Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrinmediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP-and Src kinasedependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. Expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad-rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination.

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 kinasedependent 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), 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 (2-6). 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).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 (8,9). Met...

Epidermal growth factor (EGF) receptor (EGFR) is frequently elevated in epithelial ovarian cancer, and E-cadherin expression is often reduced in advanced disease. In this study, we investigated a mechanism by which EGFR activation promotes disruption of adherens junctions through induction of matrix metalloproteinase 9 (MMP-9). We show that EGFR activation down-modulates E-cadherin, and broad spectrum MMP inhibition ameliorates EGF-stimulated junctional disruption and loss of E-cadherin protein. MMP-9 involvement in EGFdependent down-regulation of E-cadherin was determined by siRNA specifically directed against MMP-9. Furthermore, treatment with recombinant MMP-9 or transient expression of MMP-9 is sufficient to reduce E-cadherin levels in differentiated ovarian tumor cells. Stable overexpression of MMP-9 led to a loss of E-cadherin and junctional integrity, and promoted a migratory and invasive phenotype. Thus, elevated MMP-9 protein expression is sufficient for junctional disruption and loss of E-cadherin in these cells. The associations between EGFR activation, MMP-9 expression, and E-cadherin were investigated in human ovarian tumors and paired peritoneal metastases wherein immunohistochemical staining for activated (phospho) EGFR and MMP-9 colocalized with regions of reduced E-cadherin. These data suggest that regulation of MMP-9 by EGFR may represent a novel mechanism for downmodulation of E-cadherin in ovarian cancer. [Cancer Res 2008;68(12):4606-13]

Benign metastasizing leiomyoma is a rare condition affecting women with a history of uterine leiomyomata and is characterized by multiple histologically benign pulmonary smooth muscle tumors. Speculations on its pathogenesis include a benign uterine leiomyoma colonizing the lung, a metastatic low-grade uterine leiomyosarcoma, and primary pulmonary leiomyomatosis. To elucidate its pathogenesis, we analyzed the clinical, pathological and immunohistochemical features, clonality, and telomere length of multiple lung and uterine tumors in three patients with benign metastasizing leiomyoma. In all cases, pulmonary tumors had benign histology and immunohistochemical profiles (estrogen receptor positive, progesterone receptor positive, and very low proliferative index) identical to uterine leiomyoma. In eight tumors from three patients, clonality was assessed by analyzing the variable length of the polymorphic CAG repeat sequence within the human androgen receptor gene. In the two informative patients pulmonary and uterine tumors showed identical patterns of androgen receptor allelic inactivation, indicating that they were clonal. The telomere length measured by fluorescence in situ hybridization in pulmonary leiomyomas of all three patients were either long or very long and were identical to the uterine counterparts, indicating significant telomere shortening is not a crucial step for developing metastases. Our evidence supports the notion that benign metastasizing leiomyoma is clonally derived from benign-appearing uterine leiomyomas.

Elevated levels of the bioactive lipid lysophosphatidic acid (LPA) are detectable in the majority of patients with both early-and late-stage ovarian cancer, suggesting that LPA promotes early events in ovarian carcinoma dissemination. LPA contributes to the development, progression, and metastasis of ovarian cancer in part by inducing the expression of genes that contribute to proliferation, survival, or invasion, including cyclooxgenase-2 (COX-2) and matrix metalloproteinase-2 (MMP-2). We have previously shown that LPA promotes proMMP-2 activation and MMP-2-dependent migration and invasion in ovarian cancer cells. The purpose of the current study was to determine whether the effect of LPA on acquisition of the metastatic phenotype in ovarian cancer cells is mediated via a COX-2-dependent mechanism. Immunohistochemical analysis of 173 ovarian tumors showed strong COX-2 immunoreactivity in 63% of tumor specimens, including 50% of borderline tumors. LPA increased COX-2 protein expression in a time-and concentration-dependent manner in two of three immortalized borderline ovarian epithelial cells as well as in four of six ovarian cancer cell lines. This was accomplished by both activation of the Edg/ LPA receptor and LPA-mediated transactivation of the epidermal growth factor receptor, which increased COX-2 expression via the Ras/mitogen-activated protein kinase pathway. COX-2 also played a role in LPA-induced invasion and migration, as treatment with the COX-2 specific inhibitor NS-398 reduced LPA-induced proMMP-2 protein expression and activation and blocked MMP-dependent motility and invasive activity. These data show that COX-2 functions as a downstream mediator of LPA to potentiate aggressive cellular behavior. (Cancer Res 2005; 65(6): 2234-42)

Advanced and metastatic ovarian cancer is a leading cause of death from gynecologic malignancies. A more detailed understanding of the factors controlling invasion and metastasis may lead to novel anti-metastatic therapies. To model cellular interactions that occur during intraperitoneal metastasis, comparative cDNA microarray analysis and confirmatory real-time reverse transcription PCR (RT-PCR) were employed to uncover changes in gene expression that may occur in late stage ovarian cancer in response to microenvironmental cues, particularly native three-dimensional collagen I. Gene expression in human ovarian carcinoma tissues was evaluated on the RNA and protein level using real-time RT-PCR and immunohistochemistry. Cell invasion and migration were evaluated in a collagen invasion assay and a scratch wound assay. Three-dimensional collagen I culture led to differential expression of several genes. The role of actinin alpha-4 (ACTN4), a cytoskeleton-associated protein implicated in the regulation of cell motility, was examined in detail. ACTN4 RNA and protein expression were associated with advanced and metastatic human ovarian carcinoma. This report demonstrates that a cytoskeletal-associated protein ACTN4 is upregulated by three-dimensional collagen culture conditions, leading to increased invasion and motility of ovarian cancer cells. Expression of ACTN4 in human ovarian tumors was found to be associated with advanced-stage disease and peritoneal metastases.

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Objective-Clear cell carcinomas of the ovary constitute approximately 5% of all ovarian neoplasms and have a distinct gene expression profile relative to other ovarian carcinoma histotypes. Tumors often present as an early stage large pelvic mass with a high degree of recurrence and frequent early metastasis. Matrix metalloproteinases (MMPs) play a role in intraperitoneal metastasis through breakdown of cell-cell and cell-matrix barriers, enabling anchoring of secondary lesions and promoting proliferation in a geometrically constrained matrix environment. The objective of this study was to evaluate MMP expression in ovarian clear cell carcinoma.Methods-Immunohistochemistry was used to evaluate expression of membrane type 1 MMP (MMP-14), MMP-2 and MMP-9 in a panel of ovarian tumors. Western blotting and gelatin zymography were used to examine MMP-14 expression and activity in the clear cell carcinoma cell line ES2. The ability of ES2 cells to invade and proliferate within three-dimensional collagen gels was evaluated.Results-High level expression of MMP-14 and MMP-2 were observed in ovarian clear cell carcinoma relative to other histotypes (94-95% strong positive). MMP-14 was expressed and active in cultured ES2 cells. ES2 cells also exhibited MMP-dependent invasion of and proliferation within three-dimensional collagen gels.Conclusions-The high level expression of MMP-14 together with in vitro functional analyses suggest that MMP-14 may contribute to both the proliferative capacity and the enhanced parenchymal metastasis of ovarian clear cell carcinoma.