The NF-κB Nucleolar Stress Response Pathway.

The nuclear organelle, the nucleolus, plays a critical role in stress response and the regulation of cellular homeostasis. P53 as a downstream effector of nucleolar stress is well defined. However, new data suggests that NF-κB also acts downstream of nucleolar stress to regulate cell growth and death. In this review, we will provide insight into the NF-κB nucleolar stress response pathway. We will discuss apoptosis mediated by nucleolar sequestration of RelA and new data demonstrating a role for p62 (sequestosome (SQSTM1)) in this process. We will also discuss activation of NF-κB signalling by degradation of the RNA polymerase I (PolI) complex component, transcription initiation factor-IA (TIF-IA (RRN3)), and contexts where TIF-IA-NF-κB signalling may be important. Finally, we will discuss how this pathway is targeted by aspirin to mediate apoptosis of colon cancer cells.

A Role for the Autophagic Receptor, SQSTM1/p62, in Trafficking NF-κB/RelA to Nucleolar Aggresomes.

Elevated NF-κB activity is a contributory factor in many hematologic and solid malignancies. Nucleolar sequestration of NF-κB/RelA represses this elevated activity and mediates apoptosis of cancer cells. Here, we set out to understand the mechanisms that control the nuclear/nucleolar distribution of RelA and other regulatory proteins, so that agents can be developed that specifically target these proteins to the organelle. We demonstrate that RelA accumulates in intranucleolar aggresomes in response to specific stresses. We also demonstrate that the autophagy receptor, SQSTM1/p62, accumulates alongside RelA in these nucleolar aggresomes. This accumulation is not a consequence of inhibited autophagy. Indeed, our data suggest nucleolar and autophagosomal accumulation of p62 are in active competition. We identify a conserved motif at the N-terminus of p62 that is essential for nucleoplasmic-to-nucleolar transport of the protein. Furthermore, using a dominant-negative mutant deleted for this nucleolar localization signal (NoLS), we demonstrate a role for p62 in trafficking RelA and other aggresome-related proteins to nucleoli, to induce apoptosis. Together, these data identify a novel role for p62 in trafficking nuclear proteins to nucleolar aggresomes under conditions of cell stress, thus maintaining cellular homeostasis. They also provide invaluable information on the mechanisms that regulate the nuclear/nucleolar distribution of RelA that could be exploited for therapeutic purpose. IMPLICATIONS: The data open up avenues for the development of a unique class of therapeutic agents that act by targeting RelA and other aberrantly active proteins to nucleoli, thus killing cancer cells.

Insights into the Relationship between Nucleolar Stress and the NF-κB Pathway.

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.

Crosstalk between NF-κB and Nucleoli in the Regulation of Cellular Homeostasis.

Nucleoli are emerging as key sensors of cellular stress and regulators of the downstream consequences on proliferation, metabolism, senescence, and apoptosis. NF-κB signalling is activated in response to a similar plethora of stresses, which leads to modulation of cell growth and death programs. While nucleolar and NF-κB pathways are distinct, it is increasingly apparent that they converge at multiple levels. Exposure of cells to certain insults causes a specific type of nucleolar stress that is characterised by degradation of the PolI complex component, TIF-IA, and increased nucleolar size. Recent studies have shown that this atypical nucleolar stress lies upstream of cytosolic IκB degradation and NF-κB nuclear translocation. Under these stress conditions, the RelA component of NF-κB accumulates within functionally altered nucleoli to trigger a nucleophosmin dependent, apoptotic pathway. In this review, we will discuss these points of crosstalk and their relevance to anti-tumour mechanism of aspirin and small molecule CDK4 inhibitors. We will also briefly the discuss how crosstalk between nucleoli and NF-κB signalling may be more broadly relevant to the regulation of cellular homeostasis and how it may be exploited for therapeutic purpose.

Identification of a novel TIF-IA-NF-κB nucleolar stress response pathway.

p53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-κB transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-κB also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-κB pathway is as yet unknown. Here we demonstrate that artificial disruption of the PolI complex stimulates NF-κB signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-κB induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of NF-κB and is associated with increased nucleolar size. It is mimicked by CDK4 inhibition and is dependent upon a novel pathway involving UBF/p14ARF and S44 of the protein. We show that blocking TIF-IA degradation blocks stress effects on nucleolar size and NF-κB signalling. Finally, using ex vivo culture, we show a strong correlation between degradation of TIF-IA and activation of NF-κB in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, TIF-IA-NF-κB nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

Ex vivo treatment of patient biopsies as a novel method to assess colorectal tumour response to the MEK1/2 inhibitor, Selumetinib.

Although an array of new therapeutics has emerged for the treatment of colorectal cancer, their use is significantly impacted by variability in patient response. Better pre-clinical models could substantially improve efficacy as it may allow stratification of patients into the correct treatment regime. Here we explore acute, ex vivo treatment of fresh, surgically resected human colorectal tumour biopsies as a novel pre-clinical model for identifying patient response to specific therapeutics. The MEK1/2 inhibitor, Selumetinib (AZD6244, ARRY-142886) was used as a tool compound. Firstly, we established an acute treatment protocol and demonstrated this protocol could differentiate phenotypic and pharmacodynamic responses to Selumetinib (0-3uM). We then used the protocol to evaluate Selumetinib response in tumours from 23 colon cancer patients. These studies revealed that the agent inhibited pERK1/2 phosphorylation in all tumours, caused a significant decrease in proliferation in 5/23 (22%) tumours, and that KRAS/BRAF mutant tumours were particularly sensitive to the anti-proliferative effects of the agent. These data are consistent with data from clinical trials of Selumetinib, suggesting that acute treatment of small tumour biopsies is worthy of further exploration as a pre-clinical model to evaluate colorectal cancer response to novel therapies.

Aspirin Prevention of Colorectal Cancer: Focus on NF-κB Signalling and the Nucleolus.

Overwhelming evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity and the potential to prevent cancer, particularly colorectal cancer. However, the mechanisms underlying this effect remain hypothetical. Dysregulation of the nuclear factor-kappaB (NF-κB) transcription factor is a common event in many cancer types which contributes to tumour initiation and progression by driving expression of pro-proliferative/anti-apoptotic genes. In this review, we will focus on the current knowledge regarding NSAID effects on the NF-κB signalling pathway in pre-cancerous and cancerous lesions, and the evidence that these effects contribute to the anti-tumour activity of the agents. The nuclear organelle, the nucleolus, is emerging as a central regulator of transcription factor activity and cell growth and death. Nucleolar function is dysregulated in the majority of cancers which promotes cancer growth through direct and indirect mechanisms. Hence, this organelle is emerging as a promising target for novel therapeutic agents. Here, we will also discuss evidence for crosstalk between the NF-κB pathway and nucleoli, the role that this cross-talk has in the anti-tumour effects of NSAIDs and ways forward to exploit this crosstalk for therapeutic purpose.

A Proteomic Approach to Analyze the Aspirin-mediated Lysine Acetylome.

Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d3, in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences of nonspecific chemical acetylations.

Identification of aspirin analogues that repress NF-κB signalling and demonstrate anti-proliferative activity towards colorectal cancer in vitro and in vivo.

Substantial evidence indicates that aspirin and related non-steroidal anti-inflammatory drugs (NSAIDs) have potential as chemopreventative/therapeutic agents. However, these agents cannot be universally recommended for prevention purposes due to their potential side-effect profiles. Here, we compared the growth inhibitory and mechanistic activity of aspirin to two novel analogues, diaspirin (DiA) and fumaryl diaspirin (F-DiA). We found that the aspirin analogues inhibited cell proliferation and induced apoptosis of colorectal cancer cells at significantly lower doses than aspirin. Similar to aspirin, we found that an early response to the analogues was a reduction in levels of cyclin D1 and stimulation of the NF-κB pathway. This stimulation was associated with a significant reduction in basal levels of NF-κB transcriptional activity, in keeping with previous data for aspirin. However, in contrast to aspirin, DiA and F-DiA activity was not associated with nucleolar accumulation of RelA. For all assays, F-DiA had a more rapid and significant effect than DiA, identifying this agent as particularly active against colorectal cancer. Using a syngeneic colorectal tumour model in mice, we found that, while both agents significantly inhibited tumour growth in vivo, this effect was particularly pronounced for F-DiA. These data identify two compounds that are active against colorectal cancer in vitro and in vivo. They also identify a potential mechanism of action of these agents and shed light on the chemical structures that may be important for the antitumour effects of aspirin.

p300-mediated acetylation of COMMD1 regulates its stability, and the ubiquitylation and nucleolar translocation of the RelA NF-κB subunit.

Nucleolar sequestration of the RelA subunit of nuclear factor (NF)-κB is an important mechanism for regulating NF-κB transcriptional activity. Ubiquitylation, facilitated by COMMD1 (also known as MURR1), acts as a crucial nucleolar-targeting signal for RelA, but how this ubiquitylation is regulated, and how it differs from cytokine-mediated ubiquitylation, which causes proteasomal degradation of RelA, is poorly understood. Here, we report a new role for p300 (also known as EP300) in controlling stimulus-specific ubiquitylation of RelA, through modulation of COMMD1. We show that p300 is required for stress-mediated ubiquitylation and nucleolar translocation of RelA, but that this effect is indirect. We also demonstrate that COMMD1 is acetylated by p300 and that acetylation protects COMMD1 from XIAP-mediated proteosomal degradation. Furthermore, we demonstrate that COMMD1 acetylation is enhanced by aspirin-mediated stress, and that this acetylation is absolutely required for the protein to bind RelA under these conditions. In contrast, tumour necrosis factor (TNF) has no effect on COMMD1 acetylation. Finally, we demonstrate these findings have relevance in a whole tissue setting. These data offer a new paradigm for the regulation of NF-κB transcriptional activity, and the multiple other pathways controlled by COMMD1.

Cigarette smoke extract (CSE) delays NOD2 expression and affects NOD2/RIPK2 interactions in intestinal epithelial cells.

BACKGROUND: Genetic and environmental factors influence susceptibility to Crohn's disease (CD): NOD2 is the strongest individual genetic determinant and smoking the best-characterised environmental factor. Carriage of NOD2 mutations predispose to small-intestinal, stricturing CD, a phenotype also associated with smoking. We hypothesised that cigarette smoke extract (CSE) altered NOD2 expression and function in intestinal epithelial cells. METHODS AND FINDINGS: Intestinal epithelial cell-lines (SW480, HT29, HCT116) were stimulated with CSE and nicotine (to mimic smoking) ±TNFα (to mimic inflammation). NOD2 expression was measured by qRT-PCR and western blotting; NOD2-RIPK2 interactions by co-immunoprecipitation (CoIP); nuclear NFκB-p65 by ELISA; NFκB activity by luciferase reporter assays and chemokines (CCL20, IL8) in culture supernatants by ELISA. In SW480 and HT29 cells the TNFα-induced NOD2 expression at 4 hours was reduced by CSE (p = 0.0226), a response that was dose-dependent (p = 0.003) and time-dependent (p = 0.0004). Similar effects of CSE on NOD2 expression were seen in cultured ileal biopsies from healthy individuals. In SW480 cells CSE reduced TNFα-induced NFκB-p65 translocation at 15 minutes post-stimulation, upstream of NOD2. Levels of the NOD2-RIPK2 complex were no different at 8 hours post-stimulation with combinations of CSE, nicotine and TNFα, but at 18 hours it was increased in cells stimulated with TNFα+CSE but decreased with TNFα alone (p = 0.0330); CSE reduced TNFα-induced NFκB activity (p = 0.0014) at the same time-point. At 24 hours, basal CCL20 and IL8 (p<0.001 for both) and TNFα-induced CCL20 (p = 0.0330) production were decreased by CSE. CSE also reduced NOD2 expression, CCL20 and IL8 production seen with MDP-stimulation of SW480 cells pre-treated with combinations of TNFα and CSE. CONCLUSIONS: CSE delayed TNFα-induced NOD2 mRNA expression and was associated with abnormal NOD2/RIPK2 interaction, reduced NFκB activity and decreased chemokine production. These effects may be involved in the pathogenesis of small-intestinal CD and may have wider implications for the effects of smoking in NOD2-mediated responses.

c-Src dependency of NSAID-induced effects on NF-κB-mediated apoptosis in colorectal cancer cells.

Long-term aspirin or related non-steroidal anti-inflammatory drugs (NSAIDs) ingestion can protect against colorectal cancer (CRC). NSAIDs have a pro-apoptotic activity and we have shown that stimulation of the nuclear factor-kappaB (NF-κB) pathway is a key component of this pro-apoptotic effect. However, the upstream pathways have yet to be fully elucidated. Here, we demonstrate that aspirin activates the c-Src tyrosine kinase pathway in CRC cells. We show that c-Src activation occurs in a time- and dose-dependent manner, preceding aspirin-mediated degradation of IκBα, nuclear/nucleolar translocation of NF-κB/RelA and induction of apoptosis. Furthermore, inhibition of c-Src activity, by chemical inhibition or expression of a kinase dead form of the protein abrogates aspirin-mediated degradation of IκBα, nuclear translocation of RelA and apoptosis, suggesting a causal link. Expression of constitutively active c-Src mimics aspirin-induced stimulation of the NF-κB pathway. The NSAIDs sulindac, sulindac sulphone and indomethacin all similarly activate a c-Src-dependent NF-κB and apoptotic response. These data provide compelling evidence that c-Src is an upstream mediator of aspirin/NSAID effects on NF-κB signalling and apoptosis in CRC cells and have relevance to the development of future chemotherapeutic/chemopreventative agents.

Nucleolar targeting of RelA(p65) is regulated by COMMD1-dependent ubiquitination.

Stimulation of the NF-kappaB pathway can have proapoptotic or antiapoptotic consequences, and one mechanism that determines the outcome is the nuclear distribution of RelA. Certain stress stimuli induce nucleolar accumulation of RelA thereby mediating apoptosis, whereas others induce nucleoplasmic accumulation and inhibition of apoptosis. Here we investigated the mechanisms that regulate the nuclear distribution of RelA, specifically, the role of the ubiquitin/proteasome system. We found that stress-induced nucleolar translocation of RelA is preceded by ubiquitination of the protein. We also found that chemical proteasome inhibitors induce the ubiquitination and nucleolar translocation of RelA and that this is required for the apoptotic response to these agents. We show that the RelA nucleolar localization signal (amino acids 27-30) is a critical domain for ubiquitination of the protein but that the lysine residue within this motif is not a direct target. We show that RelA binds COMMD1, the rate-limiting component of the RelA ubiquitin ligase complex, in response to stress. Furthermore, we show that overexpression of COMMD1 promotes stress-mediated nucleolar targeting of RelA, whereas knockdown of COMMD1 blocks this effect, causing RelA to remain in the nucleoplasm. These data identify a new role for COMMD1 in regulating the nuclear/nucleolar distribution of RelA and suggest that ubiquitination acts as a signal for transport of RelA to the nucleolus. These findings have relevance to the design of chemopreventative/anticancer agents that act by targeting RelA to the nucleolar compartment.

CDK4 inhibitors and apoptosis: a novel mechanism requiring nucleolar targeting of RelA.

Components of the cyclin D-CDK4/6-INK4-Rb pathway are key regulators of the cell cycle and are frequently disrupted in cancer. Defects in this pathway usually manifest as an increase in CDK4 activity, leading to unrestricted proliferation of tumour cells. CDK4 inhibitors have been shown to possess anti-tumour activity in vitro and agents that target the cyclin D1/CDK4 complex are currently the focus of intense scrutiny for clinical application as cancer therapeutics. However, the mechanisms by which these agents mediate their effects remains to be fully elucidated. We recently described a novel mechanism by which a CDK4 inhibitor induces apoptosis in colon cancer cells through activation of the NFkB signaling pathway. Specific inhibition of CDK4 activity induced translocation of RelA, the principal component of NFkappaB, from the cytoplasm to the nucleoplasm and then to the nucleolus. This was accompanied by a repression of NFkappaB-driven transcription and apoptosis of the cancer cells. To determine the role of RelA in apoptosis, we utilised a mutant form of the protein, where the critical domain required for nucleolar targeting had been deleted. When cells expressing this mutant protein were treated with the CDK4 inhibitor, RelA translocated from the cytoplasm to the nucleoplasm, but was excluded from the nucleolus. Furthermore, apoptosis induced by CDK4 inhibition was also abrogated in cells expressing mutant RelA protein. Here, we discuss the molecular mechanisms that regulate programmed cell death induced by disruption of the cyclin D1/CDK4 complex and consider the wider implications these findings have for the future development of novel chemotherapeutic agents.

p38-mediated inactivation of cyclin D1/cyclin-dependent kinase 4 stimulates nucleolar translocation of RelA and apoptosis in colorectal cancer cells.

Aberrant nuclear factor-kappaB (NF-kappaB) signaling plays a role in cancer initiation and progression; thus, it represents a potential therapeutic target. We previously identified a mechanism of repression of NF-kappaB transcriptional activity and induction of apoptosis in colon cancer cells involving nuclear/nucleolar translocation of the RelA (p65) component of NF-kappaB. This response was stimulated by cellular stress-inducing agents, including aspirin, but not by tumor necrosis factor. Here, we investigate the upstream molecular mechanisms responsible for nucleolar targeting of RelA and show that aspirin activates the p38 mitogen-activated protein kinase (MAPK) pathway in colorectal cancer cells. We also show that aspirin causes rapid, ubiquitin-dependent degradation of cyclin D1, a known p38 target. Aspirin-induced p38 activation preceded cyclin D1 degradation, which was then followed by activation of the NF-kappaB pathway, suggesting a causative link. Indeed, chemical p38 inhibition (PD169316) and small interfering RNA directed against p38 blocked aspirin-induced cyclin D1 degradation, nucleolar translocation of RelA, and apoptosis. Furthermore, chemical inhibition of the cyclin D1/cyclin-dependent kinase 4 (CDK4) kinase complex, used as a surrogate for cyclin D1 degradation, caused nucleolar translocation of RelA, repression of kappaB-driven transcription, and apoptosis, thereby reproducing the effects of aspirin. In addition, we found that aspirin and the CDK4 inhibitor induced nucleolar translocation of RelA and apoptosis through a common mechanism involving the NH(2)-terminal nucleolar localization signal. Collectively, these data suggest that aspirin causes inhibition of cyclin D1/CDK4 through the p38 MAPK pathway. This inhibition stimulates the NF-kappaB pathway to induce nucleolar translocation of RelA and apoptosis. These novel findings have considerable relevance to the rational design of novel chemotherapeutic and chemopreventative strategies.

Aspirin activates the NF-kappaB signalling pathway and induces apoptosis in intestinal neoplasia in two in vivo models of human colorectal cancer.

Substantial evidence indicates that aspirin has antitumour activity against large bowel cancer and modulation of the NF-kappaB (NF-kappaB) signalling pathway has been identified as a key mechanism in this effect. However, studies examining how aspirin affects the NF-kappaB pathway to promote apoptosis have been restricted to in vitro analysis in tissue culture systems and have produced contrasting results. Here, we employed two animal models of human colorectal cancer to determine aspirin effects on the NF-kappaB pathway in colorectal neoplasia in vivo, and the relationship of such effects to the induction of apoptosis. We demonstrate that aspirin induces phosphorylation and degradation of cytoplasmic IkappaBalpha in xenografted HT-29 tumours and in adenomas from APC(Min+/-) mice. Furthermore, we show that this response occurs in a time-dependent manner and is paralleled by nuclear translocation of p65 and caspase activation. Using high performance liquid chromatography analysis, we demonstrate that >0.5 mM salicylate levels are achievable in xenografted tumours after low-dose aspirin (40 mg/kg) treatment and that these levels, which are pharmacologically relevant to humans, are sufficient to stimulate an NF-kappaB and apoptotic response. We demonstrate that activation of the NF-kappaB pathway is associated with increased apoptosis in neoplastic epithelial cells, but found that aspirin has a minimal effect on nuclear p65 and apoptosis in normal intestinal mucosa from APC(Min+/-) mice. These in vivo findings further establish that aspirin induces activation of the NF-kappaB pathway in neoplastic epithelial cells and provide further support that this effect is important for the antitumour activity of the agent. These data have considerable relevance to cancer prevention and therapy.

Nucleolar sequestration of RelA (p65) regulates NF-kappaB-driven transcription and apoptosis.

The molecular mechanisms that regulate nuclear NF-kappaB to determine whether the stimulation of this pathway has a pro- or anti-apoptotic effect on cells have yet to be fully defined. Nuclear compartmentalization is increasingly recognized as an important mechanism for regulating the activity of transcription-related proteins and modulating cell growth and death. We have investigated whether such compartmentalization serves as a mechanism for regulating NF-kappaB transcriptional activity. We demonstrate that the RelA component of NF-kappaB is sequestered in the nucleolus in response to the proapoptotic NF-kappaB stimuli aspirin, serum withdrawal, and UV-C radiation. In contrast, RelA is excluded from the nucleolus in response to the cytokines tumor necrosis factor and TRAIL. We identify an N-terminal motif of RelA that is essential for the nucleolar localization of the protein and show that deleting this motif inhibits the translocation of RelA from the nucleoplasm to the nucleolus. We demonstrate that the nucleolar accumulation of RelA is paralleled by a decrease in basal levels of NF-kappaB transcriptional activity and by apoptosis. Furthermore, we show that the retention of RelA in the nucleoplasm inhibits this decrease in NF-kappaB-driven transcription and blocks apoptosis induced by aspirin and UV-C radiation. This work identifies a novel cellular mechanism for regulating NF-kappaB-driven transcription and apoptosis, involving the nucleolar sequestration of a key NF-kappaB subunit. These data contribute to the understanding of the complexities of NF-kappaB function and have considerable relevance to cancer prevention and therapy.