A neuroligin-2-YAP axis regulates progression of pancreatic intraepithelial neoplasia.

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a dismal prognosis that arises from precursor lesions called pancreatic intraepithelial neoplasias (PanINs). Progression from low- to high-grade PanINs is considered as tumor initiation, and a deeper understanding of this switch is needed. Here, we show that synaptic molecule neuroligin-2 (NLGN2) is expressed by pancreatic exocrine cells and plays a crucial role in the regulation of contact inhibition and epithelial polarity, which characterize the switch from low- to high-grade PanIN. NLGN2 localizes to tight junctions in acinar cells, is diffusely distributed in the cytosol in low-grade PanINs and is lost in high-grade PanINs and in a high percentage of advanced PDACs. Mechanistically, NLGN2 is necessary for the formation of the PALS1/PATJ complex, which in turn induces contact inhibition by reducing YAP function. Our results provide novel insights into NLGN2 functions outside the nervous system and can be used to model PanIN progression.

Role of TGFß1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells.

Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFß1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFß1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFß1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFß1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFß1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

An Overview of the Molecular Cues and Their Intracellular Signaling Shared by Cancer and the Nervous System: From Neurotransmitters to Synaptic Proteins, Anatomy of an All-Inclusive Cooperation.

We propose an overview of the molecular cues and their intracellular signaling involved in the crosstalk between cancer and the nervous system. While "cancer neuroscience" as a field is still in its infancy, the relation between cancer and the nervous system has been known for a long time, and a huge body of experimental data provides evidence that tumor-nervous system connections are widespread. They encompass different mechanisms at different tumor progression steps, are multifaceted, and display some intriguing analogies with the nervous system's physiological processes. Overall, we can say that many of the paradigmatic "hallmarks of cancer" depicted by Weinberg and Hanahan are affected by the nervous system in a variety of manners.

Modulation of Angiopoietin 2 release from endothelial cells and angiogenesis by the synaptic protein Neuroligin 2.

The synaptic protein Neuroligin 2, similarly to its isoform Neuroligin 1, is produced by endothelial cells, but its activity in the vascular context remains unknown. This study aimed at verifying the hypothesis that Neuroligin 2, in parallel with its extraneuronal involvement in pancreatic beta cells exocytosis, modulated cytokine release from endothelial cells and consequently angiogenesis. We used in vitro approaches to modulate Neuroligin 2 expression and Neuroligin 2 null mice to test our hypotheses. In vitro, upon VEGF stimulation, Neuroligin 2 silencing strongly reduces Angiopoietin 2 release in the medium and increases the endothelial cell retention of Weibel Palade Bodies, the specialized organelles that store Angiopoietin 2 and various other cytokines. On the contrary, Neuroligin 2 overexpression almost depletes cells of Weibel Palade Bodies, independent of VEGF. In vivo, both the retina and tumor xenografts grown in NLGN2- null mice display an immature vasculature, with lower pericyte coverage and lower Tie2 phosphorylation. At the molecular level NLGN2 colocalizes with its neuronal partner collibystin, a CDC42 guanine nucleotide exchange factor, which is also expressed by endothelial cells and in turn modulates Angiopoietin 2 release. Neuroligin 2, an inhibitory synaptic protein, modulates a peculiar aspect of vascular function and could represent a novel target of therapy in various fields, from tumor angiogenesis to vascular diseases.

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

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

A peptide from the extracellular region of the synaptic protein α Neurexin stimulates angiogenesis and the vascular specific tyrosine kinase Tie2.

Neurexin (NRXN) and Neuroligin (NLGN) are trans-synaptic proteins involved in vascular biology. NRXN is encoded in long (α) and short (ß) isoforms. We have shown that ßNRXN modulates blood vessel development in synergy with VEGFA and associates with NLGN. On the other hand αNRXN is also expressed in blood vessels but does not interact with NLGN or act in synergy with VEGFA, thus demonstrating a differential role. To find clues into the vascular functions of αNRXN, we chose a 7 aa motif that is part of its extracellular region and was formerly selected through a proteomic search for interactors of the vascular receptor Tie2. Next we (a) synthetized and modeled such peptide in order to determine its biological activity towards Tie2 in in vitro and in vivo angiogenesis assays and (b) evaluated if αNRXN and Tie2 physically associate in situ during vascular development. We used biochemical and cellular assays to prove that the synthetic αNRXN peptide (a) modulates the angiogenic phenotype of cultured endothelial cells and angiogenesis in vivo and (b) efficiently stimulates Tie2 phosphorylation and downstream mediators in endothelial cells. Moreover, we show that αNRXN and Tie2 can be reciprocally immunoprecipitated from chicken blood vessels at late stages of vascular development. These data have a double significance, i.e. provide a novel tool to modulate Tie2 and further suggest the involvement of the NRXN family of synaptic protein in the vascular system through their interaction with a fundamental vascular player.

The synaptic proteins ß-neurexin and neuroligin synergize with extracellular matrix-binding vascular endothelial growth factor a during zebrafish vascular development.

OBJECTIVE: The goal of this study was to determine the in vivo functions of the synaptic proteins neurexins and neuroligins in embryonic vascular system development using zebrafish as animal model. METHODS AND RESULTS: In the present study, we show that the knockdown of the α-form of neurexin 1a induces balance defects and reduced locomotory activity, whereas ß-neurexin 1a and neuroligin 1 morphants present defects in sprouting angiogenesis and vascular remodeling, in particular in the caudal plexus and subintestinal vessels. Coinjection of low doses of morpholinos for ß-neurexin 1a and neuroligin 1 together or in combination with morpholinos targeting the -heparin--binding isoforms of vascular endothelial growth factor A (encoded by the VEGFAb gene) recapitulates the observed abnormalities, suggesting synergistic activity of these molecules. Similar coinjection experiments with morpholinos, targeting the enzyme heparan sulfate 6-O-sulfotransferase 2, confirm the presence of a functional correlation between extracellular matrix maturation and ß-neurexin 1a or neuroligin 1. CONCLUSIONS: Our data represent the first in vivo evidence of the role of neurexin and neuroligin in embryonic blood vessel formation and provide insights into their mechanism of action.

RNA aptamer-mediated gene therapy of prostate cancer: lessons from the past and future directions.

INTRODUCTION: Prostate cancer (PCa) is one of the most prevalent cancers in the world, and the fifth cause of death from cancer in men. Among the non-surgical treatments for PCa, gene therapy strategies are in the early stages of development and recent clinical trials have provided new insights suggesting promising future. AREAS COVERED: Recently, the creation of targeted gene delivery systems, based on specific PCa cell surface markers, has been viewed as a viable therapeutic approach. Prostate-specific membrane antigen (PSMA) is vastly expressed in nearly all prostate malignancies, and the intensity of expression increases with tumor aggressiveness, androgen independence, and metastasis. RNA aptamers are short and single-stranded oligonucleotides, which selectively bind to a specific ligand on the surface of the cells, which makes them fascinating small molecules for target delivery of therapeutics. PSMA-selective RNA aptamers represent great potential for developing targeted-gene delivery tools for PCa. EXPERT OPINION: This review provides a thorough horizon for the researchers interested in developing targeted gene delivery systems for PCa via PSMA RNA aptamers. In addition, we provided general information about different prospects of RNA aptamers including discovery approaches, stability, safety, and pharmacokinetics.

IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21(Cip1/Waf1)-mediated mechanism.

Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21(Cip1/Waf1), an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21(Cip1/Waf1) and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21(Cip1/Waf1) induced by the combined activity of STAT1, PKCδ and p38 MAPK.

Neurexins and neuroligins: synapses look out of the nervous system.

The scientific interest in the family of the so-called nervous vascular parallels has been growing steadily for the past 15 years, either by addition of new members to the group or, lately, by deepening the analysis of established concepts and mediators. Proteins governing both neurons and vascular cells are known to be involved in events such as cell fate determination and migration/guidance but not in the last and apparently most complex step of nervous system development, the formation and maturation of synapses. Hence, the recent addition to this family of the specific synaptic proteins, Neurexin and Neuroligin, is a double innovation. The two proteins, which were thought to be "simple" adhesive links between the pre- and post-synaptic sides of chemical synapses, are in fact extremely complex and modulate the most subtle synaptic activities. We will discuss the relevant data and the intriguing challenge of transferring synaptic activities to vascular functions.

The synaptic proteins neurexins and neuroligins are widely expressed in the vascular system and contribute to its functions.

Unlike other neuronal counterparts, primary synaptic proteins are not known to be involved in vascular physiology. Here, we demonstrate that neurexins and neuroligins, which constitute large and complex families of fundamental players in synaptic activity, are produced and processed by endothelial and vascular smooth muscle cells throughout the vasculature. Moreover, they are dynamically regulated during vessel remodeling and form endogenous complexes in large vessels as well as in the brain. We used the chicken chorioallantoic membrane as a system to pursue functional studies and demonstrate that a monoclonal recombinant antibody against beta-neurexin inhibits angiogenesis, whereas exogenous neuroligin has a role in promoting angiogenesis. Finally, as an insight into the mechanism of action of beta-neurexin, we show that the anti-beta-neurexin antibody influences vessel tone in isolated chicken arteries. Our finding strongly supports the idea that even the most complex and plastic events taking place in the nervous system (i.e., synaptic activity) share molecular cues with the vascular system.

Tumoral Neuroligin 1 Promotes Cancer-Nerve Interactions and Synergizes with the Glial Cell Line-Derived Neurotrophic Factor.

Many nervous proteins are expressed in cancer cells. In this report, we asked whether the synaptic protein neuroligin 1 (NLGN1) was expressed by prostatic and pancreatic carcinomas; in addition, given the tendency of these tumors to interact with nerves, we asked whether NLGN1 played a role in this process. Through immunohistochemistry on human tissue microarrays, we showed that NLGN1 is expressed by prostatic and pancreatic cancer tissues in discrete stages and tumor districts. Next, we performed in vitro and in vivo assays, demonstrating that NLGN1 promotes cancer cell invasion and migration along nerves. Because of the established role of the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) in tumor-nerve interactions, we assessed a potential NLGN1-GDNF cooperation. We found that blocking GDNF activity with a specific antibody completely inhibited NLGN1-induced in vitro cancer cell invasion of nerves. Finally, we demonstrated that, in the presence of NLGN1, GDNF markedly activates cofilin, a cytoskeletal regulatory protein, altering filopodia dynamics. In conclusion, our data further prove the existence of a molecular and functional cross-talk between the nervous system and cancer cells. NLGN1 was shown here to function along one of the most represented neurotrophic factors in the nerve microenvironment, possibly opening new therapeutic avenues.

The neuronal protein Neuroligin 1 promotes colorectal cancer progression by modulating the APC/ß-catenin pathway.

BACKGROUND: Colorectal cancer (CRC) remains largely incurable when diagnosed at the metastatic stage. Despite some advances in precision medicine for this disease in recent years, new molecular targets, as well as prognostic/predictive markers, are highly needed. Neuroligin 1 (NLGN1) is a transmembrane protein that interacts at the synapse with the tumor suppressor adenomatous polyposis Coli (APC), which is heavily involved in the pathogenesis of CRC and is a key player in the WNT/ß-catenin pathway. METHODS: After performing expression studies of NLGN1 on human CRC samples, in this paper we used in vitro and in vivo approaches to study CRC cells extravasation and metastasis formation capabilities. At the molecular level, the functional link between APC and NLGN1 in the cancer context was studied. RESULTS: Here we show that NLGN1 is expressed in human colorectal tumors, including clusters of aggressive migrating (budding) single tumor cells and vascular emboli. We found that NLGN1 promotes CRC cells crossing of an endothelial monolayer (i.e. Trans-Endothelial Migration or TEM) in vitro, as well as cell extravasation/lung invasion and differential organ metastatization in two mouse models. Mechanistically, NLGN1 promotes APC localization to the cell membrane and co-immunoprecipitates with some isoforms of this protein stimulates ß-catenin translocation to the nucleus, upregulates mesenchymal markers and WNT target genes and induces an "EMT phenotype" in CRC cell lines CONCLUSIONS: In conclusion, we have uncovered a novel modulator of CRC aggressiveness which impacts on a critical pathogenetic pathway of this disease, and may represent a novel therapeutic target, with the added benefit of carrying over substantial knowledge from the neurobiology field.

Mature endothelium and neurons are simultaneously derived from embryonic stem cells by 2D in vitro culture system.

The connections existing between vessels and nerves go beyond the structural architecture of vascular and nervous systems to comprise cell fate determination. The analysis of functional/molecular links that interconnect endothelial and neural commitments requires a model in which the two differentiation programs take place at the same time in an artificial controllable environment. To this regard, this work presents an in vitro model to differentiate embryonic stem (ES) cells simultaneously into mature neurons and endothelial cells. Murine ES cells are differentiated within an artificial environment composed of PA6 stromal cells and a serum-free medium. Upon these basal culture conditions ES cells preferentially differentiate into neurons. The addition of basic fibroblast growth factor (FGF2) to the medium allows the simultaneous maturation of neurons and endothelial cells, whereas bone morphogenetic protein (BMP)4 drives endothelial differentiation to the disadvantage of neural commitment. The responsiveness of the system to exogenous cytokines was confirmed by genes expression analysis that revealed a significant up-regulation of endothelial genes in presence of FGF2 and a massive down-regulation of the neural markers in response to BMP4. Furthermore, the role played by single genes in determining endothelial and neural fate can be easily explored by knocking down the expression of the target gene with lentiviruses carrying the corresponding shRNA sequence. The possibility to address the neural and the endothelial fate separately or simultaneously by exogenous stimuli combined with an efficient gene silencing strategy make this model an optimal tool to identify environmental signals and genes pathways involved in both endothelial and neural specification.

Characterization of the neuroligin gene family expression and evolution in zebrafish.

Neuroligins constitute a family of transmembrane proteins localized at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. They are involved in synaptic function and maturation and recent studies have linked mutations in specific human Neuroligins to mental retardation and autism. We isolated the human Neuroligin homologs in Danio rerio. Next, we studied their gene structures and we reconstructed the evolution of the Neuroligin genes across vertebrate phyla. Using reverse-transcriptase polymerase chain reaction, we analyzed the expression and alternative splicing pattern of each gene during zebrafish embryonic development and in different adult organs. By in situ hybridization, we analyzed the temporal and spatial expression pattern during embryonic development and larval stages and we found that zebrafish Neuroligins are expressed throughout the nervous system. Globally, our results indicate that, during evolution, specific subfunctionalization events occurred within paralogous members of this gene family in zebrafish.

LPHN2 inhibits vascular permeability by differential control of endothelial cell adhesion.

Dynamic modulation of endothelial cell-to-cell and cell-to-extracellular matrix (ECM) adhesion is essential for blood vessel patterning and functioning. Yet the molecular mechanisms involved in this process have not been completely deciphered. We identify the adhesion G protein-coupled receptor (ADGR) Latrophilin 2 (LPHN2) as a novel determinant of endothelial cell (EC) adhesion and barrier function. In cultured ECs, endogenous LPHN2 localizes at ECM contacts, signals through cAMP/Rap1, and inhibits focal adhesion (FA) formation and nuclear localization of YAP/TAZ transcriptional regulators, while promoting tight junction (TJ) assembly. ECs also express an endogenous LPHN2 ligand, fibronectin leucine-rich transmembrane 2 (FLRT2), that prevents ECM-elicited EC behaviors in an LPHN2-dependent manner. Vascular ECs of lphn2a knock-out zebrafish embryos become abnormally stretched, display a hyperactive YAP/TAZ pathway, and lack proper intercellular TJs. Consistently, blood vessels are hyperpermeable, and intravascularly injected cancer cells extravasate more easily in lphn2a null animals. Thus, LPHN2 ligands, such as FLRT2, may be therapeutically exploited to interfere with cancer metastatic dissemination.

Microenvironment drives the endothelial or neural fate of differentiating embryonic stem cells coexpressing neuropilin-1 and Flk-1.

The observation that the architecture of the cardiovascular and nervous systems is drawn by common guidance cues and the closeness between neural progenitors and endothelial cells in the vascular niche strongly suggests the existence of links between endothelial and neural cell fates. We identified an embryonic stem cell-derived discrete, nonclonal cell population expressing the two vascular endothelial growth factor receptors neuropilin-1 (Nrp1) and Flk1 that differentiates in vitro toward endothelial or neural phenotypes depending on microenvironmental cues. When microinjected in the chick embryo, Nrp1(+) cells integrate within the host, developing vessels and brain, and acquire endothelial and neural markers, respectively. These results show that precursors of endothelial cells and precursors of neural cells arise from the same pool of differentiating embryonic stem cells and share the expression of Nrp1 and Flk1. These data reinforce the parallelism between vascular and nervous system at the level of cell fate and commitment and open new perspective in regenerative medicine of neurovascular diseases.

Besides adhesion: new perspectives of integrin functions in angiogenesis.

During angiogenic remodelling in embryo and adult life, endothelial cells lining blood vessel walls dynamically modify their integrin-mediated adhesive contacts with the surrounding extracellular matrix. However, besides regulating cell adhesion and migration, integrins dynamically participate in a network with soluble molecules and their receptors. Angiogenesis is characterized by opposing autocrine and paracrine loops of growth factors and semaphorins that regulate the activation of integrins on the endothelial surface through tyrosine kinase receptors (TKR) and the neuropilin/plexin system. Moreover, pro- and anti-angiogenic factors can directly bind integrins and regulate endothelial cell behaviour. This review summarizes the recent progress in understanding the reciprocal interactions between integrins, TKR, and semaphorin receptors.

High Circulating Methylated DNA Is a Negative Predictive and Prognostic Marker in Metastatic Colorectal Cancer Patients Treated With Regorafenib.

Background: Regorafenib improves progression free survival (PFS) in a subset of metastatic colorectal cancer (mCRC) patients, although no biomarkers of efficacy are available. Circulating methylated DNA (cmDNA) assessed by a five-gene panel was previously associated with outcome in chemotherapy treated mCRC patients. We hypothesized that cmDNA could be used to identify cases most likely to benefit from regorafenib (i.e., patients with PFS longer than 4 months). Methods: Plasma samples from mCRC patients were collected prior to (baseline samples N = 60) and/or during regorafenib treatment (N = 62) for the assessment of cmDNA and total amount of cell free DNA (cfDNA). Results: In almost all patients, treatment with regorafenib increased the total cfDNA, but decreased cmDNA warranting the normalization of cmDNA to the total amount of circulating DNA (i.e., cmDNA/ml). We report that cmDNA/ml dynamics reflects clinical response with an increase in cmDNA/ml associated with higher risk of progression (HR for progression = 1.78 [95%CI: 1.01-3.13], p = 0.028). Taken individually, high baseline cmDNA/ml (above median) was associated with worst prognosis (HR for death = 3.471 [95%CI: 1.83-6.57], p < 0.0001) and also predicted shorter PFS (<16 weeks with PPV 86%). In addition, high cmDNA/ml values during regorafenib treatment predicted with higher accuracy shorter PFS (<16 weeks with a PPV of 96%), therefore associated with increased risk of progression (HR for progression = 2.985; [95%CI: 1.63-5.46; p < 0.0001). Conclusions: Our data highlight the predictive and prognostic value of cmDNA/ml in mCRC patients treated with regorafenib.

Type I collagen limits VEGFR-2 signaling by a SHP2 protein-tyrosine phosphatase-dependent mechanism 1.

During angiogenesis, a combined action between newly secreted extracellular matrix proteins and the repertoire of integrins expressed by endothelial cells contributes in the regulation of their biological functions. Extracellular matrix-engaged integrins influence tyrosine kinase receptors, thus promoting a regulatory cross-talk between adhesive and soluble stimuli. For instance, vitronectin has been reported to positively regulate VEGFR-2. Here, we show that collagen I downregulates VEGF-A-mediated VEGFR-2 activation. This activity requires the tyrosine phosphatase SHP2, which is recruited to the activated VEGFR-2 when cells are plated on collagen I, but not on vitronectin. Constitutive expression of SHP2(C459S) mutant inhibits the negative role of collagen I on VEGFR-2 phosphorylation. VEGFR-2 undergoes internalisation, which is associated with dynamin II phosphorylation. Expression of SHP2(C459S) impairs receptor internalisation suggesting that SHP2-dependent dephosphorylation regulates this process. These findings demonstrate that collagen I in provisional extracellular matrix surrounding nascent capillaries triggers a signaling pathway that negatively regulates angiogenesis.