The Emerging Roles of Extracellular Vesicles in Osteosarcoma

Extracellular vesicles (EVs) are heterogeneous nanosized vesicles that are constitutively released by virtually all types of cells. They have been isolated in almost all body fluids. EVs cargo consists of various molecules (nucleic acids, proteins, lipids, and metabolites), that can be found on EVs surface and/or in their lumen. EVs structure confer stability and allow the transfer of their cargo to specific cell types over a distance. EVs play a critical role in intercellular communication in physiological and pathological settings. The broadening of knowledge on EVs improved our comprehension of cancer biology as far as tumor development, growth, metastasis, chemoresistance, and treatment are concerned. Increasing evidences suggest that EVs have a significant role in osteosarcoma (OS) development, progression, and metastatic process. The modulation of inflammatory communication pathways by EVs plays a critical role in OS and in other bone-related pathological conditions such as osteoarthritis and rheumatoid arthritis. In this review we describe the emerging data on the role of extracellular vesicles in osteosarcoma and discuss the effects and function of OS-derived EVs focusing on their future applicability in clinical practice

Extracellular nanovesicles secreted by human osteosarcoma cells promote angiogenesis

Angiogenesis involves a number of different players among which extracellular nanovesicles (EVs) have recently been proposed as an efficient cargo of pro-angiogenic mediators. Angiogenesis plays a key role in osteosarcoma (OS) development and progression. Acidity is a hallmark of malignancy in a variety of cancers, including sarcomas, as a result of an increased energetic metabolism. The aim of this study was to investigate the role of EVs derived from osteosarcoma cells on angiogenesis and whether extracellular acidity, generated by tumor metabolism, could influence EVs activity. For this purpose, we purified and characterized EVs from OS cells maintained at either acidic or neutral pH. The ability of EVs to induce angiogenesis was assessed in vitro by endothelial cell tube formation and in vivo using chicken chorioallantoic membrane. Our findings demonstrated that EVs derived from osteosarcoma cells maintained either in acidic or neutral conditions induced angiogenesis. The results showed that miRNA and protein content of EVs cargo are correlated with pro-angiogenic activity and this activity is increased by the acidity of tumor microenvironment. This study provides evidence that EVs released by human osteosarcoma cells act as carriers of active angiogenic stimuli that are able to promote endothelial cell functions relevant to angiogenesis

Exosomes: Novel effectors of human platelet lysate activity

Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies

Blocking tumor-educated MSC paracrine activity halts osteosarcoma progression

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients

Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGF\u3b2, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGF\u3b2-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGF\u3b2 are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGF\u3b2-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721-33. \ua92017 AACR

Involvement of HIF-1\u3b1 activation in the doxorubicin resistance of human osteosarcoma cells.

Osteosarcoma is the most common primary bone cancer in children and adolescents. Despite aggressive treatment regimens, survival outcomes remain unsatisfactory, particularly in patients with metastatic and/or recurrent disease. Unfortunately, treatment failure is commonly due to the development of chemoresistance, for which the underlying molecular mechanisms remain unclear. The aim of the present study was to investigate the role of hypoxia-inducible factor 1\u3b1 (HIF\u20111\u3b1) and its signalling pathways as mediators of drug-resistance in human osteosarcoma. Toward this aim, we established two osteosarcoma cell lines selected for resistance to doxorubicin, a drug of choice in the treatment of this tumour. Our results showed that the multidrug resistance (MDR) phenotype was also mediated by HIF-1\u3b1, the most important regulator of cell adaptation to hypoxia. Our data showed that this transcription factor promoted the outward transport of intracellular doxorubicin by activating the P-glycoprotein (P-gp) expression in osteosarcoma cells maintained in normoxic conditions. In addition, it hindered doxorubicin-induced apoptosis by regulating the expression of c-Myc and p21. Finally, we observed that the doxorubicin-resistant cells maintained for 2 months of continuous culture in a drug-free medium, lost their drug-resistance and this effect was associated with the absence of HIF-1\u3b1 expression. The emerging role of HIF-1\u3b1 in osteosarcoma biology indicates its use as a valuable therapeutic target

New instruments in molecular genetics for the prevention of X chromosome-linked muscular dystrophy].

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AKT come bersaglio molecolare dell'attivit\ue0 di fenretinide nel glioblastoma umano

Il glioblastoma multiforme rappresenta la forma pi\uf9 maligna dei tumori cerebrali. La prognosi \ue8 fatale con un tempo medio di sopravvivenza non superiore ai 12 mesi. Studi recenti hanno identificato nell'attivazione della chinasi Akt/PKB un ruolo importante nella tumorigenesi, crescita ed aumento del grado di malignit\ue0 in questo tumore. La chinasi Akt/PKB inibisce il processo apoptotico attraverso l'inattivazione di BAD e dei fattori di trascrizione forkhead; inoltre, essa \ue8 coinvolta nella progressione del ciclo cellulare tramite regolazione diretta della Ciclina D1. Akt/PKB pu\uf2 quindi rappresentare un nuovo importante bersaglio nella strategia terapeutica del glioblastoma umano. La Fenretinide o N-(4-idrossifenil) retinamide \ue8 un derivato sintetico dell' acido retinoico, possiede bassa epatotossicit\ue0 ed ha una buona tollerabilit\ue0 in vivo. Interessante la sua composizione altamente lipofila che le permette di attraversare la barriera emato-encefalica. Numerosi studi hanno dimostrato la capacit\ue0 di questo farmaco di indurre apoptosi in vitro e di poter esercitare effetti chemiopreventivi e terapeutici. Il meccanismo molecolare d'azione non \ue8 stato ancora del tutto chiarito. Fenretinide ha dimostrato di poter agire con meccanismi dipendenti e indipendenti dai recettori per i retinoidi e di indurre apoptosi tramite attivazione della cascata delle caspasi via ceramide e specie reattive dell'ossigeno. In leucemie, linfoblastomi, neuroblastomi e carcinomi mammari in vitro l'attivit\ue0 antiproliferativa del farmaco \ue8 associata ad una riduzione dell'espressione di Ciclina D1 e Cdk4. Entrambe queste molecole sono coinvolte nella tumorigenesi del glioblastoma e rappresentano bersagli molecolari del sistema Akt/PKB. Dati di letteratura indicano che Fenretinide inibisce la proliferazione in glioblastomi umani in vitro, con induzione di apoptosi mediata dalla caspasi 3. Qui sono riportati i risultati di uno studio teso alla individuazione dell\u2019eventuale ruolo di AKT nel meccanismo molecolare dell\u2019azione esercitata da Fenretinide nel glioblastoma umano. L'analisi \ue8 stata condotta in vitro in due linee cellulari di glioblastoma umano (CRS-A2 e A-172), esprimenti ciascuna alti livelli di Akt/PKB. E' stata valutata l'attivit\ue0 antiproliferativa di Fenretinide, non nota prima in queste linee tumorali, e confermata l'induzione di apoptosi, tramite attivazione della caspasi 3. In parallelo, mediante Western blotting, \ue8 stata condotta una analisi di espressione delle principali proteine coinvolte nel pathway di Akt/PKB. I risultati dimostrano inibizione della crescita indotta da Fenretinide, in entrambe le linee cellulari; il processo apoptotico viene attivato dal farmaco nella linea CRS-A2, con un meccanismo caspasi-3 dipendente. In parallelo all'inibizione della proliferazione cellulare si osservano una down-regolazione del complesso Ciclina D1/Cdk4, della p21CIP1 e di Akt/PKB: in questo caso, con una riduzione sia dei livelli basali di Akt, che della sua forma fosforilata. La chinasi Akt/PKB, di cui \ue8 noto il ruolo nel sostenere il fenotipo tumorale, quando costitutivamente attivata, si dimostra quindi bersaglio molecolare innovativo dell\u2019azione di Fenretinide nel glioblastoma umano, aprendo la strada a nuove prospettive terapeutiche

Mitochondrial DNA D-loop as a new target of Saporin 6 nuclease activity

The single-chain ribosome-inactivating proteins (RIPs) from plant origin, including Saporin 6 from the seeds of Saponaria officinalis, are ribotoxins known to act as N-glycosidases which depurinate the conserved alpha sarcin loop of large rRNAs. As a consequence, the eukaryotic ribosomes become inactivated, thereby arresting the protein synthesis at the elongation step. RIPs are currently under study as antiviral and antiproliferative agents. Additional in vitro activities of RIPs against either RNA or DNA have been recently described. A specific nuclease activity on plasmidic DNA was demonstrated by either purified or bacterial-recombinant molecules. We report here that human mitochondrial DNA (mtDNA) is a new specific target of Saporin 6 nuclease activity. A unique site of cleavage has been identified and mapped within the most variable part of the D-loop region of the covalently closed circular mtDNA molecule