scholarly journals Hippo pathway effectors YAP1/TAZ induce a EWS-FLI1-opposing gene signature and associate with disease progression in Ewing Sarcoma

2019 ◽  
Author(s):  
Pablo Rodríguez-Núñez ◽  
Laura Romero-Pérez ◽  
Ana T. Amaral ◽  
Pilar Puerto-Camacho ◽  
Carmen Jordán ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Fusion Protein ◽  
Cell Lines ◽  
Ewing Sarcoma ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Hippo Pathway ◽  
Protein A ◽  
Gene Signature ◽  
Cell Of Origin

AbstractYAP1 and TAZ (WWTR1) oncoproteins are the final transducers of Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing Sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involvingEWSR1gene, andFLI1as the most common partner. The fusion protein is a potent driver of oncogenesis but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome.We did not observe recurrent SNV or copy number gains/losses in Hippo pathway-related loci. However, differential CpG methylation ofRASSF1locus -a regulator of Hippo pathway- was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation ofRASSF1correlated with the transcriptional silencing of the tumor suppressor isoformRASFF1A, and transcriptional activation of the protumorigenic isoformRASSF1Cpromoting YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells, and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS-FLI1 transcriptional signature. This transcriptional antagonism could be partly explained by EWS-FLI1-mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS.

scholarly journals Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion

2019 ◽  
Vol 111 (9) ◽  
pp. 970-982 ◽  
Author(s):  
Sabine Heitzeneder ◽  
Elena Sotillo ◽  
Jack F Shern ◽  
Sivasish Sindiri ◽  
Peng Xu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Plasma Protein ◽  
Immune Evasion ◽  
Ewing Sarcoma ◽  
Antigen Processing ◽  
Expression Profiles ◽  
Statistical Tests ◽  
Protein A ◽  
Differentially Expressed

AbstractBackgroundEwing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling.MethodsBy comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided.ResultsEWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways.ConclusionThis work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.

EAF1, a novel ELL-associated factor that is delocalized by expression of the MLL-ELL fusion protein

Blood ◽  
2001 ◽  
Vol 98 (1) ◽  
pp. 201-209 ◽  
Author(s):  
Federico Simone ◽  
Paul E. Polak ◽  
Joseph J. Kaberlein ◽  
Roger T. Luo ◽  
Denise A. Levitan ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Lines ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Fusion Gene ◽  
Elongation Factor ◽  
Chimeric Protein ◽  
Leukemic Cell ◽  
Transcriptional Elongation ◽  
Associated Factor

Abstract The (11;19)(q23;p13.1) translocation in acute leukemia leads to the generation of a chimeric protein that fuses MLL to the transcriptional elongation factor ELL. A novel protein was isolated from a yeast 2-hybrid screen with ELL that was named EAF1 for ELL-associated factor 1. Using specific antibodies, the endogenous EAF1 and ELL proteins were coimmunoprecipitated from multiple cell lines. In addition, endogenous EAF1 also exhibited the capacity to interact with ELL2. Database comparisons with EAF1 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that exhibited homology with the transcriptional activation domains of several translocation partner proteins of MLL, including AF4, LAF4, and AF5q31. A similar transcriptional activation domain has been identified in this region of EAF1. By confocal microscopy, endogenous EAF1 and ELL colocalized in a distinct nuclear speckled pattern. Transfection of theMLL-ELL fusion gene delocalized EAF1 from its nuclear speckled distribution to a diffuse nucleoplasmic pattern. In leukemic cell lines derived from mice transplanted withMLL-ELL–transduced bone marrow, EAF1 speckles were not detected. Taken together, these data suggest that expression of the MLL-ELL fusion protein may have a dominant effect on the normal protein-protein interactions of ELL.

scholarly journals Transcriptional Repression of the Neurofibromatosis-1 Tumor Suppressor by the t(8;21) Fusion Protein

2005 ◽  
Vol 25 (14) ◽  
pp. 5869-5879 ◽  
Author(s):  
Genyan Yang ◽  
Waleed Khalaf ◽  
Louis van de Locht ◽  
Joop H. Jansen ◽  
Meihua Gao ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Fusion Protein ◽  
Transcriptional Repression ◽  
Genetic Disorder ◽  
Suppressor Gene ◽  
Juvenile Myelomonocytic Leukemia ◽  
Increased Risk ◽  
Myeloid Progenitor Cells ◽  
Macrophage Colony

ABSTRACT Von Recklinghausen's disease is a relatively common familial genetic disorder characterized by inactivating mutations of the Neurofibromatosis-1 (NF1) gene that predisposes these patients to malignancies, including an increased risk for juvenile myelomonocytic leukemia. However, NF1 mutations are not common in acute myeloid leukemia (AML). Given that the RUNX1 transcription factor is the most common target for chromosomal translocations in acute leukemia, we asked if NF1 might be regulated by RUNX1. In reporter assays, RUNX1 activated the NF1 promoter and cooperated with C/EBPα and ETS2 to activate the NF1 promoter over 80-fold. Moreover, the t(8;21) fusion protein RUNX1-MTG8 (R/M), which represses RUNX1-regulated genes, actively repressed the NF1 promoter. R/M associated with the NF1 promoter in vivo and repressed endogenous NF1 gene expression. In addition, similar to loss of NF1, R/M expression enhanced the sensitivity of primary myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor. Our results indicate that the NF1 tumor suppressor gene is a direct transcriptional target of RUNX1 and the t(8;21) fusion protein, suggesting that suppression of NF1 expression contributes to the molecular pathogenesis of AML.

scholarly journals Human SWI-SNF Component BRG1 Represses Transcription of the c-fos Gene

1999 ◽  
Vol 19 (4) ◽  
pp. 2724-2733 ◽  
Author(s):  
Daniel J. Murphy ◽  
Stephen Hardy ◽  
Daniel A. Engel
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Protein A ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Dependent Atpase ◽  
Adenocarcinoma Cells ◽  
Cyclic Amp Response Element ◽  
Function Expression ◽  
Transcription Factor E2f

ABSTRACT Yeast and mammalian SWI-SNF complexes regulate transcription through active modification of chromatin structure. Human SW-13 adenocarcinoma cells lack BRG1 protein, a component of SWI-SNF that has a DNA-dependent ATPase activity essential for SWI-SNF function. Expression of BRG1 in SW-13 cells potentiated transcriptional activation by the glucocorticoid receptor, which is known to require SWI-SNF function. BRG1 also specifically repressed transcription from a transfected c-fos promoter and correspondingly blocked transcriptional activation of the endogenous c-fos gene. Mutation of lysine residue 798 in the DNA-dependent ATPase domain of BRG1 significantly reduced its ability to repress c-fostranscription. Repression by BRG1 required the cyclic AMP response element of the c-fos promoter but not nearby binding sites for Sp1, YY1, or TFII-I. Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb protein, transcriptional repression by BRG1 was weak unless wild-type Rb was also supplied. Interestingly, Rb-dependent repression by BRG1 was found to take place through a pathway that is independent of transcription factor E2F.

scholarly journals Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Min Kang ◽  
Bo Tang ◽  
Jixi Li ◽  
Ziyan Zhou ◽  
Kang Liu ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Tumor Growth ◽  
Prognostic Marker ◽  
Cell Lines ◽  
Cancer Cell ◽  
Transcriptional Activation ◽  
Cancer Cell Lines ◽  
Migration And Invasion ◽  
Clinical Samples

Abstract Background Very few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133). Methods We overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation. Results miPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass. Conclusion miPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.

scholarly journals MYC-driven epigenetic reprogramming favors the onset of tumorigensis by inducing a stem cell-like state

2017 ◽  
Author(s):  
Vittoria Poli ◽  
Luca Fagnocchi ◽  
Alessandra Fasciani ◽  
Alessandro Cherubini ◽  
Stefania Mazzoleni ◽  
...  
Keyword(s):  
Stem Cell ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
De Novo ◽  
Mammary Epithelial Cells ◽  
Epigenetic Reprogramming ◽  
Breast Cancers ◽  
Cell Of Origin ◽  
Tumor Initiating Cells ◽  
Oncogenic Pathways

AbstractBreast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes resulted difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Over-expression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.

The FRA12E Fragile Site Is Localized within the SMRT Gene at 12q24.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4274-4274
Author(s):  
Lionel Coignet ◽  
Jean Dolcce ◽  
Pushpankur Ghoshal ◽  
Alain Nganga ◽  
Timothy Johnson ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Lines ◽  
Nuclear Receptors ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Fragile Site ◽  
Fragile Sites ◽  
Lymphoblastoid Cell Lines ◽  
Metaphase Chromosomes ◽  
Chromosome 3P

Abstract The Silencing Mediator of retinoid and Thyroid hormone (SMRT) is a transcription co-repressor whose association with nuclear receptors both in solution and bound to DNA response elements is destabilized by ligand. SMRT and a related co-repressor N-CoR are recruiting a transcriptional repression complex, that contains sin3A/B protein and histone deacetylases (HDAC1/2) to nuclear receptors. It is now well established that SMRT can interact with non-receptor proteins such as BCL6, for its repression activities. The ability of the “silencing complex” to deacetylate histones results in a condensed chromatin state that is inhibitory to transcription. We have previously shown that SMRT was localized at chromosome 12q24. In addition, down-regulation of the SMRT protein due to 12q24 rearrangements was recurrently associated with NHL transformation, supporting a tumor suppressor function for SMRT (Cancer Res, 65(11):4554–4561, 2005). We further investigated the reasons why the 12q24 region and SMRT was targeted by chromosomal rearrangements. Genetic breakage is one mechanism by which functional loss of tumor suppressor gene activity may occur. Chromosomal locations in which genetic breakage may be induced are known as fragile sites. Fragile sites have been shown to be involved in some malignancies in which the fragile site lies within known genes, such as the FHIT gene (chromosome 3p) in lung cancer, and where small deletions are consistently observed on chromosome 3. Two fragile sites exist on the long arm of chromosome 12. FRA12B is located at 12q24.13 and FRA12E has been located at 12q24.2–3. Interestingly the FRA12E region corresponds to the site of SMRT (12q24.2). To assess whether the FRA12E fragile site is localized within the SMRT gene, we studied normal lymphocytes cultured in the presence of aphidicolin (an inducer of fragile sites) and metaphase chromosomes were subsequently prepared following classical cytogenetics protocols. These preparations were then subjected to fluorescence in situ hybridization (FISH) using a set of SMRT-specific RPCI BAC clones. Using this approach, we were able to demonstrate that the FRA12E fragile site is localized within the SMRT gene. We further characterize several lymphoblastoid cell lines that were either carrier or not of the FRA12E fragile site. This provides us with a mechanistic explanation for recurrent 12q24 rearrangements seen in transformed NHLs. The FRA12E-carrier lymphoblastoid cell lines will provide us the opportunity to characterize the structure of this fragile site.

Tumor Suppressor RASSF2 Is Downregulated By The RUNX1-ETO Fusion Protein In t(8;21)+ Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1268-1268
Author(s):  
Samuel A Stoner ◽  
Russell Dekelver ◽  
Miao-Chia Lo ◽  
Dong-Er Zhang
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Tumor Suppressor ◽  
Fusion Protein ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Over Expression ◽  
Apoptotic Protein ◽  
Acute Myeloid

Abstract Introduction The t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), present in greater than 10% of de novo AML cases. Most of these t(8;21) AML cases are classified as FAB subtype M2. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (aka AML1) and ETO (aka MTG8 and RUNX1T1) proteins. RUNX1 is a transcription factor that is essential for regulating the differentiation of hematopoietic cells, and the fusion protein retains its DNA-binding domain. Additionally, ETO contains four Nervy homology (NH) domains which facilitate a number of protein-protein interactions, notably with the NCOR2/SMRT co-repressor complex. The identification of individual genes or biological pathways which are specifically disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21)+ AML and lead to the possibility for improved treatment for these patients. Methods/Results We analyzed publicly available gene expression microarray datasets (Oncomine, TCGA) to search for genes whose expression was significantly altered in the blood of t(8;21)+ AML patients as compared to non-t(8;21) FAB subtype M2 AML and to CD34+ cells in healthy controls. One such gene that was consistently significantly downregulated in t(8;21)+ patients was Ras-association domain family member 2 (RASSF2). RASSF2 is a putative tumor suppressor that is capable of mediating apoptosis (in a Ras dependent manner) through its interactions with the MST1/2 kinases and the cancer-specific apoptotic protein Par-4. RASSF2 has previously been shown to be frequently downregulated via hypermethylation in a wide variety of solid tumors, however little is known about its function in leukemia. Here we demonstrate that RASSF2 is a potentially interesting target for downregulation by the RUNX1-ETO fusion protein. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 is significantly downregulated in both Kasumi-1 and SKNO t(8;21)+ cell lines as compared to a similar non-t(8;21) HL-60 line. We found that exogenous expression of AML1-ETO in HL-60 leukemia cells induces a rapid downregulation of RASSF2, further supporting that it is a target of this leukemogenic fusion protein. Over-expression of RASSF2 in leukemia cells significantly inhibits their proliferative capability, indicating an important biological effect of RASSF2 in blood cells. Finally, over-expression of RASSF2 significantly inhibits the long-term self-renewal capability of RUNX1-ETO expressing hematopoietic cells as measured by their serial replating ability in a colony formation assay. Discussion Based on the analysis of patient data and our own experiments it appears that RASSF2 is a direct target for downregulation by the AML1-ETO fusion protein. Due to its potential involvement as a mediator of apoptosis in important oncogenic signaling pathways RASSF2 is a strong candidate for further investigation in the context of t(8;21)+ AML pathogenesis. In particular, it will be interesting to continue to investigate the relationship between RASSF2 and apoptotic protein Par-4, as several lines of evidence suggest Par-4 to be therapeutically relevant due to its ability to selectively induce apoptosis in cancer cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma.

1994 ◽  
Vol 14 (5) ◽  
pp. 3230-3241 ◽  
Author(s):  
R A Bailly ◽  
R Bosselut ◽  
J Zucman ◽  
F Cormier ◽  
O Delattre ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Ewing Sarcoma ◽  
Transcriptional Activation ◽  
Transcriptional Regulators ◽  
Chromosome Translocation ◽  
Sequence Specificity ◽  
Genes Encoding ◽  
Ets Family ◽  
Efficient Sequence

The 5' half of the EWS gene has recently been described to be fused to the 3' regions of genes encoding the DNA-binding domain of several transcriptional regulators, including ATF1, FLI-1, and ERG, in several human tumors. The most frequent occurrence of this situation results from the t(11;22)(q24;q12) chromosome translocation specific for Ewing sarcoma (ES) and related tumors which joins EWS sequences to the 3' half of FLI-1, which encodes a member of the Ets family of transcriptional regulators. We show here that this chimeric gene encodes an EWS-FLI-1 nuclear protein which binds DNA with the same sequence specificity as the wild-type parental FLI-1 protein. We further show that EWS-FLI-1 is an efficient sequence-specific transcriptional activator of model promoters containing FLI-1 (Ets)-binding sites, a property which is strictly dependent on the presence of its EWS domain. Comparison of the properties of the N-terminal activation domain of FLI-1 to those of the EWS domain of the fusion protein indicates that EWS-FLI-1 has altered transcriptional activation properties compared with FLI-1. These results suggest that EWS-FLI-1 contributes to the transformed phenotype of ES tumor cells by inducing the deregulated and/or unscheduled activation of genes normally responsive to FLI-1 or to other close members of the Ets family. ES and related tumors are characterized by an elevated level of c-myc expression. We show that EWS-FLI-1 is a transactivator of the c-myc promoter, suggesting that upregulation of c-myc expression is under control of EWS-FLI-1.

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