Cross-Talk Between Cell Mechanics and Biochemical Signalling Pathways via Modulation of Nucleocytoplasmic Shuttling of Transcription Factors: A Novel Approach to Study Fibroblasts in Breast Cancer Environments

Abstract

Tumor progression in breast cancer has formerly only been linked to increased integrin signaling as a result of increased extracellular matrix rigidity. In this study, we further examined the role of cell mechanics, namely cell geometry (2D) and matrix rigidity (3D) in modulating the cell signaling response to the inflammatory cytokine TNF-α secreted by cancer cells and the conditioned media from metastatic breast cancer cell line MCF-7. Signal activation was measured by quantifying the nucleocytoplasmic shuttling of transcription factors (p-65, MKL1, SMAD3) in NIH3T3 mouse embryonic fibroblasts via a custom code in software ImageJ and novel methods—Micropatterning and Engineering cells in collagen matrix were employed to achieve the varied 2D and 3D environments of cells. Cell mechanics was shown to impinge on the nuclear morphology of cells—Circular and unpatterned cells tend to have circular nuclei, while rectangular cells and cells grown on rigid matrixes tend to have elongated nuclei. Upon exposure to MCF-7 conditioned media, TNF-α—p-65 signaling pathways were further amplified in cells with circular cells and cells grown on soft matrix; TGF-β—MKL1 pathways in cells grown on soft matrix were particularly highly activated; TGF-β—SMAD3 signaling pathways in all cells were activated, though the pathway was more activated in environments with decreased matrix rigidity. Increase in SMAD3 nuclear levels also led to a proportionate increase in Vimentin levels in cells grown in 3D matrix. Our novel understanding of the cross-talk between cell mechanics and biochemical signaling pathways is important for understanding the behavior of cancer-associated-fibroblasts (CAFs) in breast cancer and the discovery of the highly activated signaling pathways induced by CAFs giving rise to tumorigenesis under various cell environments is vital for the identification and engineering of new therapeutic targets.