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.

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.