Malignant ovarian ascites are rich in cellular components, membranebound vesicles, and soluble proteins. This study focused on the structure of membrane-bound vesicles and their ability to promote invasion in cultured malignant ovarian epithelium. Membrane vesicles were derived from women with stage I-IV malignant ovarian ascites and from nonmalignant gynecologic ascites. Isolated vesicles were characterized by immunofluorescence and Western blot analysis. Using gel zymography for matrix metalloproteinase (MMP) detection and a colorimetric assay for urokinase-type plasminogen activator (uPA) analysis, we analyzed the proteinase activities of MMP-2, MMP-9, and uPA from the prepared vesicles, whole cells isolated from ascites, and the cell-free ultracentrifuged supernatant. The invasiveness of established cultured malignant ovarian epithelium on addition of ascites-derived vesicles was tested using a Matrigel-based invasion assay. Fractionation of malignant ascites revealed that extracellular matrix-degrading proteinases including MMPs and uPA are localized preferentially in membrane vesicles. All malignant vesicles tested, regardless of cancer stage, stimulated invasion. Furthermore, the combination of ovarian cancer cells and membrane vesicles resulted in greater uPA activation than that of cells or vesicles alone. Membrane vesicles from malignant ascites were also found to contain activated MMP-2, MMP-9, and uPA. Our data suggest that vesiclestimulated proteinase activation leads to increased extracellular matrix degradation, which may facilitate tumor cell invasion and metastasis.

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...

Triple negative breast cancers (TNBC) lacking estrogen, progesterone and HER2 receptors account for 10–20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly-targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial-mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide we performed an in silico screen for PAH2 domain-binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3-PAH2 interaction with MAD, induce expression of CDH1 and ESR1 and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically-targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes.

Activation of matrix metalloproteinase 2 (MMP-2) has been shown to play a significant role in the behavior of cancer cells, affecting both migration and invasion. The activation process requires multimolecular complex formation involving pro-MMP-2, membrane type 1-MMP (MT1-MMP), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because calcium is an important regulator of keratinocyte function, we evaluated the effect of calcium on MMP regulation in an oral squamous cell carcinoma line (SCC25). Increasing extracellular calcium (0.09 -1.2 mM) resulted in a dose-dependent increase in MT1-MMP-dependent pro-MMP-2 activation. Despite the requirement for MT1-MMP in the activation process, no changes in MT1-MMP expression, cell surface localization, or endocytosis were apparent. However, increased generation of the catalytically inactive 43-kDa MT1-MMP autolysis product and decline in the TIMP-2 levels in conditioned media were observed. The decrease in TIMP-2 levels in the conditioned media was prevented by a broad spectrum MMP inhibitor, suggesting that calcium promotes recruitment of TIMP-2 to MT1-MMP on the cell surface. Despite the decline in soluble TIMP-2, no accumulation of TIMP-2 in cell lysates was seen. Blocking TIMP-2 degradation with bafilomycin A1 significantly increased cell-associated TIMP-2 levels in the presence of high calcium. These data suggest that the decline in TIMP-2 is because of increased calcium-mediated MT1-MMP-dependent degradation of TIMP-2. In functional studies, increasing calcium enhanced MMP-dependent cellular migration on laminin-5-rich matrix using an in vitro colony dispersion assay. Taken together, these results suggest that changes in extracellular calcium can regulate posttranslational MMP dynamics and thus affect the cellular behavior of oral squamous cell carcinoma. Oral squamous cell carcinoma (OSCC)1 is characterized by local, regional, and distant spread of the disease; however, the cellular and molecular events that control the invasive behavior are poorly understood (1, 2). Immunohistochemical studies have implicated enzymes belonging to the matrix metalloproteinase (MMP) family in basement membrane proteolysis and tissue invasion in OSCC (3). MMPs are a large family of metallo-endopeptidases with activity directed against a variety of extracellular matrix substrates (4 -7). Expression of MMP-2 (gelatinase A, a 72-kDa type IV collagenase) is observed in invasive and metastatic cases of OSCC (3,8). Furthermore, increased expression of MMP-2 is associated with decreased staining of extracellular matrix in OSCC, suggesting that MMP-2 promotes matrix breakdown (3,8). MMP-2 is secreted from cells as a zymogen (pro-MMP-2) and is activated posttranslationally by a trans-membrane MMP designated as membrane type 1-MMP (MT1-MMP) (9 -11). MT1-MMP is also up-regulated in OSCC, and increased expression is observed in highly invasive and metastatic cases (3,8). Pro-MT1-MMP is synthesized as a 63-66-kDa zymogen and is activated intracellularly to a 55-kDa species by the serine prot...

Sequence comparisons of small subunit ribosomal RNA coding regions from 12 chlorophylls a + c-containing algae were used to infer phylogenetic relationships within the Chromophytao Three chromophyte lines of descent, delineated by the Bacillariophyceae, the Phaeophyceael Xanthophyceae, and the Chrysophyceae/Eustigmatophyceae/Synurophyceae are members of a complex evolutionary assemblage, which also includes representatives of the Oomycota ("lower" fungi). Maximum parsimony and distance matrix methods demonstrate a common evolutionary history for these lineages but their relative branching order could not be determined. Other algal species with chlorophylls a + c, including dinoflagellates and prymnesiophytes, are not members of this complex assemblage. Dinoflagellates are specifically related to apicomplexans and ciliates, and the prymnesiophyte, Emiliania huxleyi, represents an independent photosynthetic lineage that separated from other eukaryotes during the nearly simultaneous divergence of plants, animals, fungi, and a number of other protist lineages. The small subunit rRNA phylogenies of chromophytes/oomycetes were compared to those derived from comparisons of ultrastructural characters. Only tubular, tripartite mastigonemes (flagellar hairs) characterized all studied taxa of chromophytes/oomycetes as a monophyletic assemblage.