Use of spatial proteomics to dissect an emerging inflammatory pathway for targeted cancer prevention

Background Chronic inflammation is a hallmark of ageing and a major cause of age-related cancer.

A key contributor is the milieu of factors secreted by senescent cells known as the senescence associated secretory phenotype (SASP). The NF-kB transcription factor is a major driver of the SASP. However, how this pathway is triggered in senescence initiation remains elusive.

We recently demonstrated that the PolI complex component, TIF-IA(RRN3), accumulates as a very early response to senescence induction and in ageing tissue.

TIF-IA accumulation is paralleled by nucleolar coalescence (a hallmark of aging and senescence), preceding transcription of SASP factors.

Furthermore, inhibiting this TIF-IA accumulation inhibits nucleolar coalescence, expression of SASP factors and senescence. In converse, overexpression of TIF-IA promotes nucleolar coalescence, the SASP and senescence.

These data reveal an emerging nucleoli-NF-kB inflammatory pathway that has considerable relevance to chronic inflammation and cancer prevention.

Aims Blockade of the NF-kB transcription factor has been the focus of considerable research effort but, has not yet materialised.

The overarching aim of this study is to advance our understanding of NF-kB activation in senescence/ageing, to reveal novel means to intervene with this pathway to prevent the SASP and cancer.

To this end, we specifically aim to identify signalling networks triggered by TIF-IA accumulation that have relevance to nucleolar fusion and transcription of SASP factors.

Methods Changes in the subcellular localisation of signalling molecules is an important mechanism to coordinate the initial response to stimuli.

Therefore, we will use Localisation of Organelle Proteins by Isotope Tagging after Differential ultraCentrifugation (LOPIT-DC) to quantify the changes in the spatial proteome induced by expression of wild type TIF-IA, and two TIF-IA mutants we have identified to have differential effects on transcription of SASP factors.

Published, bioinformatic pipelines will be used to deep mine the data, comparing WT v mutants to identify changes associated with activation of NF-kB and the SASP. Studies will be carried out in a colonic adenoma cell line as this has relevance to cancer prevention.

How the results will be used We will use the data generated in this preliminary study to attract large scale funding to: validate the findings in senescence models in vitro and in vivo; screen drugs known to modulate identified pathways using a unique nucleoli-NF-kB signalling phenotypic assay we have developed; explore identified networks in ageing and in adenoma-to carcinoma progression of colon cancer using unique human tissue we have access to.