Sp1 mechanotransduction regulates breast cancer cell invasion in engineered viscoelastic extracellular matrices.

Breast cancer progression involves extensive remodeling of the extracellular matrix (ECM), including increased stiffness, altered viscoelasticity (stress relaxation), and elevated collagen levels. While in vitro experiments have revealed a role for each of these factors in individually promoting malignant behavior, their combined effects remain unclear. Here, we engineered alginate-collagen hydrogels with independently tunable stiffness, stress relaxation, and collagen density to dissect how the complex ECM environment regulates cancer cell phenotype. We show that high stiffness, fast stress relaxation, and high collagen density led to changes in cell morphology, marked by decreased roundness, and promoted spheroid invasion in both breast cancer and non-transformed mammary epithelial cells. Single cell migration speed and displacement were greatest in matrices of high stiffness, low collagen density, and slow stress relaxation. RNA-seq and Cleavage Under Targets and Tagmentation (CUT&Tag)-seq revealed that high stiffness and fast stress relaxing groups were enriched for Sp1 target gene expression as well as increased Sp1 binding at genomic loci. Notably, analysis of publicly available claudin-low breast cancer data showed that high expression of the Sp1-regulated genes in fast stress relaxing groups was correlated with significantly reduced patient survival. Mechanistically, we found that phosphorylated Sp1 (T453) exhibited increased nuclear localization in matrices with high stiffness and fast stress relaxation. Furthermore, Sp1 phosphorylation was regulated by PI3K and ERK1/2 activity, as well as actomyosin contractility. Our tunable hydrogel platform reveals that multiple tumor-mimicking cues within complex viscoelastic microenvironments reinforce malignant traits, with Sp1 acting as a mechanoresponsive transcription factor that transduces these signals.