Human TP53 mRNA

The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the extremely aggressive high-grade serous ovarian cancer (HGSOC) has the highest TP53 mutation rate, with mutations present in >96% of cases. Here, in vitro transcribed (IVT) wild-type (WT) p53-mRNA was injected into HGSOC cell lines, primary cells, and orthotopic mouse models to investigate its role in inhibiting tumor growth and dissemination in vitro and in vivo. Introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent and durable inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells following mRNA-based reactivation of the p53 gene revealed significant changes in the expression of genes associated with p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoration of p53 function simultaneously reduced chromosomal instability in HGSOC cells, underscoring its crucial role in maintaining genomic integrity by regulating the baseline incidence of double-strand breaks induced by replication stress. Furthermore, p53 mRNA treatment dose-dependently reduced tumor growth and inhibited intraperitoneal dissemination of tumor cells in various mouse models.

The researchers aimed to restore p53 function in different preclinical models by transfecting HGSOC cell lines and primary cells with p53-mRNA. Low doses of p53-mRNA (10-250 ng) strongly induced p53 protein expression in OVCAR-8 cells (Figure 1A). 24 hours after transfection, an increase in the number of cells in the G1 phase was observed at concentrations of 100-500 ng p53-mRNA compared to the control group (Figure 1B), supporting the reactivation of p53 activity. Strong induction of cell cycle inhibitors p21, p16, and p27 was clearly observed at low doses of p53-mRNA (≥ 0.1 μg), along with downregulation of key regulators such as PLK1, Aurora A, CDK1, and Cyclin A/B, confirming the results obtained by our vector-based p53 re-expression approach (Figure 1C). These observations were associated with a significant decrease in cell viability of approximately 2-fold after 48 h at a dose of 0.5 μg (Figure 1D), accompanied by an increase in different cell death indicators, such as Puma, Noxa and Fas, cleaved PARP, cleaved Caspase-3 (Figure 1C) and increased caspase 3/7 activity (Figure 1E) and Annexin staining (Figure 1F) compared to untreated controls. Loss of colony formation ability was induced by increasing concentrations of p53-mRNA (Figure 1G). Cytotoxicity assays were also performed, confirming the effects of IVT P53 mRNA on ovarian cancer cells observed previously using apoptosis and colony formation assays (Figure 1H). These results indicate that rescue of p53 function by transfection of WT p53-mRNA induces cell death in different HGSOC cell lines.

Figure 1. Liposomal transfection of in vitro-transcribed WT p53-mRNA induces cell cycle arrest and apoptosis in the HGSOC cell line OVCAR-8. (Raab M, et al., 2024)