| Abstract: |
Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G1) to synthesis (S) phase transition of the cell cycle by responding to mitogen signaling, promoting E2F transcription and inhibition of the anaphasepromoting complex. We found that this mechanism was still required in G2-arrested cells to prevent cell cycle exit after the S phase. This mechanism revealed a role for CDK4/6 in maintaining the G2 state, challenging the notion that the cell cycle is irreversible and that cells do not require mitogens after passing the restriction point. Exit from G2 occurred during ribotoxic stress and was actively mediated by stress-activated protein kinases. Upon relief of stress, a significant fraction of cells underwent a second round of DNA replication that led to whole-genome doubling. INTRODUCTION: Abnormal DNA content is one of the most common characteristics of cancer cells. Recent evidence suggests that 35 to 40% of tumors go through a wholegenome doubling (WGD) event in their evolutionary history.WGD is associated with genome instability, metastasis, and worse overall prognosis. However, the molecular mechanisms that drive WGD are poorly understood. One proposed model is endoreplication: two rounds of DNA replication without cell division. Endoreplication has been shown to occur upon DNA damage in a p53-dependent manner. However, most tumors lose p53 function prior to the WGD event, suggesting that other mechanisms of endoreplication remain to be discovered. RATIONALE: Cell cycle entry is thought to be an irreversible commitment to cell division, thereby coupling DNA replication with division to maintain DNA content. However, endoreplication contradicts the idea of irreversible commitment. We rationalized that although DNA replication and cell division are coupled under normal circumstances, there must be away to uncouple these events during times of cellular stress and break the commitment to cell division even after DNA replication has occurred. We sought to identify how this commitment can be broken and reconcile this knowledge with our current understanding of the human cell cycle. RESULTS: We confirmed that a variety of cellular stresses, including ribotoxic stress, osmotic stress, and UV radiation, breaks cell cycle commitment and causes cell cycle exit directly fromthe growth 2 phase (G2), after DNA replication has occurred but before cell division. Ribotoxic stress occurs when ribosome function is impaired during elongation, which results in ribosome collisions. We show that, in response to ribosome collisions, activation of the mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα and the so-called stress-activated protein kinases (SAPKs) downstream leads to widespread G2 cell cycle exit. Mechanistically, G2 cell cycle exit occurs by premature activation of the anaphase- promoting complex or cyclosome (APC/C) in G2. Notably, we found that premature APC/C reactivation occurs by persistent simultaneous inhibition of cyclin-dependent kinase 1 (CDK1) and CDK4/6 in G2 by SAPKs. When ribosome function is restored and stress signaling subsides, these cells that have already replicated their DNA are able to restart the cell cycle. This leads to a second round ofDNA replication, thus completing the WGD process. In this context, p53 function was not required, as cells deficient for the p53 gene are still able to undergo this process. Given that p53 strongly inhibits growth of polyploid cells, we propose that persistent CDK inhibition by SAPKs may be an important source of WGD in p53 deficient cells. CONCLUSION: Our study uncovers a role for CDK4/6 activity and mitogen signaling beyond the G1-S transition. These pathways are frequently targeted in cancer therapy to reduce proliferation, but our findings show that G2 cell cycle exit and endoreplication are unexpected consequences of these interventions under stress conditions. These consequences may impact treatment response and resistance and should be considered. Additionally, we demonstrated for the first time that G2 cell cycle exit and endoreplication can occur through SAPK activation in cells lacking p53. These findings change our understanding of the wiring of the human cell cycle and provide amechanistic basis for the high incidence of WGD in cancer. Moreover, given the role of SAPK signaling in aging-related stress responses, SAPK-mediated WGD may represent an important source of genetic instability during aging and tumorigenesis. [ABSTRACT FROM AUTHOR] |