Sulfated hyaluronan modulates extracellular matrix composition and miRNA expression in 3D breast cancer spheroids
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Date
2026-06-05
Authors
Δροσινού, Μαρία-Γεωργία
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Abstract
Cancer continues to be one of the most prevalent diseases worldwide, as incidence rates increase-steadily. The biological heterogeneity of breast cancer arises from the presence of different-molecular subtypes; each associated with unique clinical behaviors. The extracellular matrix-(ECM) is a three-dimensional (3D) network of macromolecules, that offers structural integrity to-cells while actively influencing their functional properties in both normal and disease-related-environments. Hyaluronan (HA), distinguished as the only non-sulfated glycosaminoglycan-(GAG), regulates breast cancer progression with its effects being dependent on its molecular size.
Previous studies from our group have shown that chemically sulfated hyaluronan (sHA) alters-functional properties and ECM-related gene expression in triple-negative breast cancer cells and-reduces tumor growth in vivo. Within the tumor microenvironment (TME), ECM becomes notably-stiffer due to the high accumulation of matrix components, which promotes tumor growth.
Furthermore, microRNAs (miRNAs) are recognized as key epigenetic regulators and constituents-of the TME, influencing cellular functions, including proliferation, migration, metastasis, invasion-and resistance to chemotherapy. miRNAs are endogenous, non-coding RNA molecules that-regulate gene expression through post-transcriptional mechanisms. Among their targets, they-regulate the expression of crucial matrix effectors, while vice versa interactions within the TME-influence miRNAs expression. In this context, our research group has demonstrated the functional-role of specific miRNAs in the regulation of matrix expression, signaling and breast cancer cell-behavior. Despite the insights provided by traditional 2D cultures in cancer research, they-inadequately model the structural and biochemical complexity of the TME. As a result, cancer-research focuses on 3D culture models that better reproduce the dynamic interactions between-tumor cells and the extracellular matrix observed in solid tumors. This MSc thesis aimed to-evaluate the effects of 50 kDa fragments of non-sulfated HA and sHA (200 μg/mL) on the-expression of key matrix macromolecules, encompassing matrix metalloproteinases,-proteoglycans, and HA receptors, as well as specific miRNAs implicated in breast cancer-progression (e.g., miR-10b-5p, miR-200b-3p, let-7d-5p). In addition, we investigated the impact-of sHA on spheroid morphology and functional properties in 3D cultures of breast cancer cells-with distinct estrogen receptor (ER) status. Our findings demonstrated that sHA exerts significant-inhibitory effects on breast cancer progression in 3D models. Specifically, sHA treatment reduced-spheroid growth after prolonged culture and markedly suppressed cancer cell dissemination, with-a more pronounced effect in ERβ-suppressed (shERβ MDA-MB-231) spheroids, where migratory-capacity was completely abrogated. At the molecular level, sHA modulated the expression of key-extracellular matrix regulators, including HAS2 and RHAMM, as well as promoted alterations in-MMPs expression, underscoring its impact on ECM remodeling. Finally, sHA induced distinct-miRNA expression profiles depending on ER status. Particularly, in shERβ MDA-MB-231 cells,-a coordinated upregulation of tumor-suppressive miRNAs was observed, suggesting enhanced-regulatory plasticity and reduced tumorigenic potential. Notably, these effects were more-prominent in 3D cultures compared to 2D systems, further highlighting their importance as-physiologically relevant tumor models.
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Keywords
Activation function, Two-dimensional, Three-dimensional, Breast cancer gene 1/2, Cancer-associated fibroblasts, Cyclin d1, DNA-binding domain, Disseminating tumor cell, 17β-Estradiol, Extracellular matrix, Epithelial-to-mesenchymal transition, Estrogen receptor, Estrogen responsive elements, Glycosaminoglycans, Growth factor receptors, G protein-coupled estrogen receptor 1, Hyaluronan, Human epidermal growth factor receptor 2, Ligand-binding domain, Mesenchymal–epithelial transition, microRNAs, miRNA-induced silencing complex, Matrix metalloproteinases, Poly(A)-specific ribonuclease, Proteoglycans, Progesterone receptor, Precursor microRNAs, Receptor tyrosine kinases, Transcription factor, Tissue inhibitors of metalloproteinases, Tumor microenvironment, Triple-negative breast cancer, World Health Organization