PO-126 Exploration of novel regulators of mutant P53

Abstract

IntroductionMutations in the tumour suppressor gene, TP53 occur in more than 50% of human cancers. Mutant p53 proteins not only lose their tumour suppressive capacities, but they also gain oncogenic functions broadly referred to as gain of function (GOF). Cancer cells frequently accumulate mutant p53 (mt-p53) and a subset of these cancer cells become addicted to it for their survival. While mt-p53 is inherently labile, as is wild type p53, in cancer cells mt-p53 becomes constitutively stable. In part, this is due to the interrupted auto-regulatory loop with Mdm2. However, this alone cannot explain the stability of mt-p53 in many cancer contexts. We therefore argued that additional factors are responsible for its stability in cancer cells.Material and methodsWe performed a genome wide high-throughput RNAi screen to identify major regulators of mt-p53 levels in two different mt-p53 expressing cell lines. This was carried out by siRNA transfections in cancer cell lines and quantitating mt-p53 levels through immunostaining. We used GeneGo and panther for pathway and network analyses and gene ontology analysis. To study the interaction between mt-p53 and its candidate regulatory genes, we employed bimolecular fluorescence complementation assay. We also created Doxycyclin inducible stable cell lines overexpressing shRNA to an identified regulator of mt-p53.Results and discussionsUsing network, pathway analyses and extensive knowledge mining, we selected 37 candidate genes from 169 potential candidates for validation through p53 immunoblotting. Out of 37 genes, we chose to investigate a potential regulator, which acts as an adapter to a family of E3 ligases. In addition to observing spatial colocalization and interaction, we observed that this regulator is capable of regulating mt-p53 protein expression but not mRNA expression. Moreover, we detected reduction in mt-p53 ubiquitination in mouse embryonic fibroblasts knocked out for this E3-adapter. Upon overexpression of this E3-adapter, we observed an increase in mt-p53 levels in the extracellular vesicles. We hypothesise that this E3-adapter regulates the release of mt-p53, which potentially plays a role in modulating tumour microenvironment.ConclusionOur results highlight a previously unknown mode of mt-p53 regulation in cancer cells, thus opening up opportunities for targeting aggressive cancers with mt-p53. Future studies aim to explore the functional consequences of this E3-adapter mediated mt-p53 regulation.