Marina Koutsioumpa

Integrin ανβ3 is a pleiotrophin receptor required for pleiotrophin-induced endothelial cell migration through receptor protein tyrosine phosphatase β/ζ

We have previously shown that the angiogenic growth factor pleiotrophin (PTN) induces migration of endothelial cells through binding to its receptor protein tyrosine phosphatase β/£ (RPTPβ/ζ). In this study, we show that a monoclonal antibody against αvβ3 but not α5β1 integrin abolished PTN‐induced human endothelial cell migration in a concentration‐dependent manner. Integrin αvβ3 was found to directly interact with PTN in an RGD‐independent manner, whereas a synthetic peptide corresponding to the specificity loop of the α3 integrin extracellular domain (177CYD‐ MKTTC184) inhibited PTN‐αvβ3 interaction and totally abolished PTN‐induced endothelial cell migration. Interestingly, αvβ3 was also found to directly interact with RPTPβ/ζ, and PTN‐induced Y773 phosphorylation of β3 integrin was dependent on both RPTPβ/ζ and the downstream c‐src kinase activation. Midkine was found to interact with RPTPβ/ζ, but not with αvβ3, and caused a small but statistically significant decrease in cell migration. In the same line, PTN decreased migration of different glioma cell lines that express RPTPP/ζ but do not express αvβ3, while it stimulated migration of U87MG cells that express αvβ3 on their cell membrane. Overexpression or down‐regulation of β3 stimulated or abolished, respectively, the effect of PTN on cell migration. Collectively, these data suggest that αvβ3 is a key molecule that determines the stimulatory or inhibitory effect of PTN on cell migration.— Mikelis, C., Sfaelou, E., Koutsioumpa, M., Kieffer, N., Papadimitriou, E. Integrin ovP3 is a pleiotrophin receptor required for pleiotrophin‐induced endothelial cell migration through receptor protein tyrosine phosphatase P/£. FASEBJ. 23, 1459–1469 (2009)

Roles of pleiotrophin in tumor growth and angiogenesis

Pleiotrophin (PTN) is a heparin-binding growth factor with diverse biological activities, the most studied of these being those related to the nervous system, tumor growth and angiogenesis. Although interest in the involvement of PTN in tumor growth is increasing, many questions remain unanswered, particularly concerning the receptors and the signaling pathways involved. In this review, we briefly introduce PTN, and summarize data on its involvement in tumor growth and angiogenesis, and on what is known to date concerning the receptors and pathways involved.

Cell surface nucleolin as a target for anti-cancer therapies.

A large number of mostly recent reports show enhanced expression of the multi-functional protein nucleolin (NCL) on the surface of activated lymphocytes, angiogenic endothelial and many different types of cancer cells. Translocation of NCL at the external side of the plasma membrane occurs via a secretory pathway independent of the endoplasmic reticulum-Golgi complex, requires intracellular intact actin cytoskeleton, and seems to be mediated by a variety of factors. Cell surface NCL serves as a binding partner of several molecules implicated in cell differentiation, adhesion, and leukocyte trafficking, inflammation, angiogenesis and tumor development, mediating their biological activities and in some cases, leading to their internalization. Accumulating evidence validates cell surface NCL as a strategic target for treatment of cancer, while its property of tumor-specific uptake of targeted ligands seems to be useful for the development of non-invasive imaging tools for the diagnosis of cancer and for the targeted release of chemotherapeutic drugs. The observation that cell surface NCL exists in complexes with several other proteins implicated in tumorigenesis and angiogenesis suggests that targeting cell surface NCL might trigger multi-inhibitory effects, depending on the cell type. This review summarizes papers and patents related to the redistribution and the biological functions of cell surface NCL, with emphasis on the potential importance and advantages of developing efficient anti-cell surface NCL strategies.

Interplay between αvβ3 Integrin and Nucleolin Regulates Human Endothelial and Glioma Cell Migration

Background: Cell surface nucleolin (NCL) is a promising target for development of anticancer agents. Results: A novel pathway that includes ανβ3 integrin and leads to cell surface NCL localization and cell migration has been identified. Conclusion: ανβ3 can be used as a biomarker for the use of NCL antagonists. Significance: This pathway is active in endothelial and glioma cells, as well as in human glioblastomas. The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because αvβ3 integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and αvβ3 by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing αvβ3 and depended on the phosphorylation of β3 at Tyr773 through receptor protein-tyrosine phosphatase β/ζ (RPTPβ/ζ) and c-Src activation. Downstream of αvβ3, PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both αvβ3 and RPTPβ/ζ. Positive correlation of cell surface NCL and αvβ3 expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing αvβ3. Collectively, these data suggest that both expression and β3 integrin phosphorylation at Tyr773 determine the cell surface localization of NCL downstream of the RPTPβ/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.

Pleiotrophin as a possible new target for angiogenesis-related diseases and cancer.

Pleiotrophin (PTN) is an 18 kDa growth factor that has high affinity for heparin and together with midkine form a family of structurally related heparin binding growth factors. Screening of various human tumour cell lines and tumour specimens of different origin revealed that PTN is expressed in many types of cancer, such as gliomas, melanomas, meningiomas, neuroblastomas, choriocarcinomas, leukemias and cancers of pancreas, prostate, stomach, colon, breast, ovaries and lungs. Concerning the biological activity of PTN in cancer, there is ample evidence that it is a tumour-promoting factor, while it has also been suggested that it may be implicated in cellular quiescence rather than an oncogenic phenotype. Besides a direct effect of PTN on tumour cells, there is also a plethora of reports indicating a positive correlation between PTN and in vivo or in vitro angiogenesis, a key step in the progress of many tumours. The identification of PTN domains responsible for its angiogenic and transforming activities is considered important, and data existing so far suggest distinct or even opposite effects for different PTN regions. This review summarises papers and patents dealing with the present understanding of PTN biochemistry, actions, mechanism(s) of action and implication in several diseases, with a special emphasis on its role in diverse tumour types.

Pleiotrophin expression and role in physiological angiogenesis in vivo: potential involvement of nucleolin

BackgroundPleiotrophin (PTN) is a heparin-binding growth factor with significant role(s) in tumour growth and angiogenesis. Although implication of endogenous PTN has been studied in several in vivo models of tumour angiogenesis, its role in physiological angiogenesis has not been addressed. In the present work, we studied expression and functional significance of endogenous PTN during angiogenesis in the chicken embryo chorioallantoic membrane (CAM).MethodsUsing molecular, cellular and biochemical assays, we studied the expression pattern of PTN in CAM and human endothelial cells and its possible interaction with nucleolin (NCL). CAM cells were transfected with a pCDNA3.1 vector, empty (PC) or containing full length cDNA for PTN in antisense orientation (AS-PTN). Angiogenesis was estimated by measuring total vessel length. In vitro, human endothelial cells migration was studied by using a transwell assay, and down-regulation of NCL was performed by using a proper siRNA.ResultsEndogenous PTN mRNA and protein levels, as well as protein levels of its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) were maximal at early stages, when CAM angiogenesis is active. Application of AS-PTN onto CAM at days of active angiogenesis was not toxic to the tissue and led to dose-dependent decreased expression of endogenous PTN, ERK1/2 activity and angiogenesis. Interestingly, endogenous PTN was also immunolocalized at the endothelial cell nucleus, possibly through interaction with NCL, a protein that has a significant role in the nuclear translocation of many proteins. Down-regulation of NCL by siRNA in human endothelial cells significantly decreased nuclear PTN, verifying this hypothesis. Moreover, it led to abolishment of PTN-induced endothelial cell migration, suggesting, for the first time, that PTN-NCL interaction has a functional significance.ConclusionsExpression of endogenous PTN correlates with and seems to be involved in angiogenesis of the chicken embryo CAM. Our data suggest that NCL may have a role, increasing the number of growth factors whose angiogenic/tumorigenic activities are mediated by NCL.

A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro.

Pleiotrophin (PTN) is a heparin‐binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both ανβ3 integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C‐terminal region of PTN (PTN112–136) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN‐induced migration and tube formation of human endothelial cells in vitro. PTN112–136 inhibited binding of PTN to ανβ3 integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β3. Moreover, it abolished PTN‐induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing ανβ3‐selective antibody. PTN112–136 did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β3 Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down‐regulation of RPTPβ/ζ by siRNA or by c‐src inhibition, suggesting that PTN112–136 may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN112–136 was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123–132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN112–136 induces some of the signaling pathways triggered by PTN, it inhibits PTN‐induced angiogenic activities through inhibition of PTN binding to ανβ3 integrin. J. Cell. Biochem. 112: 1532–1543, 2011.

Aprotinin stimulates angiogenesis and human endothelial cell migration through the growth factor pleiotrophin and its receptor protein tyrosine phosphatase β/ζ

Pleiotrophin is an 18 kDa secreted polypeptide growth factor with direct pro-angiogenic and tumorigenic properties. Pleiotrophin is a substrate for proteolytic enzymes, such as plasmin, leading to proteolytic fragments with distinct activities on endothelial cell activation in vitro or angiogenesis in vivo. Aprotinin is a naturally occurring broad spectrum protease inhibitor, used widely in cardiac surgery due to its ability to inhibit plasmin and reduce perioperative bleeding. Since we have seen that aprotinin inhibits proteolysis of pleiotrophin by plasmin, the aim of the present study was to evaluate the possible role of pleiotrophin in the effects of aprotinin on angiogenesis and human endothelial cell migration. Our data demonstrate that aprotinin, in a concentration-dependent manner, is angiogenic in the chicken embryo chorioallantoic membrane assay in vivo and induces human endothelial cell migration in vitro. Aprotinin inhibits pleiotrophin proteolysis and induces expression and secretion of pleiotrophin through an AP-1-dependent transcriptional activation of the pleiotrophin gene, and pleiotrophin seems to mediate the stimulatory effects of aprotinin on cell migration through its receptor protein tyrosine phosphatase beta/zeta. The stimulatory effect of aprotinin on pleiotrophin expression and cell migration may explain, at least partly, the problems observed with the clinical use of aprotinin.

Vascular endothelial growth factor A (VEGF-A) decreases expression and secretion of pleiotrophin in a VEGF receptor-independent manner.

Vascular endothelial growth factor A (VEGF-A) is a key molecule in angiogenesis acting through VEGF receptors (VEGFRs), ανβ3 integrin, receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and cell surface nucleolin (NCL). Pleiotrophin (PTN) stimulates endothelial cell migration and limits the angiogenic effects of VEGF-A165 to the levels of its own effect, possibly acting as a VEGF-A165 modifier. Since PTN and VEGF-A165 share receptors and actions on endothelial cells, in the present work we studied whether and how VEGF-A165 affects PTN expression or secretion. VEGF-A165 decreased PTN mRNA and protein levels acting at the transcriptional level. Bevacizumab, a selective VEGFR2 tyrosine kinase inhibitor and down-regulation of VEGFR2 expression by siRNA did not affect this decrease, suggesting that it is VEGFR-independent. VEGF-A121 also decreased PTN mRNA and protein levels, suggesting that heparin binding of VEGF-A165 is not involved. Blockage of cell surface NCL, lack of expression or mutation of β3 integrin and down-regulation of RPTPβ/ζ abolished the inhibitory effect of VEGF-A165 on PTN expression and secretion. Down-regulation of endogenous PTN in endothelial cells enhanced VEGF-A165-induced increase in migration and tube formation on matrigel. Collectively, these data suggest that VEGF-A down-regulates PTN expression and secretion through the RPTPβ/ζ-ανβ3-NCL axis to enhance its own effect on cell migration and further highlight the role of RPTPβ/ζ in VEGF-A actions.

Developments in miRNA gene signaling pathways in pancreatic cancer.

Pancreatic cancer is a devastating malignancy that ranks as the fourth leading cause of cancer-related deaths worldwide. Dismal prognosis is mainly attributable to limited knowledge of the molecular pathogenesis of the disease. miRNAs have been found to be deregulated in pancreatic cancer, affecting several steps of initiation and aggressiveness of the disease by regulating important signaling pathways, such as the KRAS and Notch pathways. Moreover, the effect of miRNAs on regulating cell cycle events and expression of transcription factors has gained a lot of attention. Recent studies have highlighted the application of miRNAs as biomarkers and therapeutic tools. The current review focuses on latest advances with respect to the roles of miRNAs in pancreatic ductal adenocarcinoma associated signaling pathways and miRNA-based therapeutics.