Regulation and function of fibronectin expression in breast cancer cells in 2-D and 3-D cultures

Abstract

Fibronectin is a crucial component of the extracellular matrix and has been considered as an Epithelial-Mesenchymal Transition marker protein and changes in its expression have been correlated with pathological progression of different types of cancers. Using the triple-negative breast cancer cell line, MDA-MB-231 and its derivative LM2 that preferentially metastasizes to lung, I find up-regulated messenger RNA and protein level of fibronectin when the cells are grown as multicellular spheroids in 3-dimensional (3D) culture system for 48hr, compared with that of 2D monolayer culture system. In addition, I find the levels of integrin $\beta$-1, $\beta$-3, and $\beta$-5 known fibronectin receptors also showed increase protein levels in 3D. Knock-down of fibronectin with siRNA reduced the rate of attachment of both cells and migration of parental cells when cells were transferred from 3D to 2D surfaces. In addition attachment rate of the cells treated with siRNA against integrin $\beta$-5 siRNAs were also reduced on fibronectin Ⅳ coated plates as well as uncoated culture plates, suggesting that integrin $\beta$-5 is important for cell attachment. I also find that Src plays a role in cell attachment because treatment of cells with the Src inhibitor SU6656 caused a decrease in the cell attachment when cells were transferred from 3D to 2D conditions. In addition, it was observed that snail was increased in 3D and transfection with siRNA against snail downregulates of attachment rate of cells from 3D to 2D in MDA-MB-231-LM2 cells. With these experimental results, I suggest that increased expression of fibronectin in 3D conditions provides advantages to circulating tumor cells for the attachment of cells to 2D surfaces to find a new metastatic niche. Furthermore Src and integrin $\beta$-5 may represent important targets to block metastasis.