Publications by authors named "Lucia Napione"

17 Publications

  • Page 1 of 1

Pazopanib and Trametinib as a Synergistic Strategy against Osteosarcoma: Preclinical Activity and Molecular Insights.

Cancers (Basel) 2020 Jun 10;12(6). Epub 2020 Jun 10.

Division of Medical Oncology, Candiolo Cancer Institute, FPO-IRCCS, Str. Prov. 142 km 3.95, 10060 Candiolo (TO), Italy.

Receptor tyrosine kinases (RTKs) inhibitors' activity in advanced osteosarcoma is significant but short-lived. To prevent or at least delay drug resistance, we explored a vertical inhibition by combining drugs acting at different levels of the RTK pathways (pazopanib + trametinib). We studied pazopanib + trametinib antitumor activity both in vitro and in vivo (MNNG-HOS and KHOS xenografts in NOD/SCID mice) investigating the molecular mechanisms and potential escapes. The involvement of MAPK-PI3K pathways was validated by Nanostring technology, western blot and by silencing/overexpression experiments. Pazopanib targets were expressed on seven osteosarcoma cell lines and their pathways were activated. Pazopanib + trametinib exhibited synergistic antitumor activity by inducing apoptosis and inhibiting ERK1/2 and Akt. In vivo antitumor activity was shown in osteosarcoma-bearing mice. The drug combination significantly down-modulated RTK Ephrin Type-A Receptor 2 (EphA2) and Interleukin-7 Receptor (IL-7R), whereas induced mitogen-activated protein-kinase kinase (MAPKK) MEK6. EphA2 silencing significantly reduced osteosarcoma cell proliferation and migration, while impeding MEK6 up-regulation in the treated cells significantly increased the antitumor effect of the studied drugs. Moreover, the up-regulation of MEK6 reduced combination activity. Pazopanib + trametinib demonstrated synergistic antitumor effects in osteosarcoma models through ERK and Akt inhibition and EphA2 and IL-7R down-modulation. MEK6 up-regulation might evoke escaping mechanism.
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http://dx.doi.org/10.3390/cancers12061519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352822PMC
June 2020

SerpinB3 Differently Up-Regulates Hypoxia Inducible Factors -1α and -2α in Hepatocellular Carcinoma: Mechanisms Revealing Novel Potential Therapeutic Targets.

Cancers (Basel) 2019 Dec 4;11(12). Epub 2019 Dec 4.

Department of Clinical and Biological Sciences, Unit of Experimental Medicine & Clinical Pathology, University of Torino, 10125 Torino, Italy.

Background: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2α-dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1α and HIF-2α. SB3 acts by (i) up-regulating HIF-1α transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2α stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2α transcripts; (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2α transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2α transcripts. Conclusions These data outline either HIF-2α and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.
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http://dx.doi.org/10.3390/cancers11121933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966556PMC
December 2019

Bloch surface wave enhanced biosensor for the direct detection of Angiopoietin-2 tumor biomarker in human plasma.

Biomed Opt Express 2018 Feb 8;9(2):529-542. Epub 2018 Jan 8.

Department of Basic and Applied Science for Engineering, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy.

Quantitative detection of angiogenic biomarkers provides a powerful tool to diagnose cancers in early stages and to follow its progression during therapy. Conventional tests require trained personnel, dedicated laboratory equipment and are generally time-consuming. Herein, we propose our developed biosensing platform as a useful tool for a rapid determination of Angiopoietin-2 biomarker directly from patient plasma within 30 minutes, without any sample preparation or dilution. Bloch surface waves supported by one dimensional photonic crystal are exploited to enhance and redirect the fluorescence arising from a sandwich immunoassay that involves Angiopoietin-2. The sensing units consist of disposable and low-cost plastic biochips coated with the photonic crystal. The biosensing platform is demonstrated to detect Angiopoietin-2 in plasma samples at the clinically relevant concentration of 6 ng/mL, with an estimated limit of detection of approximately 1 ng/mL. This is the first Bloch surface wave based assay capable of detecting relevant concentrations of an angiogenic factor in plasma samples. The results obtained by the developed biosensing platform are in close agreement with enzyme-linked immunosorbent assays, demonstrating a good accuracy, and their repeatability showed acceptable relative variations.
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http://dx.doi.org/10.1364/BOE.9.000529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854056PMC
February 2018

PARP1 expression drives the synergistic antitumor activity of trabectedin and PARP1 inhibitors in sarcoma preclinical models.

Mol Cancer 2017 04 28;16(1):86. Epub 2017 Apr 28.

Sarcoma Unit, Medical Oncology, Candiolo Cancer Institute - FPO, IRCCS, Candiolo, Torino, Italy.

Background: Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death.

Methods: We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role.

Results: Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing.

Conclusions: PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.
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http://dx.doi.org/10.1186/s12943-017-0652-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410089PMC
April 2017

SPAD aptasensor for the detection of circulating protein biomarkers.

Biosens Bioelectron 2015 Jun 19;68:500-507. Epub 2015 Jan 19.

Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, 38123 Povo (Trento), Italy.

The need for decentralized clinical tests together with the concept of time and cost saving are pushing the development of portable, miniaturized, compact biosensors with diagnostic and prognostic purpose. Here, we propose an innovative detection system based on a Single Photon Avalanche Diode (SPAD) with high sensitivity and low noise, crucial features for an efficient chemiluminescence biosensor. The SPAD detector, having 60 µm diameter, has a Photon Detection Efficiency higher than 55% at 425 nm and a Dark Count Rate lower than 100 Hz at room temperature. Our design allows a good optical coupling efficiency between sample and detector. A specific biofunctional surface was implemented taking advantage of aptamers, short DNA sequences having high selectivity and affinity toward their targets. We successfully detected physiological levels of Vascular Endothelial Growth Factor (VEGF), a circulating protein biomarker highly correlated with cancer. The SPAD aptasensor showed a Limit of Detection (LoD) in the pM range, stability (up to 42 days) and re-usability (up to seven cycles). This compact biosensor is therefore a promising step toward the actual use of portable microdevices in diagnostics.
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http://dx.doi.org/10.1016/j.bios.2015.01.042DOI Listing
June 2015

A fluorescent one-dimensional photonic crystal for label-free biosensing based on BLOCH surface waves.

Sensors (Basel) 2013 Feb 5;13(2):2011-22. Epub 2013 Feb 5.

Dipartimento di Scienza Applicata e Tecnologia, Politecnico di Torino, corso Duca degli Abruzzi 24, Turin, Italy.

A one-dimensional photonic crystal (1DPC) based on a planar stack of dielectric layers is used as an optical transducer for biosensing, upon the coupling of TE-polarized Bloch Surface Waves (BSW). The structure is tailored with a polymeric layer providing a chemical functionality facilitating the covalent binding of orienting proteins needed for a subsequent grafting of antibodies in an immunoassay detection scheme. The polymeric layer is impregnated with Cy3 dye, in such a way that the photonic structure can exhibit an emissive behavior. The BSW-coupled fluorescence shift is used as a means for detecting refractive index variations occurring at the 1DPC surface, according to a label-free concept. The proposed working principle is successfully demonstrated in real-time tracking of protein G covalent binding on the 1DPC surface within a fluidic cell.
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http://dx.doi.org/10.3390/s130202011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3649429PMC
February 2013

IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism.

Angiogenesis 2012 Dec 15;15(4):713-25. Epub 2012 Jul 15.

Department of Oncological Sciences, Institute for Cancer Research and Treatment, University of Torino, 10060, Candiolo, Torino, Italy.

Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.
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http://dx.doi.org/10.1007/s10456-012-9286-9DOI Listing
December 2012

Unraveling the influence of endothelial cell density on VEGF-A signaling.

Blood 2012 Jun 17;119(23):5599-607. Epub 2012 Apr 17.

Institute for Cancer Research and Treatment, Torino, Italy.

Vascular endothelial growth factor-A (VEGF) is the master determinant for the activation of the angiogenic program leading to the formation of new blood vessels to sustain solid tumor growth and metastasis. VEGF specific binding to VEGF receptor-2 (VEGFR-2) triggers different signaling pathways, including phospholipase C-γ (PLC-γ) and Akt cascades, crucial for endothelial proliferation, permeability, and survival. By combining biologic experiments, theoretical insights, and mathematical modeling, we found that: (1) cell density influences VEGFR-2 protein level, as receptor number is 2-fold higher in long-confluent than in sparse cells; (2) cell density affects VEGFR-2 activation by reducing its affinity for VEGF in long-confluent cells; (3) despite reduced ligand-receptor affinity, high VEGF concentrations provide long-confluent cells with a larger amount of active receptors; (4) PLC-γ and Akt are not directly sensitive to cell density but simply transduce downstream the upstream difference in VEGFR-2 protein level and activation; and (5) the mathematical model correctly predicts the existence of at least one protein tyrosine phosphatase directly targeting PLC-γ and counteracting the receptor-mediated signal. Our data-based mathematical model quantitatively describes VEGF signaling in quiescent and angiogenic endothelium and is suitable to identify new molecular determinants and therapeutic targets.
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http://dx.doi.org/10.1182/blood-2011-11-390666DOI Listing
June 2012

Integration of microfluidic and cantilever technology for biosensing application in liquid environment.

Biosens Bioelectron 2010 Dec 5;26(4):1565-70. Epub 2010 Aug 5.

LATEMAR-Politecnico di Torino, Dipartimento di Scienza dei Materiali ed Ingegneria Chimica, Corso Duca degli Abruzzi 24, I-10129 Torino, Italy.

Microcantilever based oscillators have shown the possibility of highly sensitive label-free detection by allowing the transduction of a target mass into a resonant frequency shift. Most of such measurements were performed in air or vacuum environment, since immersion in liquid dramatically deteriorates the mechanical response of the sensor. Besides, the integration of microcantilever detection in a microfluidic platform appears a highly performing technological solution to exploit real time monitoring of biomolecular interactions, while limiting sample handling and promoting portability and automation of routine diagnostic tests (Point-Of-Care devices). In the present paper, we report on the realization and optimization of a microcantilever-based Lab-on-Chip, showing that microplates rather than microbeams exhibit largest mass sensitivity in liquid, while pirex rather than polymers represents the best choice for microfluidic channels. Maximum Q factor achieved was 140 (for fifth resonance mode of Pirex prototype), as our knowledge the highest value reported in literature for cantilever biosensors resonating in liquid environment without electronic feedback. Then, we proved the successfully detection of Angiopoietin-1 (a putative marker in tumor progression), showing that the related frequency shifts coming from non-specific interactions (negative controls) are roughly one order of magnitude lower than typical variations due to specific protein binding. Furthermore, we monitored the formation of antibody-antigen complex on MC surface in real-time. The proposed tool could be extremely useful for the comprehension of complex biological systems such as angiogenic machinery and cancer progression.
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http://dx.doi.org/10.1016/j.bios.2010.07.114DOI Listing
December 2010

Development of microcantilever-based biosensor array to detect Angiopoietin-1, a marker of tumor angiogenesis.

Biosens Bioelectron 2010 Jan 4;25(5):1193-8. Epub 2009 Nov 4.

LATEMAR-Politecnico di Torino, Dipartimento di Scienza dei Materiali ed Ingegneria Chimica, Torino, Italy.

Microcantilever biosensors have been proposed in the last years as very sensitive mass detectors, but few works focused on the precision and specificity of such tools. We measured the repeatability and reproducibility of our cantilever-based system, proponing the combination of results coming from both the first and second mode of vibration. Then, we optimized two biodesigns (a receptor-based and an antibody-based) to the detection of Angiopoietin-1, a possible marker in tumor progression. The reported results show that our microcantilever-based system can detect Angiopoietin-1 masses of the order of few hundreds of picograms with less than 0.5% of relative uncertainty. We showed that the evaluation of the protein surface density (number of molecules per cm(2)) could reveal interesting features concerning the multimerization state of the targeted protein. We also performed negative controls (dipping the sample in PBS without proteins) and specificity tests (dipping the sample in PBS with a "false" antigen). The related frequency shifts coming from non-specific interactions were found to be at least one order of magnitude lower than typical variations due to specific protein binding. Thanks to its fine precision and optimal specificity, our microcantilever-based system can be successfully applied as a quantitative tool for systems biology studies such as the comprehension of angiogenic machinery and cancer progression.
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http://dx.doi.org/10.1016/j.bios.2009.10.006DOI Listing
January 2010

Integrins team up with tyrosine kinase receptors and plexins to control angiogenesis.

Curr Opin Hematol 2008 May;15(3):235-42

Department of Oncological Sciences, Institute for Cancer Research and Treatment, University of Torino, Candiolo, Italy.

Purpose Of Review: Understanding the role of integrins in the formation of vascular bed is important for designing new therapeutic approaches to ameliorate or inhibit pathological vascularization. Besides regulating cell adhesion and migration, integrins dynamically participate in a network with soluble molecules and their receptors. This study summarizes recent progress in the understanding of the reciprocal interactions between integrins, tyrosine kinase, and semaphorin receptors.

Recent Findings: During angiogenic remodeling, endothelial cells that line blood vessel walls dynamically modify their integrin-mediated adhesive contacts with the surrounding extracellular matrix. During angiogenesis, opposing autocrine and paracrine loops of growth factors and semaphorins regulate endothelial integrin activation and function through tyrosine kinase receptors and the neuropilin/plexins system. Moreover, proangiogenic and antiangiogenic factors can directly bind integrins and regulate endothelial cell behavior. Studies describing these intense research areas are discussed.

Summary: Alteration in the balance between the angiogenic growth factors and semaphorins results in an impairment of integrin functions and could account for cardiovascular malformation and structural and functional abnormalities of the tumor vasculature.
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http://dx.doi.org/10.1097/MOH.0b013e3282fa745bDOI Listing
May 2008

Besides adhesion: new perspectives of integrin functions in angiogenesis.

Cardiovasc Res 2008 May 19;78(2):213-22. Epub 2008 Feb 19.

Department of Oncological Sciences, Institute for Cancer Research and Treatment, University of Torino, Sp 142, Km 3.95, 10060 Candiolo, Torino, Italy.

During angiogenic remodelling in embryo and adult life, endothelial cells lining blood vessel walls dynamically modify their integrin-mediated adhesive contacts with the surrounding extracellular matrix. However, besides regulating cell adhesion and migration, integrins dynamically participate in a network with soluble molecules and their receptors. Angiogenesis is characterized by opposing autocrine and paracrine loops of growth factors and semaphorins that regulate the activation of integrins on the endothelial surface through tyrosine kinase receptors (TKR) and the neuropilin/plexin system. Moreover, pro- and anti-angiogenic factors can directly bind integrins and regulate endothelial cell behaviour. This review summarizes the recent progress in understanding the reciprocal interactions between integrins, TKR, and semaphorin receptors.
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http://dx.doi.org/10.1093/cvr/cvn045DOI Listing
May 2008

Integrins: a flexible platform for endothelial vascular tyrosine kinase receptors.

Autoimmun Rev 2007 Nov 3;7(1):18-22. Epub 2007 Apr 3.

Dipartimento di Scienze Oncologiche, Università di Torino, 10060 Candiolo, Italy; Istituto per la Ricerca e la Cura del Cancro, Università di Torino, 10060 Candiolo, Italy. Electronic address:

Compared to lower metazoans, vertebrates built up an exclusively new set of adhesion-related genes involved in the tissue development and in their functions. They include a large variety of extracellular matrix proteins and their heterodimeric integrin adhesive receptors. Integrins control the adhesive state of the cell through complex molecular mechanisms. Outside-in signalling informs the cell about the extracellular matrix environment, while Inside-out signalling results in changes in integrin functional activity. In the last 10 years it has well established a reciprocal integration of signals originating from integrins and receptors for soluble growth factors. This review summarizes the current understanding of this connection in vascular endothelial cells and highlights how integrins regulate a genetic program triggered by angiogenic inducers during embryo development and in adult life.
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http://dx.doi.org/10.1016/j.autrev.2007.03.007DOI Listing
November 2007

Stable interaction between alpha5beta1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1.

J Cell Biol 2005 Sep;170(6):993-1004

Department of Oncological Sciences and Institute for Cancer Research and Treatment, University of Turin, 10060 Candiolo, Italy.

During angiogenic remodeling, Ang-1, the ligand of Tie2 tyrosine kinase, is involved in vessel sprouting and stabilization through unclear effects on nascent capillaries and mural cells. In our study, we hypothesized that the Ang-1/Tie2 system could cross-talk with integrins, and be influenced by the dynamic interactions between extracellular matrix and endothelial cells (ECs). Here, we show that alpha5beta1 specifically sensitizes and modulates Tie2 receptor activation and signaling, allowing EC survival at low concentrations of Ang-1 and inducing persistent EC motility. Tie2 and alpha5beta1 interact constitutively; alpha5beta1 binding to fibronectin increases this association, whereas Ang-1 stimulation recruits p85 and FAK to this complex. Furthermore, we demonstrate that Ang-1 is able to mediate selectively alpha5beta1 outside-in FAK phosphorylation. Thus, Ang-1 triggers signaling pathways through Tie2 and alpha5beta1 receptors that could cross-talk when Tie2/alpha5beta1 interaction occurs in ECs plated on fibronectin. By using blocking antibodies, we consistently found that alpha5beta1, but not alphavbeta3 activation, is essential to Ang-1-dependent angiogenesis in vivo.
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http://dx.doi.org/10.1083/jcb.200507082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2171441PMC
September 2005

Adaptor ShcA protein binds tyrosine kinase Tie2 receptor and regulates migration and sprouting but not survival of endothelial cells.

J Biol Chem 2004 Mar 9;279(13):13224-33. Epub 2003 Dec 9.

Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment (IRCC), School of Medicine, University of Torino, 10060 Candiolo, Italy.

Angiopoietin-1 can promote migration, sprouting, and survival of endothelial cells through activation of different signaling pathways triggered by the Tie2 tyrosine kinase receptor. ShcA adapter proteins are targets of activated tyrosine kinases and are implicated in the transmission of activation signals to the Ras/mitogen-activated protein kinase pathway. Here we report the identification of an interaction between the adapter protein ShcA and the cytoplasmic domain of Tie2 through in vitro co-immunoprecipitation analysis. Stimulation of endogenous Tie2 in endothelial cells with its ligand angiopoietin-1 increased its association with ShcA and phosphorylation of the adapter protein. The interaction requires the SH2 domain of ShcA and the tyrosine phosphorylation of Tie2 as shown by pull-down experiments. Furthermore, Tyr-1101 of Tie2 was identified as the primary binding site for the SH2 domain of ShcA. Overexpression of a dominant-negative form of ShcA affects angiopoietin-1-induced chemotaxis and sprouting, although it has no effect on survival of endothelial cells. Furthermore, this mutant partially reduces the tyrosine phosphorylation of the regulatory p85 subunit of phosphatidylinositol 3-kinase. Together, our results identified a novel interaction between Tie2 with the adapter molecule ShcA and suggested that this interaction may play a role in the regulation of migration and three-dimensional organization of endothelial cells induced by angiopoietin-1.
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http://dx.doi.org/10.1074/jbc.M307456200DOI Listing
March 2004

Temporal and spatial modulation of Rho GTPases during in vitro formation of capillary vascular network. Adherens junctions and myosin light chain as targets of Rac1 and RhoA.

J Biol Chem 2003 Dec 12;278(50):50702-13. Epub 2003 Sep 12.

Division of Molecular Angiogenesis, Institute for Cancer Research and Treatment (IRCC), Cadiolo, Italy.

Endothelial cells (ECs) self-organize into capillary networks when plated on extracellular matrix. In this process, Rho GTPases-mediated cytoskeletal dynamics control cell movement and organization of cell-to-matrix and cell-to-cell contacts. Time course analysis of RhoA and Rac1 activation matches specific morphological aspects of nascent pattern. RhoA-GTP increases early during EC adhesion and accumulates at sites of membrane ruffling. Rac1 is activated later and localizes in lamellipodia and at cell-to-cell contacts of organized cell chains. When ECs stretch and remodel to form capillary structures, RhoA-GTP increases again and associates with stress fibers running along the major cell axis. N17Rac1 and N19RhoA mutants impair pattern formation. Cell-to-cell contacts and myosin light chains (MLC) are targets of Rac1 and RhoA, respectively. N17Rac1 reduces the shift of beta-catenin and vascular endothelial cadherin to Triton X-100-insoluble fraction and impairs beta-catenin distribution at adherens junctions, suggesting that Rac1 controls the dynamics of cadherin-catenin complex with F-actin. During the remodeling phase of network formation, ECs show an intense staining for phosphorylated MLC along the plasma membrane; in contrast, MLC is less phosphorylated and widely diffused in N19RhoA ECs. Both N17Rac1 and N19RhoA have been used to investigate the role of wild type molecules in the main steps characterizing in vitro angiogenesis: (i) cell adhesion to the substrate, (ii) cell movement, and (iii) mechanical remodeling of matrix. N17Rac1 has a striking inhibitory effect on haptotaxis, whereas N19RhoA slightly inhibits EC adhesion and motility but more markedly Matrigel contraction. We conclude that different Rho GTPases control distinct morphogenetic aspects of vascular morphogenesis.
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http://dx.doi.org/10.1074/jbc.M307234200DOI Listing
December 2003

Tie-2-dependent activation of RhoA and Rac1 participates in endothelial cell motility triggered by angiopoietin-1.

Blood 2003 Oct 19;102(7):2482-90. Epub 2003 Jun 19.

Department of Oncological Sciences, University of Torino, Candiolo, Italy.

Angiopoietin-1 is implicated in the maturation and remodeling of the vascular network during embryo development and in adult life. Through its tyrosine kinase receptor Tie-2 it stimulates endothelial cells to migrate and change shape. Here we show that angiopoietin-1 elicits chemokinesis of endothelial cells by a phosphoinositide 3-OH kinase/son of sevenless-dependent modulation of Rac1 and RhoA. The resulting temporal events are associated with cytoskeletal rearrangements and occur in discrete zones of the cell. Endothelial cells carrying dominant-negative mutants of RhoA and Rac1 or treated with LY294002, an inhibitor of phosphoinositide 3-OH kinase, dramatically decrease their chemokinetic velocity. Taken together, these results further expand our understanding of angiopoietin-1-mediated endothelial cell motility during vascular network assembly and angiogenesis.
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http://dx.doi.org/10.1182/blood-2003-03-0670DOI Listing
October 2003