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.

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DREAM/G2M 通路在衰老细胞非致瘤永生化中的双重作用

抗衰老和肿瘤发生具有共同的基因和途径，因此将这些基因作为抗衰老干预措施的一部分会带来肿瘤发生的风险。了解平衡肿瘤发生和衰老的基因特征至关重要。为了实现这一目标，我们分析了三种自发逃避衰老的非致瘤性永生化细胞系的 RNA 测序数据。通过单样本基因集富集分析（ssGSEA）和GSEA分析，我们发现细胞生长信号（E2F靶标、MYC靶标）和肿瘤监测机制（DNA修复、G2M检查点、有丝分裂纺锤体）在所有三种细胞中均上调线，表明这些基因是非致瘤性永生化的潜在特征。进一步分析发现，这三个细胞系中182个常见上调基因与DREAM/G2M通路重叠，已知DREAM/G2M通路是其细胞中E2F、Myc靶标、DNA修复、G2M检查点和有丝分裂纺锤体通路的上游调节因子循环激活或抑制形式。通过蛋白质印迹、定量 PCR 和免疫共沉淀，我们验证了 DREAM 通路的两种形式在所有三种细胞系中均上调；该途径有助于控制细胞周期进程，支持非致瘤永生化的新机制。因此，我们提出DREAM/G2M途径在永生化过程中预防肿瘤发生方面发挥着重要的双重作用。我们的数据可以作为识别非致瘤永生化的新特征途径或基因生物标志物的基础，并可能有助于发现无肿瘤抗衰老药物筛选的新靶点。