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.

Global histone modification fingerprinting in human cells using epigenetic reverse phase protein array.

The balance between acetylation and deacetylation of histone proteins plays a critical role in the regulation of genomic functions. Aberrations in global levels of histone modifications are linked to carcinogenesis and are currently the focus of intense scrutiny and translational research investments to develop new therapies, which can modify complex disease pathophysiology through epigenetic control. However, despite significant progress in our understanding of the molecular mechanisms of epigenetic machinery in various genomic contexts and cell types, the links between epigenetic modifications and cellular phenotypes are far from being clear. For example, enzymes controlling histone modifications utilize key cellular metabolites associated with intra- and extracellular feedback loops, adding a further layer of complexity to this process. Meanwhile, it has become increasingly evident that new assay technologies which provide robust and precise measurement of global histone modifications are required, for at least two pressing reasons: firstly, many approved drugs are known to influence histone modifications and new cancer therapies are increasingly being developed towards targeting histone deacetylases (HDACs) and other epigenetic readers and writers. Therefore, robust assays for fingerprinting the global effects of such drugs on preclinical cell, organoid and in vivo models is required; and secondly, robust histone-fingerprinting assays applicable to patient samples may afford the development of next-generation diagnostic and prognostic tools. In our study, we have used a panel of monoclonal antibodies to determine the relative changes in the global abundance of post-translational modifications on histones purified from cancer cell lines treated with HDAC inhibitors using a novel technique, called epigenetic reverse phase protein array. We observed a robust increase in acetylation levels within 2-24 h after inhibition of HDACs in different cancer cell lines. Moreover, when these cells were treated with N-acetylated amino acids in addition to HDACs, we detected a further increase in histone acetylation, demonstrating that these molecules could be utilized as donors of the acetyl moiety for protein acetylation. Consequently, this study not only offers a novel assay for diagnostics and drug screening but also warrants further research of the novel class of inexpensive, non-toxic natural compounds that could potentiate the effects of HDAC inhibitors and is therefore of interest for cancer therapeutics.

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.