Elucidating the Role of Cholesterol Pathways in the Epithelial-to Mesenchymal Transition in Breast Cancer Cells

Abstract

Breast cancer is the most prevalent cancer affecting women, globally. Metastasis, which is the spread of tumour cells from the primary tumour to a secondary site, accounts for approximately 75.6% of cancer-related mortalities. The epithelial-to-mesenchymal transition (EMT) is a critical cellular program underlying the metastatic spread of cancer cells. The role of cholesterol in EMT and metastasis has not yet been established; however, there is a link between cholesterol and cancer cell growth and proliferation. This study serves to determine the role of cholesterol in the induction of EMT and metastasis and delineate the cholesterol related pathways affected by EMT induction. This study also aimed to assess the efficacy of cholesterol-lowering treatment on cells undergoing EMT. An in vitro model of EMT was established using the NMuMG cell line, a well-established model for studying EMT, treated with recombinant TGFβ. Induction of EMT was confirmed by assessing the expression of pre- and post-EMT markers. Cholesterol levels were measured using the Cholesterol Quantitation Kit (Sigma Aldrich, UK) and immunocytochemical lipid stains. Cellular cholesterol was lowered using cholesterol depleting agents: methyl-β-cyclodextrin (MβCD), 2-hydroxypropyl-β-cyclodextrin (HPβCD), and simvastatin treatment. Changes in proliferative capacity after EMT induction and treatment with cholesterol depleting agents were measured using the 2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay and immunocytochemistry. Changes in gene expression were measured using Western blotting and RT-qPCR. Invasive potential was measured using a Transwell assay. The induction of EMT in NMuMG cells resulted in a change from epithelial to mesenchymal morphology, along with an increase in expression of EMT-associated proteins, such as Vimentin and N-cadherin, and a decrease in E-cadherin expression. EMT induction resulted in an increase in invasive potential and an overall decrease in cellular cholesterol content. Cholesterol depletion with MβCD, HPβCD, and simvastatin treatment reversed EMT-related expression changes and restored expression of epithelial marker E-cadherin. In addition, cholesterol-lowering treatment reduced the invasive potential of cells after EMT induction. Due to its ability to reverse EMT-related phenotype changes, cholesterol depletion may serve as a therapeutic strategy for metastatic cancer.