NOX4-dependent ROS production by stromal mammary cells modulates epithelial MCF-7 cell migration

BACKGROUND: The influence of the stromal microenvironment on the progression of epithelial cancers has been demonstrated. Unravelling the mechanisms by which stromal cells affect epithelial behaviour will contribute in understanding cellular malignancy. It has been proposed that redox environment has a role in the acquisition of malignancy. In this work, we studied the influence of epithelial cells on the stromal redox status and the consequence of this phenomenon on MCF-7 cell motility. METHODS: We analysed in a co-culture system, the effect of RMF-EG mammary stromal cells on the migratory capacity of MCF-7 cell line. To test whether the NOX-dependent stromal redox environment influences the epithelial migratory behaviour, we knocked down the expression of NOX4 using siRNA strategy. The effect of TGF-β1 on NOX4 expression and activity was analysed by qPCR, and intracellular ROS production was measured by a fluorescent method. RESULTS: Migration of MCF-7 breast epithelial cells was stimulated when co-cultured with RMF-EG cells. This effect depends on stromal NOX4 expression that, in turn, is enhanced by epithelial soluble factors. Pre-treatment of stromal cells with TGF-β1 enhanced this migratory stimulus by elevating NOX4 expression and intracellular ROS production. TGF-β1 seems to be a major component of the epithelial soluble factors that stimulate NOX4 expression. CONCLUSIONS: Our results have identified that an increased stromal oxidative status, mainly provided by an elevated NOX4 expression, is a permissive element in the acquisition of epithelial migratory properties. The capacity of stromal cells to modify their intracellular ROS production, and accordingly, to increase epithelial motility, seems to depend on epithelial soluble factors among which TGF-β1 have a decisive role.