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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0857
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1181-1188
Copyright © 2005 by The Endocrine Society

Progesterone Increases Tissue Factor Gene Expression, Procoagulant Activity, and Invasion in the Breast Cancer Cell Line ZR-75-1

Sumie Kato, Mauricio Pinto, Andrés Carvajal, Natalia Espinoza, Carolina Monso, Anil Sadarangani, Manuel Villalon, Jan J. Brosens, John O. White, Jennifer K. Richer, Kathryn B. Horwitz and Gareth I. Owen

Unidad de Reproducción y Desarrollo (S.K., M.P., A.C., N.E., C.M., A.S., M.V., G.I.O.), Facultad de Ciencias Biológicas and Departamento de Obstetricia y Ginecologia (S.K.), Facultad de Medicina, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile; Institute of Reproductive and Developmental Biology (J.J.B., J.O.W.), Imperial College London, Hammersmith Hospital, London W12 0NN, United Kingdom; and Department of Medicine (J.K.R., K.B.H.), Division of Endocrinology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Address all correspondence and requests for reprints to: Gareth I. Owen, Ph.D., Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile. E-mail: gowen{at}bio.puc.cl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone in hormonal preparations increases the incidence of breast cancer. Tissue factor (TF), the initiator of the extrinsic coagulation pathway, is associated with metastasis in a wide variety of cancers. We demonstrate herein that TF mRNA and protein are up-regulated by progesterone in the breast cancer cell line ZR-75. Epidermal growth factor, also associated with increased breast cancer risk, did not regulate TF. The increase in TF is both rapid and transient; increasing after 6 h, reaching a maximum at 24 h, before decreasing to basal levels at 72 h. Sucrose gradient experiments demonstrated that TF is located in the heavy fraction of the plasma membrane, although caveolin-1 is not expressed in ZR-75. To understand the physiological implications of an increase in TF, we performed coagulation and invasion assays. An increase in TF corresponded to an increase in procoagulant activity. Furthermore, progesterone increased the invasion of ZR-75 cells through a matrigel, an effect that was blocked by an antibody against TF. Because TF expression is associated with an enhanced risk of metastasis, we postulate that the progesterone-dependent up-regulation of TF provides a survival advantage to burgeoning breast cancer cells and may contribute to the increased risk of cancer associated with combined hormone replacement therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TISSUE FACTOR (TF) is a transmembrane glycoprotein responsible for the initiation of the extrinsic coagulation pathway serving as the cofactor and receptor for coagulation factor VII (FVII) (1). Microarray analysis in the breast cancer line T47D demonstrated an 18-fold increase in mRNA expression after 6 h of progesterone treatment, suggesting that this gene may play a significant role in breast cancer (2). TF is located and functionally active within the plasma membrane. TF pathway inhibitor (TFPI) down-regulates TF activity and causes the redistribution of the TF, FVIIa, and TFPI in a stable and inactive quaternary complex with factor Xa to membrane associated lipid rafts-caveolae regions (3, 4). TF, which structurally shares homology with class II cytokine receptors (5), has an intracellular domain, which contains three putative phosphorylation sites. Targeted phosphorylation of these sites on Phorbol-12-myristate-13-acetate treatment are the hallmarks of a signal transduction-mediating receptor surface molecule (6). Furthermore, high TF expression is associated with increased invasive and metastatic potential of many types of malignancy (1). In breast cancer, elevated TF concentrations correlate with increased incidence of metastasis and poor prognosis (7, 8). In addition, there appears a strong relationship between TF expression and the secretion of angiogenic mediators such as vascular endothelial cell growth factor (VEGF) (9). TF bound to its ligand FVII provides protection against apoptosis, demonstrating a further potential role of TF in the development and survival of cancer cells (10). TF also plays a role in the pathological conditions of coronary artery disease, deep vein thrombosis, and athlerosclerosis (11). Whole-blood TF expression levels are increased in hypercholesterolemic patients (12), and overexpression of TF has been associated with the hypercoagulable state present in cancer patients (1).

Ovarian progesterone is a critical steroid hormone that controls cell proliferation and differentiation in the female reproductive tract (13, 14). In normal mammary tissue, high levels of progesterone during the luteal phase mediate proliferation and differentiation in milk ducts. However in the uterus, progesterone antagonizes estrogen-stimulated proliferation of endometrial epithelial cells and promotes their differentiation (15, 16). Clinical studies demonstrated that postmenopausal women taking unopposed estrogen replacement therapy are at increased risk of endometrial disorders including cancer. Although coadministration of progesterone counteracts the mitogenic action of exogenous estrogen on the endometrium, it increases dramatically the incidence of breast cancer (17, 18). In agreement with these in vivo observations, progesterone metabolites have been found to induce proliferation in certain breast cancer cell lines (19, 20).

The signaling pathways activated by epidermal growth factor (EGF) and EGF receptors (EGFRs) have been associated with breast cancer development and progression. Furthermore, hormone-resistant breast cancer is associated with an increased expression of both EGFR and EGFR ligand. This coexpression suggests the presence of an autocrine pathway of uncontrolled cell growth sustaining neoplastic transformation (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Progesterone is also known to cross-talk with the EGF signaling pathway in the control of breast cancer cell growth (25, 26, 27).

We previously demonstrated that TF is regulated by progesterone in T47D breast cancer cell line and further demonstrate herein that progesterone and not EGF potently up-regulates TF in the breast cancer cell line ZR-75. This increase in expression converts to an increase in procoagulant activity of this cell line. Furthermore, progesterone increases the invasiveness of this cell line through matrigel, an effect that is blocked by TF antibody. As mentioned, TF is involved in tumor proliferation, promoting angiogenesis and metastasis. We postulate that the progesterone enhancement of TF expression confers a survival advantage to burgeoning breast cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and hormonal treatment

ZR-75-1 breast cancer cells (28) and Ishikawa endometrial cancer cells (29, 30) were maintained in DMEM/F12 media supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). For protein and RNA experiments, cells were plated at 50% confluence in 10-cm2 petri dishes (Falcon, Becton Dickinson, Lincoln Park, NJ) and 6-cm2 petri dishes for the coagulation assay, and then the medium was changed to charcoal-treated medium containing 5% serum for 24 h before hormone or EGF treatment. 17ß-Estradiol (E, estrogen) and progesterone (both Sigma-Aldrich, St. Louis, MO) were dissolved in ethanol and added to the cells, individually or in combination, to a final concentration of 10 nM. EGF (Upstate Biotechnology, Lake Placid, NY) was added at a final concentration 10 nM. An equal volume of ethanol was used as control and in EGF treatments. The presence or absence of phenol red in the DMEM/F12 medium did not alter ovarian hormone or EGF regulation.

RT-PCR

Total RNA was isolated using the Chomczynski method (31). cDNA was generated using reverse transcriptase (Superscript II, Invitrogen). Using TF primers (sense, 5'-ttc aag aca att ttg gag tgg-3'; antisense, 5'-tct cct ggc cca tac act c-3') (BiosChile, Santiago, Chile), semiquantitative PCRs were performed from cDNA generated from hormone- and EGF-treated samples, using Taq polymerase (Invitrogen). Cycle curves were performed for all sets of PCR primers, with the number of cycles used for each primer set being in the linear range of the curve. As an internal control, primers amplifying a region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used. Semiquantitative densitometry of the bands was performed using the NIH Image 1.62c software package for Macintosh.

Western blotting

Cells were harvested in cold PBS and the pellet resuspended in lysis buffer [0.4 M KCl, 20 mM HEPES (pH 7.4), 1 mM dithiothreitol, 20% glycerol]. After sonification on ice, the lysate was centrifuged at 14,000 x g for 20 min at 4 C to separate membrane (pellet) and cytosolic (supernatant) fractions. The crude membrane fraction was resuspended in the above-mentioned lysis buffer and protein concentration determined by Bradford assay. One hundred micrograms of crude membrane extract was loaded in each lane, separated by 10% PAGE in the presence of sodium dodecylsulfate, transferred to nitrocellulose membranes, and incubated overnight with anti-TF affinity purified antibodies (1:2000 Calbiochem, EMD Biosciences, Germany; 1:1000 Genentech, South San Francisco, CA). Goat antimouse IgG secondary antibody coupled to hydrogen peroxidase (1:5000, Bio-Rad Laboratories, Hercules, CA) was applied for 1 h at room temperature. For the antiglycosylation experiment, tunicamycin (10 mg/ml, Calbiochem, EMD Biosciences) was applied 30 min before hormone treatments for 24 h. The reaction was developed with chemiluminescence using ECL Western blot analysis system (NEN Life Science Products, Western Lightning, Perkin-Elmer, Boston, MA). Semiquantitative densitometry of the bands was performed using NIH Image 1.62c software package for Macintosh to determine whether significant differences between treatments existed.

Immunocytochemistry

ZR-75 cells were cultured on glass slides in DMEM/F12 media supplemented with 10% fetal bovine serum. After 24 h in 5% charcoal-treated fetal bovine serum, cells were treated for a further 24 h with progesterone (10 nM) or ethanol vehicle. The slides were washed in PBS and fixed in methanol for 10 min at –20 C. Endogenous peroxidase activity was blocked by incubation with 10% hydrogen peroxidase, and nonspecific immunoglobulin binding was blocked incubation with protein block (serum free, Dako, Carpinteria, CA). The primary TF antibody (1:100; Calbiochem, EMD Biosciences) was added for 18 h. The slides were subsequently rinsed and incubated for 30 min with biotinylated secondary antibody (Dako). The slides were rinsed in Tris-PBS buffer and incubated for 30 min with horseradish peroxidase-conjugated streptavidin (Dako). The samples were treated with 0.1% (wt/vol) 3–3'-diaminobenzidine as a chromogen in Tris-PBS buffer containing 0.05% horseradish peroxidase. The slides were then counterstained with Harris’s hematoxylin to distinguish the nucleus. Negative controls were performed in the absence of primary antibody.

Separation of Triton X-100-insoluble complexes by sucrose gradient ultracentrifugation

Caveolae-like membrane microdomains were isolated by sucrose-gradient fractionation in the presence of Triton X-100 as previously described (32) with some modifications. Briefly, ZR-75 and Ishikawa cells of two confluent 75-cm2 flasks (Falcon, Becton Dickinson) were lysed in 2 ml of MN buffer [25 mM 2-(N-morpholine) ethane sulfonic acid (pH 6.5), 150 mM NaCl] containing 0.5% Triton X-100 and protease inhibitors (10 µg/ml benzamidine, 2 µg/ml antipain, 1 µg/ml leupeptin) for 20 min on ice. Cells were homogenized further with 10 gentle strokes of a loose-fitting type A pestle in a Dounce homogenizer. The homogenate was mixed with 2 ml of 90% (wt/vol) sucrose in MN buffer. A discontinuous sucrose gradient was formed by sequentially overlaying 4 ml of 35% (wt/vol) sucrose and 4 ml of 5% (wt/vol) sucrose, both in MN buffer, and subsequent centrifugation at 200,000 x g at 4 C for 20 h in a SW40 swinging bucket rotor (Beckman-Spinco). Starting from the top of the gradient, 11 x 1 ml gradient fractions were collected. The equivalent of 40–100 µg of total protein was separated by SDS/PAGE on 10% polyacrylamide gels and analyzed by Western blotting. Localization of caveolae was determined by Western blotting for the presence of caveolin-1 (Transduction Laboratories, Lexington, KY).

Measurement of TF procoagulant activity

TF activity was measured as the ability of cell lysates to accumulate factor X in the presence of FVII (33). Measurement of TF activity was as follows for ZR-75 and Ishikawa cell lysates. Lysate corresponding to 50,000 cells in 15 mM n-octyl-ß-D glucopyranoside (Sigma-Aldrich) was diluted in a solution of 50 mM HEPES buffer, 25 mM NaCl 0.1% BSA (pH 7.4). This mixture was incubated with a reagent mixture containing FVII (1 U/ml), factor X (1.2 U/ml), and CaCl2 (25 mM; all final concentrations) and Chromozym X (1 mM, Roche Molecular Biochemicals, Indianapolis, IN) in a 96-well plate. Incubation was for 40 min at 37 C and color development measured at 405 nm on a microplate reader (Molecular Devices, GMI Inc., Sunnyvale, CA). Recombinant rabbit TF (thromboplastin, Hemoliance Recombiplastin, Beckman Coulter Inc., Fullerton, CA) was used in the construction of a standard curve. Factor Xa generation, as measured by color change, was converted to TF procoagulant activity (units per milliliter).

Invasion assays

ZR-75 cells were seeded over porous inserts (8 µm pores, Nunc, Glastrup, Denmark) covered with matrigel (25 µg, Sigma-Aldrich). Progesterone (10 nM) or ethanol vehicle was added for 24 h. Anti-TF antibody (Genentech, 1:1000) was added 6 h after progesterone addition. The inserts were fixed in cold methanol and immunocytochemistry performed against cytokeratin and counterstained with Harris’s hematoxylin. The slides were observed under a light microscope and the number of cells in each field that had degraded the matrigel and passed through the porous membrane counted. In each experiment 15 fields (x400) were counted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Measurement of TF mRNA by RT-PCR

To determine the TF regulation, ZR-75 cells were treated with E, progesterone, and EGF (all 10 nM), alone or in combination. After isolation of mRNA, semiquantitative RT-PCR was performed after 9 h of hormonal or EGF treatment. Treatment with progesterone increased TF mRNA (Fig. 1Go). Cotreatment with EGF or estrogen did not alter the progesterone regulation, nor did they display activity alone. Densitometry units, normalized against the GAPDH loading control in three independent experiments, demonstrated that the progesterone increase in TF RNA was 7- to 10-fold over the vehicle control (data not shown).



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  		FIG. 1. RT-PCR of TF and GAPDH. Total RNA extracted from ZR-75 cells was used for the reverse transcription reaction. The obtained cDNA was used for PCR with specific primers for TF (556 bp) and GAPDH (678 bp). RT-PCR products were separated in 1.5% agarose gel and stained with ethidium bromide. All treatments were 9 h. C, Control, ethanol; P, progesterone.

 
TF expression is regulated by progesterone but not EGF

Because progesterone, but not EGF, increased the levels of TF mRNA, we determined whether this regulation was manifested at the protein level. The presence of TF in membrane fractions (all standardized to 100 µg per treatment) was determined by Western blotting. Anti-TF monoclonal antibody (Calbiochem, EMD Biosciences, or Genentech) produced diffuse but specific cluster of bands that migrated at approximately 47 kDa. TF expression significantly increased in response to treatment with progesterone in accordance with the regulation observed at the mRNA level (Fig. 2Go). E or EGF had no effect and cotreatment with EGF or E did not alter the progesterone-mediated expression. To confirm that these bands were TF, we took advantage of previous reports demonstrating that TF is a protein of 31 kDa that migrates at 47 kDa on a Western blot only after posttranslational modifications. TF possesses four potential N-linked glycosylation sites that produce a slightly altered migration pattern, depending on the degree of glycosylation (34). As expected, addition of a glycosylation inhibitor, tunicamycin, to ZR-75 cells treated with progesterone changed the migration pattern of each TF band from approximately 47 kDa to a band at 31 kDa (Fig. 3Go). The change in intensity from the several bands migrating around 47 kDa, in comparison with a 31-kDa band, may be due to a signal concentrated band emitting a lower signal, more than a change in quantity; however, these results cannot rule out that the stability of TF is changed by deglycosylation. Anti-TF antibodies generated from different epitopes (Calbiochem, EMD Biosciences, see those used in Figs. 3Go and 6Go; Genentech, see those used in Figs. 2Go and 5Go) yielded the same results, further confirming the authenticity of the TF bands. Under certain conditions a nonspecific band is located just below the TF band when using the antibody from Calbiochem (not shown). Lowering the primary antibody titer from 1:1000 to 1:2000 eliminates this band, which is not regulated or changed by the addition of tunicamycin.



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  		FIG. 2. Effects of ovarian hormones and EGF on TF expression. Breast cancer ZR-75 cells were incubated for 24 h in DMEM/F12 with 5% charcoal-treated serum containing vehicle (C, ethanol), 10 nM 17-ß-estradiol (E), 10 nM progesterone (P), 10 nM EGF, and the combined treatments. Cells were harvested and 100 µg of crude membrane extract separated by SDS-PAGE. TF expression was determined by Western blotting using a TF monoclonal antibody (upper panel, dilution 1:2000, Genentech). These are representative blots of a minimum seven performed. The expression of ß-actin (lower panel, dilution 1:3000, Sigma-Aldrich) represents equal loading.

 


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  		FIG. 3. Progesterone up-regulates the glycosylated form of TF. The glycosylation inhibitor tunicamycin displaces the 47-kDa TF band to 31 kDa in ZR-75 cells. Cells were incubated for 24 h in DMEM/F12 with 5% charcoal-treated serum containing vehicle (C, ethanol) or 10 nM progesterone (P) in the presence or absence of the inhibitor of N-glycosylation tunicamicyn (Tun, 10 ng/ml). Cells were harvested and 100 µg of crude membrane extract separated by SDS-PAGE. TF expression was determined by Western blotting using a TF monoclonal antibody (upper panel, dilution 1:2000, Calbiochem). The expression of ß-actin (lower panel, dilution 1:3000, Sigma-Aldrich) represents protein loading.

 


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  		FIG. 6. Immunocytochemical analysis of TF expression (dilution 1:50, Calbiochem) in the presence of progesterone in ZR-75 cells. Cells were treated with progesterone (10 nM) or ethanol vehicle (control) for 24 h.

 


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  		FIG. 5. Western blots of time courses of TF expression in response to progesterone in ZR-75 cells. Experimental protocols for cell culture were as described in Fig. 1Go. Cells were harvested at stated time points between 0 and 120 h. TF expression was determined by Western blotting using a TF monoclonal antibody (upper panel, dilution 1:2000, Genentech). The expression of ß-actin (lower panel, dilution 1:3000, Sigma-Aldrich) represents equal loading.

 
Progesterone receptor-mediated and transient regulation of TF by progesterone

To determine whether progesterone was using the progesterone receptor in the up-regulation of TF, we treated the cells with the competitive inhibitor RU486 (Roche) 30 min before the addition of progesterone (Fig. 4Go). As anticipated, if progesterone needed binding to its receptor, the inhibitor completely abolished progesterone regulation. To further characterize the regulation of TF by progesterone in ZR-75, we performed a time course from 0 to 120 h after progesterone addition. An increase in protein levels was observed as early as 6 h, consistent with previous reports categorizing TF as an early response gene, reaching maximum levels at 24 h of treatment, before declining and disappearing completely at 72 h (Fig. 5Go). This demonstrates the induction by progesterone is both rapid and transient.



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  		FIG. 4. RU486 inhibits the progesterone-mediated increase in TF. Western blot of TF expression in the presence and absence of progesterone and the progesterone receptor inhibitor RU486. Progesterone (10 nM) was added for 24 h, and RU486 (100 nM) was added 30 min before progesterone addition. The expression of ß-actin (lower panel, dilution 1:3000, Sigma-Aldrich) represents equal loading.

 
Immunocytochemistry

To confirm the Western blot analysis demonstrating a progesterone-mediated increase in TF in ZR-75 cells and evaluate TF localization, immunocytochemistry was performed (Fig. 6Go). Cells grown on coverslips were treated under the same conditions as applied for Western blotting. Strong staining of TF was observed after 24 h of progesterone treatment. As anticipated, this protein appears to be localized primarily to the plasma membrane, with no significant staining observed in the Harris’s hematoxylin-counterstained nucleus.

Membrane distribution of TF in ZR-75 cells

It is reported that an activation of TF function is accompanied by TF translocation from lipid raft-associated caveolae to the heavy fraction of the plasma membrane (4). To determine the location of TF in the presence of progesterone, we performed sucrose gradient experiments that separated the membrane component into 11 fractions (Fig. 7Go). Lanes 4–6 correspond to Caveolae-type membrane microdomains (light buoyant density, detergent-insoluble caveolin-1-containing membrane fractions) and lanes 7–11 encompass the heavier (noncaveolin-1-containing) fraction of the plasma membrane (32). Figure 7Go demonstrates that in ZR-75 cells no TF is detected by Western blotting in the absence of progesterone, whereas in its presence, TF is restricted to the heavy fraction after 24 h of treatment. Caveolin-1 is not expressed in ZR-75 under any treatment condition (see Discussion). To demonstrate that the location of the caveolin-1 is concentrated in fractions 4–6 in our fractionation, we performed the experiment simultaneously in the caveolin-1-expressing endometrial cancer cell line, Ishikawa (Fig. 7Go, lower panel).



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  		FIG. 7. Membrane distribution of TF by sucrose gradient ultracentrifugation and Ishikawa cells (lower panel). ZR-75 cells were incubated for 24 h in DMEM/F12 with 5% charcoal-treated serum and then for a further 24 h in the same medium containing either ethanol vehicle (control) or 10 nM progesterone (Prog). Western blot analysis was carried out with anti-TF monoclonal antibody (dilution 1:2,000, Calbiochem) and anti-caveolin-1 antibody (1:10,000; Transduction Laboratories). Lanes 1–3 correspond to light fraction, lanes 4–6 to caveolae-type membrane microdomains, and lanes 8–11 to heavy fraction of the plasma membrane.

 
Procoagulant activity of TF in ZR-75 cells

To determine whether the induction of TF by progesterone has biological activity and physiological significance, we performed a procoagulant assay in hormone- and EGF-treated cell line extracts. This coagulation assay determined cell surface TF procoagulant activity as measured by the generation of factor Xa (33). The levels of TF protein expression, in the presence and absence of treatment, corresponded with the procoagulant activity (Fig. 8Go).



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  		FIG. 8. Effect of ovarian hormones and EGF on the induction of TF activity in ZR-75 cells. Confluent monolayers of ZR-75 were incubated with vehicle (ethanol control, C), 10 nM 17ß-estradiol (E), 10 nM progesterone (P), 10 nM EGF, and the combined treatments for 24 h. TF activity in cell lysates was measured by the ability to activate factor X in presence of FVII. The data shown correspond to the mean value and the positive SD of six samples (n = 6). This is a representative experiment of four performed. Statistical analysis was performed by the Student t test method against control values (*, P < 0.0001; #, P < 0.0002; ··, P < 0.0001).

 
These results demonstrate that TF protein is up-regulated by progesterone in a breast cancer cell line, and this increase corresponds to an increase in biological activity.

Invasion assay

As previously mentioned, TF has been associated with an increase of metastatic potential in breast cancer tumors. To determine whether the increase in TF expression observed by progesterone corresponded to an increase in metastatic activity in our system, we performed invasion assays. Cells were seeded over porous inserts covered with matrigel and treated with progesterone or ethanol vehicle for 24 h. As demonstrated by a representative experiment in Fig. 9Go, progesterone increases the invasion of ZR-75 cells in comparison with vehicle control (nine independent experiments, P < 0.0001), and this increase was reversed by the inclusion of an anti-TF antibody (Genentech) (five independent experiments, P < 0.0026), demonstrating that TF was involved in the progesterone-mediated increase in invasion. Two independent assays demonstrated that antibody alone did not effect basal invasion. In each experiment 15 fields (x400) were counted.



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  		FIG. 9. Representative matrigel invasion assay. ZR-75 cells were seeded over matrigel-covered porous inserts and treated with progesterone (10 nM) or ethanol vehicle for 24 h. Monoclonal antibody (Ab) against TF (Genentech 1:1000) was added simultaneously with progesterone. Immunocytochemistry against cytokeratin determined that in nine independent experiments, progesterone increased the number of cells degrading the matrigel and passing through the pores (P < 0.0001), and this was inhibited by the addition of TF antibody (five independent experiments, P < 0.0026). Two independent experiments demonstrated that the presence of antibody alone did not alter basal levels of invasion. In each experiment 15 fields (x400) were counted.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TF, the initiator of the extrinsic coagulation cascade, is expressed in many malignancies, including lung, prostate, and colorectal cancers. It is thought to play an important role in cancer progression, metastasis, and prothrombotic state (7, 35, 36, 37). Previous microarray analysis identified TF as gene under control of the activated progesterone receptor in T47D breast cancer cells (2). This report demonstrated only an increase by progesterone on a microarray. We have now expanded these initial observations and demonstrated the increase in TF on progesterone treatment also occurs at the protein level in T47D (not shown), and this process is not confined to this breast cancer cell line. In ZR-75 cells progesterone also increases TF mRNA levels after 6 (not shown) and 9 h of treatment, and this was mirrored by a transient increase in protein expression levels. Experiments using the inhibitor of RNA polymerase, actinomycin-D, have demonstrated that the TF regulation of progesterone is occurring at the level of transcription. This is in accordance with the presented result that the progesterone receptor inhibitor RU486 completely abolishes the progesterone regulation of TF. To pinpoint the site and mechanism of progesterone regulation, we cloned the TF promoter and are currently mapping the sequence conferring progesterone regulation (Pinto, M., S. Kato, N. Espinoza, C. Monso, J. J. Brosens, G. I. Owen, manuscript in preparation). Herein we show that the glycosylated form of TF is up-regulated by progesterone in ZR-75 breast cancer cells. Estrogen did not effect TF expression levels or procoagulant activity, which may be at first surprising because dogma dictates that estrogen pretreatment is required to increase progesterone receptor levels to facilitate progesterone action. However, progesterone receptors are present in ZR-75 cells in the absence of an estrogenic stimulus, and previous work in the T47-D breast cancer line (26) demonstrates that progesterone can still mediate protein regulation, even when PR levels are chronically down-regulated. EGF, previously reported to be influential in breast cancer cell fate, did not regulate TF or alter progesterone-mediated regulation. Further experimentation in our laboratory demonstrated that EGFR is expressed in ZR-75 cells, and EGF treatment causes phosphorylation of MAPK within several minutes of addition, demonstrating that the EGF pathway is active in this cell line. Although further experimentation is required, results presented herein suggest that progesterone has a role in the metastatic properties of breast cancer cell progression, whereas EGF confers enhanced proliferation.

Clinical studies have demonstrated that the presence of progestins in hormone replacement therapy (HRT) preparations increases the incidence of breast cancer (18). Signaling of the cytoplasmic tail of TF via phosphorylation has been shown to increase the production of VEGF secretion, a potent mediator of angiogenesis (38). VEGF in turn up-regulates TF mRNA and protein expression in endothelial cells, thereby further promoting vascular development (39). Our hypothesis, that progesterone confers a survival advantage to burgeoning cancer cells, is given further weight by the recent publication demonstrating that the phase of the menstrual cycle can dramatically alter the metastatic potential of melanoma cells transplanted in mice (40). We postulate that in breast cancer cells the progesterone up-regulation of TF and other aspects of the metastatic process increases the likelihood that these cancer cells will survive and metastasize. This in turn confers the increase in breast cancer incidence observed in the presence of progesterone containing HRT preparations. Within this model, we previously demonstrated that progesterone also increases glucose transporters in this cell line (41). The corresponding increase in glucose uptake could provide the extra energy required by the breast cancer tumor for potential angiogenesis and metastasis (41, 42). In regard to this hypothesis of direct clinical relevance is the timing of surgery in premenopausal women with breast cancer. If the surgery is performed during the luteal phase of the menstrual cycle, any breast cancer cells that escape into circulation during this procedure have an enhanced metastatic and procoagulant activity due to the presence of high levels of circulating progesterone. This would therefore increase the risk of tumor reappearance and induce a hypercoagulable state, both related to poorer patient prognosis.

Caveolae are present in the plasma membrane as caveolin-coated invaginations involved in transport and signaling functions influencing cell growth, apoptosis, angiogenesis, and transvascular exchange (43). Although not strictly required for the inhibition of TF activity by TFPI, previous studies demonstrated that the association of TF with caveolae-rich invaginations of the plasma membrane attenuates TF activity (44). We found that caveolin-1 is not expressed in ZR-75, which is in agreement with previous reports from the MCF-7 breast cancer cell line (45). Because no caveolin-1 is present in ZR-75, the presence of TF in the heavy membrane portion may suggest this receptor is in an active state, awaiting its ligand to initiate coagulation. We are in the process of stably introducing caveolin-1 into this cell line to test this hypothesis.

Many cancer patients have a hypercoagulable or prothrombotic state leading to recurrent thrombosis due to the impact of cancer cells on the coagulation cascade (46). It has been estimated that hypercoagulation accounts for a significant percentage of mortality and morbidity in cancer patients (47). Because TF is the initiator of the extrinsic coagulation cascade, it may be expected that a significant increase in the risk of thromboembolic events would occur in response to an increase in TF production. To demonstrate that the observed increases in TF in our cell line related to an increase in biological activity, we performed coagulation assays on pretreated cell extracts. As expected, TF expression correlated with procoagulant activity, increasing more than 4-fold with progesterone in ZR-75. Our results suggest that the increase in TF expression and the accompanying increase in the extrinsic coagulation cascade activity by local or exogenous progesterone may increase the risk of thromboembolic events in breast cancer patients.

In cancer, TF is already known to be a marker of metastatic potential. FVII has been shown to induce metastasis in pancreatic cancer cell lines, a process that is blocked by anti-TF antibody (48). We demonstrate herein that TF increased by progesterone can induce coagulation in the presence of FVII, and this effect is blocked by TF antibody in breast cancer cells. Based on clinical reports that demonstrate that progesterone-containing HRT reduces TFPI (49, 50), an inhibitor of coagulation, and that estrogen can regulate TFPI type 2 in the ZR-75 cell line (51), further experiments are ongoing to determine whether these proteins are altered by progesterone and play a role in invasion in our cell lines.

In summary, progesterone increases TF in the two breast cancer cell lines tested, ZR-75 and T47D. The majority of breast cancer deaths are attributable to metastases and thrombosis. Based on the correlation presented herein among TF expression, invasion (metastasis), and procoagulant activity (thrombosis), this work presents a mechanism for the clinical observation that the presence of progesterone correlates with the increased risk of breast cancer incidence. We suggest that TF may be an important future target for breast cancer treatment. Future applications may include the use of antiprogestins, anti-TF antibodies, or TF inhibitors in the treatment of breast cancer to reduce the risk of metastasis and reduce the associated hypercoagulable state that leads to cardiovascular mortalities in cancer patients. Further elucidation of the TF pathway may lead to the identification of potential therapeutic targets in steroid hormone-responsive breast cancers, especially in tumor s that display high metastatic potential.


   Acknowledgments
 
We gratefully acknowledge the kind donation of the anti-TF monoclonal antibody (Genentech). We thank Dr. Alfonso Gonzalez (Pontificia Universidad Catolica de Chile) for the donation of the tunicamycin and the caveolin-1 antibody; the laboratory of Dr. Andrew Quest (Universidad de Chile) for its help and assistance with sucrose gradient experiments; and Dr. Alfredo Germain, Dr. Diego Mezzano, and Olga Panes (Pontificia Universidad Catolica de Chile) for their assistance with the procoagulant assays. We also thank Cecilia Chacón and Jenny Corthon (Centro de Investigaciones Medicas, the Pontificia Universidad Catolica de Chile) for their assistance with the immunocytochemistry studies.


   Footnotes
 
This work was supported by Grant 1020715 from the Wellcome Trust (GR071469), the Chilean Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 1020715), Wellcome Trust (GR071469), and Fundación Andes (C-1368).

First Published Online November 23, 2004

Abbreviations: E, 17ß-Estradiol; EGF, epidermal growth factor; EGFR, EGF receptor; FVII, factor VII; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRT, hormone replacement therapy; MN, buffer of 2-(N-morpholine) ethane sulfonic acid and NaCl; TF, tissue factor, TFPI, TF pathway inhibitor; VEGF, vascular endothelial growth factor.

Received May 10, 2004.

Accepted October 18, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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