Abstract
Oncogene-induced senescence (OIS) is a tumor suppressor response that induces permanent cell cycle arrest in response to oncogenic signaling. Through the combined activation of the p53-p21 and p16-Rb suppressor pathways, OIS leads to the transcriptional repression of proliferative genes. Although this protective mechanism has been essentially described in primary cells, we surprisingly observed in this study that the OIS program is conserved in established colorectal cell lines. In response to the RAS oncogene and despite the inactivation of p53 and p16(INK4), HT29 cells enter senescence, up-regulate p21(WAF1), and induce senescence-associated heterochromatin foci formation. The same effect was observed in response to B-RAF(v600E) in LS174T cells. We also observed that p21(WAF1) prevents the expression of the CDC25A and PLK1 genes to induce cell cycle arrest. Using ChIP and luciferase experiments, we have observed that p21(WAF1) binds to the PLK1 promoter to induce its down-regulation during OIS induction. Following 4-5 weeks, several clones were able to resume proliferation and escape this tumor suppressor pathway. Tumor progression was associated with p21(WAF1) down-regulation and CDC25A and PLK1 reexpression. In addition, OIS and p21(WAF1) escape was associated with an increase in DNA damage, an induction of the epithelial-mesenchymal transition program, and an increase in the proportion of cells expressing the CD24(low)/CD44(high) phenotype. Results also indicate that malignant cells having escaped OIS rely on survival pathways induced by Bcl-xL/MCL1 signaling. In light of these observations, it appears that the transcriptional functions of p21(WAF1) are active during OIS and that the inactivation of this protein is associated with cell dedifferentiation and enhanced survival.
Highlights
In vivo as an early protection against carcinogenesis
Oncogene-induced senescence (OIS) Is Still Functional in HT29 Cells Despite the Inactivation of p53 and p16INK4—Several studies have shown that oncogenes induce growth arrest and senescence, but it is generally believed that this protective pathway occurs essentially in primary cells to restrain the initial events of cell transformation [24, 25]
We generated HT29 cell lines expressing the H-RASV12 oncogene under the control of a doxycycline-inducible promoter. p53 is mutated in this cell line, but p16INK4 is not expressed, probably as a consequence of promoter methylation
Summary
In vivo as an early protection against carcinogenesis. Its induction involves the combined activities of p53 and p21WAF1 to inhibit cell cycle progression and of p16INK4 and Rb to induce the transcriptional repression of proliferative genes through heterochromatin formation [2]. Proliferative genes are compacted within these foci to prevent cell cycle progression, generally as a consequence of Rb-mediated silencing Through their combined inhibitory effects on cyclin-cdk complexes, the p16INK4 and p21WAF1 inhibitors play an essential role in OIS induction and in the consequent tumor suppression. Transcriptional functions of p21WAF1 have been described following overexpression or in response to chemotherapy treatment, it remains to be determined whether this occurs during OIS and which promoters are targeted by p21WAF1 to restrain an abnormal oncogenic activity This activity has already been demonstrated for the p14ARF tumor suppressor because this protein can interact with the MYC oncogene to prevent the activation of proliferative genes. These malignant cells show enhanced dependence on Bcl-xL/MCL1 signaling, suggesting that proapoptotic pathways are generated during OIS escape and EMT induction
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