Proteomic analysis of migrasomes derived from IL-6 amplifier-induced human breast cancer cells.

Integrative Proteomic Analysis Implicates Inhibition of Intracellular Protein Trafficking in Therapy-Induced Migrastasis in Prostate Cancer

Novel Roles of Splice-Derived Tumor Mirtrons in the Regulation of Macrophage Dichotomy 2310054

Background: Tumor-derived exosomes play key roles in cell-cell communication within the tumor microenvironment and have been shown to drive macrophage polarization towards an M2-like phenotype.We recently identified a novel class of splicing generated small RNAs, known as mirtrons, are highly packed in exosomes and potentially modulate macrophage plasticity. However, molecular mechanisms for this exosome-driven phenomenon are not completely clear. Human macrophages (U937) were co-cultured with exosomes derived from human lung (H23), prostate (PC3), and a variety subtypes of breast cancer cell lines (T47D, SKBR3, and MDA-MB-468). Expression of mirtrons including miR-7110, -cv002, and -6756 in cancer cells and in exosomes was assessed by qPCR. Immunofluorescent imaging was used to assess exosomal small RNA uptake in macrophages and their intracellular localization. Genomic characterization of M2-associated mirtrons in association with exosome exposure was performed using ChIP-seq. All three mirtrons–particularly cv002–were consistently enriched in tumor-derived exosomes relative to their parent tumor cells across all cancer cell lines examined. However, mirtron expression levels in exosomes or the cytoplasm did not correlate with tumor aggressiveness in breast cancer. Immunofluorescence imaging demonstrated that mirtrons carried by tumor-derived exosomes are taken up by human monocyte U937 cells and are predominantly localized to the cytoplasm, consistent with the behavior of conventional miRNAs. Notably, a subset of mirtrons was also observed to translocate into the nucleus and interact with monocyte genomic DNA, as supported by ChIP-seq analyses. Importantly, these mirtron—DNA interactions were enriched at M2-associated gene loci, suggesting a novel regulatory mechanism underlying macrophage polarization. Our results showed a novel mechanism through exosomal delivery of tumor derived mirtrons to manipulate M2-like macrophage polarization. NIGMS (R16GM153648) Immune Response Regulation: Molecular Mechanisms (IRM)

Proteomic and Functional Comparison of Extracellular Vesicles from Wild-Type and Lyn-Deficient Stromal Cells.

BRD4 contributes to the cytokine-induced acquisition of an IL-13RA2-positive inflammatory fibroblast phenotype

Abstract Background and Aims Inflammatory bowel diseases (IBDs) are characterized by chronic intestinal inflammation, in which inflammatory fibroblasts contribute to epithelial damage and amplification of mucosal inflammation. However, the molecular mechanisms regulating their pathogenic activity remain incompletely understood. This study investigated the role of the epigenetic reader BRD4 in the differentiation and function of IL-13RA2-expressing inflammatory fibroblasts in IBD. Methods Single-cell RNA sequencing datasets from IBD patients and controls were analyzed to define BRD4 expression across stromal populations. Protein expression was validated by immunofluorescence and flow cytometry in intestinal tissues and primary fibroblasts. Primary fibroblasts from IBD patients were stimulated with interleukin (IL)-4, IL-13, and tumor necrosis factor-alpha (TNF-α) to induce an inflammatory phenotype, and BRD4 function was assessed using the BRD4-targeting PROTAC AT1. Cytokine and chemokine expression was evaluated by RNA sequencing, real-time polymerase chain reaction (PCR), and ELISA, while neutrophil chemotaxis assays assessed the functional effects of fibroblast-derived mediators. Results BRD4 was significantly upregulated in inflammatory fibroblasts from IBD patients compared with other stromal subsets and controls. Inflamed tissues showed enrichment of BRD4⁺ and IL-13RA2⁺ fibroblasts. In vitro, cytokine stimulation induced IL-13RA2 expression in the majority of fibroblasts, all of which co-expressed BRD4. BRD4 degradation significantly reduced the proportion of IL-13RA2⁺ fibroblasts. BRD4⁺ fibroblasts exhibited a pro-inflammatory transcriptional profile, including CCL2, CXCL1, CXCL2, IL-11, IL-24, IL-32, and CSF3. Degradation of BRD4 markedly decreased the expression of these mediators and reduced neutrophil migration. Conclusions BRD4 contributes to cytokine-induced inflammatory activation and pro-inflammatory functions of intestinal fibroblasts in IBD. Targeting BRD4 may represent a potential strategy to modulate stromal cell-driven inflammation.

Oncostatin M orchestrates collective epithelial migration via HIF1A activation.

Extracellular signals strongly influence cell behavior, yet the mechanisms by which specific ligands mediate changes in phenotype remain unclear. The cytokine Oncostatin M (OSM) regulates homeostasis, wound healing, inflammation, and cancer progression. We previously found that OSM induces collective cell migration (CCM), a process whereby cells move as cohesive units while retaining cell-cell contacts, in MCF10A mammary epithelial cells. Here, we investigated how OSM drives CCM by comparing its effects with those elicited by epidermal growth factor (EGF) and interferon gamma (IFNG), defining ligand-specific phenotypes and molecular networks. Integrative transcriptomic and proteomic analyses identified hypoxia-inducible factor-1 (HIF1A) and signal transducer and activator of transcription 3 (STAT3) as central regulators of OSM responses. Functional validation revealed that HIF1A drives transcriptional programs associated with hypoxia, metabolic reprogramming, and immune pathways. Complement signaling emerged as a downstream effector of HIF1A, and its inhibition disrupted OSM-induced clustering and CCM. These findings establish a mechanistic link between OSM signaling, HIF1A activation, and CCM, demonstrating how cytokine-driven transcriptional reprogramming coordinates epithelial cell migration. Analysis of public breast cancer data indicate that this pathway is active in human tumors and may contribute to tissue remodeling, repair, and metastasis.

Upregulation of macrophage UPP1 promotes lung adenocarcinoma metastasis through an mtROS-cGAS-NLRP3 inflammasome axis.

Metastasis and immunosuppression remain major barriers to effective treatment of lung adenocarcinoma (LUAD), yet the metabolic mechanisms governing the pro-tumor functions of tumor-associated macrophages are incompletely understood. In this study, we identified Uridine Phosphorylase 1 (UPP1) as a macrophage-enriched metabolic regulator associated with LUAD progression. By integrating single-cell RNA sequencing with clinical cohort analyses, we found that UPP1 was preferentially expressed in tumor-associated macrophages and was associated with adverse clinical outcomes. Functional and mechanistic studies demonstrated that dysregulated UPP1 disrupted nucleotide homeostasis, leading to mitochondrial reactive oxygen species accumulation and mitochondrial DNA leakage. These mitochondrial stress signals activated the cGAS-STING pathway, which preferentially engaged NLRP3 inflammasome signaling rather than canonical antiviral responses. Consequently, macrophages underwent pyroptosis and released elevated levels of interleukin-1β (IL-1β). Through paracrine signaling, macrophage-derived IL-1β promoted epithelial-mesenchymal transition in LUAD cells and enhanced their invasive capacity in vitro. Consistent with these findings, co-injection of UPP1-overexpressing macrophages significantly increased spontaneous lung metastasis in vivo. Clinically, elevated UPP1 expression served as an independent predictor of poor survival. Furthermore, pharmacological blockade of this signaling cascade or neutralization of IL-1β attenuated macrophage-induced malignant phenotypes in tumor cells, highlighting the therapeutic relevance of this pathway. Collectively, our findings identify a macrophage-specific immunometabolic circuit in which UPP1-driven mitochondrial stress activates the mtROS-cGAS-NLRP3 axis, promoting IL-1β-dependent macrophage-tumor crosstalk and metastatic progression. These results suggest that UPP1 may serve as both a prognostic biomarker and a potential therapeutic target in LUAD.