Link between GRHL2 Motifs and Intratumor Heterogeneity of Cis-Regulatory Elements Results in Poor Outcome in Luminal Breast Cancer: A Single-cell Chromatin Accessibility Analysis
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PROVIDER: PRJNA816201 | ENA |
REPOSITORIES: ENA
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Project description:In breast cancer patients, tumor heterogeneity is associated with prognosis and therapeutic response; however, the epigenetic diversity that exists in primary tumors remains unknown. Using a single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), we obtained the chromatin accessibility profiles of 12,452 cells from 16 breast cancer patients including 11 luminal, 1 luminal-HER2, 1 HER2+, and 3 triple-negative subtypes. Via this profiling process, tumors were classified into cancer cells and the tumor microenvironment, highlighting the heterogeneity of disease-related pathways including estrogen receptor (ER) signaling. Furthermore, the coexistence of cancer cell clusters with different ER binding motif enrichments was identified in a single ER+ tumor. In a cluster with reduced ER motif enrichment, we identified GRHL2, a transcription factor, as the most enriched motif, and it cooperated with FOXA1 to initiate endocrine resistance. Coaccessibility analysis revealed that GRHL2 binding elements potentially regulate genes associated with endocrine resistance, metastasis, and poor prognosis in patients that received hormonal therapy. Overall, our study suggests that epigenetic heterogeneity could lead to endocrine resistance and poor prognosis in breast cancer patients and it offers a large-scale resource for further cancer research.
2022-03-16 | GSE198639 | GEO
Project description:In breast cancer patients, tumor heterogeneity is associated with prognosis and therapeutic response; however, the epigenetic diversity that exists in primary tumors remains unknown. Using a single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), we obtained the chromatin accessibility profiles of 12,452 cells from 16 breast cancer patients including 11 luminal, 1 luminal-HER2, 1 HER2+, and 3 triple-negative subtypes. Via this profiling process, tumors were classified into cancer cells and the tumor microenvironment, highlighting the heterogeneity of disease-related pathways including estrogen receptor (ER) signaling. Furthermore, the coexistence of cancer cell clusters with different ER binding motif enrichments was identified in a single ER+ tumor. In a cluster with reduced ER motif enrichment, we identified GRHL2, a transcription factor, as the most enriched motif, and it cooperated with FOXA1 to initiate endocrine resistance. Coaccessibility analysis revealed that GRHL2 binding elements potentially regulate genes associated with endocrine resistance, metastasis, and poor prognosis in patients that received hormonal therapy. Overall, our study suggests that epigenetic heterogeneity could lead to endocrine resistance and poor prognosis in breast cancer patients and it offers a large-scale resource for further cancer research.
| S-EPMC9177858 | biostudies-literature
Project description:The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation-related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies, we identified the NK2 homeobox 1 transcription factor (Nkx2-1) as a direct transcriptional target of Grhl2. By binding and transcriptional assays, and by confocal microscopy we showed that these two transcription factors form a positive feed-back loop in vivo and in cell lines, and are co-expressed in lung bronchiolar and alveolar type II cells. The morphological changes observed in flattening lung alveolar type II cells in culture are associated with down-regulation of Grhl2 and Nkx2-1. Reduction of Grhl2 in lung epithelial cell lines results in lower expression levels of Nkx2-1 and of known Grhl2 target genes. By microarray analysis we identified that in addition to Cadherin1 and Claudin4, Grhl2 regulates other cell interaction genes such as semaphorins and their receptors, which also play a functional role in developing lung epithelium. Impaired collective cell migration observed in Grhl2 knockdown cell monolayers is associated with reduced expression of these genes and may contribute to the altered epithelial phenotype reported in Grhl2 mutant mice. Thus, Grhl2 functions at the nexus of a novel regulatory network, connecting lung epithelial cell identity, migration and cell-cell interactions. To identify genes regulated by GRHL2 in lung epithelial cells, we performed cDNA microarray analyses in MLE15 cells transduced with Grhl2-shRNA and compared to a non-silencing control. Independent transductions of MLE15 cells using Grhl2-shRNA (n=3) and a non-silencing control (n=2) were analyzed.
2012-09-10 | E-GEOD-40729 | biostudies-arrayexpress
Project description:Healthy placental development is essential for reproductive success; failure of the feto-maternal interface results in preeclampsia and intrauterine growth retardation. We found that grainyhead-like 2 (GRHL2), a CP2-type transcription factor, is highly expressed in chorionic trophoblast cells, including basal chorionic trophoblast (BCT) cells located at the chorioallantoic interface in murine placentas. Placentas from Grhl2-deficient mouse embryos displayed defects in BCT cell polarity and basement membrane integrity at the chorioallantoic interface, as well as a severe disruption of labyrinth branchingmorphogenesis.Selective Grhl2 inactivation only in epiblastderived cells rescued all placental defects but phenocopied intraembryonic defects observed in global Grhl2 deficiency, implying the importance of Grhl2 activity in trophectoderm-derived cells. ChIPseq identified 5282 GRHL2 binding sites in placental tissue. By integrating these data with placental gene expression profiles, we identified direct and indirect Grhl2 targets and found a marked enrichment of GRHL2 binding adjacent to genes downregulated in Grhl2−/− placentas, which encoded known regulators of placental development and epithelial morphogenesis. These genes included that encoding the serine protease inhibitor Kunitz type 1 (Spint1), which regulates BCT cell integrity and labyrinth formation. In human placenta, we found that human orthologs of murine GRHL2 and its targets displayed co-regulation and were expressed in trophoblast cells in a similar domain as in mouse placenta. Our data indicate that a conserved Grhl2-coordinated gene network controls trophoblast branching morphogenesis, thereby facilitating development of the site of feto-maternal exchange. This might have implications for syndromes related to placental dysfunction. In vivo genome-wide examination of binding sites of the transcription factor GRHL2 by ChIP-seq using wild-type murine E17.5 placenta tissue. Two samples in total: one GRHL2 ChIP sample and one IgG ChIP sample using wild-type placentas tissue as antibody control.
2015-03-10 | E-GEOD-65962 | biostudies-arrayexpress
Project description:Using a retrovirus-mediated cDNA expression cloning approach we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant-negative GRHL2 proteins induce downregulation of ERBB3 gene expression, a striking reduction in cell proliferation and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying dual roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc-finger-enhancer binding protein 1 (ZEB1) which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph-node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis. To evaluate the role of the GRHL2 transcription factor in carcinogenessis we overexpressed the human GRHL2 protein in NIH3T3 cells. Parental NIH3T3 cells do not express the orthologic protein. Alterations in genome-wide transcriptional profiles were determined by microarray analysis.
2013-09-05 | E-GEOD-43610 | biostudies-arrayexpress
Project description:Breast cancer is a heterogeneous disease, and breast cancer cell lines are invaluable for studying this heterogeneity. However, the epigenetic diversity across these cell lines remains poorly understood. In this study, we performed genome-wide chromatin accessibility analysis on 23 breast cancer cell lines, including 2 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative (ER+/HER2−), 3 ER+/HER2+, 2 HER2+, and 15 triple-negative breast cancer (TNBC) lines. These cell lines were classified into three groups based on their chromatin accessibility: the receptor-positive group (Group-P), TNBC basal group (Group-B), and TNBC mesenchymal group (Group-M). Motif enrichment analysis revealed that only Group-P exhibited coenrichment of forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2) motifs, whereas Group-B was characterized by the presence of the GRHL2 motif without FOXA1. Notably, Group-M did not show enrichment of either FOXA1 or GRHL2 motifs. Furthermore, gene ontology analysis suggested that group-specific accessible regions were associated with their unique lineage characteristics. To investigate the epigenetic landscape regulatory roles of FOXA1 and GRHL2, we performed knockdown experiments targeting FOXA1 and GRHL2, followed by assay for transposase-accessible chromatin sequencing analysis. The findings revealed that FOXA1 maintains Group-P–specific regions while suppressing Group-B–specific regions in Group-P cells. In contrast, GRHL2 preserves commonly accessible regions shared between Group-P and Group-B in Group-B cells, suggesting that FOXA1 and GRHL2 play a pivotal role in preserving distinct chromatin accessibility patterns for each group. Specifically, FOXA1 distinguishes between receptor-positive and TNBC cell lines, whereas GRHL2 distinguishes between basal-like and mesenchymal subtypes in TNBC lines.
2024-02-01 | GSE254218 | GEO
Project description:Breast cancer is a heterogeneous disease, and breast cancer cell lines are invaluable for studying this heterogeneity. However, the epigenetic diversity across these cell lines remains poorly understood. In this study, we performed genome-wide chromatin accessibility analysis on 23 breast cancer cell lines, including 2 estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative (ER+/HER2−), 3 ER+/HER2+, 2 HER2+, and 15 triple-negative breast cancer (TNBC) lines. These cell lines were classified into three groups based on their chromatin accessibility: the receptor-positive group (Group-P), TNBC basal group (Group-B), and TNBC mesenchymal group (Group-M). Motif enrichment analysis revealed that only Group-P exhibited coenrichment of forkhead box A1 (FOXA1) and grainyhead-like 2 (GRHL2) motifs, whereas Group-B was characterized by the presence of the GRHL2 motif without FOXA1. Notably, Group-M did not show enrichment of either FOXA1 or GRHL2 motifs. Furthermore, gene ontology analysis suggested that group-specific accessible regions were associated with their unique lineage characteristics. To investigate the epigenetic landscape regulatory roles of FOXA1 and GRHL2, we performed knockdown experiments targeting FOXA1 and GRHL2, followed by assay for transposase-accessible chromatin sequencing analysis. The findings revealed that FOXA1 maintains Group-P–specific regions while suppressing Group-B–specific regions in Group-P cells. In contrast, GRHL2 preserves commonly accessible regions shared between Group-P and Group-B in Group-B cells, suggesting that FOXA1 and GRHL2 play a pivotal role in preserving distinct chromatin accessibility patterns for each group. Specifically, FOXA1 distinguishes between receptor-positive and TNBC cell lines, whereas GRHL2 distinguishes between basal-like and mesenchymal subtypes in TNBC lines.
2024-02-01 | GSE254216 | GEO
Project description:Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMTs) occurring in wound healing processes and the cancer stem cell-like compartment of tumors, including TGF-β-dependence, we investigated the role of a Grainyhead gene (GRHL2) in oncogenic EMT. Grainyhead was specifically down-regulated in the claudin-low subclass of mammary tumors and in the basal-B subclass of breast cancer cell lines. Functionally, GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis-sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated, in part, by its suppression of ZEB1 expression, through direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription, and up-regulated mir200b/c as well as the TGF-β receptor antagonist, BMP2. The expression of GRHL2 in the breast cancer cell line MDA-MB-231 triggered a mesenchymal-to-epithelial transition and sensitized the cells to anoikis. These results indicate that GRHL2 is a suppressor of the oncogenic EMT. 3 biologic replicates for each cell line. Comparison of HMLE+Twist-ER cells expressing GRHL2/pMIG vs. HMLE+Twist-ER cells expressing empty pMIG.
2012-02-25 | E-GEOD-36081 | biostudies-arrayexpress
Project description:Healthy placental development is essential for reproductive success; failure of the feto-maternal interface results in preeclampsia and intrauterine growth retardation. We found that grainyhead-like 2 (GRHL2), a CP2-type transcription factor, is highly expressed in chorionic trophoblast cells, including basal chorionic trophoblast (BCT) cells located at the chorioallantoic interface in murine placentas. Placentas from Grhl2-deficient mouse embryos displayed defects in BCT cell polarity and basement membrane integrity at the chorioallantoic interface, as well as a severe disruption of labyrinth branchingmorphogenesis.Selective Grhl2 inactivation only in epiblastderived cells rescued all placental defects but phenocopied intraembryonic defects observed in global Grhl2 deficiency, implying the importance of Grhl2 activity in trophectoderm-derived cells. ChIPseq identified 5282 GRHL2 binding sites in placental tissue. By integrating these data with placental gene expression profiles, we identified direct and indirect Grhl2 targets and found a marked enrichment of GRHL2 binding adjacent to genes downregulated in Grhl2−/− placentas, which encoded known regulators of placental development and epithelial morphogenesis. These genes included that encoding the serine protease inhibitor Kunitz type 1 (Spint1), which regulates BCT cell integrity and labyrinth formation. In human placenta, we found that human orthologs of murine GRHL2 and its targets displayed co-regulation and were expressed in trophoblast cells in a similar domain as in mouse placenta. Our data indicate that a conserved Grhl2-coordinated gene network controls trophoblast branching morphogenesis, thereby facilitating development of the site of feto-maternal exchange. This might have implications for syndromes related to placental dysfunction.
2015-03-10 | GSE65960 | GEO
Project description:Using a retrovirus-mediated cDNA expression cloning approach we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant-negative GRHL2 proteins induce downregulation of ERBB3 gene expression, a striking reduction in cell proliferation and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying dual roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc-finger-enhancer binding protein 1 (ZEB1) which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph-node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis.
2013-09-05 | GSE43610 | GEO