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• W2080303025 abstract "Late stage ovarian cancer is characterized by disseminated intraperitoneal metastasis as secondary lesions anchor in the type I and III collagen-rich submesothelial matrix. Ovarian carcinoma cells preferentially adhere to interstitial collagen, and collagen-induced integrin clustering up-regulates the expression of the transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP). Collagenolytic activity is important in intraperitoneal metastasis, potentiating invasion through the mesothelial cell layer and colonization of the submesothelial collagen-rich matrix. The objective of this study was to elucidate a potential mechanistic link between collagen adhesion and MT1-MMP expression. Our results indicate that culturing cells on three-dimensional collagen gels, but not thin layer collagen or synthetic threedimensional hydrogels, results in rapid induction of the transcription factor EGR1. Integrin signaling through a SRC kinase-dependent pathway is necessary for EGR1 induction. Silencing of EGR1 expression using small interfering RNA abrogated collagen-induced MT1-MMP expression and inhibited cellular invasion of three-dimensional collagen gels. These data support a model for intraperitoneal metastasis wherein collagen adhesion and clustering of collagen binding integrins activates integrin-mediated signaling via SRC kinases to induce expression of EGR1, resulting in transcriptional activation of the MT1-MMP promoter and subsequent MT1-MMP-catalyzed collagen invasion. This model highlights the role of unique interactions between ovarian carcinoma cells and interstitial collagens in the ovarian tumor microenvironment in inducing gene expression changes that potentiate intraperitoneal metastatic progression. Late stage ovarian cancer is characterized by disseminated intraperitoneal metastasis as secondary lesions anchor in the type I and III collagen-rich submesothelial matrix. Ovarian carcinoma cells preferentially adhere to interstitial collagen, and collagen-induced integrin clustering up-regulates the expression of the transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP). Collagenolytic activity is important in intraperitoneal metastasis, potentiating invasion through the mesothelial cell layer and colonization of the submesothelial collagen-rich matrix. The objective of this study was to elucidate a potential mechanistic link between collagen adhesion and MT1-MMP expression. Our results indicate that culturing cells on three-dimensional collagen gels, but not thin layer collagen or synthetic threedimensional hydrogels, results in rapid induction of the transcription factor EGR1. Integrin signaling through a SRC kinase-dependent pathway is necessary for EGR1 induction. Silencing of EGR1 expression using small interfering RNA abrogated collagen-induced MT1-MMP expression and inhibited cellular invasion of three-dimensional collagen gels. These data support a model for intraperitoneal metastasis wherein collagen adhesion and clustering of collagen binding integrins activates integrin-mediated signaling via SRC kinases to induce expression of EGR1, resulting in transcriptional activation of the MT1-MMP promoter and subsequent MT1-MMP-catalyzed collagen invasion. This model highlights the role of unique interactions between ovarian carcinoma cells and interstitial collagens in the ovarian tumor microenvironment in inducing gene expression changes that potentiate intraperitoneal metastatic progression. Epithelial ovarian carcinoma is the leading cause of death from gynecologic malignancy (1.Jemal A. Murray T. Samuels A. Ghafoor A. Ward E. Thun M.J. CA-Cancer J. Clin. 2003; 53: 5-26Crossref PubMed Scopus (3363) Google Scholar), due primarily to the fact that the majority of patients are diagnosed at late stage (III and IV) when metastasis has already occurred. Approximately 10% of all epithelial ovarian carcinomas are hereditary and may be facilitated by gene mutations, whereas the remaining 90% are sporadic (2.Narod S.A. Ford D. Devilee P. Barkardottir R.B. Lynch H.T. Smith S.A. Ponder B.A. Garber J.E. Birch J.M. Cornelis R.S. Kelsell D.P. Spurr N.K. Smyth E. Haites N. Sobol H. Bignon Y.-J. Chang-Claude J. Hamann U. Lindblom A. Borg A. Piver M.S. Gallion H.H. Struewing J.P. Whittemore A. Tonin P. Goldgar D.E. Easton D.F. the Breast Cancer Linkage ConsortiumAm. J. Hum. Genet. 1995; 56: 254-264PubMed Google Scholar, 3.Claus E.B. Schildkraut J.M. Thompson W.D. Risch N.J. Cancer. 1996; 77: 2318-2324Crossref PubMed Scopus (611) Google Scholar, 4.Houlston R.S. Collins A. Slack J. Campbell S. Collins W.P. Whitehead M.I. Morton N.E. Ann. Hum. Genet. 1991; 55: 291-299Crossref PubMed Scopus (54) Google Scholar, 5.Narod S.A. Madlensky L. Bradley L. Cole D. Tonin P. Rosen B. Risch H.A. Cancer. 1994; 74: 2341-2346Crossref PubMed Scopus (80) Google Scholar, 6.Auranen A. Iselius L. Br. J. Cancer. 1998; 77: 1537-1541Crossref PubMed Scopus (7) Google Scholar). Tumors are thought to arise from the single cell layer of the ovarian epithelium and metastasis occurs via direct extension into the peritoneal cavity, where shed cells from the primary tumor are found as a component of malignant ascites. Secondary tumors arise as a consequence of CD44- and integrin-mediated intra-peritoneal adhesion and localized invasion into the interstitial collagen-rich submesothelial matrix. Proteolytic activity is important at multiple stages in intraperitoneal metastasis, including disruption of cell-cell interactions, migration and invasion into the mesothelial cell layer, and the submesothelial matrix to anchor secondary lesions, and for subsequent tumor-mediated angiogenesis (7.Ghosh S. Wu Y. Stack M.S. Cancer Treat. Res. 2002; 107: 331-351PubMed Google Scholar). Multiple studies have shown that microenvironmental contacts with stromal cells or extracellular matrix elements may play a major role in tumor progression by inducing epigenetic changes in transformed cells (8.Roskelley C.D. Bissell M.J. Semin. Cancer Biol. 2002; 12: 97-104Crossref PubMed Scopus (105) Google Scholar, 9.Bissell M.J. Radisky D. Nat. Rev. Cancer. 2001; 1: 46-54Crossref PubMed Scopus (1714) Google Scholar). Metastasizing ovarian cancer cells encounter a collagen-rich environment, as the submesothelial matrix is comprised primarily of interstitial collagens (types I and III) and ovarian tumors induce a fibro-proliferative response characterized by increased synthesis of collagen in the peritoneal cavity (10.Harvey W. Amlot P.L. J. Pathol. 1983; 139: 337-347Crossref PubMed Scopus (53) Google Scholar, 11.Stylianou E. Jenner L.A. Davies M. Coles G.A. Williams J.D. Kidney Int. 1990; 37: 1563-1570Abstract Full Text PDF PubMed Scopus (349) Google Scholar, 12.Zhu G.G. Risteli J. Puistola U. Kauppila A. Risteli L. Cancer Res. 1993; 53: 5028-5032PubMed Google Scholar). Malignant ovarian epithelial cells preferentially adhere to type I collagen via α2β1 and α3β1 integrins (13.Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar, 14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Model experiments using microbeads containing immobilized integrin subunit-specific antibodies, to mimic matrix-induced integrin aggregation, showed that clustering of β1-integrins induced expression of the transmembrane collagenase designated membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) 3The abbreviations used are: MT1-MMP, membrane type 1 matrix metalloproteinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; EGR1, early growth response protein 1; RT, reverse transcriptase; siRNA, small interfering RNA; MAPK, mitogen-activated protein kinase.3The abbreviations used are: MT1-MMP, membrane type 1 matrix metalloproteinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; EGR1, early growth response protein 1; RT, reverse transcriptase; siRNA, small interfering RNA; MAPK, mitogen-activated protein kinase. as well as MT1-MMP-mediated activation of pro-MMP2 (14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). As invasion of three-dimensional collagen matrices by ovarian cancer cells is potentiated by MT1-MMP collagenolytic activity (14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), these data support integrin-mediated collagen adhesion as an important microenvironmental regulator of ovarian cancer metastasis. Because of its role as an in vivo collagenase, the activity of MT1-MMP is stringently regulated via multiple mechanisms including transcriptional and post-translational control (15.Munshi H.G. Stack M.S. Cancer Metastasis Rev. 2006; 25: 45-56Crossref PubMed Scopus (103) Google Scholar). Previous data from our laboratory suggested that collagen-induced expression of MT1-MMP in ovarian cancer cells was regulated, in part, at the level of transcription (5.Narod S.A. Madlensky L. Bradley L. Cole D. Tonin P. Rosen B. Risch H.A. Cancer. 1994; 74: 2341-2346Crossref PubMed Scopus (80) Google Scholar); however, in contrast to other members of the MMP family, relatively little is known regarding transcriptional control of MT1-MMP expression (20.Ning W. Li C.J. Kaminski N. Feghali-Bostwick C.A. Alber S.M. Di Y.P. Otterbein S.L. Song R. Hayashi S. Zhou Z. Pinsky D.J. Watkins S.C. Pilewski J.M. Sciurba F.C. Peters D.G. Hogg J.C. Choi A.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14895-14900Crossref PubMed Scopus (285) Google Scholar, 21.Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (121148) Google Scholar, 22.Davidson B. Goldberg I. Gotlieb W.H. Kopolovic J. Ben-Baruch G. Nesland J.M. Berner A. Bryne M. Reich R. Clin. Exp. Metastasis. 1999; 17: 799-808Crossref PubMed Scopus (168) Google Scholar, 23.Afzal S. Lalani E.N. Poulsom R. Stubbs A. Rowlinson G. Sato H. Seiki M. Stamp G.W. Hum. Pathol. 1998; 29: 155-165Crossref PubMed Scopus (83) Google Scholar). The current study was undertaken to elucidate a potential mechanistic link between collagen adhesion and MT1-MMP expression. Our results support a model wherein β1 integrin signaling via a SRC kinase-dependent pathway can potentiate intra-peritoneal metastasis by rapid induction of the early growth response protein (EGR1), inducing transcriptional activation of the MT1-MMP promoter and subsequent MT1-MMP-mediatated collagen invasion. Immunohistochemistry−Immunohistochemical analysis was done retrospectively on tumor tissue microarrays prepared by the Pathology Core Facility of the Robert H. Lurie Comprehensive Cancer Center at Northwestern University assembled from tissue originally taken for routine diagnostic purposes. The microarray tissue specimens included 149 human ovarian carcinomas (77 serous, 45 endometroid, 9 mucinous, and 18 clear cell). Samples were cut 3-4-μm thick and deparaffinized. The cores were 1 mm in diameter. Antigen retrieval was accomplished by heat induction at 99 °C for ∼45 min. Immunohistochemical staining with antibodies to MT1-MMP (Neomarkers, Kalamazoo, MI, clone RB-1544B raised against a peptide from the second quarter of MT1-MMP) at 1:100 dilution was done according to standard procedures. Breast carcinoma was used as a positive control for MT1-MMP. Analysis of tissue sections was done by light microscopy by an anatomic pathologist (B. P. A.) without prior knowledge of the clinical variables. Scoring of MT1-MMP was assigned according to the average overall intensity of the staining and was graded as follows: 0, no staining; 1, fine granular staining; 2, somewhat coarse staining, but less than positive control tissue (human placenta); 3, very coarse staining, similar to positive control tissue. Staining <10% of tumor cells, regardless of intensity, was considered negative. Staining of between 10 and 75% of tumor cells was considered focal positive, and staining of greater than 75% of tumor cells was considered diffuse positive. Statistical analyses were performed by the Biostatistics Core Facility of the Robert H. Lurie Comprehensive Cancer Center. Materials−Polyclonal anti-early growth response protein 1 (EGR1) recognizing 58-kDa human EGR1 was purchased from Aviva Systems Biology (San Diego, CA). Monoclonal anti-β-tubulin (clone TUB2.1), polyclonal anti-matrix metalloproteinase-14 (hinge region), anti-rabbit and anti-mouse immunoglobulin G horseradish peroxidase-conjugated antibodies, and bacterial collagenase were obtained from Sigma. Phospho-SRC family (Tyr-416) and c-SRC polyclonal antibodies were from Cell Signaling Technology and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Rat tail type I collagen and BDPuraMatrix Peptide Hydrogel were purchased from BD Biosciences. The kinase inhibitors PP2, PP3, PD98059, SB203580, wortmannin, and LY294002 were purchased from Calbiochem. The broad-spectrum inhibitor of metalloproteinases GM6001 and a function blocking mouse anti-human integrin β1 monoclonal antibody (mAb 1959) were obtained from Chemicon International (Temecula, CA). Cell Culture−The ovarian carcinoma cell line DOV13 was provided by Dr. R. Bast, Jr. (M. D. Anderson Cancer Center, Houston, TX). DOV13 cell culture was maintained in minimal essential medium (Invitrogen), 10% fetal bovine serum (Invitrogen), penicillin/streptomycin (Cellgro, Mediatech), amphotericin B (Cellgro, Mediatech), nonessential amino acids (Cellgro, Mediatech), sodium pyruvate (Cellgro, Mediatech), and insulin from bovine pancreas (10 mg/liter; Sigma) at 37 °C in 5% CO2 (16.Moser T.L. Pizzo S.V. Bafetti L.M. Fishman D.A. Stack M.S. Int. J. Cancer. 1996; 67: 695-701Crossref PubMed Scopus (103) Google Scholar). Normal immortalized ovarian epithelial cell line IOSE398 was provided by Dr. N. Auersperg (University of British Columbia, Vancouver, Canada) and maintained for no longer then 10 passages in a 1:1 mixture of media 199 (Sigma) and MCDB105 (Sigma), 5% fetal bovine serum, and 50 μg/ml gentamicin (Sigma). Three-dimensional Culture Model of Ovarian Cancer Metastasis−To mimic initial stages of submesothelial matrix invasion by ovarian cancer cells, cells in suspension were plated atop of a three-dimensional collagen I gel. Three-dimensional collagen I gels were prepared by mixing rat tail type I collagen to the final concentration of 0.8 mg/ml with serum-free medium and solidified by polymerization for 30 min at 37 °C. Control cells were plated on thin layer collagen I (two-dimensional collagen I) prepared by coating tissue culture plates by passive adsorption with a dilute (10 μg/ml) solution of rat tail collagen I overnight. Prior to inducing matrix contact, cells were subjected to 20-24 h serum starvation, suspended with trypsin, neutralized with soybean trypsin inhibitor, resuspended in serum-free media, and subsequently plated atop three-dimensional or two-dimensional type I collagen surfaces for the indicated time points. Before collecting cells, media was removed by aspiration, plates were placed on ice and treated with 0.1 mg/ml collagenase solution in phosphate-buffered saline to remove collagen followed by scraping from the plate. The remaining collagen gel was sheared by pipeting and further disrupted by brief incubation (up to 5 min at 37 °C) with the above collagenase solution. Cells were collected by centrifugation for 5 min at 2000 rpm at 4 °C, washed twice with phosphate-buffered saline, and centrifuged as above to collect. Inhibitors−The kinase inhibitors PP2, PP3, PD98059, SB203580, wortmannin, and LY294002 were used at concentrations 10, 10, 50, 25, 0.1, and 25 μm, respectively, for all experiments. As determined by trypan blue (Sigma) staining, cells were viable at these conditions. Where applicable, inhibitors were also co-polymerized into three-dimensional collagen gels. Prior to the experiment, starved cells were pretreated with the inhibitors for 1 h. Gelatin Zymography−Gelatinase activities in conditioned media were determined by SDS-PAGE zymography as previously described (17.Moser T.L. Young T.N. Rodriguez G.C. Pizzo S.V. Bast Jr., R.C. Stack M.S. Int. J. Cancer. 1994; 56: 552-559Crossref PubMed Scopus (116) Google Scholar). Gels were prepared with 9% acrylamide and 0.1% gelatin, samples were electrophoresed without reduction (18.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). SDS was removed from the gel through a 30-min incubation in 2.5% Triton X-100. To initiate gelatinase activity, gels were incubated in 100 ml of 20 mm glycine, 10 mm CaCl2, and 1 μm ZnCl2, pH 8.3, at 37 °C for 24-48 h prior to staining for protein. Developed gels were dried and scanned using Epson Perfection 1640SU and Adobe Photoshop 7.0 software. Western Blotting−Cells incubated under various conditions were collected as described above and lysed with buffer (TBST) containing 50 mm Tris, 150 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 1% Nonidet P-40, proteinase inhibitor mixture (Roche). Cell lysates (20 μg) were electrophoresed on 9% SDS-polyacrylamide gels under reducing conditions (18.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar), electroblotted to a polyvinylidene difluoride membrane (19.Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar), blocked with 5% skim milk in TBST (25 mmol/liter of Tris, pH 7.5, 150 mmol/liter of NaCl, 0.1% Tween 20) for 1 h at room temperature (20 °C). Membranes were incubated for 1-2 h at room temperature with antibodies derived against proteins of interest. The antibodies were used at the following dilutions: 1:1000 for anti-human hinge region MT1-MMP polyclonal antibody in 3% bovine serum albumin in TBST, 1:500 for anti-human EGR1 polyclonal antibody in 5% skim milk in TBST, 1:500 for anti-phospho-SRC (Tyr-416) polyclonal antibody in 3% bovine serum albumin in TBST, 1:500 for anti-total SRC polyclonal antibody in 5% skim milk in TBST, 1:1000 for anti-β-tubulin monoclonal antibody in 5% skim milk in TBST. Immunoreactive bands were visualized with an anti-(rabbit IgG)-peroxidase or anti-(mouse IgG)-peroxidase (1:1000 in 5% skim milk in TBST) and enhanced chemiluminescence using LAS3000 (Fujifilm) and LAS3000 ImageReader software. Band intensities were determined using LAS3000 ImageGauge software according to the manufacturer’s instructions. mRNA Extraction and cDNA Synthesis−Total mRNA was purified from 1 to 2 × 106 cells using the Aurum Total RNA Mini Kit (Bio-Rad). cDNA was synthesized from 10 μg of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad). mRNA purification and cDNA synthesis experiments were repeated three times. Quantitative Real Time PCR−Real time PCR was carried out with the LightCycler RT-PCR System (Bio-Rad) according to the manufacturer’s instructions. SYBR Green was used for quantitative PCR as a double-stranded DNA-specific fluorophore. The PCR was conducted by initial denaturation for 10 min at 95 °C followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s using the iTaq SYBR Green Supermix (Bio-Rad). To determine the specificity of the PCR primers, melting curves were collected by heating the products at 95 °C, then cooling down to 65 °C, and then slowly melting at 0.5 °C/s up to 95 °C. Primer sequences for EGR1 mRNA detection were according to the previously published sequences (20.Ning W. Li C.J. Kaminski N. Feghali-Bostwick C.A. Alber S.M. Di Y.P. Otterbein S.L. Song R. Hayashi S. Zhou Z. Pinsky D.J. Watkins S.C. Pilewski J.M. Sciurba F.C. Peters D.G. Hogg J.C. Choi A.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14895-14900Crossref PubMed Scopus (285) Google Scholar). Sequences of other primers used for the real-time RT-PCR were constructed according to the requirements for primers used for real time RT-PCR (supplemental Table 1). Efficiency of amplification was determined using the standard curves method. Relative quantification of gene expression between experimental (three-dimensional collagen I) and control (two-dimensional collagen I) samples was measured by normalization against endogenous RPL-19 using the ΔCT method (21.Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (121148) Google Scholar). Prior to using RPL-19 as a control, it has been established that its expression correlated well with the total RNA concentration and did not change with the time and treatment used in our studies. -Fold changes were quantified as 2-(ΔCTsample-ΔCTcontrol) as described previously (21.Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (121148) Google Scholar). Transient Transfections−Transient transfections were performed using lipofection method with ExGene (Fermentas) as a vehicle. EGR1 and control siRNA (Santa Cruz Biotechnology) were transiently transfected into DOV13 cells according to the manufacturer’s instructions. Cellular Invasion Assay−Invasion assays were performed using Transwell chambers (0.8 μm, BD Biosciences) as described before (14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In brief, Transwell inserts were coated on the bottom with a thin layer of human collagen (Sigma) as a chemoattractant for 1 h at 37 °C. The inner well of the filters contained 20 μg of human collagen that was allowed to air dry overnight. Cells (70,000) were plated in the collagen-coated inserts, and allowed to invade the collagen gel for 18-24 h at 37 °C. Filters were collected and the cells adhering to the lower surface were fixed and stained using the Diff-Quik staining kit (Dade Behring) according to the manufacturer’s instructions. Cells in several random fields were counted and averaged. Invading cells were expressed as a percent of total cells added to the invasion chamber. Analysis of MT1-MMP Expression in Human Ovarian Carcinoma−The normal ovarian surface epithelium does not express MMPs and MT1-MMP is not detected in benign tumors; however, MT1-MMP has been detected in malignant ovarian tumors (22.Davidson B. Goldberg I. Gotlieb W.H. Kopolovic J. Ben-Baruch G. Nesland J.M. Berner A. Bryne M. Reich R. Clin. Exp. Metastasis. 1999; 17: 799-808Crossref PubMed Scopus (168) Google Scholar, 23.Afzal S. Lalani E.N. Poulsom R. Stubbs A. Rowlinson G. Sato H. Seiki M. Stamp G.W. Hum. Pathol. 1998; 29: 155-165Crossref PubMed Scopus (83) Google Scholar, 24.Auersperg N. Maines-Bandiera S.L. Kruk P.A. Sharp F. Mason P. Blacket T. Berek J. Ovarian Cancer III. Chapman Hall, London1994: 157-169Google Scholar). To evaluate MT1-MMP expression in a large patient cohort, samples from 149 patients were examined for MT1-MMP immunoreactivity. Of these samples, 77 (52%) were serous carcinoma, 45 (30%) were endometrioid carcinoma, 18 (12%) were clear cell carcinoma, and 9 (6%) were mucinous carcinoma. The vast majority of ovarian tumors (78%) displayed positive MT1-MMP immunoreactivity (Table 1, Fig. 1). Staining, with the exception of 17 serous carcinomas, 11 endometrioid carcinomas, and one mucinous carcinoma, was typically diffuse (expression in greater than 75% of tumor cells). MT1-MMP expression was high (3+ or 2+) in 52% of patients. Representative examples of serous, endometrioid, clear cell, and mucinous ovarian tumors with intense MT1-MMP immunoreactivity are shown in Fig. 1. It is interesting to note that in clear cell carcinoma, the most invasive histotype, MT1-MMP, is expressed at high levels in 94% of cases (Table 1). No significant MT1-MMP staining was observed in the stromal compartment. Strong MT1-MMP positivity (3+ or 2+) was not differentially distributed according to Fédération Internationale des Gynaecologistes et Obstetristes stage.TABLE 1Immunohistochemical analysis of MT1-MMP expression in epithelial ovarian tumorsHistotype3+2+1+0Serous (77)8 (10%)24 (31%)24 (31%)21 (28%)Endometrioid (45)4 (9%)19 (42%)10 (22%)12 (27%)Clear cell (18)3 (17%)14 (78%)0 (0%)1 (5%)Mucinous (9)2 (22%)3 (34%)2 (22%)2 (22%) Open table in a new tab β1 Integrin-mediated MT1-MMP Expression on Three-dimensional Type I Collagen−Ovarian carcinomas metastasize intra-peritoneally through implantation into the collagen-rich submesothelium, providing a mechanism for anchoring of secondary lesions to enable proliferation at multiple sites in the peritoneal cavity. We have previously demonstrated preferential adhesion of primary and established ovarian cancer cells to interstitial collagen relative to other matrix proteins including fibronectin, laminin, type IV collagen, and vitronectin (16.Moser T.L. Pizzo S.V. Bafetti L.M. Fishman D.A. Stack M.S. Int. J. Cancer. 1996; 67: 695-701Crossref PubMed Scopus (103) Google Scholar, 25.Fishman D.A. Kearns A. Chilukuri K. Bafetti L.M. O'Toole E.A. Georgacopoulos J. Ravosa M.J. Stack M.S. Invasion Metastasis. 1998; 18: 15-26Crossref PubMed Scopus (78) Google Scholar). Modeling matrix-induced integrin aggregation using microbead-immobilized β1 integrin antibodies demonstrated that integrin clustering enhanced MT1-MMP expression and this stimulation of MT1-MMP collagenolytic activity was a rate-limiting step for invasion of three-dimensional collagen gels (14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). To evaluate further the molecular mechanisms that regulate MT1-MMP expression, a model of intraperitoneal metastasis comprised of tumor cells on three-dimensional collagen gels was utilized. Cells cultured on three-dimensional collagen type I gels adopt a distinct elongated morphology relative to cells cultured on thin layer (two-dimensional) collagen (Fig. 2A). These morphological changes are abrogated by pretreatment with β1 integrin function blocking antibodies (Fig. 2A, right panel), supporting the role of β1 integrins in ovarian cancer cell interaction with three-dimensional collagen gels. Cells generate internal tension via cytoskeletal modulations, balanced in part by adhesion to the ECM, and ample evidence suggests that integrin-mediated ECM adhesion is inherently a mechanosensory process (26.Cukierman E. Pankov R. Yamada K.M. Curr. Opin. Cell Biol. 2002; 14: 633-639Crossref PubMed Scopus (766) Google Scholar, 27.Miyamoto S. Akiyama S.K. Yamada K.M. Science. 1995; 267: 883-885Crossref PubMed Scopus (790) Google Scholar, 28.Miyamoto S. Teramoto H. Coso O.A. Gutkind J.S. Burbelo P.D. Akiyama S.K. Yamada K.M. J. Cell Biol. 1995; 131: 791-805Crossref PubMed Scopus (1103) Google Scholar). It has been proposed that it is the ability of the ECM to resist tension and promote cell distortion that controls cellular behavior (29.Ingber D.E. Differentiation. 2002; 70: 547-560Crossref PubMed Scopus (139) Google Scholar). To test this hypothesis, ovarian cancer cells were cultured in a three-dimensional peptide hydrogel (PuraMatrix Peptide Hydrogel, BD Biosciences) to modify cell shape in the absence of collagen-induced integrin clustering. Control experiments included three-dimensional collagen gels and thin layer type I collagen. Induction of MT1-MMP activity was evaluated by monitoring MT1-MMP-catalyzed activation of pro-MMP2 via gelatin zymography (13.Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar, 14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Consistent with previous reports (13.Ellerbroek S.M. Fishman D.A. Kearns A.S. Bafetti L.M. Stack M.S. Cancer Res. 1999; 59: 1635-1641PubMed Google Scholar, 14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), thin layer collagen culture did not induce MT1-MMP activity and corresponding changes in pro-MMP2 activation (Fig. 2B). Similarly, altering cell shape by three-dimensional culture in PuraMatrix Peptide Hydrogel in the absence of collagen contact was not sufficient to induce MT1-MMP activity (Fig. 2C). In contrast, cells cultured in three-dimensional type I collagen gels exhibited a time-dependent increase in MT1-MMP activity (Fig. 2, D and E). This is supported by Western blot analysis of MT1-MMP protein levels, showing a similar time-dependent increase in MT1-MMP protein (Fig. 2, F and G). Levels of pro-MMP2 were not reproducibly altered by any of the above culture conditions. MT1-MMP Induction in Three-dimensional Collagen I Is Transcriptionally Regulated by EGR1−MT1-MMP is subject to considerable post-translational control via protein trafficking, zymogen activation, and autolysis; however, mechanisms of transcriptional regulation have not been extensively evaluated. We previously reported that collagen-induced MT1-MMP activity was blocked, in part, by actinomycin D, an inhibitor of RNA synthesis (14.Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The promoter region of MT1-MMP has many putative transcription factor binding sites (30.Folgueras A.R. Pendas A.M. Sanchez L.M. Lopez-Otin C. Int. J. Dev. Biol. 2004; 48: 411-424Crossref PubMed Scopus (481) Google Scholar), several of which have been demonstrated to regulate MT1-MMP expression (31.Haas T.L. Stitelman D. Davis S.J. Apte S.S. Madri J.A. J. Biol. Chem. 1999; 274: 22679-22685Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 32.Lohi J. Lehti K. Valtanen H. Parks W.C. Keski-Oja J. Gene (Amst.). 2000; 242: 75-86Crossref PubMed" @default.
• W2080303025 created "2016-06-24" @default.
• W2080303025 creator A5013293580 @default.
• W2080303025 creator A5034090671 @default.
• W2080303025 creator A5049102733 @default.
• W2080303025 creator A5061824879 @default.
• W2080303025 creator A5065417262 @default.
• W2080303025 date "2007-02-01" @default.
• W2080303025 modified "2023-10-15" @default.
• W2080303025 title "Microenvironmental Regulation of Membrane Type 1 Matrix Metalloproteinase Activity in Ovarian Carcinoma Cells via Collagen-induced EGR1 Expression" @default.
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