cGAS/STING/TBK1/IRF3 innate immunity pathway can regulate chromosomal instability in a p21-dependent manner

Abstract

Chromosomal instability (CIN) in cancer cells has been reported to activate the cGAS-STING innate immunity pathway via micronuclei formation, thus affecting tumor immunity and tumor progression. However, converse effects of the cGAS/STING pathway per se on CIN have not yet been investigated. I addressed this issue using knockdown and add-back of each component of the cGAS/STING/TBK1/IRF3 pathway, and monitored the extent of CIN by measuring micronuclei formation after release from nocodazole-induced mitotic arrest. Interestingly, knockdown of cGAS (cyclic GMP-AMP synthase) along with induction of mitotic arrest in HeLa and U2OS cancer cells clearly resulted in an increased micronucleus phenotype and chromosome missegregation. Knockdown of STING (stimulator of interferon genes), TBK1 (TANK binding kinase-1), or IRF3 (interferon regulatory factor-3) also increased micronuclei formation. Moreover, transfection of cGAMP, the product of cGAS enzymatic activity, as well as add-back of cGAS WT (but not catalytic-dead mutant cGAS), or WT or constitutively active STING (but not an inactive STING mutant) rescued the micronuclei phenotype, demonstrating that all components of the cGAS/STING/TBK1/IRF3 pathway play a role in preventing CIN. Moreover, p21 levels were decreased in cGAS-, STING-, TBK1- and IRF3-knockdown cells in association with precocious G2/M transition and an enhanced micronuclei phenotype. Overexpression of p21 or inhibition of CDK1 in cGAS-depleted cells reduced micronuclei formation and abrogated precocious G2/M transition, indicating that the decrease in p21 and subsequent precocious G2/M transition is the main mechanism underlying the induction of CIN by defective cGAS/STING signaling.