Dynamic remodeling of spiny synapses is crucial for cortical circuit development, refinement, and plasticity, while abnormal morphogenesis is associated with neuropsychiatric disorders. Here we show in cultured rat cortical neurons that activation of Epac2, a PKA-independent cAMP target and Rap guanine-nucleotide exchange factor (GEF), induces spine shrinkage, increases spine motility, removes synaptic GluR2/3-containing AMPA receptors, and depresses excitatory transmission, while its inhibition promotes spine enlargement and stabilization. Epac2 is required for dopamine D1-like receptor-dependent spine shrinkage and GluR2 removal from spines. Epac2 interaction with neuroligin promotes its membrane recruitment and enhances its GEF activity. Rare missense mutations in the EPAC2 gene, previously found in individuals with autism, affect basal and neuroligin-stimulated GEF activity, dendritic Rap signaling, synaptic protein distribution, and spine morphology. Thus, we identify a novel mechanism that promotes dynamic remodeling and depression of spiny synapses, whose mutations may contribute to some aspects of disease.

Dendritic spine morphogenesis contributes to brain function, cognition, and behavior, and is altered in psychiatric disorders. Kalirin is a brain-specific guanine-nucleotide exchange factor (GEF) for Rac-like GTPases and is a key regulator of spine morphogenesis. Here, we show that KALRN -knockout mice have specific reductions in cortical, but not hippocampal, Rac1 signaling and spine density, and exhibit reduced cortical glutamatergic transmission. These mice exhibit robust deficits in working memory, sociability, and prepulse inhibition, paralleled by locomotor hyperactivity reversible by clozapine in a kalirin-dependent manner. Several of these deficits are delayed and age-dependent. Our study thus links spine morphogenic signaling with age-dependent, delayed, disease-related phenotypes, including cognitive dysfunction.

Objective Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. Methods To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119, UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. Results Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. Conclusions Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community.

Enzymes in the matrix metalloproteinase (MMP) family have been linked to key events in developmental biology for almost 50 years. Biochemical, cellular and in vivo analyses have established that pericellular proteolysis contributes to numerous aspects of ontogeny including ovulation, fertilization, implantation, cellular migration, tissue remodeling and repair. Surface anchoring of proteinase activity provides spatial restrictions on substrate targeting. This review will utilize membrane type 1 MMP (MT1-MMP) as an example to highlight substrate diversity in pericellular proteolysis catalyzed by a membrane anchored MMP.

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...

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]

The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

Wnt signalling pathways have been shown to play key roles in both normal development and tumorigenesis. Progression of many human cancers is associated with defined mutations in Wnt pathway components that result in dysregulated β-catenin-mediated gene transcription. Although Wnt pathway mutations are rare in epithelial ovarian cancer (with the exception of the endometrioid histotype), accumulating evidence supports a role for Wnt signalling in ovarian tumorigenesis in the absence of genetic mutations. The present review summarizes evidence in support of activated Wnt signalling in ovarian tumours and discusses alternative mechanisms for Wnt pathway activation in the ovarian tumour microenvironment.