a role for adam12 in breast tumor progression and stromal ... · a role for adam12 in breast tumor...

A Role for ADAM12 in Breast Tumor Progression and

Stromal Cell Apoptosis

Marie Kveiborg,1Camilla Frohlich,

1Reidar Albrechtsen,

1Verena Tischler,

1Nikolaj Dietrich,

1

Peter Holck,1Pauliina Kronqvist,

3Fritz Rank,

2Arthur M. Mercurio,

4and Ulla M. Wewer

1

1Institute of Molecular Pathology, University of Copenhagen; 2Department of Pathology, Rigshospitalet University Hospital, Copenhagen,Denmark; 3Department of Pathology, Turku University, Turku, Finland; and 4Beth Israel Deaconess Center, Harvard Medical School,Boston, Massachusetts

Abstract

As in developmental and regenerative processes, cell survivalis of fundamental importance in cancer. Thus, a tremendouseffort has been devoted to dissecting the molecular mecha-nisms involved in understanding the resistance of tumorcells to programmed cell death. Recently, the importance ofstromal fibroblasts in tumor initiation and progression hasbeen elucidated. Here, we show that stromal cell apoptosisoccurs in human breast carcinoma but is only rarely seen innonmalignant breast lesions. Furthermore, we show thatADAM12, a disintegrin and metalloprotease up-regulated inhuman breast cancer, accelerates tumor progression in amouse breast cancer model. ADAM12 does not influencetumor cell proliferation but rather confers both decreasedtumor cell apoptosis and increased stromal cell apoptosis.This dual role of ADAM12 in governing cell survival isunderscored by the finding that ADAM12 increases theapoptotic sensitivity of nonneoplastic cells in vitro whilerendering tumor cells more resistant to apoptosis. Together,these results show that the ability of ADAM12 to influenceapoptosis may contribute to tumor progression. (Cancer Res2005; 65(11): 4754-61)

Introduction

ADAM12, a disintegrin and metalloprotease, is a member of theADAMs family, which has >33 members with diverse expressionprofiles and cellular functions (1–3). This group of proteins ischaracterized by a multidomain structure comprised of prodo-main, metalloprotease, disintegrin, and cysteine-rich domains,as well as a transmembrane and cytoplasmic domain. ADAM12is involved in several pathologic conditions characterized byabnormal cell growth (4). Mice deficient in ADAM12 exhibitnormal prenatal development, but f30% died within the firstpostnatal week for unknown reasons (5). Mice surviving beyondthe first postnatal week seemed normal, but some had deficienciesin development of interscapular brown adipose tissue as well asreduced neck and interscapular muscle tissue (5). In a subsequentstudy, we showed that mice overexpressing ADAM12 exhibitincreased adipogenesis (6).In humans, two alternatively spliced forms of ADAM12 are

expressed, the prototype transmembrane form, ADAM12-L, and a

shorter secreted form, ADAM12-S (7). Interestingly, both forms arehighly expressed in placenta (7) and levels of ADAM12 in the serumof pregnant women seem to be a valuable marker for abnormalplacental growth and could potentially serve as a useful marker inclinical prenatal diagnostics (8). ADAM12 is expressed at low levelsin most normal adult tissues, but it is expressed at higher levels ina large proportion of human carcinomas, including breast, gastric,colon carcinomas, and liver metastases (9–11). A recent proteomicapproach identified ADAM12 in the urine of breast cancer patientsand showed that its levels in urine correlated with tumor pro-gression, suggesting that ADAM12 could be used as a noninvasivediagnostic test for breast cancer (12). ADAM12 expression in hu-man glioblastomas was shown to correlate with cell proliferationand shedding of pro-heparin-binding epidermal growth factor(pro-HB-EGF; ref. 13). Finally, it should be mentioned thatmicroarray analysis showed that ADAM12 is up-regulated inaggressive fibromatosis (14).In the present study, we investigated the function of ADAM12

in breast cancer. We show that ADAM12 promotes tumor pro-gression by a mechanism involving the ability of ADAM12 todifferentially influence the apoptotic potential of tumor versusstromal cells.

Materials and Methods

Tissue samples. Breast tissue samples from individuals with breast

carcinomas (20 individuals) and from individuals with nonmalignantbreast tissue (15 individuals) were obtained from patients who underwent

surgery at the Rigshospitalet University Hospital, Copenhagen, Denmark.

In addition, tissue samples from individuals with breast carcinoma

(102 individuals) were obtained from patients who underwent surgeryat the Turku University Hospital or the Jyvaskyla Central Hospital (48) or

purchased as tissue arrays (54) from Zymed Laboratories (South San

Francisco, CA).The experimental use of the surgical specimens was in accordance

with the guidelines of the Danish Breast Cancer Group and the hospital

ethical guidelines of Turku University Hospital and Jyvaskyla Central

Hospital, respectively.Transgenic mice. To generate mammary gland–specific ADAM12

transgene expression, a modified version of the mouse mammary tumor

virus long terminal repeat promoter/enhancer (MMTV-LTR)-Sv40-Bssk

vector (a generous gift from R.G. Oshima, The Burnham Institute, La Jolla,CA) was used. Briefly, the EcoRI site of the vector’s multiple cloning site

was replaced by PacI. cDNAs encoding either the transmembrane human

ADAM12-L lacking the cytoplasmic tail (ADAM12-Dcyt) or the secretedform of human ADAM12 (ADAM12-S) were cloned into the PacI site of

Sv40-Bssk vector. PvuI and SpeI were used to excise MMTV-ADAM12-S

and MMTV-ADAM12-Dcyt fragments, which were injected into the male

pronucleus of fertilized zygotes isolated from superovulated donor mice.Injected embryos were implanted into pseudopregnant recipients and

allowed to develop to term. For the ADAM12-S transgenic mice, C57BL/6J �CBA, F1 mice were used as donors and recipients and for backcrossing,

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

M. Kveiborg, C. Frohlich, and R. Albrechtsen contributed equally to this work.Requests for reprints: Ulla M. Wewer, Institute of Molecular Pathology, University

of Copenhagen, Frederik V’s vej 11, 2100 Copenhagen, Denmark. Phone: 45-3532-6056;Fax: 45-3532-6081; E-mail: [email protected].

I2005 American Association for Cancer Research.

Cancer Res 2005; 65: (11). June 1, 2005 4754 www.aacrjournals.org

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whereas the inbred mouse strain, FVB/n, was used for the MMTV-ADAM12-Dcyt mice. C57BL/6J and FVB/n mice were purchased from M&B

(Copenhagen, Denmark).

Heterozygous transgenic FVB/N-TgN(MMTV-PyMT)634Mul mice ex-

pressing polyomavirus middle T antigen (PyMT) in the mammary glandunder transcriptional control of the MMTV-LTR were obtained from The

Jackson Laboratory (Bar Harbor, ME). MMTV-PyMT males were crossed

with females from the transgenic lines MMTV-ADAM12-S (C57BL/6J �CBA, F1) and MMTV-ADAM12-Dcyt (FVB/n) to generate ADAM12-S/PyMTand ADAM12-Dcyt/PyMT mice, respectively, and their corresponding PyMT

littermates. All animals analyzed in this study were females.

The MMTV-ADAM12-S and MMTV-ADAM12-Dcyt transgenic mice were

identified by PCR on tail genomic DNA using the human ADAM12forward primer 5V-CAGGATCCAGAGAGACCCTCAAG-3V and the reverse

primer 5V-CAACTCGAGCGGCAGGTTAAACAG-3V. The PyMT transgene was

identified using the sense primer 5V-GGAAGCAAGTACTTCACAAGGG-3Vand the antisense primer 5V-GGAAAGTCACTAGGAGCAGGG-3V.

After weaning, all animals were both weighed and examined for mam-

mary gland tumors by palpation twice a week. The date that the first tumor

appeared was noted. Mice were sacrificed at 12 weeks of age. One hourbefore sacrifice, 5-bromo-2V-deoxyuridine (BrdUrd; 50 Ag/g body weight)

was injected i.p. The tumors were dissected, weighed, and divided into

pieces, which were either frozen in liquid nitrogen or fixed in formalin for

further analysis. The lungs were removed, fixed in formalin, and embeddedin paraffin for further evaluation of metastasis.

All experiments were conducted in accordance with the guidelines of

the Animal Experiment Inspectorate, Denmark.Cell culture. Mouse 3T3-L1 fibroblastic preadipocytes (CL-173), CHO-K1

Chinese hamster ovarian cells (CCL-61), human MG-63 osteosarcoma

(CRL-1427), human A-431 squamous cell carcinoma (CRL-1555), human

HeLa adenocarcinoma (CCL-2), and normal human lung fibroblasts MRC-5(CCL-171) were obtained from the American Type Culture Collection

(Rockville, MD). HEK293-EBNA cells were obtained from Invitrogen

(Tastrup, Denmark). All cell lines except CHO-K1 were cultured in DMEM

with Glutamax I and 4,500 mg/L glucose, 50 units/mL penicillin, 50 Ag/mLstreptomycin, and 10% fetal bovine serum (FBS); for MRC-5 cells, 1 mmol/L

sodium pyrovate (Invitrogen) was also included in the culture media.

CHO-K1 cells were maintained in DMEM/F12 (1:1; Invitrogen) supple-mented with 50 units/mL penicillin, 50 Ag/mL streptomycin, and 10% FBS.

3T3-L1 cells stably expressing hADAM12-Dcyt or vector control were

generated by retroviral transduction as described previously (15) and kept

in selection media (2.0 Ag/mL puromycin). CHO-K1 cells were transfectedusing the FuGene 6 Transfection Reagent (Roche Diagnostics, Mannheim,

Germany) according to the manufacturer’s instructions.

Antibodies and reagents. Monoclonal antibodies (6E6, 8F8, 6C10) and

polyclonal antiserum (rb 122, rb 109) to ADAM12 were generated as

previously described (7, 16). Rabbit monoclonal antibodies to cyclin D1

(clone SP4) were obtained from Lab Vision (Fremont, CA); rabbit polyclonal

IgG to Bax (sc-493) was purchased from Santa Cruz Biotechnology, Inc.

(Santa Cruz, CA); monoclonal antibody to actin (MAB 1501R) was

purchased from Chemicon (Temecula, CA); rabbit anti-BrdUrd antibody

was obtained from Roche (Indianapolis, IN); and rabbit antibody to active

caspase3 (ab2302) was purchased from Abcam, Ltd. (Cambridge, United

Kingdom). Secondary antibodies were obtained from Dako A/S (Glostrup,

Denmark). Recombinant ADAM12-S was generated by transfecting human

HEK293-EBNA cells with cDNA encoding human ADAM12-S and purified

using fast protein liquid chromatography cation exchange and concanavalin

A affinity chromatography as described previously (17). Recombinant

human tumor necrosis factor (TNF) a and mouse mTNFa were purchased

from Strathmann Biotec AG (Hamburg, Germany). Cycloheximide and

BrdUrd were purchased from Sigma-Aldrich (Brøndby, Denmark). The

broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH2F (zVAD-fmk)

was obtained from Bachem (Bubendorf, Switzerland).

Histologic analysis and immunohistochemical staining. Tissuesamples were fixed in formalin and embedded in paraffin; 4 Am sections

were stained with H&E for morphologic analysis. Immunostaining

procedures were done as previously described (10). To examine tissue

sections for apoptosis, ApoTag reagents were used according to themanufacturer’s instructions (Invitrogen). Each tissue section was scanned,

enlarged, and the approximate area of the tissue section estimated using

square transparent paper overlay imprinted with squares measuring

25 mm2. The numbers of apoptotic and necrotic cells present in eachsection of tissue were counted and the numbers per mm3 estimated

essentially as previously described (18).

Samples from mammary gland tumors were obtained from 10 ADAM12-

S/PyMT compared with 10 PyMT littermates and 10 ADAM12-Dcyt/PyMTcompared with 10 PyMT littermates and processed as described above.

Malignant (early and late carcinoma) and premalignant (hyperplasia and

adenoma) areas were marked and the areas estimated. The measurements

in both cases were carried out independently by two observers.Western blotting. Breast tumor tissue from humans and mice and

cell extracts were examined by Western blotting as described previously

(6, 15, 16). In brief, proteins were extracted in radioimmunoprecipitationassay buffer [50 mmol/L Tris-HCl (pH 7.4), 1% Triton X-100, 25 mmol/L

HEPES, 150 mmol/L NaCl, 0.2% deoxycholate, 5 mmol/L MgCl2, 1 mmol/L

Na3VO4, 1 mmol/L NaF, and a protease inhibitor cocktail (complete, EDTA-

free protease inhibitor cocktail tablets; Roche Molecular Biochemical,Hvidovre, Denmark)]. In some cases, immunoprecipitation with monoclonal

antibodies (6E6, 8F8, 6C10) and protein-G sepharose beads (Amersham

Pharmacia Biotech, Uppsala, Sweden) was done. Detection of ADAM12 was

done using rb 122 as a primary antibody, horseradish peroxidase–conjugatedsecondary antibody, and the chemiluminescence SuperSignal reagent

(Pierce, Cheshire, United Kingdom). Detection of Bax and actin was done

by Western blotting using specific primary antibodies as described above.Construction of plasmids for in vitro experiments. cDNAs encoding

human ADAM12-L truncated at nucleotide 2,498, resulting in a membrane-

inserted protein lacking the cytoplasmic tail (ADAM12-Dcyt), and ADAM12-

Dcyt with a E351-Q catalytic site mutation were cloned into the pEGFP-N1vector (Clontech Laboratories, Heidelberg, Germany) to create COOH-

terminal fusion proteins with enhanced green fluorescence protein (EGFP)

or into pBABEpuro for retroviral transduction as previously described (15).

cDNA for ADAM12-S was cloned into the pCEP4 vector (Invitrogen) forepisomal expression in HEK293-EBNA cells.

Cytotoxicity assay and detection of condensed nuclei of culturedcells. Apoptosis was induced by treating cells with varying concentrations

of TNFa in the presence of 5 Amol/L cycloheximide for 24 hours, except

for 3T3-L1 cells that were treated for 5 hours. Alternatively, cell media was

removed and the cells were subjected to UVC irradiation (254 nm) with

60 J/m2. After irradiation, fresh media was added and the cells were

incubated for 24 hours. Cell cytotoxicity was analyzed using the Cytotoxicity

Detection kit (Roche) as described by the manufacturer. Briefly, 5,000 to

10,000 cells were cultured on 96-well plates 24 hours before induction.

Following induction, cytotoxicity was quantitated by measuring the lactate

dehydrogenase activities in the culture media (M) and in the attached cells

after lysis in media with 1% Triton X-100 (A). Calculation of cytotoxicity

was done using the ratio M/(M + A). Nuclear condensation was detected

by staining the cells with membrane permeable Hoechst 33342 (Molecular

Probes, Leiden, the Netherlands). To avoid washing away poorly attached

cells, media was carefully removed from the cell layer. Next, cells were fixed

and stained simultaneously by adding 4% paraformaldehyde in PBS

containing 2.5 Ag/mL Hoechst 33342 and incubating for 30 minutes at

room temperature. Finally, cells were washed once with cold PBS before

visualization by fluorescence microscopy and counting the percentage

of cells with condensed and brightly stained nuclei. When counting the

percentage of green cells with condensed nuclei, >100 cells were counted.

When counting the percentage cells with condensed nuclei out of all cells,

at least four different fields were counted. The numbers represent

independent results from two individuals that were obtained in a blinded

manner in at least two independent experiments.Annexin V/propidium iodide analysis. HeLa cells were treated with

purified recombinant ADAM12-S or vehicle for 24 hours followed by

18-hour treatment with 10 ng/mL hTNFa and 5 Amol/L cycloheximide in

the presence of recombinant ADAM12-S. The percentage of apoptotic cellswas determined using an annexin V-FITC detection kit (Oncogene, Inc.,

ADAM12 and Tumor Stromal Cell Apoptosis

www.aacrjournals.org 4755 Cancer Res 2005; 65: (11). June 1, 2005



Boston, MA) according to manufacturer’s instructions. Briefly, cells weretrypsinized, resuspended in the culture medium, and washed in PBS. Cells

were stained with annexin V-FITC for 15 minutes and washed; propidium

iodide was then added before the cells were analyzed with a FACScalibur

machine (BD Biosciences, Palo Alto, CA). The cells were kept on ice untilthe fluorescence-activated cell sorting (FACS) analysis was completed.

Cell proliferation assays in vitro . DNA synthesis was determined

using the 5-bromo-2V-deoxyuridine labeling and detection kit from

Boehringer Mannheim (Mannheim, Germany). Briefly, cells were incubatedfor 60 minutes at 37jC with 10 Amol/L BrdUrd and subsequently fixed in

methanol. DNA was denatured by incubating in 2 N HCl for 15 minutes

followed by neutralization in 0.1 mol/L borate buffer (pH 8.5). BrdUrd

incorporation was detected using an anti-BrdUrd antibody as described bythe manufacturer. Short-term growth rates were determined by seeding

10,000 cells/well in 24-well plates. At days 1, 2, 3, and 4 after seeding, cells

in four individual wells were trypsinized and counted. Trypan blue dye(0.4% w/v) exclusion was used to ensure equal viability of the cells.

Statistical analysis. Statistical analysis was done using Kaplan-Meier to

determine tumor-free periods, and the log-rank test, the Mann Whitney test,

or the Student’s t test for comparing two independent groups. P < 0.05 wereconsidered statistically significant.

Results and Discussion

To evaluate a possible role for ADAM12 in breast carcinoma,we first assessed its expression in human breast tumors.Immunostaining of tissue specimens from human breast carci-nomas and nonneoplastic lesions using a polyclonal antiserum(rb 122) to ADAM12 revealed that most of the malignant breasttumors tested exhibited ADAM12 immunoreactivity, whereasnonmalignant breast lesions (i.e., fibroadenomas) exhibited faintor no immunostaining (Fig. 1A and C ; ref. 10). We confirmed thattumor cells express ADAM12 using another polyclonal antiserum(rb 109) raised against the cytoplasmic tail of ADAM12 and amonoclonal antibody to ADAM12 (6E6; data not shown). Westernblotting identified expression of both the 110 kDa proform andthe 90 kDa mature form of ADAM12-L in human breastcarcinoma extracts (Fig. 1B). Based on previous results (15), wehypothesized that ADAM12 may influence tumor cells apoptosis.For this reason, we examined the pattern of apoptosis in thesame tumors using the ApoTag assay. We detected the presenceof apoptotic tumor cells in breast carcinomas as expected, butimportantly also observed apoptotic stromal cells in breastcarcinomas (Fig. 1D and E). In contrast, apoptosis was observedonly occasionally in the stroma of normal and nonmalignantbreast lesions (Fig. 1F). Quantitative assessment showed anapoptotic index of 2.0 F 1.0% in malignant tumors (n = 14) and0.3 F 0.5% in nonneoplastic lesions (n = 11; P < 0.0001; Fig. 1G).The presence of apoptotic cells in the stroma was confirmedusing antibodies to caspase 3 (data not shown).ADAM12 exists in two alternatively spliced forms, the prototype

transmembrane form, ADAM12-L, and the shorter secreted form,ADAM12-S (7). The main functions of ADAM12, includingproteolysis and interaction with syndecan and integrins, aremediated via its extracellular part, whereas the cytoplasmic tailmight tightly regulate cellular localization (16, 19). Thus, to testthe role of ADAM12 in breast tumor progression, we made two linesof transgenic mice using the MMTV-LTR (Fig. 2A): one expressingmembrane-anchored ADAM12-L lacking the cytoplasmic tail toenrich cell surface expression (ADAM12-Dcyt; ref. 19) and the otherexpressing the secreted form, ADAM12-S, mimicking the findingof ADAM12-S in the urine of breast cancer patients (12). MMTV-ADAM12-Dcyt and MMTV-ADAM12-S transgenic mice exhibited no

apparent differences in the mammary gland during developmentcompared with their control littermates (data not shown). Also,neither nulliparous nor multiparous females observed for>18 months developed spontaneous breast tumors. These micewere bred with mice carrying the polyomavirus middle Toncogene (MMTV-PyMT). PyMT expression in the femalemammary gland results in rapid development of multifocalmammary adenocarcinomas (20) sharing many features withhuman breast cancer (21). Using Western blotting, PyMT tumorswere found to express little endogenous ADAM12, whereas largeamounts of ADAM12 was detected in PyMT breast tumor tissuein which the ADAM12 gene was introduced (Fig. 2B). In femaleoffspring, tumors in ADAM12-Dcyt/PyMT mice emerged by day48 (F10), 10 days earlier than PyMT mice (day 58 F 12;P = 0.0823; Fig. 2C), whereas ADAM12-S/PyMTmice had palpabletumors by day 41 (F5), 9 days earlier than PyMT littermates (day 50F 7; P = 0.0003; Fig. 2D). All mice were euthanized at 12 weeks ofage, at which time all had developed palpable tumors. The averagetotal tumor mass in ADAM12-Dcyt/PyMT mice was 2.09 F 0.62 gcompared with 1.22 F 0.67 g in PyMT littermates (P = 0.0153;Fig. 2E). The average total tumor mass in ADAM12-S/PyMT micewas 2.06 F 0.97 g compared with 1.15 F 1.06 g in PyMT littermates(P = 0.0005; Fig. 2F). Thus, enhanced ADAM12 expression in PyMTtumors accelerates tumor development and increases tumorburden.The histopathologic appearance of breast tumors was evaluated

using previously described criteria (21). Premalignant areas werecharacterized by hyperplasia or adenoma exhibiting scattered andmoderate cyclin D staining and located in a loose connective tissuerich in adipocytes. Malignant areas were characterized by thepresence of less-differentiated tumor cells exhibiting polymor-phism and frequent mitoses that had invaded the surroundingconnective tissue. These areas exhibited widespread and intensecyclin D staining (not shown), were densely packed with tumorcells, and contained less adipocyte-containing loose connectivetissue than the premalignant areas (Fig. 2G and H). Multiplesections of ADAM12-Dcyt– and ADAM12-S–expressing PyMTtumors were compared with PyMT tumors to estimate the relativeareas characterized as premalignant or malignant lesions. ADAM12-expressing tumors had significantly larger areas of malignanttumor tissue than did PyMT littermates (P < 0.004) and a reductionin premalignant areas (Fig. 2I and J). The frequency of lungmetastasis was also evaluated. In ADAM12-Dcyt/PyMT mice(n = 12), 77.8% showed lung metastases compared with 25% intheir PyMT littermates (n = 12; P < 0.03). In ADAM12-S/PyMTmice,the frequency of metastasis was not significantly different from thePyMT littermates (not shown). These results support the notionthat ADAM12 confers increased malignancy in this mouse model.We next evaluated the degree of cell proliferation and apoptosis

in PyMT breast tumors. For 5-bromo-2V-deoxyuridine (BrdUrd)incorporation into tumor cells, no difference was observedbetween ADAM12-expressing PyMT mice and their littermatecontrols (11.6 F 1.8% versus 11.7 F 1.3% labeled cells, P = 0.42),suggesting the overall rate of tumor cell proliferation was notsignificantly influenced by ADAM12 expression (Fig. 3A and B).Because the effect of ADAM12 on tumor progression might notbe primarily caused by changes in tumor cell proliferation, weasked whether ADAM12 might influence cell survival byexamining the level of apoptosis in both tumor and stromalcells. First, we estimated the percent of apoptotic tumor cellsusing the ApoTag assay (Fig 3C); 1.59 F 0.29% of tumor cells in

Cancer Research




ADAM12-Dcyt/PyMT mice and 4.54 F 1.01% of tumor cells inPyMT mice were found to be apoptotic (P = 0.0015), suggestingthe overall level of tumor cell apoptosis was reduced in ADAM12-expressing PyMT breast tumors. These results correlated withWestern blot data demonstrating a reduced level of proapoptoticBax protein expression in ADAM12-Dcyt/PyMT tumor tissuecompared with tumor tissue of PyMT littermates (Fig. 3D). Next,we examined the influence of ADAM12 on stromal cell apoptosis.Interestingly, we found an increase in apoptotic cells in the stromaof ADAM12-expressing tumors compared with their littermatecontrols as assessed by hematoxylin staining (data not shown), by

ApoTag staining (Fig. 3E-G), and by immunostaining withantibodies to caspase 3 (Fig. 3H). A statistically significantdifference in the number of apoptotic stromal cells was observedin ADAM12-Dcyt/PyMT mice compared with that of PyMTlittermates (P = 0.003; Fig. 3I) and in ADAM12-S/PyMT tumorscompared with that of PyMT littermates (P = 0.049; Fig. 3J). Theseresults strongly suggest that ADAM12 may accelerate tumorprogression by reducing tumor cell apoptosis and by increasingstromal cell apoptosis.To extend these in vivo findings, we examined the effect of

ADAM12 on cell proliferation and on sensitivity to apoptotic

Figure 1. ADAM12 and stromal cell apoptosis inhuman breast carcinomas. A, microscopic image ofADAM12 immunostaining in the tumor cells (arrows ) of ahuman breast carcinoma (intraductal c. not otherwisespecified). Normal duct cells (arrowhead) do not exhibitimmunostaining. B, Western blotting of ADAM12 inextracts of human breast carcinoma. C, quantitativeassessment of ADAM12-positive and ADAM12-negativebreast carcinomas. D and E, ApoTag staining ofapoptotic cells (brownish color ) in breast carcinomas.Note apoptotic cells in the stroma (arrows ) in additionto apoptotic tumor cells. F, ApoTag staining of afibroadenoma with no signs of apoptosis in the stroma,but with a few apoptotic cells in the epithelium (arrow ).G, quantitative assessment of stromal apoptosis innonmalignant breast lesions and malignant breastcarcinoma. **, P < 0.01. In (A ) and (D -F ), nuclei arecounterstained with hematoxylin. Bars , 40 Am (A);10 Am (D ); 15 Am (E); and 40 Am (F ).





stimuli in vitro . To mimic in vivo situations, we exposed bothnonmalignant and malignant cells to either membrane-anchoredADAM12 (ADAM12-Dcyt) or secreted ADAM12-S. Cell proliferationassays showed that ADAM12 decreased growth of nonmalignantcells but had no effect on tumor cell growth (Supplementary Fig. S1).Interestingly, nonmalignant cells of both mesenchymal andepithelial origin (3T3-L1 and CHO-K1, respectively) expressingADAM12-Dcyt were more sensitive to apoptosis induced by treat-

ment with TNFa or UV-irradiation than their control cells notoverexpressing ADAM12 (Fig. 4A and B). As a specificity control,cells were treated with the broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-CH2F (zVad-fmk), which completely abolished apo-ptosis (Fig. 4B). We next tested the effect of adding purifiedhuman full-length ADAM12-S on TNFa-induced apoptosis.Nonmalignant cells treated with ADAM12-S exhibited anincreased sensitivity to apoptosis compared with that of cells

Figure 2. ADAM12 accelerates tumor progressionin a mouse breast cancer model. A, schematicdemonstration of the MMTV-ADAM12-Dcyt and theMMTV-ADAM12-S transgene constructs. B, Westernblot analysis of the expression of ADAM12 in breasttumor tissue of ADAM12-Dcyt/PyMT and ADAM12-S/PyMT transgenic mice, compared with their respectivelittermate PyMT controls. C and D, a Kaplan-Meiertumor free duration curve of PyMT mice (n = 13)compared with ADAM12-Dcyt/PyMT (n = 10) andPyMT mice (n = 15) compared with ADAM12-S/PyMT(n = 16). E and F, total tumor mass (g) isolated fromPyMT mice (n = 10 in each group; black columns )compared with ADAM12-Dcyt/PyMT (n = 10) andADAM12-S/PyMT transgenic mice (n = 10; whitecolumns ). G and H, histopathology at lowmagnification of H&E-stained sections of a PyMTtumor compared with an ADAM12-Dcyt/PyMT tumor,respectively. Light area in the PyMT tumor sectionindicates connective tissue rich in adipocytes. Bar ,1.7 mm. I and J, histopathologic assessment of therelative percent of premalignant or malignant areas ofmultiple sections of tumors from PyMT mice comparedwith tumors from ADAM12-Dcyt/PyMT and ADAM12-S/PyMT mice, respectively. The measurements werecarried out independently by two observers. For eachgroup, n = 10. *, P < 0.05, **, P < 0.01.

Cancer Research




not exposed to ADAM12-S (Fig. 4C and D). In sharp contrast, theaddition of ADAM12-S to malignant cells (HeLa, A-431, and MG-63) rendered them resistant to TNFa-induced apoptosis asassessed by FACS analysis of annexin V– and propidiumiodide–positive cells and by quantifying the percentage ofcondensed nuclei (Fig. 4E and F). These results show thatADAM12 had opposing effects on nonmalignant and malignanttumor cells; nonmalignant cells became more sensitive toapoptosis, whereas malignant cells became more resistant.The molecular mechanism for the apparent paradoxical effect

of ADAM12 on tumor cells and tumor-associated stromal cells is

not clear, but overexpressing a protease-deficient form ofADAM12 with a catalytic site mutation was sufficient to conferincreased apoptotic sensitivity to nonmalignant cells in vitro(Supplementary Fig. S2), thereby suggesting that ADAM12protease activity at least under these in vitro conditions is notlikely to be involved. Instead, ADAM12 may possibly mediate itseffects through its binding partners h1 integrin and possiblysyndecans, also known to be important in tumor invasion andmetastasis (22–25).Our findings provide novel insight into the role of ADAM12

in malignant tumors. ADAM12 increased tumor aggressiveness

Figure 3. ADAM12 decreases tumor cell apoptosis andincreases stromal apoptosis. A and B, immunostainingwith antibodies to BrdUrd in PyMT and ADAM12-Dcyt/PyMT mice, respectively. C, quantitative assessment ofApoTag-positive apoptotic tumor cells in PyMT andADAM12-Dcyt/PyMT mice. D, Western blotting of tumorextracts with anti-Bax antibody. An actin antibody wasused to assess equivalent loading. E and F, microscopicimages of ApoTag staining in tissue sections fromADAM12-Dcyt/PyMT mice. G, microscopic image ofApoTag staining in tumors from PyMT mice. Arrows pointto apoptotic stromal cells characterized by shrinkage,nuclear condensations, and apoptotic bodies (E and F )and to an apoptotic tumor cell (G ). H, caspase3immunostaining of stromal cells of ADAM12-Dcyt/PyMTmice. Arrows point to caspase3-positive fibroblasticstromal cells. I and J, quantitative assessment of stromalapoptotic cells in tumors from PyMT and ADAM12-Dcyt/PyMT mice and in tumors from PyMT and ADAM12-S/PyMTmice, respectively. Bars , 40 Am (A and B ); 15 Am(E and F ); and 25 Am (G and H ).





(by decreasing time for tumor onset, increasing tumor burdenand metastasis, and increasing the degree of malignancyhistologically) and, importantly, decreased tumor cell apoptosis.Unexpectedly, we observed that ADAM12 increased stromal cellapoptosis both in vivo and in vitro . We suggest that increasedstromal cell apoptosis might work in concert with increasedtumor cell resistance to apoptosis, both favoring tumorprogression. More specifically, we propose that increased stromalcell apoptosis contributes to the ongoing remodeling of thestromal compartment. It has been recently suggested thatstromal cell apoptosis in human breast carcinomas might be anew biomarker of malignancy (26). In conclusion, we show thatADAM12 facilitates breast cancer progression and we propose

that the mechanism involved relates to the ability of ADAM12 toinfluence the apoptotic potential of tumor and stromal cellsdifferentially.

AcknowledgmentsReceived 1/26/2005; revised 3/1/2005; accepted 3/30/2005.

Grant support: Danish Cancer Society; Danish Medical Research Council; DanskKræftforskningsfond; Neye, Velux, Novo Nordisk, Lundbeck, Munksholm, Friis,Thaysen, and Haensch Foundations; and Bayer AG.

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 Dr. Teijo Kuopio (Jyvaskyla Central Hospital, Jyvaskyla, Finland) for helpwith tissue samples; Xiufeng Xu and Eva Engvall for help with generating the firstADAM12 transgenic mice; Brit Valentin, Jacqueline Tybjerg, and Signe Breum for tech-nical assistance; and Linda Raab and Dana Beitner-Johnson for editing the manuscript.

Figure 4. ADAM12 increases apoptoticsensitivity in cultured nonmalignant cellsbut renders cultured tumor cells moreresistant to apoptosis. A, percentage ofcytotoxicity in ADAM12-Dcyt expressing3T3-L1 cells (white columns ) or controlcells (black columns ) after 5-hourtreatment with 1 ng/mL mTNFa and5 Amol/L cycloheximide. B, CHO-K1 cellswere transiently transfected with constructsencoding EGFP (black columns ) orADAM12-Dcyt fused to EGFP (whitecolumns ). The percentage of green cellswith condensed nuclei 24 hours after UVCirradiation (60 J/m2) with or withoutzVad-fmk (100 Amol/L) or after 24-hourtreatment with 1 ng/mL mTNFa and5 Amol/L cycloheximide with or withoutzVad-fmk (100 Amol/L). C, percentage ofMRC-5 cells nuclei in cultures treated withcontrol vehicle (black columns ) or 1 ng/mLpurified recombinant ADAM12-S (whitecolumns ) for 24 hours followed by 24-hourtreatment with or without 100 ng/mLhTNFa and 10 Amol/L cycloheximide.D, percentage of CHO-K1 cells withcondensed nuclei in cultures treated withcontrol vehicle (black columns ) or 1 Ag/mLpurified recombinant ADAM12-S (whitecolumns ) 24 hours after UVC irradiation(254 nm, 60 J/m2) or after 24-hourtreatment with 1 ng/mL mTNFa and5 Amol/L cycloheximide. E, annexinV-FITC–positive (FL1-H ) and propidiumiodide–positive (FL2-H ) HeLa cells incultures treated with 1 ng/mL purifiedrecombinant ADAM12-S or control vehiclefor 24 hours followed by 18-hour treatmentwith or without 10 ng/mL hTNFa and5 Amol/L cycloheximide. F, percentage ofcells with condensed nuclei in culturestreated with control vehicle (black columns)or 1 ng/mL purified recombinantADAM12-S (white columns ) for 24 hoursfollowed by 24-hour treatment with orwithout hTNFa and cycloheximide asfollows. HeLa: 10 ng/mL hTNFa and5 Amol/L cycloheximide; A-431: 100 ng/mLhTNFa and 10 Amol/L cycloheximide;MG-63: 100 ng/mL hTNFa and 10 Amol/Lcycloheximide. A , B , D , and E, average ofat least three independent experiments.C and F, cells in at least four independentfields were counted in each experiment;a representative of two independentexperiments is shown. The numbersrepresent independent results from twoindividuals obtained in a blinded manner.*, P < 0.05, **, P < 0.01.

Cancer Research




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2005;65:4754-4761. Cancer Res Marie Kveiborg, Camilla Fröhlich, Reidar Albrechtsen, et al. Stromal Cell ApoptosisA Role for ADAM12 in Breast Tumor Progression and

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