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.

Cell Fusion-Mediated Tissue Regeneration as an Inducer of Polyploidy and Aneuploidy

“…Numerous studies further showed that the frequency of cell fusion events was increased upon acute tissue damage or chronic inflammation [7,12,18,32,63,64,[72][73][74][75], which is plausible with regard to efficient BMSC-based and cell fusion-mediated tissue regeneration. BMSCs not only have to be converted into a pro-fusogenic state for subsequent hybridization with target cells but also have to be recruited to the site of tissue damage.…”

“…For instance, the fusion of trophoblasts to syncytiotrophoblasts is chiefly regulated by Syncytin-1 and -2, which are transmembrane proteins of retroviral origin [59,60]. Syncytin-1 and -2 are still the best characterized cell fusion mediating proteins in humans, and they might also be involved in human osteoclast fusion [61] and in the fusion of cancer cells with endothelial cells [62,63] or mesenchymal stem cells [64]. In contrast, fusion of myoblasts to multinucleated myofibers depends on remodeling of the actin cytoskeleton and formation of podosome-like structures, which penetrate the target cell, thereby causing the merging of plasma membranes [65,66].…”

“…In this context, it has been shown that the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) might also be a mediator of cell fusion. Osteoclastogenesis [70,74,75], as well as the fusion of cancer cells with endothelial cells [63,76], mesenchymal stem cells [64], or breast epithelial cells [72,73,77] can be induced by TNF-α. Some data revealed that TNF-α could mediate fusion due to induction of MMP-9 expression [73,75], which plays a role in osteoclastogenesis and giant cell formation [75,78].…”

Die Rolle der Zellfusion im Tumorkontext. Fakt oder Fiktion?

“…Mehrere Studien deuten auf einen Zusammenhang zwischen Inflammation/ inflammatorische Mediatoren und Zellfusion hin [19,47,61,65,81,90]. Sowohl Johansson et al als auch Davies et al konnten in tierexperimentellen Studien belegen, dass die Fre quenz von Zellfusionsereignissen in Gegenwart von inflammatori schen Konditionen mitunter bis um den Faktor 100 erhöht war im Vergleich zu entsprechenden Kontrollstudien [19,47].…”

“…Sowohl Johansson et al als auch Davies et al konnten in tierexperimentellen Studien belegen, dass die Fre quenz von Zellfusionsereignissen in Gegenwart von inflammatori schen Konditionen mitunter bis um den Faktor 100 erhöht war im Vergleich zu entsprechenden Kontrollstudien [19,47]. Speziell für das pro-inflammatorische Zytokin TNF-α konnte ein pro-fusioge ner Effekt beobachtet werden, was sowohl die Fusion von Makro phagen als auch die Fusion von Tumorzellen mit anderen Zellen be trifft [61,65,81,90]. Auch in chronisch entzündetem Gewebe kommt es zu einer erhöhten Frequenz von Zellfusionsereignissen [47,59,69], da die Zellfusion, wie oben erwähnt, eine Rolle in der Geweberegeneration/ Wundheilung spielt und durch inflammato rische Prozesse begünstigt wird.…”