Insights into the Relationship between Nucleolar Stress and the NF-κB Pathway
Authors: Jingyu Chen, Lesley A. Stark
Journal: Trends in Genetics (2019)

Nucleoli play a critical role in maintaining cellular homeostasis in health, ageing, and disease, as does the NF-κB transcription factor pathway. Emerging data suggest that interplay between these two cellular factors regulates cell growth and death.An atypical nucleolar stress response pathway, characterized by degradation of the PolI complex component, TIF-IA, and nucleolar enlargement, lies upstream of NF-κB pathway activation.Components of the NF-κB complex accumulate in nucleoli in response to stress. The presence of RelA in nucleoli triggers cytoplasmic relocation of nucleophosmin which mediates apoptosis by transporting BAX to mitochondria.The chemopreventive agent aspirin triggers TIF-IA–NF-κB signalling in human colorectal tumours, which is important for its antitumour activity. The nuclear organelle the nucleolus and the transcription factor nuclear factor of κ-light-chain-enhancer of activated B cells (NF-κB) are both central to the control of cellular homeostasis, dysregulated in common diseases and implicated in the ageing process. Until recently, it was believed that they acted independently to regulate homeostasis in health and disease. However, there is an emerging body of evidence suggesting that nucleoli and NF-κB signalling converge at multiple levels. Here we will review current understanding of this crosstalk. We will discuss activation of the NF-κB pathway by nucleolar stress and induction of apoptosis by nucleolar sequestration of NF-κB/RelA. We will also discuss the role of TIF-IA, COMMD1, and nucleophosmin, which are key players in this crosstalk, and the therapeutic relevance, particularly with respect to the antitumour effects of aspirin. The nuclear organelle the nucleolus and the transcription factor nuclear factor of κ-light-chain-enhancer of activated B cells (NF-κB) are both central to the control of cellular homeostasis, dysregulated in common diseases and implicated in the ageing process. Until recently, it was believed that they acted independently to regulate homeostasis in health and disease. However, there is an emerging body of evidence suggesting that nucleoli and NF-κB signalling converge at multiple levels. Here we will review current understanding of this crosstalk. We will discuss activation of the NF-κB pathway by nucleolar stress and induction of apoptosis by nucleolar sequestration of NF-κB/RelA. We will also discuss the role of TIF-IA, COMMD1, and nucleophosmin, which are key players in this crosstalk, and the therapeutic relevance, particularly with respect to the antitumour effects of aspirin. There are many parallels between nucleoli and the nuclear factor of κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor. For example, both are stimulated by the same plethora of stresses (Table 1), both regulate the same downstream physiological processes, both are dysregulated in a variety of common diseases which contributes to disease aetiology, and both are implicated in senescence and ageing [1.Chen J. et al.Identification of a novel TIF-IA-NF-kappaB nucleolar stress response pathway.Nucleic Acids Res. 2018; 46: 6188-6205Crossref PubMed Scopus (4) Google Scholar, 2.Perkins N.D. Gilmore T.D. Good cop, bad cop: the different faces of NF-kappaB.Cell Death Differ. 2006; 13: 759-772Crossref PubMed Scopus (346) Google Scholar, 3.Boulon S. et al.The nucleolus under stress.Mol. Cell. 2010; 40: 216-227Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 4.Nunez Villacis L. et al.New roles for the nucleolus in health and disease.Bioessays. 2018; 40e1700233Crossref PubMed Scopus (10) Google Scholar, 5.Osorio F.G. et al.NF-kappaB signaling as a driver of ageing.Int. Rev. Cell Mol. Biol. 2016; 326: 133-174Crossref PubMed Scopus (19) Google Scholar, 6.Tiku V. et al.Small nucleoli are a cellular hallmark of longevity.Nat. Commun. 2016; 8: 16083Crossref PubMed Scopus (23) Google Scholar]. Until recently, it was believed that these factors act independently to regulate cellular homeostasis in health, ageing, and disease. However, there is a growing body of evidence to suggest that nucleolar stress activates the NF-κB pathway, and that nucleolar sequestration of NF-κB components is a critical apoptotic regulator. Here we will review the various levels of convergence between NF-κB and the nucleolus. We will also discuss the relevance of nucleoli–NF-κB crosstalk to the antitumour mechanisms of aspirin and the potential relevance to senescence and ageing.Table 1Stress Stimuli Disrupt Nucleoli and Activate NF-κBaAdapted from [1].Stress typeStimulusActivation of NF-κBRefsNucleolar disruptionRefsDNA damage/genotoxic stressUV-CYes109.Wu Z.H. Miyamoto S. Many faces of NF-kappaB signaling induced by genotoxic stress.J. Mol. Med. (Berl). 2007; 85: 1187-1202Crossref PubMed Scopus (0) Google Scholar, 110.Li N. Karin M. Ionizing radiation and short wavelength UV activate NF-kappaB through two distinct mechanisms.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13012-13017Crossref PubMed Scopus (0) Google ScholarYes63.Al Baker E.A. et al.A p53-independent pathway regulates nucleolar segregation and antigen translocation in response to DNA damage induced by UV irradiation.Exp. 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Topoisomerase poisons activate the transcription factor NF-kappaB in ACH-2 and CEM cells.Nucleic Acids Res. 1996; 24: 4242-4248Crossref PubMed Scopus (0) Google ScholarYesBleomycinYesYesOxidative stressReactive oxygen intermediateYes115.Schreck R. et al.Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review).Free Radic. Res. Commun. 1992; 17: 221-237Crossref PubMed Scopus (1216) Google ScholarYes116.Wang K. et al.Heat shock protein 70 inhibits hydrogen peroxide-induced nucleolar fragmentation via suppressing cleavage and down-regulation of nucleolin.Cell Stress Chaperones. 2012; 17: 121-130Crossref PubMed Scopus (15) Google Scholar, 117.Mekhail K. et al.Restriction of rRNA synthesis by VHL maintains energy equilibrium under hypoxia.Cell Cycle. 2006; 5: 2401-2413Crossref PubMed Google ScholarHypoxiaYes118.Kenneth N.S. Rocha S. Regulation of gene expression by hypoxia.Biochem. J. 2008; 414: 19-29Crossref PubMed Scopus (154) Google ScholarYesChemotherapeutic/chemopreventive agentsDoxorubicinYes119.Campbell K.J. et al.Cisplatin mimics ARF tumor suppressor regulation of RelA (p65) nuclear factor-kappaB transactivation.Cancer Res. 2006; 66: 929-935Crossref PubMed Scopus (0) Google ScholarYes120.Burger K. et al.Chemotherapeutic drugs inhibit ribosome biogenesis at various levels.J. Biol. Chem. 2010; 285: 12416-12425Crossref PubMed Scopus (188) Google ScholarEtoposideYes114.Piret B. Piette J. Topoisomerase poisons activate the transcription factor NF-kappaB in ACH-2 and CEM cells.Nucleic Acids Res. 1996; 24: 4242-4248Crossref PubMed Scopus (0) Google Scholar112.Boisvert F.M. et al.A quantitative proteomics analysis of subcellular proteome localization and changes induced by DNA damage.Mol. Cell. Proteomics. 2010; 9: 457-470Crossref PubMed Scopus (127) Google ScholarAspirin/nonsteroidal anti-inflammatory drugsYes81.Stark L.A. Dunlop M.G. Nucleolar sequestration of RelA (p65) regulates NF-kappaB-driven transcription and apoptosis.Mol. Cell. Biol. 2005; 25: 5985-6004Crossref PubMed Scopus (0) Google Scholar, 82.Loveridge C.J. et al.The proapoptotic effects of sulindac, sulindac sulfone and indomethacin are mediated by nucleolar translocation of the RelA(p65) subunit of NF-kappaB.Oncogene. 2008; 27: 2648-2655Crossref PubMed Scopus (0) Google ScholarYes1.Chen J. et al.Identification of a novel TIF-IA-NF-kappaB nucleolar stress response pathway.Nucleic Acids Res. 2018; 46: 6188-6205Crossref PubMed Scopus (4) Google ScholarNutrient stressSerum starvationYes81.Stark L.A. Dunlop M.G. Nucleolar sequestration of RelA (p65) regulates NF-kappaB-driven transcription and apoptosis.Mol. Cell. Biol. 2005; 25: 5985-6004Crossref PubMed Scopus (0) Google ScholarYes54.Mayer C. et al.mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability.Genes Dev. 2004; 18: 423-434Crossref PubMed Scopus (294) Google ScholarOncogenic stressRas/BCR-ABLYes2.Perkins N.D. Gilmore T.D. Good cop, bad cop: the different faces of NF-kappaB.Cell Death Differ. 2006; 13: 759-772Crossref PubMed Scopus (346) Google ScholarYes121.Sriskanthadevan-Pirahas S. et al.The EGF/Ras pathway controls growth in Drosophila via ribosomal RNA synthesis.Dev. Biol. 2018; 439: 19-29Crossref PubMed Scopus (4) Google Scholara Adapted from 1.Chen J. et al.Identification of a novel TIF-IA-NF-kappaB nucleolar stress response pathway.Nucleic Acids Res. 2018; 46: 6188-6205Crossref PubMed Scopus (4) Google Scholar. Open table in a new tab The nucleolus is a highly dynamic, multifunctional, subnuclear organelle [3.Boulon S. et al.The nucleolus under stress.Mol. Cell. 2010; 40: 216-227Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 4.Nunez Villacis L. et al.New roles for the nucleolus in health and disease.Bioessays. 2018; 40e1700233Crossref PubMed Scopus (10) Google Scholar, 7.Grummt I. The nucleolus—guardian of cellular homeostasis and genome integrity.Chromosoma. 2013; 122: 487-497Crossref PubMed Scopus (92) Google Scholar]. Its most established role is as the hub of ribosome biogenesis, the process by which rRNA is transcribed, cleaved, and assembled into ribosomes (Figure 1). This process is the most energy consuming in the cell and as such, is tightly linked to metabolic and proliferative activity. The starting point, and arguably the rate-limiting step, is transcription of ribosomal DNA (rDNA) by the RNA polymerase I (PolI) preinitiation complex (PIC). This complex includes upstream binding factor (UBF), which acts as derepressor and coactivator; SL1 proteins, which confer promotor specificity; and TIF-IA (gene name RRN3), which is essential as it tethers PolI to the rDNA promoter [8.Yuan X. et al.Multiple interactions between RNA polymerase I, TIF-IA and TAF(I) subunits regulate preinitiation complex assembly at the ribosomal gene promoter.EMBO Rep. 2002; 3: 1082-1087Crossref PubMed Scopus (68) Google Scholar]. The output of rDNA transcription is the 47S preribosomal RNA transcript, which is cotranscriptionally matured through nucleolytic processing and assembly with ribosomal proteins and cofactors, to eventually generate the 40S and 60S ribosomal subunits [9.Kressler D. et al.Driving ribosome assembly.Biochim. Biophys. Acta. 2010; 1803: 673-683Crossref PubMed Scopus (280) Google Scholar]. 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This process is broadly termed nucleolar stress and can take many forms, dependent on cell context and the nature of the insult [14.Nemeth A. Grummt I. Dynamic regulation of nucleolar architecture.Curr. Opin. Cell Biol. 2018; 52: 105-111Crossref PubMed Scopus (0) Google Scholar, 17.van Sluis M. McStay B. Nucleolar reorganization in response to rDNA damage.Curr. Opin. Cell Biol. 2017; 46: 81-86Crossref PubMed Scopus (6) Google Scholar]. Outcomes associated with nucleolar stress include differentiation, cell cycle arrest, autophagy, DNA repair, senescence, and apoptosis [4.Nunez Villacis L. et al.New roles for the nucleolus in health and disease.Bioessays. 2018; 40e1700233Crossref PubMed Scopus (10) Google Scholar, 16.Tsai R.Y. Pederson T. Connecting the nucleolus to the cell cycle and human disease.FASEB J. 2014; 28: 3290-3296Crossref PubMed Scopus (42) Google Scholar, 18.Pfister A.S. Emerging role of the nucleolar stress response in autophagy.Front. Cell. 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New research has revealed activation of the NF-κB pathway as an alternative key mechanism by which nucleolar stress regulates cell growth and death. NF-κB is a family of highly conserved, inducible transcription factors that play a critical role not only in innate and adaptive immunity, but also in stress response and the maintenance of cellular homeostasis [2.Perkins N.D. Gilmore T.D. Good cop, bad cop: the different faces of NF-kappaB.Cell Death Differ. 2006; 13: 759-772Crossref PubMed Scopus (346) Google Scholar, 25.Gilmore T.D. Introduction to NF-kappaB: players, pathways, perspectives.Oncogene. 2006; 25: 6680-6684Crossref PubMed Scopus (1378) Google Scholar]. The family comprise five members, namely, RelA (p65), RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52 (NF-κB2). All members have a Rel homology domain which allows dimerization, translocation to the nucleus, and DNA binding, while only RelA, RelB, and C-Rel have the ability to drive transcription. The most abundant form of NF-κB, and the primary mediator of NF-κB-dependent stress response, is the RelA:P50 heterodimer. In resting cells, this complex is held in the cytoplasm by the inhibitor of NF-κB (IκB) protein, IκBα [26.Taniguchi K. Karin M. NF-kappaB, inflammation, immunity and cancer: coming of age.Nat. Rev. Immunol. 2018; 18: 309-324Crossref PubMed Scopus (140) Google Scholar, 27.Karin M. How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex.Oncogene. 1999; 18: 6867-6874Crossref PubMed Google Scholar]. When the cell is exposed to a wide array of stimuli, for example, inflammatory cytokines, bacterial pathogens, cytotoxic agents, DNA damaging agents, nutrient deprivation, hypoxia, and physical insult, IκBα is phosphorylated and targeted for degradation by the 26S proteasome. This unmasks the nuclear localization signal on RelA, allowing NF-κB dimers to translocate to the nucleus and influence expression of over 150 target genes [28.Pahl H.L. Activators and target genes of Rel/NF-kappaB transcription factors.Oncogene. 1999; 18: 6853-6866Crossref PubMed Google Scholar]. These genes control a broad range of physiological processes including proliferation, differentiation, senescence, cell cycle progression, and apoptosis. A number of upstream pathways have been described that lead to degradation of IκB. The most established of these is the canonical pathway. This is induced by classic NF-κB stimuli [such as tumour necrosis factor alpha (TNFα)] and is characterized by rapid phosphorylation of IκBα by the inhibitor of κB kinase (IKK) complex [27.Karin M. How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex.Oncogene. 1999; 18: 6867-6874Crossref PubMed Google Scholar]. By contrast, stress stimuli generally induce IκB phosphorylation/degradation with a delayed and slow kinetic. Specific mechanisms have been proposed for this delayed response [29.Wu S. Tong L. 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Cell. 2004; 13: 853-865Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar]. However, how multiple heterogeneous stresses converge on the NF-κB pathway has been far from clear. Intuitively, given the parallels between stresses that disrupt ribosome biogenesis and those that activate NF-κB (Table 1), nucleolar disruption could provide a unifying mechanism. Indeed, a number of nucleolar proteins are known to regulate NF-κB signalling. Furthermore, an atypical form of nucleolar stress has recently been shown to lie upstream of NF-κB pathway activation. The nucleolar proteome contains over 4500 proteins that shuttle dynamically between this and other cellular compartments, depending on cell context [34.Andersen J.S. et al.Nucleolar proteome dynamics.Nature. 2005; 433: 77-83Crossref PubMed Scopus (818) Google Scholar]. Several studies have shown the importance of such nucleolar proteins in the regulation of NF-κB signalling, especially in response to stress. For example, casein kinase type 2 (CK2), which is bound to TIF-IA in the PolI complex [35.Bierhoff H. et al.Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.Mol. Cell. Biol. 2008; 28: 4988-4998Crossref PubMed Scopus (0) Google Scholar], has previously been shown to phosphorylate IκBα in response to UV-C, potentiating NF-κB pathway activation [30.Kato Jr., T. et al.CK2 is a C-terminal IkappaB kinase responsible for NF-kappaB activation during the UV response.Mol. Cell. 2003; 12: 829-839Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 36.Cirigliano S.M. et al.The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines.Cancer Cell Int. 2017; 17: 42Crossref PubMed Scopus (0) Google Scholar]. 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The ribosomal protein L3 prevents the degradation of IκB upon 5-fluorouracil treatment, thus repressing NF-κB activity [44.Russo A. et al.Enhancement of 5-FU sensitivity by the proapoptotic rpL3 gene in p53 null colon cancer cells through combined polymer nanoparticles.Oncotarget. 2016; 7: 79670-79687Crossref PubMed Google Scholar, 45.Russo A. et al.rpL3 promotes the apoptosis of p53 mutated lung cancer cells by down-regulating CBS and NFkappaB upon 5-FU treatment.Sci. Rep. 2016; 6: 38369Crossref PubMed Scopus (30) Google Scholar], while S3 promotes activity by interacting with NF-κB complexes in the nucleus [46.Wan F. et al.Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.Cell. 2007; 131: 927-939Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar]. Hence, there are a number of signalling pathways by which stress-mediated nucleolar disruption could alter NF-κB nuclear translocation and transcriptional activity. The first direct evidence that disruption of nucleolar homeostasis may activate NF-κB signalling came from studies showing that depletion of the multifunctional nucleolar protein, nucleophosmin (NPM, B23), induces degradation of IκB and nuclear translocation of RelA in colon cancer cells [47.Hariharan N. Sussman M.A. Stressing on the nucleolus in cardiovascular disease.Biochim. Biophys. Acta. 2014; 1842: 798-801Crossref PubMed Scopus (21) Google Scholar]. Depletion of the small nucleolar RNA host gene 15 (SNHG15) has also been shown to influence NF-κB signalling in a renal cell carcinoma model [48.Du Y. et al.Knockdown of SNHG15 suppresses renal cell carcinoma proliferation and EMT by regulating the NF-kappaB signaling pathway.Int. J. Oncol. 2018; 53: 384-394PubMed Google Scholar]. More recently, small interfering (si)RNA depletion of the PolI complex components UBF, TIF-IA, or RPA194 (a component of PolI) was shown to induce degradation of IκBα, S536 phosphorylation of RelA (a marker for activation), nuclear translocation of RelA, increased NF-κB transcriptional activity, and increased transcription of NF-κB target genes in multiple cell types [1.Chen J. et al.Identification of a novel TIF-IA-NF-kappaB nucleolar stress response pathway.Nucleic Acids Res. 2018; 46: 6188-6205Crossref PubMed Scopus (4) Google Scholar]. Interestingly, this effect was not mimicked by actinomycin D, CX5461, or BMH-21 which block PolI transcription initiation (CX5461 and BMH-21) and elongation (Actinomycin D) [49.Drygin D. et al.Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth.Cancer Res. 2011; 71: 1418-1430Crossref PubMed Scopus (223) Google Scholar, 50.Peltonen K. et al.A targeting modality for destruction of RNA polymerase I that possesses anticancer activity.Cancer Cell. 2014; 25: 77-90Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar]. Hence, unlike p53 nucleolar stress response, the link between nucleolar disruption and NF-κB activation is independent of rDNA transcription [21.Bursac S. et al.Activation of the tumor suppressor p53 upon impairment of ribosome biogenesis.Biochim. Biophys. Acta. 2014; 1842: 817-830Crossref PubMed Scopus (33) Google Scholar]. Recent studies, aimed at further exploration of nucleoli–NF-κB crosstalk, uncovered a novel pathway by which nucleolar function is altered by stress and revealed this atypical nucleolar stress pathway lies upstream of NF-κB signalling [1.Chen J. et al.Identification of a novel TIF-IA-NF-kappaB nucleolar stress response pathway.Nucleic Acids Res. 2018; 46: 6188-6205Crossref PubMed Scopus (4) Google Scholar]. This new pathway is centred around the PIC component TIF-IA. TIF-IA is the master regulator of PolI-driven transcription [35.Bierhoff H. et al.Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.Mol. Cell. Biol. 2008; 28: 4988-4998Crossref PubMed Scopus (0) Google Scholar, 51.Jin R. Zhou W. TIF-IA: an oncogenic target of pre-ribosomal RNA synthesis.Biochim. Biophys. Acta. 2016; 1866: 189-196PubMed Google Scholar]. Not only is it essential for anchoring PolI to the rDNA promotor, it is also the component of the PIC that transduces environmental signals to the PolI transcriptional machinery. Upon exposure of cells to exogenous stresses, TIF-IA is targeted by a complex network of kinases and phosphatases including mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and protein phosphatase 2A (PP2A), which activate or inactivate the protein to fine-tune the transcriptional output [51.Jin R. Zhou W. TIF-IA: an oncogenic target of pre-ribosomal RNA synthesis.Biochim. Biophys. Acta. 2016; 1866: 189-196PubMed Google Scholar, 52.Bodem J. et al.TIF-IA, the factor mediating growth-dependent control of ribosomal RNA synthesis, is the mammalian homolog of yeast Rrn3p.EMBO Rep. 2000; 1: 171-175Crossref PubMed Google Scholar, 53.Zhao J. et al.ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth.Mol. Cell. 2003; 11: 405-413Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 54.Mayer C. et al.mTOR-dependent activation of the transcription factor TIF-IA links rRNA synthesis to nutrient availability.Genes Dev. 2004; 18: 423-434Crossref PubMed Scopus (294) Google Scholar]. The consequences of TIF-IA inactivation are context dependent but in general, its loss leads to cel