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 kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-κB pathway subunits NF-κB2 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-κB2 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-κB 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-κB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-κB 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-κB can inhibit tumor suppressor function and promote tumorigenesis. Author Summary Although the classical NF-κB pathway is frequently associated with the induction of cellular senescence and the senescence associated secretory phenotype (SASP), the role of the alternative NF-κB pathway, which is frequently activated in hematological malignancies as well as some solid tumors, has not been defined. We therefore investigated the role of the alternative NF-κB pathway in this process. Here we report that NF-κB2 and RelB, the effectors of the alternative NF-κB pathway, suppress senescence through inhibition of p53 activity. Using primary human fibroblasts, we demonstrate that this is accomplished through NF-κB2/RelB dependent control of a previously unknown pathway, incorporating regulation of CDK4 and 6 expression as well as regulators of p21WAF1 and p53 protein stability. Loss of NF-κB2/RelB results in suppression of retinoblastoma (Rb) tumour suppressor phosphorylation, which in turn leads to inhibition of EZH2 expression and de-repression of p53 activity. Interestingly, we find that CD40 ligand stimulation of cells from Chronic Lymphocytic Leukemia patients, which strongly induces the alternative NF-κB pathway, also induces EZH2 expression. We propose that the alternative NF-κB pathway can promote tumorigenesis through suppression of p53 dependent senescence, a process that may have relevance to cancer cells retaining wild type p53.
【초록키워드】 Tumor, Crosstalk, Hematological malignancy, Hematological malignancies, chronic lymphocytic leukemia, oxygen, senescence, CD40L, cell cycle, stability, western blot, siRNA, Phosphorylation, pathway, phenotype, ROS, protein stability, Degradation, repression, expression, quantitative PCR, wild type, patients, mechanism, reactive oxygen species, NF-κB, NF-κB pathway, NF-κB2, cellular senescence, Target genes, Cancer cells, p53, Analysis, fibroblasts, regulate, solid tumors, tumors, chronic, leads, the cell, target gene, retinoblastoma, Activation, Complexes, Regulation, subunit, tumorigenesis, suppression, NEMO, microarray analysis, Lymphocytic leukemia, knockdown, CD40, RELA, subunits, CD40 ligand, ChIP, effector, effectors, chromatin immunoprecipitation, retinoblastoma protein, Western blot analyses, alternative NF-κB pathway, ANAPC1, CDK4, CDK6, cell senescence, EZH2, IKKα, IκBα, microenvironment, NF-κB inducing kinase, p100, p21WAF1, polycomb protein, PSMA5, RacGAP1, RelB, SASP, tumor suppressor, Effect, Loss, Cell, stimulated, defined, classical, investigated, inhibit, provide, activated, dependent on, promote, induce, suppress, senescent, subset, turn, reactive oxygen specy, cancer cell, c-Rel, de-repression, DEK, IKK, IκB, NIK, or canonical, p52, regulatory subunit, tumour suppressor, Western blot analysis, 【제목키워드】 p53,