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TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites in Breast Cancer Cells [ChIP-seq]

ABSTRACT: The interplay between mitogenic and proinflammatory signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. We have used global nuclear run-on coupled with deep sequencing to characterize the immediate transcriptional responses of MCF-7 breast cancer cells treated with estradiol, TNF?, or both. In addition, we have integrated these data with chromatin immunoprecipitation coupled with deep sequencing for estrogen receptor alpha (ER?), the pioneer factor FoxA1 and the p65 subunit of the NF-?B transcription factor. Our results indicate extensive transcriptional interplay between these two signaling pathways, which is observed for a number of classical mitogenic and proinflammatory protein-coding genes. In addition, GRO-seq has allowed us to capture the transcriptional crosstalk at the genomic locations encoding for long non-coding RNAs, a poorly characterized class of RNAs which have been shown to play important roles in cancer outcomes. The synergistic and antagonistic interplay between estrogen and TNF? signaling at the gene level is also evident in the patterns of ER? and NF-?B binding, which relocalize to new binding sites that are not occupied by either treatment alone. Interestingly, the chromatin accessibility of classical ER? binding sites is predetermined prior to estrogen treatment, whereas ER? binding sites gained upon co-treatment with TNF? require NF-?B and FoxA1 to promote chromatin accessibility de novo. Our data suggest that TNF? signaling recruits FoxA1 and NF-?B to latent ER? enhancer locations and directly impact ER? enhancer accessibility. Binding of ER? to latent enhancers upon co-treatment, results in increased enhancer transcription, target gene expression and altered cellular response. This provides a mechanistic framework for understanding the molecular basis for integration of mitogenic and proinflammatory signaling in breast cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE59530 | GEO | 2015/03/05

SECONDARY ACCESSION(S): PRJNA255509

REPOSITORIES: GEO

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Project description:The interplay between mitogenic and proinflammatory signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. We have used global nuclear run-on coupled with deep sequencing to characterize the immediate transcriptional responses of MCF-7 breast cancer cells treated with estradiol, TNF?, or both. In addition, we have integrated these data with chromatin immunoprecipitation coupled with deep sequencing for estrogen receptor alpha (ER?), the pioneer factor FoxA1 and the p65 subunit of the NF-?B transcription factor. Our results indicate extensive transcriptional interplay between these two signaling pathways, which is observed for a number of classical mitogenic and proinflammatory protein-coding genes. In addition, GRO-seq has allowed us to capture the transcriptional crosstalk at the genomic locations encoding for long non-coding RNAs, a poorly characterized class of RNAs which have been shown to play important roles in cancer outcomes. The synergistic and antagonistic interplay between estrogen and TNF? signaling at the gene level is also evident in the patterns of ER? and NF-?B binding, which relocalize to new binding sites that are not occupied by either treatment alone. Interestingly, the chromatin accessibility of classical ER? binding sites is predetermined prior to estrogen treatment, whereas ER? binding sites gained upon co-treatment with TNF? require NF-?B and FoxA1 to promote chromatin accessibility de novo. Our data suggest that TNF? signaling recruits FoxA1 and NF-?B to latent ER? enhancer locations and directly impact ER? enhancer accessibility. Binding of ER? to latent enhancers upon co-treatment, results in increased enhancer transcription, target gene expression and altered cellular response. This provides a mechanistic framework for understanding the molecular basis for integration of mitogenic and proinflammatory signaling in breast cancer. Using GRO-seq and ChIP-seq (ER, FoxA1 and p65) to assay the molecular crosstalk of MCF-7 cells treated with E2, TNFa or both E2+TNFa.

Project description:The interplay between mitogenic and proinflammatory signaling pathways play key roles in determining the phenotypes and clinical outcomes of breast cancers. We have used global nuclear run-on coupled with deep sequencing to characterize the immediate transcriptional responses of MCF-7 breast cancer cells treated with estradiol, TNFM-NM-1, or both. In addition, we have integrated these data with chromatin immunoprecipitation coupled with deep sequencing for estrogen receptor alpha (ERM-NM-1), the pioneer factor FoxA1 and the p65 subunit of the NF-M-NM-:B transcription factor. Our results indicate extensive transcriptional interplay between these two signaling pathways, which is observed for a number of classical mitogenic and proinflammatory protein-coding genes. In addition, GRO-seq has allowed us to capture the transcriptional crosstalk at the genomic locations encoding for long non-coding RNAs, a poorly characterized class of RNAs which have been shown to play important roles in cancer outcomes. The synergistic and antagonistic interplay between estrogen and TNFM-NM-1 signaling at the gene level is also evident in the patterns of ERM-NM-1 and NF-M-NM-:B binding, which relocalize to new binding sites that are not occupied by either treatment alone. Interestingly, the chromatin accessibility of classical ERM-NM-1 binding sites is predetermined prior to estrogen treatment, whereas ERM-NM-1 binding sites gained upon co-treatment with TNFM-NM-1 require NF-M-NM-:B and FoxA1 to promote chromatin accessibility de novo. Our data suggest that TNFM-NM-1 signaling recruits FoxA1 and NF-M-NM-:B to latent ERM-NM-1 enhancer locations and directly impact ERM-NM-1 enhancer accessibility. Binding of ERM-NM-1 to latent enhancers upon co-treatment, results in increased enhancer transcription, target gene expression and altered cellular response. This provides a mechanistic framework for understanding the molecular basis for integration of mitogenic and proinflammatory signaling in breast cancer. Using GRO-seq and ChIP-seq (ER, FoxA1 and p65) to assay the molecular crosstalk of MCF-7 cells treated with E2, TNFM-NM-1 or both E2+TNFM-NM-1.

Project description:This model is from the article: Heterogeneity Reduces Sensitivity of Cell Death for TNF-Stimuli Schliemann M, Bullinger E, Borchers S, Allgower F, Findeisen R, Scheurich P. BMC Syst Biol. 2011 Dec 28;5(1):204. 22204418 , Abstract: BACKGROUND: Apoptosis is a form of programmed cell death essential for the maintenance of homeostasis and the removal of potentially damaged cells in multicellular organisms. By binding its cognate membrane receptor, TNF receptor type 1 (TNF-R1), the proinflammatory cytokine Tumor Necrosis Factor (TNF) activates pro-apoptotic signaling via caspase activation, but at the same time also stimulates nuclear factor kappaB (NF-kappaB)-mediated survival pathways. Differential dose-response relationships of these two major TNF signaling pathways have been described experimentally and using mathematical modeling. However, the quantitative analysis of the complex interplay between pro- and anti-apoptotic signaling pathways is an open question as it is challenging for several reasons: the overall signaling network is complex, various time scales are present, and cells respond quantitatively and qualitatively in a heterogeneous manner. RESULTS: This study analyzes the complex interplay of the crosstalk of TNF-R1 induced pro- and anti-apoptotic signaling pathways based on an experimentally validated mathematical model. The mathematical model describes the temporal responses on both the single cell level as well as the level of a heterogeneous cell population, as observed in the respective quantitative experiments using TNF-R1 stimuli of different strengths and durations. Global sensitivity of the heterogeneous population was quantified by measuring the average gradient of time of death versus each population parameter. This global sensitivity analysis uncovers the concentrations of Caspase-8 and Caspase-3, and their respective inhibitors BAR and XIAP, as key elements for deciding the cell's fate. A simulated knockout of the NF-kappaB-mediated anti-apoptotic signaling reveals the importance of this pathway for delaying the time of death, reducing the death rate in the case of pulse stimulation and significantly increasing cell-to-cell variability. CONCLUSIONS: Cell ensemble modeling of a heterogeneous cell population including a global sensitivity analysis presented here allowed us to illuminate the role of the different elements and parameters on apoptotic signaling. The receptors serve to transmit the external stimulus; procaspases and their inhibitors control the switching from life to death, while NF-kappaB enhances the heterogeneity of the cell population. The global sensitivity analysis of the cell population model further revealed an unexpected impact of heterogeneity, i.e. the reduction of parametric sensitivity. Note: SBML model generated from Matlab system description on 12-July-2011 21:08:15 by exportSBML Copyright Eric Bullinger 2007-2011

Project description:Aberrant activation of the forkhead protein FOXA1 is observed in advanced hormone-related cancers. However, to date, key mediators of high FOXA1 signaling remain elusive. We demonstrate that ectopic high FOXA1 (H-FOXA1) expression promotes estrogen receptor-positive (ER+) breast cancer (BC) metastasis in a xenograft mouse model. Mechanistically, H-FOXA1 reprograms ER-chromatin binding to elicit a core gene signature (CGS) highly enriched in ER+ endocrine-resistant (EndoR) cells. We identified Secretome14, a CGS subset encoding ER-dependent cancer secretory proteins, strongly predicts poor outcomes of ER+ BC and is elevated in ER+ metastases vs. primary tumors, irrespective to the ESR1 mutations. Parental (P) ER+ BC cells and their endocrine-resistant (EndoR) derivatives, and ER+ BC cells expressing doxycycline (Dox)-inducible ectopic FOXA1 were used in this study. Differential gene expression analysis was performed in EndoR vs. P cells and P cells +Dox vs. -Dox. We found that the FOXA1-CGS was highly enriched in the altered transcriptomes of two ER+ BC cell models (ZR75-1 and T47D) expressing ectopic H-FOXA1. The enriched hallmark gene sets, shared by the H-FOXA1 cell models, include “inflammatory response”, “complement”, and “interferon gamma response” for the H-FOXA1-induced, and “estrogen response early” and “estrogen response late” for the H-FOXA1-repressed genes. These findings point to the common transcriptional profile exhibiting an immune- over estrogen-responsive signature induced by H-FOXA1. In addition, we found that both the tamoxifen-resistant (TamR) and estrogen deprivation-resistant (EDR) derivatives of the 600MPE P cells were enriched for the H-FOXA1-induced CGS and FOXA1/ER-activated Secretome14. Our findings uncover H-FOXA1-induced ER reprogramming driving EndoR and metastasis, possibly via a H-FOXA1/ER-dependent secretome that warrants further studies to clarify its involvement in disease progression of ER+ metastatic BC.

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TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites in Breast Cancer Cells [ChIP-seq]

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