Cortactin and fascin-1 regulate extracellular vesicle release by controlling endosomal trafficking or invadopodia formation and function

ABSTRACTInvadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. We used our high-content screening assay to identify kinases impacting invadopodia formation. Among the top hits we selected TAO3, a STE20-like kinase of the GCK subfamily, for further analysis. TAO3 was over-expressed in many human cancers, and regulated invadopodia formation in melanoma, breast and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in 3-dimensional matrices and in vivo. We developed potent catalytic inhibitors of TAO3 that inhibited invadopodia formation and function, and tumor cell extravasation and growth. Using these inhibitors, we determined that TAO3 activity was required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action.SIGNIFICANCETargeting tumor invasive behavior represents an understudied opportunity. We used an unbiased screening approach to identify kinases required for invadopodia formation and function. We validated TAO3, both genetically and with a novel inhibitor, and determined TAO3 function. Our data support clinical development of this class of target.

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The Necroptosis Effector MLKL drives Small Extracellular Vesicle Release and Tumour Growth in Glioblastoma

10.1101/2021.01.12.426398 ◽

2021 ◽

Author(s):

Gwennan André-Grégoire ◽

Tiphaine Douanne ◽

An Thys ◽

Clément Maghe ◽

Kathryn Jacobs ◽

...

Keyword(s):

Tumour Growth ◽

Tumour Progression ◽

Therapeutic Option ◽

Extracellular Vesicle ◽

Endosomal Trafficking ◽

Tumour Burden ◽

Vesicle Release ◽

Constitutive Secretion

AbstractExtracellular vesicles (EVs) are lipid-based nano-sized particles that convey biological material from donor to recipient cells. They play key roles in tumour progression, notably in glioblastoma in which the subpopulation of Glioblastoma Stem-like Cells (GSCs) might represent a meaningful source of tumour-derived EVs. However, the mechanisms involved in the production and release of EVs by GSCs are still poorly understood. Here, we report the identification of MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of small EVs from GSCs. The targeting of MLKL by genetic, protein depletion or chemical approaches alters endosomal trafficking and EV release and reduces GSC expansion in vitro. This function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL triggers a reduction of both the tumour burden in xenografted mice and of the level of plasmatic EVs. This work reinforces the idea of a non-deadly role for MLKL in endosomal trafficking and suggests that interfering with EV biogenesis is a promising therapeutic option to sensitize glioblastoma cells to death.

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STEM-18. STROMAL EXTRACELLULAR VESICLES DRIVE GLIOMA STEM CELL REPROGRAMMING

Neuro-Oncology ◽

10.1093/neuonc/noab196.092 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi24-vi25

Author(s):

Lata Adnani ◽

Brian Meehan ◽

Jordan Kassouf ◽

Cristiana Spinelli ◽

Nadim Tawil ◽

...

Keyword(s):

Molecular Subtype ◽

Extracellular Vesicle ◽

Host Cells ◽

Tumour Mass ◽

Membrane Structures ◽

And Function ◽

Rate Limiting ◽

The Brain

Abstract Glioblastoma multiforme (GBM) represents the most frequent and lethal form of brain tumors originating from glioma stem cells (GSCs). GBM remains lethal because the rate limiting patho-mechanisms remain poorly understood. In this regard, few limitations involve the diversity 'between' cellular states and the molecular/cellular complexity 'within' the tumour mass. Using cell based- and mouse- models, we explored extracellular vesicle (EV) mediated interactions between cancer and stromal cells impacting phenotypes of GSCs as a function of their molecular subtype. EVs are spherical membrane structures that cells release to expel different molecular cargo (lipids, proteins, RNA, DNA), which can be transported across a distance in the brain and taken up by various ‘recipient’ cells resulting in reprogramming of the recipient cell's content and function. In vivo, GSCs altered their pattern of NOTCH signalling and molecular phenotype as a function of proximity to non-transformed host cells in the brain. In vitro stromal EVs altered GSC sphere forming capacity, proteome and expression of mesenchymal markers. Thus, EV mediated tumour-stromal interactions could represent a biological switch and a novel targeting point in the biology of GBM.

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Extracellular vesicle interplay in cardiovascular pathophysiology

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00925.2020 ◽

2021 ◽

Author(s):

Sherin Saheera ◽

Vivek P Jani ◽

Kenneth W Witwer ◽

Shelby Kutty

Keyword(s):

Plasma Membrane ◽

Cardiovascular System ◽

Lipid Bilayer ◽

Cellular Responses ◽

Extracellular Vesicle ◽

Cargo Proteins ◽

And Function ◽

Intercellular Communications ◽

Rna And Dna

Extracellular vesicles (EVs) are nanosized lipid bilayer-delimited particles released from cells that mediate intercellular communications and play a pivotal role in various physiological and pathological processes. Subtypes of EVs may include plasma-membrane ectosomes or microvesicles and endosomal-origin exosomes, although functional distinctions remain unclear. EVs carry cargo proteins, nucleic acids (RNA and DNA), lipids, and metabolites. By presenting or transferring this cargo to recipient cells, EVs can trigger cellular responses. Here, we summarize what is known about EV biogenesis, composition, and function, with an emphasis on the role of EVs in cardiovascular system. Additionally, we provide an update on the function of EVs in cardiovascular pathophysiology, further highlighting their potential for diagnostic and therapeutic applications.

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Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

eLife ◽

10.7554/elife.41460 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 7

Author(s):

Albert Lu ◽

Paulina Wawro ◽

David W Morgens ◽

Fernando Portela ◽

Michael C Bassik ◽

...

Keyword(s):

Nucleic Acid ◽

Molecular Mechanisms ◽

Membrane Vesicles ◽

Extracellular Vesicle ◽

Biologically Active ◽

Vesicle Release ◽

Guide Rnas ◽

Disease States ◽

Genome Wide ◽

First Time

Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles.

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Mesenchymal Stem Cell-Derived Extracellular Vesicle-Based Therapy for Alzheimer’s Disease: Progress and Opportunity

Membranes ◽

10.3390/membranes11100796 ◽

2021 ◽

Vol 11 (10) ◽

pp. 796

Author(s):

Yi-An Chen ◽

Cheng-Hsiu Lu ◽

Chien-Chih Ke ◽

Ren-Shyan Liu

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Neurodegenerative Disorder ◽

Therapeutic Potential ◽

Extracellular Vesicle ◽

Synaptic Loss ◽

Molecular Machinery ◽

And Function ◽

Preclinical And Clinical Studies ◽

Ad Therapy

Alzheimer’s disease (AD), as a neurodegenerative disorder, is characterized by mass neuronal and synaptic loss and, currently, there are no successful curative therapies. Extracellular vesicles (EVs) are an emerging approach to intercellular communication via transferring cellular materials such as proteins, lipids, mRNAs, and miRNAs from parental cells to recipient cells, leading to the reprogramming of the molecular machinery. Numerous studies have suggested the therapeutic potential of EVs derived from mesenchymal stem cells (MSCs) in the treatment of AD, based on the neuroprotective, regenerative and immunomodulatory effects as effective as MSCs. In this review, we focus on the biology and function of EVs, the potential of MSC-derived EVs for AD therapy in preclinical and clinical studies, as well as the potent mechanisms of MSC-derived EVs actions. Finally, we highlight the modification strategies and diagnosis utilities in order to make advance in this field.

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Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714085115 ◽

2018 ◽

Vol 115 (6) ◽

pp. E1127-E1136 ◽

Cited By ~ 26

Author(s):

Katharina B. Beer ◽

Jennifer Rivas-Castillo ◽

Kenneth Kuhn ◽

Gholamreza Fazeli ◽

Birgit Karmann ◽

...

Keyword(s):

Plasma Membrane ◽

Extracellular Vesicle ◽

Endosomal Trafficking ◽

Vesicle Budding ◽

Sorting Nexins ◽

Dnaj Protein ◽

Membrane Budding ◽

Pleiotropic Functions

Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans. However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

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CIP4 Targeted to Recruit GTP-Cdc42 Involving in Invadopodia Formation Through NF-κB Signaling Pathway Promotes Invasion and Metastasis of Colorectal Cancer

10.21203/rs.3.rs-433625/v1 ◽

2021 ◽

Author(s):

Zhiyan Hu ◽

Jiaxian Zhu ◽

Yidan Ma ◽

Ting Long ◽

Lingfang Gao ◽

...

Keyword(s):

Colorectal Cancer ◽

Signaling Pathway ◽

Tumor Metastasis ◽

Migration And Invasion ◽

Downstream Signaling ◽

And Function ◽

Downstream Signaling Pathway ◽

Invadopodia Formation

Abstract Background CIP4 (Cdc42-interacting protein 4), a member of the F-BAR family which plays an important role in regulating cell membrane and actin, has been reported to interact with Cdc42 and closely associated with tumor invadopodia formation. However, the specific mechanism of the interaction between CIP4 and Cdc42 as well as the downstream signaling pathway in response in colorectal cancer (CRC) remains unknown, which is worth exploring for its impact on tumor infiltration and metastasis. Methods Immunohistochemistry and western blot analyses were performed to detect the expression of CIP4 and Cdc42. Their relationship with CRC clinicopathological characteristics was further analyzed. Wound-healing, transwell migration and invasion assays tested the effect of CIP4 on cells migration and invasion ability in vitro, and the orthotopic xenograft colorectal cancer mouse mode evaluated the tumor metastasis in vivo. The invadopodia formation and function were assessed by immunofluorescence, scanning electron microscopy (SEM) and matrix degradation assay. The interaction between CIP4 and Cdc42 was confirmed by co-immunoprecipitation (co-IP) and GST-Pull down assays. Immunofluorescence was used to observed the colocalization of CIP4, GTP-Cdc42 and invadopodia. The related downstream signaling pathway was investigated by western blot and immunofluorescence. Results CIP4 expression was significantly higher in human colorectal cancer tissues and correlated with the CRC infiltrating depth and metastasis as well as the lower survival rate in patients. In cultured CRC cells, knockdown of CIP4 inhibited cell migration and invasion ability in vitro and the tumor metastasis in vivo, while overexpression of CIP4 confirmed the opposite situation by promoting invadopodia formation and matrix degradation ability. In addition, we identified GTP-Cdc42 as a directly interactive protein of CIP4, which was upregulated and recruited by CIP4 to participate in this process. Furthermore, activated NF-κB signaling pathway was found in CIP4 overexpression CRC cells contributing to invadopodia formation while inhibition of either CIP4 or Cdc42 led to suppression of NF-κB pathway resulted in decrease quantity of invadopodia. Conclusion Our findings suggested that CIP4 targets to recruit GTP-Cdc42 and directly combines with it to accelerate invadopodia formation and function by activating NF-κB signaling pathway, thus promoting CRC infiltration and metastasis.

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