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
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.
KeywordsCellular mechanics Extracellular matrix rigidity Cell geometry Nuclear morphology TNF-α treatment MCF-7 conditioned media Transcription factors Signaling pathways TNF-α—p-65 TGF-β—MKL1 TGF-β—SMAD3 Cancer-associated-fibroblasts Tumorigenesis
I would like to specially thank Ms. Saradha Venkathachalapathy along with other members of G. V. S. Lab of Mechanobiology Institute, NUS, as well as NUS High School of Math and Science for their unwavering support and guidance throughout this project.
- 1.Kostic, A., Lynch, C. D., & Sheetz, M. P. (2009). Differential matrix rigidity response in breast cancer cell lines correlates with the tissue tropism. PLoS ONE, 4(7). https://doi.org/10.1371/journal.pone.0006361.
- 3.Jain, N., Iyer, K. V., Kumar, A., & Shivashankar, G. V. (2013). Cell geometric constraints induce modular gene-expression patterns via redistribution of HDAC3 regulated by actomyosin contractility. Proceedings of the National Academy of Sciences, 110(28), 11349–11354. https://doi.org/10.1073/pnas.1300801110.CrossRefGoogle Scholar
- 7.Wu, Y., Zhang, X., Salmon, M., Lin, X., & Zehner, Z. E. (2007). TGFβ1 regulation of vimentin gene expression during differentiation of the C2C12 skeletal myogenic cell line requires Smads, AP-1 and Sp1 family members. Biochimica Et Biophysica Acta (BBA)—Molecular Cell Research, 1773(3), 427–439. https://doi.org/10.1016/j.bbamcr.2006.11.017.
- 8.Rueden, C. T., Schindelin, J., Hiner, M. C., Dezonia, B. E., Walter, A. E., Arena, E. T., et al. (2017). ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics, 18(1). https://doi.org/10.1186/s12859-017-1934-z.
- 9.The R Project for Statistical Computing. (n.d.). Retrieved from http://www.R-project.org/.
- 12.Sero, J. E., Sailem, H. Z., Ardy, R. C., Almuttaqi, H., Zhang, T., & Bakal, C. (2015). Cell shape and the microenvironment regulate nuclear translocation of NF-κB in breast epithelial and tumor cells. Molecular Systems Biology, 11(3), 790. https://doi.org/10.15252/msb.20145644.CrossRefPubMedGoogle Scholar
- 16.Millet, C., & Zhang, Y. E. (2007). Roles of Smad3 in TGF-β signaling during carcinogenesis. Critical Reviews™ in Eukaryotic Gene Expression, 17(4), 281–293. https://doi.org/10.1615/critreveukargeneexpr.v17.i4.30.
- 19.Jaganathan, H., Gage, J., Leonard, F., Srinivasan, S., Souza, G. R., Dave, B., et al. (2014). Three-dimensional in vitro co-culture model of breast tumor using magnetic levitation. Scientific Reports, 4(1). https://doi.org/10.1038/srep06468.