scholarly journals Trp63 is regulated by STAT5 in mammary tissue and subject to differentiation in cancer

2014 ◽  
Vol 21 (3) ◽  
pp. 443-457 ◽  
Author(s):  
Shahin Assefnia ◽  
Keunsoo Kang ◽  
Svenja Groeneveld ◽  
Daisuke Yamaji ◽  
Sarah Dabydeen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Triple Negative ◽  
Cultured Cells ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Proximal Promoter ◽  
Mammary Tissue ◽  
Mrna Levels ◽  
Rna Seq ◽  
Mesenchymal Transition

Transformation-related protein 63 (Trp63), the predominant member of the Trp53 family, contributes to epithelial differentiation and is expressed in breast neoplasia.Trp63features two distinct promoters yielding specific mRNAs encoding two major TRP63 isoforms, a transactivating transcription factor and a dominant negative isoform. Specific TRP63 isoforms are linked to cell cycle arrest, apoptosis, survival, and epithelial mesenchymal transition (EMT). Although TRP63 overexpression in cultured cells is used to elucidate functions, little is known aboutTrp63regulation in normal and cancerous mammary tissues. This study used ChIP-seq to interrogate transcription factor binding and histone modifications of theTrp63locus in mammary tissue and RNA-seq and immunohistochemistry to gauge gene expression. H3K4me2 and H3K4me3 marks coincided only with the proximal promoter, supporting RNA-seq data showing the predominance of the dominant negative isoform. STAT5 bound specifically to theTrp63proximal promoter andTrp63mRNA levels were elevated upon deletingStat5from mammary tissue, suggesting its role as a negative regulator. The dominant negative TRP63 isoform was localized to nuclei of basal mammary epithelial cells throughout reproductive cycles and retained in a majority of the triple-negative cancers generated from loss of full-lengthBrca1. Increased expression of dominant negative isoforms was correlated with developmental windows of increased progesterone receptor binding to the proximalTrp63promoter and decreased expression during lactation was correlated with STAT5 binding to the same region. TRP63 is present in the majority of triple-negative cancers resulting from loss ofBrca1but diminished in less differentiated cancer subtypes and in cancer cells undergoing EMT.

Translating transcriptome to immunophenotype in head and neck squamous cell carcinoma (HNSCC) to identify pathways promoting T-cell infiltration.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e17542-e17542
Author(s):  
Theodoros Rampias ◽  
Christos K. Kontos ◽  
Alexandros Polyzos ◽  
Aris Giotakis ◽  
Evangelos Giotakis ◽  
...  
Keyword(s):  
T Cell ◽  
Checkpoint Inhibitors ◽  
Predictive Biomarkers ◽  
Keratinocyte Differentiation ◽  
Cell Infiltration ◽  
Mrna Levels ◽  
Rna Seq ◽  
Mesenchymal Transition ◽  
Invasive Margin ◽  
T Cell Infiltration

e17542 Background: We sought to analyze the transcriptional landscape of HNSCC in an attempt to identify tumor-intrinsic oncogenic pathways that appear to mediate T-cell infiltration of tumor tissue. In this direction, we employ a methodology that integrates histopathology data of the tumor microenvironment with its corresponding transcriptome. Methods: 32 frozen HNSCCs were subjected to RNA-seq and corresponding FFPE were scored for plasma cells, tertiary lymphoid structures and CD8a+ TILs (center, invasive margin). RNA-seq data were analyzed to identify differentially expressed genes (DEGs) between tumors scored by immunohistochemistry (IHC) as CD8a high and CD8a low. Gene ontology analysis (GO) was performed based on DEGs > 1.5 fold expression change between CD8a high and CD8a low groups. Candidate genes were investigated by hierarchical clustering in TCGA RNA-seq data and further validated by IHC and quantitative RT-PCR in our cohort. Results: 32 HNSCCs were either scored as CD8a high or CD8a low based on IHC detection of CD8a+ cells in invasive margin of tumors. Comparative analysis of mRNA expression data between CD8a high and CD8a low groups in our cohort revealed that Muc1/16 overexpression and glycosylation was highly enriched in T cell infiltrated group of tumors. This finding was further validated using antibodies that detect glycosylated epitopes for both mucins. Analysis of TCGA RNA-seq data indicated that Muc1/16 overexpressing tumors share signatures of early keratinocyte differentiation and stem cell identity and co-express high levels of enzymes that promote Muc1/16 glycosylation. Interestingly, loss of CDH1 and acquisition of epithelial mesenchymal transition (EMT) markers in the cluster of Muc1/16 overexpressing tumors is strongly correlated with elevated CD8a, IDO1, CD274 and CXCL10 mRNA levels (P < 0.0001). Conclusions: Muc1/16 overexpressing tumors represent a very immunogenic HNSCC cluster. Previous studies have shown that mucins 1 and 16 in cancer cells expose glycosylated-specific epitopes that are recognized by T cells as cancer antigens. To this end, MUC1/16 expression may serve as predictive biomarkers for response to immunotherapy and MUC-targeted immunotherapy may function as an attractive partner to checkpoint inhibitors in HNSCC.

scholarly journals Perturbation of β1-Integrin Function in Involuting Mammary Gland Results in Premature Dedifferentiation of Secretory Epithelial Cells

2002 ◽  
Vol 13 (10) ◽  
pp. 3521-3531 ◽  
Author(s):  
Marisa M. Faraldo ◽  
Marie-Ange Deugnier ◽  
Sylvie Tlouzeau ◽  
Jean Paul Thiery ◽  
Marina A. Glukhova
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Dominant Negative Mutant ◽  
Β1 Integrin ◽  
Mrna Levels ◽  
Pregnancy And Lactation

To study the mechanism of β1-integrin function in vivo, we have generated transgenic mouse expressing a dominant negative mutant of β1-integrin under the control of mouse mammary tumor virus (MMTV) promoter (MMTV-β1-cyto). Mammary glands from MMTV-β1-cyto transgenic females present significant growth defects during pregnancy and lactation and impaired differentiation of secretory epithelial cells at the onset of lactation. We report herein that perturbation of β1-integrin function in involuting mammary gland induced precocious dedifferentiation of the secretory epithelium, as shown by the premature decrease in β-casein and whey acidic protein mRNA levels, accompanied by inactivation of STAT5, a transcription factor essential for mammary gland development and up-regulation of nuclear factor-κB, a negative regulator of STAT5 signaling. This is the first study demonstrating in vivo that cell–extracellular matrix interactions involving β1-integrins play an important role in the control of milk gene transcription and in the maintenance of the mammary epithelial cell differentiated state.

CREB trans-activates the murine H+-K+-ATPase α2-subunit gene

2004 ◽  
Vol 287 (4) ◽  
pp. C903-C911 ◽  
Author(s):  
Xiangyang Xu ◽  
Wenzheng Zhang ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Transcriptional Control ◽  
Luciferase Activity ◽  
Collecting Duct ◽  
Binding Activity ◽  
Dominant Negative ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Dna Binding Activity

Despite its key role in potassium homeostasis, transcriptional control of the H+-K+-ATPase α2-subunit (HKα2) gene in the collecting duct remains poorly characterized. cAMP increases H+-K+-ATPase activity in the collecting duct, but its role in activating HKα2 transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HKα2 promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca2+-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HKα2 transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HKα2 mRNA levels, and CREB overexpression stimulated the activity of HKα2 promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HKα2 promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HKα2 promoter-luciferase activity, suggesting that constitutive CREB participates in basal HKα2 transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HKα2 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. In vitro DNase I footprinting and gel shift/supershift analysis of the proximal promoter with recombinant glutathione S-transferase (GST)-CREB-1 and mIMCD-3 cell nuclear extracts revealed sequence-specific DNA-CREB-1 complexes at −86/−60. Mutation at three CRE-like sequences within this region abolished CREB-1 DNA-binding activity and abrogated CREB-VP16 trans-activation of the HKα2 promoter. In contrast, mutation of the neighboring −104/−94 κβ element did not alter CREB-VP16 trans-activation of the HKα2 promoter. Thus CREB-1, binding to one or more CRE-like elements in the −86/−60 region, trans-activates the HKα2 gene and may represent an important link between rapid and delayed effects of cAMP on HKα2 activity.

scholarly journals Epithelial Transcription Factor FOXA1 Regulates Prostate Cancer Immune Response

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A1017-A1017
Author(s):  
Lourdes T Brea ◽  
Xiaohai Wang ◽  
Jindan Yu
Keyword(s):  
Prostate Cancer ◽  
Immune Response ◽  
Transcription Factor ◽  
Inflammatory Response ◽  
Epithelial Mesenchymal Transition ◽  
Checkpoint Inhibitors ◽  
Macrophage Infiltration ◽  
Dependent Manner ◽  
Rna Seq ◽  
Mesenchymal Transition

Abstract Background : While localized prostate cancer (PCa) can be mitigated by surgery and radiation, metastatic PCa remains a challenge to treat. Androgen deprivation therapies and androgen receptor (AR) pathway inhibitors are mainstay treatments for advanced PCa. Yet, resistance often develops leading to castration-resistant prostate cancer (CRPC). Forkhead Box A1 (FOXA1) is a pioneer transcription factor that plays pivotal roles in regulating AR activity and promoting epithelial differentiation. Studies have shown that FOXA1 is frequently downregulated in CRPC tumors. Congruently, FOXA1 loss is reported to induce aberrant AR signaling, epithelial-mesenchymal transition, and PCa de-differentiation. However, the role of FOXA1 in regulating PCa immune response, an area of much interest recently, has not been reported. CRPC has shown poor response to immune checkpoint inhibitors, due to its immunosuppressive nature. A better understanding of the tumor intrinsic mechanisms regulating PCa tumor immunity will inform the design of better targeted immunotherapeutic approaches. Methods: We performed RNA-seq, ChIP-seq, qPCR, western blot, and ELISA analyses to evaluate how FOXA1 regulates inflammatory response genes. We utilized an in vitro macrophage infiltration transwell assay, in which M2-like macrophages were added to the upper chamber and PCa cells were plated in the lower chamber, to examine how perturbations to PCa cells affect macrophage migration. Finally, we performed bioinformatic analyses of patient datasets to confirm the clinical relevance of FOXA1 repression of inflammatory genes in PCa. Results: Through integration of RNA-seq and ChIP-seq data, we uncovered a novel function of FOXA1 in suppressing inflammatory response pathways. In accordance, patient data analyses revealed that inflammatory response genes were upregulated in FOXA1-low PCa tumors. Mechanistically, we showed that FOXA1 proteins bound an intragenic enhancer of Hypoxia-inducible factor 1-alpha (HIF1A) gene to directly repress its expression, such that FOXA1 loss induced HIF1A upregulation. We further showed that Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) became upregulated upon FOXA1 depletion in a HIF1A-dependent manner. This led to infiltration by immunosuppressive, tumor promoting M2-like macrophages. Inhibiting this HIF1A-CCL2 axis with a HIF1A inhibitor or CCL2 neutralizing antibody blocked macrophage infiltration. Future studies using immunocompetent mouse models are needed to confirm the effect of FOXA1 on macrophage infiltration in vivo and evaluate the preclinical potential of targeting the FOXA1-HIF1A-CCL2 axis in CRPC. Conclusion: This study proposes a novel role for FOXA1 loss in promoting macrophage infiltration via the HIF1A-CCL2 axis. Moreover, our findings suggest that targeting this axis may be a promising approach for the treatment of FOXA1-low CRPC tumors.

scholarly journals PD-L1 Blockade by Atezolizumab Downregulates Signaling Pathways Associated with Tumor Growth, Metastasis, and Hypoxia in Human Triple Negative Breast Cancer

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1050 ◽  
Author(s):  
Reem Saleh ◽  
Rowaida Z. Taha ◽  
Varun Sasidharan Nair ◽  
Nehad M. Alajez ◽  
Eyad Elkord
Keyword(s):  
Breast Cancer ◽  
Tumor Growth ◽  
Signaling Pathways ◽  
Triple Negative Breast Cancer ◽  
Molecular Mechanisms ◽  
Triple Negative ◽  
Epithelial Mesenchymal Transition ◽  
Cancer Therapies ◽  
Rna Seq ◽  
Mesenchymal Transition

Triple negative breast cancer (TNBC) is the most aggressive type of breast cancer, which shows resistance to common breast cancer therapies, as it lacks the expression of the most common breast cancer targets. Therefore, TNBC treatment remains a challenge. Targeting programmed cell death-ligand 1 (PD-L1) by monoclonal antibodies (mAbs), for example, atezolizumab, has revolutionized the treatment for various cancer types. However, the therapeutic efficacy of targeting PD-L1 in TNBC is currently under investigation. In this study, we investigated the molecular mechanisms by which the human TNBC cell line MDA-MB-231, expressing PD-L1, responds to atezolizumab, using RNA-Seq. Transcriptome analysis revealed 388 upregulated and 362 downregulated genes in response to atezolizumab treatment. The expression of selected genes, from RNA-Seq data, was subsequently validated using RT-qPCR in the MDA-MB-231 and MDA-MB-468 TNBC cells following atezolizumab treatment. Bioinformatics analysis revealed that atezolizumab downregulates genes promoting cell migration/invasion and metastasis, epithelial-mesenchymal transition (EMT), cell growth/proliferation/survival, and hypoxia. On the contrary, genes associated with apoptosis and DNA repair were upregulated in response to atezolizumab treatment. Gene set enrichment analyses revealed that a significant number of these genes are related to the NF-kB, PI3K/Akt/mTOR, MAPK, and CD40 signaling pathways. Using functional assays, we confirmed that atezolizumab increases MDA-MB-231 cell apoptosis/necrosis, and reduces their proliferation and viability. Collectively, our findings provide novel insights into the molecular mechanisms/signaling pathways by which atezolizumab exerts inhibitory effects on TNBC, thereby inhibiting EMT/metastasis, tumor growth/survival, and the induction of hypoxia.

scholarly journals Novel muscle-specific enhancer sequences upstream of the cardiac actin gene.

1994 ◽  
Vol 14 (5) ◽  
pp. 3504-3513 ◽  
Author(s):  
C Biben ◽  
B J Kirschbaum ◽  
I Garner ◽  
M Buckingham
Keyword(s):  
Cultured Cells ◽  
Proximal Promoter ◽  
Actin Gene ◽  
Mrna Levels ◽  
Distal Enhancer ◽  
Cardiac Actin ◽  
E Box ◽  
C3h Mice ◽  
Myogenic Factors ◽  
E Boxes

A DNase I-hypersensitive site analysis of the 5'-flanking region of the mouse alpha-cardiac actin gene with muscle cell lines derived from C3H mice shows the presence of two such sites, at about -5 and -7 kb. When tested for activity in cultured cells with homologous and heterologous promoters, both sequences act as muscle-specific enhancers. Transcription from the proximal promoter of the alpha-cardiac actin gene is increased 100-fold with either enhancer. The activity of the distal enhancer in C2/7 myotubes is confined to an 800-bp fragment, which contains multiple E boxes. In transfection assays, this sequence does not give detectable transactivation by any of the myogenic factors even though one of the E boxes is functionally important. Bandshift assays showed that MyoD and myogenin can bind to this E box. However, additional sequences are also required for activity. We conclude that in the case of this muscle enhancer, myogenic factors alone are not sufficient to activate transcription either directly via an E box or indirectly through activation of genes encoding other muscle factors. In BALB/c mice, in which cardiac actin mRNA levels are 8- to 10-fold lower, the alpha-cardiac actin locus is perturbed by a 9.5-kb insertion (I. Garner, A. J. Minty, S. Alonso, P. J. Barton, and M. E. Buckingham, EMBO J. 5:2559-2567, 1986). This is located at -6.5 kb, between the two enhancers. The insertion therefore distances the distal enhancer from the promoter and from the proximal enhancer of the bona fide cardiac actin gene, probably thus perturbing transcriptional activity.

scholarly journals STAT3β, a Splice Variant of Transcription Factor STAT3, Is a Dominant Negative Regulator of Transcription

1996 ◽  
Vol 271 (22) ◽  
pp. 13221-13227 ◽  
Author(s):  
Eric Caldenhoven ◽  
Thamar B. van Dijk ◽  
Roberto Solari ◽  
John Armstrong ◽  
Jan A. M. Raaijmakers ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Splice Variant ◽  
Dominant Negative ◽  
Negative Regulator

scholarly journals Loss of function of bHLH transcription factor Nrd1 in tomato induces an arabinogalactan protein-encoding gene and enhances resistance to Pseudomonas syringae pv. tomato

2021 ◽  
Author(s):  
Ning Zhang ◽  
Chloe Hecht ◽  
Xuepeng Sun ◽  
Zhangjun Fei ◽  
Gregory B Martin
Keyword(s):  
Transcription Factor ◽  
Pseudomonas Syringae ◽  
Transcript Abundance ◽  
Negative Regulator ◽  
Arabinogalactan Protein ◽  
Bhlh Transcription Factor ◽  
Dependent Manner ◽  
Rna Seq ◽  
Loss Of Function ◽  
Encoding Gene

Basic helix-loop-helix (bHLH) transcription factors constitute a superfamily in eukaryotes but their roles in plant immunity remain largely uncharacterized. We found that the transcript abundance in tomato leaves of one bHLH transcription factor-encoding gene, Nrd1 (negative regulator of resistance to DC3000 1), was significantly increased after treatment with the immunity-inducing flgII-28 peptide. Plants carrying a loss-of-function mutation in Nrd1 (Δnrd1) showed enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 although early pattern-triggered immunity responses such as generation of reactive oxygen species and activation of mitogen-activated protein kinases after treatment with flagellin-derived flg22 and flgII-28 peptides were unaltered compared to wild-type plants. An RNA-Seq analysis identified a gene, Agp1, whose expression is strongly suppressed in an Nrd1-dependent manner. Agp1 encodes an arabinogalactan protein and overexpression of the Agp1 gene in Nicotiana benthamiana led to ~10-fold less Pst growth compared to the control. These results suggest that the Nrd1 protein promotes tomato susceptibility to Pst by suppressing the defense gene Agp1. RNA-Seq also revealed that loss of Nrd1 function has no effect on the transcript abundance of immunity-associated genes including Bti9, Core, Fls2, Fls3 and Wak1 upon Pst inoculation, suggesting that the enhanced immunity observed in the Δnrd1 mutants is due to the activation of key PRR signaling components as well as loss of Nrd1-regulated suppression of Agp1.

IL-17 stimulates MMP-1 expression in primary human cardiac fibroblasts via p38 MAPK- and ERK1/2-dependent C/EBP-β, NF-κB, and AP-1 activation

2007 ◽  
Vol 293 (6) ◽  
pp. H3356-H3365 ◽  
Author(s):  
Dolores M. Cortez ◽  
Marc D. Feldman ◽  
Srinivas Mummidi ◽  
Anthony J. Valente ◽  
Bjorn Steffensen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
P38 Mapk ◽  
Western Blotting ◽  
Cardiac Fibroblasts ◽  
Critical Role ◽  
Dominant Negative ◽  
Expression Vectors ◽  
Type I ◽  
Mrna Levels ◽  
Human Cardiac Fibroblasts

Matrix metalloproteinases (MMPs) degrade collagen and mediate tissue remodeling. The novel cytokine IL-17 is expressed during various inflammatory conditions and modulates MMP expression. We investigated the effect of IL-17 on MMP-1 expression in primary human cardiac fibroblasts (HCF) and delineated the signaling pathways involved. HCF were treated with recombinant human IL-17. MMP-1 expression was analyzed by Northern blotting, RT-quantitative PCR, Western blotting, and ELISA; transcriptional induction and transcription factor binding by EMSA, ELISA, and reporter assay; and p38 MAPK and ERK1/2 activation by protein kinase assays and Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA), and adenoviral dominant-negative expression vectors. IL-17 stimulated MMP-1 gene transcription, net mRNA levels, protein, and promoter-reporter activity in HCF. This response was blocked by IL-17 receptor-Fc chimera and IL-17 receptor antibodies, but not by IL-6, TNF-α, or IL-1β antibodies. IL-17-stimulated type I collagenase activity was inhibited by the MMP inhibitor GM-6001 and by siRNA-mediated MMP-1 knockdown. IL-17 stimulated activator protein-1 [AP-1 (c-Fos, c-Jun, and Fra-1)], NF-κB (p50 and p65), and CCAAT enhancer-binding protein (C/EBP)-β DNA binding and reporter gene activities, effects attenuated by antisense oligonucleotides, siRNA-mediated knockdown, or expression of dominant-negative signaling proteins. Inhibition of AP-1, NF-κB, or C/EBP activation attenuated IL-17-stimulated MMP-1 expression. IL-17 induced p38 MAPK and ERK1/2 activation, and inhibition by SB-203580 and PD-98059 blunted IL-17-mediated transcription factor activation and MMP-1 expression. Our data indicate that IL-17 induces MMP-1 in human cardiac fibroblasts directly via p38 MAPK- and ERK-dependent AP-1, NF-κB, and C/EBP-β activation and suggest that IL-17 may play a critical role in myocardial remodeling.

scholarly journals Heat Stress Factors Expressed during Seed Maturation Differentially Regulate Seed Longevity and Seedling Greening

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 335 ◽  
Author(s):  
Concepción Almoguera ◽  
Pilar Prieto-Dapena ◽  
Raúl Carranco ◽  
José Luis Ruiz ◽  
Juan Jordano
Keyword(s):  
Transcription Factor ◽  
Heat Stress ◽  
Blue Light ◽  
Seed Longevity ◽  
Dominant Negative ◽  
Seedling Stage ◽  
Stress Factors ◽  
Rna Seq ◽  
Loss Of Function ◽  
Light Responses

Heat Stress Factor A9 (A9), a seed-specific transcription factor contributing to seed longevity, also enhances phytochrome-dependent seedling greening. The RNA-seq analyses of imbibed-seed transcripts here reported indicated potential additional effects of A9 on cryptochrome-mediated blue-light responses. These analyses also suggested that in contrast to the A9 effects on longevity, which require coactivation by additional factors as A4a, A9 alone might suffice for the enhancement of photomorphogenesis at the seedling stage. We found that upon its seed-specific overexpression, A9 indeed enhanced the expected blue-light responses. Comparative loss-of-function analyses of longevity and greening, performed by similar expression of dominant-negative and inactive forms of A9, not only confirmed the additional greening effects of A9, but also were consistent with A9 not requiring A4a (or additional factors) for the greening effects. Our results strongly indicate that A9 would differentially regulate seed longevity and photomorphogenesis at the seedling stage, A9 alone sufficing for both the phytochrome- and cryptochrome-dependent greening enhancement effects.

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