Laboratoire Microenvironnement Tumoral

Publication de l’équipe

Année de publication : 2015

Giorgio Seano, Luca Primo (2015 Mar 20)

Podosomes and invadopodia: tools to breach vascular basement membrane.

Cell cycle (Georgetown, Tex.) : 1370-4 : DOI : 10.1080/15384101.2015.1026523 En savoir plus
Résumé

The vascular basement membrane (BM) is a thin and dense cross-linked extracellular matrix layer that covers and protects blood vessels. Understanding how cells cross the physical barrier of the vascular BM will provide greater insight into the potentially critical role of vascular BM breaching in cancer extravasation, leukocyte trafficking and angiogenic sprouting. In the last year, new evidence has mechanistically linked the breaching of vascular BM with the formation of specific cellular micro-domains known as podosomes and invadopodia. These structures are specialized cell-matrix contacts with an inherent ability to degrade the extracellular matrix. Specifically, the formation of podosomes or invadopodia was shown as an important step in vascular sprouting and tumor cell extravasation, respectively. Here, we review and comment on these recent findings and explore the functions of podosomes and invadopodia within the context of pathological processes such as tumor dissemination and tumor angiogenesis.

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Eleanor I Ager, Sergey V Kozin, Nathaniel D Kirkpatrick, Giorgio Seano, David P Kodack, Vasileios Askoxylakis, Yuhui Huang, Shom Goel, Matija Snuderl, Alona Muzikansky, Dianne M Finkelstein, Daniel T Dransfield, Laetitia Devy, Yves Boucher, Dai Fukumura, Rakesh K Jain (2015 Feb 25)

Blockade of MMP14 activity in murine breast carcinomas: implications for macrophages, vessels, and radiotherapy.

Journal of the National Cancer Institute : DOI : 10.1093/jnci/djv017 En savoir plus
Résumé

Matrix metalloproteinase (MMP) 14 may mediate tumor progression through vascular and immune-modulatory effects.

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Meenal Datta, Laura E Via, Walid S Kamoun, Chong Liu, Wei Chen, Giorgio Seano, Danielle M Weiner, Daniel Schimel, Kathleen England, John D Martin, Xing Gao, Lei Xu, Clifton E Barry, Rakesh K Jain (2015 Jan 28)

Anti-vascular endothelial growth factor treatment normalizes tuberculosis granuloma vasculature and improves small molecule delivery.

Proceedings of the National Academy of Sciences of the United States of America : 1827-32 : DOI : 10.1073/pnas.1424563112 En savoir plus
Résumé

Tuberculosis (TB) causes almost 2 million deaths annually, and an increasing number of patients are resistant to existing therapies. Patients who have TB require lengthy chemotherapy, possibly because of poor penetration of antibiotics into granulomas where the bacilli reside. Granulomas are morphologically similar to solid cancerous tumors in that they contain hypoxic microenvironments and can be highly fibrotic. Here, we show that TB-infected rabbits have impaired small molecule distribution into these disease sites due to a functionally abnormal vasculature, with a low-molecular-weight tracer accumulating only in peripheral regions of granulomatous lesions. Granuloma-associated vessels are morphologically and spatially heterogeneous, with poor vessel pericyte coverage in both human and experimental rabbit TB granulomas. Moreover, we found enhanced VEGF expression in both species. In tumors, antiangiogenic, specifically anti-VEGF, treatments can « normalize » their vasculature, reducing hypoxia and creating a window of opportunity for concurrent chemotherapy; thus, we investigated vessel normalization in rabbit TB granulomas. Treatment of TB-infected rabbits with the anti-VEGF antibody bevacizumab significantly decreased the total number of vessels while normalizing those vessels that remained. As a result, hypoxic fractions of these granulomas were reduced and small molecule tracer delivery was increased. These findings demonstrate that bevacizumab treatment promotes vascular normalization, improves small molecule delivery, and decreases hypoxia in TB granulomas, thereby providing a potential avenue to improve delivery and efficacy of current treatment regimens.

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Laura di Blasio, Paolo Armando Gagliardi, Alberto Puliafito, Roberto Sessa, Giorgio Seano, Federico Bussolino, Luca Primo (2015 Jan 16)

PDK1 regulates focal adhesion disassembly by modulating endocytosis of αvβ3 integrin.

Journal of cell science : 863-77 : DOI : 10.1242/jcs.149294 En savoir plus
Résumé

Non-amoeboid cell migration is characterised by dynamic competition among multiple protrusions to establish new adhesion sites at the cell’s leading edge. However, the mechanisms that regulate the decision to disassemble or to grow nascent adhesions are not fully understood. Here we show that, in endothelial cells, 3-phosphoinositide-dependent protein kinase 1 (PDK1) promotes focal adhesion (FA) turnover by controlling endocytosis of integrin αvβ3 in a PI3K-dependent manner. We demonstrate that PDK1 binds and phosphorylates integrin αvβ3. Downregulation of PDK1 increases FA size and slows down their disassembly. This process requires both PDK1 kinase activity and PI3K activation but does not involve Akt. Moreover, PDK1 silencing stabilises FA in membrane protrusions decreasing migration of endothelial cells on vitronectin. These results indicate that modulation of integrin endocytosis by PDK1 hampers endothelial cell adhesion and migration on extracellular matrix, thus unveiling a novel role for this kinase.

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Année de publication : 2014

Giorgio Seano, Giulia Chiaverina, Paolo Armando Gagliardi, Laura di Blasio, Alberto Puliafito, Claire Bouvard, Roberto Sessa, Guido Tarone, Lydia Sorokin, Dominique Helley, Rakesh K Jain, Guido Serini, Federico Bussolino, Luca Primo (2014 Sep 15)

Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis.

Nature cell biology : 931-41, 1-8 : DOI : 10.1038/ncb3036 En savoir plus
Résumé

The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α6β1. In contrast, the binding of α6β1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α6β1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis.

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Giorgio Seano, Thomas Daubon, Elisabeth Génot, Luca Primo (2014 Sep 10)

Podosomes as novel players in endothelial biology.

European journal of cell biology : 405-12 : DOI : 10.1016/j.ejcb.2014.07.009 En savoir plus
Résumé

Podosomes and invadopodia, collectively known as invadosomes, are specialized cell-matrix contacts with an inherent ability to degrade extracellular matrix. Their occurrence in either normal (podosomes) or cancer cells (invadopodia) is thus traditionally associated with cell invasiveness and tissue remodelling. These specialized micro-domains of the plasma membrane are characterized by enrichment of F-actin, cortactin and metalloproteases. Recent developments in the field show that, under some circumstances, vascular endothelial cells (ECs) can be induced to form this kind of peculiar structures. Cultured ECs contain either 0.5-1-μm-wide individual podosomes or 5 to 10 μm wide ring-like clusters of podosomes (podosome rosettes). The formation of individual podosomes or podosome rosettes in ECs can be induced by soluble factors, such as TGFβ, VEGF, TNFα or pharmacological agents, such as phorbol esters. Recently, the evidence of the existence of such structures in vascular endothelium has been provided by ex vivo observation. Endothelial podosome rosettes have recently been functionally linked to arterial remodelling and sprouting angiogenesis. Concerted efforts aim now at confirming the relevance of endothelial podosomes in these patho-physiological processes in vivo. In the current review, we will introduce some general considerations regarding ECs in the vascular system. From there on, we will review the various EC types where podosomes have been described and the state-of-art knowledge hitherto generated regarding endothelial podosome features.

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Paolo Armando Gagliardi, Laura di Blasio, Alberto Puliafito, Giorgio Seano, Roberto Sessa, Federica Chianale, Thomas Leung, Federico Bussolino, Luca Primo (2014 Aug 6)

PDK1-mediated activation of MRCKα regulates directional cell migration and lamellipodia retraction.

The Journal of cell biology : 415-34 : DOI : 10.1083/jcb.201312090 En savoir plus
Résumé

Directional cell migration is of paramount importance in both physiological and pathological processes, such as development, wound healing, immune response, and cancer invasion. Here, we report that 3-phosphoinositide-dependent kinase 1 (PDK1) regulates epithelial directional migration and invasion by binding and activating myotonic dystrophy kinase-related CDC42-binding kinase α (MRCKα). We show that the effect of PDK1 on cell migration does not involve its kinase activity but instead relies on its ability to bind membrane phosphatidylinositol (3,4,5)-trisphosphate. Upon epidermal growth factor (EGF) stimulation, PDK1 and MRCKα colocalize at the cell membrane in lamellipodia. We demonstrate that PDK1 positively modulates MRCKα activity and drives its localization within lamellipodia. Likewise, the retraction phase of lamellipodia is controlled by PDK1 through an MRCKα-dependent mechanism. In summary, we discovered a functional pathway involving PDK1-mediated activation of MRCKα, which links EGF signaling to myosin contraction and directional migration.

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Anna Valeria Samarelli, Elena Riccitelli, Laura Bizzozero, Tatiana Nunes Silveira, Giorgio Seano, Margherita Pergolizzi, Grazia Vitagliano, Ilaria Cascone, Gilles Carpentier, Alessia Bottos, Luca Primo, Federico Bussolino, Marco Arese (2014 May 27)

Neuroligin 1 induces blood vessel maturation by cooperating with the α6 integrin.

The Journal of biological chemistry : 19466-76 : DOI : 10.1074/jbc.M113.530972 En savoir plus
Résumé

The synaptic protein Neuroligin 1 (NLGN1), a cell adhesion molecule, is critical for the formation and consolidation of synaptic connectivity and is involved in vascular development. The mechanism through which NLGN1 acts, especially in vascular cells, is unknown. Here, we aimed at deepening our knowledge on the cellular activities and molecular pathways exploited by endothelial NLGN1 both in vitro and in vivo. We analyzed the phenotypic consequences of NLGN1 expression modulation in endothelial cells through in vitro angiogenesis assays and the mouse postnatal retinal angiogenesis model. We demonstrate that NLGN1, whereas not affecting endothelial cell proliferation or migration, modulates cell adhesion to the vessel stabilizing protein laminin through cooperation with the α6 integrin, a specific laminin receptor. Finally, we show that in vivo, NLGN1 and α6 integrin preferentially colocalize in the mature retinal vessels, whereas NLGN1 deletion causes an aberrant VE-cadherin, laminin and α6 integrin distribution in vessels, along with significant structural defects in the vascular tree.

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Année de publication : 2013

Giorgio Seano, Giulia Chiaverina, Paolo Armando Gagliardi, Laura di Blasio, Roberto Sessa, Federico Bussolino, Luca Primo (2013 Mar 9)

Modeling human tumor angiogenesis in a three-dimensional culture system.

Blood : e129-37 : DOI : 10.1182/blood-2012-08-452292 En savoir plus
Résumé

The intrinsic complexity of the process of vessel formation limits the efficacy of cellular assays for elucidation of its molecular and pharmacologic mechanisms. We developed an ex vivo three-dimensional (3D) assay of sprouting angiogenesis with arterial explants from human umbilical cords. In this assay, human arterial rings were embedded in basement membrane extract gel, leading to a network of capillarylike structures upon vascular endothelial growth factor (VEGF) A stimulation. The angiogenic outgrowth consisted of endothelial cells, which actively internalized acetylated-low-density lipoprotein, surrounded by pericytes. Computer-assisted quantification of this vascular network demonstrated considerable sensitivity of this assay to several angiogenic inhibitors, including kinase inhibitors and monoclonal antibodies. We also performed targeted gene knockdown on this model by directly infecting explanted umbilical arteries with lentiviruses carrying short-hairpin RNA. Downregulation of VEGFR2 resulted in a significant reduction of the sprouting capability, demonstrating the relevance of human vascular explants for functional genomics studies. Furthermore, a modification of this assay led to development of a 3D model of tumor-driven angiogenesis, in which angiogenic outgrowth was sustained by spheroids of prostate cancer cells in absence of exogenous growth factors. The human arterial ring assay bridges the gap between in vitro endothelial cell and animal model, and is a powerful system for identification of genes and drugs that regulate human angiogenesis.

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Année de publication : 2012

Paolo Armando Gagliardi, Laura di Blasio, Francesca Orso, Giorgio Seano, Roberto Sessa, Daniela Taverna, Federico Bussolino, Luca Primo (2012 Sep 7)

3-phosphoinositide-dependent kinase 1 controls breast tumor growth in a kinase-dependent but Akt-independent manner.

Neoplasia (New York, N.Y.) : 719-31 En savoir plus
Résumé

3-phosphoinositide-dependent protein kinase 1 (PDK1) is the pivotal element of the phosphatidylinositol 3 kinase (PI3K) signaling pathway because it phosphorylates Akt/PKB through interactions with phosphatidylinositol 3,4,5 phosphate. Recent data indicate that PDK1 is overexpressed in many breast carcinomas and that alterations of PDK1 are critical in the context of oncogenic PI3K activation. However, the role of PDK1 in tumor progression is still controversial. Here, we show that PDK1 is required for anchorage-independent and xenograft growth of breast cancer cells harboring either PI3KCA or KRAS mutations. In fact, PDK1 silencing leads to increased anoikis, reduced soft agar growth, and pronounced apoptosis inside tumors. Interestingly, these phenotypes are reverted by PDK1 wild-type but not kinase-dead mutant, suggesting a relevant role of PDK1 kinase activity, even if PDK1 is not relevant for Akt activation here. Indeed, the expression of constitutively active forms of Akt in PDK1 knockdown cells is unable to rescue the anchorage-independent growth. In addition, Akt down-regulation and pharmacological inhibition do not inhibit the effects of PDK1 overexpression. In summary, these results suggest that PDK1 may contribute to breast cancer, even in the absence of PI3K oncogenic mutations and through both Akt-dependent and Akt-independent mechanisms.

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Roberto Sessa, Giorgio Seano, Laura di Blasio, Paolo Armando Gagliardi, Claudio Isella, Enzo Medico, Franco Cotelli, Federico Bussolino, Luca Primo (2012 Aug 8)

The miR-126 regulates angiopoietin-1 signaling and vessel maturation by targeting p85β.

Biochimica et biophysica acta : 1925-35 : DOI : 10.1016/j.bbamcr.2012.07.011 En savoir plus
Résumé

Blood vessel formation depends on the highly coordinated actions of a variety of angiogenic regulators. Vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1) are both potent and essential proangiogenic factors with complementary roles in vascular development and function. Whereas VEGF is required for the formation of the initial vascular plexus, Ang-1 contributes to the stabilization and maturation of growing blood vessels. Here, we provide evidence of a novel microRNA (miRNA)-dependent molecular mechanism of Ang-1 signalling modulation aimed at stabilizing adult vasculature. MiRNAs are short non-coding RNA molecules that post-trascriptionally regulate gene expression by translational suppression or in some instances by cleavage of the respective mRNA target. Our data indicate that endothelial cells of mature vessels express high levels of miR-126, which primarily targets phosphoinositide-3-kinase regulatory subunit 2 (p85β). Down-regulation of miR-126 and over-expression of p85β in endothelial cells inhibit the biological functions of Ang-1. Additionally, knockdown of miR-126 in zebrafish resulted in vascular remodelling and maturation defects, reminiscent of the Ang-1 loss-of-function phenotype. Our findings suggest that miR-126-mediated phosphoinositide-3-kinase regulation, not only fine-tunes VEGF-signaling, but it strongly enhances the activities of Ang-1 on vessel stabilization and maturation.

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Alessio Noghero, Alessia Perino, Giorgio Seano, Elisa Saglio, Giuseppe Lo Sasso, Franco Veglio, Luca Primo, Emilio Hirsch, Federico Bussolino, Fulvio Morello (2012 Jun 23)

Liver X receptor activation reduces angiogenesis by impairing lipid raft localization and signaling of vascular endothelial growth factor receptor-2.

Arteriosclerosis, thrombosis, and vascular biology : 2280-8 : DOI : 10.1161/ATVBAHA.112.250621 En savoir plus
Résumé

Liver X receptors (LXRα, LXRβ) are master regulators of cholesterol homeostasis. In the endothelium, perturbations of cell cholesterol have an impact on fundamental processes. We, therefore, assessed the effects of LXR activation on endothelial functions related to angiogenesis in vitro and in vivo.

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Lucia Napione, Simona Pavan, Andrea Veglio, Andrea Picco, Guido Boffetta, Antonio Celani, Giorgio Seano, Luca Primo, Andrea Gamba, Federico Bussolino (2012 Apr 19)

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

Blood : 5599-607 : DOI : 10.1182/blood-2011-11-390666 En savoir plus
Résumé

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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Année de publication : 2010

Luca Primo, Giorgio Seano, Cristina Roca, Federica Maione, Paolo Armando Gagliardi, Roberto Sessa, Marianna Martinelli, Enrico Giraudo, Laura di Blasio, Federico Bussolino (2010 Jun 24)

Increased expression of alpha6 integrin in endothelial cells unveils a proangiogenic role for basement membrane.

Cancer research : 5759-69 : DOI : 10.1158/0008-5472.CAN-10-0507 En savoir plus
Résumé

The integrin alpha6 subunit is part of the alpha6beta1 and alpha6beta4 integrin complexes, which are known to be receptors for laminins and to mediate several biological activities such as embryogenesis, organogenesis, and invasion of carcinoma cells. However, the precise role of alpha6 integrin in angiogenesis has not yet been addressed. We observed that both vascular endothelial growth factor-A and fibroblast growth factor-2 strongly upregulate alpha6 integrin in human endothelial cells. Moreover, alpha6 integrin was positively modulated in angiogenic vessels in pancreatic neuroendocrine carcinoma. In this transgenic mouse model of spontaneous tumorigenesis, alpha6 integrin expression increased in the angiogenic stage, while being expressed at low levels in normal and hyperplastic tissue. We studied the functional role of alpha6 integrin during angiogenesis by lentivirus-mediated gene silencing and blocking antibody. Cell migration and morphogenesis on basement membrane extracts, a laminin-rich matrix, was reduced in endothelial cells expressing low levels of alpha6 integrin. However, we did not observe any differences in collagen matrices. Similar results were obtained in the aortic ring angiogenesis assay. alpha6 integrin was required for vessel sprouting on basement membrane gels but not on collagen gels, as shown by stably silencing this integrin in the murine aorta. Finally, a neutralizing anti-alpha6 integrin antibody inhibited in vivo angiogenesis in chicken chorioallantoic membrane and transgenic tumor mouse model. In summary, we showed that the alpha6 integrin participated in vascular endothelial growth factor-A and fibroblast growth factor-2-driven angiogenesis in vitro and in vivo, suggesting that it might be an attractive target for therapeutic approaches in angiogenesis-dependent diseases such as tumor growth.

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Année de publication : 2009

Federica Maione, Fabiola Molla, Claudia Meda, Roberto Latini, Lorena Zentilin, Mauro Giacca, Giorgio Seano, Guido Serini, Federico Bussolino, Enrico Giraudo (2009 Oct 8)

Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models.

The Journal of clinical investigation : 3356-72 : DOI : 10.1172/JCI36308 En savoir plus
Résumé

Tumor growth and progression rely upon angiogenesis, which is regulated by pro- and antiangiogenic factors, including members of the semaphorin family. By analyzing 3 different mouse models of multistep carcinogenesis, we show here that during angiogenesis, semaphorin 3A (Sema3A) is expressed in ECs, where it serves as an endogenous inhibitor of angiogenesis that is present in premalignant lesions and lost during tumor progression. Pharmacologic inhibition of endogenous Sema3A during the angiogenic switch, the point when pretumoral lesions initiate an angiogenic phase that persists throughout tumor growth, enhanced angiogenesis and accelerated tumor progression. By contrast, when, during the later stages of carcinogenesis following endogenous Sema3A downmodulation, Sema3A was ectopically reintroduced into islet cell tumors by somatic gene transfer, successive waves of apoptosis ensued, first in ECs and then in tumor cells, resulting in reduced vascular density and branching and inhibition of tumor growth and substantially extended survival. Further, long-term reexpression of Sema3A markedly improved pericyte coverage of tumor blood vessels, something that is thought to be a key property of tumor vessel normalization, and restored tissue normoxia. We conclude, therefore, that Sema3A is an endogenous and effective antiangiogenic agent that stably normalizes the tumor vasculature.

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