Exosome release is regulated by mTORC1 via a Rab27A-dependent mechanism
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA470637 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7138585.fastq.gz | Fastqsanger.gz | |||
| SRR7138586.fastq.gz | Fastqsanger.gz |
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Project description:Exosomes are small membrane-bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA and miRNA and are involved in cell waste management and intercellular communication. In the present study we show that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). We find that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63-positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug-stimulated release is blocked by siRNA-mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin treated cells reveals that inhibition of mTORC1 does not alter its protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions
2019-03-05 | GSE114235 | GEO
Project description:Exosomes are small membrane-bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA, and miRNA, and are involved in cell waste management and intercellular communication. In the present study, it is shown that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). It is found that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63-positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug-stimulated release is blocked by siRNA-mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin-treated cells reveals that inhibition of mTORC1 does not significantly alter its majority protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions.
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Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-^Slow/low/high/lowM-^T or M-^SLLHLM-^T, which allowed the identification of genes regulated by mTORC1 by performing the appropriate comparisons
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Project description:Ductal carcinoma in situ (DCIS) is a precancerous condition that increases the risk of invasive breast cancer (IBC), but not all DCIS cases progress to IBC. The molecular factors driving this transition remain unclear. Small extracellular vesicles (sEVs), or exosomes, play a role in advanced cancer progression, though their function in DCIS is poorly understood. This study explores the role of sEVs and their RNA content in DCIS progression. We found that Rab27A, a key regulator of exosome release, is upregulated in DCIS and IBC tissues compared to normal breast tissue. Inhibiting sEV release by knocking down Rab27A disrupted pro-invasive signaling and reduced invasion in a DCIS mouse model. Using the MCF10 breast cancer progression series, we observed increased microRNA (miRNA) content in sEVs as cells transitioned from normal to malignant, with the most significant differential miRNA expression seen in IBC-derived sEVs. In vivo, DCIS progression raised circulating sEV miRNA levels, which were reduced by Rab27A knockdown. Reintroducing miR-205, enriched in IBC-derived sEVs, suppressed DCIS cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) markers. Co-expression of miR-205 with Rab27A knockdown also suppressed TGF-β signaling, activated MAPK p38, and induced cell cycle arrest and apoptosis. These findings show that the RNA cargo of sEVs changes during malignancy, with specific miRNAs driving DCIS progression. Re-expression of miR-205 offers a promising therapeutic approach to prevent DCIS from becoming invasive.
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Project description:Ductal carcinoma in situ (DCIS) is a precancerous condition that increases the risk of invasive breast cancer (IBC), but not all DCIS cases progress to IBC. The molecular factors driving this transition remain unclear. Small extracellular vesicles (sEVs), or exosomes, play a role in advanced cancer progression, though their function in DCIS is poorly understood. This study explores the role of sEVs and their RNA content in DCIS progression. We found that Rab27A, a key regulator of exosome release, is upregulated in DCIS and IBC tissues compared to normal breast tissue. Inhibiting sEV release by knocking down Rab27A disrupted pro-invasive signaling and reduced invasion in a DCIS mouse model. Using the MCF10 breast cancer progression series, we observed increased microRNA (miRNA) content in sEVs as cells transitioned from normal to malignant, with the most significant differential miRNA expression seen in IBC-derived sEVs. In vivo, DCIS progression raised circulating sEV miRNA levels, which were reduced by Rab27A knockdown. Reintroducing miR-205, enriched in IBC-derived sEVs, suppressed DCIS cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) markers. Co-expression of miR-205 with Rab27A knockdown also suppressed TGF-β signaling, activated MAPK p38, and induced cell cycle arrest and apoptosis. These findings show that the RNA cargo of sEVs changes during malignancy, with specific miRNAs driving DCIS progression. Re-expression of miR-205 offers a promising therapeutic approach to prevent DCIS from becoming invasive.
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Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a “low/low/high/low” or “LLHL”, which allowed the identification of genes regulated by mTORC1 by performing the appropriate comparisons
2011-08-03 | GSE27982 | GEO
Project description:Extracellular vesicles (EVs) are small membrane-derived vesicles that shuttle proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the CNS. Exosomes are small EVs (40-150 nm) that are derived from multi-vesicular bodies (MVBs) of the endo-lysosomal pathway, formed by inward budding of the limiting membrane into the MVB lumen and released into the extracellular space upon fusion of the MVB with the plasma membrane (PM). Previous research revealed certain effector molecules to be required for exosome release. For instance, RAB GTPases have been shown to control exosome release in a cell- and tissue-specific manner. In addition, exosome release appears to be evoked by membrane depolarization in conjunction with calcium influx and to depend on members of the SNARE family of proteins. However, the specific cellular and molecular factors that regulate neuronal exosome release and segregate it from the release of neurotransmitter vesicles are currently unknown. Here, we used a combination of molecular biology, patch-clamp electrophysiology and pH-sensitive dye imaging to examine the effect of the neuronal growth and differentiation factors basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) on neuronal EV release. We found that bFGF-treatment of cultured hippocampal neurons increased the abundance of EVs in the culture medium as measured by Western blot and nanoparticle tracking analysis (NTA), without affecting the number or size of neuronal MVBs. The effect of bFGF depended on calcium and on receptor tyrosine kinase (RTK) activity as the calcium chelating agent BAPTA and the tyrosine kinase inhibitor genistein both abolished the bFGF-induced increase in EV release. In accord with these results, untreated neurons had a low rate of spontaneous and stimulus-evoked MVB-PM fusion events as measured by pH-sensitive dye imaging in conjunction with patch-clamp electrophysiology, but treatment with bFGF significantly increased the number of neurons exhibiting MVB-PM fusion events in response to a high-frequency stimulus. Proteomic analysis of neuronal EVs by Liquid Chromatography Mass Spectrometry (LC-MS) demonstrated bFGF to increase the abundance of the v-SNARE vesicle-associated membrane protein 3 (VAMP3, cellubrevin) on EVs, whereas VAMP2 was decreased. Conversely, knocking-down VAMP3 in cultured neurons abolished the effect of bFGF on EV release. Similar to bFGF, the classical neurotrophins BDNF and NGF increased neuronal EV release in a VAMP3-dependent manner. In summary, our results thus reveal a new function for neurotrophic factor signalling in controlling neuronal exosome release and support the investigation of growth factor-mediated signal transduction via EVs in the healthy and diseased CNS.
2020-02-06 | PXD014401 | Pride