Ute Reuning

A novel method for the in vivo isolation of circulating tumor cells from peripheral blood of cancer patients using a functionalized and structured medical wire

The isolation of circulating tumor cells (CTCs) from the blood of patients afflicted with solid malignant tumors becomes increasingly important as it may serve as a ‘liquid biopsy’ with the potential of monitoring the course of the cancer disease and its response to cancer therapy, with subsequent molecular characterization. For this purpose, we functionalized a structured medical Seldinger guidewire (FSMW), normally used to obtain safe access to blood vessels and other organ cavities, with a chimeric monoclonal antibody directed to the cell surface expressed epithelial cell surface adhesion molecule (EpCAM). This medical device was optimized in vitro and its biocompatibility was tested according to the regulations for medical devices and found to be safe with no noteworthy side effects. Suitability, specificity and sensitivity of the FSMW to catch and enrich CTCs in vivo from circulating peripheral blood were tested in 24 breast cancer or non-small cell lung cancer (NSCLC) patients and in 29 healthy volunteers. For this, the FSMW was inserted through a standard venous cannula into the cubital veins of healthy volunteers or cancer patients for the duration of 30 min. After removal, CTCs were identified by immunocytochemical staining of EpCAM and/or cytokeratins and staining of their nuclei and counted. The FSMW successfully enriched EpCAM-positive CTCs from 22 of the 24 patients, with a median of 5.5 (0–50) CTCs in breast cancer (n=12) and 16 (2–515) CTCs in NSCLC (n=12). CTCs could be isolated across all tumor stages, including early stage cancer, in which distant metastases were not yet diagnosed, while no CTCs could be detected in healthy volunteers. In this observatory study, no adverse effects were noted. Evidently, the FSMW has the potential to become an important device to enrich CTCs in vivo for monitoring the course of the cancer disease and the efficacy of anticancer treatment.

Prognostic significance of urokinase (uPA) and its inhibitor PAI-1 for survival in advanced ovarian carcinoma stage FIGO IIIc

SummaryStrong evidence has accumulated on the prognostic value of tumour-associated proteolytic factors in patients afflicted with solid malignant tumours, including advanced ovarian cancer. We evaluated the prognostic impact of the protease urokinase plasminogen activator (uPA) and its inhibitor PAI-1 on overall survival in patients with advanced ovarian cancer stage FIGO IIIc in order to select patients at risk. uPA and PAI-1 antigen were determined by ELISA in primary tumour tissue extracts of 86 ovarian cancer patients FIGO stage IIIc enrolled in a prospective study. Univariate and multivariate analyses were performed using the Cox proportional hazard model. The time-varying coefficient model of Gray was used to assess the time-dependent strength of prognostic factors tumour mass, uPA and PAI-1 on overall survival. In all patients, uPA and PAI-1 (optimized cut-offs of 2.0 and 27.5 ng mg-1 protein respectively), in addition to the traditional prognostic parameters of residual tumour mass, nodal status, grading and ascites volume, were of prognostic significance in univariate analysis for overall survival. Even in patients with residual tumour mass (n = 43), the statistically independent prognostic impact of PAI-1 persisted, allowing further discrimination between low- and high-risk patients. In multivariate analysis, residual tumour mass (P < 0.001, relative risk (RR) 4.5), PAI-1 (P < 0.001; RR 3.1) and nodal status (P = 0.022, RR 2.6) turned out to be strong, statistically independent prognostic parameters. Evaluation of the time-dependent prognostic impact of residual tumour mass and PAI-1 on overall survival (n = 86, 50 months) revealed that the prognostic power of these factors increased with time. In patients with advanced ovarian cancer, both residual tumour mass and PAI-1 are statistically independent strong prognostic factors. Even within patient subgroups with or without residual tumour mass, PAI-1 allowed selection of patients at risk who might benefit from individualized therapy protocols.

Ovarian cancer cell proliferation and motility is induced by engagement of integrin alpha(v)beta3/Vitronectin interaction.

Abstract During tumor metastasis, a fine-tuned balance between the formation and loosening of adhesive cell contacts has to occur, a process based on the regulated expression of integrins. Human ovarian OV-MZ-6 cancer cells express the integrin αvβ3, which associates with vitronectin (VN) and correlates with ovarian cancer progression. Adhesion and spreading of OV-MZ-6 cells on VN was accompanied by the formation of focal adhesion contacts and the recruitment of activated tyrosine-phosphorylated focal adhesion kinase. Cultivation of OV-MZ-6 cells on VN resulted in a significantly induced cell proliferation. This VN effect could be mimicked by cultivating cells on the immobilized αvβ3-directed peptide cyclo-Arg-Gly-Asp-D-Phe-Val (cRGDfV). VN-dependent OV-MZ-6 cell adhesion and proliferation was significantly enhanced by overexpression of αvβ3, and was accompanied by rapid and transient tyrosinephosphorylation of p44erk 1/p42erk 2 mitogen-activated protein kinase. Moreover, overexpression of αvβ3 and OV-MZ-6 cell attachment to VN increased cell motility up to 5-fold accompanied by prominent changes in cytoskeletal organization and cell morphology. Upon αvβ3/VN interaction, by cDNA expression microarray analysis we identified altered mRNA levels of cmyc, epidermal growth factor receptor (EGF-R), transcription factor Fra-1, prothymosin-α (PTMA), integrin-linked kinase (ILK), and the cell adhesion molecule SQM-1, candidates which are possibly involved in changes of the adhesive, migratory, and proliferative phenotype of human ovarian cancer cells.

Urokinase-type Plasminogen Activator (uPA) and its Receptor (uPAR): Development of Antagonists of uPA / uPAR Interaction and their Effects In Vitro and In Vivo

In cancer, increased levels of the tumor-associated serine protease uPA (urokinase-type plasminogen activator) and its receptor uPAR (CD87) are linked to tumor progression, metastasis, and shortened survival in patients afflicted with this disease. Strong clinical and experimental evidence has accumulated that the cell surface interaction of uPA with uPAR facilitates extravasation and intravasation of cancer cells by regulating local proteolysis and attachment of the cells to components of the extracellular matrix. Moreover, the uPA/uPAR system is also implicated in proliferation of some tumor cells and migration of tumor and endothelial cells. Thus, metastasis formation is facilitated via tumor cell spread through the blood circulation system and neovascularization at the metastatic site. This multifunctional potential has rendered the uPA/uPAR system an attractive novel target for anti-metastatic therapy. Consequently, inhibitors of the uPA/uPAR interaction have been and are currently developed for suppression of tumor growth and angiogenesis. In addition to antibodies and recombinant uPA- or uPAR-derived proteins, various linear and cyclic peptides as well as small molecules have been designed and synthesized which potently interfere with the uPA/uPAR interaction, leading to reduced tumor progression in experimental animals. Such compounds affecting the uPA/uPAR system represent novel tumor biology-based therapeutic agents, thereby opening new ways for patient optimized and individualized cancer therapy.

Molecular and functional interdependence of the urokinase-type plasminogen activator system with integrins.

Abstract The serine protease urokinase-type plasminogen activator (uPA), its inhibitor PAI-1, and its cellular receptor uPA-R (CD87) are of crucial importance during cellular invasion and migration, required for a variety of physio- and pathophysiological processes. It has become increasingly evident in recent years that the uPA/uPA-R-system has far more functional properties than plasminogen activation alone. This is reflected by its involvement in cellular events such as proliferation, adhesion, migration, and chemotaxis. Since uPA-R lacks a transmembrane domain and thus on its own is not capable of transmitting signals into cells, association and functional cooperation with other signaling molecules/receptors is needed. In this respect, one group of adhesion and signaling receptors, the integrins, have been identified which constitute, together with the uPA/uPA-R-system, an interdependent biological network by which the uPA/uPA-R-system broadly affects integrin functions and vice versa. Moreover, there is a growing body of evidence that cellular uPA, uPA-R, and PAI-1 expression is under control of specific ECM/integrin interactions and also that integrins are regulated by components of the uPA/uPARsystem. By this multifaceted crosstalk, cells may modulate their proteolytic, adhesive, and migratory activities and monitor ECM integrity in their microenvironment.

Urokinase induces proliferation of human ovarian cancer cells: characterization of structural elements required for growth factor function

Ovarian cancer metastasis is associated with an increase in the urokinase‐type plasminogen activator (uPA) and its receptor uPAR. We present evidence that binding of uPA to uPAR provokes a mitogenic response in the human ovarian cancer cell line OV‐MZ‐6 in which endogenous uPA production had been significantly reduced by stable uPA ‘antisense’ transfection. High molecular weight (HMW) uPA, independent of its enzymatic activity, produced an up to 95% increase in cell number concomitant with 2‐fold elevated [3H]thymidine incorporation as did the catalytically inactive but uPAR binding amino‐terminal fragment of uPA, ATF. uPA‐induced cell proliferation was significantly decreased by blocking uPA/uPAR interaction by the monoclonal antibody IIIF10 and by soluble uPAR. The efficiency of the uPAR binding synthetic peptide cyclo19,31uPA19–31 to enhance OV‐MZ‐6 cell growth proved this molecular domain to be the minimal structural determinant for uPA mitogenic activity. Dependence of uPA‐provoked cell proliferation on uPAR was further demonstrated in Raji cells which do not express uPAR and were thus not induced by uPA. However, upon transfection with full‐length uPAR, Raji cells acquired a significant growth response to HMW uPA and ATF.

Inhibition of the Interaction of Urokinase-Type Plasminogen Activator (uPA) with Its Receptor (uPAR) by Synthetic Peptides

Focusing of the serine protease urokinase-type plasminogen activator (uPA) to the cell surface via interaction with its specific receptor (uPAR, CD87) is an important step for tumor cell invasion and metastasis. The ability of a synthetic peptide derived from the uPAR-binding region of uPA (comprising amino acids 16-32 of uPA; uPA(16-32)) to inhibit binding of fluorescently labeled uPA to uPAR on human promyeloid U937 cells was assessed by quantitative flow cytofluorometric analysis (FACS) and compared to the inhibitory capacities of other synthetic peptides known to interfere with uPA/uPAR-interaction. An about 3000-fold molar excess of uPA(16-32) resulted in 50% inhibition of pro-uPA binding to cell surface-associated uPAR. Using a solid-phase uPA-ligand binding assay employing recombinant soluble uPAR coated to microtiter plates, the minimal binding region of wild-type uPA was determined. The linear peptide uPA(19-31) and its more stable disulfide-bridged cyclic form (cyclo(19,31)uPA(19-31)) displayed uPAR-binding activity whereas other peptides such as uPA(18-30), uPA(20-32) or uPA(20-30) did not react with uPAR. Cyclic peptide derivatives of cyclo(19,31)uPA(19-31) in which certain amino acids were deleted and/or replaced by other amino acids as well as uPAR-derived wild-type peptides did also not inhibit uPA/uPAR-interaction. Therefore, the present investigations identified cyclo(19,31)uPA(19-31) as a potential lead structure for the development of uPA-peptide analogues to block uPA/uPAR-interaction.

beta(3)A-integrin downregulates the urokinase-type plasminogen activator receptor (u-PAR) through a PEA3/ets transcriptional silencing element in the u-PAR promoter

ABSTRACT Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that β3-integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition β3-integrin regulates u-PAR expression. Overexpression of β3-integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (−1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced β3-integrin expression. A region between −398 and −197 bp of the u-PAR promoter was critical for β3-integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at −248 bp substantially impaired the ability of β3-integrin to downregulate the u-PAR promoter, suggesting that thePEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when β3-integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the β3A-integrin isoform but not by other β3-integrin isoforms and required the cytoplasmic membrane NITY759 motif. Moreover, overexpression of the short but not the long isoform of the β3-integrin adapter protein β3-endonexin blocked u-PAR promoter activity through the PEA3/etsbinding site. Thus, besides the physical interaction of β3-integrin and u-PAR at the cell surface, β3 signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

Clinical utility of level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1

The prognostic and/or predictive value of the cancer biomarkers, urokinase-type plasminogen activator (uPA) and its inhibitor (plasminogen activator inhibitor [PAI]-1), determined by ELISA in tumor-tissue extracts, was demonstrated for several cancer types in numerous clinically relevant retrospective or prospective studies, including a multicenter breast cancer therapy trial (Chemo-N0). Consequently, for the first time ever for any cancer biomarker for breast cancer, uPA and PAI-1 have reached the highest level of evidence, level-of-evidence-1. At present, two other breast cancer therapy trials, NNBC-3 and Plan B, also incorporating uPA and PAI-1 as treatment-assignment tools are in effect. Furthermore, small synthetic molecules targeting uPA are currently in Phase II clinical trials in patients afflicted with advanced cancer of the ovary, breast or pancreas.

Tissue inhibitor of metalloproteinases-1 induces a pro-tumourigenic increase of miR-210 in lung adenocarcinoma cells and their exosomes

Tissue inhibitor of metalloproteinases-1 (TIMP-1) recently emerged as a pro-metastatic factor highly associated with poor prognosis in a number of cancers. This correlation seemed paradox as TIMP-1 is best described as an inhibitor of pro-tumourigenic matrix metalloproteinases. Only recently, TIMP-1 has been revealed as a signalling molecule that can regulate cancer progression independent of its inhibitory properties. In the present study, we demonstrate that an increase of both exogenous and endogenous TIMP-1 led to the upregulation of miR-210 in a CD63/PI3K/AKT/HIF-1-dependent pathway in lung adenocarcinoma cells. TIMP-1 induced P110/P85 PI3K-signalling and AKT phosphorylation. It also led to increase of HIF-1α protein levels positively correlating with HIF-1-regulated mRNA expression and upregulation of the microRNA miR-210. Downstream targets of miR-210, namely FGFRL1, E2F3, VMP-1, RAD52 and SDHD, were decreased in the presence of TIMP-1. Upon the overexpression of TIMP-1 in tumour cells, miR-210 was accumulated in exosomes in vitro and in vivo. These exosomes promoted tube formation activity in human umbilical vein endothelial cell (HUVECs), which was reflected in increased angiogenesis in A549L-derived tumour xenografts. Activation and elevation of PI3K, AKT, HIF-1A and miR-210 in tumours additionally confirmed our in vitro data. This new pro-tumourigenic signalling function of TIMP-1 may explain why elevated TIMP-1 levels in lung cancer patients are highly correlated with poor prognosis.