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 wildtype, phosphomimetic (T567E-MT1-MMP), and phosphodefective (T567A-MT1-MMP) MT1-MMP show that MT1-MMP promotes MCA formation. Confluent T567E-MT1-MMPexpressing cells exhibit rapid detachment kinetics, spontaneous release as cell-cell adherent sheets concomitant with MT1-MMP-catalyzed A 3 integrin ectodomain shedding, and robust MCA formation. Expansive growth within threedimensional 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. [Cancer Res 2009;69(17): 7121-9]

Increasing evidence suggests that the cytoplasmic tail of membrane type 1 matrix metalloproteinase (MT1-MMP) is subject to phosphorylation and that this modification may influence its enzymatic activity at the cell surface. In this study, phosphorylated MT1-MMP is detected using a phospho-specific antibody recognizing a protein kinase C consensus sequence (phospho-TXR), and a MT1-MMP tail peptide is phosphorylated by exogenous protein kinase C. To characterize the potential role of cytoplasmic residue Thr 567 in these processes, mutants that mimic a state of either constitutive (T567E) or defective phosphorylation (T567A) were expressed and analyzed for their functional effects on MT1-MMP activity and cellular behavior. Phospho-mimetic mutants of Thr 567 exhibit enhanced matrix invasion as well as more extensive growth within a three-dimensional type I collagen matrix. Together, these findings suggest that MT1-MMP surface action is regulated by phosphorylation at cytoplasmic tail residue Thr 567 and that this modification plays a critical role in processes that are linked to tumor progression.Largely composed of a mixture of collagens, laminins, and vitronectin, the extracellular matrix (ECM) 2 serves as both a physical scaffold and a barrier against cell invasion. It has become increasingly evident that the structural condition of the ECM plays a unique role in regulating cell behavior. Proteolysis of integral components of the basement membrane disturbs the barrier provided by the ECM. Without physical restriction, cells invade the surrounding environment in an unregulated manner. The ability of matrix metalloproteinases (MMPs) to collectively degrade nearly all ECM constituents allows this class of enzymes to function in a diverse range of physiological processes (1, 2). Of the anchored MMPs, membrane type 1 matrix metalloproteinase (MT1-MMP) was the first to be discovered and has been most thoroughly characterized. Unlike soluble MMPs, MT1-MMP has a stretch of hydrophobic amino acids that traverse the cell membrane, followed by a short cytoplasmic tail composed of 20 amino acids (3). The advantage of cell surface localization is 2-fold. Surface restriction allows MT1-MMP to modify the immediate pericellular environment, overcoming physical constraints imposed by the ECM (2). Localization at the cell surface also places tethered MMPs in an optimal position to function at invadapodia, highly specialized areas of the cell membrane that form during focalized cell invasion (4). Although information regarding the role of the cytoplasmic tail is relatively limited (5, 6), this domain may function as a bridge to the intracellular machinery.MT1-MMP has an essential role in matrix remodeling during physiological processes (7,8). Conversely, its enzymatic activity is key to acquiring a metastatic phenotype in a variety of tumor cells, including lung, colon, breast, and cervical carcinomas (2, 9 -11). The ability to alter the physical structure of the pericellular environment, while triggering the activation and modific...

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Mucin16 (MUC16/cancer antigen 125 (CA-125)), a high molecular weight glycoprotein expressed on the ovarian tumor cell surface, potentiates metastasis via selective binding to mesothelin on peritoneal mesothelial cells. Shed MUC16/CA-125 is detectable in sera from ovarian cancer patients. We investigated the potential role of membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane collagenase highly expressed in ovarian cancer cells, in MUC16/CA-125 ectodomain shedding. An inverse correlation between MT1-MMP and MUC16 immunoreactivity was observed in human ovarian tumors and cells. Further, when MUC16-expressing OVCA433 cells were engineered to overexpress MT1-MMP, surface expression of MUC16/CA-125 was lost, while cells expressing the inactive E240A mutant retained surface MUC16/CA-125. As a functional consequence, decreased adhesion of cells expressing catalytically active MT1-MMP to three-dimensional mesomimetic cultures and intact ex vivo peritoneal tissue explants was observed. Nevertheless, mesomimetic invasion is enhanced in MT1-MMP-expressing cells. Together these data support a model wherein acquisition of catalytically active MT1-MMP expression in ovarian cancer cells induces MUC16/CA-125 ectodomain shedding, reducing adhesion to meso-mimetic cultures and to intact peritoneal explants. However, proteolytic clearing of MUC16/CA-125, catalyzed by MT1-MMP, may then expose integrins for high affinity cell binding to peritoneal tissues, thereby anchoring metastatic lesions for subsequent proliferation within the collagen-rich sub-mesothelial matrix.

The epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas and promotes cellular responses that contribute to ovarian cancer pathobiology. In addition to modulation of mitogenic and motogenic behavior, emerging data identify EGFR activation as a novel mechanism for rapid modification of the cell surface proteome. The transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a major contributor to pericelluar proteolysis in the ovarian carcinoma microenvironment and is subjected to extensive posttranslational regulation. In the present study, the contribution of EGFR activation to control of MT1-MMP cell surface dynamics was investigated. Unstimulated ovarian cancer cells display caveolar colocalization of EGFR and MT1-MMP, whereas EGFR activation prompts internalization via distinct endocytic pathways. EGF treatment results in phosphorylation of the MT1-MMP cytoplasmic tail, and cells expressing a tyrosine mutated form of MT1-MMP (MT1-MMP-Y 573 F) exhibit defective MT1-MMP internalization. As a result of sustained cell surface MT1-MMP activity, a phenotypic epithelial-mesenchymal transition is observed, characterized by enhanced migration and collagen invasion, whereas growth within three-dimensional collagen gels is inhibited. These data support an EGFR-dependent mechanism for regulation of the transition between invasive and expansive growth of ovarian carcinoma cells via modulation of MT1-MMP cell surface dynamics.

Ovarian carcinoma is most frequently detected when disease has already disseminated intraabdominally, resulting in a 5-year survival rate of less than 20% owing to complications of metastasis. Peritoneal ascites is often present, establishing a unique microenvironmental niche comprised of tumor and inflammatory cells, along with a wide range of bioactive soluble factors, several of which stimulate the EGF-receptor (EGFR). Elevated EGFR is associated with less favorable disease outcome in ovarian cancer, related in part to EGFR activation of signaling cascades that lead to enhanced matrix metalloproteinase expression and/or function. The available data suggest that modulating the expression or activity of the EGFR and/or matrix metalloproteinases offers opportunity for targeted intervention in patients with metastatic disease. KeywordsEGF-receptor; invasion; metastasis; MMP-9; MT1-MMP/MMP-14; ovarian carcinoma Epithelial ovarian carcinoma (EOC) is the leading cause of death from gynecologic malignancy, resulting in 15,520 deaths in the USA in 2008 [201]. When disease is confined to the ovary, 5-year survival is greater than 90%. Unfortunately, the majority of women with EOC are diagnosed with already disseminated intra-abdominal disease and have a low 5-year survival rate of less than 20%. Thus, while early detection is a pressing clinical problem, analysis of factors that promote metastatic dissemination can ultimately improve the survival of women with ovarian cancer. Ovarian tumor microenvironmentA dualistic model for ovarian tumorigenesis has been proposed that is characterized by specific genetic alterations and unique molecular signatures [1][2][3]. In this classification system, lowgrade carcinomas are often confined to the ovary, arise from a recognized precursor lesion, †Author (Figure 1) [9][10][11]. These shed tumor cells may also block peritoneal lymphatics [12], contributing to the accumulation of peritoneal ascites, which can further facilitate metastatic implantation. Single cells and MCAs adhere to peritoneal mesothelium, where upon adhesive interactions and localized proteolysis enable anchoring of proliferative secondary lesions [9,10]. An exception is found in atypical proliferative serous tumors or micropapillary proliferative serous tumors (grouped as 'borderline' or 'low malignant potential'), the prognosis of which requires evaluation of peritoneal implants that often accompany these tumors. While patients with 'noninvasive implants' have a 10-year survival of practically 100%, a 34% mortality within 7 years is observed for those patients with invasive implants [12].An unique microenvironmental niche is thereby established in the peritoneal cavity, comprised of tumor cells, inflammatory cells and a plethora of soluble factors secreted by -or in response to -tumor cells, including growth factors, inflammatory mediators, bioactive lipids and proteolytic enzymes [13][14][15][16][17][18][19][20]. In addition, products of matrix invasion or partially resolved fibrosis, as well as shed...

Supplementary Figure 2 from Ovarian Cancer Cell Detachment and Multicellular Aggregate Formation Are Regulated by Membrane Type 1 Matrix Metalloproteinase: A Potential Role in I.p. Metastatic Dissemination

Supplementary Data from Epidermal Growth Factor Receptor–Mediated Membrane Type 1 Matrix Metalloproteinase Endocytosis Regulates the Transition between Invasive versus Expansive Growth of Ovarian Carcinoma Cells in Three-Dimensional Collagen