scholarly journals The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Margaret E Torrence ◽  
Michael R MacArthur ◽  
Aaron M Hosios ◽  
Alexander J Valvezan ◽  
John M Asara ◽  
...  
Keyword(s):  
Target Genes ◽  
Human Cancer ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Glutathione Synthesis ◽  
Human Cancer Cell Lines ◽  
Downstream Target ◽  
Mtorc1 Signaling ◽  
Transcriptional Changes ◽  
Established Role

The mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, its broader roles as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon insulin-stimulated mTORC1 signaling to those activated by the ISR. In multiple mouse embryo fibroblast (MEF) and human cancer cell lines, the mTORC1-ATF4 pathway stimulated expression of only a subset of the ATF4 target genes induced by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.

scholarly journals The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals

2020 ◽  
Author(s):  
Margaret E. Torrence ◽  
Michael R. MacArthur ◽  
Aaron M. Hosios ◽  
Alexander J. Valvezan ◽  
John M. Asara ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Target Genes ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Glutathione Synthesis ◽  
Downstream Target ◽  
Mechanistic Target Of Rapamycin ◽  
Transcriptional Changes ◽  
Growth Promoting ◽  
Established Role

AbstractThe mechanistic target of rapamycin complex 1 (mTORC1) stimulates a coordinated anabolic program in response to growth-promoting signals. Paradoxically, recent studies indicate that mTORC1 can activate the adaptive stress-responsive transcription factor ATF4 through mechanisms distinct from its canonical induction by the integrated stress response (ISR). However, how ATF4 functions in response to these distinct modes of regulation and its broader role as a downstream target of mTORC1 are unknown. Therefore, we directly compared ATF4-dependent transcriptional changes induced upon activation of mTORC1 or the ISR. The mTORC1-ATF4 pathway stimulated the expression of only a subset of the ATF4 target genes stimulated by the ISR, including genes involved in amino acid uptake, synthesis, and tRNA charging. We demonstrate that ATF4 is a metabolic effector of mTORC1 involved in both its established role in promoting protein synthesis and in a previously unappreciated function for mTORC1 in stimulating cellular cystine uptake and glutathione synthesis.

scholarly journals Research Resource: Expression Profiling Reveals Unexpected Targets and Functions of the Human Steroid Receptor RNA Activator (SRA) Gene

2010 ◽  
Vol 24 (5) ◽  
pp. 1090-1105 ◽  
Author(s):  
Charles E. Foulds ◽  
Anna Tsimelzon ◽  
Weiwen Long ◽  
Andrew Le ◽  
Sophia Y. Tsai ◽  
...  
Keyword(s):  
Target Genes ◽  
Human Cancer ◽  
Myogenic Differentiation ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Small Subset ◽  
Small Interfering Rnas ◽  
Human Cancer Cell Lines ◽  
Downstream Target ◽  
Bona Fide

Abstract The human steroid receptor RNA activator (SRA) gene encodes both noncoding RNAs (ncRNAs) and protein-generating isoforms. In reporter assays, SRA ncRNA enhances nuclear receptor and myogenic differentiation 1 (MyoD)-mediated transcription but also participates in specific corepressor complexes, serving as a distinct scaffold. That SRA RNA levels might affect some biological functions, such as proliferation, apoptosis, steroidogenesis, and myogenesis, has been reported. However, the breadth of endogenous target genes that might be regulated by SRA RNAs remains largely unknown. To address this, we depleted SRA RNA in two human cancer cell lines with small interfering RNAs and then assayed for changes in gene expression by microarray analyses. The majority of significantly changed genes were reduced upon SRA knockdown, implicating SRA RNAs as endogenous coactivators. Unexpectedly, only a small subset of direct estrogen receptor-α target genes was affected in estradiol-treated MCF-7 cells. Eight bona fide SRA downstream target genes were identified (SLC2A3, SLC2A12, CCL20, TGFB2, DIO2, TMEM65, TBL1X, and TMPRSS2), representing entirely novel SRA targets, except for TMPRSS2. These data suggest unanticipated roles for SRA in glucose uptake, cellular signaling, T3 hormone generation, and invasion/metastasis. SRA depletion in MDA-MB-231 cells reduced invasiveness and expression of some genes critical for this process. Consistent with the knockdown data, overexpressed SRA ncRNA coactivates certain target promoters and may enhance the activity of some coregulatory proteins. This study is a valuable resource because it represents the first genome-wide analysis of a mammalian RNA coregulator.

scholarly journals FOXA1 defines cancer cell specificity

2016 ◽  
Vol 2 (3) ◽  
pp. e1501473 ◽  
Author(s):  
Gaihua Zhang ◽  
Yongbing Zhao ◽  
Yi Liu ◽  
Li-Pin Kao ◽  
Xiao Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cancer Cell ◽  
Cancer Progression ◽  
Target Genes ◽  
Human Cancer ◽  
Cell Types ◽  
Human Cancer Cell Lines ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Pioneer Transcription Factor ◽  
Specific Regulation

A transcription factor functions differentially and/or identically in multiple cell types. However, the mechanism for cell-specific regulation of a transcription factor remains to be elucidated. We address how a single transcription factor, forkhead box protein A1 (FOXA1), forms cell-specific genomic signatures and differentially regulates gene expression in four human cancer cell lines (HepG2, LNCaP, MCF7, and T47D). FOXA1 is a pioneer transcription factor in organogenesis and cancer progression. Genomewide mapping of FOXA1 by chromatin immunoprecipitation sequencing annotates that target genes associated with FOXA1 binding are mostly common to these cancer cells. However, most of the functional FOXA1 target genes are specific to each cancer cell type. Further investigations using CRISPR-Cas9 genome editing technology indicate that cell-specific FOXA1 regulation is attributable to unique FOXA1 binding, genetic variations, and/or potential epigenetic regulation. Thus, FOXA1 controls the specificity of cancer cell types. We raise a “flower-blooming” hypothesis for cell-specific transcriptional regulation based on these observations.

microRNA Control of Hematopoietic Stem Cell Fitness and Myelopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 373-373 ◽  
Author(s):  
Chinavenmeni S. Velu ◽  
Sarah Porteous ◽  
Haiming Xu ◽  
Avinash M. Baktula ◽  
Philip Roehrs ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Lines ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Human Cancer ◽  
Fetal Liver ◽  
Embryonic Stem ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Human Cancer Cell Lines

Abstract The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell (HSC) self renewal and myeloid differentiation. Gfi1 null mice eventually die of HSC exhaustion, and Gfi1−/− HSC are not competitive in transplantation assays. Here we show that Gfi1 is a master regulator of microRNAs and that transcriptional control of a microRNA gene is critical for Gfi1-directed stem cell competitiveness and myelopoiesis. First, we show that the gene encoding miR21 is a direct transcriptional target of Gfi1. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal Gfi1 binding to specific DNA sequences upstream of the miR21 stem loop. Moreover, the expression of Gfi1 and miR21 is reciprocal in 1) wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), and 3) during treatment-induced differentiation of human myeloid leukemia cell lines. Forced expression of Gfi1 lowers miR21 levels in wild type Lin− bone marrow cells and human cancer cell lines. Knockdown of Gfi1 expression with shRNA in human cancer cell lines increases miR21 expression. Moreover, conditional deletion of Gfi1 induces miR21 expression in primary murine hematopoietic cells, including sorted CMP and GMP. Thus, Gfi1 transcriptionally regulates miR21 in both human and murine hematopoietic cells. Interestingly, we find that the Ski oncoprotein/transcriptional corepressor is a direct target of miR-21. Subsequently, Ski is dramatically reduced in Gfi1−/− HSC and in wild type bone marrow Lin− cells forced to express miR21. Gfi1 may repress miR21 to maintain functional competence. Specifically, we find that Ski is a previously undescribed Gfi1 corepressor. Both endogenous Ski and Gfi1 physically interact. Synthetic Ski and Gfi1 proteins reveal that the interaction is both strong and specific. Chromatin immunoprecipitation reveals Ski and Gfi1 occupy several Gfi1 target genes. However, Ski function is critical as a corepressor on only a subset of Gfi1 target genes. To determine the importance of Ski corepression to Gfi1 induced biology, we examined two well established phenotypes of Gfi1 loss of function; HSC competitiveness and myelopoiesis. When Gfi1−/− embryonic stem cells are injected into a wild type blastocyst, they do not participate in hematopoiesis. Similarly, we find that when Ski−/− embryonic stem cells are injected into a blastocyst, they infrequently participate in hematopoeisis. Next, because Ski−/− animals die at or before birth, we examined the fitness of Ski−/− fetal liver HSC. In competitive transplantation assays, Ski−/− fetal liver HSC were significantly impaired in reconstitution compared to congenic wild-type competitor fetal-liver HSC. Moreover, Ski null HSC generated significantly less myeloid progeny. Thus, Ski−/− HSC display a partial phenocopy of Gfi1−/− hematopoiesis. We conclude that Gfi1 directly targets miR21 to control the expression of Ski, a corepressor for Gfi1, and that the Gfi1/Ski complex is critical to regulate a subset of Gfi1 target genes important for HSC fitness and myeloid cell production.

scholarly journals Development and interlaboratory evaluation of a NIST Reference Material RM 8366 for EGFR and MET gene copy number measurements

2019 ◽  
Vol 57 (8) ◽  
pp. 1142-1152 ◽  
Author(s):  
Hua-Jun He ◽  
Biswajit Das ◽  
Megan H. Cleveland ◽  
Li Chen ◽  
Corinne E. Camalier ◽  
...  
Keyword(s):  
Reference Material ◽  
Copy Number ◽  
Target Genes ◽  
Human Cancer ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Cancer Diagnostics ◽  
Gene Copy ◽  
Human Cancer Cell Lines ◽  
Copy Numbers

Abstract Background The National Institute of Standards and Technology (NIST) Reference Material RM 8366 was developed to improve the quality of gene copy measurements of EGFR (epidermal growth factor receptor) and MET (proto-oncogene, receptor tyrosine kinase), important targets for cancer diagnostics and treatment. The reference material is composed of genomic DNA prepared from six human cancer cell lines with different levels of amplification of the target genes. Methods The reference values for the ratios of the EGFR and MET gene copy numbers to the copy numbers of reference genes were measured using digital PCR. The digital PCR measurements were confirmed by two additional laboratories. The samples were also characterized using Next Generation Sequencing (NGS) methods including whole genome sequencing (WGS) at three levels of coverage (approximately 1 ×, 5 ×  and greater than 30 ×), whole exome sequencing (WES), and two different pan-cancer gene panels. The WES data were analyzed using three different bioinformatic algorithms. Results The certified values (digital PCR) for EGFR and MET were in good agreement (within 20%) with the values obtained from the different NGS methods and algorithms for five of the six components; one component had lower NGS values. Conclusions This study shows that NIST RM 8366 is a valuable reference material to evaluate the performance of assays that assess EGFR and MET gene copy number measurements.

scholarly journals Somatic mutations in early metazoan genes disrupt regulatory links between unicellular and multicellular genes in cancer

2018 ◽  
Author(s):  
Anna S. Trigos ◽  
Richard B. Pearson ◽  
Anthony T. Papenfuss ◽  
David L. Goode
Keyword(s):  
Regulatory Networks ◽  
Target Genes ◽  
Point Mutations ◽  
Primitive Elements ◽  
Clinical Value ◽  
Recurrent Point ◽  
Downstream Target ◽  
Transcriptional Alterations ◽  
Transcriptional Changes ◽  
Regulator Genes

AbstractExtensive transcriptional alterations are observed in cancer, many of which activate core biological processes established in unicellular organisms or suppress differentiation pathways formed in metazoans. Through rigorous, integrative analysis of genomics data from a range of solid tumours, we show many transcriptional changes in tumours are tied to mutations disrupting regulatory interactions between unicellular and multicellular genes within human gene regulatory networks (GRNs). Recurrent point mutations were enriched in regulator genes linking unicellular and multicellular subnetworks, while copy-number alterations affected downstream target genes in distinctly unicellular and multicellular regions of the GRN. Our results depict drivers of tumourigenesis as genes that created key regulatory links during the evolution of early multicellular life, whose dysfunction creates widespread dysregulation of primitive elements of the GRN. Several genes we identified as important in this process were associated with drug response, demonstrating the potential clinical value of our approach.

scholarly journals Loss of the Notch effector RBPJ promotes tumorigenesis

2014 ◽  
Vol 212 (1) ◽  
pp. 37-52 ◽  
Author(s):  
Iva Kulic ◽  
Gordon Robertson ◽  
Linda Chang ◽  
Jennifer H.E. Baker ◽  
William W. Lockwood ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Growth ◽  
Target Genes ◽  
Human Cancer ◽  
Global Analysis ◽  
Notch Pathway ◽  
Gene Promoters ◽  
Human Cancer Cell Lines ◽  
Immunodeficient Mice ◽  
Cell Death Pathway

Aberrant Notch activity is oncogenic in several malignancies, but it is unclear how expression or function of downstream elements in the Notch pathway affects tumor growth. Transcriptional regulation by Notch is dependent on interaction with the DNA-binding transcriptional repressor, RBPJ, and consequent derepression or activation of associated gene promoters. We show here that RBPJ is frequently depleted in human tumors. Depletion of RBPJ in human cancer cell lines xenografted into immunodeficient mice resulted in activation of canonical Notch target genes, and accelerated tumor growth secondary to reduced cell death. Global analysis of activated regions of the genome, as defined by differential acetylation of histone H4 (H4ac), revealed that the cell death pathway was significantly dysregulated in RBPJ-depleted tumors. Analysis of transcription factor binding data identified several transcriptional activators that bind promoters with differential H4ac in RBPJ-depleted cells. Functional studies demonstrated that NF-κB and MYC were essential for survival of RBPJ-depleted cells. Thus, loss of RBPJ derepresses target gene promoters, allowing Notch-independent activation by alternate transcription factors that promote tumorigenesis.

scholarly journals p53-Related Transcription Targets of TAp73 in Cancer Cells—Bona Fide or Distorted Reality?

2020 ◽  
Vol 21 (4) ◽  
pp. 1346
Author(s):  
Chao Wang ◽  
Cui Rong Teo ◽  
Kanaga Sabapathy
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Target Genes ◽  
Growth Promotion ◽  
Human Cancer ◽  
Cancer Cell Lines ◽  
Cellular Growth ◽  
Developmental Defects ◽  
Human Cancer Cell Lines ◽  
Cellular Survival

Identification of p73 as a structural homolog of p53 fueled early studies aimed at determining if it was capable of performing p53-like functions. This led to a conundrum as p73 was discovered to be hardly mutated in cancers, and yet, TAp73, the full-length form, was found capable of performing p53-like functions, including transactivation of many p53 target genes in cancer cell lines. Generation of mice lacking p73/TAp73 revealed a plethora of developmental defects, with very limited spontaneous tumors arising only at a later stage. Concurrently, novel TAp73 target genes involved in cellular growth promotion that are not regulated by p53 were identified, mooting the possibility that TAp73 may have diametrically opposite functions to p53 in tumorigenesis. We have therefore comprehensively evaluated the TAp73 target genes identified and validated in human cancer cell lines, to examine their contextual relevance. Data from focused studies aimed at appraising if p53 targets are also regulated by TAp73—often by TAp73 overexpression in cell lines with non-functional p53—were affirmative. However, genome-wide and phenotype-based studies led to the identification of TAp73-regulated genes involved in cellular survival and thus, tumor promotion. Our analyses therefore suggest that TAp73 may not necessarily be p53’s natural substitute in enforcing tumor suppression. It has likely evolved to perform unique functions in regulating developmental processes and promoting cellular growth through entirely different sets of target genes that are not common to, and cannot be substituted by p53. The p53-related targets initially reported to be regulated by TAp73 may therefore represent an experimental possibility rather than the reality.

scholarly journals The FKH domain in FOXP3 mRNA frequently contains mutations in hepatocellular carcinoma that influence the subcellular localization and functions of FOXP3

2020 ◽  
Vol 295 (16) ◽  
pp. 5484-5495 ◽  
Author(s):  
Jianwei Ren ◽  
Yi Liu ◽  
Shanshan Wang ◽  
Yu Wang ◽  
Wende Li ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Subcellular Localization ◽  
Genomic Dna ◽  
Target Genes ◽  
Human Cancer ◽  
Luciferase Assay ◽  
Mouse Tumor ◽  
Foxp3 Mrna ◽  
Human Cancer Cell Lines

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

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