Abstract
p53 (TP53) is the most commonly mutated tumor suppressor in cancer. How mutant forms of p53 can contribute to disease progression to influence patient outcomes is important to understand. Previous in-vitro and in-vivo data have shown p53 mutants can drive hyperproliferation, resistance to cell death, and promote drug resistance. However, most of the literature has focused on a small subset of frequently occurring missense mutations in p53, namely hotspot mutations. With the advent of next-generation sequencing technology, many additional mutations in p53 have been identified. Therefore, to improve our understanding of p53 mutants in cancer, we studied the association of uncommon p53 mutants on patient outcomes. To characterise the biological impact of these mutants, we also created experimental tools to test in-vitro.
An analysis of the TCGA Pan-Cancer dataset was used to explore uncommon mutations in p53 present in >50 tumors compared to a hotspot mutation. Kaplan-Meier survival analyses were used to explore whether patients with tumors expressing uncommon TP53 mutations influence disease free survival (DFS). Site-directed mutagenesis was conducted to produce DNA constructs expressing two uncommon p53 missense mutations in H179, associated with poor DFS. Using isogenic in-vitro cell lines, H1299 lung carcinoma cells stably expressing mutant p53 (H179R, H179Y, and R175H) or no p53 (Empty) were created. SYBR-GreenTM assay was carried out on the created stable cell lines, to investigate whether one of these uncommon mutants (H179R/Y) was more efficient at promoting cell proliferation compared to the hotspot mutant (R175H) or p53 null (Empty).
Of the 8 uncommon p53 mutations included in this study, only zinc-binding mutations at C176 and H179 were associated with poorer DFS. Site-directed mutagenesis was effective at creating one hotspot mutant and two uncommon mutations in the pCMV- Neo-Bam-wtp53 plasmid. Restriction digest was used to remove the TP53 insert from the pCMV-Neo-Bam-wtp53 plasmid to generate pCMV-Empty. These plasmids were transfected in H1299 cells to confirm that the plasmids either expressed either no p53 (Empty) or mutant p53. After confirmation, H1299 cells stably expressing these plasmids were created via antibiotic selection. Interestingly, 2/3 cell proliferation replicates, suggests that H179R and H179Y missense mutations in p53 can drive hyperproliferation, compared to cells with no p53 expression and cells with a hotspot mutant. Cell survival, apoptosis, DNA damage assays, and RNA-sequencing will be carried out in the future to characterise these mutations, enabling a deeper understanding of the biological pathways associated with aggressive disease leading to poor patient outcomes.