This factor for polyadenylation is heterotrimeric G protein, some of which were reported reported to change the length of the 30-UTR of mRNA to have important roles in carcinogenesis (Supplementary and thus the binding sites of microRNAs (miRNA), which Tables S3 and S4). Although further detailed analyses tend to bind to these regions (48). Loss of miRNA are necessary to determine the direct targets of CSTF2, the complementary sites in 30-UTR of some oncogenes is information implies the biological importance of CSTF2 one of the mechanisms of oncogene activation. In cancer in human carcinogenesis. cells, 30-UTR of some oncogenes is likely to be shortened In this study, we showed that CSTF2 gene was over- by alternative cleavage and polyadenylation, resulting expressedinmostlungcancersandlikelytoplayan in avoidance of silencing by miRNA and activation of important role in the growth of lung cancers. Moreover, oncogenes in cancer cells (49). It is still unknown why clinicopathologic data through our tissue microarray CSTF2 was overexpressed in testis and cancer cells; experiments showed that patients with NSCLC, in which however, CSTF2 may play important roles for activation CSTF2 was highly expressed, had shorter survival periods of some oncogenes by changing the length of 30-UTR of than those with CSTF2 weak positive/negative tumors. their mRNAs in cancer cells. This is the first study that proves a potential of tissue

5898 Clin Cancer Res; 17(18) September 15, 2011 Clinical Cancer Research

CSTF2 Activation in Lung Cancer

CSTF2 expression as a prognostic biomarker for human In summary, CSTF2 might play an important role in the cancers. growth and progression of lung cancers. CSTF2 overexpres- Somatic epidermal growth factor receptor (EGFR) sion in resected specimens may be a useful indicator of mutation was reported to be biologically important for adjuvant therapy to lung cancer patients who are likely to the clinical response of NSCLCs to EGFR tyrosine kinase have poor prognosis. inhibitors such as gefitinib or erlotinib (5, 50). To examine the relevance of CSTF2 expression to this subgroup of Disclosure of Potential Conflicts of Interest NSCLCs with EGFR mutation, we examined the correlation between CSTF2 overexpression and EGFR mutation status No potential conflicts of interest were disclosed. in NSCLC tissues using specific antibodies to E746-A750 deletion and those to a point mutation (L858R) in EGFR, Grant Support and found that EGFR mutation was not associated with P ¼ This work was supported in part by Grant-in-Aid for Scientific Research (B) CSTF2 strong expression ( 0.2555, Fisher’s exact test; and Grant-in-Aid for Scientific Research on Innovative Areas from The Japan Supplementary Table S5; Supplementary Fig. S3). The Society for the Promotion of Science. Y. Daigo is a member of Shiga Cancer combined results suggest that further functional analysis Treatment Project supported by Shiga Prefecture (Japan). The costs of publication of this article were defrayed in part by the and screening to selectively inhibit CSTF2 function by payment of page charges. This article must therefore be hereby marked small molecule compounds and/or nucleic acid drugs advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate could be a potential therapeutic strategy that is expected this fact. to have a powerful biological activity against broad clini- Received January 26, 2011; revised June 22, 2011; accepted July 23, 2011; copathologic types of lung cancers. published OnlineFirst August 3, 2011.

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5900 Clin Cancer Res; 17(18) September 15, 2011 Clinical Cancer Research

Characterization of a Cleavage Stimulation Factor, 3′ pre-RNA, Subunit 2, 64 kDa (CSTF2) as a Therapeutic Target for Lung Cancer

Masato Aragaki, Koji Takahashi, Hirohiko Akiyama, et al.

Clin Cancer Res 2011;17:5889-5900. Published OnlineFirst August 3, 2011.

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