RESUMEN
BACKGROUND: Extracellular vesicles (EVs) are released by nearly every cell type and have attracted much attention for their ability to transfer protein and diverse RNA species from donor to recipient cells. Much attention has been given so far to the features of EV short RNAs such as miRNAs. However, while the presence of mRNA and long noncoding RNA (lncRNA) transcripts in EVs has also been reported by multiple different groups, the properties and function of these longer transcripts have been less thoroughly explored than EV miRNA. Additionally, the impact of EV export on the transcriptome of exporting cells has remained almost completely unexamined. Here, we globally investigate mRNA and lncRNA transcripts in endothelial EVs in multiple different conditions. RESULTS: In basal conditions, long RNA transcripts enriched in EVs have longer than average half-lives and distinctive stability-related sequence and structure characteristics including shorter transcript length, higher exon density, and fewer 3' UTR A/U-rich elements. EV-enriched long RNA transcripts are also enriched in HNRNPA2B1 binding motifs and are impacted by HNRNPA2B1 depletion, implicating this RNA-binding protein in the sorting of long RNA to EVs. After signaling-dependent modification of the cellular transcriptome, we observed that, unexpectedly, the rate of EV enrichment relative to cells was altered for many mRNA and lncRNA transcripts. This change in EV enrichment was negatively correlated with intracellular abundance, with transcripts whose export to EVs increased showing decreased abundance in cells and vice versa. Correspondingly, after treatment with inhibitors of EV secretion, levels of mRNA and lncRNA transcripts that are normally highly exported to EVs increased in cells, indicating a measurable impact of EV export on the long RNA transcriptome of the exporting cells. Compounds with different mechanisms of inhibition of EV secretion affected the cellular transcriptome differently, suggesting the existence of multiple EV subtypes with different long RNA profiles. CONCLUSIONS: We present evidence for an impact of EV physiology on the characteristics of EV-producing cell transcriptomes. Our work suggests a new paradigm in which the sorting and packaging of transcripts into EVs participate, together with transcription and RNA decay, in controlling RNA homeostasis and shape the cellular long RNA abundance profile.
Asunto(s)
Vesículas Extracelulares , MicroARNs , ARN Largo no Codificante , Movimiento Celular , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Exosomes are small extracellular vesicles of around 100 nm of diameter produced by most cell types. These vesicles carry nucleic acids, proteins, lipids, and other biomolecules and function as carriers of biological information in processes of extracellular communication. The content of exosomes is regulated by the external and internal microenvironment of the parent cell, but the intrinsic mechanisms of loading of molecules into exosomes are still not completely elucidated. In this study, by the use of next-generation sequencing we have characterized in depth the RNA composition of healthy endothelial cells and exosomes and provided an accurate profile of the different coding and noncoding RNA species found per compartment. We have also discovered a set of unique genes preferentially included (or excluded) into vesicles. Moreover, after studying the enrichment of RNA motifs in the genes unequally distributed between cells and exosomes, we have detected a set of enriched sequences for several classes of RNA. In conclusion, our results provide the basis for studying the involvement of RNA-binding proteins capable of recognizing RNA sequences and their role in the export of RNAs into exosomes.
Asunto(s)
Exosomas/metabolismo , ARN/clasificación , Secuencia de Bases , Exosomas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Motivos de Nucleótidos/genética , ARN/genética , ARN/metabolismo , Análisis de Secuencia de ARNRESUMEN
Tumour-derived extracellular vesicles (EVs) participate in tumour progression by deregulating various physiological processes including angiogenesis and inflammation. Here we report that EVs released by endothelial cells in a mammary tumour environment participate in the recruitment of macrophages within the tumour, leading to an immunomodulatory phenotype permissive for tumour growth. Using RNA-Seq approaches, we identified several microRNAs (miRNAs) found in endothelial EVs sharing common targets involved in the regulation of the immune system. To further study the impact of these miRNAs in a mouse tumour model, we focused on three miRNAs that are conserved between humans and mouse, that is, miR-142-5p, miR-183-5p and miR-222-3p. These miRNAs are released from endothelial cells in a tumour microenvironment and are transferred via EVs to macrophages. In mouse mammary tumour models, treatment with EVs enriched in these miRNAs leads to a polarization of macrophages toward an M2-like phenotype, which in turn promotes tumour growth.
Asunto(s)
Vesículas Extracelulares , MicroARNs , Neoplasias , Animales , Modelos Animales de Enfermedad , Células Endoteliales , Vesículas Extracelulares/genética , Ratones , MicroARNs/genética , Microambiente Tumoral , Macrófagos Asociados a TumoresRESUMEN
In endothelial cells, binding of vascular endothelial growth factor (VEGF) to the receptor VEGFR2 activates multiple signaling pathways that trigger processes such as proliferation, survival, and migration that are necessary for angiogenesis. VEGF-bound VEGFR2 becomes internalized, which is a key step in the proangiogenic signal. We showed that the urokinase plasminogen activator receptor (uPAR) interacted with VEGFR2 and described the mechanism by which this interaction mediated VEGF signaling and promoted angiogenesis. Knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) impaired VEGFR2 signaling, and uPAR deficiency in mice prevented VEGF-induced angiogenesis. Upon exposure of HUVECs to VEGF, uPAR recruited the low-density lipoprotein receptor-related protein 1 (LRP-1) to VEGFR2, which induced VEGFR2 internalization. Thus, the uPAR-VEGFR2 interaction is crucial for VEGF signaling in endothelial cells.
Asunto(s)
Neovascularización Fisiológica/fisiología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Factor A de Crecimiento Endotelial Vascular/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Endocitosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrina beta1/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Unión Proteica , Transducción de SeñalRESUMEN
The interaction between tumor cells and their microenvironment is an essential aspect of tumor development. Therefore, understanding how this microenvironment communicates with tumor cells is crucial for the development of new anti-cancer therapies. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression. They are secreted into the extracellular medium in vesicles called exosomes, which allow communication between cells via the transfer of their cargo. Consequently, we hypothesized that circulating endothelial miRNAs could be transferred to tumor cells and modify their phenotype. Using exogenous miRNA, we demonstrated that endothelial cells can transfer miRNA to tumor cells via exosomes. Using miRNA profiling, we identified miR-503, which exhibited downregulated levels in exosomes released from endothelial cells cultured under tumoral conditions. The modulation of miR-503 in breast cancer cells altered their proliferative and invasive capacities. We then identified two targets of miR-503, CCND2 and CCND3. Moreover, we measured increased plasmatic miR-503 in breast cancer patients after neoadjuvant chemotherapy, which could be partly due to increased miRNA secretion by endothelial cells. Taken together, our data are the first to reveal the involvement of the endothelium in the modulation of tumor development via the secretion of circulating miR-503 in response to chemotherapy treatment.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Exosomas/metabolismo , MicroARNs/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Quimioterapia Adyuvante , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células Endoteliales de la Vena Umbilical Humana , Humanos , MicroARNs/administración & dosificación , MicroARNs/genética , Terapia NeoadyuvanteRESUMEN
The N-terminal fragment of prolactin (16K PRL) inhibits tumor growth by impairing angiogenesis, but the underlying mechanisms are unknown. Here, we found that 16K PRL binds the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1), which is known to contextually promote tumor angiogenesis and growth. Loss of PAI-1 abrogated the antitumoral and antiangiogenic effects of 16K PRL. PAI-1 bound the ternary complex PAI-1-urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR), thereby exerting antiangiogenic effects. By inhibiting the antifibrinolytic activity of PAI-1, 16K PRL also protected mice against thromboembolism and promoted arterial clot lysis. Thus, by signaling through the PAI-1-uPA-uPAR complex, 16K PRL impairs tumor vascularization and growth and, by inhibiting the antifibrinolytic activity of PAI-1, promotes thrombolysis.
Asunto(s)
Fibrinólisis , Neovascularización Patológica , Inhibidor 1 de Activador Plasminogénico/fisiología , Prolactina/fisiología , Animales , División Celular , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Neoplasias/irrigación sanguínea , Neoplasias/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Prolactina/químicaRESUMEN
The 16-kDa angiostatic N-terminal fragment of human prolactin (16K hPRL) has been reported to be a new potent anticancer compound. This protein has already proven its efficiency in several mouse tumor models in which it prevented tumor-induced angiogenesis and delayed tumor growth. In addition to angiogenesis, tumors also stimulate the formation of lymphatic vessels, which contribute to tumor cell dissemination and metastasis. However, the role of 16K hPRL in tumor-induced lymphangiogenesis has never been investigated. We establish in vitro that 16K hPRL induces apoptosis and inhibits proliferation, migration, and tube formation of human dermal lymphatic microvascular endothelial cells. In addition, in a B16F10 melanoma mouse model, we found a decreased number of lymphatic vessels in the primary tumor and in the sentinel lymph nodes after 16K hPRL treatment. This decrease is accompanied by a significant diminished expression of lymphangiogenic markers in primary tumors and sentinel lymph nodes as determined by quantitative RT-PCR. These results suggest, for the first time, that 16K hPRL is a lymphangiostatic as well as an angiostatic agent with antitumor properties.
Asunto(s)
Proteínas Angiostáticas/farmacología , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Ganglios Linfáticos/efectos de los fármacos , Linfangiogénesis/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Prolactina/farmacología , Proteínas Angiostáticas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Células Endoteliales/patología , Femenino , Humanos , Ganglios Linfáticos/patología , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/patología , Ratones , Neovascularización Patológica/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Prolactina/uso terapéuticoRESUMEN
BACKGROUND: Angiogenesis, the formation of new blood vessels from existing vasculature, plays an essential role in tumor growth, invasion, and metastasis. 16K hPRL, the antiangiogenic 16-kDa N-terminal fragment of human prolactin was shown to prevent tumor growth and metastasis by modifying tumor vessel morphology. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated the effect of 16K hPRL on tumor vessel maturation and on the related signaling pathways. We show that 16K hPRL treatment leads, in a murine B16-F10 tumor model, to a dysfunctional tumor vasculature with reduced pericyte coverage, and disruption of the PDGF-B/PDGFR-B, Ang/Tie2, and Delta/Notch pathways. In an aortic ring assay, 16K hPRL impairs endothelial cell and pericyte outgrowth from the vascular ring. In addition, 16K hPRL prevents pericyte migration to endothelial cells. This event was independent of a direct inhibitory effect of 16K hPRL on pericyte viability, proliferation, or migration. In endothelial cell-pericyte cocultures, we found 16K hPRL to disturb Notch signaling. CONCLUSIONS/SIGNIFICANCE: Taken together, our data show that 16K hPRL impairs functional tumor neovascularization by inhibiting vessel maturation and for the first time that an endogenous antiangiogenic agent disturbs Notch signaling. These findings provide new insights into the mechanisms of 16K hPRL action and highlight its potential for use in anticancer therapy.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Vasos Sanguíneos/crecimiento & desarrollo , Neoplasias/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Prolactina/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos/farmacología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Técnicas de Cocultivo , Células Endoteliales , Ratones , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Pericitos , Prolactina/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short peptides known to destabilize membranes and lipid cores and characterized by an asymmetric distribution of hydrophobic residues along the axis when helical. We have previously shown that 16-kDa fragments of the human prolactin/growth hormone (PRL/GH) family members are potent angiogenesis inhibitors. Here, we demonstrate that all these fragments possess a 14-aa sequence having the characteristics of a tilted peptide. The tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo. These antiangiogenic effects are abolished when the peptides' hydrophobicity gradient is altered by mutation. We further demonstrate that the well known tilted peptides of simian immunodeficiency virus gp32 and Alzheimer's beta-amyloid peptide are also angiogenesis inhibitors. Taken together, these results point to a potential new role for tilted peptides in regulating angiogenesis.