y537s erα  (Addgene inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PARP-1 as a novel target in endocrine-resistant breast cancer

doi: 10.1186/s13046-025-03441-4

Figure Lengend Snippet: PARP-1 expression correlates with poor outcomes in ERα-positive breast cancer (BC) patients and is up-regulated by estrogen in ERα-positive BC cells. Kaplan-Meier curves depicting the correlation of PARP-1 levels with breast cancer survival ( A ) and relapse-free survival ( B ) in ERα-positive BC patients in the METABRIC dataset. BC patients who died from other diseases were not included in the analyses. Box plots showing the expression levels PARP-1 in ERα-positive BC patients in the METABRIC cohort, stratified by the Nottingham Prognostic Index ( C ) and the tumor grade ( D ). The number of patients is indicated in the panels. (*) p -value < 0.001. PARP-1 expression (green signal) evaluated by immunofluorescence experiments in ERα wild type (wt) MCF7 and T47D cells ( E , G ) or ERα Y537S mutated cells ( F , H ) treated with vehicle or 10 nM 17β-estradiol (E 2 ) for 8 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, blue signal). The images represent 10 random fields from three independent experiments. Scale bar: 50 μm. The side panels represent the fold induction of mean fluorescent intensity of PARP-1 expression in E 2 respect to vehicle-treated cells, calculated on at least 10 random fields of each sample. Data represent the average of three biological replicates, error bars indicate SEM. (*) p < 0.05

Article Snippet: MCF7 ERα wild type cells and MCF7 ERα Y537S cells were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University [ ].

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PARP-1 as a novel target in endocrine-resistant breast cancer

doi: 10.1186/s13046-025-03441-4

Figure Lengend Snippet: ERα is involved in estrogen-induced expression of PARP-1. ( A , D ) PARP-1 expression evaluated by immunoblotting assays in ERα wild type (wt) or Y537S mutated MCF7 or T47D cells treated for 8 h with vehicle or 10 nM 17β-estradiol (E 2 ) alone or in combination with 1µM fulvestrant. Immunoblots of PARP-1 ( B , E ) and ERα ( C , F ) levels in ERα wt or Y537S mutated MCF7 or T47D cells transfected with siRNA or siESR1 (encoding for ERα) and treated with vehicle or 10 nM E 2 for 8 h, as indicated. Right panels show densitometric analysis of the blots normalized to β-Actin, which was used as a loading control. Recruitment of ERα and Sp1 to PARP-1 promoter by ChIP assay in ERα wt or Y537S mutated MCF7 ( G ) and T47D ( H ) cells. In control samples, nonspecific IgGs were used instead of the primary antibody. The amplified sequences were evaluated by real-time PCR. Data represent the average of three biological replicates with error bars indicating SEM. ( I ) Diagram depicting PARP-1 chromatin site assessed for ERα and Sp1 binding using chromatin immunoprecipitation (ChIP) created with Biorender.com. (*) p < 0.05; (**) p < 0.005; (***) p < 0.0005; (****) p < 0.0001

Article Snippet: MCF7 ERα wild type cells and MCF7 ERα Y537S cells were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University [ ].

Techniques: Expressing, Western Blot, Transfection, Control, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PARP-1 as a novel target in endocrine-resistant breast cancer

doi: 10.1186/s13046-025-03441-4

Figure Lengend Snippet: PARP-1 regulates the transcriptional activity of ERα in BC cells. Heatmaps of mRNA expression of main ERα target genes evaluated by real-time PCR in ERα wild type (wt) or Y537S mutated MCF7 ( A-B ) and T47D ( C-D ) cells treated for 24 h with vehicle or 10 nM E 2 alone or in combination with 1µM fulvestrant or niraparib, as indicated. mRNA expression of major ERα target genes in ERα wt ( E , I ) and Y537S mutated ( G , K ) MCF7 and T47D cells. Cells were transiently transfected for 36 h with negative control (siRNA) or siPARP-1 and treated with vehicle or 10 nM 17β-estradiol (E 2 ), as indicated. Values are normalized to human beta-2-microglobulin (B2M) endogenous control expression and shown as fold changes of mRNA expression. Efficacy of PARP-1 silencing in ERα wt ( F , J ) and Y537S mutated ( H , L ) MCF7 and T47D cells. Side panels show a densitometric analysis of the blots normalized to β Actin, which was used as a loading control. ( M-P ) ERα wt and Y537S mutated MCF7 and T47D cells were transfected with an ER luciferase reporter gene (ERE-luc) along with the internal transfection control Renilla Luciferase ( M , O ) or with Gal4 reporter gene GK1, the Gal4 fusion protein encoding the Ligand Binding Domain (LBD) of ERα (GalERα) and the internal transfection control Renilla Luciferase ( N , P ), and next were treated for 12 h with vehicle or 10 nM E 2 in the presence or absence of 1µM fulvestrant or niraparib, as indicated. The normalized luciferase activity values of cells treated with vehicle (-) were set as 1-fold induction, upon which the activity induced by treatments was calculated. Data represent the average of three biological replicates with error bars indicating SEM. (**) p < 0.005; (***) p < 0.0005; (****) p < 0.0001

Article Snippet: MCF7 ERα wild type cells and MCF7 ERα Y537S cells were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University [ ].

Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Control, Luciferase, Ligand Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PARP-1 as a novel target in endocrine-resistant breast cancer

doi: 10.1186/s13046-025-03441-4

Figure Lengend Snippet: PARP-1 influences the DNA binding between ERα and FoxA1. ( A ) Efficacy of PARP-1 silencing in ERα Y537S mutated MCF7 cells. Side panel shows a densitometric analysis of the blots normalized to β-Actin, which was used as a loading control. ( B ) ChIP-seq analysis showing ERα and FoxA1 binding in MCF7 ERα Y537S of the PGR gene. The peaks of ChIP-seq were analyzed via IGV.org platform. Analysis of ERα ( C ) or FoxA1 ( D ) DNA binding peaks in MCF7 ERα Y537S cells transfected with siRNA or siPARP-1. Data represent the average of 3 biological replicates with error bars indicating SEM. (*) p < 0.05; (***) p < 0.0005; (****) p < 0.0001. Heat maps of global evaluation enrichment around the Transcription Start Site (TSS) in ChIP of ERα ( E ) or FoxA1 ( F ) in MCF7 ERα Y537S cells transfected for 30 h with siRNA or siPARP-1. Sequencing data was analyzed via the galaxy.org platform

Article Snippet: MCF7 ERα wild type cells and MCF7 ERα Y537S cells were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University [ ].

Techniques: Binding Assay, Control, ChIP-sequencing, Transfection, Sequencing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PARP-1 as a novel target in endocrine-resistant breast cancer

doi: 10.1186/s13046-025-03441-4

Figure Lengend Snippet: Effects of PARP-1 and ERα inhibition on the progression of BC tumors expressing ERα Y537S mutation. ( A ) Representative in vivo luminescence images of mice bearing tumors expressing ERα Y537S mutated MCF7 cells at day 90 after treatment initiation. ( B ) Total photon flux of luminescence signals measured by in vivo imaging for tumors expressing ERα Y537S mutated MCF7 cells ( n = 8). ( C ) The average weight of mammary glands at the time of sacrifice ( n = 8 glands). ( D ) Representative IHC sections of PanCK staining for each treatment mice group; right box plot of PanCK % in the mammary gland determined with QuPath for Windows. Data represent the average of biological replicates with error bars indicating SEM. (*) p < 0.05; (**) p < 0.005; (****) p < 0.0001. ( E ) Volcano plot illustrating the differentially expressed genes (DEGs) in niraparib respect to vehicle-treated ERα Y537S MCF7-derived tumors. ( F ) Volcano plot showing the DEGs in lasofoxifene respect to vehicle-treated ERα Y537S MCF7-derived tumors. ( G ) Volcano plot illustrating the DEGs in niraparib + lasofoxifene respect to vehicle-treated ERα Y537S MCF7-derived tumors. Significantly down-regulated genes (log2FC ≤ -0.5 and padj < 0.05) are shown in blue, significantly up-regulated genes (log2FC ≥ 0.5 and padj < 0.05) are shown in red, non-significant genes are shown in grey (padj ≥ 0.05). ( H ) Venn diagram showing the intersection of the genes down-regulated in niraparib and lasofoxifene-treated ERα Y537S MCF7-derived tumors respect to the vehicle-treated counterpart. ( I ) Venn diagram showing the intersection of the genes commonly down-regulated by niraparib, lasofoxifene and niraparib + lasofoxifene respect to vehicle in ERα Y537S MCF7-derived tumors

Article Snippet: MCF7 ERα wild type cells and MCF7 ERα Y537S cells were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University [ ].

Techniques: Inhibition, Expressing, Mutagenesis, In Vivo, In Vivo Imaging, Staining, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: PARP-1 as a novel target in endocrine-resistant breast cancer

doi: 10.1186/s13046-025-03441-4

Figure Lengend Snippet: Proposed mechanisms involved in the regulation of PARP-1 by E 2 and ERα transcriptional activity by PARP-1. ( A ) E 2 -induced (in ERα wt BC cells) or constitutive active (in ERα Y537S mutated BC cells) ERα dimerizes, translocate into the nucleus and interacts with the Sp1 protein on specific Sp1 DNA binding sites located within the promoter region of PARP-1, toward its transcription. ( B ) In ERα Y537S mutated BC cells, PARP-1 is involved in the regulation of the ERα and FoxA1-mediated gene expression machinery that is associated with proliferative effects

Article Snippet: MCF7 ERα wild type cells and MCF7 ERα Y537S cells were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University [ ].

Techniques: Activity Assay, Binding Assay, Gene Expression

Journal: NPJ Breast Cancer

Article Title: ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

doi: 10.1038/s41523-023-00601-7

Figure Lengend Snippet: Representative confocal images of PLA (red puncta) and DAPI (blue nuclei)-stained cells after vehicle treatment in a MCF7 and b T47D cells. Scale bars represent 5 um. Quantification of average cytoplasmic PLA puncta counts per cell for c MCF7 and d T47D cells. Quantification of average nuclear PLA puncta counts per cell for e MCF7 and f T47D cells. Data represents 3 replicates with error bars indicating standard error of the mean (SEM). P -values comparing cell variants are indicated. Asterisks within bars indicate statistically significant differences between HD and PR-stimulated treatments within a given cell variant (* p < 0.05, ** p < 0.005).

Article Snippet: MCF7 parent cells (MCF7 ERα WT) and MCF7 ERα Y537S-het were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University .

Techniques: Staining, Variant Assay

Journal: NPJ Breast Cancer

Article Title: ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

doi: 10.1038/s41523-023-00601-7

Figure Lengend Snippet: Plots of log2(fold change) for differentially expressed transcripts (|log2(FC)|>1, p-adj. < 0.05) in a – d MCF7 cells and e – h T47D cells expressing ERα Y537S-het ( a , c , e , g ) or ERα Y537S-hom ( b , d , f , h ) relative to ERα WT, after HD ( a , b , e , f ) or PR-stimulated ( c , d , g , h ) treatment. Data represents 3 replicates with error bars indicating SEM. Differentially expressed ERα (pink) and PR (teal) target genes are noted above or below their corresponding bars.

Article Snippet: MCF7 parent cells (MCF7 ERα WT) and MCF7 ERα Y537S-het were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University .

Techniques: Expressing

Journal: NPJ Breast Cancer

Article Title: ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

doi: 10.1038/s41523-023-00601-7

Figure Lengend Snippet: Log 2 (fold change) of differentially expressed genes shared between ERα Y537S-expressing patient tumor transcriptome data and a MCF7 and b T47D cell lines. Differentially expressed genes with potential shared ERα/PR regulatory binding sites, as defined by Khushi et al., are outlined in pink dashed lines. Differentially expressed genes are those with p < 0.05 and |log 2 (FC)|>1, where fold change is relative to matched tumors or cell lines expressing ERα WT. c Quantification of IRS1 mRNA expression in MCF7 and T47D cell variants, relative to T47D ERα WT expression levels. Data represent the average of 3 biological replicates with error bars indicating SEM. P -values comparing cell variants are indicated. Asterisks within bars indicate statistically significant differences between HD and PR-stimulated treatments within a given cell variant (* p < 0.05).

Article Snippet: MCF7 parent cells (MCF7 ERα WT) and MCF7 ERα Y537S-het were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University .

Techniques: Expressing, Binding Assay, Variant Assay

Journal: NPJ Breast Cancer

Article Title: ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

doi: 10.1038/s41523-023-00601-7

Figure Lengend Snippet: a Diagram depicting IRS1 chromatin sites assessed for ERα and PR binding using ChIP-qPCR. Potential binding conformations at each site are outlined in dashed lines. Graphic created with Biorender.com. Chromatin binding of ERα and PR at distinct regions of IRS1 is depicted in a for b , d MCF7, and c , e T47D cell variants. For all chromatin immunoprecipitation analyses ( b – e ), data represents the % of input chromatin analyzed. Data represent the average of 3 biological replicates with error bars indicating SEM. P -values comparing cell variants are indicated. Asterisks within bars indicate statistically significant differences between HD and PR-stimulated treatments within a given cell variant (* p < 0.05, ** p < 0.005, ** p < 0.001, ** p < 0.0001).

Article Snippet: MCF7 parent cells (MCF7 ERα WT) and MCF7 ERα Y537S-het were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University .

Techniques: Binding Assay, ChIP-qPCR, Chromatin Immunoprecipitation, Variant Assay

Journal: NPJ Breast Cancer

Article Title: ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

doi: 10.1038/s41523-023-00601-7

Figure Lengend Snippet: Quantification of IRS1 protein in a – c MCF7 and d – f T47D cell variants. Representative lane images from ProteinSimple WES quantification for a MCF7 and d T47D cell variants. Paired lanes indicate β-actin loading control and IRS1 expression for cell variants transfected with a negative control siRNA or si ESR1 . b , e Quantification of IRS1 expression based on signal/noise ratio from WES quantification, with normalization to β-actin loading control and hormone-deprived ERα WT. c , f Comparison of relative IRS1 expression in siCtrl and si ESR1 samples, with normalization to β-actin loading control and to paired siCtrl, to assess the effect of ERα knockdown on IRS1 expression in each cell variant and treatment. Data represent the average of 3 biological replicates with error bars indicating SEM. P -values comparing cell variants are indicated. Asterisks within bars indicate statistically significant differences between HD and PR-stimulated treatments within a given cell variant (* p < 0.05).

Article Snippet: MCF7 parent cells (MCF7 ERα WT) and MCF7 ERα Y537S-het were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University .

Techniques: Control, Expressing, Transfection, Negative Control, Comparison, Knockdown, Variant Assay

Journal: NPJ Breast Cancer

Article Title: ERα/PR crosstalk is altered in the context of the ERα Y537S mutation and contributes to endocrine therapy-resistant tumor proliferation

doi: 10.1038/s41523-023-00601-7

Figure Lengend Snippet: Proliferation, as measured by % cell confluence relative to the initial timepoint (t 0 ), upon siRNA knockdown of IRS1 is shown in a MCF7 and b T47D cell lines. Proliferation of c MCF7 and d T47D cells treated with Vehicle, 4OHT, laso, or ful, alone or in combination with NT-157. Graphs show % confluence after 5 days of treatment, normalized to vehicle. Data represent the average of 3 biological replicates with error bars indicating SEM. P -values indicate a significant change in proliferation at endpoint compared to each respective single drug treatment (NT-157, 4OHT, laso, or ful alone). Asterisks within bars indicate a significant change in proliferation compared to vehicle treatment (* p < 0.05, ** p < 0.005, ** p < 0.001, ** p < 0.0001).

Article Snippet: MCF7 parent cells (MCF7 ERα WT) and MCF7 ERα Y537S-het were generated and gifted by Ben Ho Park, originally at Johns Hopkins University and now at Vanderbilt University .

Techniques: Knockdown