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Ovarian cancer cell detachment and multicellular aggregate formation are regulated by membrane type 1 matrix metalloproteinase: a potential role in I.p. metastatic dissemination.

ABSTRACT: An early event in the metastasis of epithelial ovarian carcinoma is shedding of cells from the primary tumor into the peritoneal cavity followed by diffuse i.p. seeding of secondary lesions. Anchorage-independent metastatic cells are present as both single cells and multicellular aggregates (MCA), the latter of which adhere to and disaggregate on human mesothelial cell monolayers, subsequently forming invasive foci. Although this unique metastatic mechanism presents a distinct set of therapeutic challenges, factors that regulate MCA formation and dissemination have not been extensively evaluated. Proteolytic activity is important at multiple stages in i.p. metastasis, catalyzing migration through the mesothelial monolayer and invasion of the collagen-rich submesothelial matrix to anchor secondary lesions, and acquisition of membrane type 1 matrix metalloproteinase (MT1-MMP; MMP-14) expression promotes a collagen-invasive phenotype in ovarian carcinoma. MT1-MMP is regulated posttranslationally through multiple mechanisms including phosphorylation of its cytoplasmic tail, and the current data using ovarian cancer cells expressing wild-type, phosphomimetic (T567E-MT1-MMP), and phosphodefective (T567A-MT1-MMP) MT1-MMP show that MT1-MMP promotes MCA formation. Confluent T567E-MT1-MMP-expressing cells exhibit rapid detachment kinetics, spontaneous release as cell-cell adherent sheets concomitant with MT1-MMP-catalyzed alpha(3) integrin ectodomain shedding, and robust MCA formation. Expansive growth within three-dimensional collagen gels is also MT1-MMP dependent, with T567E-MT1-MMP-expressing cells exhibiting multiple collagen invasive foci. Analysis of human ovarian tumors shows elevated MT1-MMP in metastases relative to paired primary tumors. These data suggest that MT1-MMP activity may be key to ovarian carcinoma metastatic success by promoting both formation and dissemination of MCAs.

SUBMITTER: Moss NM

PROVIDER: S-EPMC2737080 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Ovarian cancer cell detachment and multicellular aggregate formation are regulated by membrane type 1 matrix metalloproteinase: a potential role in I.p. metastatic dissemination.

Moss Natalie M NM Barbolina Maria V MV Liu Yueying Y Sun Limin L Munshi Hidayatullah G HG Stack M Sharon MS

Cancer research 20090825 17

An early event in the metastasis of epithelial ovarian carcinoma is shedding of cells from the primary tumor into the peritoneal cavity followed by diffuse i.p. seeding of secondary lesions. Anchorage-independent metastatic cells are present as both single cells and multicellular aggregates (MCA), the latter of which adhere to and disaggregate on human mesothelial cell monolayers, subsequently forming invasive foci. Although this unique metastatic mechanism presents a distinct set of therapeutic ...[more]

PMID: 19706774

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Project description:During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multicellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich submesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior; however, the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin-expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin-expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the submesothelial collagen matrix. Acquisition of Ncad by Ecad+ cells increased mesothelial clearance activity but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a 'leader-follower' mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad in preclinical models of EOC metastasis.

Project description:Matrix metalloproteinase-2 (MMP-2) expression is often up-regulated in advanced cancers and known to play an important role in tumor angiogenesis. We previously showed that adenoviral-mediated delivery of siRNA for MMP-2 (Ad-MMP-2-Si) inhibited lung cancer growth, angiogenesis, and metastasis. In this study, we investigated the signaling mechanisms involved in Ad-MMP-2-Si-mediated inhibition of angiogenesis. Ad-MMP-2-Si treatment inhibited neovascularization in vivo as determined by mouse dorsal air sac model, and conditioned medium from Ad-MMP-2-Si-infected A549 lung cancer cells (Ad-MMP-2-Si-CM) inhibited endothelial tube formation in vitro. Ad-MMP-2-Si-CM decreased proliferation as determined by Ki-67 immunofluorescence and induced apoptosis in endothelial cells as determined by terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) assay. Furthermore, Ad-MMP-2-Si-CM inhibited AKT phosphorylation and induced phosphorylation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase in endothelial cells. Overexpression of constitutively active AKT reversed the Ad-MMP-2-Si-CM-mediated inhibition of tube formation and induction of ERK phosphorylation. Conversely, Ad-MMP-2-Si-CM induced tissue inhibitor of metalloproteinase (TIMP) 3 expression, and the interaction of vascular endothelial growth factor 2 and TIMP-3 was determined by coimmunoprecipitation experiments. TIMP-3 induction was mediated by ERK activation. In addition, electrophoretic mobility shift and chromatin immunoprecipitation assays show that Sp1 transcription factor mediated Ad-MMP-2-Si-CM-stimulated increase of TIMP-3. Vasculature destruction was confirmed with colocalization studies with TUNEL and an endothelial marker, CD31, in tumor sections of Ad-MMP-2-Si-treated mice. Our data collectively suggest that MMP-2 inhibition induces endothelial apoptosis in vivo and inhibits endothelial tube formation. These experiments provide the first evidence that inhibition of p-AKT and induction of p-ERK1/2 are crucial events in the induction of TIMP-3-mediated endothelial apoptosis in MMP-2 inhibited lung tumors.

Dataset Information

Ovarian cancer cell detachment and multicellular aggregate formation are regulated by membrane type 1 matrix metalloproteinase: a potential role in I.p. metastatic dissemination.

Publications

Ovarian cancer cell detachment and multicellular aggregate formation are regulated by membrane type 1 matrix metalloproteinase: a potential role in I.p. metastatic dissemination.

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