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

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.