The role of ZEB1-induced epithelial-mesenchymal transition (EMT) in the mechanobiology of breast cancer cells

Abstract

Tumour metastasis remains a major focus of cancer research, contributing to chemoresistance and poor prognosis, accounting for more than 90% of all cancer related mortalities. The process by which tumour cells escape from their primary site and migrate to distant parts of the body to form a secondary tumour is known as epithelial-mesenchymal transition (EMT). This process is regulated by the microenvironment of the tumour cells, extracellular matrix (ECM) stiffness in particular, and accounts for the poor prognosis and chemotherapy resistance in cancer patients. In this project we used isogenic adenocarcinoma cell lines; MDA231 and MDA231-shZEB1 to investigate the effect of matrix stiffness on metastatic and non-metastatic breast cancer cells using polyacrylamide hydrogels as a substrate. MDA231 is a triple-negative cell line, commonly used to model chemotherapy resistant late-stage breast cancer. Cellular properties such as proliferation, migration, spread area, and apoptotic response to common therapeutic drugs (doxorubicin and taxol) were assayed and changes in EMT markers were analysed as a function of substrate stiffness. We also investigated the displacement induced by the two cell lines on soft polyacrylamide hydrogels to understand the interaction between the cells and their ECM. Additionally, we explored variations in gene expression from RNA sequencing. Our results show that increased matrix stiffness promotes metastatic properties such as proliferation, cell spreading, migration, higher expression of EMT markers and apoptosis resistance to Taxol in MDA231 breast cancer cells. Having investigated the displacement exerted by both cells on soft hydrogels, we conclude that although both cell types induce similar levels of displacement at a single-cell level, epithelial MDA231-shZEB1 cells collectively exert larger displacement than metastatic MDA231 cells at higher seeding densities. This is because epithelial cells form colonies so neighbouring cells would contribute to the feel of stiffness received by other cells whiting the colony, probing them to react in a way they would normally do on stiff substrates. Cells sense and respond to changes in stiffness by altering their cytoskeletal assembly as well as gene and protein levels, determining cellular phenotypes. We developed a suitable model system to study the relationship between breast cancer cells, and their substrates and identified critical genes associated with the most resistant population (MDA231 on stiff) such as BMF. This will help develop a novel cancer treatment specifically targeting taxol-resistant metastatic breast cancer cells. Using our model we also hope to help development of new strategies for early detection and prevention.

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Identifiers

ORCID for Aya Abdulhakim Ben Issa: orcid.org/0000-0002-5344-9915

ORCID for Nick Evans: orcid.org/0000-0002-3255-4388

ORCID for Bram Sengers: orcid.org/0000-0001-5859-6984

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