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Elevated exosomal Nm23-H1 subdues the pro-migratory potential of breast cancer cell-derived exosomes by altering downstream signaling cascades

ABSTRACT: Purpose: Although Nm23-H1 lacks secretion signal peptide, its presence in the extracellular space and extracellular vesicles (EVs) is widely reported. Proteomics profiling revealed that EVs released by cancer cells may contain Nm23-H1 proteins but the role of vesicular Nm23-H1 remains unknown. Here, we investigated the function of vesicular Nm23-H1 by performing transcriptome analysis of the recipient post 24 h of PBS (control) or exosome treatment. Methods: MCF-7 cells were treated with PBS or exosomes for 24 h. Cells were lysed in TRIzol. RNA quality was verified with Agilent 5400 fragment analyzer prior to library preparation. SMART-Seq2 library construction was performed, and sequencing was carried out in 2×100 cycle paired-end mode on a BGI DNBSEQ-G400 sequencer. FASTQ raw read files were trimmed using cutadapt to remove adapter sequences, primers, poly-A tails, and other types of unwanted sequences. The trimmed reads were then be mapped to the genome (UCSC hg38) using STAR with default parameters. Reads were quantified and differentially expressed genes (DEGs) were identified using DEBrowser (v1.19.1) as a graphical interface to DESeq2 with significance considered at q-value ≤ 0.05 and absolute fold change ≥ 1.2. qRT–PCR validation was performed using SuperScript® III First-Strand Kit and LightCycler 480 SYBR Green I assays. Results: We examined the changes in the transcriptome of MCF-7 cells post exosome treatment. Polyadenylated RNA-sequencing was performed on RNA isolated from the recipient cells post 24 h of PBS (control) or exosome treatment. Upon 231 exo treatment, changes in the transcript levels of 122 differentially expressed genes (DEGs) were observed, whereas 231/H1 exo treatment induced a differential expression of 449 genes. A common set of 45 genes was found to be regulated by both 231 exo and 231/H1 exo treatments. More importantly, the comparison between 231/H1 exo and 231 exo treatments revealed 107 differentially expressed genes. Conclusions: Our transcriptome analysis of the recipient cells post exosome treatment revealed multiple genes that are regulated upon exosome treatment. Exosomes with an elevated level of Nm23-H1 caused alterations in the transcript level of multiple cancer-related genes in the recipient cells. Moreover, it stimulated type I interferon signaling via STAT1 phosphorylation. These results indicate an atypical signaling pathway mediated by the uptake of exosomes with an elevated level of Nm23-H1, which may account for the anti-metastatic effect of Nm23-H1 in the tumor microenvironment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189685 | GEO | 2025/12/30

REPOSITORIES: GEO

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Project description:Background: Docosahexaenoic acid (DHA) is a natural compound with anticancer and anti-angiogenesis activity that is currently under investigation as both a preventative agent and an adjuvant to breast cancer therapy. However, the precise mechanisms of DHA’s anticancer activities are unclear. It is understood that the intercommunication between cancer cells and their microenvironment is essential to tumor angiogenesis. Exosomes are extracellular vesicles that are important mediators of intercellular communication and play a role in promoting angiogenesis. However, very little is known about the contribution of breast cancer exosomes to tumor angiogenesis or whether exosomes can mediate DHA’s anticancer action. Results: Exosomes were collected from MCF7 and MDA-MB-231 breast cancer cells after treatment with DHA. We observed an increase in exosome secretion and exosome microRNA contents from the DHA-treated cells. The expression of 83 microRNAs in the MCF7 exosomes was altered by DHA (>2-fold). The most abundant exosome microRNAs (let-7a, miR-23b, miR-27a/b, miR-21, let-7, and miR-320b) are known to have anti-cancer and/or anti-angiogenic activity. These microRNAs were also increased by DHA treatment in the exosomes from other breast cancer lines (MDA-MB-231, ZR751 and BT20), but not in exosomes from normal breast cells (MCF10A). When DHA-treated MCF7 cells were co-cultured with or their exosomes were directly applied to endothelial cell cultures, we observed an increase in the expression of these microRNAs in the endothelial cells. Furthermore, overexpression of miR-23b and miR-320b in endothelial cells decreased the expression of their pro-angiogenic target genes (PLAU, AMOTL1, NRP1 and ETS2) and significantly inhibited tube formation by endothelial cells, suggesting that the microRNAs transferred by exosomes mediate DHA’s anti-angiogenic action. These effects could be reversed by knockdown of the Rab GTPase, Rab27A, which controls exosome release. Conclusions: We conclude that DHA alters breast cancer exosome secretion and microRNA contents, which leads to the inhibition of angiogenesis. Our data demonstrate that breast cancer exosome signaling can be targeted to inhibit tumor angiogenesis and provide new insight into DHA’s anticancer action, further supporting its use in cancer therapy. Examination of small RNA populations in MCF7 cells and exosomes after DHA treatment.

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Elevated exosomal Nm23-H1 subdues the pro-migratory potential of breast cancer cell-derived exosomes by altering downstream signaling cascades

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