detection-aggregation-prone-behavior-mutant-p53-v157f-breast-cancer

Detection of Aggregation-Prone Behavior in Mutant P53 V157F Breast Cancer Cells Using Multipoint Thioflavin T Fluorescence

The tumor suppressor p53, encoded by the TP53 gene, plays a central role in maintaining genomic stability. TP53 mutations, particularly hotspot variants, ...

I-Shou University

(1) Background: A premature termination codon (PTC) can be induced by a type of point mutation known as a nonsense mutation, which occurs within the coding region. Approximately 3.8% of human cancer patients have nonsense mutations of p53. However, the non-aminoglycoside drug PTC124 has shown potential to promote PTC readthrough and rescue full-length proteins. The COSMIC database contains 201 types of p53 nonsense mutations in cancers. We built a simple and affordable method to create different nonsense mutation clones of p53 for the study of the PTC readthrough activity of PTC124. (2) Methods: A modified inverse PCR-based site-directed mutagenesis method was used to clone the four nonsense mutations of p53, including W91X, S94X, R306X, and R342X. Each clone was transfected into p53 null H1299 cells and then treated with 50 μM of PTC124. (3) Results: PTC124 induced p53 re-expression in H1299-R306X and H1299-R342X clones but not in H1299-W91X and H1299-S94X clones. (4) Conclusions: Our data showed that PTC124 more effectively rescued the C-terminal of p53 nonsense mutations than the N-terminal of p53 nonsense mutations. We introduced a fast and low-cost site-directed mutagenesis method to clone the different nonsense mutations of p53 for drug screening.

A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon Readthrough Activity of PTC124.

Head and neck squamous cell carcinoma (HNSCC) cells have a high p53 mutation rate, but there were rare reported about the p53 gain of function through the prion-like aggregated form in p53 mutated HNSCC cells. Thioflavin T (ThT) is used to stain prion-like proteins in cells. Previously, we found that ThT and p53 staining were co-localized in HNSCC cells (Detroit 562 cells) with homozygous p53 R175H mutation. NAMPT inhibitor can repress ThT staining in Detroit 562 cells. In our previous study, co-treatment with p73 activator NSC59984 and NAMPT inhibitor FK886 synergistically repressed Detroit 562 cell proliferation. In this study, we found that two heterozygous p53-R280T mutation HNSCC cell lines, TW01 and HONE-1, also have the ThT staining signal. Treatment with chlorophyllides and p73 activator or NAMPT inhibitor did not synergistically repress cell proliferation in either Detroit 562 or HONE-1 cells. Chlorophyllides reduced the ThT aggregation signal in both Detroit 562 and HONE-1 cells. Chlorophyllides also induced p73 and caspase 3/7 expression and repressed NAMPT expression in both Detroit 562 and HONE-1 cells. Chlorophyllides reduced tumor size in vivo in Detroit 562 cells injected into a xenograft nude mice model, but this in vivo tumor repression effect was not found in p73 knockdown Detroit 562 cells. Moreover, NAMPT was repressed by chlorophyllides independent of p73 status in vivo. We thus concluded that chlorophyllides have a dual anticancer function when applied to HNSCC cells with p53 gain-of-function mutation, via activation of p73 and repression of p53 aggregation.

Chlorophyllides repress gain-of-function p53 mutated HNSCC cell proliferation via activation of p73 and repression of p53 aggregation in vitro and in vivo.

: The mutation rate of p53 in breast cancer is around 20%. Specific p53 mutations exhibit prion-like abnormal misfolding and aggregation and gain oncogenic function, causing resistance to the chemotherapy drug doxorubicin. In this study, we identified key upstream regulatory molecules that inhibit the aggregation of p53 with the aim of increasing the anticancer effect of doxorubicin. : Thioflavin T was employed as a fluorescent probe to detect prion-like protein aggregates within cells, the response to various inhibitors was evaluated using CCK8 assay, and the coefficient of drug interaction was calculated. The cell apoptosis ratio was evaluated using Caspase-3/7 based flow cytometry assay. : MDA-MB-231 cells (with p53 R280K mutation) and T47D cells (with p53 L194F mutation) had a strong Thioflavin T staining signal, but MDA-MB-468 cells (with p53 R273H mutation) had a weak Thioflavin T signal. Compared to MDA-MB-468 cells, which had a good response to doxorubicin, both MDA-MB-231 and T47D showed high doxorubicin drug resistance. Co-treatment with various misfolding p53 aggregation inhibitors and doxorubicin found that only the HSP70 inhibitor and doxorubicin had synergistic anticancer activity in both MDA-MB-231 and T47D cells. Furthermore, this co-treatment induced cell apoptosis in MDA-MB-231, which was reversed by a pan-caspase inhibitor. : Doxorubicin resistance caused by specific p53 mutants can be resolved by co-treatment with a HSP70 inhibitor in breast cancer cells.

Article	Year
A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon Readthrough Activity of PTC124. Biomedicines\| PubMed ID: 37238980	2023
Chlorophyllides repress gain-of-function p53 mutated HNSCC cell proliferation via activation of p73 and repression of p53 aggregation in vitro and in vivo. Biochimica et biophysica acta. Molecular basis of disease\| PubMed ID: 39788216	2025
Synergistic Anticancer Activity of HSP70 Inhibitor and Doxorubicin in Gain-of-Function Mutated p53 Breast Cancer Cells. Biomedicines\| PubMed ID: 40426862	2025

Kuan-Yo Wu

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