IFNγ induces oxidative stress, DNA damage and tumor cell senescence via TGFβ/SMAD signaling-dependent induction of Nox4 and suppression of ANT2

Cellular senescence provides a biological barrier against tumor progression, often associated with oncogene-induced replication and/or oxidative stress, cytokine production and DNA damage response (DDR), leading to persistent cell-cycle arrest. While cytokines such as tumor necrosis factor-alpha (TNF alpha) and interferon gamma (IFN gamma) are important components of senescence-associated secretome and induce senescence in, for example, mouse pancreatic beta-cancer cell model, their downstream signaling pathway(s) and links with oxidative stress and DDR are mechanistically unclear. Using human and mouse normal and cancer cell models, we now show that TNF alpha and IFN gamma induce NADPH oxidases Nox4 and Nox1, reactive oxygen species (ROS), DDR signaling and premature senescence. Unlike mouse tumor cells that required concomitant presence of IFN gamma and TNF alpha, short exposure to IFN gamma alone was sufficient to induce Nox4, Nox1 and DDR in human cells. siRNA-mediated knockdown of Nox4 but not Nox1 decreased IFN gamma-induced DDR. The expression of Nox4/Nox1 required Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling and the effect was mediated by downstream activation of transforming growth factor-beta (TGF beta) secretion and consequent autocrine/paracrine activation of the TGF beta/Smad pathway. Furthermore, the expression of adenine nucleotide translocase 2 (ANT2) was suppressed by IFN gamma contributing to elevation of ROS and DNA damage. In contrast to mouse B16 cells, inability of TC-1 cells to respond to IFN gamma/TNF alpha by DDR and senescence correlated with the lack of TGF beta and Nox4 response, supporting the role of ROS induced by NADPH oxidases in cytokine-induced senescence. Overall, our data reveal differences between cytokine effects in mouse and human cells, and mechanistically implicate the TGF beta/SMAD pathway, via induction of NADPH oxidases and suppression of ANT2, as key mediators of IFN gamma/TNF alpha-evoked genotoxicity and cellular senescence.