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[Cancer Research 64, 652-658, January 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Pro-Matrix Metalloproteinase-2 Transfection Increases Orthotopic Primary Growth and Experimental Metastasis of MDA-MB-231 Human Breast Cancer Cells in Nude Mice

Angus M. Tester1, Mark Waltham1,2, Se-Jeong Oh4,6, Seog-Nyeon Bae5,6, Margaret M. Bills1, Emma C. Walker1, Francis G. Kern6, William G. Stetler-Stevenson7, Marc E. Lippman6 and Erik W. Thompson1–3,6

1 Victorian Breast Cancer Research Consortium (VBCRC) Invasion and Metastasis Unit, St. Vincent’s Institute of Medical Research,
2 Department of Surgery, University of Melbourne, and
3 Bernard O’Brien Institute for Microsurgery, St. Vincent’s Hospital, Melbourne, Australia;
4 Department of Surgery, The Catholic University of Korea, Our Lady of Mercy Hospital, Inchon, Korea;
5 Departments of Obstetrics and Gynecology, Kangnam St. Mary’s Hospital, The Catholic University of Korea Medical College, Seoul, Korea;
6 Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC; and
7 Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade all known extracellular matrix proteins, as well as a growing number of growth factors, growth factor receptors, cytokines, and cytokine receptors (1) . These can have either positive or negative effects on important cellular processes, including growth, migration, angiogenesis, and invasion (1) . MMPs have been implicated in the induction and progression of mammary cancers in transgenic mice (reviewed in Ref. 2 ), and MMPs are abundant in the stroma surrounding invasive human breast cancers (reviewed in Ref. 3 ).

MMP-2 (gelatinase A) is believed to play a critical role in basement membrane degradation. It is able to hydrolyze elastin, laminin, fibronectin, proteoglycans, fibrillin, and most notably collagen type IV, which is a major structural component of basement membranes (4, 5, 6) . Additional MMP-2 substrates include Fas ligand, tumor necrosis factor, fibroblast growth factor receptor 1, heparin binding epidermal growth factor, interleukin 8, interleukin 1ß, insulin-like growth factor binding proteins, fibrinogen, factor XII, the CC chemokine MCP-3, and the CXC chemokines SDF-1{alpha} and ß (reviewed in Ref. 2 ). Strong associations have been reported between MMP-2, cancer cell invasiveness, and metastatic progression of a variety of cancers, including lung, prostate, breast, colon, and neuroblastoma (7, 8, 9, 10, 11, 12) . MMP-2-deficient mice show decreased experimental lung metastasis with cells that either do or do not make their own MMP-2 (13) . As with most MMPs, MMP-2 is synthesized and secreted as a latent zymogen, requiring the proteolytic removal of the propeptide for activity. MMP-2 activation is initiated by membrane type (MT) MMPs (14) , and MT1-MMP appears to be a preeminent activator of MMP-2 (15, 16, 17) compared with MT2-, MT3-, MT5-, and MT6-MMP (18, 19, 20, 21, 22) . MMP-2 is largely produced by the reactive stromal cells adjacent to breast tumors (3 , 23 , 24) ; however, the ability to activate pro-MMP-2 is a hallmark of invasive human breast cancer cell lines (25) and correlates with mesenchymal attributes, including vimentin and MT1-MMP expression, thought to arise through epithelial to mesenchymal transition (26, 27, 28) .

Patients with node-positive breast cancer have a high propensity for recurrence, the most common first sites being locally in the chest wall, locoregional lymph nodes, and bone. Liver, lung, and central nervous system are less common sites of first metastasis but are often effected in patients with well-advanced disease (29) . Bone is an especially favored site, and >25% of breast cancer patients with invasive cancer will develop clinically relevant bone metastases (30) . At autopsy, >80% of women who die from the disease show evidence of skeletal involvement (31) . This study investigates the involvement of MMP-2 in the metastatic spread of human MDA-MB-231 human breast cancer cells. This invasive, estrogen receptor-negative, vimentin-positive cell line reproducibly forms primary tumors when implanted into the mammary fat pads of nude mice and metastasizes to their lungs, livers, and brains over a 2-month period (32) . Bone metastasis is not seen in this model, and intracardiac inoculation (29 , 33 , 34) was used to study bone and soft tissue metastasis of these cells. We report here that overexpression of pro-MMP-2 by MDA-MB-231 cells enhanced their invasiveness in the in vitro Matrigel-based assays, stimulated their growth in the mammary fat pad in nude mice, and enhanced the degree of experimental metastasis to a variety of organs after intracardiac inoculation in the nude mouse. Thus, this study directly implicates MMP-2 in the metastatic progression of breast cancer cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Originally the MDA-MB-231 human breast cancer cell line was obtained from the American Type Culture Collection (Manassas, VA) and genetically tagged by transduction of the bacterial ß-galactosidase (Lac Z) retroviral vector to generate the MDA-MB-231 BAG cell line (35) . Cells were routinely passaged in Richter’s improved minimum essential medium [IMEM (Life Technologies, Inc., Gaithersburg, MD)] supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Cells were confirmed to be free of Mycoplasma contamination using the Gen-Probe kit (Gen-Probe, San Diego, CA).

RNase Protection Analysis.
RNase protection analysis using total RNA was performed as described previously (36) . The MMP-2 RNase protection probe was prepared by subcloning an internal 320-bp fragment of the pGel 186.2 clone (kindly provided by Dr. Greg Goldberg, St. Louis, MO; Ref. 37 ) into the pGEM7zf+ expression vector (Promega; Madison, WI). A probe for 36B4, a nonestrogen-regulated ribosomal protein, which shows similar expression in a variety of breast cancer cell lines, was used as a loading control (38) .

Stable Transfection of Pro-MMP-2 into MDA-MB-231 BAG Cells.
Full-length human proMMP-2 cDNA (clone pIV-16; Ref. 39 ) was cloned into the mammalian expression vector pCHC6 (36) . This vector places the cDNA under the constitutive control of the cytomegalovirus intermediate gene promoter, which shows good activity in a variety of human breast cancer cells. The pCHC6-pro-MMP-2 or pCHC6 (vector control) plasmids were transfected into MDA-MB-231 BAG cells using the Chen and Okayama protocol (40) . The cells were cultured under Hygromycin B selection (150 µg/ml; Sigma Chemical Co., St. Louis, MO). Hygromycin B-resistant colonies were cloned using cloning cylinders, expanded, and cultured under continued Hygromycin B selection.

Analysis of Pro-MMP-2 Expression and Activation.
Cells (5 x 104) were plated in a 24-well plate in IMEM containing 10% fetal bovine serum. The cells were allowed to adhere overnight, then washed in unsupplemented media twice before incubation in 200 µl of serum-free medium [improved minimum essential medium containing 1x ITS (10 µg/ml insulin, 5.5 µg/ml transferin, and 5 ng/ml selenium], 1x nonessential amino acids, 2 mg/ml fibronectin, 1 mM sodium pyruvate, 1x vitamins, and 0.1% BSA (all from Sigma Chemical Co.) for 24 h. Concanavalin A (conA; 25 µg/ml) was included in some cultures to induce activation of pro-MMP-2 as described previously (41) . Conditioned media (24 h) was analyzed by gelatin zymography to detect activation of pro-MMP-2 as previously described (26) and also by Western blot. Western blot analysis was carried out on total protein lysates prepared by harvesting cells in radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA], with added protease inhibitors (10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na3VO4, 1 mM NaF, and 1 mM phenylmethylsulfonyl fluoride). Protein concentration was estimated using BCA protein assay kit (Pierce, Rockford, IL). Lysates were separated on a 10% SDS-PAGE gel and subsequently transferred onto polyvinylidene difluoride membrane Immobilon-P (Millipore, Bedford, MA). Membranes were blocked in 5% milk powder and incubated with anti-MMP2 antibody PAb 753 (kindly provided by Dr. Jack Windsor, University of Alabama), at a concentration of 10 µg/ml. Detection of the horseradish peroxidase-conjugated goat antirabbit antibody was carried out using SuperSignal West Pico Chemiluminescent Substrate (Pierce).

In Vitro Invasion Assays.
Boyden chamber chemoinvasion and Matrigel outgrowth analysis was performed as described previously (42) . Cells (7.5 x 104) were loaded into individual 13-mm diameter blind well Boyden chambers (Neuroprobe, Cabin John, MD) on 12-µm polyvinylpyrrolidone-free polycarbonate filters (Nucleopore, Pleasanton, CA) coated with 25 µg of Matrigel (kindly provided by Dr. Hynda Kleinman, NIH, Bethesda, MD). Each clone was tested in triplicate, and the assay was repeated five times. Assay duration was 16 h, and the degree of invasion was scored using image analysis on an IBAS Image Processing System (Kontron Image Analysis Division, Hamamatsu, Japan) after staining the cells that had traversed the filters. Five separate fields were randomly selected on each triplicate filter from all five experiments, and the data subjected to ANOVA with generalized estimating equations to adjust for two sources of dependence (intrafilter and intra-assay; Ref. 43 ). For Matrigel outgrowth assays, cells (2 x 104) were dispersed in 48-well plates in 75 µl of undiluted Matrigel (10 mg/ml) and then overlaid onto 100 µl of polymerized Matrigel. Once the top layer had polymerized, the cells were cultured in IMEM supplemented with 10% fetal bovine serum for 10 days and photographed at x20 magnification.

Mammary Fat Pad Inoculation of MDA-MB-231 Cells.
These experiments were conducted with full ethical approval of the St. Vincent’s Hospital Animal Ethics Committee and in accordance with the Australian National Health and Medical Research Councils Guidelines for the Care and Use of Laboratory Animals. Groups of 8–10 mice received mammary fat pad inoculation of either MMP-2-transfected (n = 4) or vector control (n = 4) MDA-MB-231-BAG clones (5 x 105 cells/15 µl), respectively, as described by Price et al. (44) . Tumor growth was assessed by measuring the length and width of tumors with electronic calipers every 3–4 days continuously from the third week after injection. Volumes were calculated using the formula (length) x (width)2/2. Mice were sacrificed between 38 and 60 days after inoculation when the tumors approached 2000 mm3. Statistical analysis was by two-way repeated measures ANOVA (GraphPad Prism, San Diego, CA).

Intracardiac Inoculation of Cells.
These experiments were conducted at Georgetown University in accordance with the NIH Guide for the Care and Use of Laboratory Animals. MDA-MB-231 BAG clones were washed twice with phosphate buffered saline (PBS) and then resuspended at 106 cells/ml PBS. Mice (6–8 week old intact female NCr nu/nu; NCI, Frederick, MD) were anesthetized by inhalation of methoxyflurane (Pittman Moore, Mundelein, NJ), and 105 cells were inoculated into the left ventricle as described previously (34) . The mice were allowed to recover from the anesthesia before being returned to their cages and maintained under pathogen-free conditions. Mouse health was monitored daily, and mice were sacrificed at the first signs of discomfort or after 4–5 weeks.

Analysis of Metastatic Burden in Mice.
At the time of harvest, culled mice were subjected to high resolution X-ray analysis (Faxitron; 20-µm focal source) to record skeletal defects and thinning, particularly in the growth plate region of the hind limbs. Mice were then dissected, and intact femur and tibia (carefully trimmed of skin, muscle, and excess tissue), lung, brain, kidney, and liver were snap frozen in liquid nitrogen. Total genomic DNA was isolated from each organ for real-time TaqMan PCR detection of the ß-galactosidase-tagged human breast cancer cells using the 5700 Sequence Detection System (PE Applied Biosystems, Melbourne, Australia) as described previously (34) . Each sample was analyzed by TaqMan in triplicate (minimum). The number of metastatic cells present in each organ was interpolated from a standard curve constructed with known numbers of cultured MDA-MB-231 BAG cells homogenized and spiked into control naïve mouse bones and organs.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transfection of Pro-MMP-2 into MDA-MB-231 BAG Cells.
The expression of MMP-2 by a panel of human breast cancer cell lines was investigated by RNase protection analysis. Of the cell lines analyzed, only MDA-MB-436 and Hs578T expressed MMP-2 (Fig. 1)Citation , and these have been shown previously to secrete MMP-2 by zymography (25) . We selected MDA-MB-231 cells to transfect with the pro-MMP-2 cDNA to analyze the biological significance of MMP-2 in cancer progression. The MDA-MB-231 cell line does not constitutively express MMP-2 but is able to activate exogenous pro-MMP-2 to its mature form after stimulation with conA (41) or collagen (25) and has previously been shown to form distant metastatic colonies after intracardiac injection of the cells into immunocompromised mice (33 , 34 , 45) . Pro-MMP-2 cDNA was transfected into MDA-MB-231 BAG cells as described in "Materials and Methods." Five stable MMP-2-expressing clones and 10 vector control clones were selected for additional analysis. Fig. 2ACitation shows that each of the pro-MMP-2-transfected clones produced and secreted pro-MMP-2 (72 kDa), whereas the clones transfected with vector alone (representative of all control clones tested and parental cells) did not produce MMP-2. The identification of pro-MMP-2 secreted by the pro-MMP-2-transfected clones was confirmed by Western blot analysis (Fig. 2B)Citation . When cultured in the presence of conA, the pro-MMP-2-transfected clones were able to activate the pro-MMP-2 to the intermediate (64 kDa) and fully active (62 kDa) forms (Fig. 2C)Citation to a similar extent to that seen previously with parental MDA-MB231-BAG cells and exogenous proMMP-2 (25) . As expected. conA had no effect on proMMP-2 secretion or activation by the cells transfected with vector alone.



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  		Fig. 1. Lack of matrix metalloproteinase-2 expression in MDA-MB-231 human breast cancer cells. RNase protection analysis of total cellular RNA (40 µg) from a panel of human breast cancer cell lines was performed to compare for steady-state levels of matrix metalloproteinase-2 mRNA expression (283 bp). 36B4 (205 bp), a nonestrogen-regulated ribosomal protein, was used as a loading control.

 


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  		Fig. 2. Promatrix metalloproteinase-2 (pro-MMP-2)-transfected MDA-MB-231 clones secrete and activate MMP-2. Conditioned media (24 h) from cells transfected with pro-MMP-2 or vector control (VC) were analyzed for secreted pro-MMP-2 by gelatin zymography (A) or Western blot (B). C, representative clones (MMP-2 no. 19, VC no. 9) were cultured in the presence or absence of concanavalin A (conA), conditioned media (24 h) was then subjected to gelatin zymography to detect latent (72 kDa), intermediate (64 kDa), and fully mature MMP-2 (62 kDa).

 
In Vitro Invasive Potential of Cells.
To determine whether the cells were able to use the transfected proMMP-2, their in vitro invasive potential was analyzed in Matrigel-based invasion assays. Boyden chamber chemoinvasion analysis (Fig. 3A)Citation showed that a significantly greater number (P < 0.001, ANOVA) of pro-MMP-2 transfected cells invaded the Matrigel compared with the vector control clones. This was seen in all of the clones analyzed (see figure legend). Attempts to additionally stimulate this with agents known to enhance MMP-2 activation (conA, phorbol ester, or fibrillar collagen) were unsuccessful because of their own acute effects on the invasion assays (data not shown).



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  		Fig. 3. Increased in vitro invasive potential of promatrix metalloproteinase-2 (pro-MMP-2)-transfected cells. A, the ability of cells to invade Matrigel (25 µg/cm2) was analyzed by chemoinvasion in blind well Boyden chambers. The assay was performed over 16 h. Error bars represent the mean ±SD; each clone was analyzed five times in triplicate. Statistical analysis was performed by ANOVA, and * indicates a P <0.0001 compared with the vector control clones. B, cells were cultured within a Matrigel matrix for 10 days. Images were captured from a phase microscope at x20 magnification. Panels show photomicrographs of parental (A), vector control clones no. 9 (B) and no. 10 (C), and pro-MMP-2 clones no. 16 (D), no. 17 (E), and no. 19 (F).

 
The ability of the cells to grow within a three-dimensional Matrigel matrix was also analyzed in the Matrigel outgrowth assay. Fig. 3BCitation shows that the three pro-MMP-2 clones analyzed produced a more extensive stellate network of outgrowth compared with the parental cells and vector control clones. It is noteworthy that our previous study in which MCF-7 cells were transfected with the same pro-MMP-2 vector did not show any enhanced invasion, as was recently confirmed by others (47) . MCF-7 cells have retained epithelial characteristics and lack MT1-MMP and, consequently, lack the ability to activate the proMMP-2 (27) .

Tumorigenicity of the Cells in the Mouse Mammary Fat Pad.
Parental polyclonal MDA-MB-231-BAG cells show a latency period after inoculation into the mice of 10–15 days before they become palpable and then grow exponentially over a 6–8-week period when they reach ethical limits for tumor burden, as also seen in the current study (Fig. 4)Citation . Tumors arising from each of the four MMP-2-transfected MDA-MB-231 clones grew faster than any of the four vector control clones. Two of the MMP-2-transfected clones outgrew the parental population, whereas the other two did not. We have seen in a number of such experiments that clonal MDA-MB-231 sublines perform poorly compared with the parent, and we attribute this to lack of heterotypic, interclonal stabilization. However, all MMP-2-transfected clones outgrew all vector control clones (two-way RM-ANOVA, P = 0.003).



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  		Fig. 4. Mammary fat pad analysis. Matrix metalloproteinase (MMP)-2-transfected clones and vector control clones were injected into mammary fat pads of nude mice. Growth of the MMP-2-transfected clones are shown in {square}, whereas the vector control clones are shown in {bullet}, and individual clones are indicated by each line. Parental cells (MDA-MB-231 BAG) cells are shown in {blacktriangleup}. Statistical analysis showed that MMP-2-transfected clones grew faster than their vector control counterparts (P = 0.0003).

 
In Vivo Analysis of the Metastatic Potential of the Cells.
An arterial seeding model involving direct intracardiac inoculation of the human breast cancer cells into nude mice was used to analyze the in vivo effect of transfected pro-MMP-2 on experimental metastasis to bone and soft organs. In a pilot experiment, pools of three vector control clones (nos. 9, 10, and 19) and three pro-MMP-2-transfected clones (nos. 16, 17, and 19) were used, and a notably higher degree of bone damage was detected radiographically in the MMP-2-transfected pool (Fig. 5)Citation . The three individual clones of each type were additionally tested in 4 mice each for bone and organ colonization after intracardiac injection using PCR, and mice were monitored radiologically for osteolysis. Overall, there was an increase in metastasis with the three MMP-2 clones compared with the vector control clones tested. Exact Pearson {chi}2 analysis indicated that MMP-2 facilitated either lung or bone metastasis in conjunction with additional clonal traits (P < 0.05; data not shown). In a follow up experiment designed for greater statistical power, five pro-MMP-2 clones and 10 vector controls were inoculated intracardiacally into 10 mice/clone, and the incidence of metastasis to bone and visceral organs was quantitated by real-time PCR. Faxitron analysis was not available; however, we have previously demonstrated a direct correlation between the degree of osteolytic damage observed and the metastatic burden detected by real-time PCR within the bone matrix (34) .



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  		Fig. 5. Radiographic analysis of osteolytic lesions after intracardiac injection. Bone damage was analyzed by high-resolution X-ray analysis (Faxitron; 20-µm focal source). Representative images of the long bones from mice after intracardiac inoculation of a pooled population of vector control clones (nos. 9, 10, and 19) are depicted in the left panel, and the pooled population of matrix metalloproteinase-2-transfected clones (nos. 16, 17, and 19) are depicted in the right panel. White arrowheads indicate lesions.

 
The individual five pro-MMP-2-transfected clones showed differing metastatic potential (Table 1)Citation . Clones MMP-2 nos. 16 and 17 were the most metastatic, showing a high level of metastatic progression to all of the organs analyzed. Clone MMP-2 no. 19 showed a high rate of metastasis to brain and liver compared with the other organs, whereas clones MMP-2 nos. 7 and 11 showed metastasis to all organs but to a lesser level. Overall the 10 vector control clones displayed a lower metastatic potential (Table 1)Citation . However, vector control (VC) clones VC nos. 2, 5, and 9 did exhibit a high incidence of metastasis to one or more visceral organ.


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  		Table 1 Incidence of metastasis after intracardiac injection of cells into mice

Five pro-matrix metalloproteinase (MMP)-2-transfected (MMP-2) clones and 10 vector control (VC) clones were injected into the left ventricle of nude mice (n = 10/clone). Total genomic DNA was isolated from each organ, and metastatic human breast cancer cells were detected by real time PCR. Values in parentheses represent the number of mice that survived until organ harvest.

 
The data from the individual clones was compared in two ways; Fig. 6ACitation shows that those mice injected with pro-MMP-2-transfected cells exhibited a significant increase (P < 0.001, Mann-Whitney test) in the incidence of metastasis to bone (4-fold) as well as significant increases to brain (P = 0.001), liver (P = 0.002), and kidney (P = 0.028; all ~2-fold) compared with the clones transfected with vector alone. Pro-MMP-2 transfection did not significantly affect the incidence of metastasis to the lung. Real-time PCR enables quantification of the metastatic burden in the different tissues. Fig. 6BCitation shows that the pro-MMP-2-transfected clones produced significantly larger tumors in the lungs of mice (P < 0.02, Mann-Whitney test) and showed a trend toward larger burdens in bone (which failed to achieve statistical significance), but appeared not to show increased metastatic burden in the other soft organs (liver, kidney, and brain).



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  		Fig. 6. In vivo metastatic analysis of cells. Five promatrix metalloproteinase (pro-MMP)-2-transfected clones and 10 clones transfected with vector alone were injected into the left ventricle of nude mice (n = 10/clone). After 4–5 weeks, the presence of the genetically tagged human breast cancer cells was detected by TaqMan real-time PCR. Clones transfected with pro-MMP-2 are represented by the {blacksquare} and vector control clones by the {square}. Error bars represent the mean ± SD. A, the incidence of metastasis to bone and visceral organs is expressed as the percentage of animals with detectable metastatic cells in those organs. Cells transfected with pro-MMP-2 showed a significant (*) increase (Mann-Whitney test) in the incidence of metastasis to bone (P < 0.001), brain (P = 0.001), liver (P = 0.002), and kidney (P = 0.028). B, quantitation of the metastatic burden in the secondary organs by real-time PCR indicated that MMP-2-transfected cells produced significantly (*) larger tumors in lung (P < 0.02, Mann-Whitney test).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MMP-2 catalytic activity requires the removal of the propeptide. MDA-MB-231 cells express MT-MMP (27) , the cell surface activator of pro-MMP-2, and were able to activate the pro-MMP-2 into the fully mature enzyme when cultured in the presence of conA. We demonstrated that the cells were able to use the pro-MMP-2, resulting in an enhanced in vitro invasive phenotype. Pro-MMP-2-transfected clones were able to invade a Matrigel barrier at a greater rate and also exhibited more rapid stellate outgrowth within a Matrigel matrix. Outgrowth within Matrigel (a gel composed of basement membrane components) demonstrates the cells proliferative, migratory, adhesive, and invasive abilities (47) . The ability of the cancer cell to activate latent pro-MMP-2 is paramount to the cell using the transfected pro-MMP-2. Transfection of MMP-2 into the noninvasive MCF-7 human breast cancer cell line did not result in an increase in the invasive phenotype of the cells, presumably because of a lack of MT-MMP expression.9 The mechanism of MMP-2 activation used by the MMP-2-transfected cells in the above assays is not known because our culture systems did not contain any of the known stimuli for MT1-MMP-mediated MMP-2 activation such as fibrillar collagen or conA (29) . The results, however, clearly imply that the transfected MMP-2 is functioning, and there is sufficient pericellular activated MMP-2 generated. The stimulation seen in the mammary fat pad tumors after MMP-2-transfection additionally indicates that the MMP-2 is functional and perhaps stimulates the generation of autocrine or paracrine growth signals and/or stimulates angiogenesis of the primary tumors.

These data are consistent with additional studies in MDA-MB-231 cells with Prinomastat (AG3340), a gelatinase-selective MMP inhibitor, which dramatically inhibited the growth of parental MDA-MB-231 BAG xenografts when administered at an early stage.10 Our results suggest that MMP-2 is at least one candidate target for this inhibition, although MMP-9 and MT1-MMP, against which this inhibitor is also selectively effective, cannot be ruled out (48) . Abundant MMP-2 localization of MMP-2 protein in the tumor parenchyma has been reported at the protein level in many breast cancer studies, although the mRNA for MMP-2, and its activator MT1-MMP, is almost exclusively produced by the surrounding stroma (3 , 16 , 49) . Analysis of the MDA-MB-231 xenografts with species-specific reverse transcription-PCR primers show abundant mouse stromal MMP-2 and MT1-MMP, and confirm the lack of expression of human MMP-2 or MMP-9 by the parental MDA-MB-231 cells.11 Thus, the pro-MMP-2-transfected MDA-MB-231 cells provide a model for the MMP-2 usually provided by stromal cells. Transfection of MCF-7 cells with MT1-MMP alone (46) or in conjunction with integrin ß3 (50) causes increased MMP-2-activation and invasion in vitro, and MT1-MMP alone caused up-regulation of VEGF in MCF-7 cells, which increased the take rate of these cells in nude mice (46) . This was not enhanced significantly by added transfection of MMP-2 and was not seen with MMP-2 transfection alone and so is unlikely to explain the growth stimulation we have seen in this study.

As a model for certain aspects of human breast cancer metastasis, cancer cells inoculated directly into the left ventricle of immunocompromised mice are able to form distant metastatic colonies in bone and visceral organs (34 , 45 , 51) . Our use of MDA-MB-231 BAG cells genetically tagged with the bacterial ß-galactosidase retroviral vector enabled quantitation of metastatic cells in bone and visceral organs by PCR (34) . We can detect micrometastases as small as 100 cells in lung tissue and have demonstrated a strong correlation between the degree of osteolytic bone damage and the size of the metastatic burden detected in bone (34) . The metastatic profile of the clones differed, with individual clones exhibiting preferential metastasis toward one organ over another. Along these lines, a bone-seeking clone was recently shown to exhibit different properties to a brain-seeking clone derived by sequential passages to the respective organs in nude mice (52) .

Cells overexpressing pro-MMP-2 demonstrated an enhanced metastatic potential, with a significant increase in the incidence as measured by the number of mice showing a threshold number of cells in each organ. We found increased detection of metastasis to bone, brain, liver, and kidney and an increase in the average amount of metastatic burden in lung, although the incidence of lung metastases did not increase. In bone, the trend to increased metastatic burden did not achieve statistical significance. Given the increases seen in the primary tumor and in lung, it is possible that the study was insufficiently powered to detect similar increases in the other soft organs.

The spread of cancer cells from the primary tumor to distant metastatic sites is a complex and multistep process. Experimental metastasis in this model requires the cells to adhere to target endothelia, invade, and migrate into and finally colonize the secondary site or organ (53) . MMPs have been shown to facilitate these processes (reviewed in Ref. 54 ), and mice lacking MMP-2, MMP-7, or MMP-11 exhibit reduced tumorigenesis and angiogenesis (13 , 55 , 56) . This would suggest that MMP-2 confers a more invasive phenotype on the cells, enabling them to locate to and/or establish in secondary organs, and may possibly confer a growth advantage once the cells have begun to proliferate in these end organs. Pro-MMP-2-transfected cells did not show any increased incidence of metastasis to the lungs of mice after intracardiac inoculation, but here, significantly larger tumor burdens were detected. This site-specific difference could be due to higher initial burdens in some of the lungs from injection artifacts such that any advantage coming from pro-MMP-2 transfection for incidence in the lungs was not observed.

Bone metastases result in osteolytic lesions and this bone damage is responsible for the painful conditions experienced by patients. A consensus has formed that osteoclasts and not the cancer cells are responsible for the increased bone resorption (57) , although some challenge this (58) . Breast cancer cells have been shown to stimulate the formation and resorptive activity of osteoclasts by the production of cytokines (59 , 60) . The data presented here suggest that MMP-2 may influence the manifestation of osteolytic lesions. Again, the mechanism behind the increased metastatic incidence with the MMP-2-transfected clones is not clear—it could influence the homing of the cells to the bone, their ability to bind target endothelium and/or extravasate, or perhaps enhance the invasive migration of these cells through the marrow cavity. Recent studies both in breast (61) and prostate cancer (58) cell lines have indicated the potential benefit of MMP inhibitors in limiting bone resorption associated with experimental bone metastasis. In addition, our own data with Prinomastat (Agouron AG 3340; Agouron Pharmaceuticals) in the model described here also showed a delayed onset of osteolytic metastasis.11 The reduction of osteolytic damage in these studies could be attributable to inhibition of host MMPs involved in the bone resorption process and/or those produced by the cancer cells or by reactive bone stromal cells in response to the presence of the cancer cells. Certainly, a number of MMPs have been implicated in osteoclast recruitment, activation, and function, including MMP-1, MMP-3, MMP-9, and MMP-14. The benefit of added pro-MMP-2 in this process suggests that the tumor MMP contribution could be important for homing to the bone, extravasation, migration and invasion, and/or growth of the cancer cells. Alternatively, the additional MMP-2 in the bone marrow cavity could supplement that required for osteoclast maturation—again, additional experimentation is required to address this. This could be an important therapeutic niche because blocking the passage and/or establishment of breast cancer cells to bone could lead to a reduction in painful skeletal metastases.

We provide evidence here that MMP-2 can stimulate the growth and metastasis of human breast cancer cells. Cells overexpressing pro-MMP-2 showed a significantly higher invasiveness in vitro, primary tumor growth in the mammary fat pad, and incidence of metastasis to brain, liver bone, and kidney in an established mouse model. It did not stimulate spontaneous metastasis to soft tissues from the mammary tumors. Additional delineation of the role of MMP-2 in both the primary tumor establishment and experimental metastasis, along with continued refinement of this class of inhibitors, may lead to better prevention and/or treatment options for breast cancer control.


   ACKNOWLEDGMENTS
 
We thank Dr. John Handfelt (Lombardi Cancer Center) for his statistical help, the Statistical Consulting Center at the University of Melbourne, Australia, and Gloria Arand and Nicolle Gibson for their technical support.


   FOOTNOTES
 
Grant support: The Victorian Breast Cancer Research Consortium and the Thomaiy Breast Cancer Research Fund, Melbourne, Australia, as well as the NIH Breast Cancer SPORE Grant 2P50-CA58185-04 in the United States. This work was also supported in part by the Lombardi Cancer Center Shared Resources for Macromolecular Synthesis and Sequencing, Tissue Culture, Animals, and Cytochemistry and Microscopy, USPHS Grant 2P30-CA-51008.

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 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Erik W. Thompson, Invasion and Metastasis Unit, University of Melbourne, Department of Surgery, St. Vincent’s Hospital, 29 Regent Street, Fitzroy, 3065, Australia. Phone: 61-3-9288-2569; Fax: 61-3-9416-0926; E-mail: rik{at}medstv.unimelb.edu.au

9 S. Bae, F. Kern, M. Lippman, and E. Thompson, unpublished observations. Back

10 M. Waltham, A. Tester, N. Ruangpanit, M. Bills, D. Shalinsky, and E. Thompson, unpublished observations. Back

11 M. Lafleur, A. Drew, E. de Sousa, E. Walker, M. Waltham, M. Bills, T. Blick, and E. Thompson, manuscript in preparation. Back

Received 2/11/02. Revised 8/28/03. Accepted 11/11/03.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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