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Matrix stiffness induces a tumorigenic phenotype in mammary epithelium through changes in chromatin accessibility

ABSTRACT: In breast cancer, the increased stiffness of the extracellular matrix (ECM) is a key driver of malignancy. Yet little is known about the epigenomic changes that underlie the tumorigenic impact of ECM mechanics. Here, we show in a 3D culture model of breast cancer that stiff ECM induces a tumorigenic phenotype through changes in chromatin state. We found that increased stiffness yielded cells with more wrinkled nuclei and with increased lamina-associated chromatin, that cells cultured in stiff matrices displayed more accessible chromatin sites, which exhibited footprints of Sp1 binding, and that Sp1 acts along with histone deacetylases (HDAC) 3 and 8 to regulate the induction of stiffness-mediated tumorigenicity. Just as cell culture on soft environments or in them rather than on tissue-culture plastic better recapitulates the acinar morphology observed in mammary epithelium in vivo, mammary epithelial cells cultured on soft microenvironments or in them also more closely replicate the in vivo chromatin state. Our results emphasize the importance of culture conditions for epigenomic studies, and reveal that chromatin state is a critical mediator of mechanotransduction.

ORGANISM(S): Homo sapiens

PROVIDER: GSE131968 | GEO | 2019/05/31

REPOSITORIES: GEO

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Project description:Background: The differentiation of pericytes into myofibroblasts causes microvascular degeneration, extracellular matrix (ECM) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel two-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Methods: Primary microvessels were cultured in the TGM2D medium. Stiff ECM was prepared by incubating ECM solution in regular culture dishes for one hour followed by PBS wash. Soft ECM with Young’s modulus of approximately 6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, qRT-PCR, western blotting, and knockdown experiments were performed on the cells. Results: Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As Integrins are the major mechanosensor, we performed qRT-PCR screening of Integrin family members selected from RNA sequencing data. We found that Integrin β1 (Itgb1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF, to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. Conclusions: Microvascular ECs mediate ECM stiffness-induced pericyte-myofibroblast differentiation through paracrine signaling.

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Matrix stiffness induces a tumorigenic phenotype in mammary epithelium through changes in chromatin accessibility

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