targeted biallelic inactivation of pten in the mouse …...targeted biallelic inactivation of pten...

Targeted Biallelic Inactivation of Pten in the Mouse Prostate Leads to

Prostate Cancer Accompanied by Increased Epithelial Cell

Proliferation but not by Reduced Apoptosis

Xiaoqian Ma,1Angelique C. Ziel-van der Made,

1Binha Autar,

1Hetty A. van der Korput,

1

Marcel Vermeij,1Petra van Duijn,

1Kitty B. Cleutjens,

1Ronald de Krijger,

1Paul Krimpenfort,

2

Anton Berns,2Theo H. van der Kwast,

1and Jan Trapman

1

1Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Center, Rotterdam and 2Division of Molecular Genetics andCentre of Biomedical Genetics, Netherlands Cancer Institute, Amsterdam, the Netherlands

Abstract

The PTEN tumor suppressor gene is frequently inactivated inhuman tumors, including prostate cancer. Based on the Cre/loxP system, we generated a novel mouse prostate cancermodel by targeted inactivation of the Pten gene. In this model,Cre recombinase was expressed under the control of theprostate-specific antigen (PSA) promoter. Conditional bial-lelic and monoallelic Pten knock-out mice were viable andPten recombination was prostate-specific. Mouse cohorts weresystematically characterized at 4 to 5, 7 to 9, and 10 to 14months. A slightly increased proliferation rate of epithelialcells was observed in all prostate lobes of monoallelic Ptenknock-out mice (PSA-Cre;Pten-loxP/+), but minimal pathologicchanges were detected. All homozygous knock-out mice (PSA-Cre;Pten-loxP/loxP) showed an increased size of the luminalepithelial cells, large areas of hyperplasia, focal prostateintraepithelial neoplasia lesions and an increased prostateweight at 4 to 5 months. More extensive prostate intra-epithelial neoplasia and focal microinvasion occurred at 7 to 9months; invasive prostate carcinoma was detected in all malePSA-Cre;Pten-loxP/loxP mice at 10 to 14 months. At 15 to 16months, a rare lymph node metastasis was found. Inhyperplastic cells and in tumor cells, the expression ofphospho-AKT was up-regulated. In hyperplastic and tumorcells, expression of luminal epithelial cell cytokeratins was up-regulated; tumor cells were negative for basal epithelial cellcytokeratins. Androgen receptor expression remained detect-able at all stages of tumor development. The up-regulation ofphospho-AKT correlated with an increased proliferation rateof the epithelial cells, but not with a reduced apoptosis.(Cancer Res 2005; 65(13): 5730-9)

Introduction

The PTEN tumor suppressor gene, also known as MMAC1 orTEP1 , is one of the most frequently inactivated genes in humancancer (1–4). The main molecular function of PTEN is counter-acting phosphoinositide-3-kinase signaling by removing the D3phosphate from the inositol ring of phosphatidylinositol (3,4,5)-phosphate. The kinase AKT/PKB is the major downstream target ofphosphatidylinositol (3,4,5)-phosphate (5, 6). Functional loss ofPTEN results in the accumulation of phosphatidylinositol (3,4,5)-

phosphate and up-regulation of phospho-AKT, consequentlymodulating the activities of its downstream effectors. Thesemolecular changes can affect many biological functions of cellssuch as proliferation, apoptosis, cell size, cell adhesion, andmigration (7–12).Germ line mutations of PTEN are the cause of Cowden’s disease,

Bannayan-Zonana syndrome and Lhermitte-Duclos disease, whichare characterized by the development of hamartomas and anincreased risk of cancer (13–15). Somatic mutations of PTEN havebeen detected at high frequency in many sporadic cancers,including glioblastoma, endometrial cancer and prostate cancer(4). Complete PTEN inactivation has been found in f15% ofprimary prostate tumors, and in up to 60% of prostate cancermetastases and prostate cancer xenografts (16–19). Immunohisto-chemical studies showed the absence of PTEN in 20% of primaryprostate tumors (20).Pten is essential in mouse development (21). Conventional

Pten�/� mice show developmental defects and die at embryonicdays 6.5 to 9.5 (22–24). Pten+/� mice are viable and display avariety of hyperplastic and dysplastic changes in different tissues.Female heterozygous mice mainly develop endometrial and breasttumors (25), most male Pten+/� mice develop lymphoma (23–25),and at lower frequency, hyperplasia and dysplasia of several othertissues, including the prostate. Tumor development might beassociated with loss of the wild-type Pten allele (23).Biallelic conditional knock-out mice completely lacking Pten

function might develop tumors in specific tissues (26). Recently, ithas been shown that prostate-targeted Pten knock-out micedevelop hyperplasia, and at older age, metastatic tumors in theprostate (27–29). In these studies, Cre expression was regulated bythe mouse mammary tumor virus (MMTV) promoter or a modifiedprobasin promoter. We generated a novel targeted Pten mouseprostate cancer model, applying the well-characterized prostate-specific antigen (PSA) promoter (30, 31) to drive prostate-specificCre expression. The Pten gene was inactivated by PSA-Cre targeteddeletion of exon 5, resulting in the expression of a truncatedprotein lacking the phosphatase domain. The same deletion haspreviously been found in a human prostate cancer xenograft (18).Cohorts of male PSA-Cre;Pten-loxP/loxP and PSA-Cre;Pten-loxP/+mice, and control littermates were systematically characterized atdifferent ages. All PSA-Cre;Pten-loxP/loxP mice showed the gradualdevelopment of prostate hyperplasia, followed by prostate intra-epithelial neoplasia (PIN) lesions and prostate cancer. Hyperplasia,PIN, and cancer were accompanied by increased cell size andepithelial cell proliferation, but not by reduced apoptosis. Prostateepithelial cells in all stages of tumor development showedincreased expression of cytokeratins. The effect of monoallelic

Requests for reprints: Jan Trapman, Department of Pathology, Josephine NefkensInstitute, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, the Netherlands.Phone: 31-10-408-7933; Fax: 31-10-408-9487; E-mail: [email protected].

I2005 American Association for Cancer Research.

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prostate-specific deletion of Pten was limited. At old age, focaldysplasia was observed in some mice, but none of the PSA-Cre;Pten-loxP/+ mice developed a prostate tumor.

Materials and Methods

Generation of prostate-targeted Pten knock-out mice. The PSA-Creconstruct was generated by linkage of the 6 kbp HindIII/HindIII fragment ofthe human PSA promoter to Cre cDNA. The PSA promoter has been provento target transgene expression to the luminal epithelial cells of the prostate(30). The mouse protamine gene provided an intron and the 3V-untranslatedregion, including the polyadenylation signal (Fig. 1A). Transgenic PSA-Crefounders were generated by injection of a 7.5 kbp PSA-Cre-Prot fragmentinto the pronuclei of fertilized oocytes of FVB mice. Prostate-specificexpression of the transgene was assessed by crossing with CAG-loxP-CAT-loxP-LacZ indicator mice (ref. 32; data not shown). The founder line PSA-CreD4 showing high, prostate-specific Cre activity was used in theexperiments described in this study.

The generation of mice (strain 129Ola) carrying the Pten-loxP allele has

been described elsewhere (33). In these mice, Pten was targeted by

introduction of a loxP recognition sequence and a flanking diagnosticEcoR1 site in intron 5, and a neomycin (neo) cassette flanked by loxP sites in

intron 4 (Fig. 1A). A 9 kbp fragment, encompassing the targeted Pten exon 5,

was electroporated into the embryonic stem cell line E14 subclone IB10 (34).

By crossbreeding PSA-Cre transgenic mice with Pten-loxP/loxP mice andsubsequent breeding of PSA-Cre;Pten-loxP/+ F1 offspring with Pten-loxP/

loxP mice, biallelic and monoallelic prostate-targeted Pten knock-out mice

were generated. Cre negative Pten-loxP/loxP littermates were kept ascontrols. All experiments were done in a 129Ola/FVB background. Mice

were housed according to institutional guidelines, and procedures were

carried out in compliance with the standards for use of laboratory animals.

PCR for genotyping and detection of specific recombination.Genomic DNA was isolated from tail tips by standard procedures. PSA-Cre

mice were genotyped by standard PCR, using two sets of primers: 5V-CCTGCTGGAAGATGGCGATTAG-3V (Cre-F) and 5V-GGACTTGC-TATTCTGTGCATCTAG-3V (Pro-R) for amplification of Cre cDNA and theprotamine 3V-untranslated region, and 5V-CTTGTAGGGTGACCAGAGCAG-3V(PSA-F) and 5V-GCAGGCATCCTTGCAAGATG-3V(PSA-R) to amplify the PSA

promoter.The primers used for genotyping of Pten-loxP mice by standard PCR were

5V-GCCTTACCTAGTAAAGCAAG-3V (In5F2) and 5V-GGCAAAGAATC-TTGGTGTTAC-3V (In5R3). For detection of recombination of Pten exon 5

in genomic DNA, we used the primers 5V-TGGCATAAGTTAGGAAAGATG-3V(In4F2) and In5R3. Primers for amplification of the control genomic Gapdh

fragment: 5V-TCCGCCCCTTCCGCTGATGCC-3V (GAPDH1) and 5V-TAGTGGGCCCTCGGCCGCCGCCTG-3V (GAPDH2). For long-range PCR,

we used the PCR core kit of Qiagen (Hilden, Germany).Southern blotting. The Gentra DNA isolation kit (BIOzym, Landgraaf,

the Netherlands) was used for isolation of genomic DNA. BglII-digested

DNA was loaded on a 1% agarose gel in TAE buffer. Next, the DNA was

blotted on Hybond-N+ membrane (Amersham Biosciences, Little Chalfont,United Kingdom), and hybridized with a 32P-labeled Pten probe. Following

washing under high stringent conditions, the filter was exposed to X-ray

film. The 320 bp hybridization probe was generated by PCR on Pten intron 5in vector DNA (Fig. 1A). PCR primers: 5V-CTAGATATCAGTGGACCTGTG-3V(Pten-5A-F) and 5V-TGGCCCTAAGAGTCCTCCACA-3V (Pten-5A-R).

Histology and immunohistochemistry. Tissues for histology were fixedin freshly prepared buffered 4% formalin for f16 hours at 4jC, dehydratedand embedded in paraffin. Five micrometer sections were attached to AAS-

coated slides and H&E-stained. The antibodies used for immunostaining

were directed against basal epithelial cell cytokeratins (mouse monoclonal

antibody 34hE12, diluted 1:100; DAKO, Glostrup, Denmark), phospho-AKT(rabbit polyclonal antiserum, 1:200; Cell Signaling Technology, Beverly, MA),

androgen receptor [rabbit polyclonal antiserum SP197, direct against the NH2

terminus (1:1,500)], and luminal epithelial cytokeratin (mouse monoclonalantibody, 1:50; BD, San Diego, CA). Microwave pretreatment for antigen

unmasking was applied by boiling in 10 mmol/L sodium citrate for 15

minutes and cooling at room temperature for 1 hour. Primary antibodieswere incubated overnight at 4jC, and biotin-labeled secondary antibodies

(1:400, DAKO) were incubated at room temperature for 1 hour. Immunore-

activity was visualized by the streptavidin-peroxidase (1:50, BioGenex, San

Ramon, CA) or streptavidin-alkaline phosphatase (1:50, BioGenex) systems( for phospho-AKT).

Western blotting. Frozen prostate lobes were pulverized under liquid

nitrogen and tissue powder was transferred into 1� Laemmli sample buffer

(10 AL sample buffer per microgram of tissue powder). The samples weresonicated for 2 � 20 seconds, boiled for 5 minutes, and centrifuged

(5 minutes at 14,000 rpm, Eppendorf centrifuge). Equal amounts of protein

were subjected to 10% SDS-PAGE. After electrophoresis, proteins were

transferred to protran membrane (Schleicher & Schuell, Dassel, Germany)and immunoblotted. Primary antibodies used were rabbit polyclonal

antibodies to AKT, phospho-AKT (Ser473, Cell Signaling Technology),

and PTEN (Neomarkers, Fremont, CA; all at 1:1,000) and mouse monoclonalh-actin antibody (Sigma, St. Louis, MO; 1:10,000).

Analysis of cell proliferation and apoptosis. For assessing in vivo cell

proliferation, mice were subjected to i.p. injection of bromodeoxyuridine

(BrdUrd, Sigma; 40 mg/kg body weight) at 30 minutes prior to sacrifice.Formalin-fixed, paraffin-embedded tissue slides from prostate lobes were

treated with 1% protease (Sigma) for 10 minutes at 37jC, rinsed with cold

PBS and incubated in 2 mol/L HCl for 30 minutes at 37jC. Primary mouse

monoclonal BrdUrd antibody IIB5 (1:20, donated by Dr. Frans Ramaekers,Department of Pathology, University of Maastricht, Maastricht, the Nether-

lands) was applied for overnight incubation at 4jC. Secondary antibody

incubation and visualization were as described above. The proliferationindex was calculated as number of BrdUrd positive cells per 1,000 adjacent

luminal epithelial cells from three independent counts. The Student’s t test

was applied for statistical analyses.

For assessment of apoptosis, tissue sections of prostate lobes wereimmunostained for active caspase-3 positive cells (R&D System, Minneap-

olis, MN; 1:1,500; ref. 35). The apoptotic index was calculated as the number

of active caspase-3-positive cells per 1,000 luminal epithelial cells. Tissue

pretreatment, visualization of immunoreactivity, calculations, and statisticswere as described above. The frequently applied terminal nucleotidyl

transferase–mediated nick end labeling (TUNEL) assay gave less reproduc-

ible results, in accordance with previously published findings (36).

Results

Pten recombination occurs in the prostates of conditionalknock-out mice and gives rise to high-level phospho-AKT inbiallelic knock-out mice. At 4 to 5 months, genomic DNA wasisolated from 19 different tissues of Cre-positive mice and PCR wasdone to determine Pten recombination. As visualized by thepresence of a 330 bp band, Cre-specific recombination of Pten exon5 was detected in the anterior, dorsolateral, and ventral prostatelobes of PSA-Cre;Pten-loxP/loxP and PSA-Cre;Pten-loxP/+ mice(Fig. 1B , lanes 1-3). Pten recombination efficiency was somewhatvariable in individual mice, however, all Cre-positive mice displayedPten recombination in each prostate lobe. None of the otherinvestigated tissues contained the recombined band (Fig. 1B , lanes4-19), except for occasional recombination in adrenal glands (datanot shown). We were unable to detect a recombined band in thefirst weeks after birth (data not shown). To obtain insight into thePten recombination efficiency, long-range PCR over exon 5 wascarried out. Primers detected both the recombined and notrecombined allele (Fig. 1C). In prostate DNA of both PSA-Cre;Pten-loxP/+ and PSA-Cre;Pten-loxP/loxP mice, the recombined band wasclearly visible (Fig. 1C , lanes 4 and 6). Southern blotting of prostateDNA from mice of each genotype confirmed the PCR data (Fig. 1D).In PSA-Cre;Pten-loxP/loxP mice, >50% of Pten was recombined inthe prostate (Fig. 1D , lane 3); as expected in PSA-Cre;Pten-loxP/+mice, the recombined 5.7 kbp band was less pronounced (Fig. 1D ,

Prostate-Targeted Pten Mutant Mice

www.aacrjournals.org 5731 Cancer Res 2005; 65: (13). July 1, 2005



lane 2). The 2.3 kbp fragment of the Pten wild-type allele in PSA-Cre;Pten-loxP/loxP mice was most likely derived from cells lackingCre expression, e.g., stromal cells.As shown by immunoblotting, Pten expression was almost

completely absent in PSA-Cre;Pten-loxP/loxP prostate lobes; thePten expression level in PSA-Cre;Pten-loxP/+ prostates was notobviously different from control prostates (Fig. 1E). In controlmice, Pten expression seemed to be different in the distinctprostate lobes, lowest expression was observed in the anteriorprostate. Phospho-AKT expression was substantially increased inPSA-Cre;Pten-loxP/loxP prostates; it was low in mice carrying oneor two functional Pten alleles.Monoallelic inactivation of Pten slightly affects the prolif-

eration rate of prostate epithelial cells, but is not sufficient forprostate cancer development. We systematically examined the

prostates of the cohort PSA-Cre;Pten-loxP/+ mice for morphologicand biological alterations at different ages (4-5, 7-9, and 10-14months). The prostate weight of PSA-Cre;Pten-loxP/+ miceseemed to be slightly increased compared with that of controllittermates, however, this was not statistically significant (Fig. 2A).Comparing the proliferation rate of luminal epithelial cells of thePSA-Cre;Pten-loxP/+ mice and the wild-type littermates, we foundan approximately 2-fold higher number of BrdUrd-positive cells inPSA-Cre;Pten-loxP/+ mice (Fig. 2B) at each time point. The prostatelobes of PSA-Cre;Pten-loxP/+ mice at 4 to 5 months and at 7 to 9months showed no obvious morphologic changes as comparedwith the wild-type littermates (Fig. 2C and D ; and data not shown).Infrequent focal dysplasia was found at 10 to 14 months (2 out of10 mice, four different foci in total; see Fig. 2E and F ; Table 1).Several PSA-Cre;Pten-loxP/+ mice were investigated at 17 months.

Figure 1. Properties of prostate-specific targeted Pten knock-out mice. A, the strategy of the generation of prostate-specific Pten -deficient mice. The PSA-Cre-Protfragment containing the 6 kbp PSA promoter, Cre cDNA and the mouse protamine intron and polyadenylation sequences were used for the generation of transgenicmice, expressing Cre in the luminal epithelial cells of the prostate. The Pten exon 5 targeting fragment used for electroporation of embryonic stem cells containsthe neo cassette flanked by a loxP site in intron 4 and a loxP site plus a diagnostic EcoRI site in intron 5. Pten exons 4 and 5 are indicated. The neo cassette wasremoved by transient Cre expression in embryonic stem cells. Prostate-specific recombination mediated by Cre expression driven by the PSA promoter resulted in thedeletion of Pten exon 5. The probe used in Southern blotting is shown as a horizontal bar. The Bgl II site f4 kb upstream of exon 4 in the mouse genome is notindicated. B, prostate-specific recombination of Pten exon 5 in PSA-Cre;Pten-loxP conditional knock-out mice. PCR and agarose gel electrophoresis are describedin Materials and Methods. Top, the recombined band from a PSA-Cre;Pten-loxP/loxP mouse; bottom, a PSA-Cre;Pten-loxP/+ mouse. Recombination-specificfragments were detected in the prostate lobes, not in the remaining tissues. Gapdh was used as a loading control: 1, anterior prostate; 2, ventral prostate; 3, dorsolateralprostate; 4, epididymis; 5, vas deferens; 6, testis; 7, seminal vesicle; 8, kidney; 9, bladder; 10, liver; 11, spleen; 12, lung; 13, heart; 14. thymus; 15, salivarygland; 16, brain; 17, thyroid; 18, pituitary gland; 19, adrenal. C, long-range PCR over Pten exon 5. Wild-type control mice (lanes 1 and 2), PSA-Cre;Pten-loxP/+mice (lanes 3 and 4), and PSA-Cre;Pten-loxP/loxP mice (lanes 5 and 6). Lanes 1, 3, and 5, DNA from spleen; lanes 2, 4 and 6, DNA from the anterior prostate lobe. The330-bp fragment represents Pten recombination; the 2 kbp band indicates no recombination. D, Southern blot for detection of recombination efficiency. Lanes 1, 2,and 3, DNA from a control, PSA-Cre;Pten-loxP/+ , and PSA-Cre;Pten-loxP/loxP mouse, respectively. All DNA samples were from anterior prostate lobes.Recombination was detected as a 5.7 kbp Bgl II fragment, the nonrecombined Bgl II fragment was 2.3 kbp. E, immunoblotting for detection of PTEN and phospho-AKTexpression. The protein lysates from prostate lobes of each genotype at 4 to 5 months were separately blotted and stained. Almost complete absence of PTENexpression and highly up-regulated phospho-AKT expression were detected in PSA-Cre;Pten-loxP/loxP mice. Total AKT expression level is essentially identical in theprostate lobes of three different genotypes of mice. There was no obvious down-regulation of PTEN or up-regulation of phospho-AKT in the prostate lobes ofPSA-Cre;Pten-loxP/+ mice. Actin was used as a loading control. VP, ventral; AP, anterior; LP, lateral; and DP, dorsal prostate lobes.

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However, even at this age, no obvious pathologic differences werefound between PSA-Cre;Pten-loxP/+ mice and control littermates(data not shown), indicating that monoallelic loss of Pten gave riseto only minor changes in the prostate, which were insufficient todrive prostate cancer development.Complete inactivation of Pten in the mouse prostate gives

rise to hyperplasia, prostate intraepithelial neoplasia, andprostate cancer. H&E staining was done on prostate sections

from at least 10 PSA-Cre;Pten-loxP/loxP mice at three time points(see Fig. 3). The PSA-Cre;Pten-loxP/loxP mice showed progressivetumor development, including hyperplasia and focal PIN lesions at4 to 5 months, more widespread PIN and focal microinvasion at 7to 9 months, and invasive tumors at 10 to 14 months (see Table 1).All prostate lobes were affected, however, the pathologic changes indorsolateral and anterior lobes were more prominent than inventral lobes.At 4 to 5 months, most glands of the different prostate lobes

of PSA-Cre;Pten-loxP/loxP mice displayed hyperplasia manifestedby an accumulation of luminal cells within the glandular lumen

(Fig. 3A-D). This was associated with a distension of the

individual preexisting glands, whereas the borders of the glandsremained well-circumscribed. Compared with the regularly

layered columnar epithelial lining of prostate tubules in the

control littermates, a lining by disorganized multilayered ductalepithelial cells with tufting and/or cribriform growth pattern was

observed. The epithelial cells in the prostates of knock-out mice

were generally enlarged. Some foci showed distinguishedepithelial dysplasia characterized by a number of distinct

atypical epithelial cells possessing dense cytoplasm, enlarged

and somewhat vesicular nuclei with more conspicuous nucleoli

(Fig. 3A-D). These lesions shared the histologic features ofhuman PIN that is widely accepted as a precursor lesion of

prostate cancer (37).At 7 to 9 months, the process of hyperplasia and distension of

preexisting glands continued, whereas dysplasia became more

diffuse (about 40-50% luminal epithelial cells involved) with moreextensive cytonuclear atypia (Fig. 3E and F). In some cases, the

border of the hyperplastic glands was more irregular as a

consequence of outpouchings of glandular structures, displayingsome dysplasia. The latter phenomenon might be considered as

microinvasive carcinoma. This was also found in association with

hyperplastic glands.In all 10- to 14-month-old PSA-Cre;Pten-loxP/loxP mice,

carcinoma was observed in common with more extensive

involvement of the prostate lobes (Table 1). All tumors were ofepithelial origin; histopathologic features classified to varied stages

of adenocarcinoma like relatively low and moderately grade-

differentiated (Fig. 3G-J), and poorly differentiated adenocarcino-ma (Fig. 3K and L). In general, a rapid transition was seen from

glandular formations of cancer cells characteristic of low-grade

adenocarcinoma to solid fields of carcinoma lacking features ofglandular differentiation, suggestive of a rapid process of

dedifferentiation.To further characterize the prostates of PSA-Cre;Pten-loxP/loxP

knock-out mice, several immunohistochemical stainings were

done on formalin-fixed tissue sections. Phospho-AKT expression

Figure 2. The effect of monoallelic inactivation of Pten on the mouse prostate.A, comparison of prostate weight between PSA-Cre;Pten-loxP/+ and controlmice. One of each prostate lobe was measured; 9, 6, and 17 mice were analyzedfor control mice ages 4 to 5, 7 to 9, and 10 to 14 months, respectively; and 12, 7,and 13 mice for PSA-Cre;Pten-loxP/+ mice ages 4 to 5, 7 to 9, and 10 to 14months, respectively; bars, SE. B, proliferation rate of prostate epithelial cells.BrdUrd-positive cells per 1,000 epithelial cells were counted. There were 9, 5,and 9 mice for the control group ages 4 to 5, 7 to 9, and 10 to 14 months,respectively; and 10, 6, 9 mice for PSA-Cre;Pten-loxP/+ mice ages 4 to 5, 7 to 9,and 10 to 14 months, respectively; bars, SE. C-F, comparison of histologicfeatures of prostates from PSA-Cre;Pten-loxP/+ mice and control littermates at10 to 14 months. C and E, prostate section from control mice, representinganterior and dorsolateral lobes, respectively. D and F, prostate sectionsfrom PSA-Cre;Pten-loxP/+ mice, representing anterior and dorsolaterallobes, respectively. There are no obvious pathologic changes in mostPSA-Cre;Pten-loxP/+ prostates (D ). Arrows, focal epithelial dysplasia (F ).Magnification, �200 (C-F).

Table 1. Morphologic alterations in the prostates of targeted Pten knock-out mice

Mouse genotype 4 to 5 months 7 to 9 months 10 to 14 months

Control 10 of 10 normal 10 of 10 normal 8 of 8 normal

PSA-Cre; Pten-loxP/+ 12 of 12 normal 10 of 10 normal 2 of 10 focal

hyperplasia/PIN

PSA-Cre; Pten-loxP/loxP 12 of 12 hyperplasia,7 of 12 focal PIN

11 of 11 hyperplasia/PIN,5 of 11 focal microinvasion

17 of 17 carcinoma





was up-regulated in hyperplasia/PIN lesions and prostate cancer(Fig. 4A-C), confirming the immunoblot results shown in Fig. 1Eand indicating that inactivation of Pten efficiently triggered directdownstream targets. Basal cell cytokeratin staining showed thatthe continuous basal cell layers in the prostate of control micebecame fragmented in PIN lesions at 4 to 5 months. Basal cellsalmost disappeared in prostate cancer, although they were clearlydetectable in adjacent benign glands (Fig. 4D-F). As expected, theluminal epithelial cells in normal prostate were androgen

receptor–positive. We also observed f90% positive staining ofepithelial cells both in hyperplasia/PIN and cancer (Fig. 4G-I),indicating that the tumors might be sensitive to hormonedeprivation treatment. The expression level and pattern of luminalepithelial cytokeratins were changed in PSA-Cre;Pten-loxP/loxPprostates, showing dominant membrane staining in hyperplasia/PIN and prostate adenocarcinoma as compared with the localstaining at the epithelial cell surface in the prostate acini ofnormal control mice (Fig. 4J-L).

Figure 3. Morphology of prostates from PSA-Cre;Pten-loxP/loxP mice. A and B, anterior prostates from 4- to 5-month-old control mice showing a well-defined singlelayer of luminal epithelial cells. C and D, anterior prostates from 4- to 5-month-old PSA-Cre;Pten-loxP/loxP mice showing massive luminal epithelial cell hyperplasia.The PIN foci show atypical epithelial cells possessing enlarged nuclei with nuclear pleomorphism and hyperchromasia, and the presence of one or more prominentnucleoli (arrows in D ). E and F, anterior prostates from 7- to 9-month-old PSA-Cre;Pten-loxP/loxP mice. The luminal epithelial cells show a cribriform growth patternwith small intraepithelial blood vessels (arrowhead in E). There were more PIN foci (about 40-50% glands) with much more extensive cellular atypia than in 4- to5-month-old PSA-Cre;Pten-loxP/loxP mice (F ). G-L, invasive adenocarcinoma in prostates of PSA-Cre;Pten-loxP/loxP mice at 10 to 14 months. G and H, smallmalignant glands extending into surrounding stromal (arrows ). I and J, pronounced invasion of glands into the surrounding stroma. K and L, poorly differentiatedcarcinoma with growth pattern of massive malignant epithelial cell sheet. Magnifications, �200 (A, C, E, G, I, and K); �400 (B, D, F, H, J, and L).

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Prostate hyperplasia and cancer are associated withincreased proliferation rate but do not correlate with adecreased apoptotic index in biallelic Pten knock-out mice.Macroscopic examination showed an obvious enlargement of eachprostate lobe in PSA-Cre;Pten-loxP/loxP mice at the age of 4 to 5months. Compared with the control mice, the average weight ofPSA-Cre;Pten-loxP/loxP prostates was more than 2-fold increased at4 to 5 months and almost 3-fold increased at 7 to 9 months. Inprostate cancer at 10 to 14 months, the average weight had rapidlyincreased (Fig. 5A).BrdUrd and activated caspase-3 immunostaining were done to

examine whether the increased prostate weight was associatedwith stimulation of proliferation and inhibition of apoptosis. Thenumber of BrdUrd-positive cells in prostates of PSA-Cre;Pten-loxP/loxP mice was significantly increased as compared with thecontrols at all three time points, the highest number of BrdUrd-positive cells was detected in the 10- to 14-month-old group(Fig. 5B, D-F). The proliferation rate correlated with the increase ofprostate weight. Interestingly, we detected more apoptotic cells inthe prostates of PSA-Cre;Pten-loxP/loxP mice as well. The averagenumber of activated caspase-3-positive cells was significantlyhigher in the prostates of PSA-Cre;Pten-loxP/loxP mice than incontrol mice at all three time points (Fig. 5C, G-I). The apoptotic

index did not vary much at different ages, indicating that enhancedapoptosis already occurred at early stages of tumor development.Complete inactivation of Pten in the prostate leads to rare

metastatic cancer at old age. In order to identify any metastasesin multiple organs, including lymphoid nodes, liver and lung, ninePSA-Cre;Pten-loxP/loxP mice (10-16 months old) were systemati-cally screened. Serial sections of each organ were collected andH&E stained. We found one case of metastatic adenocarcinomaseeding at the subcapsular sinusoidal region of a caudal para-aorticlymph node (Fig. 6A and B). Phospho-AKT and androgen receptorstaining showed a moderate expression level of both proteins (Fig.6C and D). The metastatic locus was tiny, hence, we might havemissed even smaller metastases in other mice.

Discussion

PTEN inactivation is the most frequent genetic alteration inhuman prostate cancer (16, 18, 19, 38, 39). The main molecularfunction of PTEN is to counteract phosphoinositide-3-kinase inregulating the phosphatidylinositol (3,4,5)-phosphate level in thecell. In vitro and in vivo studies have shown that PTEN affects manybiological functions of the cell, including proliferation, differenti-ation, apoptosis, polarity, and size (21, 40). Because of the

Figure 4. Immunohistochemical characterization of prostates from PSA-Cre;Pten-loxP/loxP mice. A-C, immunohistochemical analysis of phospho-AKT in prostatesections. Compared with the low expression level of phospho-AKT in prostate epithelial cells from normal adult mice (A), the phospho-AKT expression level wasup-regulated in epithelial cells in hyperplasia/PIN lesions in prostates from 4- to 5-month-old PSA-Cre;Pten-loxP/loxP mice (B ), phospho-AKT expression remained highin most malignant epithelial cells in adenocarcinoma from 10- to 14-month-old PSA-Cre;PtenloxP/loxP mice (C ). D-F, immunohistochemical staining of basal cellcytokeratins. The normal adult prostate gland shows a regular stained basal cell layer (arrows in D ), which became more fragmented in hyperplasia/PIN lesions in theprostates from PSA-Cre;PtenloxP/loxP mice at 4 to 5 months (E) and totally vanished in malignant epithelial cells of adenocarcinoma (F ). Positive stained cells exist inan adjacent residual benign gland (see arrowhead in F ). G-I, immunohistochemical staining for androgen receptor. G, androgen receptor expression in prostateepithelial cells from normal adult mice; H and I, androgen receptor staining in hyperplasia/PIN lesions and adenocarcinoma, respectively. J-L, cytokeratin staining forluminal epithelial cells. In the normal adult prostate, cytokeratin expression was mostly located at the epithelial cell surface facing the lumen (J ). The intensity andpattern of staining was dramatically changed in hyperplasia/PIN lesions with dominant membrane staining (K ). The malignant epithelial cells in prostate cancer showedstrong cytoplasm and membrane staining (L). Magnifications, �400 (A-L).





importance of PTEN in tumorigenesis and the complexity of itsbiological function, much effort has been put in the characteriza-tion of Pten knock-out mice. Initially, several groups reported theproperties of conventional Pten knock-out mice (9, 22–24, 41). Inall studies, Pten�/� conferred embryonic lethality whereasPten+/� mice developed hyperplasia, and to a variable extentdysplasia and tumors in a variety of organs. Pten+/� mice mostfrequently developed lymphoma and intestinal polyps, and infemale mice, breast cancer and endometrial cancer, indicating thetissue-dependent role of Pten inactivation in tumorigenesis.The reported pathologic changes in the prostates of Pten+/�

mice were variable, and included various stages of hyperplasia anddysplasia. Prostate cancer was rare, possibly because mice diedearlier from more rapidly developing malignancies, e.g., lymphoma,

hampering more detailed analyses of prostate cancer development.Importantly, hypomorphic Pten (Pten hy/�) mutant mice showingan even lower level of Pten expression than Pten+/� mice morerapidly developed prostate hyperplasia that in a proportion of casesprogressed to focal invasive carcinoma at older age, indicating Ptendose-dependent development of prostate lesions (27). Obviously, adrawback of conventional knock-out mice is that all cell types in alltissues are affected.Embryonic lethality of Pten�/� mice and pleiotropic effects in

Pten+/� mice have been overcome by tissue-specific Pteninactivation. Tissue-specific biallelic Pten inactivation has beenreported for thymus, mammary gland, testis, liver, spleen, andbrain (33, 42–47). In some tissues, Pten inactivation inducedtumors, including T cell lymphoma, germ cell tumors, mammary

Figure 5. Increased prostate weight is associated with an increased proliferation rate and an increased apoptotic index in PSA-Cre;Pten-loxP/loxP mice. A , theaverage weight of prostates from normal adult mice and biallelic Pten knock-out mice. There were 9, 7, and 17 mice analyzed for control mice ages 4 to 5, 7 to 9,and 10 to 14 months; and 9, 12 and 18 mice analyzed for PSA-Cre;Pten-loxP/loxP mice ages 4 to 5, 7 to 9, and 10 to 14 months, respectively; bars, SE. B, theaverage number of BrdUrd-positive epithelial cells. There were 9, 5, and 9 animals analyzed for control mice ages 4 to 5, 7 to 9, and 10 to 14 months,respectively; and 10, 10, and 9 animals analyzed for PSA-Cre;Pten-loxP/loxP mice ages 4 to 5, 7 to 9, and 10 to 14 months, respectively, Numbers indicateBrdUrd-positive cells per 1,000 epithelial cells; bars, SE. C, the average number of positive cells for active caspase-3 staining. There were 10, 6, and 6 mice forcontrol littermates ages 4 to 5, 7 to 9, and 10 to 14 months, respectively; and there were 10, 10, and 8 mice for PSA-Cre;Pten-loxP/loxP mice ages 4 to 5,7 to 9, and 10 to 14 months. The number of caspase-3-positive cells is indicated as positive cells per 1,000 epithelial cells; bars, SE. D-F, examples of BrdUrdstaining in a control mouse prostate (D), a PSA-Cre;Pten-loxP/loxP mouse prostate at 4 to 5 months (E ) and a prostate from PSA-Cre;Pten-loxP/loxPmouse at 10 to 14 months (F ). Compared with the number of positive epithelial cells in the prostate of normal 4- to 5-month-old littermates (D ), an increasednumber of positively stained epithelial cells was observed in hyperplasia/PIN lesions from 4- to 5-month-old PSA-Cre;Pten-loxP/loxP mice (E). An evenfurther increased number of positive staining epithelial cells was observed in adenocarcinoma from 10- to 14-month-old PSA-Cre;Pten-loxP/loxP mice (F ).G-I, examples of active caspase-3-staining cells in the prostates of control mice (G) and prostates of PSA-Cre;Pten-loxP/loxP mice at 4 to 5 months (H ) and at10 to 14 months (I ). There are hardly any positive cells in normal adult prostate (G), the number of staining cells increased during tumorigenesis (H and I ).Magnifications, (D-I) �400.

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tumors, and hepatocellular carcinoma. Interestingly, in othertissues, like spleen and brain, complete Pten inactivation did notgive rise to tumor formation, although inducing a variety ofmorphologic abnormalities and molecular alterations (33, 44).Taken together, it seems that tumorigenesis or developmentalabnormalities caused by Pten inactivation are rather dependent ontempo-spatial inactivation in certain tissue/cell types.Complementary to the prostate cancer model presented here,

two models have recently been described, based on prostate-specific inactivation of Pten in the Cre-loxP system, one using theMMTV promoter and a second using a (modified) rat probasinpromoter for Cre expression, respectively (27–29). Although thereare parallels, the models differ in various aspects. Differences mightbe explained by differences in activity and cell specificity of thepromoters driving Cre expression, and different genetic back-grounds. MMTV-Cre;Pten-loxP/loxP mice have a C57BL6/129background, PB-Cre4;Pten-loxP/loxP mice have a complex C57Bl/6xDBA2/129/BalbC background, and PSA-Cre;Pten-loxP/loxP miceare 129Ola/FVB. MMTV-Cre-based knock-out mice showed limitedtissue specificity, resulting in Pten recombination not only in theprostate but also in several other tissues, including skin, thymus,and mammary gland (29). In MMTV-Cre;Pten-loxP/loxP mice,hyperplastic prostates were detected a few days after birth; PINlesions were present as early as 2 weeks postnatally. Focal invasionwas noticed in >50% of the prostates at 14 weeks. Nevertheless,MMTV-Cre;Pten-loxP/loxP mice died from other malignancies,precluding investigation of later stages of prostate cancerdevelopment. The PB-Cre-based model is more related to thePSA-Cre model: both are very prostate-specific (27, 28). However, incontrast to the PSA-Cre model, in the PB-Cre-based model theventral prostate is one of the strongest affected lobes and theanterior lobe is least affected. The process of prostate cancerdevelopment in PB-Cre4;Pten-loxP/loxP mice is more aggressivethan observed in the PSA-Cre;Pten-loxP/loxP model. PIN lesionsoccurred at 6 weeks and invasive adenocarcinoma at 9 weeks of

age. Metastases were detected in half of the older mice. In ourstudy, detectable Pten recombination appeared at f4 weeks (datanot shown). PSA-Cre;Pten-loxP/loxP mice showed massive hyper-plasia and focal PIN at 16 to 20 weeks. At older age, more extensivePIN developed, followed by invasive carcinoma (Table 1); onelymph node metastasis was found. We propose that in addition tothe different genetic backgrounds, the differences in activity andcell specificity of the probasin and PSA promoters are the mainunderlying mechanisms responsible for the phenotypic differencesbetween our PSA-Cre-based model and PB-Cre-based model. It canbe presumed that the probasin promoter is active in moreprimitive progenitor cells of mature epithelial cells than the PSApromoter. Although the slower tumor development in the PSA-Cre;Pten-loxP/loxP model has its practical disadvantage, it has theimportant advantage above the PB-Cre4-based model that theconsecutive stages of prostate cancer development can beidentified more easily. Recently, the PSA promoter has also beenused for prostate-specific inactivation of Nkx3.1 (48).Although there was a slight increase in proliferation rate in

prostate epithelial cells in PSA-Cre;Pten-loxP/+ mice, we did notfind any apparent pathologic changes in adult prostates until oldage, when low-penetrant focal dysplasia was detected, indicatingthat monoallelic inactivation of Pten in prostate epithelial cells isinsufficient for prostate cancer development. However, it is quitewell possible that monoallelic Pten loss plus alterations in othersignaling routes lead to tumor development (see below). Themore pronounced prostate phenotypes observed in conventionalPten+/� knock-out mice (9, 22) could be explained by the germline inactivation of one Pten allele, affecting prenatal andpostnatal prostate development. In addition, the absence of oneactive allele in adult prostate stromal cells and in other tissuesmight indirectly influence the properties of prostate epithelialcells.In our prostate cancer model, as well as in the other models,

biallelic Pten inactivation caused an increased proliferation rate ofprostate epithelial cells, indicating an important contribution totumorigenesis (28, 29). Thus far, little is known about the effect ofPten inactivation on apoptosis in the prostate. We did not observe,as might have been expected, decreased apoptosis, but in contrast,increased apoptosis in parallel with the increased proliferation ratein all stages of the tumor model, coupling the apoptotic process tothe proliferation program in PSA-Cre;Pten-loxP/loxP mice. Thisfinding apparently contradicts previous in vitro and in vivoobservations, which correlate Pten inactivation with decreasedapoptosis (42, 49). However, in a few other in vivo studies, Pteninactivation did not lead to reducing apoptosis in agreement withour findings. Podsypanina and colleagues (24) showed absence ofreduced apoptosis in some subsets of thymus and spleenlymphocytes, although there was significantly reduced apoptosisin a variety of bone marrow cell lineages in the same Pten knock-out mouse model. Backman et al. (50) reported the absence ofdifferences between control and Pten-deficient brain cells inTUNEL staining pattern, and Groszer et al. (51) showed asignificant increase in the number of TUNEL-positive cells in thetelencephalon of Pten knock-out mice. Most importantly, similar toour prostate cancer model, in a testis-targeted Pten knock-outmouse model, increased proliferation of primordial germ cells wasassociated with increased apoptosis (43). Several hypotheses mightexplain these observations. Increased apoptosis might be asecondary event following stimulated cell proliferation or changedcell polarity in the Pten-deficient cells. Alternative, Pten-regulated

Figure 6. Complete inactivation of Pten in the prostate leads to rare metastaticdisease. A-D, metastatic prostate adenocarcinoma in a caudal para-aortic lymphnode. A and B, H&E staining showing two foci of cribriform malignant prostatecells at the subcapsular sinusoidal region of the lymphoid node. C, phospho-AKTstaining shows intermediate positive malignant epithelial cells. D, androgenreceptor staining shows positive nuclei of the malignant epithelial cells.Magnifications, �100 (A ); �400 (B-D ).





apoptosis might be a cell or tissue type–specific event. Clearly, therole of Pten in apoptosis in vivo seems more complex thanoriginally envisaged.Tumor initiation and progression generally need multiple

genetic and epigenetic events. In the Pten model, we candiscriminate several clearly defined morphologic stages leadingto prostate cancer, starting with hyperplasia, followed by PINlesions, local invasion, invasive growth in surrounding tissues, andfinally metastasis. We speculate that hyperplasia might solely resultfrom Pten inactivation. Later stages of tumor development mightinclude secondary Pten-dependent or independent genetic orepigenetic alterations. The PSA-Cre;Pten-loxP/loxP model is verywell suited to study these different biological and molecular stagesof prostate tumorigenesis.Although monoallelic Pten inactivation in our model does not

lead to dramatic pathologic changes in prostate, PSA-Cre;Pten-loxP/+ mice are important for experiments aimed at investigatingthe functional synergism between Pten and other tumorsuppressor genes and oncogenes in prostate cancer. Recently,conventional Nkx3.1 knock-out mice as well as TRAMP mice havebeen crossed with Pten+/� knock-out mice. Pten+/�;Nkx3.1.+/�and Pten+/�;Nkx3.1�/� compound mice displayed an increased

incidence of PIN lesions as compared with Nkx3.1+/� andNkx3.1�/� mice (52). In Pten+/� ;TRAMP compound mice theprogression of prostate cancer was accelerated (53). Hence, bothmodels indicate that monoallelic loss of Pten promotes theprogressive growth of prostate cancer under specific conditions.Studies with Pten+/� ;Ink4a/Arf�/� and Pten+/�;Cdkn1b�/�compound mouse models reach similar conclusions (54, 55).The tissue-targeted Pten prostate tumor model described in thepresent study permits careful definition of the synergism betweenPten loss and other genetic changes at well-defined phases oftumor development. This can be achieved by crossing our modelwith other mutant mice as well as by careful monitoring ofadditional genetic changes occurring during the distinct phases ofprostate cancer development we have described here.

Acknowledgments

Received 12/20/2004; revised 3/29/2005; accepted 4/13/2005.Grant support: This study was supported by a grant of the Dutch Cancer Society

Koningin Wilhelmina Fonds (grant EUR 2000-2262).The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Hanneke Korsten for assistance and for stimulating discussion.

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2005;65:5730-5739. Cancer Res Xiaoqian Ma, Angelique C. Ziel-van der Made, Binha Autar, et al. Epithelial Cell Proliferation but not by Reduced ApoptosisLeads to Prostate Cancer Accompanied by Increased

in the Mouse ProstatePtenTargeted Biallelic Inactivation of

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