Valeria De Pasquale

HGF/c-MET Axis in Tumor Microenvironment and Metastasis Formation

Tumor metastases are responsible for approximately 90% of all cancer-related deaths. Metastasis formation is a multistep process that requires acquisition by tumor cells of a malignant phenotype that allows them to escape from the primary tumor site and invade other organs. Each step of this mechanism involves a deep crosstalk between tumor cells and their microenvironment where the host cells play a key role in influencing metastatic behavior through the release of many secreted factors. Among these signaling molecules, Hepatocyte Growth Factor (HGF) is released by many cell types of the tumor microenvironment to target its receptor c-MET within the cells of the primary tumor. Many studies reveal that HGF/c-MET axis is implicated in various human cancers, and genetic and epigenetic gain of functions of this signaling contributes to cancer development through a variety of mechanisms. In this review, we describe the specific types of cells in the tumor microenvironment that release HGF in order to promote the metastatic outgrowth through the activation of extracellular matrix remodeling, inflammation, migration, angiogenesis, and invasion. We dissect the potential use of new molecules that interfere with the HGF/c-MET axis as therapeutic targets for future clinical trials in cancer disease.

Intracellular signaling cascades triggered by the NK1 fragment of hepatocyte growth factor in human prostate epithelial cell line PNT1A

Hepatocyte Growth Factor (HGF)/c-MET signaling has an emerging role in promoting cell proliferation, survival, migration, wound repair and branching in a variety of cell types. HGF plays a crucial role as a mediator of stromal-epithelial interactions in the normal prostate but the precise biological function of HGF/c-Met interaction in the normal prostate and in prostate cancer is not clear. HGF has two naturally occurring splice variants and NK1, the smallest of these HGF variants, consists of the HGF amino terminus through the first kringle domain. We evaluated the intracellular signaling cascades and the morphological changes triggered by NK1 in human prostate epithelial cell line PNT1A which shows molecular and biochemical properties close to the normal prostate epithelium. We demonstrated that these cells express a functional c-MET, and cell exposure to NK1 induces the phosphorylation of tyrosines 1313/1349/1356 residues of c-MET which provide docking sites for signaling molecules. We observed an increased phosphorylation of ERK1/2, Akt, c-Src, p125FAK, SMAD2/3, and STAT3, down-regulation of the expression of epithelial cell-cell adhesion marker E-cadherin, and enhanced expression levels of mesenchymal markers vimentin, fibronectin, vinculin, α-actinin, and α-smooth muscle actin. This results in cell proliferation, in the appearance of a mesenchymal phenotype, in morphological changes resembling cell scattering and in wound healing. Our findings highlight the function of NK1 in non-tumorigenic human prostatic epithelial cells and provide a picture of the signaling pathways triggered by NK1 in a unique cell line.

The Murine Model of Mucopolysaccharidosis IIIB Develops Cardiopathies over Time Leading to Heart Failure.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal disease due to the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The disease is characterized by mild somatic features and severe neurological disorders. Very little is known on the cardiac dysfunctions in MPS IIIB. In this study, we used the murine model of MPS IIIB (NAGLU knockout mice, NAGLU-/-) in order to investigate the cardiac involvement in the disease. Echocardiographic analysis showed a marked increase in left ventricular (LV) mass, reduced cardiac function and valvular defects in NAGLU-/- mice as compared to wild-type (WT) littermates. The NAGLU-/- mice exhibited a significant increase in aortic and mitral annulus dimension with a progressive elongation and thickening of anterior mitral valve leaflet. A severe mitral regurgitation with reduction in mitral inflow E-wave-to-A-wave ratio was observed in 32-week-old NAGLU-/- mice. Compared to WT mice, NAGLU-/- mice exhibited a significantly lower survival with increased mortality observed in particular after 25 weeks of age. Histopathological analysis revealed a significant increase of myocardial fiber vacuolization, accumulation of HS in the myocardial vacuoles, recruitment of inflammatory cells and collagen deposition within the myocardium, and an increase of LV fibrosis in NAGLU-/- mice compared to WT mice. Biochemical analysis of heart samples from affected mice showed increased expression levels of cardiac failure hallmarks such as calcium/calmodulin-dependent protein kinase II, connexin43, α-smooth muscle actin, α-actinin, atrial and brain natriuretic peptides, and myosin heavy polypeptide 7. Furthermore, heart samples from NAGLU-/- mice showed enhanced expression of the lysosome-associated membrane protein-2 (LAMP2), and the autophagic markers Beclin1 and LC3 isoform II (LC3-II). Overall, our findings demonstrate that NAGLU-/- mice develop heart disease, valvular abnormalities and cardiac failure associated with an impaired lysosomal autophagic flux.

New Anticancer Agents Mimicking Protein Recognition Motifs

The novel tetrasubstituted pyrrole derivatives 8g, 8h, and 8i showed selective cytotoxicity against M14 melanoma cells at low micromolar concentration. Structure-activity relationships (SARs) indicated the presence of three aromatic substituents on the pyrrole core as necessary for biological activity. Computational studies strongly suggest that the peculiar 3D orientation of these substituents is able to reproduce the hydrophobic side chains in LxxLL-like protein recognition motifs. Biological results showed altered p53 expression and nuclear translocation in cells sensitive to the compounds, suggesting p53 involvement in their anticancer mechanism of action. Unfortunately, because of poor solubility of the active analogues, it was not possible to perform further investigation by NMR techniques. Pharmacophore models were generated and used to perform 3D searches in molecular databases. Results indicated that two compounds share the same pharmacological profile and the same pharmacophoric features with our new derivatives, and one of them inhibited MDM2-MDM4 heterodimer formation.

Role of serotonergic system in the pathogenesis of fibrosis in canine idiopathic inflammatory myopathies

Idiopathic inflammatory myopathies are muscle diseases characterized by inflammation, necrosis, and fibrosis. The neurotransmitter serotonin (5-HT) has been shown to promote fibrosis in many tissues and organs by activating TGFβ-1 signaling. In this study, we evaluated the potential role of 5-HT in the pathogenesis of fibrosis in canine idiopathic inflammatory myopathies. Muscle biopsies from dogs affected by masticatory muscle myositis or polymyositis and from healthy dogs were processed for immunohistochemistry and Western blotting. The immunohistochemical analysis showed a strong expression of 5-HT in muscle tissues of affected dogs, whereas the amine was absent in the muscles of healthy dogs. Biochemical analysis showed increased expression levels of the selective 5-HT2A receptor in the muscle specimens of the most severely affected dogs versus controls. Further, increased phosphorylation levels of the TGFβ-1 signaling mediators SMAD2/3 and ERK1/2 were detected in tissue samples from affected dogs as compared to tissues from healthy dogs. Although further studies are needed, our findings highlight for the first time a potential role of 5-HT in the development of fibrosis in canine idiopathic inflammatory myopathies, thus supporting other evidence that 5-HT pro-fibrotic activity occurs via activation of TGFβ-1 signaling pathway.

Fate Map of Serotonin Transporter-Expressing Cells in Developing Mouse Thyroid

A Cre/loxP‐based fate mapping approach was used to follow the regions of the mouse thyroid labeled by the serotonin transporter SERT. Reporter gene expression (lacZ) is activated by Cre expression from the SERT locus in SERTCre/+;ROSA26R compound mouse embryos. Cell labeling, first detected in the thyroid primordium at the E10.5 prenatal stage, was followed until the postnatal day P30. The co‐localization of lacZ staining in the same cells that express the transcription factors Nkx2.1 and Pax8 at the E12.5 stage confirms their identity as thyroid cell precursors. SERT immunohistochemistry on thyroid sections of E18.5 embryos showed SERT expression in thyroid follicular cells. Western blotting analysis confirmed the expression of the protein in adult thyroid tissue and cultured FRTL‐5 cells. These results describe the fate of SERT‐expressing cells during thyroid development, suggesting an active role of SERT in the development and functions of mammalian thyroid. They also highlight the possibility to use the SERT‐Cre mouse line as a good Cre driver in early thyroid development. Anat Rec, 2011.

Serotonin regulates contractile activity of the uterus in non-pregnant rabbits

Serotonin (5-HT) can stimulate the cholinergic system of the uterus by indirect actions on the modulation of reflexes and a direct action on smooth muscles. We investigated the role of 5-HT in the regulation of the cholinergic activity in the uterine parts of non-pregnant rabbits. The right vagus or pelvic nerve and the left sympathetic trunk were stimulated by an electrical field, and the uterine contractile activity was evaluated by measuring the amplitude and frequency of slow wave electromyogram (EMG), with the surface of microelectrodes applied to the uterus bottom, body, and cervix, respectively. Double stimulation of the vagus or pelvic nerve and the sympathetic trunk increased the frequency and the amplitude of the slow wave EMG in all the uterine parts. Furthermore, the administration of exogenous 5-HT increased the vagus or pelvic induced EMG activity in all parts of the uterus. Overall our results demonstrate that 5-HT enhances the vagus contractile activity with a magnitude of the effect decreasing from the bottom to the cervix, whereas 5-HT enhances the pelvic nerve contractile functions with a magnitude of the response increasing from the bottom to the cervix. The administration of droperidol, a 5-HT3 and 4 receptor inhibitor, and spiperone, a 5-HT2 receptor antagonist, inhibited the effect of the serotoninergic fibers of the sympathetic trunk to increase the vagus and pelvic nerve EMG activity. These data suggest that 5-HT stimulation of the parasympathetic nerves results in the induction of uterine contraction via the activation of 5-HT2, 3, and 4 receptor subfamilies.

Health and disease, an orchestra of three players: Serotonin, orexins, and nitric oxide

Over the past century, a huge amount of evidence has emerged pointing to the role of the neurotransmitter serotonin, the neuropeptides orexin A and B, and the small gaseous molecule nitric oxide (NO) (Fig. 1) as crucial regulators of both physiological functions and diseases of the central nervous system (CNS) and peripheral organs as well (Berger et al., 2009; Zhou and Zhu, 2009; Shekhar, 2012). Serotonin, orexins, and NO produced in the brain centrally regulate a great number of neural activities including synaptic plasticity, sleep–wake cycle, energy balance, food intake, hormone secretion, immune responses, cardiovascular functions, and others (Szczepanska-Sadowska et al., 2010; Hu and Zhu, 2014; Shan et al., 2015). Interestingly, these regulatory factors are even involved in the complex neurobiological phenomenon relying on trust, belief, pleasure, and reward activities such as love (Esch and Stefano, 2005). They play major roles in the control of partner preference, sexual desire, erection, copulation, ejaculation, orgasm, and sexual satiety. Serotonin, orexins, NO, and their synthetic and signaling machineries are not limited to the neural tissues and brain, but they are also expressed in peripheral tissues, where their prominent role begins from the time of the morphogenesis in utero and lasts through all of adult life. Peripheral locally produced serotonin, orexins, and NO regulate cardiovascular system physiology, bowel motility, steroidogenesis in the adrenal glands and the genital tract, urethrogenital reflex, vascular smooth muscle contractility, platelet aggregation, immunity, and many other physiological processes (F€ orstermann and Sessa, 2012; Amireault et al., 2013; Li et al., 2014). The regulatory activities of the three neuromodulators depend on the place of their release and the availability of specific receptors; thus, they may exert either stimulatory or inhibitory effects depending on the site of action. An elevated number of disorders affecting either CNS or peripheral organs including psychiatric (depression, alcoholism, suicidal behavior, schizophrenia, autism), neurological (Alzheimer, Huntington, and Parkinson diseases), neuroimmune, and cardiovascular diseases, metabolic pathologies (diabetes, obesity, anorexia), gastrointestinal diseases (e.g., irritable bowel syndrome, Crohn disease, functional dyspepsia), tumors, sexual dysfunctions, and many other human pathologies have been associated with deregulation of serotonin and/or orexin and/or NO systems. To date, the majority of studies aimed at investigating the pathogenesis of the above diseases have mainly focused on highlighting the critical role of a single neuromodulator of the three, well establishing the molecular mechanisms underlying its action. By contrast, a notably low number of studies have been devoted to clarifying the mutual interactions among the three players in a specific physiological and/or pathological condition. Few examples include the evidence of a functional link among serotonin, orexin, and NO systems in the regulation of sleep and wakefulness (Monti, 2010), sexual behavior and drug addiction (Hull, 2011), migraine (Goadsby, 2005; Tso and Goadsby, 2014), gastrointestinal disorders (Okumura and Nozu, 2011; Szlachcic et al., 2013; Vermeulen et al., 2014), and neuropathic pain (Gilron and Dickenson, 2014). On the other hand, the occurrence of an interplay among serotonin, orexin, and NO systems in human health and disease is supported by the recurrent coincidence of different disorders that are mainly ascribed to the dysfunction of one of the three neuromodulator action,

The Multifaceted Role of the Lysosomal Protease Cathepsins in Kidney Disease

Kidney disease is worldwide the 12th leading cause of death affecting 8–16% of the entire population. Kidney disease encompasses acute (short-lasting episode) and chronic (developing over years) pathologies both leading to renal failure. Since specific treatments for acute or chronic kidney disease are limited, more than 2 million people a year require dialysis or kidney transplantation. Several recent evidences identified lysosomal proteases cathepsins as key players in kidney pathophysiology. Cathepsins, originally found in the lysosomes, exert important functions also in the cytosol and nucleus of cells as well as in the extracellular space, thus participating in a wide range of physiological and pathological processes. Based on their catalytic active site residue, the 15 human cathepsins identified up to now are classified in three different families: serine (cathepsins A and G), aspartate (cathepsins D and E), or cysteine (cathepsins B, C, F, H, K, L, O, S, V, X, and W) proteases. Specifically in the kidney, cathepsins B, D, L and S have been shown to regulate extracellular matrix homeostasis, autophagy, apoptosis, glomerular permeability, endothelial function, and inflammation. Dysregulation of their expression/activity has been associated to the onset and progression of kidney disease. This review summarizes most of the recent findings that highlight the critical role of cathepsins in kidney disease development and progression. A better understanding of the signaling pathways governed by cathepsins in kidney physiopathology may yield novel selective biomarkers or therapeutic targets for developing specific treatments against kidney disease.

EGFR activation triggers cellular hypertrophy and lysosomal disease in NAGLU-depleted cardiomyoblasts, mimicking the hallmarks of mucopolysaccharidosis IIIB

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease caused by the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The defective lysosomal clearance of undigested HS results in dysfunction of multiple tissues and organs. We recently demonstrated that the murine model of MPS IIIB develops cardiac disease, valvular abnormalities, and ultimately heart failure. To address the molecular mechanisms governing cardiac dysfunctions in MPS IIIB, we generated a model of the disease by silencing NAGLU gene expression in H9C2 rat cardiomyoblasts. NAGLU-depleted H9C2 exhibited accumulation of abnormal lysosomes and a hypertrophic phenotype. Furthermore, we found the specific activation of the epidermal growth factor receptor (EGFR), and increased phosphorylation levels of extracellular signal-regulated kinases (ERKs) in NAGLU-depleted H9C2. The inhibition of either EGFR or ERKs, using the selective inhibitors AG1478 and PD98059, resulted in the reduction of both lysosomal aberration and hypertrophy in NAGLU-depleted H9C2. We also found increased phosphorylation of c-Src and a reduction of the hypertrophic response in NAGLU-depleted H9C2 transfected with a dominant-negative c-Src. However, c-Src phosphorylation remained unaffected by AG1478 treatment, posing c-Src upstream EGFR activation. Finally, heparin-binding EGF-like growth factor (HB-EGF) protein was found overexpressed in our MPS IIIB cellular model, and its silencing reduced the hypertrophic response. These results indicate that both c-Src and HB-EGF contribute to the hypertrophic phenotype of NAGLU-depleted cardiomyoblasts by synergistically activating EGFR and subsequent signaling, thus suggesting that EGFR pathway inhibition could represent an effective therapeutic approach for MPS IIIB cardiac disease.