A long pentraxin-3-derived pentapeptide for the therapy of FGF8b-driven steroid hormone-regulated cancers

Fibroblast growth factor-8b (FGF8b) affects the epithelial/stromal compartments of steroid hormone-regulated tumors by exerting an autocrine activity on cancer cells and a paracrine pro-angiogenic function, thus contributing to tumor progression. The FGF8b/FGF receptor (FGFR) system may therefore represent a target for the treatment of steroid hormone-regulated tumors. The soluble pattern recognition receptor long pentraxin-3 (PTX3) binds various FGFs, including FGF2 and FGF8b, thus inhibiting the angiogenic and tumorigenic activity of androgen-regulated tumor cells. Nevertheless, the complex/proteinaceous structure of PTX3 hampers its pharmacological exploitation. In this context, the acetylated pentapeptide Ac-ARPCA-NH2 (ARPCA), corresponding to the N-terminal amino acid sequence PTX3(100-104), was identified as a minimal FGF2-binding peptide able to antagonize the biological activity of FGF2. Here, we demonstrate that ARPCA binds FGF8b and inhibits its capacity to form FGFR1-mediated ternary complexes with heparan sulphate proteoglycans. As a FGF8b antagonist, ARPCA inhibits FGFR1 activation and signalling in endothelial cells, hampering the angiogenic activity exerted in vitro and in vivo by FGF8b. Also, ARPCA suppresses the angiogenic and tumorigenic potential of prototypic androgen/FGF8b-dependent Shionogi 115 mammary carcinoma cells and of androgen/FGF8b/FGF2-dependent TRAMP-C2 prostate cancer cells. In conclusion, ARPCA represents a novel FGF8b antagonist with translational implications for the therapy of steroid hormone-regulated tumor

Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR

The subcutaneous Matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic molecules. However, quantification of the angiogenic response in the plug remains a problematic task. Here we report a simple, rapid, unbiased and reverse transcription-quantitative PCR (RT-qPCR) method to investigate the angiogenic process occurring in the Matrigel plug in response to fibroblast growth factor-2 (FGF2). To this purpose, a fixed amount of human cells were added to harvested plugs at the end of the in vivo experimentation as an external cell tracer. Then, mRNA levels of the pan endothelial cell markers murine CD31 and vascular endothelial-cadherin were measured by species-specific RT-qPCR analysis of the total RNA and data were normalized for human GAPDH or β-actin mRNA levels. RT-qPCR was used also to measure the levels of expression in the plug of various angiogenesis/inflammationrelated genes. The procedure allows the simultaneous, quantitative evaluation of the newly-formed endothelium and of non endothelial/inflammatory components of the cellular infiltrate in the Matrigel implant, as well as the expression of genes involved in the modulation of the angiogenesis process. Also, the method consents the quantitative assessment of the effect of local or systemic administration of anti-angiogenic compounds on the neovascular response triggered by FGF2

Auditory cortex hypoperfusion: a metabolic hallmark in Beta Thalassemia

Abstract Background Sensorineural hearing loss in beta-thalassemia is common and it is generally associated with iron chelation therapy. However, data are scarce, especially on adult populations, and a possible involvement of the central auditory areas has not been investigated yet. We performed a multicenter cross-sectional audiological and single-center 3Tesla brain perfusion MRI study enrolling 77 transfusion-dependent/non transfusion-dependent adult patients and 56 healthy controls. Pure tone audiometry, demographics, clinical/laboratory and cognitive functioning data were recorded. Results Half of patients (52%) presented with high-frequency hearing deficit, with overt hypoacusia (Pure Tone Average (PTA) > 25 dB) in 35%, irrespective of iron chelation or clinical phenotype. Bilateral voxel clusters of significant relative hypoperfusion were found in the auditory cortex of beta-thalassemia patients, regardless of clinical phenotype. In controls and transfusion-dependent (but not in non-transfusion-dependent) patients, the relative auditory cortex perfusion values increased linearly with age (p < 0.04). Relative auditory cortex perfusion values showed a significant U-shaped correlation with PTA values among hearing loss patients, and a linear correlation with the full scale intelligence quotient (right side p = 0.01, left side p = 0.02) with its domain related to communication skills (right side p = 0.04, left side p = 0.07) in controls but not in beta-thalassemia patients. Audiometric test results did not correlate to cognitive test scores in any subgroup. Conclusions In conclusion, primary auditory cortex perfusion changes are a metabolic hallmark of adult beta-thalassemia, thus suggesting complex remodeling of the hearing function, that occurs regardless of chelation therapy and before clinically manifest hearing loss. The cognitive impact of perfusion changes is intriguing but requires further investigations

Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk

Bruton's tyrosine kinase (Btk) is required for B-cell development. Btk deficiency causes X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk lacks a negative regulatory domain and may rely on cytoplasmic proteins to regulate its activity. Consistently, we identified an inhibitor of Btk, IBtk, which binds to the PH domain of Btk and down-regulates the Btk kinase activity. IBtk is an evolutionary conserved protein encoded by a single genomic sequence at 6q14.1 cytogenetic location, a region of recurrent chromosomal aberrations in lymphoproliferative disorders; however, the physical and functional organization of IBTK is unknown. Here, we report that the human IBTK locus includes three distinct mRNAs arising from complete intron splicing, an additional polyadenylation signal and a second transcription start site that utilizes a specific ATG for protein translation. By northern blot, 5′RACE and 3′RACE we identified three IBTKα, IBTKβ and IBTKγ mRNAs, whose transcription is driven by two distinct promoter regions; the corresponding IBtk proteins were detected in human cells and mouse tissues by specific antibodies. These results provide the first characterization of the human IBTK locus and may assist in understanding the in vivo function of IBtk

Stromal expression of long Pentraxin-3 impairs tumor growth and metastasis

Long Pentraxin-3 (PTX3) is a soluble pattern recognition receptor expressed by endothelial and immune cells in inflammatory contexts. We have previously demonstrated that PTX3 binds to different members of the FGF family, thus inhibiting their biological activity. The FGF/FGFR system strongly contributes to cancer progression by inducing tumor growth and neovascularization. To date, recombinant PTX3 protein or PTX3-overexpressing tumor cell lines have been exploited to assess the antitumor effects of this natural FGF trap. Here we generated C57BL/6 transgenic mice expressing human (h)PTX3 under the control of the endothelial specific Tie2/Tek transcription regulatory sequences. These animals were used to investigate the impact of PTX3 overexpression by the host stroma on tumor growth, vascularization and metastasis. Transgenic Tie2-hPTX3 mice were generated by cloning the hPTX3 cDNA into the late-generation, self-inactivating lentiviral vector Tie2p/e to obtain the Tie2-hPTX3 lentiviral transfer vectors that were injected into fertilized oocytes. Expression of the transgene was confirmed by RT-PCR and western blot analyses of different organs from Tie2-hPTX3 mice that showed increased levels of circulating PTX3 (80-180 ng/ml) when compared to wild type (wt) animals (<1.8 ng/ml). Also, histological analysis confirmed the perivascular accumulation of hPTX3 in transgenic animals. To assess the anti-angiogenic activity of endothelium-derived hPTX3, we performed ex vivo aorta ring and in vivo matrigel plug assays. Both assays revealed a significant inhibition of FGF2-driven angiogenesis in Tie2-hPTX3 mice that maintained their responsiveness to VEGF-A, thus confirming the specificity of the effect. Next, different syngeneic FGF-dependent tumor cell lines, including TRAMP-C2 prostate carcinoma, B16-F10 melanoma and Lewis Lung carcinoma cells, were subcutaneously injected in Tie2-hPTX3 mice. Notably, the growth of all tumor grafts was significantly reduced in Tie2-hPTX3 mice when compared to wt animals. Also, histological analysis of TRAMP-C2 tumors grown in Tie2-hPTX3 mice showed a strong perivascular expression of hPTX3 and a significant reduction of FGFR1 phosphorylation. This was paralleled by a significant decrease of tumor vascularity and tumor cell proliferation whereas no difference in tumor growth was observed for TRAMP-C2 grafts expressing a constitutively activated form of FGFR1. Finally, B16-F10 melanoma and M5076 ovarian sarcoma cells showed a dramatic decrease of their capacity to form experimental metastases in the lung and liver, respectively, after intravenous injection in Tie2-hPTX3 mice. PTX3 is a natural FGF ligand trap. Our findings demonstrate for the first time that the production of PTX3 by the host stroma may exert a dramatic impact on tumor growth, vascularization and metastasis with potential exploitation for the therapy of FGF-dependent tumors

Stromal delivery of long Pentraxin-3 impairs FGF/FGFR-dependent tumor growth and metastasis

The FGF/FGFR system contributes to cancer progression by inducing tumor growth and neovascularization, thus representing an emerging therapeutic target. Long Pentraxin-3 (PTX3) is a soluble pattern recognition receptor expressed by endothelial and immune cells in inflammatory contexts. Among various ligands, PTX3 binds different members of the FGF family, acting as a natural FGF ligand trap. Here, we generated transgenic mice expressing human (h)PTX3 under the control of endothelial specific Tie2/Tek transcription regulatory sequences (Tie2-hPTX3 mice). These animals accumulate significant levels of hPTX3 in perivascular stroma and in the blood stream. On this basis, Tie2-hPTX3 mice were used to investigate the impact of stroma delivery of hPTX3 on tumor growth, vascularization and metastasis. The anti-angiogenic activity of endothelium-derived hPTX3 was confirmed by ex vivo aorta ring and in vivo matrigel plug assays. Next, different syngeneic FGF-dependent tumor cell lines, including TRAMP-C2 prostate carcinoma, B16-F10 melanoma and Lewis Lung carcinoma cells, were subcutaneously injected in Tie2-hPTX3 mice. Notably, the growth of all tumor grafts was significantly reduced in Tie2-hPTX3 mice when compared to wild type animals and was accompanied by a significant reduction of FGFR1 phosphorylation, decrease of tumor vascularity and tumor cell proliferation. Also, B16-F10 melanoma and M5076 ovarian sarcoma cells showed a dramatic decrease of their capacity to form experimental metastases in the lung and liver, respectively, after intravenous injection in Tie2-hPTX3 mice. Also, the orthotopic growth of syngeneic pancreatic and mammary tumor cells was significantly reduced after injection in Tie2-hPTX3 mice and led to increased survival compared to control mice. Finally, double transgenic TRAMP/Tie2-hPTX3 mice showed a significant delay of multistage prostate tumor onset and progression in respect to TRAMP mice. Our findings demonstrate for the first time that in vivo delivery of PTX3 exerts a dramatic impact on tumor growth, vascularization and metastasis. These results have set the basis for the identification of a low molecular weight PTX3-derived molecule that recapitulates the FGF-trap activities of PTX3 and exhibits promising therapeutic potential for FGF-dependent tumors

Long Pentraxin-3 inhibits FGF-dependent angiogenesis andgrowth of steroid hormone-regulated tumors

Steroid hormone-regulated tumors, including breast and prostate cancers, represent a class of epithelial lesions whose growth is finely tuned by steroid hormones. However, as these tumors progress, they may become independent from steroid hormones for growth, limiting the effectiveness of hormonal ablation therapies. Fibroblast growth factors (FGFs) are potent angiogenic factors that exert non-redundant autocrine/paracrine functions in various tumors types. Experimental and clinical evidences indicate that the FGF/FGF receptor (FGFR) axis can drive the progression of steroid hormone-dependent cancers to a hormone-independent state, thus representing a possible alternative target for the treatment of hormonal cancers. Previous observations had shown that the soluble pattern recognition receptor long pentraxin-3 (PTX3) is a natural selective antagonist for a restricted number of FGF family members, inhibiting FGF2 but not FGF1 and FGF4 activity. Here, we extended these findings and assessed the capacity of PTX3 to antagonize also FGF8b and to inhibit the vascularization and growth of steroid hormone-regulated breast and prostate cancers. Surface plasmon resonance analysis demonstrates that PTX3 binds FGF8b with high affinity. As a consequence, PTX3 prevents the binding of FGF8b to its receptors, inhibits FGF8b-driven ERK1/2 activation, cell proliferation and chemotaxis in endothelial cells, and suppresses FGF8b-induced neovascularization in vivo. Also, PTX3 inhibits testosterone and FGF8b-driven proliferation in androgen-regulated Shionogi 115 (S115) mouse breast and LNCaP human prostate tumor cells and the FGF8b/FGF2-driven proliferation of TRAMP-C2 murine prostate cancer cells. Furthermore, when transfected into S115 or TRAMP-C2 cells, PTX3 impairs FGF/testosterone induced cell proliferation and angiogenic activity in the chick embryo chorioallantoic membrane (CAM) assay. Accordingly, hPTX3_S115 and hPTX3_TRAMP-C2 cells show a dramatic decrease of their tumorigenic capacity in vivo. These results identify PTX3 as a novel FGF2/FGF8b antagonist endowed with antiangiogenic and antineoplastic activity with possible implications for the therapy of breast and prostate steroid hormone-regulated tumors

Long pentraxin-3 inhibits epithelial-mesenchymal transition in melanoma cells.

During melanoma progression, malignant melanocytes are reprogrammed into mesenchymal-like cells through to an epithelial-mesenchymal transition (EMT) process associated with the acquisition of an invasive, prometastatic phenotype. The fibroblast growth factor-2 (FGF2)/FGF receptor (FGFR) system plays a pivotal role in melanoma, leading to autocrine/paracrine induction of tumor cell proliferation and angiogenesis. Long pentraxin-3 (PTX3) interacts with FGF2, and other FGF family members, inhibiting FGF-dependent neovascularization and tumor growth. Here, PTX3 protein and the PTX3-derived acetylated pentapeptide Ac-ARPCA-NH2 inhibit FGF2-driven proliferation and downstream FGFR signaling in murine melanoma B16-F10 cells. Moreover, human PTX3-overexpressing hPTX_B16-F10 cells are characterized by the reversed transition from a mesenchymal to an epithelial-like appearance, inhibition of cell proliferation, loss of clonogenic potential, reduced motility and invasive capacity, downregulation of various mesenchymal markers, and upregulation of the epithelial marker E-cadherin. Accordingly, PTX3 affects cell proliferation and EMT transition in human A375 and A2058 melanoma cells. Also, hPTX_B16-F10 cells showed a reduced tumorigenic and metastatic activity in syngeneic C57BL/6 mice. In conclusion, PTX3 inhibits FGF/FGFR-driven EMT in melanoma cells, hampering their tumorigenic and metastatic potential. These data represent the first experimental evidence about a nonredundant role of the FGF/FGFR system in the modulation of the EMT process in melanoma and indicate that PTX3 or its derivatives may represent the basis for the design of novel therapeutic approaches in FGF/FGFR-dependent tumors, including melanoma

Long Pentraxin 3/TSG-6 Interaction: A Biological Rheostat for Fibroblast Growth Factor 2–Mediated Angiogenesis

Objective—Angiogenesis is regulated by the balance between pro- and antiangiogenic factors and by extracellular matrix protein interactions. Fibroblast growth factor 2 (FGF2) is a major proangiogenic inducer inhibited by the interaction with the soluble pattern recognition receptor long pentraxin 3 (PTX3). PTX3 is locally coexpressed with its ligand tumor necrosis factor-stimulated gene-6 (TSG-6), a secreted glycoprotein that cooperates with PTX3 in extracellular matrix assembly. Here, we characterized the effect of TSG-6 on PTX3/FGF2 interaction and FGF2-mediated angiogenesis. Methods and Results—Solid phase binding and surface plasmon resonance assays show that TSG-6 and FGF2 bind the PTX3 N-terminal domain with similar affinity. Accordingly, TSG-6 prevents FGF2/PTX3 interaction and suppresses the inhibition exerted by PTX3 on heparan sulfate proteoglycan/FGF2/FGF receptor complex formation and on FGF2-dependent angiogenesis in vitro and in vivo. Also, endogenous PTX3 exerts an inhibitory effect on vascularization induced by FGF2 in a murine subcutaneous Matrigel plug assay, the inhibition being abolished in Ptx3-null mice or by TSG-6 treatment in wild-type animals. Conclusion—TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of FGF2/PTX3 interaction. This may provide a novel mechanism to control angiogenesis in those pathological settings characterized by the coexpression of TSG-6 and PTX3, in which the relative levels of these proteins may fine-tune the angiogenic activity of FGF2