Identification and characterisation of novel mutant p53 regulators

Abstract

The p53 protein is a transcription factor and has been considered as a master tumour suppressor. TP53 gene is frequently mutated in human cancers (more than 50%). Mutation in p53 not only loses its tumour suppressive functions but also gains new oncogenic properties, including increased cell proliferation, higher resistance to therapeutics and enhanced metastasis. These are consistent with the association of mutant p53 cancers with poor clinical prognosis. Importantly, some cancer cells are dependent on mutant p53 expression for survival and proliferation. This opens an exciting therapeutic window for the treatment of multiple cancer types. Despite the prominent roles of mutant p53 in malignant progression, its regulation remains partially understood.

The major goal of this study is to gain novel insight into the regulation of mutant p53. Our approach has been to identify novel regulators of mutant p53 by utilising whole-genome RNAi discovery screens. These include protein-coding siRNA, microRNA (miRNA) and long noncoding RNA (lncRNA) screens. We have developed a high content assay for these screens. The libraries were subjected to multi-tiered screens on mutant p53-expressing cell lines, including MDA-MB-468, JH-EsoAd1, MDA-MB-231 and AU565. Of 18,120 protein-coding siRNAs, the screen yielded 50 potential mutant p53 candidate regulators. Six of these were conserved between MDA-MB-468 and JH-EsoAd1, and 44 were cell-line specific. Primary lncRNA screen yielded 154 hits, in which 39 and 44 were validated in MDA-MB- 468 and JH-EsoAd1 cell lines, respectively.

The miRNA screen yielded a total of 211 mimics and 46 inhibitor hits. Of these, the top 20 miRNA mimics were pursued further. Three potential candidates were identified to down-regulate mutant p53 expression. One of these is miR-504-5p, which has been previously identified to target and down-regulate wild-type p53. Further investigation of these miRNAs led to the identification of MAP1B as a potential regulator of mutant p53. Our result demonstrated that knockdown of MAP1B sensitises mutant p53-expressing cells. The relevance of MAP1B expression to overall survival (OS) of gastric cancer patients (which harbour high TP53 mutation incidence, up to 77 %) was evaluated. Consistently, high MAP1B mRNA expression correlates with poor overall patient survival. Reciprocally, overall survival is improved in patients with low MAP1B mRNA expression. These results suggest a potential role for MAP1B in the oncogenicity of mutant p53 cancer. The mechanism by which MAP1B affects mutant p53 is yet to be elucidated. Overall, this thesis demonstrates the utility of RNAi screen in the exploration of mutant p53 regulation. This study has identified a list of potential regulators that were not previously shown to regulate mutant p53. Future study of these potential novel mutant p53 regulators will help to dissect the regulatory network of mutant p53 and potentially inform on novel therapeutic approaches.