MiR-145 emerges as a driver and biomarker of the fibrotic tumor microenvironment

For years, microRNA-145 has been something of a puzzle in cancer biology. Depending on the study, this small non-coding RNA has appeared as a tumor suppressor, a metastasis promoter, or simply a bystander. Now, a team at Thomas Jefferson University’s Sidney Kimmel Comprehensive Cancer Center argues that the confusion stems from a fundamental oversight: most research has examined miR-145 in isolated carcinoma cells, while ignoring the tumor microenvironment—the fibroblasts, endothelial cells, immune cells, and extracellular matrix that surround cancer cells and increasingly determine clinical outcomes. Their new study, published in Frontiers in Cell and Developmental Biology, positions miR-145 as both a driver and a biomarker of the fibrotic tumor microenvironment, a subtype associated with poor prognosis across multiple malignancies.

The research team, led by Zhao Lin and Ubaldo Martinez-Outschoorn, with contributions from Megan Bennett, Patrick Tassone, and Joseph M. Curry, mined The Cancer Genome Atlas databases for head and neck squamous cell carcinoma (HNSCC) and breast cancer (BRCA). They stratified tumors into four microenvironment subtypes based on immune and stromal characteristics: immune-enriched non-fibrotic, immune-enriched fibrotic, fibrotic, and immune-depleted. The fibrotic subtype—characterized by epithelial-mesenchymal transition (EMT), fibrosis, angiogenesis, and immune exclusion—showed the poorest overall survival in both cancer types. Critically, miR-145 expression was highest in these fibrotic tumors, suggesting that this microRNA could serve as a molecular fingerprint of the most aggressive microenvironmental state.

Gene Set Variation Analysis revealed that high miR-145 tumors were positively correlated with EMT, angiogenesis, NOTCH, hypoxia, TGF-β, Hedgehog, myogenesis, and Wnt/β-catenin signaling, while showing negative correlations with oxidative phosphorylation and interferon-γ pathways. Gene Set Enrichment Analysis across the top and bottom 25% of miR-145 expression identified 12 Hallmark, 37 Reactome, and 322 Gene Ontology pathways significantly enriched in miR-145-high tumors across both cancer types. Reactome analysis highlighted extensive extracellular matrix remodeling, including ECM organization, proteoglycan and collagen formation, integrin and laminin interactions, and matrix metalloproteinase activation. Gene Ontology analysis further revealed enrichment of angiogenic and lymphangiogenic programs, including sprouting angiogenesis, vasculogenesis, endothelial cell migration, and VEGF and PDGFR signaling.

Deconvolution of tumor microenvironment cell types using xCell analyses showed that high miR-145 expression was associated with increased endothelial cells and decreased immune cell infiltration in both HNSCC and BRCA. Innate immune populations, including mast cells and plasmacytoid dendritic cells, were reduced, with more pronounced depletion of adaptive immune cells, particularly B and T lymphocytes. Single-sample GSEA of the TCGA HNSCC dataset confirmed that tumors with high miR-145 displayed a highly fibrotic, immune-excluded phenotype, enriched for angiogenesis, cancer-associated fibroblasts, and extracellular matrix remodeling. Malignant cells in these tumors exhibited enhanced EMT and immune-evasive features, including increased myeloid-derived suppressor cell recruitment, reduced antigen processing and presentation, and diminished infiltration of M1 macrophages, Th1 cells, NK cells, and CD8-positive cytotoxic T cells.

EcoTyper deconvolution further refined the picture. MiR-145-high tumors were enriched for Carcinoma Ecotype 1 (CE1), which is associated with poor prognosis, low lymphocyte infiltration, and abundant periostin-positive fibroblasts, and depleted for CE9 and CE10 ecotypes, which are linked to improved survival and enhanced immune activity. Consistent with CE1, miR-145-high tumors were enriched for periostin-positive fibroblasts, partial EMT-like epithelial cells, and ANGPTL2-positive/NID2-positive tip-like endothelial cells, all associated with tumor progression and neovascularization. In contrast, cytotoxic GZMB-positive and GZMK-positive CD8 T cells, PRF1-positive NK cells, and CE9/CE10-associated T cells were reduced. Macrophage polarization also shifted toward an unfavorable phenotype, with decreased M1 macrophages and increased M2 macrophages.

To investigate the mechanism of miR-145 expression, the team established a coculture system of fibroblasts and carcinoma cells at a 4:1 ratio. After four days, exosomes were isolated from conditioned media and analyzed by miRNA array. miR-145 was highly enriched in fibroblast-derived exosomes compared with those from breast carcinoma cells under homotypic culture. Intracellular miR-145 expression was markedly higher in fibroblasts than in breast or head and neck carcinoma cells. Two coculture systems were then compared: a transwell setup with physical separation and a direct-contact coculture followed by GFP/RFP-based cell sorting. Only direct cell-cell juxtacrine interaction markedly induced miR-145 expression in carcinoma cells, pointing to membrane-bound NOTCH signaling as the likely mediator.

Proteomic analysis of Detroit562 cells ectopically expressing precursor miR-145 identified 21 proteins showing greater than 1.5-fold differential expression as candidate miR-145-regulated targets. Among them, phosphoserine phosphatase (PSPH) was the most upregulated protein, a key enzyme in the serine biosynthesis pathway metabolically linked to glycolysis. Protein-protein interaction network analysis revealed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) occupied the most connected node within the network, with cell death, proliferation, ATP production, and oxidative stress as the principal associated biological processes. Elevated GAPDH expression was associated with reduced overall survival in the TCGA HNSCC cohort (p = 0.01) and linked to a non-inflamed tumor microenvironment including fibrotic and immune-depleted subtypes. Conversely, miR-145 overexpression downregulated two autophagy-related proteins, CAPZB and SQSTM1, and increased LysoTracker and LysoSensor signals across multiple cancer cell lines, consistent with enhanced autophagy.

Mitochondrial activity assays using MitoTracker Orange showed that miR-145 increased mitochondrial mass and membrane hyperpolarization in MCF7, MDA231, FaDu, and Detroit562 cells. ATP production and glucose uptake were also elevated following miR-145 overexpression. Seahorse XF Cell Mito Stress Tests in MOC1 and Detroit562 cells revealed that miR-145 overexpression increased both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), indicating enhanced mitochondrial oxidative phosphorylation and glycolysis. In vivo, miR-145-overexpressing MOC1 cells coinjected with mouse embryonic fibroblasts formed larger tumors than controls, promoted tumor angiogenesis and lymphangiogenesis, increased expression of VEGFR2 and Jagged1, reduced CD8A-positive cytotoxic T-cell infiltration, and significantly increased fibrosis. Detroit562 cells overexpressing miR-145 co-injected with hTERT-BJ1 fibroblasts similarly promoted tumor growth with suppressed SQSTM1 and induced TOMM20 expression, consistent with elevated mitochondrial activity.

The requirement for carcinoma-fibroblast juxtacrine interaction to induce miR-145 suggested involvement of membrane-bound NOTCH signaling. Coculture markedly increased NOTCH1 expression in carcinoma cells, as shown by immunofluorescence and flow cytometry. Western blot showed that miR-145 overexpression induced the functional, cleaved intracellular domain of the Notch1 receptor (NICD). In the TCGA-HNSC dataset, the fibrotic subtype exhibited the highest expression of both miR-145 and the NOTCH signaling pathway, as well as the highest expression of the cell-surface Notch ligand JAG1, which initiates NOTCH signaling in neighboring cells. NOTCH1ΔNT overexpression significantly elevated miR-145 levels across multiple cell lines compared with controls, demonstrating a positive feedback loop between NOTCH signaling and miR-145.

Epigenetic changes, particularly DNA methylation, occur early in tumor development and can regulate gene expression. Bioinformatics and methylation-specific PCR analyses showed that miR-145 promoter methylation was markedly reduced in cocultured cancer cells compared with homotypic cultures, while fibroblasts exhibited lower methylation levels than carcinoma cells. Treatment with the DNA methylation inhibitor azacitidine (5-Aza) significantly increased miR-145 expression in cancer cells. Western blot showed increased TP53 and reduced DNMT1 and COMMD1 expression in hTERT-BJ1 and MCF7 cells. p53 can promote miR-145 post-transcriptional maturation, while DNMT1 encodes the primary enzyme responsible for maintaining DNA methylation patterns during cell division, and COMMD1 is a negative regulator of NFκB-HIF signaling. These findings indicate that miR-145 upregulation in tumors is driven by both NOTCH signaling and promoter DNA hypomethylation. The authors acknowledge that functional validation using miR-145 knockdown or knockout models would provide additional mechanistic insights, but note that miR-145 expression is markedly downregulated in tumor tissues compared with normal controls, so they focused on characterizing its baseline expression and functional associations across microenvironment subtypes. This study identifies miR-145 as a potential biomarker of the fibrotic tumor microenvironment, providing a foundation for future mechanistic studies and therapeutic strategies.

Subject of Research: Cancer Cell Biology

Article Title: MiR-145 reprogramming facilitates fibrotic trajectory in the tumor microenvironment

Article References: Lin, Z., Bennett, M., Tassone, P., Curry, J. M., & Martinez-Outschoorn, U. (2026). MiR-145 reprogramming facilitates fibrotic trajectory in the tumor microenvironment. Frontiers in Cell and Developmental Biology, 14, Article 1850756. https://doi.org/10.3389/fcell.2026.1850756

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1850756

Keywords: miR-145, tumor microenvironment, fibrosis, NOTCH signaling, immune evasion, EMT, cancer-associated fibroblasts, GAPDH, mitochondrial metabolism, hypomethylation, reprogramming, facilitates