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

 is involved in the synthesis of embryonic stem (ES) cell marker glycan, stage-specific embryonic antigen-3 (SSEA3). This gene has three native promoters and an integrated retroviral long terminal repeat (LTR) promoter. We found that -LTR is expressed at high levels in human ES cells. -LTR is also involved in the synthesis of the cancer-associated glycan, sialyl Lewis a. Sialyl Lewis a is expressed in ES cells and its expression decreases upon differentiation. Retinoic acid induced differentiation of ES cells, decreased the short form of NFYA (NFYAs), increased phosphorylation of STAT3, and decreased B3GALT5-LTR expression. NFYAs activated, and constitutively-active STAT3 (STAT3C) repressed B3GALT5-LTR promoter. The NFYAs and STAT3C effects were eliminated when their binding sites were deleted. Retinoic acid decreased the binding of NFYA to B3GALT5-LTR promoter and increased phospho-STAT3 binding. Lamin A repressed NFYAs and SSEA3 expression. SSEA3 repression mediated by a SIRT1 inhibitor was reversed by a STAT3 inhibitor. Repression of SSEA3 and sialyl Lewis a synthesis mediated by retinoic acid was partially reversed by lamin A short interfering RNA (siRNA) and a STAT3 inhibitor. In conclusion, -LTR is regulated by lamin A-NFYA and SIRT1-STAT3 signaling that regulates SSEA3 and sialyl Lewis a synthesis in ES cells, and sialyl Lewis a is also a ES cell marker.

SSEA3 and Sialyl Lewis a Glycan Expression Is Controlled by B3GALT5 LTR through Lamin A-NFYA and SIRT1-STAT3 Signaling in Human ES Cells.

Human Toll-like receptor (TLR) signaling plays a vital role in intestinal inflammation by activating the NF-B pathway. By querying GENT2 datasets, we identified the gene expression level of TLR2 and TLR4 as being substantially increased in colorectal cancer. Introduction of shRNAs for TLR4 but not TLR2 dramatically recovered disialyl Lewis and sialyl 6-sulfo Lewis glycans, which are preferentially expressed in non-malignant colonic epithelial cells and could serve as ligands for the immunosuppressive molecule Siglec-7. We screened several TLR4 ligands and found that among them BGN is highly expressed in cancers and is involved in the epigenetic silencing of Siglec-7 ligands. Suppression of BGN expression substantially downregulated NF-B activity and the marker H3K27me3 in the promoter regions of the SLC26A2 and ST6GalNAc6 genes, which are involved in the synthesis of those glycans, and restored expression of normal glycans as well as Siglec-7 binding activities. We show that in the presence of TLR4, inflammatory stimuli initiate a positive loop involving NF-B that activates BGN and further enhances TLR4 activity. Present findings indicate a putative mechanism for the promotion of carcinogenesis by loss of immunosuppressive ligands by the BGN/TLR4/ NF-B pathway.

BGN/TLR4/NF-B Mediates Epigenetic Silencing of Immunosuppressive Siglec Ligands in Colon Cancer Cells.

The p53 family has the following three members: p53, p63 and p73. p53 is a tumor suppressor gene that frequently exhibits mutation in head and neck cancer. Most p53 mutants are loss-of-function (LoF) mutants, but some acquire some oncogenic function, such as gain of function (GoF). It is known that the aggregation of mutant p53 can induce p53 GoF. The p73 activators RETRA and NSC59984 have an anti-cancer effect in p53 mutation cells, but we found that p73 activators were not effective in all head and neck squamous cell carcinoma (HNSCC) cell lines, with different p53 mutants. A comparison of the gene expression profiles of several regulator(s) in mutant HNSCC cells with or without aggregation of p53 revealed that nicotinamide phosphoribosyltransferase (NAMPT) is a key regulator of mutant p53 aggregation. An NAMPT inhibitor, to reduce abnormal aggregation of mutant p53, used in combination with a p73 activator, was able to effectively repress growth in HNSCC cells with p53 GoF mutants. This study, therefore, suggests a potential combination therapy approach for HNSCC with a p53 GoF mutation.

NAMPT Inhibitor and P73 Activator Represses P53 R175H Mutated HNSCC Cell Proliferation in a Synergistic Manner.

The members of the p53 family comprise p53, p63, and p73, and full-length isoforms of the p53 family have a tumor suppressor function. However, p53, but not p63 or p73, has a high mutation rate in cancers causing it to lose its tumor suppressor function. The top and second-most prevalent p53 mutations are missense and nonsense mutations, respectively. In this review, we discuss possible drug therapies for nonsense mutation and a missense mutation in p53. p63 and p73 activators may be able to replace mutant p53 and act as anti-cancer drugs. Herein, these p63 and p73 activators are summarized and how to improve these activator responses, particularly focusing on p53 gain-of-function mutants, is discussed.

P63 and P73 Activation in Cancers with p53 Mutation.

(1) Background: PTC124 (Ataluren) is an investigational drug for the treatment of nonsense mutation-mediated genetic diseases. With the exception of the  tumor suppressor gene, there has been little research on cancers with nonsense mutation. By conducting a database search, we found that another two tumor suppressor genes,  and , have a high nonsense mutation rate in head and neck squamous cell carcinoma (HNSCC). PTC124 may re-express the functional  or  in nonsense mutation  or  in HSNCC (2) Methods: DOK (with  Y550X) or HO-1-u-1 (with  E378X) HNSCC cells were treated with PTC124, and the  or  expression, cell viability, and - or -related downstream gene profiles were assayed. (3) Results: PTC124 was able to induce  or  expression in DOK and HO-1-u-1 cells. PTC124 was able to upregulate  downstream genes , , and  in DOK cells. PTC124 enhanced DDIT4, which is under the control of the -YAP1 pathway, in HO-1-u-1 cells. FLI-06 (a NOTCH signaling inhibitor) reversed PTC124-mediated cell growth inhibition in DOK cells. PTC124 could reverse TT-10 (a YAP signaling activator)-mediated HO-1-u-1 cell proliferation. (4) Conclusions: PTC124 can rescue nonsense mutation of  and  to repress HNSCC cell proliferation.

PTC124 Rescues Nonsense Mutation of Two Tumor Suppressor Genes  and  to Repress HNSCC Cell Proliferation.

The protein p53 is a well-known tumor suppressor that plays a crucial role in preventing cancer development [...].

The Competition of Yin and Yang: Exploring the Role of Wild-Type and Mutant p53 in Tumor Progression.

(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 prognostic impact of internal carotid artery (ICA) invasion in nasopharyngeal cancer (NPC) patients is not well established. Thus, we conducted a retrospective study to analyze the prognostic factors for ICA invasion by NPC.

Analysis of Prognostic Factors for Internal Carotid Artery Invasion by Nasopharyngeal Carcinoma.

: 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.

Synergistic Anticancer Activity of HSP70 Inhibitor and Doxorubicin in Gain-of-Function Mutated p53 Breast Cancer Cells.

This Opinion summarizes the strategies for anticancer treatment in p53-mutated head and neck squamous cell carcinoma (HNSCC). It examines six strategies for anticancer treatment in p53-mutated HNSCC: 1. direct reactivation of mutated p53; 2. activation of p63; 3. activation of p73; 4. degradation of mutated p53; 5. blocking the p53-regulated oncogenic microRNA; and 6. blocking the p53-regulated oncogenic long non-coding RNA. Since HNSCC has a high p53 mutation rate compared to other types of cancers, these strategies for combating p53-mutated HNSCC may prove useful for generating new ideas or methods for developing treatments for other cancers with p53 mutations. This article also explores other factors that may impact the effectiveness of anticancer therapies in p53-mutated HNSCC.

Article	Year
SSEA3 and Sialyl Lewis a Glycan Expression Is Controlled by B3GALT5 LTR through Lamin A-NFYA and SIRT1-STAT3 Signaling in Human ES Cells. Cells\| PubMed ID: 31936807	2020
BGN/TLR4/NF-B Mediates Epigenetic Silencing of Immunosuppressive Siglec Ligands in Colon Cancer Cells. Cells\| PubMed ID: 32050430	2020
NAMPT Inhibitor and P73 Activator Represses P53 R175H Mutated HNSCC Cell Proliferation in a Synergistic Manner. Biomolecules\| PubMed ID: 35327630	2022
P63 and P73 Activation in Cancers with p53 Mutation. Biomedicines\| PubMed ID: 35884795	2022
PTC124 Rescues Nonsense Mutation of Two Tumor Suppressor Genes and to Repress HNSCC Cell Proliferation. Biomedicines\| PubMed ID: 36428516	2022
The Competition of Yin and Yang: Exploring the Role of Wild-Type and Mutant p53 in Tumor Progression. Biomedicines\| PubMed ID: 37189810	2023
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
Analysis of Prognostic Factors for Internal Carotid Artery Invasion by Nasopharyngeal Carcinoma. Cancers\| PubMed ID: 39941853	2025
Synergistic Anticancer Activity of HSP70 Inhibitor and Doxorubicin in Gain-of-Function Mutated p53 Breast Cancer Cells. Biomedicines\| PubMed ID: 40426862	2025
Strategies for Anticancer Treatment in p53-Mutated Head and Neck Squamous Cell Carcinoma. Biomedicines\| PubMed ID: 40426992	2025

Bi-He Cai

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