Role of nucleolar stress in the anti-tumour activity of non-steroidal anti-inflammatory drugs (NSAIDs).
Lobb, Ian Thomas
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Overwhelming evidence indicates that aspirin (ASA) and related non-steroidal anti-inflammatory drugs (NSAIDs) have anti-tumour activity against colorectal cancer (CRC). Although the underlying mechanisms have yet to be fully elucidated, the host laboratory have shown that nucleolar sequestration of the NF-κB component RelA is critical. In the course of these studies, it was noted that alongside effects on the NF- κB pathway, ASA has a profound effect on nucleoli, including a dramatic increase in nucleolar size. These data were particularly interesting as, in addition to its role in ribosome biogenesis, the nucleolus is known to act as a stress sensor and play a key role in the regulation of cell growth and apoptosis. Indeed, this organelle has been identified as a potential target for anti-tumour agents. However, how stress causes changes to nucleolar function, and how these are translated into changes in cell phenotype, remain unclear. Therefore, the aim of my thesis was to fully characterise ASA effects on nucleoli and to determine whether these effects contribute to the anti-tumour activity of this agent. I found that ASA induced an atypical form of nucleolar stress that was associated with enlargement of the organelle, relocalisation of nucleolar markers to the periphery, depletion of the critical component of the Pol I transcription factor complex, TIF-IA, and inhibition of rRNA transcription. These effects were independent of the p38 and JNK2 MAP kinase pathways. However, they were mimicked by inhibition of CDK4, which had previously been shown to lie upstream of ASA effects on the NF-κB pathway. These data describe a novel mechanism by which ASA, and CDK4 inhibition, may inhibit the growth of colon cancer cells. In addition to this candidate approach, I used Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based quantitative proteomics to obtain a global overview of ASA effects on nucleoli of colon cancer cells. Firstly, a protocol was successfully developed to isolate pure nucleoli from SW480 CRC cell lines. This protocol was then applied to SILAC labelled cells treated with ASA for three time-points (0, 6, 10 h). In collaboration with R.T Hay and M. Tatham (University of Dundee), proteomic analysis was then carried out by tandem-mass spectrometry. These data confirmed that ASA has a significant effect on the nucleolar proteome. They also revealed that ASA induces a distinct type of nucleolar stress that is associated with the accumulation of chaperones, translational regulators and members of the ubiquitin-proteasome system (UPS) in this organelle. These data were reminiscent of studies previously published on the effect of proteasome inhibition on nucleoli. I therefore used SILAC-based proteomics to compare ASA effects on nucleoli to those induced by the proteasome inhibitor, MG132. I found that similar sub-groups of proteins accumulate in nucleoli in response to both agents and that ASA induced proteotoxic stress in a similar manner to MG132. Fluorescence correlation spectroscopy in collaboration with R. Duncan and K. Martin (Heriot-Watt University) demonstrated the relative reduction in mobility of nucleolar DsRed-RelA, indicating that, similar to MG132, ASA induces formation of nucleolar aggresomes. Mechanistic studies suggested that blocking ASA-mediated proteotoxic stress blocked the apoptotic effects of the agent. Taken together, these data define a distinct type of nucleolar stress that may be involved in the cells response to proteotoxic stress and be required for the anti-tumour activity of ASA.