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Senescence of smooth muscle cells (SMCs) has a crucial role in the pathogenesis of abdominal aortic aneurysm (AAA), a disease of vascular degeneration. Perturbation of cellular ribosomal DNA (rDNA) transcription triggers nucleolar stress response. Previously we demonstrated that induction of nucleolar stress in SMCs elicited cell cycle arrest via the ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR)-p53 axis. However, the specific roles of nucleolar stress in vascular degeneration remain unexplored. In the present study, we demonstrated for the first time that in both human and animal AAA tissues, there were non-coordinated changes in the expression of RNA polymerase I machinery components, including a downregulation of transcription initiation factor-IA (TIF-IA). Genetic deletion of TIF-IA in SMCs in mice (smTIF-IA-/-) caused spontaneous aneurysm-like lesions in the aorta. In vitro, induction of nucleolar stress triggered a non-canonical DNA damage response, leading to p53 phosphorylation and a senescence-like phenotype in SMCs. In human AAA tissues, there was increased nucleolar stress in medial cells, accompanied by localized DNA damage response within the nucleolar compartment. Our data suggest that perturbed rDNA transcription and induction of nucleolar stress contribute to the pathogenesis of AAA. Moreover, smTIF-IA-/- mice may be a novel animal model for studying spontaneous AAA-like vascular degenerations.

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.

Ribosomes are universally important in biology and their production is dysregulated by developmental disorders, cancer, and virus infection. Although presumed required for protein synthesis, how ribosome biogenesis impacts virus reproduction and cell-intrinsic immune responses remains untested. Surprisingly, we find that restricting ribosome biogenesis stimulated human cytomegalovirus (HCMV) replication without suppressing translation. Interfering with ribosomal RNA (rRNA) accumulation triggered nucleolar stress and repressed expression of 1392 genes, including High Mobility Group Box 2 (HMGB2), a chromatin-associated protein that facilitates cytoplasmic double-stranded (ds) DNA-sensing by cGAS. Furthermore, it reduced cytoplasmic HMGB2 abundance and impaired induction of interferon beta (IFNB1) mRNA, which encodes a critical anti-proliferative, proinflammatory cytokine, in response to HCMV or dsDNA in uninfected cells. This establishes that rRNA accumulation regulates innate immune responses to dsDNA by controlling HMGB2 abundance. Moreover, it reveals that rRNA accumulation and/or nucleolar activity unexpectedly regulate dsDNA-sensing to restrict virus reproduction and regulate inflammation. (145 words)

The nucleolus is a multifunctional organelle that, in addition to its primary role in ribosome biogenesis, has emerged as a critical stress sensor and coordinator of stress response. However, the molecular nature of how nucleoli sense stress and coordinate downstream cellular consequence remains poorly understood. NF-κB signalling is a critical regulator of stress response. Many cellular stresses that disrupt nucleolar function also stimulate the NF-κB pathway. However, the role of NF-κB as a downstream effector of nucleolar stress has not yet been examined. Aspirin, a known chemopreventative agent, stimulates the NF-κB pathway to mediate apoptosis but the upstream mechanisms are unclear. In this thesis, I identified a novel nucleolar stress response pathway that culminates in activation of NF-κB signalling, and demonstrated the significance of this nucleolar pathway in the anti-tumour effects of aspirin. Using multiple approaches, I made the novel observations that disruption of the Pol I complex activates the cytoplasmic NF-κB signalling pathway. I show that multiple stress stimuli of NF-κB pathway induce degradation of the crucial Pol I complex component, rDNA transcription initiation factor IA (TIF-IA). I identified the tumour suppressor, p14ARF and the Pol I complex component, upstream binding factor (UBF) as mediators of this degradation. I revealed that inhibition of CDK4 activity lies upstream of UBF/p14ARF-facilitated TIF-IA degradation. Furthermore, using different approaches I show that blocking aspirin/CDK4i-mediated degradation of TIF-IA blocks the effects of these agents on nucleolar morphology and NF-κB signalling. Finally, I show this nucleolar stress response pathway, containing a UBF/p14ARF/TIF-IA axis, is utilized by aspirin to kill colon cancer cells. Taken together, this data presented in this thesis advances understanding of nucleolar stress response, and has therapeutic implications with regard to the anti-tumour effects of aspirin.

TIF-IA is a basal transcription factor of RNA polymerase I (Pol I) that is a major target of the JNK2 signaling pathway in response to ribotoxic stress. Using advanced fluorescence microscopy and kinetic modeling we elucidated the subcellular localization of TIF-IA and its exchange dynamics between the nucleolus, nucleoplasm and cytoplasm upon ribotoxic stress. In steady state, the majority of (GFP-tagged) TIF-IA was in the cytoplasm and the nucleus, a minor portion (7%) localizing to the nucleoli. We observed a rapid shuttling of GFP-TIF-IA between the different cellular compartments with a mean residence time of approximately 130 s in the nucleus and only approximately 30 s in the nucleoli. The import rate from the cytoplasm to the nucleus was approximately 3-fold larger than the export rate, suggesting an importin/exportin-mediated transport rather than a passive diffusion. Upon ribotoxic stress, GFP-TIF-IA was released from the nucleoli with a half-time of approximately 24 min. Oxidative stress and inhibition of protein synthesis led to a relocation of GFP-TIF-IA with slower kinetics while osmotic stress had no effect. The observed relocation was much slower than the nucleo-cytoplasmic and nucleus-nucleolus exchange rates of GFP-TIF-IA, indicating a time-limiting step upstream of the JNK2 pathway. In support of this, time-course experiments on the activity of JNK2 revealed the activation of the JNK kinase as the rate-limiting step.

We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.