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5-Fluorouridine restores functional full-length p53 in TP53 nonsense mutant human tumor cells (RNA-Seq)

ABSTRACT: The tumor suppressor gene TP53 is mutated in approximately half of all human tumors. Of those, around 10% are nonsense mutations that produce truncated and inactive p53 protein. Induction of translational readthrough is a promising approach for rescuing full-length p53 and thereby eliminate tumor cells with nonsense mutant TP53. To find novel nonsense mutant TP53 readthrough-inducing compounds with a tolerable toxicity profile, we performed an in silico screening of data at the National Cancer Institute database and identified 5-Fluorouracil (5-FU). We show here that 5-FU induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53 and that this activity is mediated by its metabolite 5-Fluorouridine (FUr). Ribo-seq analysis validated induction of translational readthrough by FUr. We also show that FUr is incorporated into RNA where it potentially allows base pairing of Arg tRNA at the R213X UGA premature termination codon. Full-length p53 rescued by FUr is transcriptionally active and triggers p53-dependent cell death. Moreover, treatment with 5-FU or FUr restores full-length p53 expression in TP53 R213X mutant human tumor xenografts in vivo. Our results suggest that induction of readthrough by 5-FU/FUr could contribute to therapeutic efficacy in patients with TP53 nonsense mutant tumors.

ORGANISM(S): Homo sapiens

PROVIDER: GSE196710 | GEO | 2022/12/01

REPOSITORIES: GEO

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5-Fluorouridine restores functional full-length p53 in TP53 nonsense mutant human tumor cells (Ribo-Seq)

Project description:The tumor suppressor gene TP53 is mutated in approximately half of all human tumors. Of those, around 10% are nonsense mutations that produce truncated and inactive p53 protein. Induction of translational readthrough is a promising approach for rescuing full-length p53 and thereby eliminate tumor cells with nonsense mutant TP53. To find novel nonsense mutant TP53 readthrough-inducing compounds with a tolerable toxicity profile, we performed an in silico screening of data at the National Cancer Institute database and identified 5-Fluorouracil (5-FU). We show here that 5-FU induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53 and that this activity is mediated by its metabolite 5-Fluorouridine (FUr). Ribo-seq analysis validated induction of translational readthrough by FUr. We also show that FUr is incorporated into RNA where it potentially allows base pairing of Arg tRNA at the R213X UGA premature termination codon. Full-length p53 rescued by FUr is transcriptionally active and triggers p53-dependent cell death. Moreover, treatment with 5-FU or FUr restores full-length p53 expression in TP53 R213X mutant human tumor xenografts in vivo. Our results suggest that induction of readthrough by 5-FU/FUr could contribute to therapeutic efficacy in patients with TP53 nonsense mutant tumors.

2022-12-01 | GSE196709 | GEO

p53 influences 5-FU-induced chromatin accessibility [ATAC-seq]

Project description:5-Fluorouracil (5-FU) is one of the most common drugs used in chemotherapy because of its efficacy and stability. However, 5-FU has been known to work on only 10~15% of colon cancer patients. Therefore, the study of 5-FU sensitivity study is necessary to increase the survival of colon cancer patients. p53 is one of the factors that effect on drug sensitivity. Main function of p53 has been focused on transcription activity in cell death. Numerous drug related studies show that expression of wild-type p53 increases drug sensitivity in various cancer types through inducing apoptotic signaling pathways. Currently, it has been reported that p53 has both transcriptional and non-transcriptional activities in apoptosis. However, most studies about p53 non-transcriptional activities in apoptosis are mainly focused on p53 induced mitochondrial outer membrane permeabilization, triggering the release of pro-apoptotic factors. The chromatin structure and organization have strongly attracted because of their impacts in cellular phenotypes. Recent studies have been shown that changes in chromatin accessibility affect cell phenotypes for external stimuli. Since p53 plays an important role in drug sensitivity and 5-FU is an external stimulus for cancer cells, we hypothesized that p53 may effect on 5-FU sensitivity through different regulation of chromatin accessible regions between TP53-WT and TP53-KO cells. To determine the effect of p53 on chromatin accessibility modification associated with 5-FU sensitivity, ATAC-seq and RNA-seq were performed under 5-FU treatment on TP53-WT as drug sensitive and TP53-KO as drug resistant HCT116 cells. In this study, we found that 5-FU induces chromatin accessibility in both TP53-WT and TP53-KO cells. Moreover, our results show that 5-FU induced chromatin accessibility increases expression of apoptotic genes in TP53-WT HCT116 cells through p53 non-transcriptional activity in nucleus.

2021-02-10 | GSE157949 | GEO

p53 influences 5-FU-induced chromatin accessibility [ChIP-seq]

2021-02-10 | GSE157950 | GEO

Gene expression analysis of U251 human glioblastoma cell line

2012-12-10 | GSE35454 | GEO

Gene expression analysis of U251 human glioblastoma cell line

Project description:The human TP53 gene is frequently mutated in tumors and cell lines. Unlike other tumor suppressors that are commonly inactivated by deletions or nonsense mutations, the majority of p53-mutations are missense point mutations that result in the expression of a full-length protein with an altered amino acid that has lost sequence specific DNA-binding. Expression of mutant p53 (mutp53) confers advantages to tumor cells and transcriptional regulation of several genes mediating the beneficial effects has been shown to play a role. However, molecular mechanisms of transcriptional regulation by mutp53 are still poorly understood. We used the glioblastoma-derived U-251 MG human cell line endogenously expressing mutp53 protein (R273H mutation) to analyze gene expression profiles on Agilent Whole Human Genome Microarray after transient and stable depletion of mutp53 expression. Gene expression data was correlated with a ChIP study on a custom tiling array to understand the contribution of endogenously expressed mutp53 to transcriptional regulation. This series of microarray experiments contains the gene expression profiles of glioblastoma-derived U-251 MG human cell lines engineered to constitutively express a p53-specific shRNA or scrambled control shRNA. To reverse the effect of mutp53 depletion, stable clones were modified by stable integration of a mutp53-R273H expression construct or empty pCDNA3 vector as a control. In addition, we performed gene expression analysis of U-251 MG cells transiently transfected with p53-specific siRNA or control siRNA (3 biological replicates each).

2012-12-10 | E-GEOD-35454 | biostudies-arrayexpress

Mutant p53 interactome study with IP-MS

Project description:Inactivating TP53 mutations lead to a loss of function of p53, but can also often result in oncogenic gain-of-function (GOF) of mutant p53 (mutp53) proteins which promote tumor development and progression. The GOF activities of TP53 mutations are well documented, but the mechanisms involved remain poorly understood. Here, we study the mutp53 interactome with IP-MS.

2023-11-27 | PXD047094 | Pride

26S proteasome is conserved, therapeutically relevant mediator of mutant p53 gain-of-function in cancer

Project description:Mutant p53 proteins, resulting form frequent TP53 tumor suppressor missense mutations, possess gain-of-function activities and are among the most widespread and robust oncoproteins in human tumors. They are potentially important but understudied therapeutic targets. No studies to date have distinguished common, therapeutically relevant mutant p53 gain-of-function effects, from effects specific to different mutant variants and cell backgrounds. Here we identify 26S proteasome machinery as the common downstream effector controlled by mutant p53s in Triple Negative Breast Cancer (TNBC - aggressive carcinomas with TP53 as the most frequently mutated locus) and conserved in other human cancers. We have identified this pathway using a combination of single-model, multi-method vertical analysis (whole cell proteome, RNA sequencing an ChIP sequencing) and multi-cell line, horizontal analysis of transcriptiomes. We found that different missense mutant p53s regardless of the cell background transcriptionaly activate whole 26S proteasome machinery. Proteasome activity is significantly increased in p53 mutant versus wild-type or knockdown/null status - in cellular and mouse models as well as in human breast tumors. Increased proteasome activity leads to inhibition of tumor suppressive pathways. The control of mutant p53 over proteasome transcription and activity results in the increased resistance to proteasome inhibitors. By combining the mutant p53 targeting agents and proteasome inhibitor we were able to overcome the “bounce-back” proteasome inhibitor resistance mechanism in mutant p53 bearing TNBC cells and xenografts in vivo.

2016-05-03 | PXD001673 | Pride

Use of NanoLuc-Based Reporters that Monitor Nonsense Suppression and Inhibition of Nonsense-Mediated mRNA Decay to Identify Potent New Agents That Partially Reverse the Effects of Nonsense Mutations

Project description:Premature termination codons (PTCs) lead to loss of protein function by 1) ending mRNA translation before a full-length protein is made, and 2) by triggering nonsense-mediated mRNA decay (NMD), a pathway that targets a PTC-containing mRNA for degradation. Nonsense suppression therapy aims to restore protein function by suppressing termination at PTCs (called readthrough, or RT). However, the efficiency of current readthrough agents is generally quite low. We reasoned that by stimulating both readthrough and increasing mRNA levels, greater protein function can be restored than with readthrough alone. In this study, we developed a series of novel NanoLuc reporters designed to identify and differentiate compounds that induce readthrough, enhance mRNA abundance (by NMD inhibition or other mechanisms), or target both mechanisms simultaneously (referred to as dual RT/NMD reporters). We show that treatments that promote readthrough and NMD inhibition led to synergistic increases in NanoLuc activity of the RT/NMD reporter over either condition alone. We then used a dual RT/NMD reporter to carry out a high throughput screen of a library of 771,345 compounds. We identified 180 compounds with the ability to increase NanoLuc activity in our dual RT/NMD reporter assay. To confirm that such synergy can also amplify protein function in a disease model, we treated cells expressing a CFTR-Y122X mini-intron construct with one hit from this screen, SRI-37240. We found that this compound produced a much larger increase in CFTR activity compared to either the RT compound G418 +/- the NMD inhibitor amlexanox, and the activity obtained could be further enhanced with CFTR modulators. These results confirm the utility of these new reporters to identify many potent new molecules that counteract the effects of nonsense mutations.

2020-12-01 | GSE144140 | GEO

Fur mutant analysis

Project description:Wildtype G27 and it's Fur mutant derivative were exposed to pH 5.0 for the indicated length of time. Bacterial cells in the exponential phase of growth (denoted log) and stationary phase of growth were used. fur1 denotes a single fur mutant while fur2 denotes an additional fur mutant. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: all_pairs

2006-02-03 | GSE4160 | GEO

Transcription profiling of H. pylori wild type G27 and Fur mutant derivative exposed to pH 5.0

2006-06-06 | E-SMDB-3498 | biostudies-arrayexpress

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