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Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier.

ABSTRACT: Brain metastasis is an important cause of mortality in breast cancer patients. A key event during brain metastasis is the migration of cancer cells through blood-brain barrier (BBB). However, the molecular mechanism behind the passage through this natural barrier remains unclear. Here we show that cancer-derived extracellular vesicles (EVs), mediators of cell-cell communication via delivery of proteins and microRNAs (miRNAs), trigger the breakdown of BBB. Importantly, miR-181c promotes the destruction of BBB through the abnormal localization of actin via the downregulation of its target gene, PDPK1. PDPK1 degradation by miR-181c leads to the downregulation of phosphorylated cofilin and the resultant activated cofilin-induced modulation of actin dynamics. Furthermore, we demonstrate that systemic injection of brain metastatic cancer cell-derived EVs promoted brain metastasis of breast cancer cell lines and are preferentially incorporated into the brain in vivo. Taken together, these results indicate a novel mechanism of brain metastasis mediated by EVs that triggers the destruction of BBB.

SUBMITTER: Tominaga N

PROVIDER: S-EPMC4396394 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier.

Tominaga Naoomi N Kosaka Nobuyoshi N Ono Makiko M Katsuda Takeshi T Yoshioka Yusuke Y Tamura Kenji K Lötvall Jan J Nakagama Hitoshi H Ochiya Takahiro T

Nature communications 20150401

Brain metastasis is an important cause of mortality in breast cancer patients. A key event during brain metastasis is the migration of cancer cells through blood-brain barrier (BBB). However, the molecular mechanism behind the passage through this natural barrier remains unclear. Here we show that cancer-derived extracellular vesicles (EVs), mediators of cell-cell communication via delivery of proteins and microRNAs (miRNAs), trigger the breakdown of BBB. Importantly, miR-181c promotes the destr ...[more]

PMID: 25828099

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Project description:Apoptosis is an important factor during the early stage of intracerebral hemorrhage. MiR-181c plays a key regulatory role in apoptosis. However, whether miR-181c is involved in apoptosis of prophase cells after intracerebral hemorrhage remains unclear. Therefore, in vitro and in vivo experiments were conducted to test this hypothesis. In vivo experiments: collagenase type VII was injected into the basal ganglia of adult Sprague-Dawley rats to establish an intracerebral hemorrhage model. MiR-181c mimic or inhibitor was injected in situ 4 hours after intracerebral hemorrhage. Neurological functional defects (neurological severity scores) were assessed 1, 7, and 14 days after model establishment. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and western blot assay were conducted 14 days after model establishment. In vitro experiments: PC12 cells were cultured under oxygen-glucose deprivation, and hemins were added to simulate intracerebral hemorrhage in vitro. MiR-181c mimic or inhibitor was added to regulate miR-181c expression. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, luciferase reporter system, and western blot assay were performed. Experimental results revealed differences in miR-181c expression in brain tissues of both patients and rats with cerebral hemorrhage. In addition, in vitro experiments found that miR-181c overexpression could upregulate the Bcl-2/Bax ratio to inhibit apoptosis, while inhibition of miR-181c expression could reduce the Bcl-2/Bax ratio and aggravate apoptosis of cells. Regulation of apoptosis occurred through the phosphoinositide 3 kinase (PI3K)/Akt pathway by targeting of phosphatase and tensin homolog deleted on chromosome ten (PTEN). Higher miR-181c overexpression correlated with lower neurological severity scores, indicating better recovery of neurological function. In conclusion, miR-181c affects the prognosis of intracerebral hemorrhage by regulating apoptosis, and these effects might be directly mediated and regulated by targeting of the PTEN\PI3K/Akt pathway and Bcl-2/Bax ratio. Furthermore, these results indicated that miR-181c played a neuroprotective role in intracerebral hemorrhage by regulating apoptosis of nerve cells, thus providing a potential target for the prevention and treatment of intracerebral hemorrhage. Testing of human serum was authorized by the Ethics Committee of China Medical University (No. 2012-38-1) on February 20, 2012. The protocol was registered with the Chinese Clinical Trial Registry (Registration No. ChiCTR-COC-17013559). The animal study was approved by the Institutional Animal Care and Use Committee of China Medical University (approval No. 2017008) on March 8, 2017.

Project description:Malignant germ-cell-tumours (GCTs) are characterised by microRNA (miRNA/miR-) dysregulation, with universal over-expression of miR-371~373 and miR-302/367 clusters regardless of patient age, tumour site, or subtype (seminoma/yolk-sac-tumour/embryonal carcinoma). These miRNAs are released into the bloodstream, presumed within extracellular-vesicles (EVs) and represent promising biomarkers. Here, we comprehensively examined the role of EVs, and their miRNA cargo, on (fibroblast/endothelial/macrophage) cells representative of the testicular GCT (TGCT) tumour microenvironment (TME). Small RNA next-generation-sequencing was performed on 34 samples, comprising representative malignant GCT cell lines/EVs and controls (testis fibroblast [Hs1.Tes] cell-line/EVs and testis/ovary samples). TME cells received TGCT co-culture, TGCT-derived EVs, and a miRNA overexpression system (miR-371a-OE) to assess functional relevance. TGCT cells secreted EVs into culture media. MiR-371~373 and miR-302/367 cluster miRNAs were overexpressed in all TGCT cells/subtypes compared with control cells and were highly abundant in TGCT-derived EVs, with miR-371a-3p/miR-371a-5p the most abundant. TGCT co-culture resulted in increased levels of miRNAs from the miR-371~373 and miR-302/367 clusters in TME (fibroblast) cells. Next, fluorescent labelling demonstrated TGCT-derived EVs were internalised by all TME (fibroblast/endothelial/macrophage) cells. TME (fibroblast/endothelial) cell treatment with EVs derived from different TGCT subtypes resulted in increased miR-371~373 and miR-302/367 miRNA levels, and other generic (eg, miR-205-5p/miR-148-3p) and subtype-specific (seminoma, eg, miR-203a-3p; yolk-sac-tumour, eg, miR-375-3p) miRNAs. MiR-371a-OE in TME cells resulted in increased collagen contraction (fibroblasts) and angiogenesis (endothelial cells), via direct mRNA downregulation and alteration of relevant pathways. TGCT cells communicate with nontumour stromal TME cells through release of EVs enriched in oncogenic miRNAs, potentially contributing to tumour progression.

Project description:Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.

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Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier.

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Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier.

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