TNF-α plays a crucial role in cancer initiation and progression by enhancing cancer cell proliferation, survival, and migration. Even though the known functional role of AWP1 (zinc finger AN1 type-6, ZFAND6) is as a key mediator of TNF-α signaling, its potential role in the TNF-α-dependent responses of cancer cells remains unclear. In our current study, we found that an AWP1 knockdown using short hairpin RNAs increases the migratory potential of non-aggressive MCF-7 breast cancer cells with no significant alteration of their proliferation in response to TNF-α. A CRISPR/Cas9-mediated AWP1 knockout in MCF-7 cells led to mesenchymal cell type morphological changes and an accelerated motility. TNF-α administration further increased this migratory capacity of these AWP1-depleted cells through the activation of NF-κB accompanied by increased epithelial-mesenchymal transition-related gene expression. In particular, an AWP1 depletion augmented the expression of Nox1, reactive oxygen species (ROS) generating enzymes, and ROS levels and subsequently promoted the migratory potential of MCF-7 cells mediated by TNF-α. These TNF-α-mediated increases in the chemotactic migration of AWP1 knockout cells were completely abrogated by an NF-κB inhibitor and a ROS scavenger. Our results suggest that a loss-of-function of AWP1 alters the TNF-α response of non-aggressive breast cancer cells by potentiating ROS-dependent NF-κB activation.
Background MSCs have long been thought to be immune-privileged with low levels of major histocompatibility complex (MHC) class I and rare expression of MHC class II. However, growing evidence indicates that these cells may not actually be hypoimmunogenic, particularly when exposed to cytokines such as IFN-γ. IFN-γ primed increase of MHC class I expression can promote the rejection of allogenic MSCs in the host recipient. A strategy to overcome this drawback is urgently required. Methods We knocked out β2-microglobulin (B2M) in MSCs, which is a component of MHC class I, using the ribonucleoprotein (RNP)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) system. The expression of MSC surface markers, MHC class I, and B2M was assayed by flow cytometry and western blotting. Upon co-culture of MSCs with CD4+ and CD8+ T cells, the survival and proliferation of both cell types were examined by cell counting kit (CCK-8) and carboxy fluorescein succinimidyl ester (CFSE), respectively. The levels of immunomodulatory molecules in MSCs were evaluated by both enzyme-linked immunosorbent assay (ELISA) and western blotting. Results B2M-knockout MSCs expressed low levels of MHC class I even upon IFN-γ priming, but maintained their native properties as evidenced by the expression of specific surface markers. CD8+ T cell proliferation was also far less stimulated by B2M-knockout MSCs than by control cells. Under these conditions, B2M-knockout MSCs had a significantly longer survival duration (> 2.4 fold) than did control cells. B2M-knockout MSCs showed significantly elevated levels of immune-modulatory molecules including indoleamine 2, 3-dioxygenase 1 (IDO-1), prostaglandin E2 (PGE2), C-C motif chemokine ligand 2 (CCL-2), and interleukin-6 (IL-6); conversely, B2M-knockout MSCs produced significantly lower levels of proinflammatory molecules (e.g., IL-1b, CXCL10) compared with control cells. Conclusion The loss of B2M in MSCs potentiated the immunomodulatory effects of IFN-γ priming while mitigating its potential inflammatory effects. B2M-knockout MSCs are a potentially promising treatment for immune-related inflammatory diseases.
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