CaSm-Mediated Cellular Transformation Is Associated with ...cancerres.aacrjournals.org/content/canres/65/14/6228.full.pdf · CaSm-Mediated Cellular Transformation Is Associated with

CaSm-Mediated Cellular Transformation Is Associated

with Altered Gene Expression and

Messenger RNA Stability

Melissa M. Fraser,1Patricia M. Watson,

3Mostafa M. Fraig,

1Joseph R. Kelley,

4

Peter S. Nelson,5Alice M. Boylan,

3David J. Cole,

4and Dennis K. Watson

1,2

Departments of 1Pathology and Laboratory Medicine, 2Biochemistry and Molecular Biology, 3Medicine,and 4Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolinaand 5Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington

Abstract

CaSm (cancer-associated Sm-like) was originally identifiedbased on elevated expression in pancreatic cancer and inseveral cancer-derived cell lines. CaSm encodes a 133 aminoacid protein that contains two Sm motifs found in thecommon small nuclear RNA proteins and the LSm (like-Sm)family of proteins. Compared with normal human prostatetissue and primary prostate epithelial cells, some primaryprostate tumors and prostate cancer–derived cell lines haveelevated CaSm expression. Expression of antisense CaSm RNAin DU145 cells results in reduced CaSm protein levels and lesstransformed phenotype, measured by anchorage-independentgrowth in vitro and tumor formation in severe combinedimmunodeficient mice in vivo. Additional data shows thatadenoviral delivery of antisense CaSm inhibits the growth ofprostate cancer cell lines by altering cell cycle progression,and is associated with reduced expression of cyclin B1 andCDK1 proteins. Consistent with failure of antisense-treatedcells to enter mitosis, microarray analysis identified alteredexpression of NEK2 and nucleophosmin/B23. Although themechanisms by which CaSm contributes to neoplastictransformation and cellular proliferation are unknown, ithas been shown that the yeast homologue (spb8/LSm1) ofCaSm is required for 5Vto 3Vdegradation of specific mRNAs.We provide data consistent with a similar role for CaSm inhuman cells, supporting the hypothesis that elevated CaSmexpression observed in cancer leads to destabilization ofmultiple gene transcripts, contributing to the mutatorphenotype of cancer cells. (Cancer Res 2005; 65(14): 6228-36)

Introduction

Prostate cancer is the most prevalent cancer in American malesand the second leading cause of their cancer death (1). Thedevelopment of many tumor types progresses from normal !dysplasia ! carcinoma in situ ! localized primary tumor !tumor with metastases. However, it is believed that the develop-ment of prostate tumors is a more stochastic process. Theprogression of prostate cancer varies considerably among patients.Indolent prostate cancers may remain localized for decades,whereas aggressive prostate cancers can metastasize rapidly to

lymph nodes and/or by hematogenous routes. Human prostatecancer is heterogeneous in appearance and genetically unstable.Multiple genotypes from the same primary prostate tumor inphenotypically similar foci are frequently identified (2). A betterunderstanding of the molecular controls that regulate prostatecancer growth and transformation is needed in order to developmore effective therapeutic approaches for aggressive prostatecancer. Furthermore, such understanding may allow insight intonew markers for patient stratification, allowing those patients mostlikely to progress to clinical prostate cancer to be treatedaggressively. Based on these observations, genetic and epigeneticstudies of prostate cancer have been directed towards elucidationof possible mechanisms that could account for the rapidprogression observed in some patients. We have hypothesized thatalteration in transcriptional control and repair offer two mecha-nisms leading to rapid changes in the transcriptome of the prostatecancer cell. Specifically, we and others have shown that the ETS2transcription factor is expressed at elevated levels in prostatecancer and is necessary for some of the transformed phenotypes ofprostate cancer cells (3–5). In addition, we have shown that thereare defects of mismatch repair genes in prostate cancer (6, 7), likelyto contribute to genomic instability observed in prostate cancer(8, 9). In addition to transcriptional changes, alterations in mRNAstability would be expected to contribute to the mutator phenotype(10) in prostate cancer.To characterize molecular alterations that lead to cancer, we

previously employed subtractive hybridization cloning in order toidentify and isolate genes whose expression pattern is associatedwith pancreatic cancer (11, 12). This approach led to the isolationand initial characterization of CaSm ( for cancer-associated Sm-like) that was identified due to its elevated expression in pancreaticcancer. CaSm is overexpressed in a majority of pancreatic tumorsand cancer-derived cell lines (12) as well as metastatic tumors (13).Furthermore, CaSm expression is required for the maintenance oftransformed phenotype of pancreatic cancer cell lines (12).Elevated CaSm expression is not limited to pancreatic cancer,being overexpressed in lung, esophageal and bladder tumors, andpleural mesothelioma.6 Increased levels of CaSm mRNA are alsoobserved in prostate, liver, lung, ovarian, rectal, bladder, and kidneycancer–derived cell lines. The current study provides evidence thatCaSm is expressed at elevated levels in prostate cancer and usesantisense and small interfering RNA (siRNA) inhibition of CaSmexpression to show that CaSm affects prostate cancer cell

Note: M.M. Fraser and P.M. Watson contributed equally to this work.Requests for reprints: Dennis K. Watson, Department of Pathology and

Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue,Charleston, SC 29403. Phone: 843-792-3962; Fax: 843-792-3940; E-mail: [email protected].

I2005 American Association for Cancer Research. 6 Unpublished observations.

Cancer Res 2005; 65: (14). July 15, 2005 6228 www.aacrjournals.org

Research Article

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proliferation and tumor development. For the first time, we provideevidence that CaSm may exert its effect by altering messagestability. These collective results support the importance of CaSmin the development and maintenance of cellular transformation.

Materials and Methods

Cell lines and tissue culture. DU145 and PC3 cell lines were purchased

from the American Type Culture Collection (Manassas, VA), and weregrown in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10%

fetal bovine serum (Life Technologies). The 267B-1 cell line (SV40 T-antigen

immortalized neonatal prostate epithelium) was generously provided by

Dr. J. Rhim (Bethesda Naval Hospital, Bethesda, MD) and cultured in P4-8Fmedium with 2% serum (Biological Research Faculty and Facility, Inc.,

Ijamsville, MD). Normal prostate epithelial cells were purchased and

cultured in PrEGM medium (Clonetics, Rockland, ME) according to the

manufacturer’s instructions. All cell lines were propagated at 37jC in anatmosphere containing 5% CO2.

Transfection of DU145 cells was done in six-well plates using 1 Ag of DNA, 6AL LipofectAMINE PLUS reagent or 4 AL of LipofectAMINE reagent (LifeTechnologies). Stable cell lines were selected in 400 Ag/mL of G418

(Calbiochem, La Jolla, CA). Cell lines were screened by Northern blot analysis

and clones expressing antisense CaSm were selected for anchorage-

dependent and -independent studies. Anchorage-dependent growth studieswere done using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT; Sigma, St. Louis, MO) assays. Parental and antisense expressing

clone cells were plated at a density of 10,000 cells per well in 96-well plates.

They were cultured for a period of 7 days and analyzed on a spectropho-tometer on days 1, 3, 5, and 7. Anchorage-independent growth was assessed

by soft agar colony formation. Parental and clone cells were plated at a

density of 20,000 cells per well in six-well plates. Cells were fed and grown for a

period of 4 weeks and colonies were counted. The bottom layer of agar was0.6% agar, whereas the top layer and feeding layers were comprised of 0.4%

agar. Both the MTT and soft agar assays were done in duplicate.

NIH-3T3 cells were kindly provided by Dr. Donald Blair (National CancerInstitute, Frederick, MD) and maintained in DMEM containing 8% calf

serum at 37jC in an atmosphere containing 10% CO2. Cells were transfected

with 1 Ag of DNA using LipofectAMINE PLUS reagent in six-well plates at

40% to 50% confluence. Two days after transfection, cells were replated in100 mm dishes, at a ratio of 1:2, and 0.25 Amol/L dexamethasone was added

to the medium. Foci were counted on day 12 at 40� magnification on an

inverted light microscope.

RNA isolation and analysis. RNA was purified from cultured cells ortissues using RNAstat (TEL-TEST) or Trizol (Invitrogen, Carlsbad, CA)

according to themanufacturer’s protocol. Northern blot analysis was done on

total RNA. The RNA was fractionated on a 1.2% agarose gel with 0.66 mol/L

formaldehyde. Gels were transferred overnight to Duralon membrane(Stratagene, La Jolla, CA) in 0.1 mol/L sodium phosphate (pH 6.8). The

Duralon membrane was UV cross-linked and hybridized with 32P-labeled

probe (Stratagene prime it II kit) in QuikHyb solution (Stratagene) according

to the manufacturer’s protocols. Quality of RNA and equivalent loading wasverified by ethidium bromide staining of 18S and 28S RNA.

Probes. An 894 bp CaSm-specific insert was prepared by EcoRI digestion

of pSGneoSK/CaSm (12). A p21/WAF1-specific insert was isolated by

HindIII-AspI digestion of pC-WAF1-S (provided by Dr. Bert Vogelstein,

Department of Oncology, Johns Hopkins University, Baltimore, MD). A B23

insert was obtained by EcoRI digestion of a nucleophosmin/B23 fragment

generated by RT-PCR (described below) and cloned into the pCRII-TA

vector. Strand-specific probes were created by linear PCR using single

primers (Table 1).RT-PCR. RNA from prostate cell lines and normal tissue was used as a

template for RT-PCR. cDNA was obtained from total RNA (5 Ag) followingthe manufacturer’s protocol (Superscript II, Invitrogen). cDNA was thenused for PCR amplification using gene-specific primers (Table 1).

Expression vectors and cloning. Antisense CaSm in pSGneoSK was

previously described (12). CaSm was amplified by PCR using sequences to

provide restriction enzyme sites for directional cloning in FpcDNA3.

FpcDNA3 is a modified pcDNA3 vector that allows incorporation of an

amino-terminal FLAG epitope (provided by Dr. Craig Hauser, The Burnham

Institute, La Jolla, CA). Orientation and sequence was verified by analysis on

ABI 373 automated sequencer (Applied Biosystems, Foster City, CA). Dr. D.

Blair provided pM1, which contains the v-mos oncogene inserted into the

EcoRI site of the pBR322 vector.

Small interfering RNA. CaSm siRNA sequences were identified using

the criteria of Paddison and Hannon (14). Two sequences were identified

that were unique to the CaSm gene and conformed to the consensus of 5V-AA19N-3V. These sequences were CaSm1-104-123, aacugguuuuggacgcgcu;

and CaSm2-328-347, aauacgggigauauuccucga. RNA in the sense and

antisense direction was synthesized and annealed to form double-stranded

RNA by Dharmacon Research, Inc. (Lafayette, CO). Transfection into

prostate cancer cell lines was done using OligofectAMINE (Invitrogen) with

dsRNA at a final concentration of 20 nmol/L. The transfection was repeated

on days 3 and 5, and cellular protein and total RNA extracted on day 7.

Western blot analysis. Cells were lysed in radioimmunoprecipitation

assay buffer [150 mmol/L NaCl, 50 mmol/L Tris (pH 7.4), 1% Triton X-100,

0.1% SDS, 1% sodium deoxycholate, and 8% protease inhibitors]. Proteinswere resolved by SDS-PAGE, transferred to a nitrocellulose membrane

(Schleicher & Schuell, Keene, NH) and incubated for 1 hour with one of the

following antibodies: h-actin (Oncogene Research Products, Boston, MA),

cyclin B (#14541A, BD Biosciences/PharMingen, San Jose, CA), CDK1(14391A, PharMingen), p21 (OP64-100UG, Oncogene Research Products),

and Nek2 (SC-7440, Santa Cruz, Santa Cruz, CA). After washing, the

Table 1. Primers used for PCR experiments

Region Application Primer sequences Ampliconsize (bp)

Cyclenumber

Annealingtemperature (jC)

B23/nucleophosmin RT-PCR cloning,Northern

5V-TGGAGGTGGTAGCAAGGTTC-3V 397 40 55

5V-CCGGAAGCAATTCTTCACAT-3VCaSm RT-PCR 5V-CTTCAAGAGCCAACAGCCTCTGG-3V 499 35, 40 60

5V-GGAGGGAACTGGTTTTGGACGC-3VS26 cDNA RT-PCR 5V-CTCCGGTCCGTGCCTCCAAG-3V 408 30, 35 62

5V-CAGAGAATAGCCTGTCTTCAGTC-3VNek2 RT-PCR 5V-TGACCAAATTGCATCTAACTGG-3V 396 40 55

5V-CATGAGCCATGCCTTTCTGT-3VCaSm sense probe Northern 5V-GAGGGATCCATGAACTATATGCCTGGCACC-3V 11 55

CaSm antisense

probe

Northern 5V-GAGGACTCGAGGGGCAAAAGATTAGTACTC-3V 11 55

CaSm Expression Is Required for Cellular Transformation

www.aacrjournals.org 6229 Cancer Res 2005; 65: (14). July 15, 2005



membrane was incubated in horseradish peroxidase–labeled secondaryantibody [HRP-goat-F(abV)2 anti-mouse-IgG (#M35007, Caltag, Burlingame,

CA) or HRP goat-F(abV)2 anti-rabbit IgG (#L43007, Caltag)] for 20 minutes.

The membrane was developed in enhanced chemiluminescence (#34080,

Pierce, Rockford, IL) for 5 minutes and exposed to Kodak MR film.The chicken anti-CaSm antibody was made using full-length bacterial

expressed protein injected into laying hens (Aves Laboratory, Portland, OR).

IgY was purified from eggs and affinity-purified with full-length protein

covalently linked to agarose (Pierce). Rabbit anti-CaSm antibody was initiallyprovided by the Luhrmann laboratory (Max-Planck Institute, Germany)

and subsequently generated using a synthetic peptide corresponding to the

20 COOH-terminal amino acids of CaSm covalently coupled to bovine serum

albumin (Rockland, Gilbertsville, PA). The antibody was affinity-purifiedas above. The specificity of this antibody was shown by the absence of

immunoreactive proteins in Western blots of extracts prepared from cell

lines that do not express CaSm mRNA. For Western blot, proteins wereseparated on a 12.5% acrylamide gel and transferred to nitrocellulose.

After blocking, the membrane was incubated overnight in 2 Ag/mL affinity-

purified anti-CaSm IgY and for 1 hour in HRP-goat anti-IgY.

Analysis of adenovirus-infected cells. DU145 and PC3 cells wereuntreated or infected with adenoviral LacZ (Ad-LacZ) or adenoviral

antisense CaSm (Ad-aCaSm; refs. 15, 16) at a multiplicity of infection

(MOI) of 100 in serum-free media for 90 minutes. The virus was then

removed and medium containing 10% serum was added. Under theseconditions, 40% and 45% of DU145 and PC3 cells were infected, respectively,

as indicated by LacZ expression. Anchorage-dependent growth was

measured by MTT assays (described above). For cell cycle analyses, infectedcells were harvested at 24, 48, or 72 hours, fixed, and dehydrated in 70%

ethanol at �20jC for at least 24 hours. Cells were stained with a solution

containing 50 Ag/mL propidium iodide (Sigma-Aldrich) and 20 Ag/mL

RNaseA (Sigma-Aldrich) in PBS and analyzed using flow cytometry.Animal studies. Animal handling and procedures were approved by the

Medical University of South Carolina Animal Studies Committee. The hind

legs of severe combined immunodeficiency (SCID)-Bg mice were shaved

and sterilized with ethanol prior to the injection of 1 � 106 DU145 cells. Thelength and width of the tumors were measured. Final tumor volume was

calculated by the following formula: V = (L � W2) � p/6.mRNA isolation and microarray analysis. Total RNA was isolated

using Trizol (Invitrogen) and 1 mg of total RNA was used to isolate mRNA

by Oligotex suspension (#70061, Qiagen, Valencia, CA) following the

manufacturers instructions. To identify genes that are differentially

expressed between two samples, cDNAs were labeled with Cy3 or Cy5

and hybridized simultaneously to an identical array. The GEM V array

(Genome Systems, Inc., St. Louis, MO) contained f9,600 expressed

sequence tags and cDNAs. The signals from each of 9,600 spots were

scanned and scored for the normalized ratio of Cy3 to Cy5. Hybridization

and analysis of the GEM V array was done by Genome Systems. The second

array comprises a nonredundant set of >6,000 prostate-derived cDNAs,

‘‘spotted’’ onto polylysine-coated microscope slides using an OmniGrid

high-precision robotic gridder (GeneMachines, Genomic Solutions, Ann

Arbor, MI; ref. 17). A fluorescent image of the microarray was collected at

both emission wavelengths using a GenePix 4000 fluorescent scanner (Axon

Instruments, Inc., Sunnyvale CA) and image analysis was done using

GenePix Pro Microarray Acquisition and Analysis Software. For each

experiment, each cDNA was represented twice on each slide, and the

experiments done in duplicate producing four data points per cDNA clone

per hybridization probe. Intensity ratios for each cDNA clone hybridized

with probes derived from the respective cell lines were calculated as log2

(experimental intensity / control intensity). Gene expression levels were

considered significantly different between the two conditions if at least

three of the four replicate spots for a given cDNA showed an average log2

ratio of >0.66 or <�0.66, and the signal intensity was >2 SD above the image

background.

Immunohistochemistry of human prostate tumor samples. Human

prostate cancer samples were obtained from the Hollings Cancer CenterTumor Bank, Medical University of South Carolina. All the patients in this

study had clinically localized, pathologically documented prostate cancer,

and were treated at the Department of Urology, Medical University ofSouth Carolina from 1999 to 2000. The average age of these patients was

68.9 years (range 48-78). All samples had a Gleason grade of 6 or 7. A total

of 10 patients were scored for CaSm expression; all specimens were

formalin-fixed and paraffin-embedded. Deparaffinized tissue sections wererehydrated, endogenous peroxidase activity was blocked using 3% H2O2 in

methanol, and antibody binding sites blocked with 1.5% normal goat

serum. For antigen retrieval, slides were microwaved in citrate buffer

antigen retrieval solution (Vector Laboratories, Burlingame, CA) thricefor 5 minutes. Immunohistochemical staining was done using Vector ABC

Elite kit (Vector Laboratories). Affinity-purified rabbit anti-CaSm anti-

bodies were used at 4 Ag/mL. Sections were stained using 3,3V-diaminobenzidine and counterstained with hematoxylin. All sections wereexamined independently by a pathologist (M. Fraig). The presence of

staining was compared with that of normal prostate tissue present in the

same section. No staining was observed with negative control samples(absence of primary antibody or incubation with rabbit IgG).

Results

CaSm expression is elevated in prostate cancer–derived celllines. To determine CaSm gene expression in prostate-derived celllines, Northern blot analysis was done using a full-length CaSmprobe. Expression was high in all prostate cancer–derived cell linestested (LNCaP, DU145, and PC3; Fig. 1A). In contrast, CaSmexpression is reduced in the immortalized fetal prostate epithelialcell line, 267B1.RT-PCR analysis (Fig. 1B) was used to evaluate the expression of

CaSm in prostate cancer cell lines compared with normal prostatetissue. High-level CaSm mRNA expression was detected in prostate

Figure 1. CaSm expression in prostate and prostate-derived cell lines.A, Northern blot was done with total RNA isolated from prostate-derived cells.The RNA was resolved on a 1.2% agarose gel, transferred to a nitrocellulosemembrane and hybridized using a 32P-labeled CaSm-specific probe. B, RNAfrom several prostate cancer cell lines and normal tissue was used as a templatefor RT-PCR. The cDNA was amplified using primers against CaSm (top ) or S26rRNA (bottom ). Amplification of S26 was done to assure the quality of RNA. S26reactions were amplified for 30 or 35 cycles, whereas CaSm reactions wereamplified for 35 and 40 cycles. C, Western blot of normal prostate tissue (NP)and prostate-derived cell lines. Fifteen micrograms of protein extract wereresolved on a 12.5% acrylamide gel, transferred to nitrocellulose and probed withanti-CaSm IgY, followed by HRP-goat anti-chicken IgY and Pierce West Picochemiluminescent substrate.

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cancer cell lines; in contrast, relatively low expression levels wereseen in normal prostate. Consistent with mRNA expression,elevated CaSm protein expression was also found in all prostatecell lines tested. Normal prostate epithelial cells express very lowlevels of CaSm compared with the cancer cell lines (Fig. 1C).CaSm expression is necessary and sufficient for cellular

transformation. To determine whether CaSm expression isnecessary for the maintenance of the transformed phenotype,DU145 cells were transfected with a pSGneoSK plasmid vectorexpressing antisense CaSm. The effect of antisense expression onthe ability of stable cell clones to grow independent ofanchorage as colonies suspended in soft agar and form tumorsin SCID mice was evaluated. The antisense-expressing cell clonesformed significantly fewer and smaller colonies than the parentalcell line, demonstrating that CaSm expression is necessary foranchorage-independent growth (Fig. 2A). The parental DU145cells formed >350 colonies, of which 65 were >280 Am indiameter. In contrast, the three antisense clones formed between15 and 44 colonies each. Protein extracts were prepared fromDU145 cells and these antisense clones and CaSm level assessedby Western blot. The antisense clones were found to consistentlyexpress less CaSm protein than parental DU145 cells (Fig. 2A,inset). We next examined the ability of DU145 clones expressingantisense CaSm to form tumors. The DU145 tumors grew to asize of 100 mm3 in an average of 31.6 days, whereas theantisense DU145 clone never grew to this size over the 60-dayobservation period. The Kaplan-Meyer survival curves gave a logrank probability of 0.0018 using survival to a tumor volume of100 mm3 as the y axis (Fig. 2B).Reduction of CaSm expression in human prostate cancer–derived

cell lines inhibits their anchorage-independent growth and tumorformation, both hallmarks of transformation. To evaluate whetherCaSm can function as an oncogene, NIH-3T3 cells were transfectedwith a vector containing the complete open reading frame of humanCaSm and analyzed for in vitro foci formation. CaSm-transfectedNIH-3T3 cells formed 80 foci of transformed cells (Fig. 2C) comparedwith 9 foci for untransfected NIH-3T3 cells. As a control fortransformation, NIH-3T3 cells transfected with the v-mos oncogeneformed 115 foci. Collectively, these studies show that CaSmexpression is necessary and sufficient for cellular transformation.Adenoviral expression of antisense CaSm inhibits cell

proliferation. We have shown that multiple stable antisenseclones fail to grow independent of anchorage. Adenoviral deliveryof antisense CaSm allows examination of altered phenotypes ofpools of infected cells rather than selected individual clones. Weused a previously characterized adenoviral vector expressingantisense CaSm RNA (Ad-anti-CaSm; refs. 15, 16) to study theeffect of reduced CaSm on the growth of prostate cancer. Northernblot analysis with a CaSm-specific sense ssDNA probe showed thatAd-anti-CaSm–infected cells expressed the appropriate antisenseconstruct. This probe revealed a signal at 900 bp corresponding toexpression of the CaSm antisense in prostate cancer lines treatedwith the virus (Fig. 3A, middle). To assess whether Ad-anti-CaSmexpression affected the level of endogenous CaSm message, aCaSm-specific antisense ssDNA probe was used that selectivelydetected CaSm mRNA. CaSm mRNA level was significantlydecreased in DU145 cells 48 hours after infection with Ad-anti-CaSm. In contrast, no change was observed in the uninfected andLacZ control infected cells (Fig. 3A, top).To determine the effect of antisense CaSm on the growth

of prostate cancer cells, DU145 and PC3 cells were infected with

Ad-LacZ (MOI 100) or Ad-anti-CaSm (MOI 100) and assayedfor anchorage-dependent cell proliferation by MTT assay. DU145Ad-anti-CaSm–infected cells showed reduced cell numbers com-pared with untreated or Ad-LacZ–treated cells (Fig. 3B). There wasno change in growth characteristics in the Ad-anti-CaSm–infectedPC3 cells (data not shown).To confirm the findings from the antisense CaSm experiments,

we did complementary loss of function studies using siRNA(14, 18). Two siRNAs were designed to use in gene silencingexperiments. Unique sequences were selected that are nothomologous to other sequences in the human genome. Transfec-tion of prostate cancer cells with either of these siRNAssignificantly reduced expression of CaSm protein, whereas controlsequences did not (Fig. 3C). In addition, cells treated with these

Figure 2. CaSm expression is associated with cellular transformation.A, parental cells and antisense-expressing clones were grown in soft agar for4 weeks. The number of colonies greater than 140 Am was determined. Numberof colonies for each transfected clone was compared with the parental cellsusing a two-sample t test. As three clones were analyzed, a Bonferroniadjustment was done, indicating a significant P value as <0.017. The P value foreach antisense clone compared with parental cells was <0.001. A, Western blotof total extracts prepared from DU145 parental cells and stable antisenseclones (19, 21, and 22) showing reduced protein expression of CaSm inantisense clones (inset ). B, CaSm is necessary for tumor formation in vivo .Kaplan-Meier curves for DU145 cells and DU145 cells stably transfected withCaSm antisense. Cells were injected into SCID-Bg mice at day 0 and tumorgrowth was monitored weekly. The DU145 cells were injected into the right flankand the antisense cells were injected into the left flank. The data were plotted inKaplan-Meier curves and analyzed using the log rank test and found to besignificantly different (P = 0.0018). C, CaSm transfection of NIH-3T3 cells leadsto loss of contact inhibition with foci formation of transformed cells. NIH-3T3 cellswere transfected with a plasmid containing the full-length CaSm or the v-mosoncogene and scored for foci formation. The v-mos oncogene serves as apositive control for transformation. Number of foci for 3T3 parental cells andv-mos and CaSm transfected were compared using a two-sample t test. The Pvalue for the v-mos oncogene was P = 3 � 10�6 and P = 3 � 10�5 for CaSm.





siRNAs have reduced proliferation, as observed after infection withAd-antisense CaSm (Fig. 3D).Ad-anti-CaSm treatment in vitro leads to a cytostatic

inhibition of the cell cycle. Based on the observed differences ingrowth capabilities observed in anti-CaSm–infected cells, cellpopulations were further analyzed by flow cytometry. DU145 andPC3 prostate cancer–derived cell lines were infected at an MOI of100with Ad-anti-CaSm or Ad-LacZ and examined by flow cytometry.Treatment with the CaSm antisense virus alters the proportion ofcells in each phase of the cell cycle, with a significant decrease inG0/G1 populations and an increase in the number of cells in the G2-Mphase. Specifically, 41% of DU145 cells treated for 48 hours withAd-anti-CaSm were arrested in the G2-M phase (30% in G1),compared with only 18% (62% G1) of control Ad-LacZ–infectedcells. PC3 cells infected with Ad-anti-CaSm virus do not exhibitsignificant alterations in the cell cycle, consistentwith theMTT data.Ad-anti-CaSm–infected cells display a phenotype similar to

endoreduplication. DU145 cells treated with Ad-anti-CaSmexhibit a large percentage of cells with >4 N DNA content,compared with the untreated or Ad-LacZ–treated cells. Forty-eighthours after infection, 26% of Ad-anti-CaSm–treated DU145 cellshad a >4 N DNA content, compared with 1% to 2% for untreated orAd-lacZ–treated cells (Fig. 4A). A >4 N population is often referredto as ‘‘aggregate cells,’’ representing two or more cells stucktogether. However, 4V,6-diamidino-2-phenylindole stained Ad-anti-CaSm–treated cells shows that these cells have enlarged nuclei(Fig. 4B). This data suggests that treatment of DU145 with Ad-anti-CaSm results in DNA synthesis in the absence of mitosis, a

phenotype often associated with endoreduplication or endorepli-cation (19). It should be noted that despite the significantdecreases in cell growth and the dramatic changes in the cellcycle, a substantial sub-G0 peak was not detected, suggesting thatapoptosis is not occurring as a result of Ad-anti-CaSm treatment(Fig. 4A).After infection with anti-CaSm–expressing virus, DU145 cells

also exhibit a marked decrease in the number of cells undergoingmitosis (Fig. 4C). In every field of view at 400� magnification, twoto four cells can be seen undergoing mitosis in both untreated andAd-LacZ–treated cells. In contrast, Ad-anti-CaSm–treated cellsrarely show any cells undergoing mitosis. Overall, we observe a 10-fold reduction of mitotic cells in Ad-anti-CaSm–treated DU145 cellsrelative to untreated or Ad-lacZ–treated cells.Altered expression of G2-M regulatory genes as a conse-

quence of antisense CaSm expression. Because cyclin B andCDK1 are essential for the G2-M transition, we examined whethertheir expression was altered following Ad-anti-CaSm infection.Cyclin B1 and CDK1 protein are reduced after infection with Ad-anti-CaSm (Fig. 4D), whereas these are not affected by Ad-LacZ

Figure 3. Loss of CaSm mRNA effects cell proliferation. A, ssDNA probeanalysis confirms that Ad-antisense-CaSm is able to effectively reduceendogenous CaSm expression in human DU145 prostate cancer cells (top ) andthat antisense CaSm RNA is expressed in the infected cells. B, DU145 cellswere maintained in RPMI or treated with Ad-LacZ or Ad-antisense-CaSm at anMOI of 100. Cells were treated for 90 minutes and cell number was assessed byMTT for 6 days. The Ad-antisense CaSm–treated cells show reduced growthcompared with control and Ad-lacZ–treated cells. C, DU145 cells weretransfected with each CaSm siRNA and a negative control scramble dsRNA ormaintained in RPMI. Western blot analysis of DU145 cells treated with CaSmsiRNA for 7 days show reduced CaSm protein levels compared with control andscramble siRNA. Loading control with actin staining (bottom ). D, DU145 cellshave a slower increase in cell number after treatment with siRNA. Cells weretransfected as above at days 1, 2, and 5 (arrows ) and cell number was assessedby MTT.

Figure 4. Ad-anti-CaSm–treated cells have cells with an increased cellpopulation with >4 N DNA and altered expression of G2-M regulatory genes.DU145 cells treated with Ad-anti-CaSm at an MOI of 100 have a largerpopulation of aggregate cells than those left untreated or treated with adenoviralLacZ. A, DU145 cells were untreated or treated with adenoviral LacZ orantisense CaSm at an MOI of 100. At 48 hours, the cells were stained withpropidium iodide and analyzed for DNA content by flow cytometry. B, cells wereinfected as in (A) and stained with 4V,6-diamidino-2-phenylindole and enlargednuclei counted under 400� magnification. Fifty out of 124 Ad-antisense-CaSm–treated cells have enlarged nuclei, whereas untreated (control) orAd-LacZ-treated cells have 5 of 63 and 0 of 69 enlarged nuclei, respectively.Number of enlarged nuclei were compared with parental cells using atwo-sample t test (P < 0.001 Ad-LacZ versus Ad antisense CaSm). C, DU145cells were left untreated or treated with Ad-LacZ or Ad-antisense-CaSm at a MOIof 100. Cells were fixed with 70% methanol in PBS. The cells were washed oncewith PBS and stained for 15 minutes at 37jC with 1 Ag/mL 4V,6-diamidino-2-phenylindole and mitotic figures counted. DU145 cells untreated or treated withadenoviral LacZ showed at least 5 of 63 cells and 6 of 69 cells, respectively,in mitosis, whereas Ad-anti-CaSm–treated cells showed 1 mitotic cell out of 124counted. The number of mitotic figures were compared with parental cells using atwo-sample t test (P < 0.01 Ad-LacZ versus Ad-antisense CaSm). D, Westernblot and RT-PCR analysis of Ad-antisense-CaSm–treated cells 48 hourspost-infection compared with untreated and Ad-LacZ-treated DU145 cells forproteins involved in cell cycle regulation. Cyclin B and CDK1 protein levels arereduced in Ad-anti-CaSm–treated DU145 cells by Western blot consistentwith a G2-M cell cycle block (top ). Nek2 mRNA expression is increased inAd-antisense-CaSm–treated DU145 cells by RT-PCR with S26 ribonucleoproteinas control (middle ). Nucleophosmin/B23 RNA level is decreased inAd-antisense-CaSm–treated DU145 by Northern blot (bottom ).

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virus. Microarray analysis was used to identify additional geneswith altered expression as a consequence of reduced CaSm level.Polyadenylic acid [poly(A)]-enriched RNA, isolated from DU145parental cells and a stable cell line expressing antisense CaSm,was used to prepare cDNA probes for analysis of two differentcDNA libraries. The Human UniGEMV (Genome Systems) arraycontained probes for 7,000 genes. The second microarray allowedassessment of >6,000 prostate-derived cDNAs (17, 20). Theexpression profiles identified differences between these cell linesand we selected two genes critical for cell cycle progression forvalidation. Nek2 [never in mitosis A (NIMA)-related kinase-2], acore component of the centrosome, peaks in S-G2 phase and isreduced when cells enter mitosis (21). Nek2 expression wasincreased in anti-CaSm expressing DU145 cells. Northern blotanalysis confirmed Nek2 mRNA overexpression in stable antisenseexpressing cells compared with parental DU145 cells (data notshown). Differential expression was also seen in Ad-anti-CaSm–infected cell lines and confirmed that Nek2 mRNA level iselevated after infection with Ad-anti-CaSm virus (Fig. 4D, middle).Nucleophosmin/B23 associates with centrosomes and is requiredfor DNA replication (22). Analysis of the prostate cDNA arrayindicated that nucleophosmin/B23 mRNA was reduced in RNAprepared from anti-CaSm expressing DU145 cells. Nucleophos-min/B23 mRNA expression is reduced in cells infected with theAd-anti-CaSm virus (Fig. 4D, bottom), validating the microarrayresults. Collectively, these alterations, along with reduced cyclinB/CDK1, could contribute to the G2-M blockade in the adenovirusantisense cell populations.Cells expressing anti-CaSm have elevated p21/Cip1 mRNA

levels. The flow cytometry profiles showed that Ad-anti-CaSminfection leads to cell cycle alterations in DU145 cells. Based on theimportance of p21/Cip1 in cell cycle control, expression of p21/Cip1 mRNA was determined following Ad-anti-CaSm infection. Thelevel of p21/Cip1 mRNA was compared between DU145, a DU145clone with stable expression of anti-CaSm, and a pool of Ad-anti-CaSm or Ad-lacZ–infected DU145 cells (Fig. 5A). Northern analysisshows that cells expressing anti-CaSm have significantly higherlevels of p21/Cip1 mRNA than the parental controls. To determinewhether the increased message level resulted in increased p21protein, total protein was extracted from untreated and Ad-LacZ orAd-aCaSm–treated DU145 cells and subjected to Western blotanalysis using a p21-specific antibody. Unexpectedly, p21 proteinlevels were not significantly altered between untreated, Ad-LacZand Ad-aCaSm–treated DU145 cells (Fig. 5B).Based on the homology between CaSm and yeast LSm1/spb8, a

protein that contributes to mRNA stability, altered mRNA stabilitymay contribute to the observed increase in p21/Cip1 mRNA. Anactinomycin D blockade experiment was done to determine ifantisense CaSm treatment led to changes in mRNA stability.Parental and antisense-expressing DU145 cell lines were treatedwith actinomycin D and total RNA was isolated at several timepoints following drug addition. The abundance of p21/Cip1 mRNAwas evaluated by Northern blot analysis (data not shown) andquantified by phosphoimaging (Fig. 5C). The half-life of p21/Cip1 inthe parental DU145 cells was 1.5 hours; in contrast, the half-life ofp21/Cip1mRNAwas increased to 3.8 hours in stable antisense CaSmexpressing DU145 cells. These results suggest that p21/Cip1 mRNAstability is dependent on CaSm expression level and are consistentwith a role for CaSm in the control of mRNA stability in human cells.Although Spb8/LSm1 mutations result in stabilized messages

that retain their 5Vcap, they are deadenylated. To assess whether

the stabilized p21 mRNA was deadenylated, poly(A) selection wasused to enrich for polyadenylated mRNA. Total RNA and poly(A)-containing mRNA from DU145 and stable antisense CaSmexpressing cells were analyzed by Northern blot. Whereas anincreased level of total p21 mRNA was observed in the antisense-expressing cells, no significant enrichment was observed in thepoly(A) p21 message population (Fig. 5D). When compared withthe parental cells, there was significantly less polyadenylated p21mRNA in the antisense CaSm-expressing cells. From these data, wehypothesize that the stabilized p21 messages are deadenylated andthus, are inefficiently translated.CaSm protein is elevated in prostate cancer tissue. Our data

suggest that CaSm protein expression may be up-regulated duringthe progression of prostate cancer. To evaluate CaSm proteinexpression in prostate tissue, we conducted immunohistochemicalstaining of human prostate cancer sections. Only those sectionscontaining both normal and tumor areas on the same slide werescored. As shown in Fig. 6, CaSm protein is found predominantly inthe cytoplasm of normal and cancer epithelial cells. However,epithelial cells present in prostate carcinomas show increasedprotein expression in 40% of patients (4 of 10) examined(representative data, Fig. 6A). Elevated CaSm protein expressionwas not observed in all carcinoma specimens examined. The

Figure 5. Antisense CaSm expression alters the stability of p21/Cip1 mRNA.A, p21/Cip1 expression is increased in cells expressing antisense CaSm. TotalRNA was isolated from DU145 cells and a stable antisense CaSm expressionclone. RNA was also isolated from DU145 cells infected with the indicatedadenovirus. RNA was resolved on a 1.2% agarose gel, transferred to anitrocellulose membrane and hybridized using a 32P-labeled p21/Cip1-specificprobe. B, p21 protein levels are not changed by antisense CaSm expression.Western blot analysis was done on cell extracts from the indicated treated cells.C, DU145 cells and a stable antisense CaSm-expressing cell line were treatedwith actinomycin D (5 Ag/mL) and total RNA was harvested at 0, 0.25, 0.5, 0.75,1.0, 1.5, 2, 4, 5, and 6 hours after the addition of the drug. Total RNA wasresolved on a 1.2% agarose gel, transferred to a nitrocellulose membrane andhybridized with a 32P-labeled specific probe for p21. Relative mRNA abundancewas determined using phosphoimaging and densitometry, comparing p21 mRNAto S26 mRNA as control. Lines were generated using an exponential equation.R2 values were 0.975 for DU145 and 0.901 for the antisense DU145 clone.D, p21 mRNA is deadenylated in antisense CaSm-expressing cells. Total andpoly(A) RNA were isolated from DU145 and a clone stably expressing antisenseCaSm RNA was resolved on a 1.2% agarose gel, transferred and hybridizedusing a 32P-labeled p21/Cip1-specific probe.





remaining cases were divided between those with equivalentexpression between tumor and normal regions (1 of 10) and thosecases where CaSm protein level is higher in the normal tissue(5 of 10; Fig. 6B).

Discussion

Our current studies show that CaSm is required to maintainthe transformed phenotype of prostate cancer cells and canfunction as an oncogene. Overexpression of this gene in NIH-3T3cells leads to foci formation in vitro . Stable expression ofantisense in DU145 cells results in reduced CaSm protein levelsand reduced ability to grow independent of anchorage in vitro oras tumors in vivo . Additionally, siRNA-mediated reduction ofCaSm protein also leads to altered cell growth. Ad-anti-CaSmtreatment of DU145 cells has a cytostatic effect, with increasedG2-M populations of cells along with a substantial increase incells with nuclei containing more than the normal 4 N content ofDNA. These phenotypic changes are associated with changes inthe expression of proteins that regulate cell cycle progressionthrough G2-M and mitosis, consistent with the model that down-regulation of the CaSm oncogene results in the blockage of thecell cycle prior to completion of mitosis.The normal G1-S-G2-M progression of the cell cycle is controlled

by a series of cyclins and cyclin-dependent kinases (23, 24). CyclinB/CDK1 functions during G2 into M phase. A change in the levels ofcyclin B or CDK proteins or CDKI activity could explain the cellcycle changes observed after treatment with CaSm antisense. It hasrecently been shown that siRNA-mediated reduction of cyclin B1inhibits cellular proliferation, with cells arrested in G2-M (25), asimilar phenotype to that found following Ad-anti-CaSm treatment.Furthermore, reduced cyclin B1 level is also observed during theendomitotic cell cycle of megakaryocytic cells (26), and suggeststhat the reduced cyclin B level in anti-CaSm–treated cells maycontribute to the accumulation of cells with >4 N DNA. Reduction inCDK1/cyclin B protein level by CaSm antisense may suggest thatCaSm overexpression could have an inverse effect and increaseCDK1 function in cancer cells. Thus, CaSm overexpressing cells maybe able to bypass the G2 checkpoint and increase their rate ofproliferation. Microarray analysis showed that anti-CaSm affects theexpression of multiple genes. Two genes important for control ofthe G2-M transition and progression through mitosis, Nek2 andnucleophosmin/B23, were selected and altered expression initiallydetected by microarray analyses was validated. Nek2 is a NIMA-related kinase implicated in regulating centrosome structure at theG2-M transition (27, 28). Two Nek2 splice variants (Nek2A andNek2B) have been identified, and Nek2A has been shown to be atarget for APC/C and proteosome-mediated degradation duringentry into mitosis (29), consistent with the observation thatproteolysis of NIMA is required for completion of Aspergillusmitosis (30). Overexpression of Nek2 has been found to induceaneuploidy, chromosome instability, and supernumerary chromo-somes, aborted mitosis or failed cytokinesis (31). Anti-CaSmexpression results in increased Nek2 mRNA expression, consistentwith altered spindle checkpoint signaling that could contribute tothe failure to enter mitosis, contributing to G2-M arrest and/or >4 NDNA (endoreduplication). Nucleophosmin/B23 is a multifunctionalprotein implicated as a target for cyclin E/cyclin-dependent kinase 2in modulating centrosome duplication and cell cycle control (22).Nucleophosmin/B23 has been considered to be a marker of cellularproliferation, is frequently overexpressed in a variety of human

malignancies, and has been shown to transform NIH-3T3 cells(reviewed in ref. 22). Antisense-mediated down-regulation ofnucleophosmin/B23 results in delayed entry into mitosis (32).Consistent with these properties, we find that nucleophosmin/B23expression is reduced in proliferation-defective, anti-CaSm–expressing, cells.A previous study reported that CaSm mRNA was down-

regulated in metastatic prostate cancer compared with normaltissue and localized prostate cancer. They also reported that CaSmwas not expressed in two prostate cancer–derived cell lines, PC3and DU145 (33). In this study, we find that CaSm proteinexpression is low in primary normal prostate epithelial cellscompared with prostate cancer–derived cell lines. Furthermore,the data presented here indicates that CaSm protein is expressedat higher levels in a subset of prostate cancer specimens comparedwith normal tissue areas. The current data also shows that CaSmmRNA and protein are both easily detectable in PC3 and DU145cells, suggesting possible clonal variation in these cell lines.

Figure 6. Immunohistochemical staining of CaSm protein in human prostatecancer. Representative sections stained with rabbit anti-CaSm antibody usingVectastain Elite ABC Kit. Normal regions (arrows ) and adjacent cancer regions(arrowheads ). A, prostate tumor tissue that has elevated CaSm staining,compared with adjacent normal tissue. B, case where CaSm expression is notelevated in tumor tissue relative to normal. All of the original microscopicmagnifications are 200�.

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CaSm has homology to a family of Sm-containing proteins (34),first identified in yeast (35) and human (36) by database searchingusing core Sm sequences. Sm and LSm proteins form heptamericcomplexes involved in RNA metabolism. The LSm2-LSm8 complexfunctions during pre-mRNA splicing, binding to U6 small nuclearRNA and necessary for U4/U6 assembly (36, 37). LSm1 to LSm7proteins are required for 5V to 3V mRNA degradation. Loss offunction studies show that the yeast CaSm homologue, spb8/LSm1,has a critical role in the decapping of mRNA (38). Consistent withsuch a role in mRNA stability, yeast LSm1-LSm7 has been shown toform a stable complex with the exonuclease Xrn1 as well asproteins that function in mRNA decapping, Dcp1/Dcp2 and Pat1(39). Pat1 (Mrt1p) has also been shown to interact with spb8/LSm1in vivo (40). Yeast LSm1, Pat1, Dcp1, Dcp2, and Xrn1 all localize todiscrete cytoplasmic foci (P bodies; ref. 41). Together, theseproteins control the stability of mRNA in yeast (34, 42). Althoughthe function(s) of hLSm1 (CaSm) has not been determined,previous publications show a correlation between hLSm1 andmRNA degradation. Components of LSm1-LSm7 colocalize withhuman Dcp1/2 and Xrn1 in discrete cytoplasmic foci (43–45),Recently, additional factors linked to mRNA decay have beenshown to be colocalized in these cytoplasmic foci (46). Collectively,the presence of similar complexes in yeast and human cells areconsistent with the model that CaSm/LSm1 may have a role inmessage stability. Our finding that antisense-mediated reduction ofCaSm is associated with increased p21/CIP mRNA stabilityprovides the first direct evidence consistent with this model.It is generally accepted that gene expression is altered in cancer.

Although many studies of gene expression examine the control oftranscriptional initiation by specific transcription factors, it isevident that mRNA turnover provides an important mechanism for

posttranscriptional control of gene expression. Thus, it is possiblethat alteration of decay rates of certain mRNAs (e.g., tumorsuppressor and oncogenes) could play a role in cancer initiationand progression. The cyclin-dependent kinase inhibitor p21 is acritical regulator of cellular proliferation, mediating cell cyclearrest. Although the p21 transcript is stabilized in antisense CaSmexpressing DU145 cell lines, the mRNA is deadenylated, and thusinefficiently translated. However, this data suggests that p21decapping is a CaSm-associated process, and supports the modelthat CaSm overexpression leads to destabilization of p21 mRNA,leading to reduced p21 levels, allowing a bypass of a criticalcheckpoint in G1-S and G2-M transitions. Although it remains tobe determined how CaSm function contributes to the control ofmessage stability, CaSm’s impact on message stability representsan alternative mechanism for affecting the transcriptome in cancercells.

Acknowledgments

Received 2/24/2005; revised 4/11/2005; accepted 4/29/2005.Grant support: Supported in part by grants from the Department of Defense

(N00014-96-1-1298 to M.M. Fraser and D.K. Watson), National Science Foundation(1SC EPSCoR 3100-Z136 to M.M. Fraser and D.K. Watson), NIH (RO1 ES011323 toP.M. Watson and A.M. Boylan; CA97186 to P.S. Nelson), and the Abney Foundation(to M.M. Fraser).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Margaret Romano (Hollings Cancer Center Tumor Bank), John Zhang(Medical University of South Carolina Antibody Facility), and Candice Bergan and RickPeppler (Medical University of South Carolina Flow Cytometry Facility) for technicalassistance; Scott Miller for assistance with statistical analysis; and Drs. Tilmann Achseland Dierk Ingelfinger of the Luhrmann Laboratory (Max Planck Institute) forproviding LSm1 antibodies used for the initial studies and for advice on conditions foroptimal electrophoretic conditions for resolution of CaSm protein.



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2005;65:6228-6236. Cancer Res Melissa M. Fraser, Patricia M. Watson, Mostafa M. Fraig, et al. Altered Gene Expression and Messenger RNA StabilityCaSm-Mediated Cellular Transformation Is Associated with

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