mutant p53 proteins  (Eppendorf AG)

Journal: NPJ breast cancer

Article Title: Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants.

doi: 10.1038/s41523-023-00582-7

Figure Lengend Snippet: Fig. 1 Effect of p53 missense mutations on hallmark phenotypes of cancer. Phenotypes were measured in three independent experiments with replicates, and the results were normalized to the log2 fold change over the phenotype of p53 WTOE cells. The error bars represent the standard error of mean (SEM) values, and significant differences (two-sided single-sample t-test) from the WTOE mean are indicated by red asterisks for indicated p values. In all plots, cells were sorted by each phenotype, from less (left) to more (right) aggressive. a Survivability of the cell line panel in absence of growth factors (serum and EGF) were measured by the CellTiter-glo assay. b Cell apoptosis was detected by the level of activity of caspase 3 and caspase 7, and the reciprocal of luminescence were used to measure the level of resistance to apoptosis. c Migration was assessed using the transwell assay. d Cell invasion was assessed using the transwell assay with Matrigel-coated chamber membranes. e To measure anoikis, the death index (ratio of dead cells and live cells) was calculated for each cell line, and the reciprocal values were used to measure the level of resistance to anoikis.

Article Snippet: METHODS Production of MCF10A cell lines expressing p53 mutant proteins The MCF10A cells (ATCC, CRL-10317) expressing WT and missense mutant p53 proteins were made using the Gateway plasmids generated by plasmid repository DNASU (dnasu.org).

Techniques: Glo Assay, Activity Assay, Migration, Transwell Assay

Journal: NPJ breast cancer

Article Title: Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants.

doi: 10.1038/s41523-023-00582-7

Figure Lengend Snippet: Fig. 2 Morphology of the MCF10A p53 mutant-induced mammospheres. a Low-magnification image (scale bar: 100 μm) of the mammospheres formed by p53 missense mutants in Matrigel by on-top method stained for nucleus (DAPI, blue), laminin (green), and β-catenin (red). The cell lines were ordered by the degree of polarization shown below in (b) which shows the hollowness and laminin staining of the mammospheres. The nuclear DAPI intensity was measured in 20 concentric bins from the center of the mammospheres (left panel). The right panel shows the laminin staining intensity profiles across the bins, where laminin signal intensity in the inner bins indicate disruption of cell polarity. The intensity value data was normalized to the total number of pixels for each spheroid.

Article Snippet: METHODS Production of MCF10A cell lines expressing p53 mutant proteins The MCF10A cells (ATCC, CRL-10317) expressing WT and missense mutant p53 proteins were made using the Gateway plasmids generated by plasmid repository DNASU (dnasu.org).

Techniques: Mutagenesis, Staining, Disruption

Journal: NPJ breast cancer

Article Title: Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants.

doi: 10.1038/s41523-023-00582-7

Figure Lengend Snippet: Fig. 3 Cellular phenotypic heterogeneity of MCF10A cell lines expressing 10 different mutant p53 proteins. a The phenotype measurements for each of the p53 mutant-expressing cells were normalized (as log2-transformed mean fold changes) over those of the control p53 WTOE cells and displayed as a heat map. The cell lines were sorted by the mean of the normalized values for all 6 phenotypes. The p53 mutants are color-coded by mutant type as indicated. The detailed results are in Supplementary Table 1. b Five-year survival rates of basal-like breast cancer patients with different missense TP53 mutations were obtained from TCGA and METABRIC datasets and visualized by the Kaplan-Meier plot. Only the mutations with 3 or more corresponding samples were used. The mutations were color-coded by the phenotypic aggressiveness (red: more aggressive, blue: less aggressive), and the WT TP53 curve is shown in black. c Correlation between the overall aggressiveness and the 5-year survival rates for different p53 mutations is shown with the Pearson’s correlation coefficient (R) and the p value. The blue bands represent the 95% confidence intervals.

Article Snippet: METHODS Production of MCF10A cell lines expressing p53 mutant proteins The MCF10A cells (ATCC, CRL-10317) expressing WT and missense mutant p53 proteins were made using the Gateway plasmids generated by plasmid repository DNASU (dnasu.org).

Techniques: Expressing, Mutagenesis, Transformation Assay, Control

Journal: NPJ breast cancer

Article Title: Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants.

doi: 10.1038/s41523-023-00582-7

Figure Lengend Snippet: Fig. 5 The Hippo/YAP/TAZ pathway is strongly associated with invasiveness of cells expressing different p53 mutants. a Biplots for a PLSR model trained on ssGSEA scores of the entire pathway terms in WikiPathways database and the vector of invasiveness is shown. The model was based on top 5 pathway terms that were identified by cross-validated forward feature selection. Cell lines (colored by invasiveness) were transformed and projected on a 2-component space, and the explained variance of invasiveness by each component is shown in the axis labels. The loadings of pathway terms were scaled to fit the data range and displayed as green lines. b Biplot for a PLSR model trained on expression values of genes in the Hippo-YAP Signaling Pathway (WikiPathways) and the vector of invasiveness is shown. Analysis performed as described for Fig. 5a. c In the WikiPathways diagrams for the Hippo/YAP/TAZ pathways, individual genes were color-coded by the Pearson correlations between gene expression levels and invasiveness across the 13 cell lines. d Transcriptional activity of TEAD proteins was measured in the 13-cell line panel in triplicates by a cell-based luciferase reporter assay. The luminescence values were normalized to the log2 fold changes over the WTOE values. The difference between a group of two more invasive cells (R273C and Y220C) and four less invasive cells (Y234C, WT, H179R, and G245S) was tested by the two-sided Student’s t-test. Pearson’s correlation between the normalized TEAD activities and the invasiveness across the cell lines was also calculated (bottom box).

Article Snippet: METHODS Production of MCF10A cell lines expressing p53 mutant proteins The MCF10A cells (ATCC, CRL-10317) expressing WT and missense mutant p53 proteins were made using the Gateway plasmids generated by plasmid repository DNASU (dnasu.org).

Techniques: Expressing, Plasmid Preparation, Selection, Transformation Assay, Gene Expression, Activity Assay, Luciferase, Reporter Assay

Journal: NPJ breast cancer

Article Title: Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants.

doi: 10.1038/s41523-023-00582-7

Figure Lengend Snippet: Fig. 8 Overview of heterogeneous functional impact of different p53 missense mutations on cancer hallmark phenotypes. The normalized phenotypic scores (as log2 fold changes over controls WTOE cells as shown in the legend) for each mutant p53 protein-expressing cells are shown as bar plots. The cell lines were positioned by their relative phenotypic profiles calculated by PCA, while the positions of phenotype names are approximately centered among the cells with more aggressive behavior.

Article Snippet: METHODS Production of MCF10A cell lines expressing p53 mutant proteins The MCF10A cells (ATCC, CRL-10317) expressing WT and missense mutant p53 proteins were made using the Gateway plasmids generated by plasmid repository DNASU (dnasu.org).

Techniques: Functional Assay, Mutagenesis, Expressing

Journal: Oncogene

Article Title: Spontaneous and inherited TP53 genetic alterations

doi: 10.1038/s41388-021-01991-3

Figure Lengend Snippet: Li Fraumeni patients inherit a mutant TP53 allele that acts at an early age of onset (six months to 20 years) indicating that the mutant p53 protein functions as an initial or truncal mutation promoting benign cell replication and clonal expansion and producing a high excess risk for cancer. TP53 mutations that give rise to cancers later in life more likely function as a later mutation in an ordered series of mutant genes that drive cancerous growth. By 50–70 years of life, cancers arise with low excess risk and the mutant p53 protein acts in the benign to malignant transformation as the last gene in the ordered series of cancer forming mutations [ , , ].

Article Snippet: AJL is a founder, board member and shareholder in PMV Pharmaceuticals that produce small molecule reactivators of p53 mutant proteins.

Techniques: Mutagenesis, Transformation Assay

Journal: Oncogene

Article Title: Spontaneous and inherited TP53 genetic alterations

doi: 10.1038/s41388-021-01991-3

Figure Lengend Snippet: The XAF-1 protein binds within the proline-rich domain (see Fig. 2) and the great majority of missense mutations that contribute to cancers reside in the DNA binding domain. The eight spontaneous mutations and two environmental mutations form the ten most common TP53 mutations (33% of cancers), which have very little or no transcriptional activity. More than 350 additional TP53 missense mutations have a weak transcriptional activity, a much lower frequency of occurrence in producing cancers over a four log distribution, and cause 67% of cancers. The hypothesis that pro-apoptotic XAF-1 binding to a weak transcriptional p53 protein promotes apoptosis so that the weak allele fails to form cancer. An XAF-1 gene transcriptionally silenced by epigenetic marks or a polymorphism that inactivates the protein would then permit weak, minor TP53 alleles to produce cancer .

Article Snippet: AJL is a founder, board member and shareholder in PMV Pharmaceuticals that produce small molecule reactivators of p53 mutant proteins.

Techniques: Binding Assay, Activity Assay

Journal: Oncogene

Article Title: Spontaneous and inherited TP53 genetic alterations

doi: 10.1038/s41388-021-01991-3

Figure Lengend Snippet: The p53 and MDM-2 proteins form a module in the cell where p53 transcribes the MDM-2 gene and the MDM-2 protein promotes the degradation of the p53 protein. Stress signals, in this case, DNA damage is sensed by the ATM protein which phosphorylates a CHEK-2 mediator which in turn phosphorylate serine -15 in the p53 protein, and other sites in p53 and MDM-2, that disrupts the MDM-2-P53 protein complex. This increases p53 protein levels and along with other epigenetic modifications of p53 and MDM-2 the p53 transcription factor makes an informed choice between cell cycle arrest, DNA repair, and return to homeostasis or programed cell death by one of five possible methods .

Article Snippet: AJL is a founder, board member and shareholder in PMV Pharmaceuticals that produce small molecule reactivators of p53 mutant proteins.

Techniques:

Journal: Oncogene

Article Title: Spontaneous and inherited TP53 genetic alterations

doi: 10.1038/s41388-021-01991-3

Figure Lengend Snippet: 1. The XAF-1 protein binds to the p53 protein in the proline domain and dissociates MDM-2 from p53, stabilizing p53. 2. The XAF-1 protein binds to the ubiquitin ligase SIAH-2, which inhibits its functions, increasing the levels and activity of HIPK-2, which in turn phosphorylates p53-serine 46, which promotes the transcription of a pro-apoptotic p53 pathway. 3. The XAF-1 protein also binds to ZNF-313, a ubiquitin ligase that promotes the degradation of the p21 protein (cell cycle arrest). This is also is a pro-apoptotic activity .

Article Snippet: AJL is a founder, board member and shareholder in PMV Pharmaceuticals that produce small molecule reactivators of p53 mutant proteins.

Techniques: Ubiquitin Proteomics, Activity Assay