Overexpression of Membrane-Type Matrix Metalloproteinase-1 Gene Induces Mammary Gland Abnormalities and Adenocarcinoma in Transgenic Mice1

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[CANCER RESEARCH 61, 984–990, February 1, 2001] Overexpression of Membrane-type Matrix Metalloproteinase-1 Gene Induces Mammary Gland Abnormalities and Adenocarcinoma in Transgenic 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 brane domains as MT-MMP-1 through -5, these enzymes have been proposed to be the master switches of ECM turnover based on the To investigate the role of membrane-type matrix metalloproteinase-1 purported ability of MT-MMPs to activate other MMPs such as (MT1-MMP) in mammary gland development and tumorigenesis, trans- proMMP-2 and MMP-13. ProMMP-2 and MMP-13 are degradative genic mice overexpressing MT1-MMP in mammary gland under the control of the mouse mammary tumor virus long terminal repeat-promoter enzymes widely implicated in tumor invasion and metastasis (10, 16, 17). were generated. The mouse mammary tumor virus/MT1-MMP transgenic As do other MMPs, MT1-MMP has also been proposed to play mice displayed abnormalities in 82% of female mammary glands. The critical roles in both physiology and pathology by remodeling the abnormalities were verified as lymphocytic infiltration, fibrosis, hyper- ECM. MT1-MMP expression is particularly high in kidney during plasia, alveolar structure disruption, dysplasia, and adenocarcinoma. mouse embryogenesis and also in the adult human (12, 18). Recent Northern and reverse transcription-PCR analyses demonstrated that data indicate that MT1-MMP may also function as a fibrinolytic MT1-MMP mRNA was overexpressed in mammary glands exhibiting enzyme in the absence of plasmin and mediate pericellular proteolysis abnormalities. Western blot analysis and immunohistochemical studies in angiogenesis (19). Recently it was reported that MT1-MMP-defi- have revealed that the protein expression level was also increased in these cient mice develop dwarfism, osteopenia, arthritis, and connective glands. In addition, the ␤-casein gene as a functional epithelial cell marker tissue disease because of inadequate collagen turnover (20). MT1- was poorly expressed in the mammary glands of transgenic mice exhibiting abnormalities. Gelatin zymography showed significantly increased MMP is also overexpressed in various tumor tissues, including human MMP-2 activation in these mammary glands. These results showed that colon, breast, and head and neck carcinoma (10, 21–24). Although overexpression of MT1-MMP induced remodeling of the extracellular MT1-MMP expression has been proved in numerous tumors, the roles matrix and tumor formation in the mammary glands of transgenic mice. assigned to MT1-MMP in tumorigenesis and tumor progression are Therefore, we suggest that overexpression of MT1-MMP may play a key relatively poorly understood. In the present study, we generated role in development and tumorigenesis in mammary glands. MMTV/MT1-MMP transgenic mice and examined premalignant abnormalities and adenocarcinoma in mammary glands. The results INTRODUCTION suggest that overexpression of MT1-MMP may play a key role in development and tumorigenesis in mammary glands. MMPs,3 which degrade the various components of ECM, play critical roles in the tissue remodeling of multicellular organisms as well as in tumor invasion (1–4). MMPs may play a role in any one of MATERIALS AND METHODS multiple critical events in tumor evolution, including tumorigenesis, tumor growth, angiogenesis, generation of reactive stroma, and tumor Generation of MMTV/MT1-MMP Transgenic Mice. To generate a vec- cell invasion and metastasis (5). For example, the lack of MMP-7 in tor pmMT1, a 1.8-kb mouse MT1-MMP cDNA of full length for the coding mice showed a reduction in intestinal tumorigenesis (6), and its sequence was ligated into the SalI and XhoI sites of the mammalian expression vector pMAM-neo (Clonetech, Palo Alto, CA; Ref. 25). A HindIII DNA overexpression in mammary tissue accelerates mammary tumor for- fragment (4.4 kb) containing MMTV-LTR, MT1-MMP cDNA, and SV40 mation in mice carrying the MMTV/ErbB-2 transgene (7). In addition, polyadenylation sequences was microinjected into the pronuclei of fertilized MMP-2-defective mice showed reduced angiogenesis and tumor pro- mouse eggs obtained from C57BL/6 ϫ DBA F1 hybrid females as described gression (8). MMP-11 knockout mice showed reduced tumorigenesis (Ref. 26; Fig. 1). The DNA-injected eggs were transferred to pseudopregnant in response to chemical mutagenesis (9). ICR female mice. Transgenic mice were identified by PCR analysis of the Whereas the majority of the MMPs are secreted as soluble enzymes genomic DNA using primers specific to mMT1-injection DNA. The oligonu- into the extracellular milieu, a subset of MMPs have been identified cleotides used for the amplification were a forward primer 5Ј-ACA-AGA- in recent years to contain additional sequences capable of anchoring GCG-CAA-CGG-ACT-CA-3Ј complementary to MMTV LTR gene sequences on plasma membrane (10–15). Named after the putative transmem- and 5Ј-ACG-GTG-TAA-GCT-CCG-GTA-3Ј specific to the MT1-MMP gene. Histological and Immunohistochemical Stain. Mammary tissues were obtained from wild-type and transgenic mice at various stages of development. Received 11/17/99; accepted 11/20/00. Tissues were fixed in neutral buffered 10% formalin overnight and embedded 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 accordance with in paraffin, sectioned at 4 m, and stained with H&E. For immunohistochem- 18 U.S.C. Section 1734 solely to indicate this fact. ical staining, the 4-␮m paraffin-embedded sections were prepared on the 1 This work was supported by grants NB0540 and NB0870 from the Ministry of Probe-on Plus slides (Fisher, Pittsburgh, PA) and deparaffinized by xylene. Science and Technology of Korea. Next, tissue sections were rehydrated in PBS solution, and then the slides were 2 To whom requests for reprints should be addressed, at Laboratory of Animal Developmental Biotechnology, Korea Research Institute of Bioscience and Biotechnol- blocked in 3% hydrogen peroxide for 10 s. The slides were washed twice in ogy, Taejon 305-333, Korea. Phone: 82-42-860-4422; Fax: 82-42-860-4608; E-mail: Immuno/DNA buffer solution (Research Genetics, Huntsville, AL) and then [email protected]. incubated in protein blocker solution (Research Genetics) for 3 min. The 3 The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane-type; sections were incubated at 4°C overnight with the monoclonal antibody, ECM, extracellular matrix; RT-PCR, reverse transcription-PCR; MMTV LTR, mouse mammary tumor virus long terminal repeat; GAPDH, glyceraldehyde-3-phosphate dehy- 113-5B7 against MT1-MMP (10), and incubated with the universal secondary drogenase. antibody (Research Genetics). The sections were incubated with diaminoben- 984

water washes, and incubated overnight in 50 mM Tris-HCl (pH 7.5) containing

10 mM CaCl2, 0.5 M NaCl, and 0.02% NaN3 at 37°C. After incubation, the gel was 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 generated by microinjecting a 4.4-kb HindIII DNA fragment containing the mouse MT1-MMP cDNA under the transcriptional control of the MMTV LTR promoter. Transgenic mice were identified by PCR analysis, and two female mice and one male founder mouse were obtained (Fig. 1). Transgenic mouse lines were established by mating transgenic founder mice to C57BL/6 mice. All of the founders were fertile and capable of transmitting the transgene to progeny. Expres- Fig. 1. Generation of MMTV/MT1-MMP transgenic mice. A, structure of pmMT1 sion of the MMTV/MT1-MMP transgene in various stages of mam- vector. The pmMT1 vector was constructed by inserting 1.8 kb of mouse MT1-MMP mary gland development was examined by RT-PCR. The expression cDNA into the pMAMneo vector (see “Materials and Methods”). The 4.4 kb of the HindIII fragment of MMTV-LTR, mouse MT1-MMP cDNA, and SV40 polyadenylation of transgene mRNA was readily detectable throughout all of the site [SV40 poly(A)] was microinjected into fertilized eggs. B, identification of MMTV/ stages (data not shown). Two lines, designated nos. 4 and 11, were MT1-MMP transgenic mice by PCR analysis. The primer sequences were described in “Materials and Methods.” The expected fragment (440 bp) was indicated in A. Founders selected for the additional experiments because the female founder no. 4 and 11 were female; no. 12 was male. showed poor lactation after the second and sixth parturitions, respec- tively. MT1-MMP Overexpression Induces Abnormalities in Trans- zidine for 10 min and washed with Immuno/DNA (Research Genetics). May- er’s hematoxylin (Research Genetics) was used as counterstain, and the slides genic Mammary Glands. To determine whether the expression of were mounted with universal mount (Research Genetics). the MT1-MMP transgene affected morphology of the transgenic mam- Northern Blot Analysis. Total RNA was isolated from tissues by the mary gland, we performed macroscopic and histological examination guanidium-thiocyanate extraction method. RNA (20 ␮g) from each tissue sample was fractionated on 1% agarose gels in the presence of 10% formamide and 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 the MT1-MMP transcript, 1.8 kb of MT1-MMP cDNA were used. The probe for ␤-casein was obtained by RT-PCR analysis with total RNA from wild-type lactating mammary gland and specific primers. The oligonucleotides for amplification were a forward primer, 5Ј-GAG-ACT-TTG- ACA-CGA-GGC-GG-3Ј, and a reverse primer, 5Ј-GAA-TGG-CCT-CGA- ATG-TG-3Ј. The probes were labeled with [␣-32P]dGTP by the random prime labeling system (Amersham Pharmacia Biotech, Piscataway, NJ). Signals were visualized by autoradiography. RT-PCR Analysis. For reverse transcription, the first strand of cDNA was synthesized 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 using MT1-MMP primers. The primers for transgene and total (endogenous ϩ transgene) MT1-MMP were designated from sequences of pmMT1. Total MT1- MMP primers produced 320 bp in electrophoresis. The oligonucleotides for amplification were the forward primer, 5Ј-AAC-TTC-AGC-CCC-GAA-GCC- TG-3Ј, and the reverse primer, 5Ј-ACG-GTG-TAA-GCT-CCG-GTA-3Ј. For transgene detection, the 321-bp fragments were detected as sequences from the SV40 polyadenylation site in pmMT1, and the primers were a forward 5Ј- GGT-AGA-AGA-CCC-CAA-GGA-CT-3Ј and a reverse 5Ј-TCT-AGT-CAA- GGC-ACT-ATA-CAT-CAA-3Ј. The primers for 451 bp of mouse GAPDH for internal control were a forward 5Ј-ACC-ACA-GTC-CAT-GCC-ATC-AC-3Ј and a reverse 5Ј-TAC-AGC-AAC-AGG-GTG-GTG-GA-3Ј. 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 protein from each tissue homogenate were subjected to 12% SDS-PAGE and then Fig. 2. Microscopic findings of mammary gland from wild-type control mice (A and B), transferred to nitrocellulose membrane. The filters were blocked with 5% BSA and MMTV/MT1-MMP transgenic mice (C–F). A, normal mammary gland with resting in Tris-buffered saline [50 mM Tris-HCl (pH 7.5) and 0.15 M NaCl] containing ducts, minimal periductal and abundant adipose tissue from a wild-type virgin mouse 14 0.1% Tween 20 (TBST) for3hatroom temperature, then washed with TBST, months of age. B, normal alveolar structure with proliferative epithelial cells from a wild-type mouse 2 month of age at day 13 of lactating. C, moderate degree of fibrosis and blotted with a monoclonal antibody, 113-15E1 against MT1-MMP (10). (arrow) with lymphocytic infiltration in mammary glands from a mouse 1.5 months of age Bands were localized with the enhanced chemiluminescence system (Amer- and weaned after 5 days. D, severe hyperplasia of mammary gland from the transgenic sham Pharmacia Biotech, Piscataway, NJ). virgin at 14 months of age. E, disrupted alveolar structures with secretory proteinous Gelatin Zymography. Samples were applied without heating or reduction materials (asterisks) from transgenic mouse no. 4 at lactation day 13. F, focal dysplasia of glandular epithelium from the multiparous transgenic mouse. Magnification: ϫ200 (A, to 10% polyacrylamide gel containing 1 mg/ml gelatin. After electrophoresis, B, C, and E); ϫ100 (D); and ϫ400 (F). AD, adipose tissues; AV, alveolar structure; DU, the gels were washed twice for 20 min with 2.5% Triton X-100, then with brief duct; EP, epithelial cells; SC, stromal cells; LY, lymphocytic infiltration; M, mitosis. 985

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 disruption Founder no. 11 No. 11 6 4 Weaning after 3 days Adenocarcinoma F1 progeny of no. 4 No. 4-1 14 3 Weaning after 3 days Adenocarcinoma F1 progeny of no. 11 No. 11-1 13 6 Weaning after 3 days Adenocarcinoma using the female mice from 7 weeks to 18 months of age. The cytes), 52% showed moderate and severe hyperplasia (proliferation of mammary glands were divided into groups of virgin, pregnancy and epithelial cell), 15% showed dysplasia (proliferation of atypical cells), lactation, after 1 or 2 times of parturition (1–2 parous), and after Ն3 and 9% showed mammary adenocarcinoma. Only 18% of all of the times of parturition (multiparous). transgenic mice were histologically normal in mammary glands. By There were several kinds of histological abnormalities in the mam- comparison, 75% of the wild-type control mice showed entirely mary glands of the MMTV/MT1-MMP transgenic mice, including normal mammary glands, and the remaining 25% showed only mild lymphocytic infiltration in the stroma, periductal fibrosis, epithelial hyperplasia or lymphocytic infiltration (Table 2). hyperplasia in the mammary ducts, alveolar structure disruption in the Multiple abnormalities as severe as alveolar structure disruption lactating glands, dysplastic change in the ductal epithelium, and and adenocarcinoma were detected in young transgenic mice of 3 or adenocarcinoma (Fig. 2). Periductal fibrosis and ductal hyperplasia 6 months of age after repetitive pregnancy and lactation, suggesting were most common, and ectactic ducts containing proteinous materi- that the abnormalities were affected by parity (frequency of preg- als with lipid droplets were occasionally found in the mice with nancy) rather than by the ages of the individual mice. Lymphocytic periductal fibrosis (Fig. 2C). Hyperplasias of the alveolar type were infiltration, fibrosis, hyperplasia, and dysplasia were present in the also seen in the transgenic mammary glands (Fig. 2D). One of the majority of the mice in the virgin, 1–2 parous, and multiparous lactating glands showed numerous disclosed collapsed alveolar struc- groups, but were not frequent in the pregnancy and lactation group of tures and large dilated ducts containing secretory materials (Fig. 2E). the transgenic mice. The dysplasias of the virgin mice were found as In contrast, wild-type lactating glands displayed disclosed-cell focal. The tumors were found in the multiparous groups, but not in the rounded alveolar structures with proliferation of the epithelial cells virgin, pregnancy and lactation, and 1–2 parous groups. (Fig. 2B). Lesions in the mammary glands of multiparous transgenic mice s.c. tumors were found in mammary glands of three multiparous were more severe than in the other groups (Table 2). The hyperplastic transgenic mice (Table 1). The major histological patterns of the and fibrotic lesions tended to be much more severe in the multiparous adenocarcinomas were acinar carcinoma, which shows in typical subset of transgenic mice. Moreover, mammary-gland tumors were MMTV-infected mice (27), and papillary carcinoma in ductal epithe- developed in three of six multiparous transgenic mice (Table 1). lium adjacent to the major tumor lesions (Fig. 3). Additionally, Expression of MT1-MMP in Mammary Glands of Transgenic hyperplastic or dysplastic lesions and fibrotic stromas were found Mice. MT1-MMP expression was investigated in mammary glands consistently adjacent to the malignant tumor. Many mitotic figures from transgenic mice by immunohistochemical analysis (Fig. 4). and necrosis were observed frequently in the tumor (Fig. 3B). The MT1-MMP was expressed in the fibrous stroma cells of the mammary tumors were divided by thick fibrous tissue and grew in a diffuse or glands from wild-type control mice (Fig. 4A). It was expressed in the nest formation. Pulmonary metastasis was found in one of three mice epithelial cells as well as in the fibrous stroma cells of the mammary with mammary carcinoma, confirming the malignant nature of the gland in lactating transgenic mice (Fig. 4B). Particularly, the expres- tumors (Fig. 3E). Tumor cell emboli were found in the lumen of the sion of MT1-MMP was apparent in the epithelial cells of the trans- blood vessels in the mammary gland (Fig. 3D). genic mammary gland, which showed disrupted alveolar structures As summarized in Table 2, 70% of the transgenic mice investigated (Fig. 4C). Additionally, MT1-MMP was markedly expressed in tu- showed lymphocytic infiltrations, 55% showed moderate and severe mors surrounding the fibrous stroma, but weakly expressed in the fibrosis (collagen and fibroblast accumulation with the loss of adipo- tumor cells of the transgenic mice (Fig. 4D).

Fig. 3. Macroscopic and microscopic findings of the mammary tumor from the MMTV/MT1-MMP transgenic mouse. A, large s.c. mass (arrow) in no. 11 founder mammary glands. B, adenocarcinoma of the mammary gland. Acinar structures are present with mitosis (M, arrowheads). C, papillary ductal carcinomas are adjacent to the main acinar carcinoma of the mammary gland. D, tumor emboli in the blood vessels. E, multiple metastasis are detected in the lung. Magnification: ϫ400 (B); ϫ100 (C and F); and ϫ40 (D). AC, acinar tumor; AD, adipose tissue; AV, alveolar structure; BR, bronchus; BV, blood vessel;. DU, duct; M, mitosis; PC, papillary carcinoma; SC, stromal cell; T, tumor cell; TE, tumor emboli.

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Table 2 Incidence of mammary pathologies in MMTV/MT1-MMP transgenic mice (no. of abnormality-detected mice/no. of mice histologically examined). Lymphocytic Alveolar structure No pathology infiltration Fibrosis Hyperplasia disruption Dysplasia Tumor Wild-type Virgin 4/5 1/5 0/5 0/5 0/5 0/5 0/5 Pregnancy and lactationa 1/2 1/2 0/2 0/2 0/2 0/2 0/2 1–2 Parusb 3/4 1/4 0/4 1/4 0/4 0/4 0/4 Multiparusc 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 0/12 Pregnancy and lactationa 4/7 2/7 1/7 0/7 1/7 0/7 0/7 1–2 Parusb 0/8 7/8 6/8 3/8 0/8 0/8 0/8 Multiparusc 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.

To examine the expression level of the transgene in transgenic 1) and 3 days after weaning (Fig. 7, Lane 3) showed high-level mammary glands, total RNA was obtained from macroscopically expression of ␤-casein mRNA. By comparison, in the same develop- normal glands and s.c. masses. The transgene and total (endoge- ment stage and at the same ages of transgenic mice, ␤-casein was not nous ϩ transgene) expression of MT1-MMP were investigated using expressed in disrupted alveolar structure and tumors (Fig. 7, Lanes 4, RT-PCR. A novel band indicating the transgene product was identi- 5, and 6) and showed very low-level expression in the normal residual fied in all of the transgenic mammary glands, but not in the wild-type glands of the transgenic mammary gland (Fig. 7, Lanes 7 and 8). The control (Fig. 5A). In addition, MT1-MMP transcripts were detected in results demonstrate that morphological changes, which were induced the tumor mammary glands, whereas negligible or no hybridization by ectopic expression of MT1-MMP, resulted in the complete aboli- was observed by Northern blot analysis in the wild-type control or in tion of expression of ␤-casein mRNA in tumor-bearing mammary- normal lesions of the transgenic mammary gland (Fig. 5B). gland tissues. To further verify the expression levels of the MT1-MMP protein, Activation of proMMP-2 in Tumor Tissues. Homogenates from we analyzed homogenates from the tumors and then performed West- the tumor were analyzed by gelatin zymography to determine the ern blotting detection with antibody against MT1-MMP. As expected, activation of proMMP-2. No active gelatinase A was present, as a major band of Mr 55,000 was detected (Fig. 6). The results dem- isolated from wild-type lactating mice, and active gelatinase A was onstrated that the expression of MT1-MMP was elevated not only at observed at 3 days after weaning (Fig. 8, Lanes 1 and 2). In tumor the transcriptional level, but also at the translational level in mammary tissues, a representative zymogram revealed a significant increase in gland tumors. the activation of progelatinase A (Fig. 8). High Level Expression of MT1-MMP Affects Expression of ␤ -Casein. To determine whether the mammary gland epithelial cells DISCUSSION were functional as well as morphologically differentiated, the expression of ␤-casein as an epithelial cell differentiation marker and a We generated transgenic mice overexpressing MT1-MMP under the pregnancy/lactation related gene was analyzed by Northern blot anal- control of the MMTV LTR promoter to examine whether the over- ysis. Wild-type mammary glands at day 13 of lactation (Fig. 7, Lane expression of MT1-MMP might affect tumorigenesis in mammary

Fig. 4. Immunohistochemical findings of MT1-MMP transgenic mice. Reactions to MT1-MMP antibody were present as brown color. A, immunoreactive cells were found in stromal cells of wild-type mammary gland at lactating day 10. Reactive cell is seldom detected in epithelial cells. B, MT1-MMP-reactive epithelial cells are found in mammary gland from the MMTV/MT1- MMP transgenic mouse at lactating day 10 (arrows). C, reactive epithelial cells are increased in the disrupted alveolar structures from the mammary glands of no. 4 at lactation day 13. D, reactions to MT1-MMP are markedly increased in the tumor surrounding the stromal tissues from (arrows) no. 11 multiparous mouse at day 3 after weaning. In contrast, the tumor cells are weakly reactive in themselves (arrow heads). Magnification: ϫ100. AD, adipose tissue; AV, alveolar structure; EP, epithelial cell; SC, stromal cells; T, tumor.

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association with the overexpression of MT1-MMP in the mammary glands of abnormality-exhibiting transgenic mice, we noted a significant reduction of ␤-casein gene expression (Fig. 7). Because ␤- casein is expressed in the ductal epithelial cell of the mammary gland, we inferred that the reduction of the gene expression resulted from the loss of cellular specificity in the MMTV/MT1-MMP transgenic mice. Mammary Gland Abnormalities Seen in MMTV/MT1-MMP Transgenic Mice Characterize the Reactive Stroma. In MMTV/ MT1-MMP transgenic mammary glands, stromal changes, such as lymphocytic infiltration and fibrosis, appeared to presage malignant epithelial changes including hyperplasia, alveolar structure disruption, dysplasia, and adenocarcinoma. In the stromal cells of these mammary glands, these abnormalities were indicated throughout all of the developmental stages of the mammary gland, even in virgin (Table 2) mice. In addition, MT1-MMP expression was elevated in tumor tissue surrounding the stromal cells of the MT1-MMP transgenic mammary Fig. 5. Expression of MT1-MMP mRNA in mammary glands. Total RNA was purified glands (Fig. 4D). from macroscopically normal mammary glands, and abnormalities were detected in The reactive stroma defined as an accumulation of collagen fiber, transgenic mammary glands. A, RT-PCR analysis. PCR amplification was performed with specific primer sets designated by sequences of pmMT1 vector for transgene and total recruitment of inflammatory cells, increased vascularization, and an (endogenous and transgene) MT1-MMP. GAPDH was used as an internal control. Lane up-regulation of MMPs (2, 31–33). There was evidence suggesting 3, alveolar structure-disrupted mammary gland from the transgenic mouse. Lanes 4-6, s.c. that the formation of the tumor mass in epithelial cancers was pro- masses from transgenic mice; Lanes 7-9, macroscopically normal mammary gland in these mice. Lane 1, wild type at lactation day 13; Lane 2, wild type at 3 days after weaning; foundly reliant on the stromal cells (34). An altered stromal environ- Lane 3, no. 4; Lanes 4 and 7, no. 11; Lanes 5 and 8, no. 4-1; Lanes 6 and 9, no. 11-1. B, ment may actually promote neoplastic transformation and alterations Northern blot analysis. 1.8 kb of MT1-MMP cDNA was used as the probe. Lane 1, wild type at 3 days after weaning; Lane 2, no. 4, alveolar structure-disrupted mammary gland; in the stromal-epithelial interactions transduced via changes in the Lane 3, no. 11, macroscopically normal gland; Lane 4, s.c. masses. As an internal control, integrity of the ECM can promote neoplastic transformation (5, 33– 28S and 18S rRNA were used after EtBr staining. 38). The dramatic alteration of stromal phenotype by overexpressed MMPs such as MMP-3 leads to tumor development (39). MT1-MMP Expression Level Relates to Activation of Sub- strates Such as MMP-2. Gelatin zymography results indicate the activation 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- Fig. 6. Western blot analysis by homogenates of mammary glands from wild-type control, the alveolar structure-disrupted transgenic mouse, and s.c. masses from transgenic lated to the tumor grade, promotion, and malignancy of many tumors mice. Lane 1, wild type at 3 days after weaning; Lane 2, wild type at lactation day 13; (3, 40, 41). MT1-MMP has been implicated as a possible activator of Lane 3, no. 4; Lane 4, no. 11; Lane 5, no. 4-1; Lane 6, no. 11-1. A major band was MMP-2 and MMP-13 (12, 42–47). In addition, MT-MMPs can also indicated at M 55,000 (arrow). r degrade a number of ECM proteins, such as gelatin, fibronectin, vitronectin, fibrillar collagens, or aggrecan (48). Therefore, it is sug- glands. The transgenic mice exhibited premalignant abnormalities and gested that the MT1-MMP expression level relates to activation of the adenocarcinoma in mammary glands as shown in Tables 1 and 2. substrates including MMP-2 and results in increased aberrant degra- These results suggest that MT1-MMP may be involved in early tumor dation of ECM, which might lead to tumor formation and metastasis. promotion in transgenic mammary glands. Therefore, we have tried to understand the mechanism that generates premalignant abnormalities and tumorigenesis in the mammary glands of MMTV/MT1-MMP transgenic mice. Aberrant Expression of MT1-MMP in Epithelial Cells May Be a Direct or Indirect Consequence of Genetic Changes in the Transformed Cells. During all of the development stages of mammary gland at virgin, pregnancy, lactation, and involution, MT1-MMP Fig. 7. Expression of ␤-casein mRNA in the mammary glands of wild-type control, protein was localized in the stromal fibrous tissue of wild-type control alveolar structure-disrupted transgenic mice, and s.c. masses of transgenic mice. The mice (Fig. 4A). In contrast, ectopic expression of MT1-MMP was probes were prepared by RT-PCR for the ␤-casein-specific primer set with total RNA of wild-type lactating mammary glands. Lanes 4–6, s.c. masses from transgenic mice; Lanes detected in ductal epithelial cells and was apparent in the disrupted 7 and 8, macroscopically normal mammary gland in transgenic mice. Lane 1, wild type alveolar of the lactation glands of transgenic mice (Fig. 4, B and C). at lactation day 13; Lane 2, no. 4, s.c. mass; Lane 3, wild type at 3 days after weaning; Other investigators have supported the idea that expression of MT1- Lanes 4 and 7, no. 4-1; Lane 5, no. 11; Lanes 6 and 8, no. 11-1. As an internal control, 28S and 18S rRNA were used after EtBr staining. MMP could not be detected in normal epithelial cells, even during wound healing, but that it can be seen in transformed epithelial carcinoma cells (28, 29). There are some examples that the transcriptional activation of the MT1-MMP gene is associated with the transformation of carcinoma cells. Human breast carcinoma cell lines with weak tumorigenicity do not express MT1-MMP, but cell lines with invasive and metastatic properties express do MT1-MMP (30). These Fig. 8. Gelatin zymography by homogenates of mammary glands from wild-type findings suggest that expression of MT1-MMP in the cells correlates control, alveolar structure-disrupted transgenic mice, and s.c. masses from transgenic mice. The figure is shown as a negative image. Lane 1, wild type at 3 days after weaning; with the loss of epithelial phenotype and the acquisition of mesen- Lane 2, wild type at lactation day 13; Lane 3, no. 4, alveolar structure disrupted; Lane 4, chymal characteristics such as the expression of vimentin (30). In no. 4-1, s.c. mass; Lane 5, no. 11, s.c. mass; Lane 6, no. 11-1, s.c. mass. 988

In conclusion, we hypothesize the ectopic expression of MT1-MMP pressed with its substrate in mouse tissue during embryogenesis. J. Cell Sci., 109: in epithelial cells might be a direct or indirect consequence of cell 953–959, 1996. 19. Hiraoka, N., Allen, E., Apel, I. J., Gyetko, M. R., and Weiss, S. J. Matrix metallo- environments, including ECM remodeling and the genetic change to proteinases regulate neovascularization by acting as pericellular fibrinolysins. Cell, transformation. We suggest that overexpression of MT1-MMP can 95: 365–377, 1998. 20. Holmbeck, K., Bianco, P., Caterina, J., Yamada, S., Kromer, M., Kuznetsov, S. A., alter its extracellular environment as a stromal product. Therefore, it Mankani, M., Robey, P. G., Poole, A. R., Pidoux, I., Ward, J. M., and Birkedal- may be partly responsible for the tumorigenic effects of an altered Hansen, H. MT1-MMP deficient mice develop dwarfism, osteopenia, arthritis, and stroma. connective tissue disease due to inadequate collagen turnover. Cell, 99: 81–92, 1999. 21. Okada, A., Bellocq, J. P., Rouyer, N. Chenard, M. P., Rio, M. C., Chambon, P., and It remains to be determined whether MT1-MMP is critical for early Basset, P. Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in tumor promotion in mammary gland. Until recently, evidence for the stromal cells of human colon, breast, and head and neck carcinoma. Proc. Natl. Acad. activity of MMPs in early tumor promotion was limited. However, Sci. USA, 92: 2730–2734, 1995. 22. Nomura, H., Sato, H., Seiki, M., Mai, M., and Okada, Y. Expression of membrane- it was reported that MMP-3 induced mammary gland changes as type metalloproteinase in human gastric carcinoma. Cancer Res., 55: 3263–3266, a natural promoter in early tumor formation in the absence of 1995. exogenous mutagens or endogenous oncogenes or suppressor gene 23. Tokuraku, M., Sato, H., Murakami, S., Okada, Y., Watanabe, Y., and Seiki, M. Activation of the precursor of gelatinase A/72 kDa type IV collagenase/MMP-2 in defects (49, 50). Therefore, the availability of MMTV/MT1-MMP lung carcinoma correlates with the expression of membrane-type metalloproteinase transgenic mice as a mammary tumor model should lead to eluci- (MT-MMP) and with lymph node metastasis. Int. J. Cancer, 64: 355–359, 1995. 24. Ohtani, H., Motohashi, H., Sato, H., Seiki, M., and Nagura, H. Dual over-expression dation of the malignant process and to an understanding of how an pattern of membrane-type metalloproteianse-1 in cancer and stromal cells in human abnormal microenvironment in mammary glands could lead to gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron cancer induction and progression. microscopy. Int. J. Cancer, 68: 565–570, 1996. 25. Lee, F., Mulligan, R., Berg, P., and Ringold, G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumor virus chimaeric plas- mids. Nature (Lond.), 294: 228–232, 1981. REFERENCES 26. Hogan, B., Costantini, E., and Lancy, F. In: M. A. Salem (ed.), Manipulating the Mouse Embryo: A Laboratory Manual, pp.127–252. Cold Spring Harbor, NY: Cold 1. Sato, H., and Seiki, M. Membrane-type matrix metalloproteinases (MT-MMPs) in Spring Harbor Laboratory Press, 1986. tumor metastasis. J. Biochem., 119: 209–215, 1996. 27. Cardiff, R. D., Anver, M. R., Guesterson, B. A., Hennighausen, L., Jensen, R. A., 2. Stetler-Stevenson, W. G., Aznavoorian, S., and Liotta, L. A. Tumor cell interactions Merino, M. J., Rehm, S., Russo, J., Tavassoli, F. A., Wakerfield, L. M., Ward, J. M., with the extracellular matrix during invasion and metastasis. Annu. Rev. Cell Biol., and Green, J. E. The mammary pathology of genetically engineered mice: the 9: 541–573, 1993. consensus report and recommendation from the Annapolis meeting. Oncogene, 19: 3. Tryggvanson, K., Hoyhthy, M., and Pyke, C. Type IV collagenase in invasive tumor. 968–988, 2000. Breast Cancer Res. Treat., 24: 209–218, 1993. 28. Seiki, M. Membrane-type matrix metalloproteinases. APMIS, 107: 137–143, 1999. 4. Matrisian, L. M. Metalloproteinases and their inhibitors in matrix remodeling. Trends 29. Okada, A., Tomasetto, C., Lutz, Y., Bellocq, J. P., Rio, M. C., and Basset, P. Genet., 6: 121–125, 1990. Expression of matrix metalloproteinases during rat skin wound healing: evidence that 5. Werb, Z., Ashkenas, J., MacAuley, A., and Wiesen, J. F. Extracellular matrix membrane type-1 matrix metalloproteinase is a stromal activator of pro-gelatinase remodeling as a regulator of stromal-epithelial interactions during mammary gland A. J. Cell Biol., 137: 67–77, 1997. development, involution and carcinogenesis. Braz. J. Med. Biol. Res., 29: 1087–1097, 30. Pulyaeva, H., Bueno, J., Polette, M., Birembaut, P., Sato, H., Seiki, M., and 1996. Thompson, E. W. MT1-MMP correlates with MMP-2 activation potential seen after 6. Wilsion, C. L., Heppner, K. J., Labosky, P. A., Hogan, B. L., and Martrisian, M. epithelial to mesenchymal transition in human breast carcinoma cells. Clin. Exp. Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrily- Metastasis, 15: 111–120, 1997. sin. Proc. Natl. Acad. Sci. USA, 94: 1402–1407, 1997. 31. van den Hooff, A. Stromal involvement in malignant growth. Adv. Cancer Res., 50: 7. Rudolph-Owen, L. A., Chan, R., Muller, W. J., and Matrisan, L. M. The matrix 159–196, 1988. metalloproteinase matrilysin influences early-stage mammary tumorigenesis. Cancer 32. Weidner, N., Folkman, J., Pozza, F., Bevilacqua, P., Allred, E. N., Moore, D. H., Mei, Res., 58: 5500–5506, 1998. S., and Gasparini, G. Tumor angiogenesis: a new significant and independent prog- 8. Itoh, T., Tanioka, M., Yoshida, H., Yoshioka, T., Nishimoto, H., and Itohara, S. nostic indicator in early stage breast carcinoma. J. Natl. Cancer Inst., 84: 1875–1887, Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer 1992. Res., 58: 1048–1051, 1998. 33. Sieweke, M. H., and Bissell, M. J. The tumor promoting effect of wounding: a 9. Masson, R., Lefebvre, O., Noel, A., Fahime, M. E., Chenard, M. P., Wendling, C., possible role for TGF-␤-induced stromal alterations. Crit. Rev. Oncog., 5: 287–311, Kebers, F., LeMeur, M., Dierich, A., Fiodart, J. M., Basset, P., and Rio, M. C. In vivo 1994. evidence that the stromelysin-3 metalloproteinase contributes in a paracrine manner 34. Ronnov-Jessen, L., Petersen, O. W., and Bissell, M. J. Cellular changes involved in to epithelial cell malignancy. J. Cell Biol., 140: 1535–1541, 1998. conversion of normal to malignant breast: importance of the stromal reaction. Physiol. 10. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. Rev., 76: 69–125, 1996. A matrix metalloproteinase expressed on the surface of invasive tumor cells. Nature 35. Jacobs, T. W., Byrne, C., Colditz, G., Connolly, J. L., and Schnitt, S. J. Radial scars (Lond.), 370: 61–65, 1994. in benign breast-biopsy specimen and the risk of breast cancer. N. Engl. J. Med., 340: 11. Will, H., and Hinzmann, B. cDNA sequence and mRNA tissue distribution of novel 430–436, 1999. human matrix metalloproteinase with a potential transmembrane segment. Eur. J. Bio- 36. Jacoby, R. F., Schlack, S., Cole, C. E., Skarbek, M., Harris, C., and Meisner, L. F. A chem., 231: 602–608, 1995. juvenile polyposis tumor suppressor locus at 10q22 is deleted from nonepithelial cells 12. Takino, T., Sato, H., Shinagawa, A., and Seiki, M. Identification of the second in the lamina propria. Gastroenterology, 112: 1398–1403, 1997. membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta 37. Kinzer, K. W., and Vogelstein, B. Landscaping the cancer terrain. Science (Wash- cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP ington DC), 280: 1036–1037, 1998. family. J. Biol. Chem., 270: 23013–23020, 1995. 38. Willenbucher, R. F., Aust, D. E., Chang, C. G., Zelman, S. J., Ferrell, L. D., Moore, 13. Puente, X. S., Pendas, A. M., Llano, E., Velasco, G., and Lopenz-Otin, C. Molecular D. H., and Waldman, F. M. Genomic instability is an early event during the cloning of a novel membrane-type matrix metalloproteinase from a human breast progression pathway of ulcerative-colitis-related neoplasia. Am. J. Pathol., 154: carcinoma. Cancer Res., 56: 944–949, 1996. 1825–1830, 1999. 14. Pei, D. Identification and characterization of the fifth membrane-type matrix metal- 39. Thomasset, N., Locheter, A., Sympson, C. J., Lund, L. R., Williams, D. R., loproteinase MT5-MMP. J. Biol. Chem., 274: 8925–8932, 1999. Behrendtson, O., Werb, Z., and Bissell, M. J. Expression of autoactivated strymely- 15. Llano, E., Pendas, A. M., Freije, J. P., Nakano, A., Knauper, V., Murphy, G., and sin-1 in mammary glands of transgenic mice leads to a reactive stroma during early Lopenz-Otin, C. Identification and characterization of human MT5-MMP, a new development. Am. J. Pathol., 153: 457–467, 1998. membrane-bound activator of progelatinase A overexpressed in brain tumors. Cancer 40. Yamamoto, M., Mohanam, S., Sawaya, R., Fuller, G. N., Seiki, M., Sato, H., Res., 59: 2570–2576, 1999. Gokaslan, Z. L., Liotta, L. A., Nicolson, G. L., and Rao, J. S. Differential expression 16. Vassalli, J. D., and Pepper, M. S. Tumour biology. Membrane proteases in focus. of membrane-type matrix metalloproteinase and its correlation with gelatinase A Nature (Lond.), 370: 14–15, 1994. activation in human malignant brain tumors in vivo and in vitro. Cancer Res., 56: 17. Knauper, V., Will, H., Lopez-Otin, C., Smith, B., Atkinson, S. J., Stanton, H., 384–392, 1996. Hembry, R. M., and Murphy, G. Cellular mechanisms for human procollagenase-3 41. Emmert-Buck, M. R., Roth, M. J., Zhuang, Z., Campo, E., Rozhin, J., Sloane, B. F., (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase A Liotta, L. A., and Stetler-Stevenson, W. G. Increased gelatinase A (MMP-2) and (MMP-2) are able to generate active enzyme. J. Biol. Chem., 271: 17124–17131, cathepsin B activity in invasive tumor regions of human colon cancer samples. Am. J. 1996. Pathol., 145: 1285–1290, 1994. 18. Kinoh, H., Sato, H., Tsunezuka, Y., Takino, T., Kawashima, A., Okada, Y., and Seiki, 42. Afzal, S., Lalani, el-N., Foulkes, W. D., Boyce, B., Tickle, S., Cardillo, M. R., Baker, M. MT-MMP, the cell surface activator of proMMP-2 (pro-gelatinase A), is ex- T., Pignatelli, M., and Stamp, G. W. H. Matrix metalloproteinase-2 and tissue 989

inhibitor of metalloproteinase inhibitor-2 expression and synthetic matrix metallo- activation cascades based on membrane-type 1 matrix metalloproteinase: associated proteinase-2 inhibitor binding in ovarian carcinomas and tumor cell lines. Lab. activation of gelatinase A, gelatinase B, and collagenase B. Biochem. J., 331: Investig., 74: 406–421, 1996. 453–458, 1998. 43. Davies, B., Miles, D. W., Happerfield, L. C., Nayer, M. S., Bobrow, L. G., Rubens, 47. Murphy, G., Stanton, H., Cowell, S., Butler, G., Knauper, V., Atkison, S., and R. D., and Balkwill, F. R. Activity of type IV collagenases in benign and malignant Gavrilovic, J. Mechanisms for pro matrix metalloproteinase activation. APMIS, 107: breast disease. Br. J. Cancer, 67: 1126–1131, 1993. 38–44, 1999. 44. Strongin, A. Y., Collier, I., Bannikov, G., Marmer, B. L., Grant, G. A., and Goldberg, 48. D’Ortho, M. P., Will, H., Atkinson, S., Butler, G., Messent, A., Gavrilovic, J., Smith, G. I. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation B., Timpl, R., Zardi, L., and Murphy, G. Membrane-type metalloproteinase 1 and 2 of the activated form of the membrane metalloproteinase. J. Biol. Chem., 270: exhibit broad spectrum proteolytic capacities comparable to many matrix metallo- 5331–5338, 1995. proteinase. Eur. J. Biochem., 250: 751–757, 1997. 45. Brown, P. D., Bloxidge, R. E., Stuart, N. S. Gatter, K. C., and Carmichael, J. 49. Sternlicht, M. D., Bissell, M. J., and Werb, Z. The matrix metalloproteinase strome- Association between expression of 72-kilodalton gelatinase and tumor spreads in lysin-1 acts as a natural tumor promoter. Oncogene, 19: 1102–1113, 2000. non-small cell lung carcinoma. J. Natl. Cancer Inst., 85: 574–578, 1993. 50. Sternlicht, M. D., Lochter, A., Sympson, C. J., Huey, B., Rougier, J. P., Gray, J. W., 46. Cowell, S., Knauper, V., Stewart, M. L., D’Ortho, M. P., Standon, H., Hembry, R. M., Pinkel, D., Bissell, M. J., and Werb, Z. The stromal proteinase MMP3/stromelysin-1 Lopez-Otin, C. Reynolds, J. J., and Murphy, G. Induction of matrix metalloproteinase promotes mammary carcinogenesis. Cell, 98: 137–146, 1999.

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