A novel tumor-promoting function resided in the 5’ untranslated region of vegf mRNA
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE8888 | GEO | 2008/04/04
SECONDARY ACCESSION(S): PRJNA102269
REPOSITORIES: GEO
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Project description:VEGF165 is one of the key regulators of tumor development. Using HCT116 cells transfected with full-length vegf mRNA, full-length vegf mRNA with mutations of H9D and L14E, mutated vegf mRNAs lacking untranslated regions (UTRs), and 5â??UTR mutated between nt 591 and nt 746, we found that vegf 5â??UTR resided an anti-apoptotic activity against chemotherapy independently of VEGF165. We next established HCT116 clones stably expressing vegf 5â??UTR or the mutated vegf 5â??UTR. The cells expressing 5â??UTR, but not the mutated UTR, showed the drug-resistant phenotype, anchorage-independent growth, and rapid tumor growth when implanted in athymic nude mice. Microarray and real-time PCR showed that the vegf 5â??UTR-expressing tumors up-regulated anti-apoptotic genes including mia and down-regulated pro-apoptotic genes including fas, pdcd1, nrg1, and bax. Microarray analysis also revealed specific down-regulation of IFN-inducible genes (43 genes) in the growing tumors. HCT116 cells stably transcribing vegf 5â??UTR decreased STAT1 expression and IFN alpha-STAT1 pathway. In addition to 5-fluorouracil treatment, the vegf 5â??UTR-expressing tumors did not respond to IFN alpha therapy at all. This novel tumor-promoting function in the vegf 5â??UTR may partly explain the insufficiency of the VEGF-VEGFR strategies and suggest a vegf-targeted silencing strategy as a more effective anti-tumor therapy. Experiment Overall Design: We established HCT116 clones stably expressing vegf 5â??UTR (vegf 5â??-G5) and vegf 5â??UTR mutated between nt 591 and nt 746 (vegf 5â?? mut-D5) and implanted into the left and the right flanks of the identical mouse, respectively. The generated tumors on day 14 after injection were removed and total RNA was extracted from each tumor using RNeasy mini (Qiagen). After removing contaminated DNA by DNaseI treatment, 10 micro grams each of RNA of vegf 5â??-G5-derived tumor from 3 individual mice was mixed and prepared for microarray analysis as sample RNA. The reference RNA of vegf 5â?? mut-D5-derived tumor was arranged in the same manner. The quality of both RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA (vegf 5â??-G5) and reference RNA (vegf 5â?? mut-D5) were converted to cDNA with oligo dT conjugated T7 promoter primer and reverse transcriptase. These cDNAs were amplified using T7 RNA polymerase while labeling with either Cy5-CTP or Cy3-CTP (Low RNA Fluorescent Linear Amplification Kit, Agilent Technologies). 750 ng of Cy3 and Cy5-labeled cRNA were mixed and hybridized on an Agilent 22k human 1A array in a hybridization oven at 65oC for 17 h as rotating. Following hybridization and washing of a slide according to manufactureâ??s protocol, the arrays were scanned using the Agilent DNA Microarray Scanner (G2565BA) and microarray images were analyzed using Agilent Feature Extraction Software (v7.5). The final signal intensity was resulted from subtracting background signal from row spot signal. The gene expression level was presented as ratio of sample intensity against reference intensity.
2008-06-16 | E-GEOD-8888 | biostudies-arrayexpress
Project description:Compared to primary human monocytes in whole blood cultures, few B cells activated STAT1 in response to stimulation of 2000 IU/ml IFN-beta for 45 minutes. Because activation of STAT1 leads to apoptosis induction, we tested the hypothesis that less pro-apoptotic genes are induced in B cells as compared to monocytes. Manuscript titled: Major differences in the responses of primary human leukocyte subsets to IFN-beta. Abstract: Treatment of cell lines with type I IFNs activates the formation of ISGF3 (STAT1/STAT2/ IRF9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-beta and used flow cytometry to analyze phosphorylated STATs1, 3 and 5 in CD4+ and CD8+ T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes, CD4+ and CD8+ T cells, few B cells activated STAT1 in response to IFN-beta, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of SOCS1 or relevant protein tyrosine phosphatases in B cells. Micro-array and real-time PCR analyses revealed the induction of STAT1-dependent pro-apoptotic mRNAs in monocytes but not in B cells. These data show that ISGF3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and especially in CD4+ T cells, IFN-beta activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-beta increases the survival of primary human B cells and CD4+ T cells, but enhances the apoptosis of monocytes, and also to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections, and by type I IFN treatment of patients with multiple sclerosis, hepatitis or cancer. Undiluted whole blood of 2 healthy individuals (HI) was used, and either stimulated with IFN-beta or left untreated (control) for 3 hrs. After 3 hrs, both B cells and monocytes were isolated from whole blood of the first healthy individual, and only B cells were isolated from whole blood of the second healthy individual.
2010-10-20 | E-GEOD-23307 | biostudies-arrayexpress
Project description:Purpose: Loss-of-function mutations of the breast cancer type 1 susceptibility protein (BRCA1) are associated with breast (BC) and ovarian cancer (OC). We assessed responses to histone deacetylase inhibitors (HDACi) and performed genome-wide RNA and chromatin immunoprecipitation (ChIP)-sequencing in isogenic OC cells UWB1.289 (carrying a BRCA1 mutation) and UWB1.289 transduced with wild type BRCA1. Experimental Design: Gene expression profiles of cells harboring mutated vs. functional BRCA1 and treated with the HDACi were integrated with chromatin mapping of histone H3 lysine 9 or 27 acetylation (H3K9ac, H3K27ac) at active promoters and enhancers. Key pathways found deregulated in BRCA1 mutated cells were validated. Results: Gene networks activated in BRCA1-mutated vs. BRCA1+ OC cells relate to Cellular Movement, Cellular Development, Cellular Growth and Proliferation and shared upstream regulators included TGFb1, TNF, and IFN-g. The IFN-g pathway was significantly altered in response to HDACi in BRCA1+ vs. BRCA1-mutated cells and in BRCA1-mutated/or low vs. BRCA1-normal ovarian tumors profiled in the TCGA database. Key IFN-γ-induced genes upregulated at baseline in BRCA1-mutated vs. BRCA1+ OC and BC cells included CXCL10, CXCL11, and IFI16. Increased localization of STAT1 to the promoters of these genes was observed in BRCA1-mutated cells, resulting in diminished cellular responses to IFN-γ or to STAT1 knockdown. The IFN-g signature was associated with improved survival among tumors profiled by the TCGA. Conclusions: Transcriptomic changes affecting IFN-γ response are associated with inactivating BRCA1 mutations in OC. These alterations could contribute to diminished anti-tumor immunity in BRCA1 mutated cells or tumors.
2019-12-31 | GSE131140 | GEO
Project description:Purpose: Loss-of-function mutations of the breast cancer type 1 susceptibility protein (BRCA1) are associated with breast (BC) and ovarian cancer (OC). We assessed responses to histone deacetylase inhibitors (HDACi) and performed genome-wide RNA and chromatin immunoprecipitation (ChIP)-sequencing in isogenic OC cells UWB1.289 (carrying a BRCA1 mutation) and UWB1.289 transduced with wild type BRCA1. Experimental Design: Gene expression profiles of cells harboring mutated vs. functional BRCA1 and treated with the HDACi were integrated with chromatin mapping of histone H3 lysine 9 or 27 acetylation (H3K9ac, H3K27ac) at active promoters and enhancers. Key pathways found deregulated in BRCA1 mutated cells were validated. Results: Gene networks activated in BRCA1-mutated vs. BRCA1+ OC cells relate to Cellular Movement, Cellular Development, Cellular Growth and Proliferation and shared upstream regulators included TGFb1, TNF, and IFN-g. The IFN-g pathway was significantly altered in response to HDACi in BRCA1+ vs. BRCA1-mutated cells and in BRCA1-mutated/or low vs. BRCA1-normal ovarian tumors profiled in the TCGA database. Key IFN-γ-induced genes upregulated at baseline in BRCA1-mutated vs. BRCA1+ OC and BC cells included CXCL10, CXCL11, and IFI16. Increased localization of STAT1 to the promoters of these genes was observed in BRCA1-mutated cells, resulting in diminished cellular responses to IFN-γ or to STAT1 knockdown. The IFN-g signature was associated with improved survival among tumors profiled by the TCGA. Conclusions: Transcriptomic changes affecting IFN-γ response are associated with inactivating BRCA1 mutations in OC. These alterations could contribute to diminished anti-tumor immunity in BRCA1 mutated cells or tumors.
2019-12-31 | GSE131085 | GEO
Project description:Stringent regulation of the interferon signaling pathway is essential for maintaining the immune response to pathogens and tumors. The transcription factor STAT1 is a crucial mediator of this response. Here we show that hCAF1/CNOT7 regulates class I and II interferon pathways at different crucial steps. In resting cells hCAF1 can control STAT1 trafficking by interacting with the latent form of STAT1 in the cytoplasm. IFN treatment induces STAT1 release, suggesting that hCAF1 may shield cytoplasmic STAT1 from undesirable stimulation. Consistent, hCAF1 silencing enhances STAT1 basal promoter occupancy associated with increased expression of a subset of STAT1-regulated genes. Consequently, hCAF1 knockdown cells exhibit an increased protection against viral infection and reduced viral replication. Furthermore, hCAF1 participates in the extinction of the IFN signal, through its deadenylase activity, by speeding up the degradation of some STAT1-regulated mRNAs. Since abnormal and unbalanced JAK/STAT activation is associated with immune disorders and cancer, hCAF1 could play a major role in innate immunity and oncogenesis, contributing to tumor escape. mRNAs from cells expressing the siRNA siRNA duplexes targeting hCAF1, corresponding to the coding region 941-961 (kd) (hCAF1 NCBI Reference Sequence: NM_013354.5) and one non-targeting control siRNA (mock).
2013-12-31 | E-GEOD-43334 | biostudies-arrayexpress
Project description:BRCA1 loss leads to tumor cell transcriptional reprogramming, resulting in a DNA damage-driven, mandatory cell-autonomous type I IFN inflammatory activation mediated by STING and TREX1/2. PARP inhibition augmented this immunoreactivity, creating contextual lethality to dual immune checkpoint blockade (ICB) in vivo. BRCA1-deficient tumor can escape T-cell inflammation through targeted deletion or methylation of the DNA sensing/IFN pathway genes, such as STING, IFNB1 or the chemokine CCL5. Alternatively, BRCA-mutated carcinomas retaining immunoreactivity upregulate their VEGF-A expression driven by STING, which mediates immune resistance and tumor progression. STING elimination attenuated tumor growth and abrogated therapeutic resistance to dual ICB. VEGF-A blockade synergized with immune checkpoint blockade and/or PARP inhibition to control outgrowth of Brca1-/- ovarian tumors, offering opportunities for rational combination therapy of cancers with homologous recombination repair deficiency (HRD).
2021-06-16 | GSE162935 | GEO
Project description:Compared to primary human monocytes in whole blood cultures, few B cells activated STAT1 in response to stimulation of 2000 IU/ml IFN-beta for 45 minutes. Because activation of STAT1 leads to apoptosis induction, we tested the hypothesis that less pro-apoptotic genes are induced in B cells as compared to monocytes. Manuscript titled: Major differences in the responses of primary human leukocyte subsets to IFN-beta. Abstract: Treatment of cell lines with type I IFNs activates the formation of ISGF3 (STAT1/STAT2/ IRF9), which induces the expression of many genes. To study this response in primary cells, we treated fresh human blood with IFN-beta and used flow cytometry to analyze phosphorylated STATs1, 3 and 5 in CD4+ and CD8+ T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes, CD4+ and CD8+ T cells, few B cells activated STAT1 in response to IFN-beta, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of SOCS1 or relevant protein tyrosine phosphatases in B cells. Micro-array and real-time PCR analyses revealed the induction of STAT1-dependent pro-apoptotic mRNAs in monocytes but not in B cells. These data show that ISGF3 or STAT1 homodimers are not the main activators of gene expression in primary B cells of healthy humans. Notably, in B cells and especially in CD4+ T cells, IFN-beta activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-beta increases the survival of primary human B cells and CD4+ T cells, but enhances the apoptosis of monocytes, and also to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections, and by type I IFN treatment of patients with multiple sclerosis, hepatitis or cancer.
2010-10-20 | GSE23307 | GEO
Project description:Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically-engineered mice expressing phospho-deficient threonine748–to-alanine (T748A) mutant Stat1 are resistant to LPS–induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point-mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase (IKK)–mediated Thr748 phosphorylation of Stat1, which promotes macrophages inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent functional dichotomy of Stat1 in innate immune responses: IFN phospho-tyrosine dependent, and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover novel pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.
2024-03-18 | GSE261824 | GEO
Project description:STAT1 is an important regulator of NK cell maturation and cytotoxicity. Although the consequences of Stat1-deficiency have been described in detail the underlying molecular functions of STAT1 in NK cells are only partially understood. Here we describe a novel non-canonical role of STAT1 that was unmasked in NK cells expressing Stat1-Y701F. This mutation prevents JAK-dependent phosphorylation, subsequent nuclear translocation and cytokine-induced transcriptional activity. As expected Stat1-Y701F mice displayed impaired NK cell maturation comparable to Stat1-/- animals. In contrast Stat1-Y701F NK cells exerted a significantly enhanced cytotoxicity in vitro and in vivo suggesting a so-far unknown cytoplasmic function. Using immunofluorescence technology we uncovered the recruitment of STAT1 to the immunological synapse during NK cell killing. A Stat1ind mouse expressing FLAG-tagged STAT1α was used to study the STAT1α interactome in NK cells. Mass spectrometry revealed that STAT1 directly binds proteins involved in cell junction formation and proteins associated to membrane or membrane-bound vesicles. We propose a novel function for STAT1 in the immunological synapse of NK cells regulating tumor surveillance and cytotoxicity.
2016-10-07 | PXD002206 | Pride
Project description:Type I interferons (IFN-I) are critical in antimicrobial and antitumor defense. Although IFN-I signal via the interferon-stimulated gene factor 3 (ISGF3) complex consisting of STAT1, STAT2 and IRF9, IFN-I can mediate significant biological effects via ISGF3-independent pathways. For example, absence of STAT1, STAT2 or IRF9 exacerbates neurological disease in transgenic mice with CNS-production of IFN-gamma. Here we determined the role of IFN-I-driven, ISGF3-independent signaling in regulating global gene expression in STAT1, STAT2 or IRF9-deficient murine mixed glial cell cultures (MGCs). Compared with WT, the expression of IFN-gamma-stimulated genes (ISGs) was reduced in number and magnitude in MGCs that lacked STAT1, STAT2 or IRF9. There were significantly fewer ISGs in the absence of STAT1 or STAT2 versus the absence of IRF9. The majority of ISGs regulated in the STAT1-, STAT2- or IRF9-deficient MGCs individually were shared with WT. However, only a minor number of ISGs were common to WT, STAT1-, STAT2- and IRF9-deficient MGCs. While signal pathway activation in response to IFN-gamma was rapid and transient in WT MGCs, this was delayed and prolonged and correlated with increased numbers of ISGs expressed at 12 h versus 4 h IFN-gamma exposure in all three IFN-I-signaling-deficient MGCs. In conclusion, (1) IFN-I can mediate ISG expression in MGCs via ISGF3-independent signaling pathways but with reduced efficiency, with delayed and prolonged kinetics and is more dependent on STAT1 and STAT2 than IRF9, and (2) signaling pathways not involving STAT1, STAT2 or IRF9 play a minor role only in mediating ISG expression in MGCs.
2017-02-15 | E-MTAB-5445 | biostudies-arrayexpress