In Vitro Fusion of Normal and Neoplastic Breast Epithelial Cells with Human Mesenchymal Stroma/Stem Cells Partially Involves Tumor Necrosis Factor Receptor Signaling.

Abstract

Formation of hybrid cells by "accidental cell fusion" of normal and neoplastic breast epithelial cells with local tissue-associated mesenchymal stroma/stem-like cells (MSC) in an inflammatory microenvironment can generate new cancer cell populations whereby molecular signaling mechanisms of this process remain unclear. Fusions of lentiviral enhanced green fluorescent protein-labeled MSC with mcherry-labeled breast epithelial cells were quantified and effects of tumor necrosis factor alpha (TNF-α) and receptor downstream signaling were investigated. Cocultures of MSC with normal human mammary epithelial cells, with neoplastic MCF10A, or with MDA-MB-231 or MCF7 breast cancer cells demonstrated hybrid cell formation between 0.1% and about 2% of the populations within 72 hours, whereby the fusion process occurred in less than 5 minutes. Addition of the pro-inflammatory cytokine TNF-α significantly enhanced MCF10A-MSC cell fusion. Small-interfering RNA (siRNA) knockdown experiments revealed an involvement of tumor necrosis factor (TNF) receptor-1 and -2 in this process. This was also substantiated by siRNA knockdown of tumor necrosis factor receptor type 1-associated death domain which abolished TNF-α-stimulated fusion. While TNF receptor signaling can be relayed via the Mitogen-activated protein kinase 8 (MAPK8), NF-κB or cell death pathways, examination of further downstream signaling exhibited little if any effects of MAPK8 or RelA (p65) on TNF-α-mediated cell fusion, respectively. These data suggested that cell fusion between MSC and MCF10A breast epithelial cells can be stimulated by TNF-α involving TNF receptor-activated cell death pathways or additional NF-κB signaling. Stem Cells 2018;36:977-989.