Abstract
TP53 encodes the tumour suppressor p53, which is mutated in ~50% of human cancers. 70% of TP53 mutations are missense, and some mutations gain novel properties that drive tumour biology. Extensively characterised TP53 “hotspot” mutants account for 25% of TP53 missense mutations, however it is unclear how the remaining 75% of mutations influence tumour biology, limiting opportunities to develop precision medicine targeting TP53 mutations.
Previous analysis of The Cancer Genome Atlas showed TP53 mutations at codon C176 are associated with poorer prognosis than hotspot mutations (LogRank test, p = 0.0075). Two frequently reported mutations at this codon are Cystine 176 to Phenylalanine (C176F) and Cystine 176 to Tyrosine (C176Y). Previous RNA-sequencing of H1299 cells expressing C176F/Y mutant p53 revealed upregulation of pro-angiogenesis genes, including angiopoietin-like 4 (ANGPTL4) which encodes a secreted protein that enhances lipid uptake and vascularisation. We aimed to explore the functional impact of these mutants on tumour biology and hypothesised that C176F/Y mutant p53 promotes angiogenesis via regulation of ANGPTL4 protein.
We collected media from H1299 cells expressing a vector control or C176F/Y mutant p53 and performed Western blots which showed higher levels of ANGPTL4 protein in media collected from cells expressing C176F/Y mutant p53 compared to vector control. The cells were additionally cultured in a 3D model and immunofluorescence quantitation also showed upregulated expression of ANGTPL4 from cells expressing C176F/Y mutant p53 compared to vector control (Mann-Whitney test, Vector vs C176F p = 0.003, Vector vs C176Y p = 0.0002). The 3D model additionally demonstrated increased immunofluorescence quantitation of total lipid (Mann-Whitney test, Vector vs C176F p = 0.0006) and F-actin levels (Mann-Whitney test, Vector vs C176F p = 0.0006, Vector vs C176Y p = 0.0002).
Together, our findings suggest C176F/Y mutant p53 likely promotes angiogenesis in the tumour microenvironment via ANGTPL4 secretion. This angiogenic activity may be the driver behind the poor prognosis associated with C176F/Y mutant p53. Ultimately, this research may inform the therapeutic potential of anti-angiogenic strategies in tumours harbouring C176F/Y mutant p53.