RESUMEN
Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.
Asunto(s)
Neoplasias Colorrectales , Inhibidores de Topoisomerasa I , Humanos , Inhibidores de Topoisomerasa I/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ligasas/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al ARNRESUMEN
Cancer/Testis Antigens (CTAs) represent a group of proteins whose expression under physiological conditions is restricted to testis but activated in many human cancers. Also, it was observed that co-expression of multiple CTAs worsens the patient prognosis. Five CTAs were reported acting in mitochondria and we recently reported 147 transcripts encoded by 67 CTAs encoding for proteins potentially targeted to mitochondria. Among them, we identified the two isoforms encoded by CT55 for whom the function is poorly understood. First, we found that patients with tumors expressing wild-type CT55 are associated with poor survival. Moreover, CT55 silencing decreases dramatically cell proliferation. Second, to investigate the role of CT55 on mitochondria, we first show that CT55 is localized to both mitochondria and endoplasmic reticulum (ER) due to the presence of an ambiguous N-terminal targeting signal. Then, we show that CT55 silencing decreases mtDNA copy number and delays mtDNA recovery after an acute depletion. Moreover, demethylation of CT55 promotor increases its expression, which in turn increases mtDNA copy number. Finally, we measured the mtDNA copy number in NCI-60 cell lines and screened for genes whose expression is strongly correlated to mtDNA amount. We identified CT55 as the second highest correlated hit. Also, we show that compared to siRNA scrambled control (siCtrl) treatment, CT55 specific siRNA (siCT55) treatment down-regulates aerobic respiration, indicating that CT55 sustains mitochondrial respiration. Altogether, these data show for first time that CT55 acts on mtDNA copy number, modulates mitochondrial activity to sustain cancer cell proliferation.
Asunto(s)
ADN Mitocondrial , Neoplasias , Proliferación Celular , Variaciones en el Número de Copia de ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Humanos , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , ARN Interferente Pequeño , Testículo/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a lethal aggressive cancer, in part due to elements of the microenvironment (hypoxia, hypoglycemia) that cause metabolic network alterations. The FDA-approved antihelminthic pyrvinium pamoate (PP) has previously been shown to cause PDAC cell death, although the mechanism has not been fully determined. We demonstrated that PP effectively inhibited PDAC cell viability with nanomolar IC50 values (9-93 nmol/L) against a panel of PDAC, patient-derived, and murine organoid cell lines. In vivo, we demonstrated that PP inhibited PDAC xenograft tumor growth with both intraperitoneal (IP; P < 0.0001) and oral administration (PO; P = 0.0023) of human-grade drug. Metabolomic and phosphoproteomic data identified that PP potently inhibited PDAC mitochondrial pathways including oxidative phosphorylation and fatty acid metabolism. As PP treatment reduced oxidative phosphorylation (P < 0.001), leading to an increase in glycolysis (P < 0.001), PP was 16.2-fold more effective in hypoglycemic conditions similar to those seen in PDAC tumors. RNA sequencing demonstrated that PP caused a decrease in mitochondrial RNA expression, an effect that was not observed with established mitochondrial inhibitors rotenone and oligomycin. Mechanistically, we determined that PP selectively bound mitochondrial G-quadruplexes and inhibited mitochondrial RNA transcription in a G-quadruplex-dependent manner. This subsequently led to a 90% reduction in mitochondrial encoded gene expression. We are preparing to evaluate the efficacy of PP in PDAC in an IRB-approved window-of-opportunity trial (IND:144822).
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antihelmínticos/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Metabolómica/métodos , Compuestos de Pirvinio/uso terapéutico , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Antihelmínticos/farmacología , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Humanos , Ratones , Compuestos de Pirvinio/farmacología , Análisis de Supervivencia , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Mitochondria contain their own genome (mtDNA), encoding 13 proteins of the enzyme complexes of the oxidative phosphorylation. Synthesis of these 13 mitochondrial proteins requires a specific translation machinery, the mitoribosomes whose RNA components are encoded by the mtDNA, whereas more than 80 proteins are encoded by nuclear genes. It has been well established that mitochondrial topoisomerase I (TOP1MT) is important for mtDNA integrity and mitochondrial transcription as it prevents excessive mtDNA negative supercoiling and releases topological stress during mtDNA replication and transcription. We recently showed that TOP1MT also supports mitochondrial protein synthesis, and thus is critical for promoting tumor growth. Impaired mitochondrial protein synthesis leads to activation of the mitonuclear stress response through the transcription factor ATF4, and induces cytoprotective genes in order to prevent mitochondrial and cellular dysfunction. In this perspective, we highlight the novel role of TOP1MT in mitochondrial protein synthesis and as potential target for chemotherapy.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , ADN Mitocondrial/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/biosíntesis , Biosíntesis de Proteínas , Factor de Transcripción Activador 4/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular/genética , Replicación del ADN/genética , ADN-Topoisomerasas de Tipo I/genética , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción GenéticaRESUMEN
SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.
Asunto(s)
Proteínas Nucleares/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Camptotecina , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Daño del ADN , ADN Helicasas/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Humanos , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Nucleares/metabolismo , Pirazinas , Pirazoles , Proteína de Replicación A/metabolismoRESUMEN
Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs abortive topoisomerase II cleavage complexes. Here, we identify a novel short isoform of TDP2 (TDP2S) expressed from an alternative transcription start site. TDP2S contains a mitochondrial targeting sequence, contributing to its enrichment in the mitochondria and cytosol, while full-length TDP2 contains a nuclear localization signal and the ubiquitin-associated domain in the N-terminus. Our study reveals that both TDP2 isoforms are present and active in the mitochondria. Comparison of isogenic wild-type (WT) and TDP2 knockout (TDP2-/-/-) DT40 cells shows that TDP2-/-/- cells are hypersensitive to mitochondrial-targeted doxorubicin (mtDox), and that complementing TDP2-/-/- cells with human TDP2 restores resistance to mtDox. Furthermore, mtDox selectively depletes mitochondrial DNA in TDP2-/-/- cells. Using CRISPR-engineered human cells expressing only the TDP2S isoform, we show that TDP2S also protects human cells against mtDox. Finally, lack of TDP2 in the mitochondria reduces the mitochondria transcription levels in two different human cell lines. In addition to identifying a novel TDP2S isoform, our report demonstrates the presence and importance of both TDP2 isoforms in the mitochondria.
Asunto(s)
Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/genética , Factores de Transcripción/genética , Empalme Alternativo/genética , Línea Celular Tumoral , Proteínas de Unión al ADN , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/antagonistas & inhibidores , Hidrolasas Diéster Fosfóricas , Isoformas de Proteínas/genética , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
The isoflavone genistein has been shown to act as topoisomerase II poison in various cell lines. Here, we address the question whether genistein is able to affect topoisomerase II in vivo. Juvenile male Wistar rats received either a single dose of genistein subcutaneously (s.c.; 10 mg/kg BW) or a lifelong isoflavone-rich diet encompassing in utero, lactation phase and 10 days of oral consumption, whereas genistein was mainly taken up as glycosides (25-50 mg/kg BW). The effects on the level of covalent topoisomerase II-DNA-complexes in the duodenum and colon were measured using the "Isolation of in vivo complexes of enzyme to DNA" (ICE)-bioassay. Simultaneously, serum as well as tissue concentrations of genistein and its metabolites were quantified by LC-MS. Genistein (s.c.) significantly increased the amount of covalent topoisomerase IIα and ß-DNA complexes in the gut, showing more persistent effects in the colon than in the duodenum. In case of a lifelong dietary isoflavone exposure, no effects on the stabilization of cleavage complexes was observed, except a slight increase of topoisomerase IIα-DNA-complexes in the colon. The differences between the exposure routes might be attributed to the higher serum concentration of the genistein aglycon after subcutaneous treatment probably due to circumvention of first-pass metabolism compared to oral consumption of an isoflavone-rich diet. These data indicate that subcutaneously administrated genistein clearly possesses topoisomerase poisoning properties in vivo, whereas an isoflavone-rich diet containing genistein only caused a slight effect which relevance has to be clarified in further studies.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Genisteína/farmacología , Intestinos/efectos de los fármacos , Animales , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Dieta/veterinaria , Relación Dosis-Respuesta a Droga , Femenino , Genisteína/sangre , Mucosa Intestinal/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries. Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria. To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl4) administration. TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation. The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl4 exposure. Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS). Steady-state levels of ATP, O2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl4-treated KO mice. To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide. Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion. Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Mitocondrias Hepáticas/enzimología , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Tetracloruro de Carbono/toxicidad , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , ADN-Topoisomerasas de Tipo I/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Glutatión/metabolismo , Células HCT116 , Hepatocitos/efectos de los fármacos , Hepatocitos/ultraestructura , Humanos , Regeneración Hepática/genética , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mitocondrias Hepáticas/genética , Mitocondrias Hepáticas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
SCOPE: Oxidative metabolism of daidzein (DAI) might result in the formation of hydroxylated metabolites. Here, we address the question whether these metabolites differ in their biological activity from the parent isoflavone, exemplified for the epidermal growth factor receptor and topoisomerase II, potentially resulting in an enhanced toxic profile. METHODS AND RESULTS: In contrast to DAI, 6-hydroxydaidzein (6-HO-DAI) and 8-hydroxydaidzein (8-HO-DAI) were found to inhibit the tyrosine kinase activity of the epidermal growth factor receptor in an ELISA-based test system, but showed no effects within cells. Further, the oxidative metabolites suppressed the catalytic activity of topoisomerase II in the decatenation assay. In the in vivo complexes of enzyme to DNA (ICE) bioassay, 6-HO-DAI and 8-HO-DAI did not affect the level of covalent topoisomerase II-DNA intermediates within HT29 cells, thus arguing for a catalytic inhibition of topoisomerase II rather than poisoning activity. In contrast to DAI, 6-HO-DAI and 8-HO-DAI significantly increased the rate of DNA strand breaks in HT29 cells after 24-h incubation and caused a cell cycle delay in S-phase. Differences were also observed between the oxidative metabolites, with only 6-HO-DAI inducing apoptosis but not 8-HO-DAI. CONCLUSION: These data indicate that oxidative metabolism of DAI generates metabolites with genotoxic properties where interference with topoisomerase II might play a role.
Asunto(s)
Daño del ADN/efectos de los fármacos , Glycine max/química , Isoflavonas/farmacología , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ensayo Cometa , ADN-Topoisomerasas de Tipo II/metabolismo , Células HT29 , Humanos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Grapevine-shoot extracts (GSE), containing trans-resveratrol and resveratrol oligomers, are commercially available as food supplements. Considering the topoisomerase-targeting properties of trans-resveratrol, the question of whether GSE affect these enzymes, thereby potentially causing DNA damage, was addressed. In a decatenation assay, GSE potently suppressed the catalytic activity of topoisomerase IIα (≥5 µg/mL). The resveratrol oligomers ε-viniferin, r2-viniferin, and hopeaphenol, isolated from GSE, were also identified as topoisomerase IIα inhibitors. In the in vivo complexes of enzyme to DNA (ICE) bioassay, neither GSE, r2-viniferin, nor hopeaphenol affected the level of enzyme-DNA intermediates in A431 cells, thus representing catalytic inhibitors rather than topoisomerase poisons. GSE caused moderate DNA strand breaks (≥25 µg/mL) in the comet assay. Taken together, GSE presumably acts as a catalytic inhibitor of topoisomerase II with r2-viniferin and hopeaphenol as potentially contributing constituents. However, the increase of FPG-sensitive sites points to an additional mechanism that may contribute to the DNA-damaging properties of GSE constituents.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Extractos Vegetales/química , Brotes de la Planta/química , Estilbenos/química , Vitis/química , Antígenos de Neoplasias/metabolismo , Biocatálisis/efectos de los fármacos , Línea Celular Tumoral , Ensayo Cometa , Daño del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Extractos Vegetales/farmacología , Resveratrol , Estilbenos/farmacologíaRESUMEN
The plant alkaloid lycobetaine has potent topoisomerase-targeting properties and shows anticancer activity. Based on these findings, several lycobetaine analogs were synthesized mainly differing in their substituents at 2, 8 and 9 position and their biological activities were evaluated. The topoisomerase-targeting properties and cytotoxicity of these structural analogs were assessed in the human gastric carcinoma cell line GXF251L. Performing a plasmid relaxation assay, an increased inhibition of topoisomerase I was found with N-methylphenanthridinium chlorides bearing a 8,9-methylenedioxy moiety or a methoxy group in 2-position. Furthermore, quaternized phenanthridinium derivatives bearing either a 2-methoxy or a 8,9-methylenedioxy moiety in conjunction with a 2-hydroxy or 2-methoxy group display potent topoisomerase II inhibition as shown by decatenation of kinetoplast DNA. In general, the N-methylphenanthridinium chlorides possess more potency in inhibiting topoisomerase I than topoisomerase II. All quaternized derivatives also exhibited potent inhibition of tumor cell growth in the low micromolar concentration range. Hence, N-methylphenanthridinium compounds were found to represent a promising class of compounds, potently inhibiting both, topoisomerases I and II, and may be further developed into clinically useful topoisomerase inhibitors.