Publications by authors named "Maria Vincenza Carriero"

48 Publications

A novel smaller β-defensin-derived peptide is active against multidrug-resistant bacterial strains.

FASEB J 2021 12;35(12):e22026

Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human β-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the β-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human β-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.202002330RRDOI Listing
December 2021

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells.

Cancers (Basel) 2021 Jun 30;13(13). Epub 2021 Jun 30.

Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS 'Fondazione G. Pascale', 80131 Naples, Italy.

Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients' outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan-Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13133298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8268435PMC
June 2021

Genome-wide analysis of copy number alterations led to the characterisation of PDCD10 as oncogene in ovarian cancer.

Transl Oncol 2021 Mar 27;14(3):101013. Epub 2021 Jan 27.

Department of Experimental and Clinical Medicine, "Magna Graecia", University Catanzaro, Italy. Electronic address:

Copy Number Alterations (CNAs) represent the most common genetic alterations identified in ovarian cancer cells, being responsible for the extensive genomic instability observed in this cancer. Here we report the identification of CNAs in a cohort of Italian patients affected by ovarian cancer performed by SNP-based array. Our analysis allowed the identification of 201 significantly altered chromosomal bands (70 copy number gains; 131 copy number losses). The 3300 genes subjected to CNA identified here were compared to those present in the TCGA dataset. The analysis allowed the identification of 11 genes with increased CN and mRNA expression (PDCD10, EBAG9, NUDCD1, ENY2, CSNK2A1, TBC1D20, ZCCHC3, STARD3, C19orf12, POP4, UQCRFS1). PDCD10 was selected for further studies because of the highest frequency of CNA. PDCD10 was found, by immunostaining of three different Tissue Micro Arrays, to be over-expressed in the majority of ovarian primary cancer samples and in metastatic lesions. Moreover, significant correlations were found in specific subsets of patients, between increased PDCD10 expression and grade (p < 0.005), nodal involvement (p < 0.05) or advanced FIGO stage (p < 0.01). Finally, manipulation of PDCD10 expression by shRNA in ovarian cancer cells (OVCAR-5 and OVCA429) demonstrated a positive role for PDCD10 in the control of cell growth and motility in vitro and tumorigenicity in vivo. In conclusion, this study allowed the identification of novel genes subjected to copy number alterations in ovarian cancer. In particular, the results reported here point to a prominent role of PDCD10 as a bona fide oncogene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tranon.2021.101013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846933PMC
March 2021

The Emerging Role of Neutrophil Extracellular Traps (NETs) in Tumor Progression and Metastasis.

Front Immunol 2020 16;11:1749. Epub 2020 Sep 16.

Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale", Naples, Italy.

Neutrophil Extracellular Traps (NETs) are net-like structures composed of DNA-histone complexes and proteins released by activated neutrophils. In addition to their key role in the neutrophil innate immune response, NETs are also involved in autoimmune diseases, like systemic lupus erythematosus, rheumatoid arthritis, psoriasis, and in other non-infectious pathological processes, as coagulation disorders, thrombosis, diabetes, atherosclerosis, vasculitis, and cancer. Recently, a large body of evidence indicates that NETs are involved in cancer progression and metastatic dissemination, both in animal models and cancer patients. Interestingly, a close correlation between cancer cell recruitment of neutrophils in the tumor microenvironment (Tumor Associated Neutrophils. TANs) and NET formation has been also observed either in primary tumors and metastatic sites. Moreover, NETs can also catch circulating cancer cells and promote metastasis. Furthermore, it has been reported that wake dormant cancer cells, causing tumor relapse and metastasis. This review will primarily focus on the pro-tumorigenic activity of NETs in tumors highlighting their ability to serve as a potential target for cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.01749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524869PMC
April 2021

MicroRNAs as Key Players in Melanoma Cell Resistance to MAPK and Immune Checkpoint Inhibitors.

Int J Mol Sci 2020 Jun 26;21(12). Epub 2020 Jun 26.

Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS 'Fondazione G. Pascale', 80131 Naples, Italy.

Advances in the use of targeted and immune therapies have revolutionized the clinical management of melanoma patients, prolonging significantly their overall and progression-free survival. However, both targeted and immune therapies suffer limitations due to genetic mutations and epigenetic modifications, which determine a great heterogeneity and phenotypic plasticity of melanoma cells. Acquired resistance of melanoma patients to inhibitors of BRAF (BRAFi) and MEK (MEKi), which block the mitogen-activated protein kinase (MAPK) pathway, limits their prolonged use. On the other hand, immune checkpoint inhibitors improve the outcomes of patients in only a subset of them and the molecular mechanisms underlying lack of responses are under investigation. There is growing evidence that altered expression levels of microRNAs (miRNA)s induce drug-resistance in tumor cells and that restoring normal expression of dysregulated miRNAs may re-establish drug sensitivity. However, the relationship between specific miRNA signatures and acquired resistance of melanoma to MAPK and immune checkpoint inhibitors is still limited and not fully elucidated. In this review, we provide an updated overview of how miRNAs induce resistance or restore melanoma cell sensitivity to mitogen-activated protein kinase inhibitors (MAPKi) as well as on the relationship existing between miRNAs and immune evasion by melanoma cell resistant to MAPKi.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21124544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352536PMC
June 2020

Next-Generation Sequencing Approaches for the Identification of Pathognomonic Fusion Transcripts in Sarcomas: The Experience of the Italian ACC Sarcoma Working Group.

Front Oncol 2020 15;10:489. Epub 2020 Apr 15.

Department of Research, Diagnosis and Innovative Technology, IRCCS Regina Elena National Cancer Institute, Rome, Italy.

This work describes the set-up of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2020.00489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175964PMC
April 2020

Inhibiting Monocyte Recruitment to Prevent the Pro-Tumoral Activity of Tumor-Associated Macrophages in Chondrosarcoma.

Cells 2020 04 24;9(4). Epub 2020 Apr 24.

Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS 'Fondazione G. Pascale', Naples 80131, Italy.

Chondrosarcomas (CHS) are malignant cartilaginous neoplasms with diverse morphological features, characterized by resistance to chemo- and radiation therapies. In this study, we investigated the role of tumor-associated macrophages (TAM)s in tumor tissues from CHS patients by immunohistochemistry. Three-dimensional organotypic co-cultures were set up in order to evaluate the contribution of primary human CHS cells in driving an M2-like phenotype in monocyte-derived primary macrophages, and the capability of macrophages to promote growth and/or invasiveness of CHS cells. Finally, with an in vivo model of primary CHS cells engrafted in nude mice, we tested the ability of a potent peptide inhibitor of cell migration (Ac-d-Tyr-d-Arg-Aib-d-Arg-NH, denoted RI-3) to reduce recruitment and infiltration of monocytes into CHS neoplastic lesions. We found a significant correlation between alternatively activated M2 macrophages and intratumor microvessel density in both conventional and dedifferentiated CHS human tissues, suggesting a link between TAM abundance and vascularization in CHS. In 3D and non-contact cu-culture models, soluble factors produced by CHS induced a M2-like phenotype in macrophages that, in turn, increased motility, invasion and matrix spreading of CHS cells. Finally, we present evidence that RI-3 successfully prevent both recruitment and infiltration of monocytes into CHS tissues, in nude mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9041062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226304PMC
April 2020

Effects of S‑adenosyl‑L‑methionine on the invasion and migration of head and neck squamous cancer cells and analysis of the underlying mechanisms.

Int J Oncol 2020 05 12;56(5):1212-1224. Epub 2020 Mar 12.

Dipartimento di Medicina di Precisione, Università della Campania 'Luigi Vanvitelli', I‑80138 Napoli, Italy.

S‑Adenosyl‑L‑methionine (AdoMet) is the principal methyl donor in transmethylation reactions fundamental to sustaining epigenetic modifications. Over the past decade, AdoMet has been extensively investigated for its anti‑proliferative, pro‑apoptotic and anti‑metastatic roles in several types of human cancer. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide, and is an aggressive type of cancer that is associated with a high recurrence rate, metastasis and poor treatment outcomes. The present study demonstrates, for the first time, to the best of our knowledge, that AdoMet induces cell cycle arrest and inhibits the migratory and invasive ability of two different HNSCC cell lines, oral Cal‑33 and laryngeal JHU‑SCC‑011 cells. In both cell lines, AdoMet attenuated cell cycle progression, decreased the protein level of several cyclins and downregulated the expression of p21 cell cycle inhibitor. Moreover, AdoMet was able to inhibit Cal‑33 and JHU‑SCC‑011 cell migration in a dose‑dependent manner after 24 and 48 h, respectively, and also induced a significant reduction in the cell invasive ability, as demonstrated by Matrigel invasion assay monitored by the xCELLigence RTCA system. Western blot analysis of several migration and invasion markers confirmed the inhibitory effects exerted by AdoMet on these processes and highlighted AKT, β‑catenin and small mothers against decapentaplegic (SMAD) as the main signaling pathways modulated by AdoMet. The present study also demonstrated that the combination of AdoMet and cisplatin synergistically inhibited HNSCC cell migration. Taken together, these findings demonstrate that the physiological compound, AdoMet, affects the motility and extracellular matrix invasive capability in HNSCC. Thus, AdoMet may prove to be a good candidate for future drug development against metastatic cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/ijo.2020.5011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115356PMC
May 2020

Tumor Associated Neutrophils. Their Role in Tumorigenesis, Metastasis, Prognosis and Therapy.

Front Oncol 2019 15;9:1146. Epub 2019 Nov 15.

Tumor Progression Unit, Department of Experimental Oncology, Istituto Nazionale Tumori Fondazione "G. Pascale" IRCCS, Naples, Italy.

Tumor Associated Neutrophils (TANs) are engaged into the tumor microenvironment by cytokines and chemokines, can be distinguished according to their activation and cytokine status and effects on tumor cell growing in N1 and N2 TANs. N1 TANs exert an antitumor activity, by direct or indirect cytotoxicity. N2 TANs stimulate immunosuppression, tumor growth, angiogenesis and metastasis by DNA instability, or by cytokines and chemokines release. In tumor patients, either a high number of TANs and Neutrophil-to-Lymphocyte Ratio (NLR) do correlate with poor prognosis, and, so far, TAN counts and NLR can be regarded as biomarkers. Owing to the pivotal role of TANs in stimulating tumor progression, therapeutic strategies to target TANs have been suggested, and two major approaches have been proposed: (a) targeting the CXCL-8/CXCR-1/CXCR-2 axis, thereby blocking TANs or (b) targeting substances produced by polymorpho-nuclear cells that promote tumor growth. Many studies have been accomplished either and in animal models, whereas clinical studies are restrained, presently, due to the risk of inducing immunosuppression. In this review, we deeply discuss the anti-tumorigenic or pro-tumorigenic activity of TANs. In particular, TANs relevance in tumor prognosis and therapeutic strategies are widely described. On-going clinical trials, aimed to inhibit neutrophil recruitment into the tumor are also accurately debated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2019.01146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874146PMC
November 2019

Targeting the Formyl Peptide Receptor type 1 to prevent the adhesion of ovarian cancer cells onto mesothelium and subsequent invasion.

J Exp Clin Cancer Res 2019 Nov 8;38(1):459. Epub 2019 Nov 8.

Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS 'Fondazione G. Pascale', Via M.Semmola, 80131, Naples, Italy.

Background: The biological behavior of epithelial ovarian cancer (EOC) is unique since EOC cells metastasize early to the peritoneum. Thereby, new anti-target agents designed to block trans-coelomic dissemination of EOC cells may be useful as anti-metastatic drugs. The Urokinase Plasminogen Activator Receptor (uPAR) is overexpressed in EOC tissues, and its truncated forms released in sera and/or ascitic fluid are associated with poor prognosis and unfavorable clinical outcome. We documented that uPAR triggers intra-abdominal dissemination of EOC cells through the interaction of its 84-95 sequence with the Formyl Peptide Receptor type 1 (FPR1), even as short linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY). While the pro-metastatic role of uPAR is well documented, little information regarding the expression and role of FPR1 in EOC is currently available.

Methods: Expression levels of uPAR and FPR1 in EOC cells and tissues were assessed by immunofluorescence, Western blot, or immunohystochemistry. Cell adhesion to extra-cellular matrix proteins and mesothelium as well as mesothelium invasion kinetics by EOC cells were monitored using the xCELLigence technology or assessed by measuring cell-associated fluorescence. Cell internalization of FPR1 was identified on multiple z-series by confocal microscopy. Data from in vitro assays were analysed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Tissue microarray data were analyzed with the Pearson's Chi-square (χ) test.

Results: Co-expression of uPAR and FPR1 by SKOV-3 and primary EOC cells confers a marked adhesion to vitronectin. The extent of cell adhesion decreases to basal level by pre-exposure to anti-uPAR84-95 Abs, or to the RI-3 peptide, blocking the uPAR84-95/FPR1 interaction. Furthermore, EOC cells exposed to RI-3 or desensitized with an excess of SRSRY, fail to adhere also to mesothelial cell monolayers, losing the ability to cross them. Finally, primary and metastatic EOC tissues express a high level of FPR1.

Conclusions: Our findings identify for the first time FPR1 as a potential biomarker of aggressive EOC and suggests that inhibitors of the uPAR84-95/FPR1 crosstalk may be useful for the treatment of metastatic EOC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13046-019-1465-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839174PMC
November 2019

Structure-function relationship of an Urokinase Receptor-derived peptide which inhibits the Formyl Peptide Receptor type 1 activity.

Sci Rep 2019 08 21;9(1):12169. Epub 2019 Aug 21.

Neoplastic Progression Unit, Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale", Naples, Italy.

The interaction between the short 88Ser-Arg-Ser-Arg-Tyr92 sequence of the urokinase receptor (uPAR) and the formyl peptide receptor type 1 (FPR1) elicits cell migration. We generated the Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide which inhibits the uPAR/FPR1 interaction, reducing migration of FPR1 expressing cells toward N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Ser-Arg-Ser-Arg-Tyr (SRSRY) peptides. To understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and SRSRY display the same agonist activation signature (i.e. the series of contacts that transmit the conformational transition throughout the complex), translating binding into signaling, RI-3 does not interact with the activation region of FPR1 and hence does not activate signaling. Indeed, fluorescein-conjugated RI-3 prevents either fMLF and SRSRY uptake on FPR1 without triggering FPR1 internalization and cell motility in the absence of any stimulus. Collectively, our data show that RI-3 is a true FPR1 antagonist and suggest a pharmacophore model useful for development of compounds that selectively inhibit the uPAR-triggered, FPR1-mediated cell migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-47900-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704176PMC
August 2019

Role of Microenvironment on the Fate of Disseminating Cancer Stem Cells.

Front Oncol 2019 21;9:82. Epub 2019 Feb 21.

IRCCS Istituto Nazionale Tumori, Fondazione G. Pascale, Naples, Italy.

Disseminating Cancer Stem Cells (CSCs) initiate growth in specific niches of the host tissues, the cellular and molecular components of which sustain signaling pathways that support their survival, self-renewal dormancy and reactivation. In the metastatic niche, tumor cells may enter in a dormant state to survive and, consequently, the metastasis can remain latent for years. Despite the clinical importance of metastatic latency, little is known about what induces CSCs to enter a dormant state and what allows them to remain viable for years in this state. CSCs exhibit genetic, epigenetic and cellular adaptations that confer resistance to classical therapeutic approaches. The identification of potential CSC targets is complicated by the fact that CSCs may arise as a consequence of their relationship with the local microenvironment into the metastatic niches. Indeed, microenvironment modulates the capability of CSCs to evade the innate immune response and survive. Some new therapeutic options that include drugs targeting microenvironment components are achieving encouraging results in reducing the number of CSCs in tumors and/or overcoming their resistance in preclinical studies. This review will focus on specific CSC features with an emphasis on the role of tumor microenvironment in supporting metastatic dissemination of CSCs. In addition, it sheds light on potential microenvironment-targeted therapies aimed to counteract seeding and survival of CSCs in the metastatic niche.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2019.00082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393337PMC
February 2019

Reprogramming miRNAs global expression orchestrates development of drug resistance in BRAF mutated melanoma.

Cell Death Differ 2019 07 25;26(7):1267-1282. Epub 2018 Sep 25.

IRCCS, Regina Elena National Cancer Institute, Rome, Italy.

Drug resistance imposes severe limitations to the efficacy of targeted therapy in BRAF-mutated metastatic melanoma. Although this issue has been mitigated by the development of combination therapies with BRAF plus MEK inhibitors, drug resistance inevitably occurs with time and results in clinical recurrences and untreatable disease. Hence, there is strong need of developing new combination therapies and non-invasive diagnostics for the early identification of drug-resistant patients. We report here that the development of drug resistance to BRAFi is dominated by a dynamic deregulation of a large population of miRNAs, leading to the alteration of cell intrinsic proliferation and survival pathways, as well as of proinflammatory and proangiogenic cues, where a prominent role is played by the miR-199b-5p/VEGF axis. Significant alterations of miRNA expression levels are detectable in tumor biopsies and plasma from patients after disease recurrence. Targeting these alterations blunts the development of drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41418-018-0205-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748102PMC
July 2019

Neutrophil Extracellular Traps as an Adhesion Substrate for Different Tumor Cells Expressing RGD-Binding Integrins.

Int J Mol Sci 2018 Aug 9;19(8). Epub 2018 Aug 9.

Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, 80145 Naples, Italy.

Neutrophil extracellular traps (NETs), in addition to their function as a host defense mechanism, play a relevant role in thrombus formation and metastatic dissemination of cancer cells. Here we screened different cancer cell lines endogenously expressing a variety of integrins for their ability to bind to NETs. To this end, we used NETs isolated from neutrophil-like cells as a substrate for adhesion assays of HT1080, U-87 MG, H1975, DU 145, PC-3 and A-431 cells. Levels of α5, αIIb, αv, β1, β3 and β5 chains were determined by western blot analysis in all cell lines and levels of whole integrins on the plasma membrane were assessed by fluorescence-activated cell sorting (FACS) analysis. We found that high levels of α5β1, αvβ3 and αvβ5 enhance cell adhesion to NETs, whereas low expression of α5β1 prevents cell attachment to NETs. Excess of cyclic RGD peptide inhibited cell adhesion to NETs by competing with fibronectin within NETs. The maximal reduction of such adhesion was similar to that obtained by DNase 1 treatment causing DNA degradation. Our findings indicate that NETs from neutrophil-like cells may be used as a substrate for large screening of the adhesion properties of cancer cells expressing a variety of RGD-binding integrins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms19082350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121671PMC
August 2018

Effect of ABT-888 on the apoptosis, motility and invasiveness of BRAFi-resistant melanoma cells.

Int J Oncol 2018 Sep 27;53(3):1149-1159. Epub 2018 Jun 27.

Istituto Nazionale Tumori -IRCCS- 'Fondazione G. Pascale', 80131 Naples, Italy.

Melanoma is a molecularly heterogeneous disease with many genetic mutations and altered signaling pathways. Activating mutations in the BRAF oncogene are observed in approximately 50% of cutaneous melanomas and the use of BRAF inhibitor (BRAFi) compounds has been reported to improve the outcome of patients with BRAF-mutated metastatic melanoma. However, the majority of these patients develop resistance within 6-8 months following the initiation of BRAFi treatment. In this study, we examined the possible use of the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, ABT-888 (veliparib), as a novel molecule that may be successfully employed in the treatment of BRAFi-resistant melanoma cells. Sensitive and resistant to BRAFi dabrafenib A375 cells were exposed to increasing concentrations of ABT-888. Cell viability and apoptosis were assessed by MTT assay and Annexin V-FITC analysis, respectively. The cell migratory and invasive ability was investigated using the xCELLigence technology and Boyden chamber assays, respectively. ABT-888 was found to reduce cell viability and exhibited pro-apoptotic activity in melanoma cell lines, independently from the BRAF/NRAS mutation status, in a dose-dependent manner, with the maximal effect being reached in the 25-50 µM concentration range. Moreover, ABT-888 promoted apoptosis in both the sensitive and resistant A375 cells, suggesting that ABT-888 may be useful in the treatment of BRAFi-resistant subsets of melanoma cells. Finally, in accordance with the involvement of PARP1 in actin cytoskeletal machinery, we found that the cytoskeletal organization, motility and invasive capability of both the A375 and A375R cells decreased upon exposure to 5 µM ABT-888 for 24 h. On the whole, the findings of this study highlight the pivotal role of PARP1 in the migration and invasion of melanoma cells, suggesting that ABT-888 may indeed be effective, not only as a pro-apoptotic drug for use in the treatment of BRAFi-resistant melanoma cells, but also in suppressing their migratory and invasive activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3892/ijo.2018.4457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065454PMC
September 2018

Controversial Role of Kisspeptins/KiSS-1R Signaling System in Tumor Development.

Front Endocrinol (Lausanne) 2018 30;9:192. Epub 2018 Apr 30.

IRCCS Istituto Nazionale Tumori "Fondazione G. Pascale", Naples, Italy.

was first described as a metastasis suppressor gene in malignant melanoma. encodes a 145 amino-acid residue peptide that is further processed, producing the 54 amino acid metastin and shorter peptides collectively named kisspeptins (KPs). KPs bind and activate KiSS-1R (GPR54). Although the KPs system has been extensively studied for its role in endocrinology of reproductive axis in mammals, its role in cancer is still controversial. Experimental evidences show that KP system exerts an anti-metastatic effect by the regulation of cellular migration and invasion in several cancer types. However, the role of KPs/KiSS-1R is very complex. Genomic studies suggest that KiSS-1/KiSS-1R expression might be different in the various stages of tumor development. Furthermore, overexpression of KiSS-1R has been reported to elicit drug resistance in triple negative breast cancer. In this review, we focused on multiple functions exerted by the KPs/KiSS-1R system in regulating tumor progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fendo.2018.00192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5936968PMC
April 2018

Targeting the cross-talk between Urokinase receptor and Formyl peptide receptor type 1 to prevent invasion and trans-endothelial migration of melanoma cells.

J Exp Clin Cancer Res 2017 Dec 8;36(1):180. Epub 2017 Dec 8.

IRCCS Istituto Nazionale Tumori 'Fondazione G. Pascale', Naples, Italy.

Background: Accumulating evidence demonstrates that the Urokinase Receptor (uPAR) regulates tumor cell migration through its assembly in composite regulatory units with transmembrane receptors, and uPAR is the minimal sequence required to induce cell motility through the Formyl Peptide Receptor type 1 (FPR1). Both uPAR and FPR1 are involved in melanoma tumor progression, suggesting that they may be targeted for therapeutic purposes. In this study, the role of the uPAR-FPR1 cross-talk to sustain melanoma cell ability to invade extracellular matrix and cross endothelial barriers is investigated. Also, the possibility that inhibition of the uPAR mediated FPR1-dependent signaling may prevent matrix invasion and transendothelial migration of melanoma cells was investigated.

Methods: Expression levels of uPAR and FPR1 were assessed by immunocytochemistry, Western Blot and qRT-PCR. Cell migration was investigated by Boyden chamber and wound-healing assays. Migration and invasion kinetics, trans-endothelial migration and proliferation of melanoma cells were monitored in real time using the xCELLigence technology. The agonist-triggered FPR1 internalization was visualized by confocal microscope. Cell adhesion to endothelium was determined by fluorometer measurement of cell-associated fluorescence or identified on multiple z-series by laser confocal microscopy. The 3D-organotypic models were set up by seeding melanoma cells onto collagen I matrices embedded dermal fibroblasts. Data were analyzed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons.

Results: We found that the co-expression of uPAR and FPR1 confers to A375 and M14 melanoma cells a clear-cut capability to move towards chemotactic gradients, to cross extracellular matrix and endothelial monolayers. FPR1 activity is required, as cell migration and invasion were abrogated by receptor desensitization. Finally, melanoma cell ability to move toward chemotactic gradients, invade matrigel or fibroblast-embedded collagen matrices and cross endothelial monolayers are prevented by anti-uPAR antibodies or by the RI-3 peptide which we have previously shown to inhibit the uPAR/FPR1 interaction.

Conclusions: Collectively, our findings identify uPAR and FPR1 as relevant effectors of melanoma cell invasiveness and suggest that inhibitors of the uPAR/FPR1 cross-talk may be useful for the treatment of metastatic melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13046-017-0650-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721612PMC
December 2017

Urokinase receptor derived peptides as potent inhibitors of the formyl peptide receptor type 1-triggered cell migration.

Eur J Med Chem 2018 Jan 1;143:348-360. Epub 2017 Dec 1.

Department of Pharmacy, University of Naples 'Federico II', Naples 80131, Italy; Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPEB) University of Naples "Federico II" and DFM-Scarl, Institute of Biostructures and Bioimaging - CNR Via Mezzocannone 16, 80134 Naples, Italy.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration. We and others have previously documented that the uPAR(84-95) sequence, interacts with the formyl peptide receptors (FPR)s, henceforth inducing cell migration of several cell lines, including leukocytes, and the synthetic shorter peptide (Ser-Arg-Ser-Arg-Tyr, SRSRY) retains chemotactic activity in vitro and in vivo. Recently, we have developed the head-to-tail cyclic analog [SRSRY], a new potent and stable inhibitor of monocyte trafficking. This prompted us to develop novel cyclic and linear analogs of [SRSRY] with the aim to broaden the knowledge about structure-activity relationships of peptide [SRSRY]. Herein we report their synthesis, effects on cell migration, conformational and docking analyses which served to envisage a new pharmacophore model for inhibitors of FPR1-triggered cell migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2017.11.030DOI Listing
January 2018

Retro-inverso Urokinase Receptor Antagonists for the Treatment of Metastatic Sarcomas.

Sci Rep 2017 05 2;7(1):1312. Epub 2017 May 2.

Peptipharma, Viale Città D'Europa 679, 00144, Rome, Italy.

The development of metastases is a multistep process that requires the activation of physiological and biochemical processes that govern migration, invasion and entry of metastatic cells into blood vessels. The urokinase receptor (uPAR) promotes cell migration by interacting with the Formyl Peptide Receptors (FPRs). Since both uPAR and FPR1 are involved in tumor progression, the uPAR-FPR1 interaction is an attractive therapeutic target. We previously described peptide antagonists of the uPAR-FPR1 interaction that inhibited cell migration and angiogenesis. To develop enzyme-resistant analogues, we applied here the Retro-Inverso (RI) approach, whereby the topology of the side chains is maintained by inverting the sequence of the peptide and the chirality of all residues. Molecular dynamics suggests that peptide RI-3 adopts the turn structure typical of uPAR-FPR1 antagonists. Accordingly, RI-3 is a nanomolar competitor of N-formyl-Met-Leu-Phe for binding to FPR1 and inhibits migration, invasion, trans-endothelial migration of sarcoma cells and VEGF-triggered endothelial tube formation. When sarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density, circulating tumor cells and pulmonary metastases were significantly reduced in animals treated daily with 6 mg/Kg RI-3 as compared to animals treated with vehicle only. Thus, RI-3 represents a promising lead for anti-metastatic drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-01425-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430962PMC
May 2017

Integrin-dependent cell adhesion to neutrophil extracellular traps through engagement of fibronectin in neutrophil-like cells.

PLoS One 2017 6;12(2):e0171362. Epub 2017 Feb 6.

Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy.

Neutrophil extracellular traps (NETs), originally recognized as a host defense mechanism, were reported to promote thrombosis and metastatic dissemination of cancer cells. Here we tested the role of integrins α5β1 and ανβ3 in the adhesion of cancer cells to NETs. Neutrophil-like cells stimulated with calcium ionophore (A23187) were used as a stable source of cell-free NETs-enriched suspensions. Using NETs as an adhesion substrate, two human K562 cell lines, differentially expressing α5β1 and ανβ3 integrins, were subjected to adhesion assays in the presence or absence of DNAse 1, blocking antibodies against α5β1 or ανβ3, alone or in combination with DNAse 1, and Proteinase K. As expected DNAse 1 treatment strongly inhibited adhesion of both cell lines to NETs. An equivalent significant reduction of cell adhesion to NETs was obtained after treatment of cells with blocking antibodies against α5β1 or ανβ3 indicating that both integrins were able to mediate cell adhesion to NETs. Furthermore, the combination of DNAse 1 and anti-integrin antibody treatment almost completely blocked cell adhesion. Western blot analysis and immunoprecipitation experiments showed a dose-dependent increase of fibronectin levels in samples from stimulated neutrophil-like cells and a direct or indirect interaction of fibronectin with histone H3. Finally, co-immunolocalization studies with confocal microscopy showed that fibronectin and citrullinated histone H3 co-localize inside the web-structure of NETs. In conclusion, our study showed that α5β1 and ανβ3 integrins mediate cell adhesion to NETs by binding to their common substrate fibronectin. Therefore, in addition to mechanical trapping and aspecific adsorption of different cell types driven by DNA/histone complexes, NETs may provide specific binding sites for integrin-mediated cell adhesion of neutrophils, platelets, endothelial and cancer cells thus promoting intimate interactions among these cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171362PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5293257PMC
August 2017

A Macroscopic Mathematical Model for Cell Migration Assays Using a Real-Time Cell Analysis.

PLoS One 2016;11(9):e0162553. Epub 2016 Sep 28.

Istituto per le Applicazioni del Calcolo "M. Picone", Consiglio Nazionale delle Ricerche, Naples, Italy.

Experiments of cell migration and chemotaxis assays have been classically performed in the so-called Boyden Chambers. A recent technology, xCELLigence Real Time Cell Analysis, is now allowing to monitor the cell migration in real time. This technology measures impedance changes caused by the gradual increase of electrode surface occupation by cells during the course of time and provide a Cell Index which is proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number. In this paper we propose a macroscopic mathematical model, based on advection-reaction-diffusion partial differential equations, describing the cell migration assay using the real-time technology. We carried out numerical simulations to compare simulated model dynamics with data of observed biological experiments on three different cell lines and in two experimental settings: absence of chemotactic signals (basal migration) and presence of a chemoattractant. Overall we conclude that our minimal mathematical model is able to describe the phenomenon in the real time scale and numerical results show a good agreement with the experimental evidences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0162553PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040252PMC
September 2016

The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells.

Oncotarget 2016 Aug;7(34):54474-54487

Neoplastic Progression Unit, Department of Experimental Oncology, IRCCS Istituto Nazionale Tumori "Fondazione G. Pascale", Naples, Italy.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.9976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342356PMC
August 2016

Mutant AKT1-E17K is oncogenic in lung epithelial cells.

Oncotarget 2015 Nov;6(37):39634-50

Department of Experimental and Clinical Medicine, University "Magna Graecia", Catanzaro, Italy.

The hotspot E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6-2% of human lung cancers. In this manuscript, we sought to determine whether this AKT1 variant is a bona-fide activating mutation and plays a role in the development of lung cancer. Here we report that in immortalized human bronchial epithelial cells (BEAS-2B cells) mutant AKT1-E17K promotes anchorage-dependent and -independent proliferation, increases the ability to migrate, invade as well as to survive and duplicate in stressful conditions, leading to the emergency of cells endowed with the capability to form aggressive tumours at high efficiency. We provide also evidence that the molecular mechanism whereby AKT1-E17K is oncogenic in lung epithelial cells involves phosphorylation and consequent cytoplasmic delocalization of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these results, cytoplasmic p27 is preferentially observed in primary NSCLCs with activated AKT and predicts poor survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.4022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741851PMC
November 2015

Cyclization of the urokinase receptor-derived ser-arg-ser-arg-tyr Peptide generates a potent inhibitor of trans-endothelial migration of monocytes.

PLoS One 2015 4;10(5):e0126172. Epub 2015 May 4.

Neoplastic Progression Unit, Department of Experimental Oncology, IRCCS Istituto Nazionale Tumori "Fondazione G. Pascale", Naples, Italy.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126172PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418665PMC
April 2016

Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype.

Oncotarget 2015 Mar;6(7):5324-41

Centro Ricerche Oncologiche Mercogliano, Istituto Nazionale Tumori Fondazione G. Pascale - IRCCS, Naples, Italy.

Proteomic analysis identified differentially expressed proteins between zoledronic acid-resistant and aggressive DU145R80 prostate cancer (PCa) cells and their parental DU145 cells. Ingenuity Pathway Analysis (IPA) showed a strong relationship between the identified proteins within a network associated with cancer and with homogeneous cellular functions prevalently related with regulation of cell organization, movement and consistent with the smaller and reduced cell-cell contact morphology of DU145R80 cells. The identified proteins correlated in publically available human PCa genomic data with increased tumor expression and aggressiveness. DU145R80 exhibit also a clear increase of alpha-v-(αv) integrin, and of urokinase receptor (uPAR), both included within the same network of the identified proteins. Interestingly, the actin-rich structures localized at the cell periphery of DU145R80 cells are rich of Filamin A, one of the identified proteins and uPAR which, in turn, co-localizes with αv-integrin, in podosomes and/or invadopodia. Notably, the invasive feature of DU145R80 may be prevented by blocking anti-αv antibody. Overall, we unveil a signaling network that physically links the interior of the nucleus via the cytoskeleton to the extracellular matrix and that could dictate PCa aggressiveness suggesting novel potential prognostic markers and therapeutic targets for PCa patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467152PMC
http://dx.doi.org/10.18632/oncotarget.2694DOI Listing
March 2015

AurkA inhibitors enhance the effects of B-RAF and MEK inhibitors in melanoma treatment.

J Transl Med 2014 Jul 31;12:216. Epub 2014 Jul 31.

Background: Aurora kinase A (AurkA) is over-expressed in melanoma and its inhibition has been observed to limit tumor growth, suggesting a potential role in melanoma treatment.

Methods: A human melanoma cell line with the B-RAF (V600E) mutation (A375mel) was exposed to B-RAF inhibitor (GSK2118436), MEK inhibitor (GSK1120212) and AurkA inhibitor (MLN8054) as single agents or in various combinations (BRAF plus AurkA inhibitor, MEK plus AurkA inhibitor or triple combination BRAF plus MEK plus AurkA inhibitor). Cell proliferation was assessed using xCELLigence technology. Total protein extracts were examined for p53 and c-Myc protein expression by Western blot analysis. Drug anti-tumor effects were further assessed using a 3D-human melanoma skin reconstruction model, in which tissues were incubated with serum-free medium containing control, B-RAF plus MEK inhibitor, MEK plus AurkA inhibitor or the triple combination.

Results: AurkA inhibitor plus B-RAF inhibitor, AurkA inhibitor plus MEK inhibitor or triple combination had a markedly greater anti-proliferative effect on A375 (BRAFV600E) melanoma cells than single agents. In the 3D human skin model, the triple combination had a greater anti-tumor effect at the epidermal/dermal junction than control or either double combination. However, S-100 and Ki-67 positively stained spindle-shaped cells were detected in the dermal stratum, suggesting the presence of alive and proliferating melanoma cells.

Conclusions: These findings provide new prospects for melanoma research, including combined B-RAF/AurkA inhibition for B-RAF mutated melanomas and MEK/AurkA inhibitor combination for patients without B-RAF mutations. Moreover, for the first time, we have shown that a B-RAF, MEK and AurkA inhibitor triple drug combination offers increased efficacy against melanoma cell growth and might be considered as a potential treatment strategy for enhancing clinical response in melanoma. However, although this triple drug combination was more effective at the epidermal/dermal junction, the suggested presence of alive and proliferating melanoma cells in the dermal stratum could result in drug resistance and disease recurrence. Molecular characterization of these dermal cells may be critical for the development of novel therapeutic strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12967-014-0216-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237855PMC
July 2014

Urokinase receptor promotes ovarian cancer cell dissemination through its 84-95 sequence.

Oncotarget 2014 Jun;5(12):4154-69

Department of Experimental Oncology Unit, IRCCS Istituto Nazionale Tumori "Fondazione G. Pascale", Naples, Italy. These authors contributed equally.

The clinical relevance of the urokinase receptor (uPAR) as a prognostic marker in ovarian cancer is well documented. We had shown that the uPAR sequence corresponding to 84-95 residues, linking D1 and D2 domains (uPAR84-95), drives cell migration and angiogenesis in a protease-independent manner. This study was aimed at defining the contribution of uPAR84-95 sequence to invasion of ovarian cancer cells. Now, we provide evidence that the ability of uPAR-expressing ovarian cancer cells to cross extra-cellular matrix and mesothelial monolayers is prevented by specific inhibitors of the uPAR84-95 sequence. To specifically investigate uPAR84-95 function, uPAR-negative CHO-K1 cells were stably transfected with cDNAs coding for uPAR D2 and D3 regions exposing (uPARD2D3) or lacking (uPAR∆D2D3) the 84-95 sequence. CHO-K1/D2D3 cells were able to cross matrigel, mesothelial and endothelial monolayers more efficiently than CHO-K1/∆D2D3 cells, which behave as CHO-K1 control cells. When orthotopically implanted in nude mice, tumor nodules generated by CHO-K1/D2D3 cells spreading to peritoneal cavity were more numerous as compared to CHO-K1/∆D2D3 cells. Ovarian tumor size and intra-tumoral microvessel density were significantly reduced in the absence of uPAR84-95. Our results indicate that cell associated uPAR promotes growth and abdominal dissemination of ovarian cancer cells mainly through its uPAR84-95 sequence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.1930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4147313PMC
June 2014

UPARANT: a urokinase receptor-derived peptide inhibitor of VEGF-driven angiogenesis with enhanced stability and in vitro and in vivo potency.

Mol Cancer Ther 2014 May 4;13(5):1092-104. Epub 2014 Apr 4.

Authors' Affiliations: Department of Experimental Oncology, Istituto Nazionale Tumori "Fondazione G. Pascale"-IRCCS; Department of Chemical Sciences, "Federico II" University of Naples; IBB-National Research Council Naples; and Department of Experimental Medicine, Second University of Naples, Naples, Italy.

This work is based on previous evidence showing that chemotactic sequence of the urokinase receptor (uPAR(88-92)) drives angiogenesis in vitro and in vivo in a protease-independent manner, and that the peptide Ac-Arg-Glu-Arg-Phe-NH(2) (RERF) prevents both uPAR(88-92)- and VEGF-induced angiogenesis. New N-acetylated and C-amidated peptide analogues containing α-methyl α-amino acids were designed and synthesized to optimize the biochemical properties for therapeutic applications. Among these, Ac-L-Arg-Aib-L-Arg-D-Cα(Me)Phe-NH2, named UPARANT, adopts in solution a turned conformation similar to that found for RERF, is stable to sterilization in 3 mg/mL sealed vials in autoclave for 20 minutes at 120°C, is stable in blood, and displays a long-time resistance to enzymatic proteolysis. UPARANT competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the formyl-peptide receptor, inhibits VEGF-directed endothelial cell migration, and prevents cytoskeletal organization and αvβ3 activation in endothelial cells exposed to VEGF. In vitro, UPARANT inhibits VEGF-dependent tube formation of endothelial cells at a 100× lower concentration than RERF. In vivo, UPARANT reduces to the basal level VEGF-dependent capillary sprouts originating from the host vessels that invaded Matrigel sponges implanted in mice, and completely prevents neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Both excellent stability and potency position UPARANT as a promising new therapeutic agent for the control of diseases fueled by excessive angiogenesis, such as cancer and inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-13-0949DOI Listing
May 2014

A urokinase receptor-derived peptide inhibiting VEGF-dependent directional migration and vascular sprouting.

Mol Cancer Ther 2013 Oct 12;12(10):1981-93. Epub 2013 Aug 12.

Corresponding Author: Maria Vincenza Carriero, Department of Experimental Oncology-National Cancer Institute of Naples, via M. Semmola, Naples 80131, Italy.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration, and uPAR₈₈₋₉₂ is the minimal sequence required to induce cell motility. We previously showed that soluble forms of uPAR elicit angiogenic responses through their uPAR₈₈₋₉₂ chemotactic sequence and that the synthetic peptide SRSRY exerts similar effects. By a drug design approach, based on the conformational analysis of the uPAR₈₈₋₉₂ sequence, we developed peptides (pERERY, RERY, and RERF) that potently inhibit signaling triggered by uPAR₈₈₋₉₂. In this study, we present evidence that these peptides are endowed also with a clear-cut antiangiogenic activity, although to different extents. The most active, RERF, prevents tube formation by human endothelial cells exposed to SRSRY. RERF also inhibits VEGF-triggered endothelial cell migration and cord-like formation in a dose-dependent manner, starting in the femtomolar range. RERF prevents F-actin polymerization, recruitment of αvβ3 integrin at focal adhesions, and αvβ3/VEGFR2 complex formation in endothelial cells exposed to VEGF. At molecular level, the inhibitory effect of RERF on VEGF signaling is shown by the decreased amount of phospho-FAK and phospho-Akt in VEGF-treated cells. In vivo, RERF prevents VEGF-dependent capillary sprouts originating from the host vessels that invaded angioreactors implanted in mice and neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Consistently, RERF reduced the growth and vascularization rate of tumors formed by HT1080 cells injected subcutaneously in the flanks of nude mice, indicating that RERF is a promising therapeutic agent for the control of diseases fuelled by excessive angiogenesis such as cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-13-0077DOI Listing
October 2013
-->