Overexpression of {alpha}vß6 integrin in serous epithelial ovarian cancer regulates extracellular matrix degradation via the plasminogen activation cascade

N. Ahmed1,2,7, F. Pansino4, Riley Clyde1, P. Murthi1,2, M.A. Quinn1,2, G.E. Rice1,2,3, M.V. Agrez5, S. Mok6 and M.S. Baker1,2

1 Gynaecological Cancer Research Centre, Royal Women's Hospital, Melbourne, Australia,
2 Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Australia,
3 Perinatal Research Centre, Royal Women's Hospital, Melbourne, Australia,
4 Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Australia,
5 Discipline of Surgical Science, University of Newcastle, NSW, Australia and
6 Laboratory of Gynecologic Oncology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent evidence suggests that integrins are involved in the multi-step process of tumour metastasis. The biological relevance of {alpha}v integrins and associated ß-subunits in ovarian cancer metastasis was examined by analysing the expression of these cell surface receptors in nine ovarian cancer cell lines and also in the primary human ovarian surface epithelial cell line (HOSE). ß1, ß3 and ß5 subunits were present in all ten ovarian cell lines. ß6 subunit was present at varying levels in eight out of nine cancer cell lines but was absent in the HOSE cell line. Immunohistochemical staining showed that ß6 was present in both non-invasive (borderline) and high-grade ovarian cancer tissues but was absent in benign and normal ovarian tissue. High {alpha}vß6 integrin expressing ovarian cancer cell lines had high cell surface expression of uPA and uPAR. Ovarian cancer cell lines expressing high to moderate level of {alpha}vß6 integrin demonstrated ligand-independent enhanced levels of high molecular weight (HMW)-uPA and pro-matrix metalloproteinase 2 and 9 (pro-MMP-2 and pro-MMP-9) expression in the tumour-conditioned medium. High and moderate expression of {alpha}vß6 integrin correlated with increased plasminogen-dependent degradation of extracellular matrix which could be inhibited by inhibitors of plasmin, uPA and MMPs or by monoclonal antibody against uPA, MMP-9 or {alpha}vß6 integrin. These results suggest that endogenous de novo expression of {alpha}vß6 integrin in ovarian cancer cells may contribute to their invasive potential, and that {alpha}vß6 expression may play a role in ovarian cancer progression and metastasis.

Abbreviations: DAB, diaminobenzidine tetrahydrochloride; HMW, high molecular weight; HOSE, human ovarian surface epithelial cell line; HUVEC, human umbilical vein endothelial cells; OCT, optical cutting temperature; pro-MMP, pro-matrix metalloproteinase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ability of tumour cells to migrate into extracellular matrices in vitro is known to be mediated by the co-operative expression of a family of adhesion receptors called integrins and cell surface proteinases such as uPA and MMPs (1,2). Integrins are non-covalently linked heterodimers composed of {alpha} and ß transmembrane glycoprotein subunits (2). Sixteen {alpha} and eight ß integrin subunits can form at least 22 different heterodimers of which 15 are receptors for matrix protein. The {alpha}v integrin subunit associates with several ß subunits, including ß1, ß3, ß5, ß6 and ß8 (3).

Several interactions between integrins and proteases have been reported suggesting that both systems may co-operate in extracellular matrix degradation. In melanoma cells, cell surface proteolysis by MMP-2 involves {alpha}vß3 integrin leading to enhanced collagen degradation (4). Moreover, it has been shown that interaction of either {alpha}5ß1 or {alpha}vß3 integrin with ligand triggers signal transduction pathways activating MMP-2 and MMP-9 expression (5,6). In keratinocytes, {alpha}3ß1 conveys signals regulating MMP-9 secretion and expression (7) suggesting that invasion through basement membrane may be regulated by some integrins through MMP-9.

Plasminogen and plasminogen activators are components of another proteolytic machinery thought to have an important role in tumour cell invasion-related matrix degradation. Co-ordinated expression of {alpha}vß3 integrin and uPA has been reported in melanoma cells (8). In ovarian cancer, clinical studies have shown correlation between uPA content and the clinical stage of the cancer (9). Consistent with that finding, membranes isolated from homogenized epithelial ovarian tumour specimens have shown enhanced uPA binding capacity relative to membranes from benign ovarian tumours (10). In vitro studies have shown that uPA secretion by normal ovarian surface epithelial cells to be 17–38-fold lower than in ovarian cancer cells (11). Furthermore, antisense oligonucleotides that block uPA production inhibit colonization of ovarian cancer cells injected intraperitoneally into nude mice (12) providing evidence that the plasminogen activation cascade facilitates ovarian cancer metastasis in vivo.

{alpha}vß6 is an epithelial cell-restricted integrin and has been shown to be expressed in malignant colonic and oral epithelium but not in normal epithelium (3,13). In healthy adult primate tissues, ß6 mRNA and protein are rarely detected (Breuss et al., unpublished data). In contrast, ß6 is expressed during fetal development, in wound healing and in a variety of epithelial tumours (14). Since ninety percent of ovarian cancers arise from malignant transformation of the surface epithelium it is critical to evaluate the role of this integrin in ovarian cancer development. {alpha}vß6 is concentrated at the invading margin of epithelial malignancies, the site where matrix-degrading enzymes appear to be concentrated (4). Recently, we and others have shown that heterologous expression of {alpha}vß6 integrin in colon cancer cells and oral keratinocytes, leads to enhanced secretion of MMP-9 and that this secretion is associated with plasminogen-dependent proteolysis of denatured collagen (15,16). These data are consistent with the hypothesis that {alpha}vß6 integrin plays an important role in tumour formation and progression by modulating cell surface proteolytic activity. Although, ß6 expression has been previously identified in secretory endometrial tissue (14), in this study we show for the first time the expression of {alpha}vß6 integrin in ovarian cancer tissues. We also show that in ovarian cancer cell lines the expression of {alpha}vß6 integrin correlates with ligand-independent but plasminogen dependent high matrix degradation consistent with high levels of matrix-degrading enzymes demonstrating the potential relevance of this integrin in ovarian cancer invasion and metastasis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Antibodies and reagents
The monoclonal antibodies against integrin {alpha}v (LM 142), ß1 (clone P5D2), integrins {alpha}vß3 (LM609), {alpha}vß5 (P1F6) and {alpha}vß6 (E7P6, 10D5) were from Chemicon International. Monoclonal anti-uPA (394) and uPAR (3936) were from American Diagnostic, USA. Phycoerythrin-conjugated goat anti-mouse IgG was obtained from Chemicon International (Temecula, CA) and peroxidase-conjugated goat anti-mouse antibodies were from Bio-Rad (Hercules, CA).

Cell lines
The epithelial ovarian cancer cell lines OVCA 429, OVCA 433, OVCA 432, DOV 13 and SKOV 3 were obtained from Dr Robert Bast, MD Anderson Centre, Houston, USA. PEO.1, PEO.36, OAW 42 were obtained from Dr Georgia Chenevix-Trench, Queensland Institute for Medical Research, Australia. Ovarian clear cell carcinoma cell line OVHS 1 was obtained from Dr Hideki Sakamoto, Nihon University School of Medicine, Japan. Primary human ovarian surface epithelial (HOSE) cell line immortalized by a retroviral vector (LXSN-16E6E7) expressing HPV-E6E7 open reading frame was established by Dr· Samuel Mok, Harvard Medical School, Boston, Massachusetts. Human umbilical vein endothelial cells (HUVEC) were purchased from the American Type Tissue Collection, USA. OVCA 429, OVCA 433, OVCA 432, OVHS 1, and DOV 13 were maintained in MEM medium, PEO.1, PEO.36, OAW 42, SKOV 3 and HOSE cell lines were maintained in RPMI-1640 and HUVEC were maintained in Medium 199. All tissue culture medium contained 10% heat inactivated FBS, 100 µg/ml penicillin and 100 µg/ml streptomycin. Medium 199 was additionally supplemented with 10 mM HEPES, 5 U/ml heparin and 50 µg/ml endothelial growth factor. HUVEC were grown in 25 cm2 flask coated with 0.1% gelatin in PBS. All cells were maintained at 37°C in the presence of 5% CO2. Viability of the cells was checked routinely by the tryptan blue exclusion using a haemocytometer.

Tissue collection
Serous epithelial ovarian cancer, benign and normal ovarian tissues were collected with informed patient consent and Royal Women's Hospital Human Ethics Committee approval from patients requiring clinically indicated surgical resection. Resected cancer tissues were histologically graded by an anatomical pathologist and plasma CA 125 antigen levels were measured by EIA as part of the routine clinical management of patients. Ovarian tissues were placed in optical cutting temperature (OCT) compound (cryomatrix) and snap frozen by immersion in isopentane that had been precooled on dry ice. Frozen tissue samples were stored at –80°C until processed.

Immunohistochemistry
Immunostaining was performed using conventional peroxidase–anti-peroxidase methods (DAKO LSAB + HRP kit, DAKO, Glostrup, Denmark). Briefly, 5 µm thick frozen tissue sections were cut at –23°C using a cryostat (Cryocut 1800, Leica Instruments, Nussloch, Germany) and mounted on poly-L-lysine-coated glass microscope slides. Tissue sections were fixed in acetone for 10 min at –20°C. Endogenous peroxidase activity in the tissues was quenched by first incubating the specimens for 5 min in 3% hydrogen peroxide in methanol. Specimens were then incubated with {alpha}vß6 specific E7P6 antibody followed by incubation with a secondary biotinylated link antibody and peroxidase labelled streptavidin. Staining was completed after incubation with the chromogenic substrate, 3,3'-diaminobenzidine tetrahydrochloride (DAB). Sections were lightly counterstained with Harris haematoxylin and mounted with xylene based DPX mounting medium. Negative control experiments included omission of primary antibody and substitution of primary antibody with immunoglobulin of the same isotype at the equivalent protein concentration.

Tissue staining was visualized by light microscopy (Zeiss Axioskop, Zeiss, West Germany) and photographed on Kodak Ektar 25 ISO professional film using a Zeiss Camera. Intensity of staining was evaluated with the histological grade and quantified with regards to the percentage of cells stained: no stain (0%, negative), <20% (+, weak), 20–50% (++, moderate) and >50% (+++, strong). Both routine haematoxylin and eosin staining and {alpha}vß6 staining were reviewed independently by trained pathologists.

FACScan analyses
Monolayer cultures of ovarian cancer cell lines were washed twice with PBS and then harvested with trypsin–versene (CSL Biosciences, Australia). 106 cells were incubated with primary antibody (LM 142, JB 1A, LM 609, P1F6, E7P6, 394 and 3936) for 20 min at 4°C and then washed twice with PBS. Cells were then stained with secondary antibody conjugated with phycoerythrin for 20 min at 4°C, washed twice with PBS and then resuspended in 0.5 ml PBS prior to FACScan analysis (Becton Dickinson, NJ).

Preparation of tumour-conditioned medium
Cells were seeded at a density of 2 x 106 cells/25 cm2 flask and allowed to grow in their normal medium in the presence of FBS for 2 h. At the end of the incubation period FBS-containing medium was removed and adherent cells were washed three times with their respective medium lacking FBS. Cells were maintained in their respective FBS-free medium for 48 h and the tumour-conditioned medium from the top was collected, cleared of cells and debris by centrifugation at 2000 r.p.m. for 10 min and further concentrated 20–25 fold using Biomax Ultrafree Centrifugal Filter Unit (Millipore, Bedford, USA) with a 10 kDa pore diameter cut-off. To ensure equal loading, protein estimation on the tumour-conditioned medium was performed using the BCA protein assay reagent (Pierce, Rockford, IL).

Western blotting
Tumour-conditioned medium from ovarian cancer cell lines was electrophoresed on a 10% SDS–PAGE gel under non-reducing conditions. Membranes were then probed with mouse anti-uPA (1:1000) followed by peroxidase-labelled secondary antibody and visualized by the enhanced chemiluminescence detection system according to the manufacturer's instruction.

Zymography
Pro-MMP-2 and pro-MMP-9 expression in tumour-conditioned medium were analysed using 10% SDS-gelatin (1 mg/ml final concentration) gels under non-reducing conditions. Tumour-conditioned medium collected under serum-free conditions was mixed with substrate gel sample buffer (0.5 M Tris pH 6.8, 5% SDS, 20% glycerol and 1% bromophenol blue) at 1:1 ratio. Following electrophoresis, gels were washed and incubated at 37°C overnight in the incubation buffer (50 mM Tris HCl and 5 mM CaCl2, pH 8.0), stained with 0.15% Coomassie blue R250 and de-stained in the same solution without Coomassie blue. The gelatin-degrading enzymes were identified as clear bands against the blue background of stained gels. Gelatinolytic activity attributed by pro-MMP-2 and MMP-9 was abolished by incubating zymograms with specific inhibitor of metalloproteinases namely, 1:10 phenanthroline and EDTA (2 mM). Pro-MMP-2 and MMP-9 activation in the samples were obtained by incubating tumour-conditioned medium with APMA (2 mM) for 4 h prior to zymography. Bands of active MMP-2 and MMP-9 at 84 and 62 kDa were observed (data not shown). BHK cell tumour conditioned-medium was used as a standard in each case.

Extracellular matrix preparation and assay
HUVEC were grown to near confluency on gelatin-coated 24-well plates (Falconware). Fresh medium containing 10 µCi [3H]amino acid mixture/ml (Amersham, UK) was added to the cells. Cells were allowed to grow for 2 days to confluency. Isotopic labelling medium was removed, the endothelial cell monolayer was washed with PBS and lysed by the addition of 30 mM NH4OH for 10 min at room temperature. The lysed cells were rinsed gently with PBS and allowed to dry at room temperature. This procedure was found to leave a thin intact extracellular matrix structure in the bottom of the wells, which resembled basement membranes as seen under electron microscopy. Plates not used immediately were stored at 4°C for no longer than 1 week.

Ovarian cancer cell lines (OVCA 433, OVCA 429, SKOV 3, OAW 42, OVCA 432, OVHS 1 and HOSE) were plated directly on to the isotopically labelled extracellular matrix at a density of 3 x 105 cells/well in 0.3 ml of serum-free culture medium. After 24 h the conditioned medium was removed, centrifuged in a Beckman microfuge for 5 min and counted in a ß-scintillation counter to monitor the release of label from the subendothelial extracellular matrix. In studies concerning inhibition of plasminogen activation, uPA and MMP activity, cells were treated with inhibitors and antibodies 30 min prior to plating onto the isotopically labelled extracellular matrix.

Statistics
For immunohistochemistry {chi}2 analyses (contingency table) was used to test the association between the intensity of staining and the histological grade of tumour. The effect of plasminogen on the matrix degradation by ovarian cancer cell lines was analysed by non-parametric analysis of variance (Mann–Whitney U-test). Statistical significance was indicated by P < 0.05. Data are presented as mean ± SEM.


    Results
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 Materials and methods
 Results
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 References
 
Multiple {alpha}v integrin species are expressed in ovarian cancer cell lines
The pattern of cell surface expression of {alpha}v integrin and associated ß subunit was assessed by flow cytometry in nine ovarian cancer cell lines (OVCA 429, OVCA 433, PEO.1, SKOV 3, DOV 13, PEO.36, OAW 42, OVCA 432 and OVHS 1) and in a HOSE cell line using a panel of integrin-specific antibodies, with the result presented in Table IGo and Figure 1Go. All ovarian cell lines expressed {alpha}v, ß1, ß3 and ß5 integrin subunits. Depending on the expression of {alpha}vß6 integrin, ovarian cancer cell lines, can be divided into four different categories: cells having high expression of {alpha}vß6 integrin such as OVCA 433, OVCA 429 and PEO.1; cells with moderate expression of {alpha}vß6 such as SKOV 3 and DOV 13; cells with low expression of {alpha}vß6 integrin like OAW 42 and OVCA 432 and those lacking {alpha}vß6 expression such as OVHS 1 and HOSE cell lines. The lack of {alpha}vß6 integrin in OVHS 1 and HOSE cell line was partly compensated by {alpha}vß5 expression which was higher than in the other cell lines studied.


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Table I. {alpha}v and ß subunit expression in normal ovarian and cancer cell lines
 


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Fig. 1. Flow-cytometric analyses of {alpha}v integrin and associated ß-subunit of OVCA 429, OVCA 433, PEO.1 and HOSE cell lines. Cells were incubated with either control IgG or primary {alpha}v, ß1, ß3, ß5 and ß6 monoclonal antibodies followed by secondary goat anti-mouse IgG conjugated with phycoerythrin. The median intensity of fluorescence (MIF, arbitary units, log scale) was measured. Results are representative of three independent experiments.

 
{alpha}vß 6 expression in ovarian tissues
In order to determine the physiological relevance of the presence of {alpha}vß6 integrin in ovarian cancer cell lines, cryostat sections of normal ovarian tissues and ovarian cancer tissues of different histological grades, were assessed by immunohistochemistry for the presence of {alpha}vß6 integrin using E7P6 antibody. In normal and benign ovarian tissues (eight of each) the expression of {alpha}vß6 integrin was not observed (Figure 2C and DGo) while all serous ovarian tumours borderline, grade I and grade III stained positive for {alpha}vß6 with antibody E7P6 (Figure 2E and FGo). Intensity of staining was significantly associated with the grade of the tumour, with {chi}2 being 18.92 (P = 0.001). Compared with borderline and grade I tumours the intensity of staining was higher in grade III tumours (Table IIGo). Strong staining (+++, >50%) was present in 60% of grade III ovarian tumours versus 0% in the borderline group (Table IGo). Weak staining (+, <20%), on the other hand, was present in 75% of the borderline versus 0% in grade III tumours. 25% of borderline, 80% of grade I and 40% of grade III tumours had moderate staining (++, 20–50%).



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Fig. 2. Haematoxylin and eosin staining of (A) normal ovary and (B) grade III serous ovarian cancer tissue. Photomicrographs showing the differences in positive reaction for {alpha}vß6 integrin in ovarian tissues. (C) normal ovary; (D) benign ovary; (E) borderline ovary and (F) grade III ovary. Eight tissues in normal, benign and borderline group were analysed. Ten tissues for grade III carcinoma were analysed.

 

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Table II. Intensity of {alpha}vß6 integrin stain (n %) and tumour grade in serous ovarian cancer
 
Expression of {alpha}vß6 in ovarian cancer correlated with increased expression and secretion of HMW-uPA, pro-MMP-2 and pro-MMP-9 in tumour-conditioned medium
Flow cytometric analysis of cell surface expression of uPA and uPAR showed that ovarian cancer cell lines with high expression of {alpha}vß6 integrin had high uPA and uPAR expression (Table IIIGo). Tumour-conditioned medium from all ten ovarian cell lines (both normal and cancer) were examined for the presence of HMW-uPA, pro-MMP-2 and pro-MMP-9 expression. Western blotting of equal protein loads revealed high levels of expression of HMW-uPA in the tumour-conditioned medium of cells expressing high levels of {alpha}vß6 integrin (OVCA 429, OVCA 433 and PEO.1, Figure 3Go). Moderate expression of HMW-uPA was observed in tumour-conditioned medium of SKOV 3, DOV 13 and PEO.36 (moderate to low expressers of {alpha}vß6) while very low to non-detectable levels of uPA were observed in OAW 42, OVCA 432, HOSE and OVHS 1 cell lines which either express very low levels or do not express any {alpha}vß6 integrin at all (Figure 3Go). Gelatin zymography on the tumour-conditioned medium of the above cell lines revealed a similar pattern (Figure 4Go). High {alpha}vß6 integrin expressers (OVCA 429, OVCA 433 and PEO.1) expressed high levels of both pro-MMP-9 and pro-MMP-2 while moderate expressers had relatively lower expression of MMPs. On the other hand, low expressers and cells lacking {alpha}vß6 integrin had very low pro-MMP-2 expression. Hence, these data demonstrate that high and moderate expression of {alpha}vß6 integrin in ovarian cancer cells correlates with increased HMW-uPA, pro-MMP-2 and pro-MMP-9 secretion in tumour-conditioned medium. Under the same conditions very low detectable levels of HMW-uPA and pro-MMPs were observed in OAW 42, OVCA 432, HOSE and OVHS 1-cell lines.


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Table III. uPA and uPAR expression in normal ovarian and cancer cell lines
 


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Fig. 3. Expression of HMV-uPA in tumour-conditioned medium of ovarian cancer and HOSE cell lines. Tumour-conditioned medium was prepared as described in Materials and methods section. The samples were analysed by equal protein loading on 10% SDS–PAGE electrophoresis (under non-reducing conditions) followed by western blotting using monoclonal anti-uPA antibody. Ukadaine was used as standard reference uPA. Results are representative of three experiments.

 


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Fig. 4. Gelatin zymography showing the amounts of pro-MMP-2 and pro-MMP-9 secreted in tumour-conditioned medium (concentrated 20-fold) from ovarian cell lines. The positions of purified pro-MMP-2 and pro-MMP-9 are shown on the left. Results are representative of three experiments.

 
Expression of {alpha}vß6 integrin in ovarian cancer correlates with enhanced extracellular matrix degradation
To assess the involvement of {alpha}vß6 integrin in the induction of extracellular matrix proteolysis, [3H]amino acid-labelled basement membrane was chosen as this isotope is incorporated into all protein components within the subendothelial basement membrane. Degradation of basement membrane was monitored by the release of tritium from [3H]amino acid labelled basement membrane. To assess whether matrix degradation is mediated by a plasmin-dependent mechanism, we tested the ability of ovarian cancer cells to degrade isotopically labelled basement membrane in the absence or presence of plasminogen. Preliminary experiments using this technique have shown that the degradation of basement membrane by a plasminogen-dependent process is complete within the initial 24 h of incubation and the most marked increase in degradation occurred when the medium was supplemented with 10–20 µg/ml of purified plasminogen (data not shown). Moreover, exposure of [3H]amino acid-labelled basement membrane to serum-free culture medium did not result in tritium release either in the presence or absence of plasminogen indicating that cell surface associated proteases or proteases in the culture supernatants are responsible for the degradation of the matrix (data not shown).

To assess if {alpha}vß6 integrin expression correlated increase in HMW-uPA and pro-MMPs contributes to the degradation of basement membrane, {alpha}vß6 expressing and non-expressing cells were compared for their ability to degrade the matrix. Even though the basal matrix degradation capacity in the absence of plasminogen for the ovarian cell lines tested was the same, high (OVCA 433 and OVCA 429) and moderate (SKOV 3 and DOV 13) {alpha}vß6 integrin expressing ovarian cancer cell lines had 1.3–1.4-fold enhanced capacity to degrade [3H]amino acid-labelled basement membrane in the presence of plasminogen compared with the low {alpha}vß6 integrin-expressing cell line (OVCA 432, OAW 42) (Figure 5AGo). OVHS 1 and HOSE that does not express {alpha}vß6 integrin behaved like OVCA 432 and OAW 42 cell lines and addition of exogenous plasminogen had no added effect on the degradation of basement membrane. Moreover, the enhanced plasminogen-dependent basement membrane degradation observed for the high {alpha}vß6-expressing cell line, OVCA 433, was abolished in the presence of Trasylol (anti-plasmin), amiloride (anti-uPA), 1,10 phenanthroline (anti-MMP) and antibodies against uPA or MMP-9 or {alpha}vß6 integrin (Figure 5BGo). These data indicate that plasmin-dependent proteolysis involved in uPA and MMP-mediated basement membrane degradation may be regulated by high to moderate expression of {alpha}vß6 integrin in ovarian cancer cells.




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Fig. 5. (A) Effect of {alpha}vß6 integrin expression on the degradation of 3H-labelled basement membrane. Cells (OVCA 433, OVCA 429, SKOV 3, OVCA 432, OAW 42, OVHS 1 and HOSE) were incubated for 24 h in the absence and presence of plasminogen (20 µg/ml) in 24-well plates coated with 3H-labelled basement membrane. Basement membrane degradation was measured by the release of tritium into the fluid phase. Results are shown as mean ± SEM of three different experiments performed in triplicate; (*P < 0.001, **P < 0.002 compared with control cells in the absence of plasminogen). (B) OVCA 433 cells were incubated for 24 h in the absence and presence of plasminogen (20 µg/ml) exposed either to Trasylol (anti-plasmin 20 µg/ml), amiloride (anti-uPA 2 mM), 1,10 phenanthroline (anti-MMPs 2 mM) or monoclonal antibodies against uPA or MMP-9 or {alpha}vß6 for the duration of the experiment. Basement membrane degradation was measured as described above. Results are shown as mean ± SEM of three different experiments performed in triplicate; (*P < 0.001 compared with control cells in the presence of plasminogen).

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The metastatic behaviour of disseminating cells is thought to be initiated by tumour cell adhesion to the subendothelial basement membrane, followed by proteolytic digestion of the basement membrane proteins and migration of tumour cells into the proteolytically modified stroma (17). The contribution of these processes to the unique intraperitoneal dissemination of ovarian carcinoma is unclear. In this study, we have utilized nine human ovarian carcinoma cell lines and one normal ovarian cell line to analyse the role of {alpha}vß6 integrin in cellular activities involved in metastatic function. Our data demonstrate the differential expression of {alpha}vß6 integrin in a range of ovarian cancer cell lines and determines, at multiple levels, the possible function of this integrin in promoting the metastatic potential of ovarian carcinoma. In addition, differences in {alpha}vß6 integrin expression between normal, benign and malignant tissues clearly indicate that the expression of {alpha}vß6 integrin is restricted to metastatic epithelial ovarian cancer cells and that little or no expression of this integrin is present in poorly metastatic (benign) and normal ovarian epithelial cells.

Numerous studies have suggested that a subset of tumour cells within a primary tumour appear with dominant characteristics that enhance their metastatic potential (18). One such characteristic in ovarian cancer might be expression of an adhesion molecule such as {alpha}vß6 integrin. Although, {alpha}5ß1 fibronectin receptor has been proposed as a major receptor responsible for fibronectin-mediated ovarian cancer binding to mesothelium (19) and {alpha}2ß1 integrin-mediated interaction with collagen type I has been shown to facilitate matrix invasion in ovarian cancer (20,21), the biochemical mechanism(s) by which intraperitoneal dissemination of ovarian cancer is mediated remains unknown. Multiple {alpha} subunits such as {alpha}1, {alpha}2, {alpha}3, {alpha}4, {alpha}5 and {alpha}6 in association with ß1 have been identified in ovarian cancer cells (22) but very little is known about the expression and role of {alpha}v integrin subfamily in this tumour type. The expression of {alpha}vß3 has been shown to be less frequent in epithelial ovarian tumours of low metastatic potential compared with high-grade ovarian carcinomas (23). In the present study, we sought to examine the expression and possible relevance of the {alpha}v integrin subfamily to the invasive process in a series of ovarian cancer cell lines. Cell surface expression of ß1 integrin was observed in all of the ten cell lines tested. ß3 and ß5 integrin subunits were observed in all ten ovarian cell lines, while ß6 expression was observed in eight out of nine ovarian cancer cell lines and was absent in normal human ovarian cell line (HOSE). Moreover, immunohistochemical staining for the presence of {alpha}vß6 in human ovarian tissue showed that this integrin was present in non-invasive (borderline), low and high-grade ovarian cancer tissues but was absent in benign and normal ovarian tissues. Furthermore, ß6 expression was enhanced significantly in high-grade ovarian cancer tissue compared with borderline and low-grade cancers.

In ovarian carcinoma, a strong correlation has been suggested between the expression of cell surface proteinases, propensity to metastasize and poor prognosis (24). Predominant among the proteinases with activities directed against extracellular matrix proteins are enzymes such as uPA and MMPs. uPA efficiently catalyses the specific cleavage of inactive plasminogen resulting in the formation of active plasmin. In addition to uPA, MMPs are predominant metastasis-associated proteinases. Expression of MMPs (particularly MMP-2 and MMP-9) and uPA have been linked to enhanced ovarian cancer invasion and metastasis (24). Cytosols prepared from a large number of human ovarian tissues of different histological grades have shown increasing levels of uPA compared with normal ovarian tissues (25). Moreover, recombinant soluble uPAR has been shown to inhibit proliferation and invasion of ovarian cancer cells, consistent with its ability to function as a scavenger for uPA (26). Similarly, both MMP-2 and MMP-9 are produced by ovarian cancer cell lines (17).

In our study, we demonstrate that ovarian cancer cells expressing high to moderate levels of {alpha}vß6 integrin have enhanced secretion of HMW-uPA, pro-MMP-2 and proMMP-9. {alpha}vß6 correlated increase in HMW-uPA and pro-MMP 2 and pro MMP 9 secretion in tumour conditioned medium of ovarian cancer cells is consistent with our studies in colon cancer cells (2729). As uPAR occupancy of uPA on the cell surface is required for plasmin generation, activation of MMPs and subsequent matrix degradation, these data suggest a potential regulatory mechanism whereby {alpha}vß6 integrin contributes to ovarian cancer progression by modulating tumour cell invasion by enhancing matrix degrading enzyme secretion and activity. When high and moderate {alpha}vß6 expressing ovarian cancer cells were cultured on a tritium-labelled basement matrix, addition of exogenous plasminogen induced enhanced 3H-release above the levels observed in the absence of plasminogen, but no such enhancement of basement membrane degradation was observed in non-ß6 expressing or low ß6 expressing ovarian cancer cell lines. The fact that plasminogen-dependent matrix-degradation was completely abolished by inhibitors and antibodies of uPA or MMP9 or {alpha}vß6 integrin suggest a functional role of this integrin in uPA and MMP mediated proteolysis of ovarian cancer. Even though, there was equal plasminogen-independent degradation of basement membrane in all the ovarian cancer cell lines studied it was only in high and moderate {alpha}vß6 expressing cell lines, that the addition of plasminogen produced substantial increases in basement membrane degradation. Previous studies have shown activation of MMPs by plasminogen through uPA-dependent plasmin generation (16,30). uPA can trigger several biological responses in cells including proliferation, migration, invasion, etc. (31). The fact that high expression of {alpha}vß6 integrin correlates with high secretion of uPA in the medium suggest that uPA in the presence of plasminogen may activate these cells in a paracrine fashion to produce more uPA and MMPs as a mechanism of cancer invasion and metastasis.

De novo expression of {alpha}vß6 integrin has been shown to modulate several processes in colon carcinoma cells including cell adhesion and spreading on fibronectin, proliferation within collagen gels, and MMP-9 regulation (27,29,32). {alpha}vß6 integrin plays a critical role in keratinocyte migration on fibronectin and that effect is enhanced by protein kinase C activation (33). Inhibitory antibodies to {alpha}vß6 integrin block keratinocyte migration (34) and fibronectin-dependent MMP-9 activation (16). This study provides further evidence of a link between {alpha}vß6 integrin and uPA-mediated proteolysis leading to enhanced matrix-degradation in ovarian cancer. The fact that {alpha}vß6 integrin expression is absent in normal ovarian tissues and cells, but is present in ovarian cancer tissues and cancer cell lines, and is involved in uPA-mediated proteolysis of basement membrane in vitro makes this integrin a potential target for therapeutic strategies in ovarian cancer. Future studies will focus on the effects of perturbation of {alpha}vß6 integrin expression and its relevance to intracellular signaling cascade and tumour invasiveness in ovarian cancer cells.


    Notes
 
7 To whom correspondence should be addressed at: Gynaecological Cancer Research Centre, Department of Obstetrics and Gynaecology, The University of Melbourne and Royal Women's Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. Email: nuzhata{at}unimelb.edu.au Back


    Acknowledgments
 
The authors wish to thank Royal Women's Hospital and the Brockhoff Foundation for supporting this work. N.A. was in receipt of a RoCan Fellowship in Ovarian Cancer. The support of the Rotary club of Williamstown is gratefully acknowledged.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 23, 2001; revised October 2, 2001; accepted November 16, 2001.