2D and 3D Cancer Models Reveal Upregulated ANGPTL4 Secretion in p53C176F/Y Mutant Cells

TP53 encodes the tumour suppressor p53 and is mutated in ~50% of human cancers. 70% of TP53 mutations are missense, and some missense mutations gain novel properties that drive tumour biology. TP53 “hotspot” mutants have been extensively characterised in the literature, however it is unclear how the remaining uncharacterised mutations influence tumour biology, limiting opportunities to develop precise, mutation-specific therapeutic strategies. Analysis of The Cancer Genome Atlas revealed TP53 mutations at zinc-binding codon C176 are associated with poorer prognosis than hotspot mutations. Two frequently reported mutations at this codon are Cysteine 176 to Phenylalanine (p53^C176F) and Cysteine 176 to Tyrosine (p53^C176Y). We aimed to explore the functional impact of these mutants on tumour biology and hypothesised that p53^C176F/Y promote a pro-tumour transcriptome resulting in aggressive tumour biology.

We found that although p53^C176F/Y mutants lose wildtype p53 (wt-p53) function, they remain stable and localise to the nucleus, indicating they may retain transcription factor properties. Previous RNA-sequencing suggested a potential association between p53^C176F/Y mutants and upregulation of angiogenesis. We validated potential gene targets of p53^C176F/Y across multiple cell cancer lines. This analysis revealed a significant association of upregulated angiopoietin-like 4 (ANGPTL4) transcript in prostate (PC3) and ovarian (SKOV3) cancer cells expressing p53^C176F, and in lung (H1299) cells expressing p53^C176F/Y. ANGPTL4 is a secreted protein that enhances biological effects including angiogenesis, lipid uptake, and resistance to cell death.

H1299 cells expressing p53^C176F/Y showed increased levels of ANGPTL4 secretion compared to vector control in both 2D monolayer and 3D bio-printed cultures, as interpreted by qualitative observation. siRNA knockdown of TP53 suggested the relationship between p53^C176F/Y and ANGPTL4 was unlikely to be under direct transcriptional regulation, and effects on ANGPTL4 secretion were more pronounced in 3D than 2D. Immunofluorescent staining revealed serum starvation significantly upregulated intracellular ANGPTL4 levels in cells expressing p53^C176F, which was variably observed in cells expressing p53^C176Y. These findings suggested p53^C176F/Y mutants may modulate ANGPTL4 secretion through non-direct mechanisms, which is likely reinforced by serum starvation in a 3D environment. Additionally, siRNA-mediated p53^C176F/Y knockdown was associated with a decrease in cell number compared to control siRNA. We also observed divergence of biology between p53^C176F and p53^C176Y. p53^C176F mutants were associated with a significant increase in neutral lipid and reduced survival, whereas p53^C176Y mutants showed a phenotype consistent with invasion, which requires further validation.

Together, these findings suggest p53^C176F/Y may contribute to pro-tumour biology through enhanced ANGPTL4 secretion. The study provides insight into how non-hotspot p53 mutations may contribute to aggressive tumour biology and poor patient outcomes and highlight the importance of individually characterising non-hotspot p53 mutations. Ultimately, this research provides a foundation for future research into therapeutic strategies to target the biology of p53^C176F/Y.