Federica Finetti

Prostaglandin E2 Regulates Angiogenesis via Activation of Fibroblast Growth Factor Receptor-1

Prostaglandin E2 (PGE2) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE2 on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE2 (100 nm) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE2 stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Gαi subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE2 did not phosphorylate FGFR-1. Upon binding to its receptor, PGE2 initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE2. Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE2 stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE2 angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.

EP2 prostanoid receptor promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways

In squamous cell carcinoma, the levels of nitric oxide (NO) derived from inducible NO synthase (iNOS) and prostaglandin E2 (PGE2) derived from cyclooxygenase‐2 (COX‐2) originated from tumor cells or tumor‐associated inflammatory cells have been reported to correlate with tumor growth, metastasis, and angiogenesis. The present study examined the role of the iNOS signaling pathway in PGE2‐mediated tumor invasiveness and proliferation in squamous cell carcinoma, A431, and SCC‐9 cells. Cell invasion and proliferation promoted by PGE2 were blocked by iNOS silencing RNA or iNOS/guanylate cyclase (GC) pharmacological inhibition. Consistently, iNOS‐GC pathway inhibitors blocked mitogen‐activated protein kinase‐ERK1/2 phosphorylation, which was required to mediate PGE2 functions. In vivo, in A431 cells implanted in nude mice, GC inhibition also decreased the tumor proliferation index and ERK1/2 activation. PGE2 effects were confined to the selective stimulation of the EP2 receptor subtype, leading to epidermal growth factor receptor (EGFR) transactivation via protein ki‐nase A (PKA) and c‐Src activation. EP2‐mediated ERK1/2 activation and cell functions were abolished by inhibitors of PKA, c‐Src, and EGFR, as well as by inhibiting iNOS pathway. Silencing of iNOS also impaired EGFR‐induced ERK1/2 phosphorylation. These results indicate that iNOS/GC signaling is a down‐stream player in the control of EP2/EGFR‐mediated tumor cell proliferation and invasion.—Donnini, S., Finetti, F., Solito, R., Terzuoli, E., Sacchetti, A., Morbidelli, L., Patrignani, P., Ziche, M. EP2 prostanoid receptor promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways. FASEB J. 21, 2418–2430 (2007)

Functional and pharmacological characterization of a VEGF mimetic peptide on reparative angiogenesis

Vascular endothelial growth factor (VEGF) is the main regulator of physiological and pathological angiogenesis. Low molecular weight molecules able to stimulate angiogenesis have interesting medical application for example in regenerative medicine, but at present none has reached the clinic. We reported that a VEGF mimetic helical peptide, QK, designed on the VEGF helix sequence 17-25, is able to bind and activate the VEGF receptors, producing angiogenesis. In this study we evaluate the pharmacological properties of peptide QK with the aim to propose it as a VEGF-mimetic drug to be employed in reparative angiogenesis. We show that the peptide QK is able to recapitulate all the biological activities of VEGF in vivo and on endothelial cells. In experiments evaluating sprouting from aortic ring and vessel formation in an in vivo angiogenesis model, the peptide QK showed biological effects comparable with VEGF. At endothelial level, the peptide up-regulates VEGF receptor expression, activates intracellular pathways depending on VEGFR2, and consistently it induces endothelial cell proliferation, survival and migration. When added to angiogenic factors (VEGF and/or FGF-2), QK produces an improved biological action, which resulted in reduced apoptosis and accelerated in vitro wound healing. The VEGF-like activity of the short peptide QK, characterized by lower cost of production and easier handling compared to the native glycoprotein, suggests that it is an attractive candidate to be further developed for application in therapeutic angiogenesis.

Prostaglandin E2 Primes the Angiogenic Switch via a Synergic Interaction With the Fibroblast Growth Factor-2 Pathway

Rationale: Prostaglandin (PG)E2 exerts temporally distinct actions on blood vessels, immediate vasodilatation, and long-term activation of angiogenesis. Objective: To study the mechanism of PGE2 induction of angiogenesis, we characterized its effect on fibroblast growth factor (FGF)-2 signaling in cultured endothelial cells and in ex vivo and in vivo assays of blood vessel formation. Methods and Results: Using Western blotting assay, we demonstrated that PGE2 induced upregulation of components of the FGF-2 pathway: FGF-2 protein, phosphorylation of FGF receptor type 1 (FGFR1), activation of FRS2&agr; (FGFR substrate 2&agr;), phospholipase C&ggr;, endothelial nitric oxide synthase, extracellular signal-regulated kinase 1/2, and the transcription factor STAT-3. Synergism between PGE2 and FGF-2 promoted endothelial cell proliferation and robust angiogenesis in vivo, in rabbit cornea and Matrigel assays. The magnitude of the angiogenic response to PGE2 was directly related to FGF-2 availability which determined the extent of FGFR1 activation. In fact, PGE2 induction of angiogenesis in vitro was impaired in FGF-2−/− endothelial cells and FGFR1 blockade abrogated PGE2 action on the endothelium, preventing the activation of FGF-2 signaling. Conclusion: We propose a model for the angiogenic switch based on the autocrine/paracrine FGF-2/FGFR1 activation by PGE2 and FGF-2 synergistic interaction. The synergism between the PGE2 and FGF-2 signaling pathways here described may explain the mechanism of action of drug combinations, the most notable being cyclooxygenase inhibitors with growth factors or growth factor receptor inhibitors.

Pharmacological inhibition of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis.

Background Blockade of Prostaglandin (PG) E2 production via deletion of microsomal Prostaglandin E synthase-1 (mPGES-1) gene reduces tumor cell proliferation in vitro and in vivo on xenograft tumors. So far the therapeutic potential of the pharmacological inhibition of mPGES-1 has not been elucidated. PGE2 promotes epithelial tumor progression via multiple signaling pathways including the epidermal growth factor receptor (EGFR) signaling pathway. Methodology/Principal Findings Here we evaluated the antitumor activity of AF3485, a compound of a novel family of human mPGES-1 inhibitors, in vitro and in vivo, in mice bearing human A431 xenografts overexpressing EGFR. Treatment of the human cell line A431 with interleukin-1beta (IL-1β) increased mPGES-1 expression, PGE2 production and induced EGFR phosphorylation, and vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) expression. AF3485 reduced PGE2 production, both in quiescent and in cells stimulated by IL-1β. AF3485 abolished IL-1β-induced activation of the EGFR, decreasing VEGF and FGF-2 expression, and tumor-mediated endothelial tube formation. In vivo, in A431 xenograft, AF3485, administered sub-chronically, decreased tumor growth, an effect related to inhibition of EGFR signalling, and to tumor microvessel rarefaction. In fact, we observed a decrease of EGFR phosphorylation, and VEGF and FGF-2 expression in tumours explanted from treated mice. Conclusion Our work demonstrates that the pharmacological inhibition of mPGES-1 reduces squamous carcinoma growth by suppressing PGE2 mediated-EGFR signalling and by impairing tumor associated angiogenesis. These results underscore the potential of mPGES-1 inhibitors as agents capable of controlling tumor growth.

The Syndecan-4/Protein Kinase Cα Pathway Mediates Prostaglandin E2-induced Extracellular Regulated Kinase (ERK) Activation in Endothelial Cells and Angiogenesis in Vivo

Background: Prostaglandin E2 (PGE2) induces tumor growth and angiogenesis. Results: PGE2-induced ERK activation in endothelial cells and angiogenesis are driven by syndecan-4-dependent PKCα activation. Conclusion: The syndecan-4/PKCα/ERK pathway is important for PGE2-induced angiogenesis in vitro and in vivo. Significance: Sdc4/PKCα activation is a novel finding in PGE2 signaling and may represent a pharmacological target. Prostaglandin E2 (PGE2) is regarded as the main mediator of inflammatory symptoms. In addition, it also plays an important role in tumor growth and angiogenesis. In this study, we examined the mechanism of PGE2-induced angiogenic response. We show that in the absence of proteoglycan syndecan-4 (Sdc4), PGE2-induced ERK activation is decreased significantly, as is endothelial cell migration and cord formation in a two-dimensional Matrigel assay. In vivo, PGE2-induced angiogenesis is reduced dramatically in Sdc4−/− mice. The mechanism was traced to Sdc4-dependent activation of protein kinase Cα (PKCα). Transduction of an Sdc4 S183E mutant (a cytoplasmic domain mutation that blocks Sdc4-dependent PKCα activation) into Sdc4−/− endothelial cells was not able to rescue the loss of PGE2-induced ERK activation, whereas a transduction with full-length Sdc4 resulted in full rescue. Furthermore, PGE2-induced angiogenesis was also reduced in PKCα−/− mice. Taken together, these results demonstrate that PGE2-induced activation of angiogenesis is mediated via syndecan-4-dependent activation of PKCα.

PKG-I inhibition attenuates vascular endothelial growth factor-stimulated angiogenesis

Vascular endothelial growth factor (VEGF) stimulates nitric oxide (NO) production, which mediates many of its angiogenic actions. However, the angiogenic pathways that operate downstream of NO following VEGF treatment are not well characterized. Herein, we used DT-2 and DT-3, two highly selective cGMP-dependent protein kinase I peptide inhibitors to determine the contribution of PKG-I in VEGF-stimulated angiogenesis. Incubation of chicken chorioallantoic membranes (CAM) with PKG-I peptide inhibitors decreased vascular length in a dose-dependent manner, with DT-3 being more effective than DT-2. Moreover, inhibition of PKG-I with DT-3 abolished the angiogenic response elicited by VEGF in the rabbit eye cornea. PKG-I inhibition also blocked VEGF-stimulated vascular leakage. In vitro, treatment of cells with VEGF stimulated phosphorylation of the PKG substrate VASP through VEGFR2 activation; the VEGF-stimulated VASP phosphorylation was reduced by DT-2. Pre-treatment of cells with DT-2 or DT-3 inhibited VEGF-stimulated mitogen-activated protein kinase cascades (ERK1/2 and p38), growth, migration and sprouting of endothelial cells. The above observations taken together identify PKG-I as a downstream effector of VEGFR2 in EC and provide a rational basis for the use of PKG-I inhibitors in disease states characterized by excessive neovascularization.

RNA‐mediated gene silencing of FUT1 and FUT2 influences expression and activities of bovine and human fucosylated nucleolin and inhibits cell adhesion and proliferation

In a previous article, we demonstrated the existence of fucosyl‐containing O‐glycans forms of nucleolin in bovine post‐capillary venular endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody found to interact strongly and exclusively with nucleolin in total protein extracts. The antibody was originally raised against a mollusc glycoprotein and was demonstrated to be directed against its O‐glycans, recently found to belong prevalently to the blood group H‐antigen type with fucose linked in alpha1, 2 to galactose. Here, we show that si‐RNA induced down‐regulation of the expression of FUT1 and FUT2, the fucosyltransferases required for the biosynthesis of the terminal glycan motif Fucα‐2‐Galβ‐R, reduced expression of the fucosylated nucleolin glycoforms and their exposure at the cell surface in CVEC. Treatment of the cells with FUT1/2 siRNA also reduced their ability to bind and internalize endostatin and their adhesion efficiency and inhibited cell growth. Expression of FUT1, FUT2, and FUT6 was also analyzed in serum‐stimulated versus serum‐starved cells and in cells treated with FUT1 and FUT2 siRNA. A reduced expression of fucosylated nucleolin and inhibition of cell growth by suppressing FUT1/2 expression was also tested and shown to be exhibited in human A431 cells. J. Cell. Biochem. 111: 229–238, 2010.

EGFR signaling upregulates expression of microsomal prostaglandin E synthase-1 in cancer cells leading to enhanced tumorigenicity

In this report we describe the contribution of prostaglandin E2 (PGE2) derived from the inducible microsomal PGE-synthase type-1 (mPGES-1) to the epidermal growth factor receptor (EGFR) oncogenic drive in tumor epithelial cells and in tumor-bearing mice. EGFR stimulation upregulated expression of mPGES-1 in HT-29, A431 and A549 cancer cells. Egr-1, a transcription factor induced by EGF, mediated this response. The Egr-1 rise provoked the overexpression of mPGES-1 messenger and protein, and enhanced PGE2 formation. These changes were suppressed either by silencing Egr-1, or by upstream blockade of EGFR or ERK1/2 signals. Further, in a clonogenic assay on tumor cells, EGF induced a florid tumorigenic phenotype, which regressed when mPGES-1 was silenced or knocked down. EGF-induced mPGES-1 overexpression in epithelial cell reduced E-cadherin expression, whereas enhancing that of vimentin, suggesting an incipient mesenchymal phenotype. Additionally, inhibiting the EGFR in mice bearing the A431 tumor, the mPGES-1 expression and the tumor growth, exhibited a parallel decline. In conclusion, these findings provide novel evidence that a tight cooperation between the EGF/EGFR and mPGES-1 leads to a significant tumorigenic gain in epithelial cells, and provide clues for controlling the vicious association.

A fucose-containing O-glycoepitope on bovine and human nucleolin

In this paper, we demonstrate the existence and localization of fucosyl-containing O-glycoforms of nucleolin in cultured bovine endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody raised against gp273, a glycoprotein ligand for the sperm-egg interaction in the mollusc bivalve Unio elongatulus. The function and immunological properties of gp273 mainly depend on clustered Lewis-like, fucose-containing O-glycans. Here an anti-gp273 antibody was used to evaluate whether glycoepitopes similar to those of gp273 are part of potential ligands of selectins in endothelial cells. We found that anti-gp273 strongly and exclusively interacted with a 110 kDa protein in CVEC and A431 tumor cells. After partial purification, mass spectrometry identified the protein as nucleolin. This was confirmed by comparing anti-gp273 and anti-nucleolin antibody immunoblotting after nucleolin depletion. We confirmed that anti-gp273 binding to nuclear and extranuclear nucleolin was against a fucose-containing O-glycoepitope by immunoblot analysis of the protein after chemically removing O-glycans and by lectin-blot analysis of control and nucleolin-depleted samples. Using anti-gp273 IgG, we detected nucleolin on the plasma membrane and cytoplasm. O-Glycosylation may regulate the plethora of functions in which nucleolin is involved.