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[Cancer Research 66, 9453-9460, October 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Activation of Cancer Cell Migration and Invasion by Ectopic Synthesis of Coagulation Factor VII

Shiro Koizume1, Ming-Shou Jin1, Etsuko Miyagi2, Fumiki Hirahara2, Yoshiyasu Nakamura1, Jin-Hua Piao1, Akio Asai3, Akira Yoshida1, Eiju Tsuchiya1, Wolfram Ruf4 and Yohei Miyagi1

1 Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute; 2 Department of Obstetrics, Gynecology and Molecular Reproductive Science, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 3 Department of Neurosurgery, Hirakata Hospital, Kansai Medical University, Osaka, Japan; and 4 Department of Immunology, The Scripps Research Institute, La Jolla, California

Requests for reprints: Yohei Miyagi, Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, 1-1-2 Nakao, Asahi-ku, Yokohama 241-0815, Japan. Phone: 81-45-391-5761; Fax: 81-45-366-3157; E-mail: miyagi{at}gancen.asahi.yokohama.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Blood coagulation factor VII (fVII) is physiologically synthesized in the liver and released into the blood. Binding of fVII to tissue factor (TF) at sites of vascular injury triggers coagulation and hemostasis. TF/fVIIa complex formation on the surface of cancer cells plays important roles in cancer biology. Although fVII is synthesized by hepatocellular carcinoma, it remained unclear how TF/fVIIa complex formation and promigratory signaling can occur for most other cancers in extravascular locations. Here, we show by reverse transcription-PCR analysis that nonhepatic cancer cell lines constitutively express fVII mRNA and that endogenously synthesized fVIIa triggers coagulation activation on these cells. fVIIa expression in cancer cells is inducible under hypoxic conditions and hypoxia-inducible factor-2{alpha} bound the promoter region of the FVII gene in chromatin immunoprecipitation analyses. Constitutive fVII expression in an ovarian cancer cell line enhanced both migration and invasion. Enhanced motility was blocked by anti-TF antibodies, factor Xa inhibition, and anti–protease-activated receptor-1 antibody treatment, confirming that TF/fVIIa stimulated migration by triggering cell signaling. This study shows that ectopic synthesis of fVII by cancer cells is sufficient to support proinvasive factor Xa–mediated protease-activated receptor-1 signaling and that this pathway is inducible under hypoxia. (Cancer Res 2006; 66(19): 9453-60)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Coagulation factor VII (fVII) is the key triggering enzyme of the extrinsic coagulation pathway (1). Tissue factor (TF) is the essential cofactor of fVIIa (1) and is an integral membrane protein that is widely expressed in normal and cancerous tissues in humans. TF triggers a procoagulant state in advanced cancer when tumor cells come in contact with the blood or when TF-positive microvesicles are increasingly shed into the circulation (2). The evidence is increasing that the TF/fVIIa complex also initiates key pathogenetic mechanisms in cancer, including angiogenesis (3–6), cell migration and invasion (3, 7), and cell survival (8, 9). Although activation of coagulation is critical for TF-dependent hematogenous cancer metastasis (10), it is likely that TF activities in cancer progression also involve direct cell signaling triggered by TF/fVIIa complex formation on cancer cells.

TF signaling pathways involve, on the one hand, the cytoplasmic domain (3, 5, 7, 11) that regulates cell adhesion and migration (12, 13); on the other hand, the TF/fVIIa complex signals by proteolytic mechanisms (14–17). TF/fVIIa cleaves and activates protease-activated receptor (PAR)-2 to induce these signaling pathways (18, 19), but the TF/fVIIa/factor Xa (fXa) coagulation initiation complex can also signal by activating either PAR-2 or PAR-1 (19).

Most plasma proteins are synthesized in the liver (20). Hepatocytes are the predominant source of fVII production under normal physiologic conditions, although fVII is also expressed by monocytes/macrophages in inflammation (21, 22) and atherosclerosis (23). By Northern blotting of human tissue mRNA, FVII gene expression was only detected in the liver (20), confirming a selective hepatic synthesis under normal conditions. However, fVII is expressed in some hepatocellular carcinoma cells (24, 25), and this ectopic fVII synthesis has been proposed to trigger liver cancer–specific invasion activity mediated by binding of fVIIa to TF pathway inhibitor-2 (26). Among cancers other than hepatocellular carcinoma, TF/fVIIa complex formation has been observed at the invasive edge of bladder cancer (27), ovarian cancer (28), and laryngeal carcinoma tissues (29). However, these studies were conducted by immunohistochemical procedures and the accumulated fVII on the cell surface was assumed to arise from the blood plasma due to extravasation from hyperpermeable blood vessels (27).

In the present study, we show that ectopic expression is frequent in various cancers by analyzing fVIIa mRNA in 46 cancer cell lines by reverse transcription-PCR (RT-PCR) analysis. We further show that endogenous synthesis of fVII by an ovarian cancer cell line is sufficient to promote cell migration and invasion activities of cancer cells. Invasion is one of the important properties of tumor cells that enable escape from hypoxic conditions, especially during active growth (30). Indeed, proinvasive molecules, such as growth factor receptor c-Met, urokinase-type plasminogen activator receptor (31), and Rab11, a vesicular trafficking-associated protein (32), are up-regulated under hypoxia. We further show that ectopic fVII expression is regulated by hypoxia and thus establish a regulatory pathway by which tumor cells become invasive by up-regulation of a promigratory protease-signaling complex.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, tissue specimens, and cell culture. A total of 46 human cancer cell lines were used in this study. All the cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Moregate, Brisbane, Australia) at 37°C in a humidified atmosphere under 5% CO2. Hepatoma cell lines HepG2, HLF, HLE, HUH-6 clone 5, HUH-7, and PLC/PRF/5, glioblastoma cell line T98G, ovarian cancer cell lines KURAMOCHI, PA-1, and TYK-nu, lung cancer cell lines SBC-2, SBC-3, and SBC-4, and breast cancer cell lines YMB-1, MRK-nu-1, and MCF-7 were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). Breast cancer cell lines OCUB-M and MDA-MB-453 were obtained from the RIKEN Bioresource Center Cell Bank (Tsukuba, Japan). Glioblastoma cell lines U87, U343, SF126, SF188, and SF763, ovarian cancer cell lines OVSAYO, OVTOKO, OVMANA, OVISE, OVSAHO, and OVKATE, prostate cancer cell lines LNCaP, PC-3, and DU-145, gastric cancer ell lines KATOIII, MKN28, MKN45, STKM1, and STKM2, and thyroid cancer cell lines KTA1, KTA2, KTA3, KTA4, TTA1, TTA2, and TTA3 were previously described (33–36). Breast cancer cell line HORI was established in our laboratory. Ovarian adenocarcinoma tissues, subclassified into the common ovarian surface epithelial-stromal tumors, were obtained from surgically removed specimens under the approval of the Ethical Committee of Yokohama City University School of Medicine. Written informed consent was obtained from all patients before the operation.

Semiquantitative RT-PCR analysis. Total RNA (2 µg) was subjected to a reverse transcription reaction and aliquots were used for PCR amplification with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) of TF (28 or 36 cycles), fVII (34 or 42 cycles), {gamma}-glutamyl carboxylase (28 or 36 cycles), PAR-1 and PAR-2 (35 cycles), and ß-actin transcripts (30 cycles). The following PCR primers were used: TF, 5'-CTACTGTTTCAGTGTTCAAGCAGTGA-3' and 5'-CAGTGCAATATAGCATTTGCAGTAGC-3'; fVII, 5'-GAGTGTCCATGGCAGGTCC-3' and 5'-CAGCGCGATGTCGTGGTTG-3'; {gamma}-glutamyl carboxylase, 5'-ATCAAGAGCAGCCTAGACAAC-3' and 5'-CAAGCTCTGTAGTGTTGACAT-3'; PAR-1, 5'-GCTTCAGTCTGTGCGGCCCGCTGTTG-3' and 5'-CAAACACTCCGGTGTACACAGATGGG-3'; PAR-2, 5'-CCTGCAGTGGCACCATCCAAGGAAC-3' and 5'-GCCAGATTGGCCATGTAAATCACAGCAGGG-3'; and ß-actin, 5'-CACCTTCTACAATGAGCTGC-3' and 5'-TCATGAGGTAGTCAGTCAGG-3'.

Analysis of FVII gene expression induced by hypoxia. For analyses of FVII gene expression under hypoxic conditions, cancer cells (9 x 105) were cultured in 50-mm diameter dishes for 16 hours. The medium was substituted with 5 mL of fresh medium containing 500 µmol/L cobalt chloride (CoCl2; Sigma, St. Louis, MO) for 0, 2, 4, 8, 12, 24, and 36 hours, followed by total RNA isolation and RT-PCR analyses. For incubation at 1% O2 condition, cells were cultured in multigas incubator (Juji Field, Inc., Tokyo, Japan). Alternatively, fVII mRNA levels were determined by real-time PCR analysis in a LightCycler (Roche, Indianapolis, IN) using a QuantiTect Probe RT-PCR kit (Qiagen, Valencia, CA). Monitoring of the PCR amplification of the fVII transcripts was done by fluorescence resonance energy transfer–based detection using hybridization probes labeled with fluorescent dyes (FITC or LC-Red 640). The copy numbers of fVII transcripts were determined using the pIRES/fVII plasmid (described below) as standard. The PCR primers used were 5'-ACCCCAAGGCCGAATTGTG-3' and 5'-CCACCCAGATGGTGTTGATC-3'. The hybridization probes used were 5'-AAGGGGAGTGTCCATGGCAGGTCC-FITC-3' and 5'-LCRed640-GTTGTTGGTGAATGGAGCTCAGTTGTGTG-3'. As an internal standard, the expression level of porphobilinogen deaminase was determined with the PCR primers 5'-ACCCTGCCAGAGAAGAGTGT-3' and 5'-CCACAGCATACATGCATTCC-3' and hybridization probes 5'-GGTGTTGAGGTTTCCCCGAATACT-FITC-3' and 5'-LCRed640-CTGAACTCCAGATGCGGGAACTT-3'. The copy numbers of porphobilinogen deaminase transcripts were used for normalization of the fVII mRNA expression levels.

Generation of cell lines stably transfected with fVII cDNA. A fVII expression vector was constructed by inserting the entire coding region of a human fVII cDNA into the bicistronic mammalian expression vector pIRES-puro2 (BD Biosciences, Palo Alto, CA). The fVII expression vector (pIRES/fVII) or the empty vector was transfected into OVSAYO ovarian cancer cells using the Lipofectamine 2000 reagent (Invitrogen) and two stable cell clones were established for each construct in the presence of 0.5 µg/mL puromycin.

Western blot analysis. Conditioned media from cloned or wild-type OVSAYO cells were concentrated and fVII/VIIa was detected by Western blotting under nonreducing conditions with a murine monoclonal antibody raised against human fVII/VIIa antibody (American Diagnostics, Inc., Greenwich, CT). Western blotting of hypoxia-inducible factors (HIF) was done under reducing conditions with anti-HIF-1{alpha} (H-206; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HIF-2{alpha}/endothelial PAS domain protein 1 (EPAS-1; NB100-122; Novus Biologicals, Littleton, CO), or anti-{alpha}-tubulin (B7; Santa Cruz Biotechnology) antibodies, followed by detection with the enhanced chemiluminescence system (Amersham Bioscience, Buckinghamshire, United Kingdom).

fXa generation assay. fXa generation assays were done as previously described (37) with slight modifications. Briefly, 7 x 104 cells were seeded in each well of a 24-well plate and cultured for 16 hours. The cells were then washed and fXa generation was initiated by adding 300 µL of reaction buffer containing 175 nmol/L fX to the washed cells. After 30 to 90 minutes, fXa in the supernatant was determined with the chromogenic substrate Spectrozyme Xa solution (American Diagnostics).

Transwell migration and invasion assays. For migration assays, 1 x 105 cells in 200 µL of RPMI 1640 containing 0.1% bovine serum albumin (BSA) were added to the upper chamber and 700 µL of RPMI 1640 containing 10% FBS were added to the lower chamber of 24-well Boyden chambers with PET-membranes of 8-µm pore size (Becton Dickinson, Bedford, MA) as previously described (38). After 18 hours (40 hours for MDA-MB-453 cell) of incubation, cells in the lower side of the membrane were fixed with methanol and stained with Giemsa. The number of cells was counted in five randomly chosen fields using a light microscope (x200). For migration-blocking assays, control normal mouse immunoglobulin G (IgG; Sigma), anti–TF 6B4 (25 µg/mL), anti–TF 5G9 (50 µg/mL), or both anti–TF 6B4 (25 µg/mL) and anti–TF 9C3 (50 µg/mL) antibodies together were added with the cells to the upper chamber. PAR cleavage was blocked with monoclonal anti-PAR-1 (50 µg/mL ATAP2 and 100 µg/mL WEDE15; ref. 39), rabbit polyclonal anti-PAR-2 (100 µg/mL), or control normal rabbit IgG (Zymed Laboratories, Inc., South San Francisco, CA). Proteases were blocked with the specific fXa inhibitor NAP-5 (1 µmol/L; ref. 5) or the thrombin inhibitor hirudin (4 units/mL; ref. 5) by addition to the upper chamber.

For invasion assays, 24-well BD Matrigel invasion chambers with membranes of 8-µm pore size (BD Biosciences) were used. Briefly, 2.5 x 105 cells in 500 µL of RPMI 1640 containing 0.1% BSA were placed in the upper chamber and 750 µL of RPMI 1640 containing 10% FBS were added to the lower chamber. After 72 hours of incubation, the numbers of cells in the lower side of membrane were counted.

Chromatin immunoprecipitation analysis of HIFs. Chromatin immunoprecipitation analyses of HIFs using cultured cells (4 x 105 per 100-mm dish) or isolated cancer cell nuclei were done using a chromatin immunoprecipitation assay kit (Upstate Biotechnology, Lake Placid, NY) with the modification that Dynabeads protein G (Dynal) was used for immunoprecipitations (40). Genomic sequences were amplified at 35 cycles with Ex Taq polymerase (TAKARA, Tokyo, Japan). The PCR-amplified regions of the FVII gene (GenBank accession no. AF466933) were nucleotides 1,376 to 1,553 (upstream region) and nucleotides 2,120 to 2,349 (promoter region) with the following PCR primers: upstream region, 5'-TGGTTAGTCACCACAGCTTG-3' and 5'-TGGGGTTTCACCGTGTTGAC-3'; promoter region, 5'-CACGCTGGCAACAAAACCGT-3' and 5'-ACATTCCCCATGGGACTGAC-3'. The PCR primers used for amplification of the vascular endothelial growth factor (VEGF) promoter region were previously described (41). The anti-HIF-1{alpha} antibodies H-206 or 100-105 and anti-HIF-2{alpha}/EPAS-1 antibodies NB100-122 or NB100-132 (Novus Biologicals) were used.

Statistics. Data are presented as the mean ± SD. Statistical analyses were done using StatView 4.02 software. P < 0.05 (two-sided Student's t test) was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
fVII, TF, and {gamma}-glutamyl carboxylase mRNA expressions in cancer cells. We tested 46 cancer cell lines for the FVII gene expression. Semiquantitative RT-PCR analyses were done using different PCR cycle numbers. As expected, fVII mRNA was detected in some hepatic cancer cells. However, no transcripts were observed in HLE and HLF cells, which are nondifferentiated hepatoma cell lines established from a hepatocellular carcinoma (Fig. 1A, liver ; ref. 42). HepG2, HUH-6/clone 5, and HUH-7 cells had high expression of fVIIa because the transcript was detectable at low-cycle number PCR (Fig. 1A). No transcripts were detected in six brain glioblastoma cell lines even when high-cycle number PCR was done (Fig. 1A, brain). In ovarian, prostate, lung, stomach, and thyroid cancer cell lines, fVII mRNA levels were high in some cell lines at low cycle number, but in general, fVII was only detected with high-cycle number PCR (Fig. 1A). Expression of fVII mRNA in KURAMOCHI and KATOIII cells seemed to be similar to that in hepatic cancer cells. On the other hand, high levels fVII mRNA expression were observed in most of the breast cancer cell lines (Fig. 1A, breast) with the highest expression levels observed in YMB-1, MDA-MB-453, and MCF-7 cells.


Figure 1
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  		Figure 1. RT-PCR analyses of fVII, TF, and {gamma}-glutamyl carboxylase (GGCX) mRNA expressions in 46 cancer cell lines (A) and 9 ovarian cancer surgical specimens (B). The numbers on the right side of each lane indicate the numbers of PCR cycles. ß-Actin expression was used as an internal standard.

 
To exclude that ectopic fVII expression is an artifact of in vitro culture, we also tested surgical specimens of ovarian cancer and found that most of them expressed fVII transcripts (Fig. 1B). In addition, we examined mRNA expressions of TF, the receptor and obligatory cofactor for fVIIa in these samples. TF was expressed in all cancer cells and, typically, high fVII-expressing cells also showed high levels of TF mRNA (Fig. 1). fVII is a vitamin K–dependent protein that requires posttranslational {gamma}-carboxylation of specific glutamic acid residues in its NH2 terminus for coagulant activity (43). All cancer cells and ovarian cancer isolates expressed {gamma}-glutamyl carboxylase, the crucial vitamin K–dependent enzyme, indicating that proper posttranslational modification of ectopically expressed fVII is possible.

Hypoxic stress up-regulates fVII mRNA expression by recruiting HIF-2{alpha}/EPAS-1 to the FVII promoter. Proinvasive cancer cells are frequently subjected to hypoxic stress and adapt to this condition by up-regulation of protective cellular mechanism (44). We tested whether cancer cells with no or low fVII mRNA expression could up-regulate fVII in response to hypoxic stress and thus increase proinvasive potential. Three nonhepatic cancer cells were exposed to CoCl2, known to induce hypoxic stress, and fVII mRNA expression was monitored by RT-PCR over time. Ovarian cancer cells (OVSAHO and OVSAYO) showed a time-dependent up-regulation of fVII mRNA levels with a peak at 8 hours (Fig. 2A ). Figure 2B confirms by quantitative real-time RT-PCR the substantial up-regulation of fVII mRNA expression under hypoxic stress in OVSAYO and the gastric cancer cell line MKN28 (Fig. 2B). We confirmed the up-regulation of fVII transcription in OVSAYO cells under actual 1% O2 condition although the amplitude of the induction was smaller than that caused by CoCl2 treatment (Fig. 2B).


Figure 2
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  		Figure 2. Hypoxic stress up-regulates fVII mRNA expression by recruiting HIF-2{alpha}/EPAS-1 to the FVII Promoter. A, total RNAs isolated from OVSAHO or OVSAYO cells at different time points of CoCl2 treatment were subjected to RT-PCR analysis of fVII mRNA expression (42 cycles). B, quantitative analysis of the hypoxia-induced fVII mRNA expression by real-time PCR. The fVII mRNA expression levels were normalized to porphobilinogen deaminase mRNA levels and increases in fVII mRNA levels are shown relative to 0 hours of CoCl2 or 1% O2. Columns, mean of triplicate (CoCl2) or duplicate (1% O2) experiments; bars, SD. C, Western blot analyses of HIF-1{alpha} and HIF-2{alpha} expressions in OVSAYO and MKN28 cells. Whole cell lysates prepared from cells at different time points of CoCl2 treatment were subjected to immunoblotting for HIF-1{alpha} or HIF-2{alpha}. Immunoblotting for {alpha}-tubulin was done as a protein loading control. D, chromatin immunoprecipitation analyses of HIF-1{alpha} or HIF-2{alpha} binding to the promoter regions of the FVII and VEGF genes in OVSAYO or MKN28 cells. Representative of multiple chromatin immunoprecipitation experiments using isolated nuclei or intact cells for cross-linking and typical results using isolated nuclei (OVSAYO cells) and intact cells (MKN28 cells). Straight arrows, three regions amplified by PCR. Bent arrows, transcription start site. M, 100-bp marker ladder. I, input PCR control using DNA prepared from sonicated chromatin without immunoprecipitation. N and H, results for normoxia (cultured without CoCl2 treatment) and hypoxia induced by CoCl2 treatment, respectively.

 
Hypoxia stabilizes HIF protein levels and thus induces HIF-dependent transcriptional responses under hypoxic conditions (44). HIF-1{alpha} and HIF-2{alpha} (also known as EPAS-1) bind to hypoxia-responsive elements within gene promoters and play crucial roles as transcriptional factors. We used chromatin immunoprecipitation analyses to test whether HIFs bind to the promoter of the FVII gene following CoCl2 treatment. Western blotting showed that HIF-1{alpha} and HIF-2{alpha} protein levels were increased in response to hypoxia in OVSAYO or MKN28 cells, but hypoxia-induced expression of HIF-2{alpha} in response was somewhat weak in MKN28 cells (Fig. 2C).

We analyzed two regions upstream of the transcription initiation site of the FVII gene (Fig. 2D). The immediate upstream promoter region is crucial for fVII mRNA expression in hepatic cells (20). We found some candidate HIF-binding sites (5'-CAGGT-3', 5'-CACAG-3', and 5'-CACGC-3'), known as HIF-1 ancillary sequences (45), in this region, although typical hypoxia-responsive elements [5'-(G/C/T)ACGTGC(G/C)-3' or 5'-RCGTG-3'; ref. 45] were absent. The second, more distant region was located ~600 bp upstream of the FVII promoter region and contained a single hypoxia-responsive element sequence (5'-GACGTG-3'). We confirmed these promoter sequences in OVSAYO cells by DNA sequencing (data not shown). As a positive control for the HIF-binding activities, we used the promoter region of the VEGF gene, to which HIF-1{alpha} and HIF-2{alpha} bind in response to hypoxia.

HIF-1{alpha} and HIF-2{alpha} were immunoprecipitated from total OVSAYO cells or isolated OVSAYO cell nuclei. Chromatin immunoprecipitation assay showed the expected binding of HIF-1{alpha} or HIF-2{alpha}/EPAS-1 with the promoter region of the VEGF gene (Fig. 2D). No binding of HIF-1{alpha} was detected to the two chosen regions of the FVII gene under hypoxic stress (Fig. 2D). In contrast, HIF-2{alpha} was found to bind to the promoter, but not the more distant upstream region of the FVII gene in response to hypoxic conditions (Fig. 2D). The depicted results with anti-HIF-2{alpha} antibody NB100-122 were confirmed by chromatin immunoprecipitation using another anti-HIF-2{alpha} antibody (NB100-132; data not shown). Similar results were obtained when MKN28 cells (Fig. 2D) or isolated MKN28 cell nuclei (data not shown) were used for chromatin immunoprecipitation analyses. Thus, HIF-2{alpha} binding to the FVII promoter was specifically induced by hypoxic stress.

Ectopically synthesized fVIIa is functionally active and involved in cell migration and invasion. To investigate whether the FVII gene expression in nonhepatic cancer cells leads to formation of a functional TF/fVIIa complex on the surface of cancer cells, we tested whether TF/fVIIa on FVII gene-expressing breast cancer cell line YMB-1 and gastric cancer cell line KATOIII can activate fX (Fig. 1A). Real-time RT-PCR showed that YMB-1 cells expressed fVII mRNA at comparable levels (~70%) to HepG2 hepatocellular carcinoma cells. We also examined MKN28 cells, a non-fVII-expressing gastric cancer cell line with significant TF expression (Fig. 1), as a negative control. Both YMB-1 cells (Fig. 3A ) and KATOIII cells (Fig. 3A) showed considerable fXa generation (6.5 and 3.6 pmol/L/min, respectively), but no activity was detected with fVII-negative MKN28 cells (Fig. 3A). fXa generation was also undetectable with OVSAYO cells, which expressed very low levels of fVII (Fig. 3A). fXa generation on YMB-1 or KATOIII cells was diminished (to 0.44 and 1.1 pmol/L/min, respectively) when the activity of the TF/fVIIa complex was blocked with the potent inhibitory antibody TF 5G9 (Fig. 3A). These results show that endogenous expression of fVII by tumor cells is sufficient to form a procoagulant TF/fVIIa complex on the tumor cell surface.


Figure 3
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  		Figure 3. Ectopic synthesis of functionally active fVIIa and its effects on migration and invasion of ovarian cancer cells. A, procoagulant activity on nontransfected or transfected OVSAYO cells was measured by fXa generation assay. Inhibition of fXa generation by anti-TF antibody (TF 5G9, 50 µg/mL) was determined relative to normal rabbit IgG (rIgG; 50 µg/mL). B, the effects of TF 5G9 antibody (25 µg/mL) on the migration and invasion activities of MDA-MB-453 cells were determined by transwell assay. The numbers of cells that had migrated through the PET-membrane (for migration) or Matrigel-coated membrane (for invasion) were determined. Columns, mean (n = 3); bars, SD. C, Western blot analysis of fVII expression in conditioned medium from nontransfected vector control (IRES.1 and IRES.5) or fVII-transfected (fVII.2 and fVII.16) OVSAYO cells. D, effects of ectopic expression of fVII on migration and invasion of OVSAYO cells. Columns, mean (n = 3); bars, SD. *, P < 0.05, different from vector control cells.

 
We next tested whether formation of the TF/fVIIa complex on fVII-expressing tumor cells enhances cell migration and/or invasion. Transwell analysis using a highly fVII-expressing breast cancer cell line (MDA-MB-453) revealed that the number of migrated or invaded cells was diminished when cells were treated with anti-TF antibody TF 5G9 (Fig. 3B), suggesting that ectopically expressed TF/fVIIa increases cell migration and invasion.

Effects of ectopic fVII expression on migration and invasion of ovarian cancer cells. To clearly show that ectopic expression is sufficient to enhance cell migration and/or invasion, we stably transfected OVSAYO cells that had very low levels of fVII expression and minimal TF/fVIIa activity on the cell surface. Two independent clones that expressed fVII (designated OVSAYO/fVII.2 and OVSAYO/fVII.16) and two vector control clones (designated OVSAYO/IRES.1 and OVSAYO/IRES.5) were established. By Western blotting, fVII was secreted into the conditioned media of both OVSAYO/fVII.2 and OVSAYO/fVII.16 cells, but no fVII was detected in the vector control clones (Fig. 3C). Real-time RT-PCR analysis showed 6- to 7-fold higher fVII mRNA expression levels in OVSAYO/fVII.2 cells relative to HepG2 cells. Both OVSAYO/fVII.2 and OVSAYO/fVII.16 clones showed strong fXa generation (107 and 101 pmol/L/min, respectively) whereas vector control clones did not activate coagulation (Fig. 3A). Transwell migration assays showed that ectopic expression of fVII induced a 3- to 4-fold increase in the number of migrating cells (Fig. 3D). Ectopic fVII expression also enhances cell invasion through Matrigel by 4- to 6-fold compared with the non-fVII-expressing cells (Fig. 3D). To confirm that migration is enhanced by formation of a TF/fVIIa complex on the tumor cell surface, antibody inhibition experiments were done. A combination of antibodies (6B4 and 9C3) that is known to efficiently block fVIIa binding to TF suppressed migration and invasion to levels observed with cells that were not transfected with fVIIa. The antibodies did not suppress migration of cells that did not express fVII, further showing specificity (Fig. 4A and B ). However, migration and invasion were also suppressed by an antibody (5G9) that has little effect on TF/fVIIa complex formation. This antibody is known to block binding of fX to TF/fVIIa and thus abolishes fXa dependent signaling (19). However, this antibody also blocks coagulation activation and thrombin generation that may have stimulated migration in the assay that was done in the presence of serum.


Figure 4
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  		Figure 4. Ectopic expression of fVIIa enhances migration and invasion through fXa-dependent cell signaling. A and B, effects of anti-TF antibody treatment on OVSAYO cell migration (A) and invasion (B). Cells were pretreated with anti-TF antibodies (6B4, 9C3, or 5G9) or normal mouse IgG (mIgG) as a negative control before addition to the migration chamber. Migration (C) and invasion (D) were also evaluated in the presence of the thrombin inhibitor hirudin or fXa inhibitor NAP-5. Columns, mean (n = 3); bars, SD. *, P < 0.05, different from noninhibited control.

 
Ectopic expression of fVIIa enhances migration and invasion through fXa-dependent cell signaling. To distinguish between signaling of the TF/fVIIa/Xa complex and downstream thrombin generation, we used specific inhibitors of fXa and thrombin. Transwell assays in the presence of a potent fXa inhibitor, NAP-5, showed a 3- to 5-fold reduction in the numbers of migrating or invading OVSAYO/fVII.2 or OVSAYO/fVII.16 cells (Fig. 4C and D, dotted columns). However, the potent thrombin inhibitor hirudin had no effects on migration or invasion (Fig. 4C and D, black columns). Thus, fXa-dependent signaling is responsible for stimulated migration in cells that ectopically express fVIIa. The ternary TF/fVIIa/Xa complex can activate both PAR-1 and PAR-2 in a fXa-dependent manner (19). RT-PCR showed that PAR-1 and PAR-2 were expressed in OVSAYO/fVII.2 cells, similarly to the parental OVSAYO cells (Fig. 5A ). Anti-PAR-1 treatment significantly reduced (50-70%) migration of fVII-expressing ovarian cancer cells (Fig. 5B, black columns). Cleavage blocking anti-PAR-2 antibody produced little (10-20%) inhibition of cell migration (Fig. 5B, dotted columns) and a slight additive inhibitory effect was seen when cells were pretreated with both anti-PAR-1 and anti-PAR-2 antibodies (Fig. 5B, hatched columns). Thus, in ovarian cancer cells, PAR-1 cleavage plays an essential role in TF/fVIIa–induced motility.


Figure 5
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  		Figure 5. Effects of anti-PAR-1 and anti-PAR-2 antibody treatment on the migration of fVII-transfected OVSAYO cells. A, RT-PCR analysis of PAR-1 and PAR-2 mRNA expressions (35 cycles). B, transwell analyses of the migration activity of OVSAYO/fVII.2 (top) and OVSAYO/fVII.16 cells (bottom) pretreated with anti-PAR-1 (50 µg/mL ATAP2 and 100 µg/mL WEDE15), anti-PAR-2 (100 µg/mL), or both anti-PAR-1 and anti-PAR-2 antibodies. Normal rabbit IgG (250 µg/mL) was used as a negative control. Columns, mean (n = 3); bars, SD. *, P < 0.05, different from the control in the absence of inhibitor.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we provide novel evidence that ectopic expression of fVII is frequent in cancer cells of different origin. The notable exception was glioblastoma in which no fVII transcript was detected in any of the established cell lines analyzed. In addition, we show that fVII-expressing cells invariably express the fVII cellular receptor, TF, and {gamma}-glutamyl carboxylase, the key enzyme for requisite posttranslational modification of fVIIa. Consistently, ectopically expressed fVIIa was functional on the surface of cancer cells to form a TF/fVIIa complex that triggered fXa generation. In previous studies, fVII was found to be associated with TF-positive tumor cells (27–29). Our data challenge the concept that fVIIa is derived from the circulating blood plasma to associate with tumor cells and introduce the regulated ectopic expression of fVIIa by tumor cells as a potentially novel pathogenetic factor that promotes cancer cell migration and invasion.

Ectopic fVII expression was further shown to be regulated by hypoxic conditions. We show hypoxia-induced association of HIF-2{alpha}, but not HIF-1{alpha}, with the FVII promoter. Under the same conditions, both HIF-1{alpha} and HIF-2{alpha} were found to associate with the promoter of the VEGF gene, further showing the specificity of the differential binding of HIFs to the FVII gene promoter. The fact that the relevant the FVII promoter region contains HIF-1 ancillary sequences, but not a typical hypoxia-responsive element, may be the underlying cause for the selective binding of HIF-2{alpha}. It is of interest that glioblastoma cells have recently been shown to up-regulate TF expression in hypoxia (2). Thus, cancer cells may use the regulation of either TF or fVIIa as a mechanism to adjust to a changing tumor microenvironment. In addition, it is worth considering that ectopic synthesis of fVII by hypoxic nonmalignant cells could play a role in TF-dependent angiogenesis and tissue regeneration.

We found that ectopic synthesis of fVII was sufficient to drive the migration and invasion of breast and ovarian cancer cells in transwell analyses. These activities of ovarian cancer cells were profoundly inhibited by fXa, but not by a thrombin inhibitor. In the transwell assay, fX is present in the serum as a chemoattractant stimulus. On the TF/fVIIa–expressing tumor cell surface, fX is then likely activated to enhance migration. In addition, we show that PAR-1 is the central signaling receptor responsible for fXa-enhanced motility. In vivo, fX may be produced ectopically by cancer cells. We detected very weak fX mRNA expression in OVSAYO cells by RT-PCR, which seemed to be insufficient to stimulate migration (data not shown). However, fX protein is frequently detected in cancer specimen (46) and may be locally delivered by stromal cells. For example, macrophages can bind fX through Mac-1 (47) and may locally deliver fX to the tumor microenvironment.

For breast cancer cells, it is controversial whether the TF/fVIIa complex by signaling through PAR-2 (48) or the ternary TF/fVIIa/Xa complex predominantly stimulates migration (49). In our ovarian cancer model with endogenously synthesized fVIIa, the predominant proinvasive stimulus comes from exogenous fX, rather than fVIIa. It is tempting to speculate that endogenous, regulated fVII expression enables the tumor cells to specifically control the interactions with host cells and respond to a changing microenvironment. Ectopic expression of fVII, as shown for various ovarian and breast cancer cells, may sensitize the tumor cells to respond to promigratory and proinvasive cues, such as extravasated fX. This sensitization will likely select tumor cells to escape from hypoxic environments and thus represents a key factor of malignant tumor progression and metastasis.


   Acknowledgments
 
Grant support: The Public Trust Haraguchi Memorial Cancer Research Fund (S. Koizume), the Smoking Research Foundation (Y. Miyagi), NHLBI (W. Ruf), and Research Resident Fellowship from the Foundation for the Promotion of Cancer Research, Japan, for the 2nd Term Comprehensive 10-Year-Strategy for Cancer Control (M-S. Jin).

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.

Received 5/18/06. Revised 7/16/06. Accepted 7/27/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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