Growth Factor-like Domain Research Articles

Detection and clinical significance of EGFL7 and P185 protein in tissues and serum of patients with breast cancer

Objective To detect the expressions of epidermal growth factor-like domain 7 (EGFL7) and P185 protein in breast cancer tissues and serum, and to analyze the correlation between the expression levels of EGFL7 and P185 in tissues and clinicopathological parameters of breast cancer patients. Methods Sixty patients with breast cancer in Hunan Cancer Hospital from March 2016 to March 2018 were collected as observation group, and 60 patients with breast benign lesions in the hospital during the same period were selec-ted as control group. The expressions of EGFL7 and P185 protein in tissues of patients in the two groups were detected by immunohistochemical two-step method, and the levels of EGFL7 and P185 protein in serum of patients in the two groups were detected by enzyme-linked immunosorbent assay (ELISA). The relationships between the expressions of EGFL7 and P185 protein and clinicopathological parameters of breast cancer patients were analyzed. Results The positive rates of EGFL7 in tissues in the observation group and the control group were 65.00% (39/60) and 28.33% (17/60), and there was a significant difference between the two groups (χ2=16.205, P<0.001). The positive rates of P185 in tissues in the two groups were 43.33% (26/60) and 15.00% (9/60), and there was a significant difference between the two groups (χ2=11.657, P=0.001). The serum levels of EGFL7 protein in the observation group and the control group were (3.39±0.38) μg/ml and (2.75±0.31) μg/ml respectively, with a significant difference (t=10.109, P<0.001). The serum levels of P185 protein in the two groups were (7.12±0.75) μg/ml and (6.08±0.62) μg/ml respectively, with a significant difference (t=8.279, P<0.001). The positive expression of EGFL7 protein was closely related to tumor size (χ2=6.128, P=0.013), TNM stage (χ2=7.781, P=0.005) and metastasis (χ2=5.444, P=0.020). The positive expression of P185 protein was closely related to tumor size (χ2=8.910, P=0.003) and TNM stage (χ2=8.024, P=0.005). Conclusion The levels of EGFL7 and P185 protein are high in breast cancer tissues and serum, and their positive expressions are related to tumor size and TNM stage. EGFL7 and P185 proteins play important roles in the progression of breast cancer. Key words: Breast neoplasms; Epidermal growth factor-like domain 7; P185

Designing tracers for PET imaging of the urokinase-type plasminogen activator receptor from a cyclic uPA-derived peptide: first in vitro evaluations.

The treatment of cancer remains a major challenge, especially after tumour cell dissemination and metastases formation. Expression of the urokinase-type plasminogen activation system including urokinase (uPA) and its receptor (uPAR) has been associated with the complex process of cell migration, a tumour's invasive potential as well as a reduced overall and disease-free survival of patients with solid cancers and haematological disorders. A cyclic peptide cyclo[21,29][d-Cys21 ,Cys29 ]-uPA21-30 was designed from the growth factor-like domain (GFD) of urokinase whose binding to uPAR was found to inhibit tumour growth and spread of human ovarian cancer cells in mice. With the aim of visualising uPAR expression using PET imaging to attempt an estimate on the tumour's aggressiveness, the cyclic peptide was modified with an either C- or N-terminally attached variable spacer and chelator. The free ligands were evaluated for their binding affinities to the isolated human uPAR and labelled with 68 Ga and 177 Lu to assess their lipophilicities and stabilities in human serum. Although retaining the full binding potential displayed by cyclo[21,29][d-Cys21 ,Cys29 ]-uPA21-30 to its target was found to be a challenging task upon both C- and N-terminal modification, chelator-bearing ligands were identified that can serve as promising starting points in the development of uPAR-addressing PET tracers.

Epidermal Growth Factor Like-domain 7 and miR-126 are abnormally expressed in diffuse Systemic Sclerosis fibroblasts

Systemic sclerosis (SSc) is characterized by microangiopathy with impaired reparative angiogenesis and fibrosis. Epidermal Growth Factor Like-domain 7 (EGFL7), firstly described in endothelial cells plays a pivotal role in angiogenesis. Fibroblasts (FBs) are involved in vascular remodeling, under physiological and pathological conditions. In this study, we investigated: (i) the expression of EGFL7 and its miR-126 in patients affected by diffuse cutaneous SSc (dcSSc); (ii) the ability of Transforming Growth Factor-beta (TGF-β) to modulate EGFL7 expression; (iii) the ability of EGFL7 to modulate COL1A1 expression and proliferation/migration, and (iv) the functional role of EGFL7 on angiogenesis. Patients were divided in 2 subsets: patients fulfilling the classification criteria in less than one year from Raynaud’s Phenomenon onset (Early Onset Subset–EOS), and all the others (Long Standing Subset–LSS). We show that EGFL7 expression is increased in EOS dcSSc skin and cultured FBs. EGFL7 is inducible by TGF-β on Healthy Controls (HC) FBs but not in SSc-FBs. EGFL7 decreases COL1A1 expression in EOS SSc-FBs while EGFL7 silencing up-regulates COL1A1 expression. EGFL7 promotes migration/invasion of EOS SSc-FBs but not proliferation. Finally, SSc-FBs, partially inhibit angiogenesis in organotypic coculture assays, and this is reversed by treatment with human recombinant (rh)EGFL7. We conclude that EGFL7 and its specific microRNA miR-126 may be involved in the pathogenesis of SSc vasculopathy and fibrosis.

Abstract 1755: PEGylated adenosine deaminase (ADA2) prevents adenosine-mediated increase in tumor growth and improves antitumor immune responses

Abstract Adenosine deaminase 2 (ADA2) is a human serum protein that catalyzes the reaction of deamination of adenosine to inosine. Besides catalytic activity, ADA2 has a heparin-binding domain and a growth factor-like domain. Adenosine accumulates in the tumor microenvironment and suppresses anti-tumor immune responses, while promoting tumor cell metastasis. Previously, we showed that treatment of mice with PEGylated forms of ADA2 (PEG ADA2) can decrease the accumulation of tumor-associated adenosine. Here, we analyzed the TCGA RNA-seq database using OncoLnc tool and observed that high expression of the ADA2 gene (CECR1) in certain immunotherapy responsive tumors correlated with increased survival of patients. We then tested different engineered ADA2 variants, using the inactive enzyme variant as a control, for the reversal of adenosine-mediated responses in human T cells, mouse peritoneal macrophages, and mouse and human tumor cell lines. ADA2 variants with higher catalytic activity were able to reverse adenosine-mediated suppression of human T-cell proliferation, cAMP accumulation in tumors and human T cells, and polarization of macrophages into an immunosuppressive, pro-tumor phenotype, whereas the inactive ADA2 variant failed to cause any of these effects. In addition, the variant that lacks the ability to bind to heparin but has improved catalytic activity (PEG NME8058) showed similar responses to those with the catalytically improved variant with intact heparin-binding domain (PEG NME8062). These results suggest that the improved immune cell activity with ADA2 in the presence of adenosine requires the enzyme activity of this molecule. Next, we assessed PEG NME8062 in MC38 and CT26 colon carcinoma syngeneic tumors in C57BL/6 and Balb/c mice, and observed delayed growth in both tumors. Using the inactive enzyme variant, we have shown that anti-tumor effect of PEG NME8062 depends on enzyme activity. Delayed tumor growth was also associated with increased density of key anti-tumor immune cell populations in both tumor models. These data suggest that PEG ADA2 can reduce tumor growth in an enzyme-activity-dependent manner and promote anti-tumor immune responses in vitro and in vivo. These findings have important implications for targeting purinergic signaling for cancer therapy. Citation Format: Luz Marina Londoño-R, Jessica Cowell, Lin Wang, Qiping Zhao, Lei Huang, Chris Thanos, Michael J. LaBarre, Xiaoming Li, Caglar Cekic. PEGylated adenosine deaminase (ADA2) prevents adenosine-mediated increase in tumor growth and improves antitumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1755.

AN INNOVATIVE PROTEIN CRYSTALLIZATION DEVICE FOR A BETTER ALZHEIMER’S TREATMENT

Researchers from Morgan State University (i.e., Dr. Kadir Aslan) and Juxtopia (i.e., Dr. Jayfus Doswell) constructed a prototype crystallization device (PCD) based on the MA-MAEC technique for the rapid crystallization of peptides and proteins within minutes. In contrast, traditional PCDs typically take up to several weeks to complete using conventional crystallization techniques. Researchers tested the resulting PCD device on the Amyloid precursor protein (APP) and its components as biological macromolecules of interest. APP is most commonly studied as the precursor molecule whose proteolysis generates beta amyloid (Aβ42) peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease patients. Purified APP and synthetic peptides [Aβ42 (42 amino acid residues), copper binding domain (CuBD, 63 amino acid residues) and growth factor-like domain (GFLD, 105 amino acid residues) of APP] were crystallized using microwave heating on the innovative PCD crystallization platforms to demonstrate the proof-of-principle use of the iCrystal system based on multi-parameter assessment: 1) Time of crystallization and microwave power; 2) Size of crystals; and 3) Quality of crystals. The control experiments included: 1) No microwave heating; 2) No SNFs and no microwave heating to assess the effectiveness of the MA-MAEC technique for the rapid crystallization of APP and its relevant components. The iCrystal PCD time was significantly less than traditional PCD with a higher quality crystal. The crystallization experiments for APP and its relevant peptides were repeated separately a minimum of 50 different 21-well PMMA platform to assess the reproducibility of the technique. In this regard, the variation in the size, crystallization time, and quality of APP crystals remain similar. The researched MA-MAEC technique, within the iCrystal system, demonstrated a potential to revolutionize the way peptides and proteins are crystallized as it relates to human diseases, which will afford for rapid development of treatments.

Two B-cell epitope vaccines based on uPA effectively inhibit fertility in male mice

uPA, a trypsin-like serine protease, was found to take active part in male reproduction. Our previous work had demonstrated the antifertility effects of its full length protein immunization, but with immune tolerance and other latent side effects. Here we discovered two effective B-cell epitopes of uPA for male contraception in growth factor-like domain and kringle domain respectively. Together with carrier protein, immunization of these two epitope peptides could induce high titers of specific antibodies in male mice. Significant reduction of fertility was observed in these two groups in mating trial without evident systemic illness or abnormal mating behavior. Epididymal sperms of immunized males exhibited impaired progressive motility and ability to fertilize eggs in vitro. The immunization of another predicted epitope in serine protease domain and the control groups showed no similar positive results. Importantly, T cells were not activated after the challenge of these B-cell epitopes itself, which suggests that these vaccines do not induce cell-mediated autoimmunity. Taken together, our study discovered two uPA B-cell epitopes as novel targets for male immunocontraception with minimum side effects. Considering their high identity with human uPA protein, these two epitope vaccines hold great promise to be developed for man use in the future.

The effect of aloes burn cream on expression of transforming growth feactor-β1 and epidermal growth factor-like domain 7 in rats deep partial burn wound

Objective To investigate the effect of aloes burn cream on expression of transforming growth factor-β1 (TGF-β1) and epidermal growth factor-like domain 7 (EGFL7) in rats deep partial burn wound. Methods Ninety-six male c57bl/6 rats were randomized into four groups (24 rats in each group), each of which was given one of burn wound with 2 cm in diameter on the back. After Saline solution, SD-Ag, blank bases, aloes burn cream applied on each wound, respectively. Tissue wound healing were observed at days 7, 10 and 14 by hematoxylin and eosin (HE) staining; TGF-β1 and EGFL7 level in wound tissues were detected at days 3, 7, 10 and 14 by enzyme-linked immuno sorbent assay (ELISA). Results Aloes burn cream can effectively restore the epithelization and promote the angiogenesis of burn wound; At days 3 and 7, TGF-β1 level in wound tissue of aloes burn cream group reach to (22.72±1.05), (31.58±3.91) μg/g and EGFL7 level reach to (99.67±3.33), (161.60±12.74) μg/g, which are significantly higher than that in control group, respectively (P<0.05). At days 10 post burn, EGFL7 level in wound tissue of aloes burn cream group maintain at (148.25±15.33) μg/g, which is significantly higher than that in SD-Ag gand blank bases groups (P<0.05). Conclusion Aloes burn cream increasing synthesis and release of TGF-β 1and EGFL7 and acceleate wound healing. Key words: Aloes burn cream; Burn; Transforming growth factor-β1; Epidermal growth factor-like domain 7

Research progress of aspartyl-(asparaginyl) β-hydroxylase in tumors

Aspartyl-(asparaginyl) β-hydroxylase (AAH) is a type 2 transmembrane protein and a member of the α-ketoglutarate-dependent dioxygenase family of proteins. AAH catalyzes hydroxylation of specific aspartyl and asparaginyl residues in epidermal growth factor-like domains of proteins. Overexpression of AAH is found in many types of cancer tissues, however, AAH expression is found only in normal placenta and paranephros tissues. AAH can translocate to cell membrane from endoplasmic reticulum membrane when it is overexpressed in cancer cell, which hydroxylate proteins such as Notch, Jagged, and cellular adhesion molecules that regulate cell migration and invasion. AAH is a potential biomarker for cancer diagnosis. Key words: Aspartyl-(asparaginyl) β-hydroxylase; Neoplasms; Diagnosis

Epidermal growth factor-like domain 7 promotes endothelial cell migration and angiogenesis by activating ERK signaling pathway

To explore the effect of epidermal growth factor-like domain 7(EGFL7) on the migration and angiogenesis of endothelial cells. EGFL7 overexpression vectors were constructed and transfected into human microvascular endothelial cells. The expression levels of EGFL7-mRNA and EGFL7 protein were examined by real-time RT-PCR and Western blot. Cell migration was analyzed by the wound healing. The capability of cell to form capillary-like tubes in vitro was evaluated on matrigel assay. Protein expression of p-AKT, AKT, p-ERK and ERK in endothelial cells was detected by Western blot upon transfection with EGFL7 overexpression vectors and vehicle control for 0, 10, 30 and 60 min. Migration and angiogenesis of endothelial cells were notably enhanced by EGFL7 overexpression. ERK pathway was strongly activated by EGFL7, whereas AKT remained constant in endothelial cells. Inhibition of ERK impaired EGFL7 induced ERK activation and endothelial cell migration and angiogenesis. EGFL7 effectively promotes migration and angiogenesis through ERK signaling pathway in endothelial cells.

The Fifth Region of Epidermal Growth Factor like-Domain of Thrombomodulin Is Essential to Produce Cytoprotection

We previously found that the fourth and fifth regions of epidermal growth factor-like domain of thrombomodulin (TME45) protects vascular endothelial cells from calcineurin inhibitor-induced cell damage in association with activation of extracellular signal-regulated kinase (ERK)/myeloid cell leukemia-1 (Mcl-1) signal pathways via activated protein C (APC) independent manner (Arterioscler Thromb Vasc Biol. 2012;32:2259-70). The present study attempted to identify the minimum region of TME45 that is essential to mediate the cytoprotective function of thrombomodulin. The blocking antibody for TME5 but not for TME4 hampered the ability of TME45 to increase levels of Mcl-1 in human umbilical vein endothelial cells (HUVECs), indicating the important roles of TME5 in cytoprotection. We next divided TME5 into three parts and synthesized oligonucleotides for each region. None of the fragment was as potent as TME5 to induce Mcl-1 in HUVECs, suggesting that TME5 was essential for induction of Mcl-1. Importantly, TME5 was not able to produce APC. Furthermore, TME5 counteracted calcineurin inhibitor-induced vascular permeability and stimulated angiogenesis in vitro as well as in vivo. Taken together, TME5 may be useful to prevent or treat endothelial cell damage associated lethal complications developing after hematopoietic stem cell transplantation such as sinusoidal obstruction syndrome and thrombotic microangiopathy. DisclosuresHonda:Asahi Kasei Pharma: Employment.

Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

A Novel Cysteine-Sparing NOTCH3 Mutation in a Chinese Family with CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset cerebral small vessel disorder caused by the mutations of the neurogenic locus notch homolog protein 3 (NOTCH3) gene. The extracellular part of NOTCH3 is composed of 34 epidermal growth factor-like (EGF-like) repeat domains. Each EGF-like domain is rich of cysteine and glycine to produce three loops that are essential for high-affinity binding to its ligand. Nearly all reported CADASIL-associated mutations result in gain or loss of a cysteine residue within the EGF-like domains. Only a few cysteine-sparing NOTCH3 mutations have been documented in the patients with CADASIL to date. Here, we reported a Chinese CADASIL family with a cysteine-sparing NOTCH3 mutation. In this family, affected patients had dizziness, memory loss, gait instability, or hemiplegia. Brain magnetic resonance imaging (MRI) showed diffuse leukoencephalopathy with confluent signal abnormalities in the periventricular white matter, basal ganglia, and centrum semiovale bilaterally. By screening the entire coding region of NOTCH3, a novel missense mutation p.G149V (c.446G>T) was found. This mutation was not detected in 400 normal controls. Considering the critical position of glycine within the C-loop of EGF-like domain and its high conservation through evolution, p.G149V mutation could be a potential pathogenic cause for CADASIL.

APP Homodimers Transduce an Amyloid-β-Mediated Increase in Release Probability at Excitatory Synapses

Accumulation of amyloid-β peptides (Aβ), the proteolytic products of the amyloid precursor protein (APP), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer's disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. Here, we show that Aβ40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Aβ40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/Gi/o-protein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Aβ40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer's disease.

Abstract 2622: TMEFF2 inhibits human prostate cancer cell migration through its G protein activating domain.

Abstract The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed mainly in brain and prostate. Expression of TMEFF2 is deregulated in a significant fraction of primary and metastatic prostate, suggesting a role in this disease. In fact, we have previously shown that TMEFF2 can function as a tumor suppressor in vitro, inhibiting monolayer and anchorage-independent cellular growth in HEK293T cells and sarcosine-induced cell migration and invasion in benign prostate epithelial RWPE-1 cells. However, the molecular mechanisms involved in the tumor suppressor phenotype of TMEFF2 are not clear. TMEFF2 has several biologically important features. The extracellular domain contains two follistatin domains and an epidermal growth factor-like (EGF) domain, while the transmembrane domain and short cytoplasmic tail have features resembling a G protein activating domain, suggesting that it may signal through a G protein. We hypothesize that the tumor suppressor activity of TMEFF2 is, at least in part, due to signaling mediated by the putative G protein activating domain. In order to test this hypothesis, here we investigate the role of TMEFF2 and a mutant lacking the cytoplasmic tail on cellular migration of prostate cancer cells, and begin analyzing the molecular mechanisms involved in this effect. Our results demonstrate that overexpression of TMEFF2 in human prostate cancer cell line RWPE-2 causes about 60% decrease in cellular migration as assessed by wound-healing assays. Interestingly, we found that RWPE-2 cells overexpressing TMEFF2 have a defect in cell spreading on culture dishes (∼20% decrease) as well as on vitronectin-coated coverslips (∼30% decrease) and that this defect is accompanied by abolished focal adhesion and stress fiber formation. Moreover, the inhibition of TMEFF2 on cellular migration is dependent on its G protein activating domain as deletion of this domain rescues migration, spreading on vitronectin, and focal adhesion formation. The specific G protein pathway(s) involved in the anti-migratory effect of TMEFF2 is currently under investigation. In summary, the data presented here indicate, for the first time, that the anti-migratory effect of TMEFF2 in human prostate cancer cells is mediated by its G protein activating domain. These results raise the possibility that TMEFF2 agonists may be used as therapeutic agents to mediate tumor suppression via this pathway. Citation Format: Xiaofei Chen, C. Justus, L Yang, M.j. Ruiz-Echevarria. TMEFF2 inhibits human prostate cancer cell migration through its G protein activating domain. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2622. doi:10.1158/1538-7445.AM2013-2622

Double heterozygous mutations Gln100Leu and His348Gln of the F7 gene in a patient with factor VII deficiency

A 25-year-old Chinese woman who had a history of easy bruising was admitted to hospital due to uncontrolled epistaxis. She showed factor VII activity level of 2% and factor VII antigen level of 4% of the normal value. We detected a novel missense mutation g.8355 A>T (p.Gln100Leu) in the second epidermal growth factor-like (EGF) domain and a g.11482 T>G (p.His348Gln) in the catalytic domain. Although the Gln100 residue is close to the junction of EGF-2 domain with the serine protease domain, we infer that the substitution of polar negatively charged Gln residue at the position 100 with introduction of nonpolar Leu residue may be likely to perturb proper folding, resulting in decreasing factor VII activity.

Soluble amyloid-β precursor protein binds its cell surface receptor in a cooperative fashion with glypican and syndecan proteoglycans

Proteolytic processing of amyloid-β precursor protein (APP) generates the amyloid-β peptide, which plays a central role in Alzheimer disease. The physiological function of APP and its proteolytic fragments, however, remains barely understood. Here we show that, on the basis of its binding characteristics, the secreted ectodomain of APP (sAPP) is a new member of the heparin-binding growth factor superfamily. Like other of its members, sAPP binds in a bivalent manner to the plasma membrane with two different subdomains. The N-terminal growth-factor-like domain (GFLD) is necessary and sufficient for protein-receptor binding, whereas the E2-domain mediates interaction with membrane-anchored heparan sulfate proteoglycans (HSPGs). The membrane-anchored HSPGs function as low-affinity co-receptors for sAPP and enhance the affinity to the sAPP receptor. Our findings provide a solid basis for the further identification of this receptor.

Potential Role of Kringle-Integrin Interaction in Plasmin and uPA Actions (A Hypothesis)

We previously showed that the kringle domains of plasmin and angiostatin, the N-terminal four kringles (K1–4) of plasminogen, directly bind to integrins. Angiostatin blocks tumor-mediated angiogenesis and has great therapeutic potential. Angiostatin binding to integrins may be related to the antiinflammatory action of angiostatin. We reported that plasmin induces signals through protease-activated receptor (PAR-1), and plasmin-integrin interaction may be required for enhancing plasmin concentration on the cell surface, and enhances its signaling function. Angiostatin binding to integrin does not seem to induce proliferative signals. One possible mechanism of angiostatin's inhibitory action is that angiostatin suppresses plasmin-induced PAR-1 activation by competing with plasmin for binding to integrins. Interestingly, plasminogen did not interact with αvβ3, suggesting that the αvβ3-binding sites in the kringle domains of plasminogen are cryptic. The kringle domain of urokinase-type plasminogen activator (uPA) also binds to integrins. The uPA-integrin interaction enhances uPA concentrations on the cell surface and enhances plasminogen activation on the cell surface. It is likely that integrins bind to the kringle domain, and uPAR binds to the growth factor-like domain (GFD) of uPA simultaneously, making the uPAR-uPA-integrin ternary complex. We present a docking model of the ternary complex.

Regulation of Cell Migration and Invasion by Specific Modules of uPA: Mechanistic Insights and Specific Inhibitors

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

Abstract 2122: Pleiotropic effects of TMEFF2 variants in tumorigenesis

Abstract TMEFF2, a gene with an expression profile limited to brain and prostate, contains several biologically important signatures. It encodes a transmembrane protein with a short cytoplasmic tail containing a potential G protein activating domain. The extracellular domain (ectodomain) is shed in an ADAM17/γ-secretase dependent fashion and consists of two follistatin (FS) modules and an epidermal growth factor-like (EGF) domain. A critical role for TMEFF2 in tumorigenesis is suggested by the fact that it is upregulated in a significant fraction of primary and metastatic prostate tumors; however, its role in this disease is still unknown. TMEFF2 has been suggested to function as a tumor suppressor, while the ectodomain has been shown to promote growth. The discrepancies likely arise from the complexity of TMEFF2 function. TMEFF2 could signal as a ligand, a membrane bound receptor and/or a binding protein for growth factors. In addition, several isoforms have been described that differ in their C-terminus and probably in their ability to signal and/or being modified. One of these isoforms constitutes a soluble form of the protein similar to the ectodomain. In the present study we investigate the possibility that different forms of TMEFF2 may have different roles in tumorigenesis. Our data indicate that constitutive or induced overexpression of TMEFF2 in HEK293T or prostate epithelial RWPE1 cells reduces cell proliferation by 20-30%, inhibits anchorage independent growth by 5 fold, sensitizes the cells to apoptotic stimuli by 2-3 fold and/or decreases cell migration and invasion by 2 fold, suggesting that TMEFF2 functions as a tumor suppressor. Conversely, overexpression of the ectodomain or an isoform of TMEFF2 that lacks the G protein activating domain (TMEFF2-ΔGA), results in a tumorigenic phenotype, i.e. increase in cell proliferation, anchorage-independent growth, cellular migration and/or invasion. In all, these results suggest functionally different roles for different TMEFF2 variants. The role of the G protein activating domain is currently being investigated. Importantly, we demonstrate that there is an association between the ability of these TMEFF2 variants to bind to SARDH and modulate sarcosine levels and their ability to suppress tumorigenesis. Using co-immunoprecipitation studies and sarcosine levels analysis, we show that the full length tumor suppressor form of TMEFF2 binds to SARDH and modulates sarcosine levels. In contrast, expression of TMEFF2-ΔGA or the ectodomain fails to modulate sarcosine levels or bind to SARDH and in both cases they exhibit tumor promoting phenotypes. This suggests that the function of TMEFF2 may rely, at least in part, on its ability to modulate sarcosine metabolism. Differential expression of these isoforms provides a potential mechanism for the pleiotropic effects of TMEFF2 during prostate cancer development and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2122. doi:10.1158/1538-7445.AM2011-2122

Maternal plasma and amniotic fluid angiogenic factors and their receptors in monochorionic twin pregnancies complicated by twin‐to‐twin transfusion syndrome

Angiogenic factors play a role in human placentation and may be aberrant in severe twin-to-twin transfusion syndrome (TTTS). The aim of this study was to investigate the maternal plasma and amniotic fluid angiogenic factor and receptor concentrations in twin pregnancies complicated by TTTS and to evaluate the effects of fetoscopic laser ablation. A prospective cohort of monochorionic (MC) twins complicated by severe TTTS (n = 23) was studied between October 2006 and December 2007. A cohort of uncomplicated dichorionic (DC) (n = 12) and MC (n = 7) pregnancies were studied for comparison. Circulating angiogenic factors and their receptors were measured in the maternal plasma and the recipient twin's amniotic fluid by enzyme-linked immunosorbent assay and/or FAST Quant human angiogenesis array. Plasma vascular endothelial growth factor (VEGF)-C concentrations were significantly lower in TTTS than in uncomplicated twin pregnancies (P < 0.0001). In contrast, plasma angiopoietin (Ang)-2 levels and the ratio of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) to placental growth factor (PlGF) levels were significantly increased in TTTS (P < 0.01). Plasma VEGF-D was significantly increased in advanced stage TTTS (Stage III/IV cohort; P < 0.01). This was independent of fetal size, amniotic fluid volumes or the number of apparent placental arteriovenous anastomoses. In TTTS pregnancies, amniotic fluid VEGF-C, VEGF-A, Ang-1 and the sVEGFR-1/PlGF ratio were increased compared to paired maternal plasma concentrations (P < 0.0001) while amniotic fluid concentrations of PlGF, Ang-2 and soluble tyrosine kinase with immunoglobulin-like/epidermal growth factor-like domains 2 (sTie-2) were significantly lower than plasma concentrations (P < 0.0001). No significant association between maternal plasma and amniotic fluid concentrations of angiogenic factors was noted. Plasma PlGF was transiently decreased after fetoscopic laser ablation, returning to baseline by 1 week (P = 0.0314). Fetoscopic laser ablation also affected plasma sVEGFR-1/PlGF ratio with a transient increase after therapy, followed by a significant reduction to below basal concentrations by 1 week (P = 0.0102). Only VEGF-D was significantly different (+8.3%; P = 0.0155) in amniotic fluid immediately after the completion of fetoscopic laser ablation. Maternal angiogenic activity is decreased in severe TTTS, with an increased sVEGFR-1/PlGF ratio and concentrations of Ang-2 and VEGF-D in the maternal plasma compared to uncomplicated MC twin pregnancies. Maternal circulating PlGF concentrations decrease and the sVEGFR-1/PlGF ratio increases transiently in response to fetoscopic laser ablation, but in general the angiogenic factor and receptor concentrations studied are altered little by this therapy.