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The role of endosomal protein DENND10/FAM45A in extracellular vesicle release and cancer cell migration

ABSTRACT: Cell migration is key to tumor metastasis, the most challenging clinical problem in cancer treatment. Here we are investigating the role of DENND10/FAM45A, a recently described endosomal protein, in cancer cell migration via modulating the release of extracellular vesicles (EVs). Bioinformatics data mining showed that high DENND10 expression is significantly associated with poor prognosis in multiple cancer types. DENND10 knockout (DENND10-KO) resulted in defective cell spreading and migration, with impaired formation of stress fibers and focal adhesions. EV secretion was reduced in DENND10-KO cells. Importantly, wild-type conditioned medium or EVs restored the migratory ability and cytoskeletal organization of DENND10-KO cells. Quantitative mass spectrometry revealed that DENND10 depletion led to distinct EV compositional profiles with remodeled constituents of adhesion molecules and extracellular matrix. Together, our study unveiled DENND10 as an intrinsic coordinator between EV release and cell migration, which could be exploited for designing new anti-cancer strategies.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Yanling Zhang

LAB HEAD: Yanling Zhang

PROVIDER: PXD038264 | Pride | 2024-06-14

REPOSITORIES: pride

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			Action	DRS
	DD051LQB1_KO1_Slot1-18_1_17422.d.zip	Other
	DD051LQB1_KO2_Slot1-19_1_17423.d.zip	Other
	DD051LQB1_KO3_Slot1-20_1_17424.d.zip	Other
	DD051LQB1_NC1_Slot1-15_1_17418.d.zip	Other
	DD051LQB1_NC2_Slot1-16_1_17419.d.zip	Other

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Project description:High expression levels of the RNA binding protein IGF2BP2/IMP2 are correlated with increased tumor cell proliferation, invasion, and poor prognosis in the clinic. Tumor cells release extracellular vesicles (EVs) that can polarize macrophages towards tumor-associated macrophages (TAMs), which play a role in tumor progression. However, there is a lack of understanding of IMP2’s effect on TAMs. EVs were isolated from colorectal cancer HCT116 parental (WT) and CRISPR/Cas9 IMP2 knockout (KO) cells via ultracentrifugation and tangential flow filtration. EVs were characterized according to MISEV guidelines and microRNA cargo was assessed by microRNA-Seq. Primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype by EVs and the expression of genes and surface markers was assessed by qPCR and flow cytometry, respectively. Morphological changes of macrophages as well as the migratory potential of cancer cells were assessed by the IncuCyte® system and macrophage matrix degradation potential by zymography. Changes in the metabolic activity of macrophages were quantified in live cells in a Seahorse® analyzer. For in vivo studies, EVs were injected into the yolk sac of zebrafish embryos, and macrophages were isolated by fluorescence-activated cell sorting. EV samples from both WT and KO EVs had a similar size and concentration and were positive or CD9, CD81, CD63, and ALIX. The expression of tumor-promoting genes was higher in macrophages polarized with WT EVs, while the expression of tumor-suppressing TNF and IL6 was reduced. WT EV-polarized macrophages showed a higher abundance of TAM-like surface markers, higher matrix degrading activity, as well as a higher promotion of cancer cell migration. They exhibited a higher basal oxygen consumption rate and a lower extracellular acidification rate. microRNA-seq revealed a significant difference in the microRNA composition of WT and KO EVs. Macrophages transfected with miR-181a-5p mimic showed a lower level of the phosphatase DUSP6. Zebrafish macrophages upon polarization with WT EVs showed lower expression of IL6 and TNF. Our results show that IMP2 can determine the cargo of EVs released by cancer cells, thereby modulating the EVs’ actions on macrophages. Expression of IMP2 is responsible for the secretion of EVs that polarize macrophages toward a tumor-promoting phenotype.

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The role of endosomal protein DENND10/FAM45A in extracellular vesicle release and cancer cell migration

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