Targeted Downregulation of MYC through G-quadruplex Stabilization by DNAi.
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ABSTRACT:
SUBMITTER: Psaras AM
PROVIDER: S-EPMC8464964 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III(1) region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III(1) region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.
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Project description:The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.
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Project description:Although mutations in the gene encoding the RNA splicing factor SF3B1 are frequent in multiple cancers, their functional effects and therapeutic dependencies are poorly understood. Here, we characterize 98 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations, identifying hundreds of cryptic 3' splice sites common and specific to different cancer types. Regulatory network analysis revealed that the most common SF3B1 mutation activates MYC via effects conserved across human and mouse cells. SF3B1 mutations promote decay of transcripts encoding the protein phosphatase 2A (PP2A) subunit PPP2R5A, increasing MYC S62 and BCL2 S70 phosphorylation which, in turn, promotes MYC protein stability and impair apoptosis, respectively. Genetic PPP2R5A restoration or pharmacologic PP2A activation impaired SF3B1-mutant tumorigenesis, elucidating a therapeutic approach to aberrant splicing by mutant SF3B1. SIGNIFICANCE: Here, we identify that mutations in SF3B1, the most commonly mutated splicing factor gene across cancers, alter splicing of a specific subunit of the PP2A serine/threonine phosphatase complex to confer post-translational MYC and BCL2 activation, which is therapeutically intervenable using an FDA-approved drug.See related commentary by O'Connor and Narla, p. 765.This article is highlighted in the In This Issue feature, p. 747.
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Project description:The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.
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Project description:The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.
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Project description:BACKGROUND:Heat shot protein 90 (HSP90) AA1 functions as an onco-protein to regulate the assembly, manipulation, folding and degradation of its client proteins, including c-MYC. However, little is known about the mechanism of HSP90AA1 regulation. METHODS:Transcriptome RNA-sequencing data of hepatocellular carcinoma (HCC) samples were used to detect the mRNA expression of FBXL6. Immunoprecipitation/Mass Spectrum (IP/MS) method was used to identify the interacting proteins of FBXL6. The co-immunoprecipitation assay was used to determine the interaction between FBXL6 and HSP90AA1. The in vivo ubiquitination assay was performed to determine the regulation of HSP90AA1 by FBXL6. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to determine the transcriptional regulation of FBXL6 by c-MYC. Immunohistochemical (IHC) staining was performed to study the correlation of FBXL6 and HSP90AA1 protein expression in 87 HCC samples. Cell counting and colony formation assays were implemented to detect the biological effects of FBXL6 on the growth of HCC cells in vitro. The effect of FBXL6 on HCC tumor growth in vivo was studied in a tumor xenograft model in mice. RESULTS:Here, we identified the orphan F-box protein FBXL6, a substrate recognition subunit of an SCF (Skp1-Cul1-F-box protein) complex, as the ubiquitin ligase for HSP90AA1. FBXL6 promoted K63-dependent ubiquitination of HSP90AA1 to stabilize it. Through analysis of the TCGA dataset, we found that FBXL6 was significantly increased in HCC tissues and positively correlated with c-MYC pathway. FBXL6 accumulation in HCC causes the stabilization and activation of c-MYC by preventing HSP90AA1 degradation. The activated c-MYC directly binds to the promoter region of FBXL6 to induce its mRNA expression. CONCLUSION:Collectively, our data revealed an unknown FBXL6-HSP90AA1-c-MYC axis which might contribute to the oncogenesis of HCC, and we propose that inhibition of FBXL6 might represent an effective therapeutic strategy for HCC treatment. Video abstract.
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Project description:Host colonization by lymphotropic ?-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of ?-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic ?-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC5S(mLANA) modulates cellular control of Myc turnover by antagonizing SCF(Fbw7) mediated proteasomal degradation of Myc, mimicking SCF(?-TrCP). The findings here reported reveal that modulation of Myc is essential for ?-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease.
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Project description:Although mutations in the RNA splicing factor SF3B1 are frequently observed in multiple human cancers, their functional role in promoting tumorigenesis and therapeutic significance remain poorly understood. Here we characterize the splicing landscape of 79 tumors and 12 isogenic cell lines harboring SF3B1 hotspot mutations, identifying hundreds of cryptic 3’ splice site (ss) events shared as well as specific to different tumor types. Regulatory network analysis shows that tumors harboring the most common hotspot mutation in SF3B1 (SF3B1K700E) activate the MYC transcriptional program. SF3B1 mutations lead to a dramatic decay of transcripts encoding the key PP2A phosphatase subunit PPP2R5A due to aberrant 3’ss usage, resulting in increased c-MYC serine 62 phosphorylation (allowing MYC to escape ubiquitin-mediated degradation) and in increased BCL2 serine 70 phosphorylation (leading to anti-apoptotic effects). This effect of SF3B1K700E on c-MYC and BCL2 activation through post-translational regulation was conserved across human and mouse cells and could be rescued by restored expression of PPP2R5A. Consonant with this, mutant SF3B1 promoted c-MYC driven tumorigenesis could be restored by the PP2A activating agent FTY-720. This study reveals the functional contribution of mutant SF3B1 to tumorigenesis through regulation of established oncogenic pathways and provides therapeutic strategies for related tumors.
2020-05-01 | GSE116391 | GEO
Project description:BRD7 functions as a crucial tumor suppressor in numerous malignancies. However, the effects of BRD7 on colorectal cancer (CRC) progression are still unknown. Here, based on the BRD7 knockout (BRD7-/-) and BRD7 flox/flox (BRD7+/+) mouse models constructed in our previous work, we established an azoxymethane/dextran sodium sulfate (AOM/DSS)-induced mouse model. BRD7+/+ mice were found to be highly susceptible to AOM/DSS-induced colitis-associated CRC, and BRD7 significantly promoted cell proliferation and cell cycle G1/S transition but showed no significant effect on cell apoptosis. Furthermore, BRD7 interacted with c-Myc and stabilized c-Myc by inhibiting its ubiquitin-proteasome-dependent degradation. Moreover, restoring the expression of c-Myc in BRD7-silenced CRC cells restored cell proliferation, cell cycle progression, and tumor growth in vitro and in vivo. In addition, BRD7 and c-Myc were both significantly upregulated in CRC patients, and high expression of these proteins was associated with clinical stage and poor prognosis in CRC patients. Collectively, BRD7 functions as an oncogene and promotes CRC progression by regulating the ubiquitin-proteasome-dependent stabilization of c-Myc protein. Targeting the BRD7/c-Myc axis could be a potential therapeutic strategy for CRC.
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Project description:Metabolic state of hematopoietic stem cells (HSCs) is an important regulator of self-renewal and lineage-specific differentiation. Posttranslational modification of proteins via O-GlcNAcylation is an ideal metabolic sensor, but how it contributes to megakaryopoiesis and thrombopoiesis remains unknown. Here, we reveal for the first time that cellular O-GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human-derived hematopoietic stem and progenitor cells (HSPCs). Inhibition of O-GlcNAc transferase (OGT) that catalyzes O-GlcNAcylation prolongedly decreases O-GlcNAcylation and induces the acquisition of CD34+ CD41a+ MK-like progenitors and its progeny CD34- CD41a+ /CD42b+ megakaryoblasts (MBs)/MKs from HSPCs, consequently resulting in increased CD41a+ and CD42b+ platelets. Using correlation and co-immunoprecipitation analyses, we further identify c-Myc as a direct downstream target of O-GlcNAcylation in MBs/MKs and provide compelling evidence on the regulation of platelets by novel O-GlcNAc/c-Myc axis. Our data indicate that O-GlcNAcylation posttranslationally regulates c-Myc stability by interfering with its ubiquitin-mediated proteasomal degradation. Depletion of c-Myc upon inhibition of OGT promotes platelet formation in part through the perturbation of cell adhesion molecules, that is, integrin-α4 and integrin-β7, as advised by gene ontology and enrichment analysis for RNA sequencing and validated herein. Together, our findings provide a novel basic knowledge on the regulatory role of O-GlcNAcylation in megakaryopoiesis and thrombopoiesis that could be important in understanding hematologic disorders whose etiology are related to impaired platelet production and may have clinical applications toward an ex vivo platelet production for transfusion.
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