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A Small Vimentin-Binding Molecule Blocks Cancer Exosome Release and Reduces Cancer Cell Mobility.

ABSTRACT: Vimentin is an intermediate filament protein with diverse roles in health and disease far beyond its structural functions. Exosomes or small extracellular vesicles (sEVs) are key mediators for intercellular communication, contributing to tissue homeostasis and the progression of various diseases, especially the metastasis of cancers. In this study, we evaluated a novel vimentin-binding compound (R491) for its anti-cancer activities and its roles in cancer exosome release. The compound R491 induced a rapid and reversible intracellular vacuolization in various types of cancer cells. This phenotype did not result in an inhibition of cancer cell growth, which was consistent with our finding from a protein array that R491 did not reduce levels of major oncoproteins in cancer cells. Morphological and quantitative analyses on the intracellular vacuoles and extracellular exosomes revealed that in response to R491 treatment, the exosomes released from the cells were significantly reduced, while the exosomes retained as intra-luminal vesicles inside the cells were subsequently degraded. Vim^+/- cells had lower amounts of vimentin and accordingly, lower amounts of both the retained and the released exosomes than Vim^+/+ cells had, while the vimentin-binding compound R491 inhibited only the release of exosomes. Further functional tests showed that R491 significantly reduced the migration and invasion of cancer cells in vitro and decreased the amount of exosome in the blood in mice. Our study suggests that vimentin promotes exosome release, and small-molecule compounds that target vimentin are able to both block cancer exosome release and reduce cancer cell motility, and therefore could have potential applications for inhibiting cancer invasive growth.

SUBMITTER: Wu J

PROVIDER: S-EPMC8297618 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The myotonic dystrophy kinase-related CDC42-binding kinases MRCK? and MRCK? regulate actin-myosin contractility and have been implicated in cancer metastasis. Along with the related ROCK1 and ROCK2 kinases, the MRCK proteins initiate signalling events that lead to contractile force generation which powers cancer cell motility and invasion. A potential strategy for cancer therapy is to reduce metastasis by blocking MRCK activity, either alone or in combination with ROCK inhibition. However, to date no potent small molecule inhibitors have been developed with selectivity towards MRCK.Screening a kinase-focused small molecule chemical library resulted in the identification of compounds with inhibitory activity towards MRCK. Medicinal chemistry combined with in vitro enzyme profiling led to the discovery of 4-chloro-1-(4-piperidyl)-N-[5-(2-pyridyl)-1H-pyrazol-4-yl]pyrazole-3-carboxamide (BDP00005290; abbreviated as BDP5290) as a potent MRCK inhibitor. X-ray crystallography of the MRCK? kinase domain in complex with BDP5290 revealed how this ligand interacts with the nucleotide binding pocket. BDP5290 demonstrated marked selectivity for MRCK? over ROCK1 or ROCK2 for inhibition of myosin II light chain (MLC) phosphorylation in cells. While BDP5290 was able to block MLC phosphorylation at both cytoplasmic actin stress fibres and peripheral cortical actin bundles, the ROCK selective inhibitor Y27632 primarily reduced MLC phosphorylation on stress fibres. BDP5290 was also more effective at reducing MDA-MB-231 breast cancer cell invasion through Matrigel than Y27632. Finally, the ability of human SCC12 squamous cell carcinoma cells to invade a three-dimensional collagen matrix was strongly inhibited by 2 ?M BDP5290 but not the identical concentration of Y27632, despite equivalent inhibition of MLC phosphorylation.BDP5290 is a potent MRCK inhibitor with activity in cells, resulting in reduced MLC phosphorylation, cell motility and tumour cell invasion. The discovery of this compound will enable further investigations into the biological activities of MRCK proteins and their contributions to cancer progression.

Project description:BackgroundExosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling.MethodsBreast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of?<?0.05 considered significant.ResultsExposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1? siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions.ConclusionsThese data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1?. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion.

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A Small Vimentin-Binding Molecule Blocks Cancer Exosome Release and Reduces Cancer Cell Mobility.

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