Abstract
The tumour suppressor p53 is encoded by the TP53 gene, which is the most frequently altered gene across human cancers. The majority of inactivating mutations in TP53 are missense mutations. Some missense mutations confer novel gain of function (GOF) effects which induce oncogenic biological pathways that drive tumour aggression and metastasis. Frequently occurring “hotspot” mutants of p53 have been extensively characterised, however, all regions of p53 have been found to undergo mutation and the majority of missense p53 mutations have not been characterised.
We analysed the pan-cancer dataset from the cancer genome atlas (TCGA) to determine patient outcome in concordance with the mutational status of TP53 in the tumour. We found that out of the ten TP53 codons most frequently implicated as missense mutations across all cancers, four codons were non-hotspot and included the C176 codon. Out of these four codons, only missense mutations at the C176 codon were significantly associated with reduced progression free survival in comparison to hotspot mutants, all other p53 missense mutants, and all tumours which retained wildtype p53. We found C176 was frequently altered to either C176F or C176Y, therefore we created transient and stable transfection experiments derived from plasmids which express these mutations. To model the biology driven by C176 mutations across tumours from various tissue types, three p53-null cell lines (PC3 prostate adenocarcinoma, SKOV3 ovarian carcinoma, and H1299 lung adenocarcinoma) were used.
We found that transiently transfected C176F/Y mutant p53 is defective of several canonical wildtype functions in H1299 cells when compared to cells that express plasmids encoding wildtype p53, the hotspot R175H mutant, or p53 null (Empty). Only wildtype p53 was able to induce cell death in our BFP reporter assay. We additionally found that only wildtype p53 was able to induce the transcription of three known gene targets of p53 including CDKN1A, MDM2 and PIG3.
We then created isogenic cell lines which stably express the C176F/Y mutants in all three cell lines and found that C176F/Y mutant p53 retained tetramerisation function in our H1299 stable cell lines. Additionally, C176F/Y mutant p53 retained nuclear localisation function in our PC3 stable cell lines.
RNA-sequencing (RNA-seq) analysis of our H1299 stable cell lines showed C176F/Y mutations drive anti-apoptosis, mitotic fusion implicated in DNA repair and epithelial-mesenchymal transition. Therefore, these mutants confer a gain of function, providing insight of how mutant p53 contributes to cancer progression. In the future, we plan to validate our findings from the RNA-seq analysis using functional assays.