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Regulation of p53 and Rb links the alternative NF-?B pathway to EZH2 expression and cell senescence.

ABSTRACT: There are two major pathways leading to induction of NF-?B subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of I?B? in a manner dependent on I?B kinase (IKK) ? and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-?B2(p52)/RelB containing complexes, and is dependent on IKK? and NF-?B inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-?B pathway subunits NF-?B2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-?B2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-?B target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-?B pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-?B also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-?B can inhibit tumor suppressor function and promote tumorigenesis.

SUBMITTER: Iannetti A

PROVIDER: S-EPMC4177746 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Regulation of p53 and Rb links the alternative NF-κB pathway to EZH2 expression and cell senescence.

Iannetti Alessio A Ledoux Adeline C AC Tudhope Susan J SJ Sellier Hélène H Zhao Bo B Mowla Sophia S Moore Adam A Hummerich Holger H Gewurz Benjamin E BE Cockell Simon J SJ Jat Parmjit S PS Willmore Elaine E Perkins Neil D ND

PLoS genetics 20140925 9

There are two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-κB2(p52)/RelB containing complexes, and is dependent on IKKα and NF-κB inducing ...[more]

PMID: 25255445

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Project description:The retinoblastoma protein RB and the transcription factor p53 are central tumor suppressors. They are often found inactivated in various tumor types. Both proteins play central roles in regulating the cell division cycle. RB forms complexes with the E2F family of transcription factors and downregulates numerous genes. Among the RB-E2F target genes, a large number code for key cell cycle regulators. Their transcriptional repression by the RB-E2F complex is released through phosphorylation of RB, leading to expression of the cell cycle regulators. The release from repression can be prevented by the cyclin-dependent kinase inhibitor p21/CDKN1A. The CDKN1A gene is transcriptionally activated by p53. Taken together, these elements constitute the p53-p21-RB signaling pathway. Following activation of p53, for example by viral infection or induction of DNA damage, p21 expression is upregulated. High levels of p21 then result in RB-E2F complex formation and downregulation of a large number of cell cycle genes. Thus, p53-dependent transcriptional repression is indirect. The reduced expression of the many regulators leads to cell cycle arrest. Examination of the p53-p21-RB targets and genes controlled by the related p53-p21-DREAM signaling pathway reveals that there is a large overlap of the two groups. Mechanistically this can be explained by replacing RB-E2F complexes with the DREAM transcriptional repressor complex at E2F sites in target promoters. In contrast to RB-E2F, DREAM can downregulate genes also through CHR transcription factor binding sites. This results in a distinct gene set controlled by p53-p21-DREAM signaling independent of RB-E2F. Furthermore, RB has non-canonical functions without binding to E2F and DNA. Such a role of RB supporting DREAM formation may be exerted by the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link. In the current synopsis, the mechanism of regulation by p53-p21-RB signaling is assessed and the overlap with p53-p21-DREAM signaling is examined.

Project description:Senescence is a tumor suppressive mechanism that induces a permanent proliferative arrest in response to an oncogenic insult or to the genotoxic stress induced by chemotherapy. We have recently described that some cells can escape this arrest, either because senescence was incomplete or as a consequence of a phenotypic adaptation. Malignant cells which resisted senescence emerged as more transformed cells that resist anoikis and rely on survival pathways activated by Akt and Mcl-1. In this study, we further characterize senescence escape, investigating how emergent cells could reproliferate. During the initial step of chemotherapy-induced senescence (CIS), we found that cyclin D1 was upregulated and that cell emergence was prevented when its main partner cdk4 was inactivated. Results indicate that this kinase induced the upregulation of the EZH2 methylase, a component of the polycomb PRC2 complex. Downregulated during the early step of treatment, the methylase was reactivated in clones that escaped senescence. The inactivation of EZH2, either by siRNA or by specific inhibitors, led to a specific inhibition of cell emergence. We used quantitative proteomic analysis to identify new targets of the methylase involved in senescence escape. We identified proteins involved in receptor endocytosis and described new functions for the AP2M1 protein in the control of chemotherapy-mediated senescence. Our results indicate that AP2M1 is involved in the transmission of secreted signals produced by senescent cells, suggesting that this pathway might regulate specific receptors involved in the control of CIS escape. In light of these results, we therefore propose that the cdk4-EZH2-AP2M1 pathway plays an important role during chemotherapy resistance and senescence escape. Since targeted therapies are available against these proteins, we propose that they should be tested in the treatment of colorectal or breast cancers that become resistant to first-line genotoxic therapies.

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Regulation of p53 and Rb links the alternative NF-?B pathway to EZH2 expression and cell senescence.

Publications

Regulation of p53 and Rb links the alternative NF-κB pathway to EZH2 expression and cell senescence.

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