Timeless prevents senescence-associated phenotypes and enhances DNA repair to promote esophageal cancer cell growth

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest forms of squamous cell carcinoma, comprising approximately 90% of all esophageal cancer cases. We previously demonstrated that the Fanconi anemia DNA repair (FA) pathway mitigates replication stress to preserve the self-renewal capacity of esophageal cancer cells, highlighting the critical role of minimizing replication stress in esophageal cancer proliferation. In this study, to further explore the role of replication stress in esophageal cancer growth, we investigated the function of Timeless, a key subunit of the replication fork protection complex essential for preventing replication stress. Our findings reveal that Timeless is upregulated in esophageal cancer cells, and its depletion increases sensitivity to DNA-damaging agents, inducing cellular senescence in esophageal keratinocytes. Timeless depletion also elevates the DNA damage response while reducing the expression of DNA repair proteins associated with the FA pathway and homologous recombination. Furthermore, the loss of Timeless impairs colony-forming ability in soft agar and diminishes the self-renewal capacity required to form 3D organoids. These results suggest that Timeless plays a critical role in facilitating DNA repair and esophageal cancer progression and may represent a promising target for developing effective therapeutic strategies to treat esophageal cancers.