The dual role of the DREAM/G2M pathway in non-tumorigenic immortalization of senescent cells.

Anti-aging and tumorigenesis share common genes and pathways, and thus targeting these genes as part of anti-aging interventions carries the risk of tumorigenesis. It is essential to understand the gene signatures that balance tumorigenesis and aging. To achieve this goal, we analyzed RNA-sequencing data from three non-tumorigenic immortalized cell lines that spontaneously escaped from senescence. By single sample gene set enrichment assay (ssGSEA) and GSEA analysis, we found that both cell growth signaling (E2F targets, MYC targets) and tumor surveillance mechanisms (DNA repair, G2M checkpoint, mitotic spindle) were up-regulated in all three cell lines, suggesting that these genes are potential signatures for non-tumorigenic immortalization. Further analysis revealed that the 182 commonly up-regulated genes in these three cell lines overlapped with the DREAM/G2M pathway, which is known to be the upstream regulator of E2F, Myc targets, DNA repair, G2M checkpoint and mitotic spindle pathways in its cell cycle activation or inhibitory form. By western blotting, quantitative PCR and co-immunoprecipitation, we verified that both forms of the DREAM pathway are up-regulated in all three cell lines; this pathway facilitates control of cell cycle progression, supporting a new mechanism for non-tumorigenic immortalization. Thus, we propose that the DREAM/G2M pathway plays important dual roles with respect to preventing tumorigenesis in the process of immortalization. Our data might serve as the basis for the identification of new signature pathways or gene biomarkers for non-tumorigenic immortalization, and may aid in the discovery of new targets for tumor-free anti-aging drug screening.