RESUMEN
The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.
Asunto(s)
Ensamble y Desensamble de Cromatina , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica , Factor de Unión a CCCTC/metabolismo , Carcinogénesis/genética , Cromatina/genética , Cromatina/metabolismo , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras GenéticasRESUMEN
The heterochromatin protein HP1 plays a central role in the maintenance of genome stability but little is known about how HP1 is controlled. Here, we show that the zinc finger protein POGZ promotes the presence of HP1 at DNA double-strand breaks (DSBs) in human cells. POGZ depletion delays the resolution of DSBs and sensitizes cells to different DNA-damaging agents, including cisplatin and talazoparib. Mechanistically, POGZ promotes homology-directed DNA repair by retaining the BRCA1/BARD1 complex at DSBs in an HP1-dependent manner. In vivo CRISPR inactivation of Pogz is embryonically lethal. Pogz haploinsufficiency (Pogz+ /delta) results in developmental delay, impaired intellectual abilities, hyperactive behaviour and a compromised humoral immune response in mice, recapitulating the main clinical features of the White Sutton syndrome (WHSUS). Pogz+ /delta mice are further radiosensitive and accumulate DSBs in diverse tissues, including the spleen and brain. Altogether, our findings identify POGZ as an important player in homology-directed DNA repair both in vitro and in vivo.
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Homólogo de la Proteína Chromobox 5 , Reparación del ADN , Discapacidad Intelectual , Reparación del ADN por Recombinación , Transposasas , Animales , Homólogo de la Proteína Chromobox 5/genética , Homólogo de la Proteína Chromobox 5/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN , Roturas del ADN de Doble Cadena , Humanos , Discapacidad Intelectual/genética , Ratones , Transposasas/genética , Transposasas/metabolismoRESUMEN
Defining the impact of missense mutations on the recognition of DNA motifs is highly dependent on bioinformatic tools that define DNA binding elements. However, classical motif analysis tools remain limited in their capacity to identify subtle changes in complex binding motifs between distinct conditions. To overcome this limitation, we developed a new tool, MoMotif, that facilitates a sensitive identification, at the single base-pair resolution, of complex, or subtle, alterations to core binding motifs, discerned from ChIP-seq data. We employed MoMotif to define the previously uncharacterized recognition motif of CTCF zinc-finger 1 (ZF1), and to further define the impact of CTCF ZF1 mutation on its association with chromatin. Mutations of CTCF ZF1 are exclusive to breast cancer and are associated with metastasis and therapeutic resistance, but the underlying mechanisms are unclear. Using MoMotif, we identified an extension of the CTCF core binding motif, necessitating a functional ZF1 to bind appropriately. Using a combination of ChIP-Seq and RNA-Seq, we discover that the inability to bind this extended motif drives an altered transcriptional program associated with the oncogenic phenotypes observed clinically. Our study demonstrates that MoMotif is a powerful new tool for comparative ChIP-seq analysis and characterising DNA-protein contacts.
Asunto(s)
Cromatina , Zinc , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Zinc/metabolismo , Cromatina/genética , ADN/química , Mutación , Sitios de UniónRESUMEN
The transcriptional upregulation of oncogenes is a driving force behind the progression of many tumours. However, until a decade ago, the concept of 'switching off' these oncogenic pathways represented a formidable challenge. Research has revealed that members of the bromo- and extra-terminal domain (BET) motif family are key activators of oncogenic networks in a spectrum of cancers; their function depends on their recruitment to chromatin through two bromodomains (BD1 and BD2). The advent of potent inhibitors of BET proteins (BETi), which target either one or both bromodomains, represents an important step towards the goal of suppressing oncogenic networks within tumours. Here, we discuss the biology of BET proteins, advances in BETi design and highlight potential biomarkers predicting their activity. We also outline the logic of incorporating BETi into combination therapies to enhance its efficacy. We suggest that understanding mechanisms of activity, defining predictive biomarkers and identifying potent synergies represents a roadmap for clinical success using BETi.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Humanos , Neoplasias/metabolismo , Neoplasias/patología , PronósticoRESUMEN
The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology has been used to inactivate viral DNA as a new strategy to eliminate chronic viral infections, including HIV-1. This utility of CRISPR-Cas9 is challenged by the high heterogeneity of HIV-1 sequences, which requires the design of the single guide RNA (sgRNA; utilized by the CRISPR-Cas9 system to recognize the target DNA) to match a specific HIV-1 strain in an HIV patient. One solution to this challenge is to target the viral primer binding site (PBS), which HIV-1 copies from cellular tRNA3Lys in each round of reverse transcription and is thus conserved in almost all HIV-1 strains. In this study, we demonstrate that PBS-targeting sgRNA directs Cas9 to cleave the PBS DNA, which evokes deletions or insertions (indels) and strongly diminishes the production of infectious HIV-1. While HIV-1 escapes from PBS-targeting Cas9/sgRNA, unique resistance mechanisms are observed that are dependent on whether the plus or the minus strand of the PBS DNA is bound by sgRNA. Characterization of these viral escape mechanisms will inform future engineering of Cas9 variants that can more potently and persistently inhibit HIV-1 infection.IMPORTANCE The results of this study demonstrate that the gene-editing complex Cas9/sgRNA can be programmed to target and cleave HIV-1 PBS DNA, and thus, inhibit HIV-1 infection. Given that almost all HIV-1 strains have the same PBS, which is copied from the cellular tRNA3Lys during reverse transcription, PBS-targeting sgRNA can be used to inactivate HIV-1 DNA of different strains. We also discovered that HIV-1 uses different mechanisms to resist Cas9/sgRNAs, depending on whether they target the plus or the minus strand of PBS DNA. These findings allow us to predict that a Cas9 variant that uses the CCA sequence as the protospacer adjacent motif (PAM) should more strongly and persistently suppress HIV-1 replication. Together, these data have identified the PBS as the target DNA of Cas9/sgRNA and have predicted how to improve Cas9/sgRNA to achieve more efficient and sustainable suppression of HIV-1 infection, therefore improving the capacity of Cas9/sgRNA in curing HIV-1 infection.
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Proteína 9 Asociada a CRISPR/metabolismo , ADN Viral/metabolismo , Edición Génica , VIH-1/genética , ARN Guía de Kinetoplastida/metabolismo , Línea Celular , ADN Viral/genética , Humanos , Mutagénesis Insercional , ARN Guía de Kinetoplastida/genética , Eliminación de SecuenciaRESUMEN
In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.
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Codón Iniciador/genética , Proteínas de Unión al ADN/genética , Factor 5 Eucariótico de Iniciación/genética , Genoma Humano , Animales , Unión Competitiva , Línea Celular , Línea Celular Tumoral , Codón Iniciador/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 5 Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Homeostasis/genética , Humanos , Unión Proteica , Biosíntesis de Proteínas/genética , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
CCCTC-binding factor (CTCF) is a key regulator of nuclear chromatin structure and gene regulation. The impact of CTCF on transcriptional output is highly varied, ranging from repression to transcriptional pausing and transactivation. The multifunctional nature of CTCF may be directed solely through remodeling chromatin architecture. However, another hypothesis is that the multifunctional nature of CTCF is mediated, in part, through differential association with protein partners having unique functions. Consistent with this hypothesis, our mass spectrometry analyses of CTCF interacting partners reveal a previously undefined association with the transcription factor general transcription factor II-I (TFII-I). Biochemical fractionation of CTCF indicates that a distinct CTCF complex incorporating TFII-I is assembled on DNA. Unexpectedly, we found that the interaction between CTCF and TFII-I is essential for directing CTCF to the promoter proximal regulatory regions of target genes across the genome, particularly at genes involved in metabolism. At genes coregulated by CTCF and TFII-I, we find knockdown of TFII-I results in diminished CTCF binding, lack of cyclin-dependent kinase 8 (CDK8) recruitment, and an attenuation of RNA polymerase II phosphorylation at serine 5. Phenotypically, knockdown of TFII-I alters the cellular response to metabolic stress. Our data indicate that TFII-I directs CTCF binding to target genes, and in turn the two proteins cooperate to recruit CDK8 and enhance transcription initiation.
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Epigénesis Genética , Genoma Humano , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Factores de Transcripción/fisiología , Factor de Unión a CCCTC , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , FosforilaciónRESUMEN
Cancer constitutes a set of diseases with heterogeneous molecular pathologies. However, there are a number of universal aberrations common to all cancers, one of these being the epigenetic silencing of tumor suppressor genes (TSGs). The silencing of TSGs is thought to be an early, driving event in the oncogenic process. With this in consideration, great efforts have been made to develop small molecules aimed at the restoration of TSGs in order to limit tumor cell proliferation and survival. However, the molecular forces that drive the broad epigenetic reprogramming and transcriptional repression of these genes remain ill-defined. Undoubtedly, understanding the molecular underpinnings of transcriptionally silenced TSGs will aid us in our ability to reactivate these key anti-cancer targets. Here, we describe what we consider to be the five most logical molecular mechanisms that may account for this widely observed phenomenon: 1) ablation of transcription factor binding, 2) overexpression of DNA methyltransferases, 3) disruption of CTCF binding, 4) elevation of EZH2 activity, 5) aberrant expression of long non-coding RNAs. The strengths and weaknesses of each proposed mechanism is highlighted, followed by an overview of clinical efforts to target these processes.
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Epigénesis Genética , Silenciador del Gen , Genes Supresores de Tumor , Animales , ADN (Citosina-5-)-Metiltransferasas/fisiología , Proteína Potenciadora del Homólogo Zeste 2 , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Complejo Represivo Polycomb 2/fisiología , ARN no Traducido/fisiología , Transcripción Genética , ADN Metiltransferasa 3BRESUMEN
The p16(INK4a) tumor suppressor gene is a frequent target of epigenetic inactivation in human cancers, which is an early event in breast carcinogenesis. We describe the existence of a chromatin boundary upstream of the p16 gene that is lost when this gene is aberrantly silenced. We show that the multifunctional protein CTCF associates in the vicinity of this boundary and absence of binding strongly coincides with p16 silencing in multiple types of cancer cells. CTCF binding also correlates with RASSF1A and CDH1 gene activation, and CTCF interaction is absent when these genes are methylated and silenced. Interestingly, defective poly(ADP-ribosyl)ation of CTCF and dissociation from the molecular chaperone Nucleolin occur in p16-silenced cells, abrogating its proper function. Thus, destabilization of specific chromosomal boundaries through aberrant crosstalk between CTCF, poly(ADP-ribosyl)ation, and DNA methylation may be a general mechanism to inactivate tumor suppressor genes and initiate tumorigenesis in numerous forms of human cancers.
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Neoplasias de la Mama/metabolismo , Cromatina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Silenciador del Gen , Proteínas Represoras/metabolismo , Antígenos CD , Neoplasias de la Mama/genética , Factor de Unión a CCCTC , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Regulación de la Expresión Génica , Humanos , Modelos Genéticos , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: PARP inhibitors have shown promising clinical results in cancer patients carrying BRCA1/2 mutations. Their clinical efficacy could logically be influenced by PARP1 protein levels in patient tumors. METHODS: We screened three cohorts of patients with ovarian cancer, totaling 313 samples, and evaluated PARP1 protein expression by immunohistochemistry with further validation by western blotting. RESULTS: We observed that up to 60 % of tumors showed little PARP1 protein expression. In serous ovarian tumors, comparing intratumoral PARP1 expression between chemo-naïve and post-chemotherapy patients revealed a decrease in intratumoral PARP1 following chemotherapy in all three cohorts (immunohistochemistry: p < 0.001, n = 239; western blot: p = 0.012, n = 74). The findings were further confirmed in a selection of matched samples from the same patients before and after chemotherapy. CONCLUSION: Our data suggest that patients should be screened for PARP1 expression prior to therapy with PARP inhibitors. Further, the observed reduction of intratumoral PARP1 post-chemotherapy suggests that treating chemo-naïve patients with PARP inhibitors prior to the administration of chemotherapy, or concurrently, might increase the responsiveness to PARP1 inhibition. Thus, a change in the timing of PARP inhibitor administration may be warranted for future clinical trials.
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Antineoplásicos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Poli(ADP-Ribosa) Polimerasas/biosíntesis , Anciano , Estudios de Cohortes , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/análisisRESUMEN
Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR), which is a pivotal metabolic regulator, and epigenetic modifications remains poorly understood. Our results show that mTORC1 activation caused by the abrogation of its negative regulator tuberous sclerosis complex 2 (TSC2) coincides with increased levels of the histone modification H3K27me3 but not H3K4me3 or H3K9me3. This selective H3K27me3 induction was mediated via 4E-BP-dependent increase in EZH2 protein levels. Surprisingly, mTOR inhibition also selectively induced H3K27me3. This was independent of TSC2, and was paralleled by reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the induction of H3K27me3 may potentiate the anti-proliferative effects of mTOR inhibitors.
RESUMEN
The pogo transposable element derived zinc finger protein, POGZ, is notably associated with neurodevelopmental disorders through its role in gene transcription. Many proteins involved in neurological development are often dysregulated in cancer, suggesting a potential role for POGZ in tumor biology. Here, we provided experimental evidence that POGZ influences the growth and metastatic spread of triple negative breast cancers (TNBC). In well-characterized models of TNBC, POGZ exerted a dual role, both as a tumor promoter and metastasis suppressor. Mechanistically, loss of POGZ potentiated TGFß pathway activation to exert cytostatic effects while simultaneously increasing the mesenchymal and migratory properties of breast tumors. Whereas POGZ levels are elevated in human breast cancers, the most aggressive forms of TNBC tumors, including those with increased mesenchymal and metastatic properties, exhibit dampened POGZ levels, and low POGZ expression was associated with inferior clinical outcomes in these tumor types. Taken together, these data suggest that POGZ is a critical suppressor of the early stages of the metastatic cascade.
RESUMEN
Triple-negative breast cancers (TNBCs) lack effective targeted therapies, and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohort of patients do respond to PARPi in combination with chemotherapies. Moreover, recent studies suggest that an increase in levels of ROS may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that, in response to therapy-induced stress, p66ShcA stimulated ROS production, which, in turn, potentiated the synergy of PARPi in combination with doxorubicin in TNBCs. This p66ShcA-induced sensitivity relied on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.
Asunto(s)
Antineoplásicos/farmacología , Poli(ADP-Ribosa) Polimerasa-1/efectos de los fármacos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Apoptosis , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Supervivencia Celular , Daño del ADN , Inestabilidad Genómica , Humanos , Células MCF-7 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but often lethal cancer that is diagnosed at a median age of 24 years. Optimal management of patients is not well defined, and current treatment remains challenging, necessitating the discovery of novel therapeutic approaches. The identification of SMARCA4-inactivating mutations invariably characterizing this type of cancer provided insights facilitating diagnostic and therapeutic measures against this disease. We show here that the BET inhibitor OTX015 acts in synergy with the MEK inhibitor cobimetinib to repress the proliferation of SCCOHT in vivo Notably, this synergy is also observed in some SMARCA4-expressing ovarian adenocarcinoma models intrinsically resistant to BETi. Mass spectrometry, coupled with knockdown of newly found targets such as thymidylate synthase, revealed that the repression of a panel of proteins involved in nucleotide synthesis underlies this synergy both in vitro and in vivo, resulting in reduced pools of nucleotide metabolites and subsequent cell-cycle arrest. Overall, our data indicate that dual treatment with BETi and MEKi represents a rational combination therapy against SCCOHT and potentially additional ovarian cancer subtypes.
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Epigénesis Genética , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Nucleótidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Azetidinas/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Epigénesis Genética/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones Endogámicos NOD , Ratones SCID , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Fase S/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type-based diagnosis and antithrombotic intervention.
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Vesículas Extracelulares , Glioblastoma , Glioma , Trombosis , Animales , Humanos , Ratones , Tromboplastina/genéticaRESUMEN
Bioenergetic perturbations driving neoplastic growth increase the production of reactive oxygen species (ROS), requiring a compensatory increase in ROS scavengers to limit oxidative stress. Intervention strategies that simultaneously induce energetic and oxidative stress therefore have therapeutic potential. Phenformin is a mitochondrial complex I inhibitor that induces bioenergetic stress. We now demonstrate that inflammatory mediators, including IFNγ and polyIC, potentiate the cytotoxicity of phenformin by inducing a parallel increase in oxidative stress through STAT1-dependent mechanisms. Indeed, STAT1 signaling downregulates NQO1, a key ROS scavenger, in many breast cancer models. Moreover, genetic ablation or pharmacological inhibition of NQO1 using ß-lapachone (an NQO1 bioactivatable drug) increases oxidative stress to selectively sensitize breast cancer models, including patient derived xenografts of HER2+ and triple negative disease, to the tumoricidal effects of phenformin. We provide evidence that therapies targeting ROS scavengers increase the anti-neoplastic efficacy of mitochondrial complex I inhibitors in breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Fenformina/farmacología , Factor de Transcripción STAT1/metabolismo , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Sinergismo Farmacológico , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Metabolismo Energético/efectos de los fármacos , Femenino , Glutatión/antagonistas & inhibidores , Glutatión/biosíntesis , Humanos , Interferón gamma/administración & dosificación , Interferón gamma/deficiencia , Interferón gamma/metabolismo , Células MCF-7 , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Naftoquinonas/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Fenformina/administración & dosificación , Poli I-C/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT1/agonistas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Transcriptional activation by nuclear hormone receptors is well characterized, but their cooperation with other signaling pathways to activate transcription remains poorly understood. Tumor necrosis factor alpha (TNFalpha) and all-trans retinoic acid (RA) induce monocytic differentiation of acute promyelocytic leukemia (APL) cells in a synergistic manner. We used the promoter of DIF2, a gene involved in monocytic differentiation, to model the mechanism underlying the cooperative induction of target genes by RA and TNFalpha. We show a functional RA response element in the DIF2 promoter, which is constitutively bound by PML/RARalpha in APL cells. RA stimulates release of corepressors and recruitment of chromatin modifying proteins and additional transcription factors to the promoter, but these changes cause only a modest induction of DIF2 mRNA. Co-stimulation with RA plus TNFalpha facilitates binding of NF-kappaB to the promoter, which is crucial for full induction of transcription. Furthermore, RA plus TNFalpha greatly enhanced the level of RNA Pol II phosphorylation on the DIF2 promoter, via synergistic recruitment of TFIIH. We propose that RA mediates remodeling of chromatin to facilitate binding of transcription factors, which cooperate to enhance Pol II phosphorylation, providing a mechanism whereby nuclear receptors interact with other signaling pathways on the level of transcription.
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Proteínas Reguladoras de la Apoptosis/genética , Ensamble y Desensamble de Cromatina , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas , Activación Transcripcional , Tretinoina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Línea Celular , Núcleo Celular/metabolismo , Cromatina/efectos de los fármacos , Sinergismo Farmacológico , Humanos , FN-kappa B/metabolismo , ARN Polimerasa II/metabolismo , Elementos de Respuesta , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción TFIIH/metabolismo , Factores de Transcripción/metabolismo , Células U937RESUMEN
Bromodomain and extra-terminal domain (BET) proteins, cyclic adenosine monophosphate response element-binding protein (CBP), and the E1A-binding protein of p300 (EP300) are important players in histone acetylation. Preclinical evidence supports the notion that small molecules targeting these proteins individually or in combination can elicit antitumor activity. Here, we characterize the antitumor activity of the pan BET/CBP/EP300 inhibitor NEO2734 and provide insights into its mechanism of action through bromodomain-binding assays, in vitro and in vivo treatments of cancer cell lines, immunoblotting, and transcriptome analyses. In a panel of 60 models derived from different tumor types, NEO2734 exhibited antiproliferative activity in multiple cell lines, with the most potent activity observed in hematologic and prostate cancers. Focusing on lymphoma cell lines, NEO2374 exhibited a pattern of response and transcriptional changes similar to lymphoma cells exposed to either BET or CBP/EP300 inhibitors alone. However, NEO2734 was more potent than single-agent BET or CBP/EP300 inhibitors alone. In conclusion, NEO2734 is a novel antitumor compound that shows preferential activity in lymphomas, leukemias, and prostate cancers.
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Leucemia , Línea Celular Tumoral , Humanos , Masculino , Dominios ProteicosRESUMEN
Small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and highly aggressive ovarian malignancy. In almost all cases, it is associated with somatic and often germline pathogenic variants in SMARCA4, which encodes for the SMARCA4 protein (BRG1), a subunit of the SWI/SNF chromatin remodeling complex. Approximately 20% of human cancers possess pathogenic variants in at least one SWI/SNF subunit. Because of their role in regulating many important cellular processes including transcriptional control, DNA repair, differentiation, cell division, and DNA replication, SWI/SNF complexes with mutant subunits are thought to contribute to cancer initiation and progression. Fewer than 500 cases of SCCOHT have been reported in the literature and approximately 60% are associated with hypercalcemia. SCCOHT primarily affects females under 40 years of age who usually present with symptoms related to a pelvic mass. SCCOHT is an aggressive cancer, with long-term survival rates of 30% in early-stage cases. Although various treatment approaches have been proposed, there is no consensus on surveillance and therapeutic strategy. An international group of multidisciplinary clinicians and researchers recently formed the International SCCOHT Consortium to evaluate current knowledge and propose consensus surveillance and therapeutic recommendations, with the aim of improving outcomes. Here, we present an overview of the genetics of this cancer, provide updates on new treatment targets, and propose management guidelines for this challenging cancer.
Asunto(s)
Carcinoma de Células Pequeñas/genética , ADN Helicasas/genética , Hipercalcemia/genética , Proteínas Nucleares/genética , Neoplasias Ováricas/genética , Factores de Transcripción/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Pequeñas/sangre , Carcinoma de Células Pequeñas/mortalidad , Carcinoma de Células Pequeñas/terapia , Quimioterapia Adyuvante/métodos , Quimioterapia Adyuvante/normas , Ensamble y Desensamble de Cromatina/genética , Femenino , Ginecología/normas , Humanos , Hipercalcemia/sangre , Hipercalcemia/patología , Hipercalcemia/terapia , Oncología Médica/normas , Mutación , Neoplasias Ováricas/sangre , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/terapia , Ovariectomía/normas , Ovario/patología , Ovario/cirugía , Guías de Práctica Clínica como Asunto , Radioterapia Adyuvante/normas , Trasplante de Células Madre/normas , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Here we identify methionine as a key nutrient affecting epigenetic reprogramming in CD4+ T helper (Th) cells. Using metabolomics, we showed that methionine is rapidly taken up by activated T cells and serves as the major substrate for biosynthesis of the universal methyl donor S-adenosyl-L-methionine (SAM). Methionine was required to maintain intracellular SAM pools in T cells. Methionine restriction reduced histone H3K4 methylation (H3K4me3) at the promoter regions of key genes involved in Th17 cell proliferation and cytokine production. Applied to the mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), dietary methionine restriction reduced the expansion of pathogenic Th17 cells in vivo, leading to reduced T cell-mediated neuroinflammation and disease onset. Our data identify methionine as a key nutritional factor shaping Th cell proliferation and function in part through regulation of histone methylation.