Loss of fragile WWOX gene leads to senescence escape and genome instability

<jats:title>Abstract</jats:title><jats:p>Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human <jats:italic>WWOX</jats:italic> gene resides within a common fragile site <jats:italic>FRA16D</jats:italic> that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary <jats:italic>Wwox</jats:italic> knockout (<jats:italic>Wwox</jats:italic><jats:sup>−/−</jats:sup>) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage <jats:italic>Wwox</jats:italic><jats:sup>−/−</jats:sup> MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused <jats:italic>p16</jats:italic><jats:sup><jats:italic>Ink4a</jats:italic></jats:sup> promoter hypermethylation, aberrant p53/p21<jats:sup>Cip1/Waf1</jats:sup> signaling axis and accelerated p27<jats:sup>Kip1</jats:sup> protein degradation, thereby leading to the failure of senescence induction in <jats:italic>Wwox</jats:italic>-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of <jats:italic>p21</jats:italic><jats:sup><jats:italic>Cip1/Waf1</jats:italic></jats:sup> in late-passage <jats:italic>Wwox</jats:italic><jats:sup>−/−</jats:sup> MEFs. Treatment of <jats:italic>N</jats:italic>-acetyl-<jats:sc>l</jats:sc>-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in <jats:italic>Wwox</jats:italic><jats:sup>−/−</jats:sup> MEFs. Our findings support an important role for fragile <jats:italic>WWOX</jats:italic> gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.</jats:p>