Overexpression of Membrane-type Matrix Metalloproteinase ......overexpression of MT1-MMP induced remodeling of the extracellular matrix and tumor formation in the mammary glands of

[CANCER RESEARCH 61, 984–990, February 1, 2001]

Overexpression ofMembrane-type Matrix Metalloproteinase-1Gene InducesMammary Gland Abnormalities and Adenocarcinoma inTransgenic Mice1

Hye-Yeong Ha, Hyung-Bae Moon, Myoung-Soo Nam, Jeong-Woong Lee, Zae-Yoong Ryoo, Tae-Hoon Lee,Kyung-Kwang Lee, Byung-Jan So, Hiroshi Sato, Motoharu Seiki, and Dae-Yeul Yu2

Laboratory of Animal Developmental Biotechnology, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-333, Korea [H-Y. H., M-S. N., T-H. L., K-K. L.,D-Y. Y.]; Research Institute of Medical Science, Catholic University of Korea, Seoul 137-040, Korea [J-W. L., Z-Y. R.]; Departments of Pathology [H-B. M.] and Surgery [B-J. S.], School of Medicine, Wonkwang University, Iksan 570-749, Korea; Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University,Kanazawa 920, Japan [H. S.]; and Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo 108-639, Japan [M. S.]

ABSTRACT

To investigate the role of membrane-type matrix metalloproteinase-1(MT1-MMP) in mammary gland development and tumorigenesis, trans-genic mice overexpressingMT1-MMP in mammary gland under the con-trol of the mouse mammary tumor virus long terminal repeat-promoterwere generated. The mouse mammary tumor virus/MT1-MMP transgenicmice displayed abnormalities in 82% of female mammary glands. Theabnormalities were verified as lymphocytic infiltration, fibrosis, hyper-plasia, alveolar structure disruption, dysplasia, and adenocarcinoma.Northern and reverse transcription-PCR analyses demonstrated thatMT1-MMP mRNA was overexpressed in mammary glands exhibitingabnormalities. Western blot analysis and immunohistochemical studieshave revealed that the protein expression level was also increased in theseglands. In addition, theb-caseingene as a functional epithelial cell markerwas poorly expressed in the mammary glands of transgenic mice exhib-iting abnormalities. Gelatin zymography showed significantly increasedMMP-2 activation in these mammary glands. These results showed thatoverexpression ofMT1-MMP induced remodeling of the extracellularmatrix and tumor formation in the mammary glands of transgenic mice.Therefore, we suggest that overexpression ofMT1-MMP may play a keyrole in development and tumorigenesis in mammary glands.

INTRODUCTION

MMPs,3 which degrade the various components of ECM, playcritical roles in the tissue remodeling of multicellular organisms aswell as in tumor invasion (1–4). MMPs may play a role in any one ofmultiple critical events in tumor evolution, including tumorigenesis,tumor growth, angiogenesis, generation of reactive stroma, and tumorcell invasion and metastasis (5). For example, the lack of MMP-7 inmice showed a reduction in intestinal tumorigenesis (6), and itsoverexpression in mammary tissue accelerates mammary tumor for-mation in mice carrying theMMTV/ErbB-2transgene (7). In addition,MMP-2-defective mice showed reduced angiogenesis and tumor pro-gression (8). MMP-11 knockout mice showed reduced tumorigenesisin response to chemical mutagenesis (9).

Whereas the majority of the MMPs are secreted as soluble enzymesinto the extracellular milieu, a subset of MMPs have been identifiedin recent years to contain additional sequences capable of anchoringon plasma membrane (10–15). Named after the putative transmem-

brane domains as MT-MMP-1 through -5, these enzymes have beenproposed to be the master switches of ECM turnover based on thepurported ability of MT-MMPs to activate other MMPs such asproMMP-2 and MMP-13. ProMMP-2 and MMP-13 are degradativeenzymes widely implicated in tumor invasion and metastasis (10, 16, 17).

As do other MMPs, MT1-MMP has also been proposed to playcritical roles in both physiology and pathology by remodeling theECM. MT1-MMP expression is particularly high in kidney duringmouse embryogenesis and also in the adult human (12, 18). Recentdata indicate that MT1-MMP may also function as a fibrinolyticenzyme in the absence of plasmin and mediate pericellular proteolysisin angiogenesis (19). Recently it was reported that MT1-MMP-defi-cient mice develop dwarfism, osteopenia, arthritis, and connectivetissue disease because of inadequate collagen turnover (20). MT1-MMP is also overexpressed in various tumor tissues, including humancolon, breast, and head and neck carcinoma (10, 21–24). AlthoughMT1-MMP expression has been proved in numerous tumors, the rolesassigned to MT1-MMP in tumorigenesis and tumor progression arerelatively poorly understood. In the present study, we generatedMMTV/MT1-MMP transgenic mice and examined premalignant ab-normalities and adenocarcinoma in mammary glands. The resultssuggest that overexpression ofMT1-MMP may play a key role indevelopment and tumorigenesis in mammary glands.

MATERIALS AND METHODS

Generation of MMTV/MT1-MMP Transgenic Mice. To generate a vec-tor pmMT1, a 1.8-kb mouse MT1-MMP cDNA of full length for the codingsequence was ligated into theSalI andXhoI sites of the mammalian expressionvector pMAM-neo (Clonetech, Palo Alto, CA; Ref. 25). AHindIII DNAfragment (4.4 kb) containing MMTV-LTR, MT1-MMP cDNA, and SV40polyadenylation sequences was microinjected into the pronuclei of fertilizedmouse eggs obtained from C57BL/63 DBA F1 hybrid females as described(Ref. 26; Fig. 1). The DNA-injected eggs were transferred to pseudopregnantICR female mice. Transgenic mice were identified by PCR analysis of thegenomic DNA using primers specific to mMT1-injection DNA. The oligonu-cleotides used for the amplification were a forward primer 59-ACA-AGA-GCG-CAA-CGG-ACT-CA-39complementary toMMTV LTRgene sequencesand 59-ACG-GTG-TAA-GCT-CCG-GTA-39specific to theMT1-MMP gene.

Histological and Immunohistochemical Stain.Mammary tissues wereobtained from wild-type and transgenic mice at various stages of development.Tissues were fixed in neutral buffered 10% formalin overnight and embeddedin paraffin, sectioned at 4mm, and stained with H&E. For immunohistochem-ical staining, the 4-mm paraffin-embedded sections were prepared on theProbe-on Plus slides (Fisher, Pittsburgh, PA) and deparaffinized by xylene.Next, tissue sections were rehydrated in PBS solution, and then the slides wereblocked in 3% hydrogen peroxide for 10 s. The slides were washed twice inImmuno/DNA buffer solution (Research Genetics, Huntsville, AL) and thenincubated in protein blocker solution (Research Genetics) for 3 min. Thesections were incubated at 4°C overnight with the monoclonal antibody,113-5B7 against MT1-MMP (10), and incubated with the universal secondaryantibody (Research Genetics). The sections were incubated with diaminoben-

Received 11/17/99; accepted 11/20/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants NB0540 and NB0870 from the Ministry ofScience and Technology of Korea.

2 To whom requests for reprints should be addressed, at Laboratory of AnimalDevelopmental Biotechnology, Korea Research Institute of Bioscience and Biotechnol-ogy, Taejon 305-333, Korea. Phone: 82-42-860-4422; Fax: 82-42-860-4608; E-mail:[email protected].

3 The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane-type;ECM, extracellular matrix; RT-PCR, reverse transcription-PCR; MMTV LTR, mousemammary tumor virus long terminal repeat; GAPDH, glyceraldehyde-3-phosphate dehy-drogenase.

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zidine for 10 min and washed with Immuno/DNA (Research Genetics). May-er’s hematoxylin (Research Genetics) was used as counterstain, and the slideswere mounted with universal mount (Research Genetics).

Northern Blot Analysis. Total RNA was isolated from tissues by theguanidium-thiocyanate extraction method. RNA (20mg) from each tissuesample was fractionated on 1% agarose gels in the presence of 10% formamideand transferred onto nylon membranes (Boehringer Mannheim, Mannheim,Germany) to which it was fixed using an optimized UV cross-linking proce-dure. As a probe for theMT1-MMP transcript, 1.8 kb of MT1-MMP cDNAwere used. The probe forb-caseinwas obtained by RT-PCR analysis with totalRNA from wild-type lactating mammary gland and specific primers. Theoligonucleotides for amplification were a forward primer, 59-GAG-ACT-TTG-ACA-CGA-GGC-GG-39, and a reverse primer, 59-GAA-TGG-CCT-CGA-ATG-TG-39. The probes were labeled with [a-32P]dGTP by the random primelabeling system (Amersham Pharmacia Biotech, Piscataway, NJ). Signals werevisualized by autoradiography.

RT-PCR Analysis. For reverse transcription, the first strand of cDNA wassynthesized from total RNA using oligo-dT primer and AMV reverse tran-scriptase according to the manufacturer’s instructions (Promega, Madison,WI). The resulting cDNA served as a template for PCR reaction usingMT1-MMP primers. The primers for transgene and total (endogenous1 trans-gene)MT1-MMP were designated from sequences of pmMT1. Total MT1-MMP primers produced 320 bp in electrophoresis. The oligonucleotides foramplification were the forward primer, 59-AAC-TTC-AGC-CCC-GAA-GCC-TG-39, and the reverse primer, 59-ACG-GTG-TAA-GCT-CCG-GTA-39. Fortransgene detection, the 321-bp fragments were detected as sequences from theSV40 polyadenylation site in pmMT1, and the primers were a forward 59-GGT-AGA-AGA-CCC-CAA-GGA-CT-39and a reverse 59-TCT-AGT-CAA-GGC-ACT-ATA-CAT-CAA-39. The primers for 451 bp of mouse GAPDH forinternal control were a forward 59-ACC-ACA-GTC-CAT-GCC-ATC-AC-39and a reverse 59-TAC-AGC-AAC-AGG-GTG-GTG-GA-39.

Western Blot Analysis. The mammary gland tissues were homogenized,total protein concentrations were determined using a Bio-Rad protein assay kit(Hercules, CA), and BSA was used as a standard. Equal amounts of proteinfrom each tissue homogenate were subjected to 12% SDS-PAGE and thentransferred to nitrocellulose membrane. The filters were blocked with 5% BSAin Tris-buffered saline [50 mM Tris-HCl (pH 7.5) and 0.15M NaCl] containing0.1% Tween 20 (TBST) for 3 h at room temperature, then washed with TBST,and blotted with a monoclonal antibody, 113-15E1 against MT1-MMP (10).Bands were localized with the enhanced chemiluminescence system (Amer-sham Pharmacia Biotech, Piscataway, NJ).

Gelatin Zymography. Samples were applied without heating or reductionto 10% polyacrylamide gel containing 1 mg/ml gelatin. After electrophoresis,the gels were washed twice for 20 min with 2.5% Triton X-100, then with brief

water washes, and incubated overnight in 50 mM Tris-HCl (pH 7.5) containing10 mM CaCl2, 0.5 M NaCl, and 0.02% NaN3 at 37°C. After incubation, the gelwas stained with 0.25% Coomassie Blue R-250 and destained with 10%methanol and 10% acetic acid. Proteolytic bands appeared clear on blue-stained background.

RESULTS

Generation of Transgenic Mice.Transgenic mice were generatedby microinjecting a 4.4-kbHindIII DNA fragment containing themouseMT1-MMP cDNA under the transcriptional control of theMMTV LTR promoter. Transgenic mice were identified by PCRanalysis, and two female mice and one male founder mouse wereobtained (Fig. 1). Transgenic mouse lines were established by matingtransgenic founder mice to C57BL/6 mice. All of the founders werefertile and capable of transmitting the transgene to progeny. Expres-sion of theMMTV/MT1-MMP transgene in various stages of mam-mary gland development was examined by RT-PCR. The expressionof transgene mRNA was readily detectable throughout all of thestages (data not shown). Two lines, designated nos. 4 and 11, wereselected for the additional experiments because the female foundershowed poor lactation after the second and sixth parturitions, respec-tively.

MT1-MMP Overexpression Induces Abnormalities in Trans-genic Mammary Glands. To determine whether the expression oftheMT1-MMPtransgene affected morphology of the transgenic mam-mary gland, we performed macroscopic and histological examination

Fig. 2. Microscopic findings of mammary gland from wild-type control mice (AandB),and MMTV/MT1-MMP transgenic mice (C–F).A, normal mammary gland with restingducts, minimal periductal and abundant adipose tissue from a wild-type virgin mouse 14months of age.B, normal alveolar structure with proliferative epithelial cells from awild-type mouse 2 month of age at day 13 of lactating.C, moderate degree of fibrosis(arrow) with lymphocytic infiltration in mammary glands from a mouse 1.5 months of ageand weaned after 5 days.D, severe hyperplasia of mammary gland from the transgenicvirgin at 14 months of age.E, disrupted alveolar structures with secretory proteinousmaterials (asterisks) from transgenic mouse no. 4 at lactation day 13.F, focal dysplasiaof glandular epithelium from the multiparous transgenic mouse. Magnification:3200 (A,B, C, andE); 3100 (D); and3400 (F).AD, adipose tissues;AV, alveolar structure;DU,duct; EP, epithelial cells;SC, stromal cells;LY, lymphocytic infiltration;M, mitosis.

Fig. 1. Generation of MMTV/MT1-MMP transgenic mice.A, structure of pmMT1vector. The pmMT1 vector was constructed by inserting 1.8 kb of mouse MT1-MMPcDNA into the pMAMneo vector (see “Materials and Methods”). The 4.4 kb of theHindIII fragment of MMTV-LTR, mouse MT1-MMP cDNA, and SV40 polyadenylationsite [SV40 poly(A)] was microinjected into fertilized eggs.B, identification of MMTV/MT1-MMP transgenic mice by PCR analysis. The primer sequences were described in“Materials and Methods.” The expected fragment (440 bp) was indicated inA. Foundersno. 4 and 11 were female; no. 12 was male.

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using the female mice from 7 weeks to 18 months of age. Themammary glands were divided into groups of virgin, pregnancy andlactation, after 1 or 2 times of parturition (1–2 parous), and after$3times of parturition (multiparous).

There were several kinds of histological abnormalities in the mam-mary glands of theMMTV/MT1-MMP transgenic mice, includinglymphocytic infiltration in the stroma, periductal fibrosis, epithelialhyperplasia in the mammary ducts, alveolar structure disruption in thelactating glands, dysplastic change in the ductal epithelium, andadenocarcinoma (Fig. 2). Periductal fibrosis and ductal hyperplasiawere most common, and ectactic ducts containing proteinous materi-als with lipid droplets were occasionally found in the mice withperiductal fibrosis (Fig. 2C). Hyperplasias of the alveolar type werealso seen in the transgenic mammary glands (Fig. 2D). One of thelactating glands showed numerous disclosed collapsed alveolar struc-tures and large dilated ducts containing secretory materials (Fig. 2E).In contrast, wild-type lactating glands displayed disclosed-cellrounded alveolar structures with proliferation of the epithelial cells(Fig. 2B).

s.c. tumors were found in mammary glands of three multiparoustransgenic mice (Table 1). The major histological patterns of theadenocarcinomas were acinar carcinoma, which shows in typicalMMTV-infected mice (27), and papillary carcinoma in ductal epithe-lium adjacent to the major tumor lesions (Fig. 3). Additionally,hyperplastic or dysplastic lesions and fibrotic stromas were foundconsistently adjacent to the malignant tumor. Many mitotic figuresand necrosis were observed frequently in the tumor (Fig. 3B). Thetumors were divided by thick fibrous tissue and grew in a diffuse ornest formation. Pulmonary metastasis was found in one of three micewith mammary carcinoma, confirming the malignant nature of thetumors (Fig. 3E). Tumor cell emboli were found in the lumen of theblood vessels in the mammary gland (Fig. 3D).

As summarized in Table 2, 70% of the transgenic mice investigatedshowed lymphocytic infiltrations, 55% showed moderate and severefibrosis (collagen and fibroblast accumulation with the loss of adipo-

cytes), 52% showed moderate and severe hyperplasia (proliferation ofepithelial cell), 15% showed dysplasia (proliferation of atypical cells),and 9% showed mammary adenocarcinoma. Only 18% of all of thetransgenic mice were histologically normal in mammary glands. Bycomparison, 75% of the wild-type control mice showed entirelynormal mammary glands, and the remaining 25% showed only mildhyperplasia or lymphocytic infiltration (Table 2).

Multiple abnormalities as severe as alveolar structure disruptionand adenocarcinoma were detected in young transgenic mice of 3 or6 months of age after repetitive pregnancy and lactation, suggestingthat the abnormalities were affected by parity (frequency of preg-nancy) rather than by the ages of the individual mice. Lymphocyticinfiltration, fibrosis, hyperplasia, and dysplasia were present in themajority of the mice in the virgin, 1–2 parous, and multiparousgroups, but were not frequent in the pregnancy and lactation group ofthe transgenic mice. The dysplasias of the virgin mice were found asfocal. The tumors were found in the multiparous groups, but not in thevirgin, pregnancy and lactation, and 1–2 parous groups.

Lesions in the mammary glands of multiparous transgenic micewere more severe than in the other groups (Table 2). The hyperplasticand fibrotic lesions tended to be much more severe in the multiparoussubset of transgenic mice. Moreover, mammary-gland tumors weredeveloped in three of six multiparous transgenic mice (Table 1).

Expression of MT1-MMP in Mammary Glands of TransgenicMice. MT1-MMP expression was investigated in mammary glandsfrom transgenic mice by immunohistochemical analysis (Fig. 4).MT1-MMP was expressed in the fibrous stroma cells of the mammaryglands from wild-type control mice (Fig. 4A). It was expressed in theepithelial cells as well as in the fibrous stroma cells of the mammarygland in lactating transgenic mice (Fig. 4B). Particularly, the expres-sion of MT1-MMP was apparent in the epithelial cells of the trans-genic mammary gland, which showed disrupted alveolar structures(Fig. 4C). Additionally, MT1-MMP was markedly expressed in tu-mors surrounding the fibrous stroma, but weakly expressed in thetumor cells of the transgenic mice (Fig. 4D).

Table 1 Summary of transgenic mice exhibiting alveolar disruption and adenocarcinoma in mammary glands

Transgenic mice Designated name Age (mo) Parity Developmental status at sacrifice Mammary gland abnormality

Founder no. 4 No. 4 3 2 Lactating 13 days Alveolar structure disruptionFounder no. 11 No. 11 6 4 Weaning after 3 days AdenocarcinomaF1 progeny of no. 4 No. 4-1 14 3 Weaning after 3 days AdenocarcinomaF1 progeny of no. 11 No. 11-1 13 6 Weaning after 3 days Adenocarcinoma

Fig. 3. Macroscopic and microscopic findings ofthe mammary tumor from the MMTV/MT1-MMPtransgenic mouse.A, large s.c. mass (arrow)in no.11 founder mammary glands.B, adenocarcinoma ofthe mammary gland. Acinar structures are presentwith mitosis (M, arrowheads).C, papillary ductalcarcinomas are adjacent to the main acinar carci-noma of the mammary gland.D, tumor emboli inthe blood vessels.E, multiple metastasis are de-tected in the lung. Magnification:3400 (B);3100(C and F); and 340 (D). AC, acinar tumor;AD,adipose tissue;AV, alveolar structure;BR, bronchus;BV, blood vessel;.DU, duct; M, mitosis;PC, pap-illary carcinoma;SC, stromal cell;T, tumor cell;TE,tumor emboli.

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To examine the expression level of the transgene in transgenicmammary glands, total RNA was obtained from macroscopicallynormal glands and s.c. masses. The transgene and total (endoge-nous1 transgene) expression of MT1-MMP were investigated usingRT-PCR. A novel band indicating the transgene product was identi-fied in all of the transgenic mammary glands, but not in the wild-typecontrol (Fig. 5A). In addition, MT1-MMP transcripts were detected inthe tumor mammary glands, whereas negligible or no hybridizationwas observed by Northern blot analysis in the wild-type control or innormal lesions of the transgenic mammary gland (Fig. 5B).

To further verify the expression levels of the MT1-MMP protein,we analyzed homogenates from the tumors and then performed West-ern blotting detection with antibody against MT1-MMP. As expected,a major band ofMr 55,000 was detected (Fig. 6). The results dem-onstrated that the expression of MT1-MMP was elevated not only atthe transcriptional level, but also at the translational level in mammarygland tumors.

High Level Expression of MT1-MMP Affects Expression ofb-Casein.To determine whether the mammary gland epithelial cellswere functional as well as morphologically differentiated, the expres-sion of b-caseinas an epithelial cell differentiation marker and apregnancy/lactation related gene was analyzed by Northern blot anal-ysis. Wild-type mammary glands at day 13 of lactation (Fig. 7,Lane

1) and 3 days after weaning (Fig. 7,Lane 3) showed high-levelexpression ofb-casein mRNA. By comparison, in the same develop-ment stage and at the same ages of transgenic mice,b-caseinwas notexpressed in disrupted alveolar structure and tumors (Fig. 7,Lanes 4,5, and6) and showed very low-level expression in the normal residualglands of the transgenic mammary gland (Fig. 7,Lanes 7and8). Theresults demonstrate that morphological changes, which were inducedby ectopic expression ofMT1-MMP, resulted in the complete aboli-tion of expression ofb-casein mRNA in tumor-bearing mammary-gland tissues.

Activation of proMMP-2 in Tumor Tissues. Homogenates fromthe tumor were analyzed by gelatin zymography to determine theactivation of proMMP-2. No active gelatinase A was present, asisolated from wild-type lactating mice, and active gelatinase A wasobserved at 3 days after weaning (Fig. 8,Lanes 1and 2). In tumortissues, a representative zymogram revealed a significant increase inthe activation of progelatinase A (Fig. 8).

DISCUSSION

We generated transgenic mice overexpressingMT1-MMPunder thecontrol of the MMTV LTR promoter to examine whether the over-expression ofMT1-MMP might affect tumorigenesis in mammary

Fig. 4. Immunohistochemical findings of MT1-MMP trans-genic mice. Reactions to MT1-MMP antibody were present asbrowncolor.A, immunoreactive cells were found in stromal cellsof wild-type mammary gland at lactating day 10. Reactive cell isseldom detected in epithelial cells.B, MT1-MMP-reactive epi-thelial cells are found in mammary gland from the MMTV/MT1-MMP transgenic mouse at lactating day 10 (arrows).C, reactiveepithelial cells are increased in the disrupted alveolar structuresfrom the mammary glands of no. 4 at lactation day 13.D,reactions to MT1-MMP are markedly increased in the tumorsurrounding the stromal tissues from (arrows) no. 11 multiparousmouse at day 3 after weaning. In contrast, the tumor cells areweakly reactive in themselves (arrow heads). Magnification:3100. AD, adipose tissue;AV, alveolar structure;EP, epithelialcell; SC, stromal cells;T, tumor.

Table 2 Incidence of mammary pathologies in MMTV/MT1-MMP transgenic mice (no. of abnormality-detected mice/no. of mice histologically examined).

No pathologyLymphocyticinfiltration Fibrosis Hyperplasia

Alveolar structuredisruption Dysplasia Tumor

Wild-typeVirgin 4/5 1/5 0/5 0/5 0/5 0/5 0/5Pregnancy and lactationa 1/2 1/2 0/2 0/2 0/2 0/2 0/21–2 Parusb 3/4 1/4 0/4 1/4 0/4 0/4 0/4Multiparusc 1/1 0/1 0/1 0/1 0/1 0/1 0/1

Total 9/12 (75%) 3/12 (25%) 0/12 (0%) 1/12 (8%) 0/12 (0%) 0/12 (0%) 0/12 (0%)Transgenic mice

Virgin 2/12 9/12 7/12 8/12 0/12 3/12 0/12Pregnancy and lactationa 4/7 2/7 1/7 0/7 1/7 0/7 0/71–2 Parusb 0/8 7/8 6/8 3/8 0/8 0/8 0/8Multiparusc 0/6 5/6 4/6 6/6 0/6 2/6 3/6

Total 6/33 (18%) 23/33 (70%) 18/33 (55%) 17/33 (52%) 1/33 (3%) 5/33 (15%) 3/33 (9%)a Mice in first pregnancy and first lactation.b Mice in first or second parturition.c Mice after three or more repetitive parturitions.

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glands. The transgenic mice exhibited premalignant abnormalities andadenocarcinoma in mammary glands as shown in Tables 1 and 2.These results suggest that MT1-MMP may be involved in early tumorpromotion in transgenic mammary glands. Therefore, we have tried tounderstand the mechanism that generates premalignant abnormalitiesand tumorigenesis in the mammary glands of MMTV/MT1-MMPtransgenic mice.

Aberrant Expression of MT1-MMP in Epithelial Cells May Bea Direct or Indirect Consequence of Genetic Changes in theTransformed Cells. During all of the development stages of mam-mary gland at virgin, pregnancy, lactation, and involution, MT1-MMPprotein was localized in the stromal fibrous tissue of wild-type controlmice (Fig. 4A). In contrast, ectopic expression of MT1-MMP wasdetected in ductal epithelial cells and was apparent in the disruptedalveolar of the lactation glands of transgenic mice (Fig. 4,B andC).Other investigators have supported the idea that expression of MT1-MMP could not be detected in normal epithelial cells, even duringwound healing, but that it can be seen in transformed epithelialcarcinoma cells (28, 29). There are some examples that the transcrip-tional activation of theMT1-MMP gene is associated with the trans-formation of carcinoma cells. Human breast carcinoma cell lines withweak tumorigenicity do not express MT1-MMP, but cell lines withinvasive and metastatic properties express do MT1-MMP (30). Thesefindings suggest that expression of MT1-MMP in the cells correlateswith the loss of epithelial phenotype and the acquisition of mesen-chymal characteristics such as the expression of vimentin (30). In

association with the overexpression ofMT1-MMP in the mammaryglands of abnormality-exhibiting transgenic mice, we noted a signif-icant reduction ofb-casein gene expression (Fig. 7). Becauseb-caseinis expressed in the ductal epithelial cell of the mammary gland,we inferred that the reduction of the gene expression resulted from theloss of cellular specificity in the MMTV/MT1-MMP transgenic mice.

Mammary Gland Abnormalities Seen in MMTV/MT1-MMPTransgenic Mice Characterize the Reactive Stroma.In MMTV/MT1-MMP transgenic mammary glands, stromal changes, such aslymphocytic infiltration and fibrosis, appeared to presage malignantepithelial changes including hyperplasia, alveolar structure disruption,dysplasia, and adenocarcinoma. In the stromal cells of these mam-mary glands, these abnormalities were indicated throughout all of thedevelopmental stages of the mammary gland, even in virgin (Table 2)mice. In addition, MT1-MMP expression was elevated in tumor tissuesurrounding the stromal cells of the MT1-MMP transgenic mammaryglands (Fig. 4D).

The reactive stroma defined as an accumulation of collagen fiber,recruitment of inflammatory cells, increased vascularization, and anup-regulation of MMPs (2, 31–33). There was evidence suggestingthat the formation of the tumor mass in epithelial cancers was pro-foundly reliant on the stromal cells (34). An altered stromal environ-ment may actually promote neoplastic transformation and alterationsin the stromal-epithelial interactions transduced via changes in theintegrity of the ECM can promote neoplastic transformation (5, 33–38). The dramatic alteration of stromal phenotype by overexpressedMMPs such asMMP-3 leads to tumor development (39).

MT1-MMP Expression Level Relates to Activation of Sub-strates Such as MMP-2.Gelatin zymography results indicate theactivation of MMP-2 was associated with overexpression of MT1-MMP in the transgenic mammary glands exhibiting abnormalities(Fig. 8). Elevated expression and activation of MMP-2 have corre-lated to the tumor grade, promotion, and malignancy of many tumors(3, 40, 41).MT1-MMPhas been implicated as a possible activator ofMMP-2 and MMP-13 (12, 42–47). In addition, MT-MMPs can alsodegrade a number of ECM proteins, such as gelatin, fibronectin,vitronectin, fibrillar collagens, or aggrecan (48). Therefore, it is sug-gested that theMT1-MMPexpression level relates to activation of thesubstrates including MMP-2 and results in increased aberrant degra-dation of ECM, which might lead to tumor formation and metastasis.

Fig. 5. Expression of MT1-MMP mRNA in mammary glands. Total RNA was purifiedfrom macroscopically normal mammary glands, and abnormalities were detected intransgenic mammary glands.A, RT-PCR analysis. PCR amplification was performed withspecific primer sets designated by sequences of pmMT1 vector for transgene and total(endogenous and transgene) MT1-MMP. GAPDH was used as an internal control.Lane3, alveolar structure-disrupted mammary gland from the transgenic mouse. Lanes 4-6, s.c.masses from transgenic mice;Lanes 7-9, macroscopically normal mammary gland in thesemice. Lane 1, wild type at lactation day 13;Lane 2, wild type at 3 days after weaning;Lane 3, no. 4;Lanes 4and7, no. 11;Lanes 5and8, no. 4-1;Lanes 6and9, no. 11-1.B,Northern blot analysis. 1.8 kb of MT1-MMP cDNA was used as the probe.Lane 1, wildtype at 3 days after weaning;Lane 2, no. 4, alveolar structure-disrupted mammary gland;Lane 3, no. 11, macroscopically normal gland;Lane 4, s.c. masses. As an internal control,28S and 18S rRNA were used after EtBr staining.

Fig. 6. Western blot analysis by homogenates of mammary glands from wild-typecontrol, the alveolar structure-disrupted transgenic mouse, and s.c. masses from transgenicmice. Lane 1, wild type at 3 days after weaning;Lane 2, wild type at lactation day 13;Lane 3, no. 4;Lane 4, no. 11;Lane 5, no. 4-1;Lane 6, no. 11-1. A major band wasindicated atMr 55,000 (arrow).

Fig. 7. Expression ofb-casein mRNA in the mammary glands of wild-type control,alveolar structure-disrupted transgenic mice, and s.c. masses of transgenic mice. Theprobes were prepared by RT-PCR for theb-casein-specific primer set with total RNA ofwild-type lactating mammary glands.Lanes 4–6, s.c. masses from transgenic mice;Lanes7 and8, macroscopically normal mammary gland in transgenic mice.Lane 1, wild typeat lactation day 13;Lane 2, no. 4, s.c. mass;Lane 3, wild type at 3 days after weaning;Lanes 4and7, no. 4-1;Lane 5, no. 11;Lanes 6and8, no. 11-1. As an internal control,28S and 18S rRNA were used after EtBr staining.

Fig. 8. Gelatin zymography by homogenates of mammary glands from wild-typecontrol, alveolar structure-disrupted transgenic mice, and s.c. masses from transgenicmice. The figure is shown as a negative image.Lane 1, wild type at 3 days after weaning;Lane 2, wild type at lactation day 13;Lane 3, no. 4, alveolar structure disrupted;Lane 4,no. 4-1, s.c. mass;Lane 5, no. 11, s.c. mass;Lane 6, no. 11-1, s.c. mass.

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In conclusion, we hypothesize the ectopic expression of MT1-MMPin epithelial cells might be a direct or indirect consequence of cellenvironments, including ECM remodeling and the genetic change totransformation. We suggest that overexpression of MT1-MMP canalter its extracellular environment as a stromal product. Therefore, itmay be partly responsible for the tumorigenic effects of an alteredstroma.

It remains to be determined whether MT1-MMP is critical for earlytumor promotion in mammary gland. Until recently, evidence for theactivity of MMPs in early tumor promotion was limited. However,it was reported that MMP-3 induced mammary gland changes asa natural promoter in early tumor formation in the absence ofexogenous mutagens or endogenous oncogenes or suppressor genedefects (49, 50). Therefore, the availability of MMTV/MT1-MMPtransgenic mice as a mammary tumor model should lead to eluci-dation of the malignant process and to an understanding of how anabnormal microenvironment in mammary glands could lead tocancer induction and progression.

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Matrix Metalloproteinase-1 Membrane-typeOverexpression of

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