Role of the cGAS-STING pathway in TIF-IA dependent induction of the senescence associated secretory phenotype

Abstract

Cellular senescence is a stress-induced program characterized by irreversible cell cycle arrest and the secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP).

While acute SASP promotes tissue repair and tumor suppression, its chronic activation contributes to many age-related diseases including cancer. NF-ₖB is recognised as a key transcriptional driver of the SASP. However, the pathways that activate NF-B following exposure to senescence triggers remains elusive. The abstract is too long so I would suggest just a couple of sentences of background highlighting the importance of the SASP and the lack of understanding of its initiation.

Previous work from the STARK Lab demonstrated that the PolI complex component, TIF-IA, accumulates in response to senescence triggers and that this is essential for NF-ₖB activation and the SASP. However, the molecular mechanism linking TIF-IA accumulation to SASP induction remain unclear. The cGAS–STING DNA-sensing pathway has been implicated in senescence effects on NF-B signalling. Therefore, I hypothesized that cGAS-STING may act as an intermediary. To explore this possibility, I used a model of therapy-induced senescence in which cells are treated with the DNA damaging agent, etoposide. Firstly, I compared A549 (STING-low) and HCT116 (STING-high) cells. Using qPCR western blot analysis and immunocytochemistry, I found that both cell lines showed comparable transcription of SASP factors and TIF-IA accumulation following etoposide exposure, despite the differential baseline expression of STING. Furthermore, siRNA depletion of TIF-IA significantly abrogated etoposide-mediated SASP factor transcription in both cell lines. To further evaluate STING’s role in SASP regulation, I performed STING siRNA knockdown in HCT116 cells. Contrary to my finding in the STING low A549 cells, qPCR indicated that depletion of STING in HCT116 cells caused a significant reduction in SASP factor expression in response to etoposide. Together, these results suggested that, at least in HCT116 cells, both TIF-IA and STING are required for the SASP.

Therefore, I next set out to understand the relationship between these two pathways. I found that depletion of TIF-IA significantly reduced mRNA and protein levels of STING both basally and in response to etoposide, while the opposite was observed upon TIF-IA overexpression. In contrast, STING depletion had no effect on TIF-IA accumulation in response to etoposide. These data suggest STING may be downstream of TIF-IA in the pathway to the SASP. In support of this suggestion, I found that depletion of STING significantly reduced the increase in SASP factor transcription observed after TIF-IA overexpression. These data identify a new pre-senescence pathway upstream of SASP activation that has significant relevance to age-related diseases, including cancer.