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Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.

Mesenchymal stromal cells (MSCs), seeded onto a scaffold and associated with platelet-gel, may represent an innovative treatment to improve bone repair. The preparation of MSCs for clinical use requires the fulfillment of Good Manufacturing Practice indications. The aim of this study was to validate a Good Manufacturing Practice-grade protocol of tissue engineering for bone regeneration, seeding platelet lysate (PL)-cultured MSCs onto an hydroxyapatite clinical-grade scaffold. Six large-scale experiments were performed. MSC expansions were performed comparing fetal bovine serum 10% and PL 5%. We demonstrated that PL lots contain high levels of growth factors possibly responsible of accelerated growth rate, since the number of colony-forming unit-fibroblast and population doublings were always significantly higher in PL cultures. MSCs were characterized for their phenotype and multilineage differentiation capacity, demonstrating appropriate features for both conditions. Gene expression analysis revealed higher expression of typical osteogenic genes of PL-cultured MSCs, when compared to fetal bovine serum MSCs. Cell transformation was excluded by analysis of karyotype, absence of growth without anchorage, and p53/c-myc gene expression. Scaffolds were precoated with retronectin before MSC seeding. MSC adhesion, distribution, and proliferation were demonstrated through the whole surface of the scaffold by scanning electron microscopy analysis or by immunofluorescence and MSC osteogenic differentiation through quantitative reverse transcriptase-polymerase chain reaction of typical osteogenic genes. The present report offers a model of an MSC-based bioengineered device, using an hydroxyapatite clinical-grade scaffold, and supports its potential use in tissue engineering to repair bone defects.

Purpose: Exosomes deliver signals to target cells and could thus be exploited as an innovative therapeutic tool. We investigated the ability of membrane TRAIL-armed exosomes to deliver proapoptotic signals to cancer cells and mediate growth inhibition in different tumor models. Experimental Methods and Results: K562 cells, transduced with lentiviral human membrane TRAIL, were used for the production of TRAIL+ exosomes, which were studied by nanoparticle tracking analysis, cytofluorimetry, immunoelectronmicroscopy, Western blot, and ELISA. In vitro, TRAIL+ exosomes induced more pronounced apoptosis (detected by Annexin V/propidium iodide and activated caspase-3) in TRAIL-death receptor (DR)5+ cells (SUDHL4 lymphoma and INT12 melanoma), with respect to the DR5−DR4+KMS11 multiple myeloma. Intratumor injection of TRAIL+ exosomes, but not mock exosomes, induced growth inhibition of SUDHL4 (68%) and INT12 (51%), and necrosis in KMS11 tumors. After rapid blood clearance, systemically administered TRAIL+ exosomes accumulated in the liver, lungs, and spleen and homed to the tumor site, leading to a significant reduction of tumor growth (58%) in SUDHL4-bearing mice. The treatment of INT12-bearing animals promoted tumor necrosis and a not statistically significant tumor volume reduction. In KMS11-bearing mice, despite massive perivascular necrosis, no significant tumor growth inhibition was detected. Conclusions: TRAIL-armed exosomes can induce apoptosis in cancer cells and control tumor progression in vivo. Therapeutic efficacy was particularly evident in intratumor setting, while depended on tumor model upon systemic administration. Thanks to their ability to deliver multiple signals, exosomes thus represent a promising therapeutic tool in cancer. Clin Cancer Res; 22(14); 3499–512. ©2016 AACR.

Although the ETV6–RUNX1 fusion is a frequent initiating event in childhood leukemia, its role in leukemogenesis is only partly understood. The main impact of the fusion itself is to generate and sustain a clone of clinically silent preleukemic B-cell progenitors (BCP). Additional oncogenic hits, occurring even several years later, are required for overt disease. The understanding of the features and interactions of ETV6–RUNX1–positive cells during this “latency” period may explain how these silent cells can persist and whether they could be prone to additional genetic changes. In this study, two in vitro murine models were used to investigate whether ETV6–RUNX1 alters the cellular adhesion and migration properties of BCP. ETV6–RUNX1–expressing cells showed a significant defect in the chemotactic response to CXCL12, caused by a block in CXCR4 signaling, as demonstrated by inhibition of CXCL12-associated calcium flux and lack of ERK phosphorylation. Moreover, the induction of ETV6–RUNX1 caused changes in the expression of cell-surface adhesion molecules. The expression of genes regulating the cytoskeleton was also affected, resulting in a block of CDC42 signaling. The abnormalities described here could alter the interaction of ETV6–RUNX1 preleukemic BCP with the microenvironment and contribute to the pathogenesis of the disease. Implications: Alterations in the expression of cytoskeletal regulatory genes and migration properties of BCP represent early events in the evolution of the disease, from the preleukemic phase to the clinical onset, and suggest new strategies for effective eradication of leukemia. Mol Cancer Res; 12(12); 1796–806. ©2014 AACR.

By means of morphological and biochemical criteria, we here provide evidence for the localization and function of premature ovarian failure, 1B (POF1B) in desmosomes. In monolayers of Caco-2 intestinal cells and in stratified HaCaT keratinocytes, endogenous POF1B colocalized with desmoplakin at desmosome plaques and in cytoplasmic particles aligned along intermediate filaments (IFs). POF1B predominantly co-fractionated with desmosomes and IF components and exhibited properties characteristic of desmosomes (i.e., detergent insolubility and calcium independence). The role of NH2 and COOH domains in the association of POF1B with desmosomes and IFs was revealed by transient expression of the truncated protein in Caco-2 cells and in cells lacking desmosomes. The function of POF1B in desmosomes was investigated in HaCaT keratinocytes stably downregulated for POF1B expression. Transmission electron microscopy analysis revealed a decrease in desmosome number and size, and desmosomes of the downregulated keratinocytes displayed weak electron-dense plaques. Desmosome alterations were associated with defects in cell adhesion, as revealed by the reduced resistance to mechanical stress in the dispase fragmentation assay. Moreover, desmosome localization of POF1B was restricted to granular layers in human healthy epidermis, whereas it largely increased in hyperproliferative human skin diseases, thus demonstrating the localization of POF1B also in desmosomes of multistratified epithelia.Journal of Investigative Dermatology advance online publication, 18 September 2014; doi:10.1038/jid.2014.327

Context: Dopamine agonists (DA) are the first choice treatment of prolactinomas. However, a subset of patients is resistant to DA, due to undefined dopamine D2 receptor (D2R) alterations. Recently, D2R was found to associate with filamin-A (FLNA), a widely expressed cytoskeleton protein with scaffolding properties, in melanoma and neuronal cells. Objective: The aim of the study was to investigate the role of FLNA in D2R expression and signaling in human tumorous lactotrophs and rat MMQ and GH3 cells. Design: We analyzed FLNA expression in a series of prolactinomas by immunohistochemistry and Western blotting.Weperformed FLNA silencing or transfection experiments in cultured cells from DA-sensitive or -resistant prolactinomas and in MMQ and GH3 cells, followed by analysis of D2R expression and signaling. Results: We demonstrated reduced FLNA and D2R expression in DA-resistant tumors. The crucial role of FLNA on D2R was demonstrated by experiments showing that: 1) FLNA silencing in DA-sensitive prolactinomas resulted in 60% reduction of D2R expression and abrogation of DA-induced inhibition of prolactin release and antiproliferative signals, these results being replicated in MMQ cells that endogenously express FLNA and D2R; and 2) FLNA overexpression in DA-resistant prolactinomas restored D2R expression and prolactin responsiveness to DA, whereas this manipulation was ineffective in GH3 cells that express FLNA but not D2R. No alteration in FLNA promoter methylation was detected, ruling out the occurrence of epigenetic FLNA silencing in DA-resistant prolactinomas. Conclusions: These data indicate that FLNA is crucial for D2R expression and signaling in lactotrophs, suggesting that the impaired response to DA may be related to the reduction of FLNA expression in DA-resistant prolactinomas. Copyright © 2012 by The Endocrine Society.

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarised epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. Here we show colocalisation of POF1B with markers of both adherens and tight junctions in human jejunum. The tight junction localisation was maintained by the human POF1B stably expressed in the MDCK polarised epithelial cell line, whereas it was lost by the POF1B R329Q variant associated with POF. Localisation of apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant. However, tight junction assembly was altered, cells were dysmorphic and the monolayer organisation was also altered in three-dimensional culture systems. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organisation of F-actin at the junctions. A role for POF1B in the regulation of the actin cytoskeleton was further verified by shRNA silencing of the endogenous protein in human intestinal Caco-2 cells. Taken together, these data indicate that localisation of POF1B to tight junctions has a key role in the organisation of epithelial monolayers by regulating the actin cytoskeleton. © 2011. Published by The Company of Biologists Ltd.

Metastatic melanoma is associated with poor prognosis and still limited therapeutic options. An innovative treatment approach for this disease is represented by targeting acidosis, a feature characterizing tumor microenvironment and playing an important role in cancer malignancy. Proton pump inhibitors (PPI), such as esomeprazole (ESOM) are prodrugs functionally activated by acidic environment, fostering pH neutralization by inhibiting proton extrusion. We used human melanoma cell lines and xeno-transplated SCID mice to provide preclinical evidence of ESOM antineoplastic activity. Human melanoma cell lines, characterized by different mutation and signaling profiles, were treated with ESOM in different pH conditions and evaluated for proliferation, viability and cell death. SCID mice engrafted with human melanoma were used to study ESOM administration effects on tumor growth and tumor pH by magnetic resonance spectroscopy (MRS). ESOM inhibited proliferation of melanoma cells in vitro and induced a cytotoxicity strongly boosted by low pH culture conditions. ESOM-induced tumor cell death occurred via rapid intracellular acidification and activation of several caspases. Inhibition of caspases activity by pan-caspase inhibitor z-vad-fmk completely abrogated the ESOM-induced cell death. ESOM administration (2.5 mg kg-1) to SCID mice engrafted with human melanoma reduced tumor growth, consistent with decrease of proliferating cells and clear reduction of pH gradients in tumor tissue. Moreover, systemic ESOM administration dramatically increased survival of human melanoma-bearing animals, in absence of any relevant toxicity. These data show preclinical evidence supporting the use of PPI as novel therapeutic strategy for melanoma, providing the proof of concept that PPI target human melanoma modifying tumor pH gradients. © 2009 UICC.

Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery.

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl-L-cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.

// Chiara Palmi 1 , Angela M. Savino 1 , Daniela Silvestri 2, 3 , Ilaria Bronzini 4 , Gunnar Cario 5 , Maddalena Paganin 4 , Barbara Buldini 4 , Marta Galbiati 1 , Martina U. Muckenthaler 6 , Cristina Bugarin 1 , Pamela Della Mina 7 , Stefan Nagel 8 , Elena Barisone 9 , Fiorina Casale 10 , Franco Locatelli 11 , Luca Lo Nigro 12 , Concetta Micalizzi 13 , Rosanna Parasole 14 , Andrea Pession 15 , Maria C. Putti 4 , Nicola Santoro 16 , Anna M. Testi 17 , Ottavio Ziino 18 , Andreas E. Kulozik 6 , Martin Zimmermann 19 , Martin Schrappe 5 , Antonello Villa 7 , Giuseppe Gaipa 1 , Giuseppe Basso 4 , Andrea Biondi 3 , Maria G. Valsecchi 2 , Martin Stanulla 19 , Valentino Conter 3 , Geertruy te Kronnie 4 , Giovanni Cazzaniga 1 1 Centro Ricerca M. Tettamanti, Clinica Pediatrica, Universita di Milano Bicocca, Fondazione MBBM/Ospedale San Gerardo, Monza, Italy 2 Center of Biostatistics for Clinical Epidemiology, Department of Health Sciences, University of Milano-Bicocca, Milan, Italy 3 Clinica Pediatrica, Universita di Milano Bicocca, Fondazione MBBM/Ospedale San Gerardo, Monza, Italy 4 Laboratory of Onco-Hematology, Department SDB, Universita di Padova, Padova, Italy 5 Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany 6 Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg and EMBL/Medical Faculty Molecular Medicine Partnership Unit, Heidelberg, Germany 7 Microscopy and Image Analysis Consortium, Universita di Milano-Bicocca, Monza, Italy 8 Department of Human and Animal Cell Lines, Leibniz-Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany 9 Pediatric Onco-Hematology, Stem Cell Transplantation and Cellular Therapy Division, Regina Margherita Children’s Hospital, Turin, Italy 10 Pediatric Oncology Service, Pediatric Department of 2nd University of Naples, Naples, Italy 11 Department of Pediatric Hematology/Oncology, IRCCS Ospedale Bambino Gesu, Rome - University of Pavia, Pavia, Italy 12 Center of Pediatric Hematology Oncology, Azienda Ospedaliero-Universitaria “Policlinico Vittorio Emanuele”, Catania, Italy 13 Hematology/Oncology Unit, G. Gaslini Children’s Hospital, Genoa, Italy 14 Department of Pediatric Hemato-Oncology, Ospedale Pausilipon, Napoli, Italy 15 Department of Pediatrics, “Lalla Seragnoli” Hematology-Oncology Unit, University of Bologna, Bologna, Italy 16 Department of Pediatrics, Division of Pediatric Hematology-Oncology, University “A. Moro” of Bari, Bari, Italy 17 Division of Hematology, Department of Biotechnologies and Hematology, “Sapienza” University of Rome, Rome, Italy 18 Pediatric Hematology and Oncology Unit, A.R.N.A.S. Civico, Di Cristina and Benfratelli Hospital, Palermo, Italy 19 Department of Paediatric Haematology and Oncology, Hannover Medical School, Hannover, Germany Correspondence to: Giovanni Cazzaniga, email: gianni.cazzaniga@hsgerardo.org Andrea Biondi, email: abiondi.unimib@gmail.com Keywords: CRLF2, pediatric leukemia, T acute lymphoblastic leukemia, prognostic marker, high risk Received: May 20, 2016 Accepted: July 01, 2016 Published: July 15, 2016 ABSTRACT Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL. We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers. Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median ( CRLF2-high ); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated. Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib. In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway.

Glucose-regulated stress protein gp96 is known to be involved in the host response to pathogens and to cancer. Our study explored the relationships between gp96 and human blood plasmacytoid dendritic cells (pDC) and proved that gp96 directly targets pDC by a receptor-dependent interaction. Competition studies identified CD91 as a gp96 receptor on pDC, and laser confocal imaging indicated that CD91 triggering was followed by gp96 endocytosis and trafficking into early endosomes and later into the endoplasmic reticulum compartment. Using two alternative Abs, we showed that human blood pDC reproducibly expressed CD91, although different levels of expression were detectable among the analyzed donors. Moreover, CpG-matured pDC displayed CD91 receptor up-regulation that correlated with an increased gp96 binding. Functionally, gp96-pDC interaction activated the NF-κB pathway, leading to the nuclear translocation of the NF-κB complex. gp96-treated pDC maintained an immature phenotype, while they down-modulated the release of IL-8, suggesting an anti-inflammatory role of this pathway, and they strongly up-regulated the cell surface expression of the gp96 receptor CD91. CpG-matured or gp96-treated pDC, expressing high levels of the gp96 receptor CD91, antagonized the gp96-induced activation of monocyte-derived dendritic cells in terms of cell surface phenotype and cytokine production. Altogether, these results suggest that gp96-pDC interaction might represent an active mechanism controlling the strength of the immune response to free, extracellular available gp96; this mechanism could be particularly relevant in wounds and chronic inflammation.