A comparison of four technologies for detecting p53 aggregates in ovarian cancer

Abstract

The tumor suppressor protein p53 is mutated in half of all cancers and has been described to form amyloid-like structures, commonly known from key proteins in neurodegenerative diseases. Still, the clinical relevance of p53 aggregates remains largely unknown, which may be due to the lack of sensitive and specific detection methods. The aim of the present study was to compare the suitability of four different methodologies to specifically detect p53 aggregates: co-immunofluorescence (co-IF), proximity ligation assay (PLA), co-immunoprecipitation (co-IP), and the p53-Seprion-ELISA in cancer cell lines and epithelial ovarian cancer tissue samples. In 7 out of 10 (70%) cell lines, all applied techniques showed concordance. For the analysis of the tissue samples co-IF, co-IP, and p53-Seprion-ELISA were compared, resulting in 100% concordance in 23 out of 30 (76.7%) tissue samples. However, Co-IF lacked specificity as there were samples, which did not show p53 staining but abundant staining of amyloid proteins, highlighting that this method demonstrates that proteins share the same subcellular space, but does not specifically detect p53 aggregates. Overall, the PLA and the p53-Seprion-ELISA are the only two methods that allow the quantitative measurement of p53 aggregates. On the one hand, the PLA represents the ideal method for p53 aggregate detection in FFPE tissue, which is the gold-standard preservation method of clinical samples. On the other hand, when fresh-frozen tissue is available the p53-Seprion-ELISA should be preferred because of the shorter turnaround time and the possibility for high-throughput analysis. These methods may add to the understanding of amyloid-like p53 in cancer and could help stratify patients in future clinical trials targeting p53 aggregation.

Oncogenic Mutant p53 Sensitizes Non-Small Cell Lung Cancer Cells to Proteasome Inhibition via Oxidative Stress-Dependent Induction of Mitochondrial Apoptosis

“…Since Onc-p53 has a high tendency to aggregate in cells (38,39), it has been hypothesized that proteotoxic stress from Onc-p53 aggregates may induce proteasome activity to maintain cellular homeostasis and manage the proteotoxic load (40). Indeed, the p53 R273H allele expressed in H1975 cells has been observed to form intracellular aggregates in breast and ovarian cancer cells using an antibody sensitive to high molecular weight protein aggregates, though the aggregation status of p53 R267P (H1437 cells) has not yet been reported (39,41). Though we did not explore 26S proteasome subunit transcription in this paper, our data indicates that Onc-p53 causes basal proteotoxic stress as indicated by elevated basal proteasome activities in Onc-p53 NSCLC cells, and this stress can be linked through basal ROS generation (13) to activation of NRF2-dependent transcriptional programs, including NRF2-dependent proteasome subunit transactivation, suggesting a positive feedback loop may sustain proteasome gene expression in Onc-p53 NSCLC cells (9).…”

Inhibition of MARK2 inhibits ovarian cancer cell proliferation by regulating PI3K/AKT/p53 axis

“…Inhibitors developed against this signaling pathway, such as LY294002, have been shown to inhibit OC progression and serve as potential therapeutics [19,20]. In addition, the positive rate of p53 in OC tissues is significantly higher than normal tissue and is positively correlated with histological grade, suggesting that abnormal expression of p53 plays an important role in the proliferation and development of tumor cells [21]. Studies on gastrointestinal tumors have found that tumors with high p53 mutation rate develop rapidly and are prone to metastasize [21].…”

“…In addition, the positive rate of p53 in OC tissues is significantly higher than normal tissue and is positively correlated with histological grade, suggesting that abnormal expression of p53 plays an important role in the proliferation and development of tumor cells [21]. Studies on gastrointestinal tumors have found that tumors with high p53 mutation rate develop rapidly and are prone to metastasize [21]. Therefore, both the PI3K and p53 pathway could affect the OC progression.…”