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Cell Growth & Differentiation Vol. 11, 221-229, April 2000
© 2000 American Association for Cancer Research


Articles

1{alpha},25-Dihydroxyvitamin D3 and Its Analogues Down-Regulate Cell Invasion-associated Proteases in Cultured Malignant Cells1

Katri Koli2 and Jorma Keski-Oja

Departments of Virology [K. K., J. K-O.] and Pathology [J. K-O.], the Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland

Abstract

Vitamin D and its derivatives (deltanoids) are potent regulators of cell proliferation and differentiation. Targeted production of proteolytic enzymes like serine proteases and metalloproteinases is an important part of the invasive process of cancer cells. Treatment with 1{alpha}25-dihydroxyvitamin D3 [1{alpha},25(OH)2D3] decreases the invasive properties of breast carcinoma cells. Here we have analyzed the effects of 1{alpha},25(OH)2D3 and its synthetic analogues on the secretion and cell surface association of the components of the plasminogen activator (PA) system and on the secretion of certain matrix metalloproteinases (MMPs) and their inhibitors in MDA-MB-231 breast carcinoma cells. Deltanoids were able to decrease the secretion of urokinase PA and tissue-type PA activity in a dose-dependent manner and to increase PA inhibitor 1 secretion, leading to reduced total PA activity. CB1093 was the most potent analogue, effective at concentrations several logarithms lower than 1{alpha},25(OH)2D3. Transient transfection of different urokinase PA promoter reporter constructs to HT-1080 fibrosarcoma indicator cells indicated that vitamin D-responsive sequences were located between nucleotides -2350 and -1870 in the 5' region of the promoter. Treatment of MDA-MB-231 cells with 1{alpha},25(OH)2D3 or other deltanoids also resulted in decreased MMP-9 levels in association with increased tissue inhibitor of MMP 1 activity. Membrane-type 1-MMP expression or proteolytic processing were not appreciably affected by deltanoids. Vitamin D and its analogues caused a decrease in Matrigel invasion assays of MDA-MB-231 cells. Cancer cell invasion is associated with coordinated secretion of proteolytic enzymes and their inhibitors. Vitamin D and its derivatives can evidently influence invasive processes by two means: (a) decreasing the expression and activity of cell invasion-associated serine proteases and metalloproteinases; and (b) inducing their inhibitors.

Introduction

Accumulating evidence indicates that deltanoids can suppress breast tumor progression in animal models, and this is due to a reduction of tumor growth as well as a reduction of the development of metastases (1, 2, 3) . The steps in the metastatic cascade that are regulated by deltanoids are not well understood. The metastatic process comprises sequential steps including the escape of cells from the primary tumor, survival and transport in the circulation, arrest in a distant organ, extravasation, and growth of cells in the new site (4) . The production of proteolytic enzymes is an important event in various steps of the metastatic process, especially in the degradation of basement membranes and cancer cell invasion into the surrounding normal tissue (5 , 6) .

There are three main groups of proteolytic enzymes, namely the serine proteases, the metalloproteinases, and the cysteine proteinases. The serine proteases uPA3 and tPA can convert plasminogen to plasmin, which is a wide-spectrum protease capable of degrading extracellular and basement membrane proteins as well as activating latent forms of metalloproteinases (7) . Plasmin production is negatively regulated by efficient inhibition of PAs by PAIs. Dysregulation of the PA system in various types of cancers is a frequent observation, and high levels of uPA, PAI-1, and uPAR in tumors correlate with poor patient prognosis (8 , 9) . MMPs are a family of proteases capable of degrading extracellular matrix and basement membrane components including collagens under physiological conditions (10) . These secreted or transmembrane proteases need zinc for catalytic function and are produced as inactive zymogens requiring extracellular activation. MMPs are susceptible to inhibition by TIMPs, which may also have other functions in MMP-mediated processes. The tight regulation of MMPs and TIMPs under normal physiological conditions is often disrupted in malignant disease, leading to increased invasion and tissue destruction (11) . Elevated activities of cysteine proteases have also been suggested to contribute to invasion and metastasis (12 , 13) .

Many hormones, growth factors, and cytokines regulate the PA system. Epidermal growth factor and fibroblast growth factor as well as the tumor promoter PMA can up-regulate uPA in different cell systems (14) . We have previously found that 1{alpha},25(OH)2D3 down-regulates the production of uPA activity in skin keratinocytes and fibroblasts independent of their antiproliferative responses (15 , 16) . In the presence of uPA stimulators epidermal growth factor or PMA, 1{alpha},25(OH)2D3 was still able to reduce secreted uPA activity in keratinocytes, suggesting an important role in the regulation of the PA system in these cells. MMPs are also under tight regulation at both the level of activation of zymogens and the level of transcription. These processes are under growth factor regulation, with PMA being the most extensively studied regulator of MMP activity. In human prostate cancer cells as well as in chondrocyte matrix vesicles, vitamin D metabolites have been suggested to regulate MMP-2 and MMP-9 activity (17 , 18) .

The breast cancer cell line MDA-MB-231 is very invasive and produces high amounts of uPA, PAI-1, and uPAR (9) . These cells also produce high constitutive levels of MMP-9 but undetectable levels of MMP-2. Expression of MMP-1, MMP-3 (stromelysin-1), TIMP-1, and TIMP-2 as well as MT1-MMP has been described previously (19 , 20) . The in vitro invasiveness of MDA-MB-231 cells can be reduced by treatment with 1{alpha},25(OH)2D3, but the mechanisms of this process are still unclear (21) . Using zymography and reverse zymography, we analyzed the effects of 1{alpha},25(OH)2D3 and its synthetic analogues on the production of the components of the PA and MMP systems. To characterize the mechanisms of regulation of uPA by 1{alpha},25(OH)2D3, we used uPA promoter constructs that were transiently expressed in HT-1080 fibrosarcoma cells. In addition, MT1-MMP expression and activity were analyzed in MDA-MB-231 cells. We report here that deltanoids are potent down-regulators of MMP and PA activity in MDA-MB-231 cells, most likely influencing their invasive behavior.

Results

Regulation of the PA System by 1{alpha},25(OH)2D3
Deltanoids are considered relatively new antitumor agents that can influence tumor growth and progression by several mechanisms. They are potent inhibitors of growth for many cancer cells and can also decrease invasion and metastasis in in vitro models as well as in mouse tumor models. Treatment with 1{alpha},25(OH)2D3 of estrogen receptor-negative, highly invasive MDA-MB-231 breast carcinoma cells results in a prominent decrease in their in vitro invasive potential. This was shown not to be based exclusively on its antiproliferative and antimigratory effects (21) . Later it was found that the in vitro invasive capacity of this cell line, as measured by Matrigel invasion assay, was largely dependent on the cell surface uPA system (9) .

A caseinolysis in agar assay indicated that treatment of MDA-MB-231 with 10-7 M 1{alpha},25(OH)2D3 cells under serum-free conditions for 48 h decreased the secreted PA activity to 40% of control values (Fig. 1A).Citation We then analyzed by zymography and reverse zymography the molecular forms of PAs responsible for this effect. MDA-MB-231 cells secreted both uPA and tPA into the culture medium (see also Fig. 2).Citation The amounts of PAs and the uPA:tPA ratio varied somewhat between experiments, but uPA was found to be the prominent PA secreted into the culture medium. 1{alpha},25(OH)2D3 down-regulated secreted uPA activity in a concentration-dependent manner, as observed after a 48-h incubation (Fig. 1B)Citation . The decrease in PA activity was associated with a concomitant increase in secreted PAI-1 activity (Fig. 1B)Citation , leading to a prominent decrease in total PA activity. Time dependence analysis of the effect indicated that a decrease in uPA activity was already detectable after a 6-h treatment and that the difference increased during the 48-h incubation (data not shown).



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Fig. 1. Down-regulation of the PA activity of MDA-MB-231 cells by 1{alpha},25(OH)2D3. MDA-MB-231 cells were incubated with 1{alpha},25(OH)2D3 under serum-free conditions for 48 h, and then conditioned media were collected and analyzed by caseinolysis assays. A, total PA activity measured by caseinolysis in agarose assay from conditioned medium of control or 1{alpha},25(OH)2D3 (10-7 M)-treated cells. Results from three separate experiments are combined, and total PA activity is presented as a percentage of control. B, zymographic (PAs) and reverse zymographic (PAI-1) assays from conditioned media of cells treated with the indicated concentrations of 1{alpha},25(OH)2D3. The lysis zones corresponding to uPA and PAI-1 are indicated on the left.

 


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Fig. 2. Potencies of vitamin D analogues in the regulation of PAs. MDA-MB-231 cells were treated with the indicated concentrations of vitamin D analogues EB1098, KH1060, and CB1093 for 48 h, followed by zymographic analysis of secreted PA and PAI-1 activity. A, top panel, zymographic assay to detect uPA and tPA activity from CB1093-treated MDA-MB-231 cell-conditioned media or acid eluates (acid dissociates receptor-bound uPA from the cells). Bottom panel, PAI-1 activity from conditioned media. The lysis zones corresponding to uPA, tPA, and PAI-1 are indicated on the left. Bands most probably representing the uPA/PAI-1 and tPA/PAI-1 complexes are indicated with an arrowhead. B, zymographic assay from MDA-MB-231 cell-conditioned media after treatment with different concentrations of EB1089 or KH1060. The lysis zones corresponding to uPA and tPA are indicated on the left.

 
Vitamin D Analogues in the Regulation of PAs
EB1089, KH1060, and CB1093 are synthetic deltanoids with a modified side chain, and they exhibit increased potency in growth regulation in combination with decreased calcemic properties (22) . All of the analogues like 1{alpha},25(OH)2D3 induce very modest (10–15%) growth inhibition in MDA-MB-231 cells (data not shown; Ref. 16 ), indicating that this breast carcinoma cell line is insensitive to the antiproliferative effects of deltanoids. This does not result from the lack of VDR, although MDA-MB-231 cells have been shown to express low levels of VDR (23) .

MDA-MB-231 cells were incubated with increasing concentrations of the analogues under serum-free conditions for 48 h. Zymographic analysis of the conditioned media indicated that all three analogues reduced the levels of both secreted uPA and tPA activity with a potency at least 100 times higher than that of 1{alpha},25(OH)2D3 (Fig. 2)Citation . CB1093 was found to be the most potent analogue in this respect. Even 10-10 M concentrations of CB1093 resulted in a prominent decrease in secreted PA activity (Fig. 2ACitation , media). CB1093 was also a potent inducer of PAI-1 activity. The concentration-response curve was different from that of uPA; PAI-1 was prominently induced in a bell-shaped manner within a concentration range from 10-10 to 10-8 M, but not with higher concentrations (Fig. 2ACitation , bottom panel). Cell surface-associated uPA activity was analyzed by eluting receptor-bound uPA with acidic buffer, followed by neutralization and analysis by zymography (acid eluates). A decrease in cell surface-bound uPA by CB1093 was evident and probably resulted from decreased secretion of uPA as well as increased PAI-1 levels. At high concentrations of CB1093, the cell surface-associated uPA activity increased again, probably reflecting lower PAI-1 activity. Higher molecular weight lysis zones in the zymogram represent the uPA/PAI-1 and tPA/PAI-1 complexes. Fig. 2BCitation illustrates the down-regulation of secreted uPA and tPA by increasing concentrations of EB1089 and KH1060.

Regulation of Invasion through Matrigel by Vitamin D Analogues
The effects of vitamin D analogues on the in vitro invasive capacity of MDA-MB-231 cells were analyzed using Matrigel-coated membranes (see "Materials and Methods"). In accordance with previous reports, we found that the cells needed a preincubation with vitamin D compounds to observe a decrease in their invasion through Matrigel. Cells were treated with 10-7 M concentrations of vitamin D analogues for 3 days before a 20-h cell invasion assay. MDA-MB-231 cells are highly invasive, and 1{alpha},25(OH)2D3 and the analogues decreased cell invasion to approximately half of the control level (Fig. 3).Citation Concentrations lower than 10-7 M of the vitamin D compounds did not decrease the invasive capacity of MDA-MB-231 cells. Unexpectedly, CB1093, EB1089, and KH1060 were not found to be superior to 1{alpha},25(OH)2D3 in this experimental design. Variability in the length of the experiment, vitamin D concentration, and composition of Matrigel as well as effects of vitamin D compounds on cell migration and cell adhesion probably add to the variability of results in in vitro invasion assays. Prostate cancer metastasis in vivo has been found to be markedly and equivalently inhibited by 1{alpha},25(OH)2D3 and EB1089 (24) . However, EB1089 was significantly less calcemic, which makes it a better candidate for cancer treatment.



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Fig. 3. Regulation of invasion through Matrigel by vitamin D analogues. MDA-MB-231 cells were pretreated with 10-7 M 1{alpha},25(OH)2D3, CB1093, EB1089, or KH1060 for 3 days before a 20-h invasion assay. Cells invading through Matrigel-coated membranes were stained and counted in a bright-field microscope. The number of invasive cells is presented as a percentage of untreated cells. The values represent the mean of two separate experiments ± SD.

 
Regulation of uPA mRNA by 1{alpha},25(OH)2D3 and CB1093
Our earlier studies with human and mouse keratinocytes suggested that down-regulation of uPA by 1{alpha},25(OH)2D3 was rather rapid and mediated through mRNA regulation (15) . MDA-MB-231 cells were treated with increasing concentrations of 1{alpha},25(OH)2D3 or CB1093 under serum-free conditions for 4 or 18 h, and then uPA mRNA levels were analyzed by Northern blotting (Fig. 4).Citation Short treatment with vitamin D analogues did not change uPA mRNA levels, but after an 18-h incubation, a clear concentration-dependent decrease was observed. CB1093 was clearly more potent in the regulation of uPA mRNA, as suggested by data from activity assays. These results suggest that vitamin D can regulate uPA at the mRNA level in MDA-MB-231 cells and that the kinetics of regulation depends on the cell type.



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Fig. 4. uPA mRNA regulation by 1{alpha},25(OH)2D3 and CB1093 in MDA-MB-231 cells. MDA-MB-231 cells were treated with increasing concentrations of 1{alpha},25(OH)2D3 or CB1093 for 18 h as indicated. Total cellular RNA was prepared and analyzed by Northern hybridization using uPA and G3DPH probes successively. Radioactivity levels were quantified with a BAS-2500 bioimaging analyzer (Fuji). The values in the chart are expressed as units relative to G3DPH.

 
Effect of 9-cis-RA and RA Alone or in Combination with CB1093 on PA Activity
Many effects of vitamin D are mediated through nuclear VDR in association with RXR. It is known that in some cases, RXR ligand 9-cis-RA can interfere negatively with 1{alpha},25(OH)2D3-mediated regulation of transcription (25) . We analyzed the interaction of these two interactive receptor systems on PA activity secreted by MDA-MB-231 cells. Treatment of cells with 9-cis-RA (10-6 M) in combination with CB1093 (10-9 M) did not affect the deltanoid-induced down-regulation of uPA activity (Fig. 5).Citation This suggests that RXR ligand is not involved in the regulation of uPA by CB1093. RA (10-6 M) alone and in combination with CB1093 decreased both secreted and cell surface-associated uPA levels.



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Fig. 5. Effect of 9-cis-RA and RA alone or in combination with CB1093 on PA activity. MDA-MB-231 cells were treated with 9-cis-RA (10-6 M), RA (10-6 M), or the deltanoid CB1093 (10-9 M) for 48 h as indicated, followed by zymographic analysis of secreted PA activity from conditioned media or cell surface-associated uPA activity from acid eluates. Lysis zones corresponding to uPA are shown.

 
1{alpha},25(OH)2D3 Regulation of uPA Promoter Activity
To analyze whether vitamin D regulation of uPA is at the transcriptional level and to search for possible regulatory regions, we performed CAT activity assays with different uPA promoter constructs. The -2350 construct is a full-length promoter/CAT construct, whereas -1870 and -818 are 5' deletion constructs (26) . HT-1080 fibrosarcoma cells were selected for the promoter activity assays because of good transfection efficiency and responsiveness to vitamin D. Cells transfected with each of these constructs in combination with a pCMVß-gal control plasmid were treated with 1{alpha},25(OH)2D3, followed by CAT activity measurements. The corrected CAT activity (see "Materials and Methods") results are expressed as a percentage of untreated control. The activity of the full-length uPA promoter construct was down-regulated by 1{alpha},25(OH)2D3 treatment (Fig. 6).Citation The -1870 and -818 constructs were not responsive to 1{alpha},25(OH)2D3, but the basal level of activity was decreased or increased, respectively. Similar results were obtained in transfected MK-2 mouse keratinocytes (data not shown). These results suggest that the 1{alpha},25(OH)2D3-responsive regulatory regions in the uPA promoter are located between nucleotides -2350 and -1870.



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Fig. 6. 1{alpha},25(OH)2D3 regulation of uPA promoter activity. HT-1080 cells were transfected with different uPA promoter-CAT constructs by electroporation, and CAT activity was measured after treatment with or without (Ctrl) 1{alpha},25(OH)2D3 (10-7 M) for 24 h. The results are expressed as a percentage of the corrected CAT activity of control. Results from two separate experiments are combined.

 
1{alpha},25(OH)2D3 and CB1093 Regulation of Gelatinolytic MMP Activity
MDA-MB-231 cells were found to secrete MMP-9 but not MMP-2 into the conditioned medium. Gelatin zymographic assays showed also bands of proteolytic activity migrating close to the Mr 50,000 marker, probably representing stromelysins (Fig. 7).Citation Treatment of cells with 10-7 M 1{alpha},25(OH)2D3 under serum-free conditions for 48 h decreased the secreted MMP-9 activity consistently to one-third of the control levels (Fig. 7)Citation . CB1093 decreased MMP-9 levels at concentrations less than 1% of those of 1{alpha},25(OH)2D3. Maximal effect was seen at 10-9 M concentration of CB1093. CB1093 was also able to strongly induce the secretion of TIMP-1 activity, with a bell-shaped response curve similar to the PAI-1 response compare (Fig. 7Citation with Fig. 2Citation ). TIMP-2 was also induced, but to a much lower extent. These results suggest that deltanoids can influence the MMP/TIMP balance in breast carcinoma cells in addition to modulating the PA system.



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Fig. 7. 1{alpha},25(OH)2D3 and CB1093 regulation of gelatinolytic MMP activity. MDA-MB-231 cells were treated with the indicated concentrations of 1{alpha},25(OH)2D3 or CB1093 for 48 h, and then the secreted MMPs or TIMPs were analyzed by gelatin zymographic or reverse zymographic assays, respectively. Lysis zones corresponding to MMP-9, as well as lysis-resistant zones (reverse zymogram) corresponding to TIMP-1 and TIMP-2, are indicated on the right. Arrowhead indicates the migration of unidentified gelatinolytic proteins most probably representing stromelysins. Note the gradual decrease of MMP-9 activity, the bell-shaped induction of TIMP-1, and the lower level of induction of TIMP-2.

 
MT1-MMP Expression and Activation in MDA-MB-231 Cells
The activation of pro-MMP-2 is considered to be important for cell migration and invasion. Treatment of cells with PMA or ConA leads to the production of active MMP-2, depending on the cell line. Recently, it has become evident that MT-MMPs have an important role in MMP-2 activation at the cell surface (27, 28, 29) . Five different MT-MMPs have been cloned, and at least three of them are able to induce processing of MMP-2 to its active forms. Expression of high levels of MT1-MMP is associated with invasive and metastatic breast carcinomas (30) . MT1-MMP itself is produced as an inactive Mr 63,000 zymogen, which is processed to the Mr 60,000 active form. The Mr 60,000 form must be membrane associated to perform its function. A Mr 43,000 form of MT1-MMP representing an inactive degradation product is produced by PMA treatment of HT-1080 fibroblasts, and it correlates well with MMP-2 activation (27) .

MDA-MB-231 cells express large amounts of MT1-MMP mRNA as well as protein. We analyzed whether 1{alpha},25(OH)2D3 could alter MT1-MMP production or activation in these cells. A 24-h treatment of cells with increasing concentrations of 1{alpha},25(OH)2D3 under serum-free conditions did not affect MT1-MMP mRNA levels (data not shown). PMA (4 nM), alone or in combination with 1{alpha},25(OH)2D3 (10-7 M), did not affect MT1-MMP protein levels when total cell lysates were analyzed by immunoblotting after a 24-h treatment. The Mr 60,000 MT1-MMP was the predominant form in MDA-MB-231 cells, and PMA did not induce the appearance of the known prominent cleavage product (Mr 43,000 form), as seen in control HT-1080 cells (Fig. 8A).Citation 1{alpha},25(OH)2D3, when added in combination with PMA, did not affect the formation of the Mr 43,000 form in HT-1080 cells (data not shown). The band of Mr 50,000 is nonspecific because blotting with preimmune rabbit serum resulted in the detection of the same band (27) . Immunoblotting analysis of purified membrane fractions of MDA-MB-231 cells revealed low levels of the inactive Mr 63,000 form in untreated cells (Fig. 8B)Citation , suggesting that although MDA-MB-231 cells produce considerable amounts of the Mr 60,000 form, it does not remain membrane associated during purification from untreated cells. PMA induced the appearance of very low levels of the Mr 60,000 active MT1-MMP, whereas ConA induced a clear increase in membrane-associated Mr 60,000 MT1-MMP. 1{alpha},25(OH)2D3 did not have any detectable effects on MT1-MMP production or activation. These results are in accordance with the results of Yu et al. (20) , which suggest that ConA but not PMA can induce MT1-MMP-dependent MMP-2 processing in MDA-MB-231 cells.



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Fig. 8. MT1-MMP expression in MDA-MB-231 cells. MDA-MB-231 and HT-1080 cells were treated with 1{alpha},25(OH)2D3 (10-7 M), PMA (4 nM), or ConA (20 µg/ml) for 24 h as indicated. A, immunoblotting of total cell lysates with antibodies against MT1-MMP. The Mr 60,000 and Mr 43,000 forms are indicated on the right. The ~Mr 50,000 band represents a nonspecific band (27) . B, immunoblotting of isolated cell membranes with antibodies against MT1-MMP reveals the characteristic Mr 63,000 and Mr 60,000 bands.

 
Discussion

There are several steps in tumor progression that could possibly be regulated by deltanoids. They are potent growth inhibitors for cells of epithelial origin, and this is achieved by induction of cell cycle arrest as well as apoptosis (2 , 31) . They also induce differentiation, which results in a less motile and thus less malignant phenotype. Cell-matrix contacts mediated by integrins are often changed in cancer cells (32 , 33) . Deltanoids have been shown to regulate the expression of certain integrins and their ligands. At least laminin and its receptor, fibronectin, and tenascin-C are regulated by vitamin D, with modification of expression depending on the cell type (34, 35, 36, 37) . In addition, deltanoids regulate the expression and activity of various growth factors that modulate the proliferative and angiogenic properties of cells. Induction of transforming growth factor ß and its receptor mediates at least in part the growth-inhibitory response in human keratinocytes and in breast carcinoma cells (38, 39, 40) . Recently, an interplay between transforming growth factor ß and vitamin D signaling pathways through SMAD transcriptional coactivators has been found (41) . Vitamin D has also been suggested to inhibit the motility and metastasis of Lewis lung carcinoma tumors via inhibition of granulocyte-macrophage colony-stimulating factor production (42) . In addition, induction of the expression of insulin-like growth factor-binding protein 3 is associated with growth-inhibitory response in some breast cancer cell lines (43) .

We report here that vitamin D and its analogues are potent regulators of the activity of invasion-associated proteases. Involvement of vitamin D in the regulation of the PA system has been reported in human keratinocytes, rat osteogenic sarcoma cells, and U-937 mononuclear phagocytes (15 , 44 , 45) . Like growth factor regulation of the PA system in general, the response to vitamin D is cell-type specific. In keratinocytes, vitamin D down-regulates both uPA and PAI-1, whereas in sarcoma cells and phagocytes, enhanced PA activity is associated with down-regulation of PAI-1 or PAI-2, respectively. Our new data indicate that in MDA-MB-231 breast carcinoma cells, vitamin D down-regulates uPA and up-regulates PAI-1, leading to a decrease in the secreted PA activity. Reduced PA activity is probably the main mechanism by which vitamin D inhibits invasiveness of this particular cell line because the results of Holst-Hansen et al. (9) suggest that the in vitro invasiveness of MDA-MB-231 cells is dependent on uPA activity.

Loss of cell-cell contacts through loss of E-cadherin expression is typical for an invasive cancer cell. It has been shown in breast carcinoma cells that blockade of E-cadherin-dependent adhesion induces the production of uPA, which could facilitate tumor cell invasion (46) . High levels of uPA in patients with breast tumors correlate with shorter disease-free interval and overall survival (47 , 48) . The therapeutic down-regulation of uPA levels might be beneficial because studies using monoclonal antibody against urokinase or antisense oligonucleotides show reduced invasion and metastasis of tumor cells in mice (49 , 50) . The role of the uPA system in the metastatic cascade is not clear, but obviously it provides localized extracellular matrix degrading activity as well as increased migratory capacity for tumor cells. The presence of uPAR at focal contacts serves to localize plasmin production to special sites at cell surfaces. Immunohistochemical studies suggest that components of the uPA system are located at the invasive edge of cancers and also in the associated stromal cells (51) . Production of plasmin may also release and activate growth factors important for tumor growth and angiogenesis. uPA levels in breast carcinomas have been shown to correlate with microvascular density, suggesting the involvement of the uPA system in neovascularization of tumors (52) .

As in human keratinocytes (15) , 1{alpha},25(OH)2D3 regulation of uPA was found to be at the transcriptional level in HT-1080 cells used in the promoter assays. HT-1080 fibrosarcoma cells were selected for the assays because of good transfection efficiency. These cells are responsive to vitamin D treatment, and their in vitro invasiveness is reduced by vitamin D (37) . More detailed analysis of the uPA promoter activity suggested that the responsive regulatory region is between nucleotides -2350 and -1870 in the 5' end of the promoter. No known vitamin D-inhibitory sequences were found from this region. Therefore, we do not know whether VDR binds directly to the uPA promoter. The 5' end of the uPA promoter contains at least Sp-1, c-ets-1, cAMP-responsive element-binding protein, CRE-BP1/c-jun, and two AP-1 sites, of which one is important for the basal expression of uPA (26) . Interference between AP-1 and VDR on osteocalcin gene expression has been described in human osteosarcoma cells (53) .

1{alpha},25(OH)2D3 has shown good promise for treatment of cancer patients, but a major problem for use has been its hypercalcemia-inducing capacity. Therefore, several vitamin D analogues with lesser hypercalcemia-inducing properties have been synthesized. The functional analyses of these analogues have resulted in the discovery of several analogues with potent antiproliferative effects in association with lower calcaemic capacity. Three analogues with side chain modifications, CB1093, EB1089, and KH1060, were found here to be at least 100 times more potent that 1{alpha},25(OH)2D3 in the regulation of uPA activity. EB1089 and CB1093 are potent in the inhibition of breast cancer cell proliferation and in producing regression of experimental mammary tumors (22) . CB1093 has been found to be 10 times more potent than EB1089 in inducing apoptosis in MCF-7 breast carcinoma cells. The potencies of the analogues in reducing uPA activity correlate well with their potencies in inducing apo-ptosis. Results from Danielsson et al. (22) suggest that CB1093 shows a preference for the activation of DR-3-type VDREs, whereas EB1089 selectively activates IP9-type VDREs. It will be interesting to find whether uPA promoter has some kind of a VDRE.

Degradation of basement membrane collagen is essential for tumor cell invasion and spread. MMPs are capable of degrading collagen, and their expression is often increased during tumor progression. MMP-9 and MMP-2 can degrade type IV collagen, which is a major component of basement membranes. MDA-MB-231 cells were found to secrete MMP-9 activity, which was down-regulated by treatment with vitamin D or CB1093. In human prostate cancer cells as well as in human mononuclear phagocytes, vitamin D has been reported to reduce MMP-9 activity (18 , 54) . A concomitant increase in the secretion of TIMP-1 and, to a slightly lower extent, TIMP-2 was observed in MDA-MB-231 cells, indicating that vitamin D can change the MMP/TIMP balance in breast carcinoma cells, probably contributing to the less invasive phenotype. TIMP-1 levels have been shown to correlate with the invasive capacity of tumor cells (55 , 56) . Recent studies have suggested that MMPs have a role not only in the breakdown of physical barriers but also in the regulation of tumor growth (for review, see Ref. 6 ). The mechanisms may involve the generation of active matrix fragments as well as the release and activation of growth factors leading to modulation of the growth environment (57) .

Expression of MT1-MMP in breast carcinomas correlates with the presence of lymph node and distant metastasis, clinical stage, and size of tumors (30) . MT1-MMP plays a key role in the activation of pro-MMP-2. In human breast carcinoma tissues, MMP-2 mRNA is predominantly localized to the stroma, possibly requiring a tumor component for activation (58) . Soluble factors produced by tumor cells may also induce fibroblast production of MT1-MMP and consequent MMP-2 activation, as suggested by Polette et al. (59) . MDA-MB-231 cells express high levels of MT1-MMP mRNA and activate exogenously added MMP-2 in response to ConA treatment (20) . We found that MT1-MMP mRNA levels were not affected by vitamin D in MDA-MB-231 cells. The predominant form produced by the cells was the active Mr 60,000 form, but it was not localized to the membrane fraction unless cells were treated with ConA. 1{alpha},25(OH)2D3 had no detectable effect on MT1-MMP production or activation, indicating that its interference with MMP-2 activation was not probable.

Our studies indicate that deltanoids are potent regulators of invasion-associated proteases. In both the PA and the MMP systems, vitamin D decreased the production of proteases and increased the production of their inhibitors. Potent vitamin D analogues in combination with tamoxifen or cisplatin will probably prove to be a good choice for the treatment of breast cancer patients, as in vitro work from Koshizuka et al. (60) and Vink-van Wijngaarden et al. (61) suggests.

Materials and Methods

Reagents
1{alpha},25(OH)2D3 and its synthetic analogues EB1089, KH1060 (62) , and CB1093 (22) were obtained from Leo Pharmaceutical Products (Copenhagen, Denmark). The uPA-CAT constructs -2350, -1870, and -818 (26) were kindly provided by Prof. Francesco Blasi (H. S. San Raffaele Scientific Institute, Milan, Italy). Polyclonal rabbit antibodies against MT1-MMP were used as described previously (27) .

Cell Culture
MDA-MB-231 breast carcinoma cells were obtained from Prof. R. Vihko (University of Oulu, Oulu, Finland) and cultured in RPMI 1640 containing 10% FCS (Life Technologies, Inc., Rockville, MD), 100 units/ml penicillin, and 50 µg/ml streptomycin. HT-1080 human fibrosarcoma cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in MEM containing 10% heat-inactivated FCS and the above-mentioned antibiotics.

Caseinolysis Assays
Caseinolysis in agarose assays were carried out as described previously (63) . The caseinolysis gels contain plasminogen (Cromogenix, Mölndal, Sweden) and casein in 1.2% agarose (FMC BioProducts, Rockland, MD). Plasminogen, when activated by the PA present in the medium sample, degrades casein and forms a clear zone of caseinolysis in the gel during the sample diffusion proportional to the PA activity of the sample and time of diffusion. Different dilutions of human high molecular weight uPA (Calbiochem, La Jolla, CA) were used to draw standard plots in which PA activity (in international units) is plotted against the diameter of the lytic sphere, which describes the area of the zone of clearing.

Zymographic assays were used to identify the molecular forms of PAs and PAI-1 (64) . Medium samples or acid eluates [cells treated with 50 mM glycine-HCl buffer (pH 3.0) containing 0.1 mM NaCl at room temperature for 3 min] were first electrophoresed in 4–15% gradient polyacrylamide gels containing SDS (Bio-Rad, Hercules, CA) under reducing (for reverse zymography) or nonreducing (for zymography) conditions. SDS was removed by extensive washing with PBS/Triton X-100 (2.5%). For reverse zymography, uPA (2 IU/ml) was added to the final wash for 15 min. The gels were then placed on caseinolysis gels and incubated at 37°C for 24 h. The position of PAs in the SDS gel was shown as lysis of the indicator gels. The position of PAI-1 was shown as lysis-resistant opaque band.

Invasion Assay
MDA-MB-231 cells were incubated with vitamin D analogues for 48 h in growth medium, after which serum-free medium supplemented with vitamin D analogues was changed for 24 h. Cells were harvested and counted, and 0.5 x 105 cells/chamber was used for each invasion assay. Cell culture inserts in a 24-well plate (Becton Dickinson Labware, Bedford, MA) were coated with 85 µg/cm2 growth factor-reduced Matrigel (Becton Dickinson Labware) for 3 h at 37°C. Unbound material was aspirated, and the chambers were rinsed gently with serum-free media before the addition of cells. Cells were added to coated inserts in serum-free media containing vitamin D analogues. The lower chambers contained medium with 5% FCS and vitamin D analogues. The inserts were incubated for 20 h at 37°C. The cells that had invaded to the lower surface of the membranes were fixed and stained with Dade Diff-Quick stain kit (Dade AG, Düdingen, Switzerland), and random fields were counted under a light microscope. The decreased number of invasive cells is expressed as a percentage of untreated cells.

Gelatin Zymography and Reverse Zymography
To analyze the gelatinolytic proteins in cell-conditioned medium, samples were run under nonreducing conditions in 10% SDS-polyacrylamide gels containing 2 mg/ml gelatin. For reverse zymography, samples were run under nonreducing conditions in 12% SDS-polyacrylamide gels containing 2 mg/ml gelatin and 40 ng/ml gelatinase A (Chemicon International Inc., Temecula, CA). After electrophoresis, the gels were washed twice with 50 mM Tris-HCl buffer (pH 7.6) containing 5 mM CaCl2, 1 µM ZnCl2, and 2.5% Triton X-100 (v/v) for 15 min to remove SDS, followed by a brief rinsing in washing buffer without Triton X-100. The gels were then incubated at 37°C for 24–48 h in washing buffer containing 1% Triton X-100. After incubation, the gels were stained with Coomassie Brilliant Blue R250 (Bio-Rad) and destained with 10% acetic acid.

Transfection and CAT Assay
HT-1080 cells were transfected with different uPA-CAT constructs by electroporation (65) . Cotransfection with pCMVß-gal plasmid (Clontech, Palo Alto, CA) was performed as an internal control. Cells were transfected with 3 µg of pCMVß-gal + 16 µg of promoter-CAT constructs using 400 V and 960 µF. The following day, the transfected cells were treated with 1{alpha},25(OH)2D3 for 24 h, and then cells were harvested, and CAT and ß-galactosidase activities were measured according to standard protocols (65) . CAT activities were corrected for ß-galactosidase activity.

RNA Isolation and Northern Blotting
Total cellular RNA was purified from cultured cells using the RNeasy total RNA kit (Qiagen, Hilden, Germany). For Northern blot analysis, 10 µg of RNA were fractionated on 1.2% agarose gels containing formaldehyde and transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech, Uppsala, Sweden) by capillary transfer. Prehybridization and hybridization were performed at 68°C in ExpressHyb hybridization solution (Clontech). cDNA probes for MT1-MMP, uPA, and G3DPH were labeled with [32P]dCTP (>3000 Ci/mmol; Amersham Pharmacia Biotech) using a Rediprime Labeling Kit (Amersham Pharmacia Biotech). Radioactivity levels were quantified with a BAS-2500 bioimaging analyzer (Fuji, Tokyo, Japan).

Cell Lysis and Immunoblotting
The cells were lysed with Triton lysis buffer [50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 1% (v/v) Triton X-100, 10 mM EDTA, 10 µg/ml aprotinin, 1 µg/ml pepstatin A, and 1 µg/ml aminoethylbenzenesulphonyl fluoride] on ice, and the lysates were clarified by centrifugation. Cell membrane extracts were prepared as described previously (27) . For immunoblotting, aliquots of cell lysates or membrane extracts were electrophoresed in 4–15% gradient SDS-polyacrylamide gels, and proteins were electrophoretically transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Immunodetection of MT1-MMP was performed as described previously (27) .

Acknowledgments

We thank Dr. Lise Binderup (Leo Pharmaceutical Products, Copenhagen, Denmark) for providing the deltanoids and Sami Starast for fine technical assistance.

Footnotes

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.

1 Supported by the Academy of Finland, the Finnish Cancer Foundation, Sigrid Juselius Foundation, Novo Nordisk Foundation, Helsinki University Hospital Fund, Biocentrum Helsinki, and the University of Helsinki. Back

2 To whom requests for reprints should be addressed, at department of Virology, P. O. Box 21, Haartman Institute, University of Helsinki, Haartmaninkatu 3, FIN-00014 Helsinki, Finland. Back

3 The abbreviations used are: uPA, urokinase plasminogen activator; tPA, tissue-type plasminogen activator; PA, plasminogen activator; PAI, PA inhibitor; MMP, matrix metalloproteinase; TIMP, tissue inhibitors of metalloproteinases; RXR, retinoid X receptor; RA, retinoic acid; 1{alpha},25(OH)2D3, 1{alpha},25-dihydroxyvitamin D3; VDR, vitamin D receptor; uPAR, uPA receptor; MT-MMP, membrane-type MMP; ConA, concanavalin A; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyltransferase; VDRE, vitamin D-responsive element; G3DPH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received for publication 9/ 7/99. Accepted for publication 3/ 3/00.

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J. Biol. Chem., June 8, 2001; 276(23): 20085 - 20092.
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