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1.
BMC Cancer ; 24(1): 928, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-39090568

RESUMEN

BACKGROUND: Osteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism. METHODS: We evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism. RESULTS: In this study, we found that extremely low concentrations of GNE-987(2-10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development. CONCLUSIONS: This research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.


Asunto(s)
Apoptosis , Neoplasias Óseas , Proteínas de Ciclo Celular , Proliferación Celular , Osteosarcoma , Factores de Transcripción , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Proteínas que Contienen Bromodominio , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones Desnudos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Osteosarcoma/genética , Osteosarcoma/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Elife ; 122024 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-39092485

RESUMEN

The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a 'timely two-ness' that allows cell division to occur in absence of a SAC-dependent mitotic delay.


Asunto(s)
Proteínas de Ciclo Celular , Centrosoma , Puntos de Control de la Fase M del Ciclo Celular , Mitosis , Centrosoma/metabolismo , Humanos , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático/metabolismo , Huso Acromático/fisiología , División Celular , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Células HeLa
3.
Nat Commun ; 15(1): 6608, 2024 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-39098906

RESUMEN

The antitumor performance of PROteolysis-TArgeting Chimeras (PROTACs) is limited by its insufficient tumor specificity and poor pharmacokinetics. These disadvantages are further compounded by tumor heterogeneity, especially the presence of cancer stem-like cells, which drive tumor growth and relapse. Herein, we design a region-confined PROTAC nanoplatform that integrates both reactive oxygen species (ROS)-activatable and hypoxia-responsive PROTAC prodrugs for the precise manipulation of bromodomain and extraterminal protein 4 expression and tumor eradication. These PROTAC nanoparticles selectively accumulate within and penetrate deep into tumors via response to matrix metalloproteinase-2. Photoactivity is then reactivated in response to the acidic intracellular milieu and the PROTAC is discharged due to the ROS generated via photodynamic therapy specifically within the normoxic microenvironment. Moreover, the latent hypoxia-responsive PROTAC prodrug is restored in hypoxic cancer stem-like cells overexpressing nitroreductase. Here, we show the ability of region-confined PROTAC nanoplatform to effectively degrade BRD4 in both normoxic and hypoxic environments, markedly hindering tumor progression in breast and head-neck tumor models.


Asunto(s)
Proteínas de Ciclo Celular , Nanopartículas , Proteolisis , Factores de Transcripción , Humanos , Proteolisis/efectos de los fármacos , Animales , Nanopartículas/química , Línea Celular Tumoral , Ratones , Factores de Transcripción/metabolismo , Femenino , Proteínas de Ciclo Celular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Profármacos/farmacología , Profármacos/química , Fotoquimioterapia/métodos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Microambiente Tumoral/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Nucleares/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones Endogámicos BALB C , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Proteínas que Contienen Bromodominio
4.
Sci Adv ; 10(34): eadp5753, 2024 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-39178260

RESUMEN

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Metilación de ADN , Nucleosomas , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Nucleosomas/metabolismo , Nucleosomas/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Microscopía por Crioelectrón , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Islas de CpG , Ubiquitinación , Evolución Molecular , ADN/metabolismo , ADN/química , ADN/genética , Dedos de Zinc , Cromatina/metabolismo , Cromatina/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/química , Eucariontes/genética , Eucariontes/metabolismo , Unión Proteica , Histonas/metabolismo , Histonas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/química
5.
Int J Mol Sci ; 25(15)2024 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-39125637

RESUMEN

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.


Asunto(s)
Proteínas de Ciclo Celular , Glucógeno Sintasa Quinasa 3 , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-akt , Humanos , Células HEK293 , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Ciclo Celular/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/genética , Proteínas Tirosina Quinasas/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/efectos de los fármacos
6.
Cells ; 13(16)2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-39195269

RESUMEN

Centrosomal Protein 55 (CEP55) exhibits various oncogenic activities; it regulates the PI3K-Akt-pathway, midbody abscission, and chromosomal instability (CIN) in cancer cells. Here, we analyzed the mechanism of how CEP55 controls CIN in ovarian and breast cancer (OvCa) cells. Down-regulation of CEP55 reduced CIN in all cell lines analyzed, and CEP55 depletion decreased spindle microtubule (MT)-stability in OvCa cells. Moreover, recombinant CEP55 accelerated MT-polymerization and attenuated cold-induced MT-depolymerization. To analyze a potential relationship between CEP55-controlled CIN and its impact on MT-stability, we identified the CEP55 MT-binding peptides inside the CEP55 protein. Thereafter, a mutant with deficient MT-binding activity was re-expressed in CEP55-depleted OvCa cells and we could show that this mutant did not restore reduced CIN in CEP55-depleted cells. This finding strongly indicates that CEP55 regulates CIN by controlling MT dynamics.


Asunto(s)
Proteínas de Ciclo Celular , Inestabilidad Cromosómica , Microtúbulos , Humanos , Inestabilidad Cromosómica/genética , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Femenino , Huso Acromático/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo
7.
Cells ; 13(16)2024 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-39195284

RESUMEN

The AurkA serine/threonine kinase is a key regulator of cell division controlling mitotic entry, centrosome maturation, and chromosome segregation. The microtubule-associated protein TPX2 controls spindle assembly and is the main AurkA regulator, contributing to AurkA activation, localisation, and stabilisation. Since their identification, AurkA and TPX2 have been described as being overexpressed in cancer, with a significant correlation with highly proliferative and aneuploid tumours. Despite the frequent occurrence of AurkA/TPX2 co-overexpression in cancer, the investigation of their involvement in tumorigenesis and cancer therapy resistance mostly arises from studies focusing only on one at the time. Here, we review the existing literature and discuss the mitotic phenotypes described under conditions of AurkA, TPX2, or AurkA/TPX2 overexpression, to build a picture that may help clarify their oncogenic potential through the induction of chromosome instability. We highlight the relevance of the AurkA/TPX2 complex as an oncogenic unit, based on which we discuss recent strategies under development that aim at disrupting the complex as a promising therapeutic perspective.


Asunto(s)
Aurora Quinasa A , Proteínas Asociadas a Microtúbulos , Neoplasias , Humanos , Aurora Quinasa A/metabolismo , Aurora Quinasa A/genética , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Animales , Mitosis/genética , Aberraciones Cromosómicas , Inestabilidad Cromosómica/genética , Regulación Neoplásica de la Expresión Génica
8.
Cell Death Dis ; 15(8): 610, 2024 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-39174499

RESUMEN

PARP1 is crucial in DNA damage repair, chromatin remodeling, and transcriptional regulation. The principle of synthetic lethality has effectively guided the application of PARP inhibitors in treating tumors carrying BRCA1/2 mutations. Meanwhile, PARP inhibitors have exhibited efficacy in BRCA-proficient patients, further highlighting the necessity for a deeper understanding of PARP1 function and its inhibition in cancer therapy. Here, we unveil PIN2/TRF1-interacting telomerase inhibitor 1 (PINX1) as an uncharacterized PARP1-interacting protein that synergizes with PARP inhibitors upon its depletion across various cancer cell lines. Loss of PINX1 compromises DNA damage repair capacity upon etoposide treatment. The vulnerability of PINX1-deficient cells to etoposide and PARP inhibitors could be effectively restored by introducing either a full-length or a mutant form of PINX1 lacking telomerase inhibitory activity. Mechanistically, PINX1 is recruited to DNA lesions through binding to the ZnF3-BRCT domain of PARP1, facilitating the downstream recruitment of the DNA repair factor XRCC1. In the absence of DNA damage, PINX1 constitutively binds to PARP1, promoting PARP1-chromatin association and transcription of specific DNA damage repair proteins, including XRCC1, and transcriptional regulators, including GLIS3. Collectively, our findings identify PINX1 as a multifaceted partner of PARP1, crucial for safeguarding cells against genotoxic stress and emerging as a potential candidate for targeted tumor therapy.


Asunto(s)
Proteínas de Ciclo Celular , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Línea Celular Tumoral , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Daño del ADN , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Etopósido/farmacología , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X
9.
Front Immunol ; 15: 1438198, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39136009

RESUMEN

Background: Ovarian carcinoma (OC) is a prevalent gynecological malignancy associated with high recurrence rates and mortality, often diagnosed at advanced stages. Despite advances in immunotherapy, immune exhaustion remains a significant challenge in achieving optimal tumor control. However, the exploration of intratumoral heterogeneity of malignant epithelial cells and the ovarian cancer tumor microenvironment is still limited, hindering our comprehensive understanding of the disease. Materials and methods: Utilizing single-cell RNA sequencing (scRNA-seq), we comprehensively investigated the cellular composition across six ovarian cancer patients with omental metastasis. Our focus centered on analysis of the malignant epithelial cells. Employing CytoTRACE and slingshot pseudotime analyses, we identified critical subpopulations and explored associated transcription factors (TFs) influencing ovarian cancer progression. Furthermore, by integrating clinical factors from a large cohort of bulk RNA sequencing data, we have established a novel prognostic model to investigate the impact of the tumor immune microenvironment on ovarian cancer patients. Furthermore, we have investigated the condition of immunological exhaustion. Results: Our study identified a distinct and highly proliferative subgroup of malignant epithelial cells, known as C2 TOP2A+ TCs. This subgroup primarily consisted of patients who hadn't received neoadjuvant chemotherapy. Ovarian cancer patients with elevated TOP2A expression exhibited heightened sensitivity to neoadjuvant chemotherapy (NACT). Moreover, the transcription factor MYBL2 in this subgroup played a critical role in ovarian cancer development. Additionally, we developed an independent prognostic indicator, the TOP2A TCs Risk Score (TTRS), which revealed a correlation between the High TTRS Group and unfavorable outcomes. Furthermore, immune infiltration and drug sensitivity analyses demonstrated increased responsiveness to Paclitaxel, Cisplatin, and Gemcitabine in the Low TTRS Group. Conclusion: This research deepens our understanding of malignant epithelial cells in ovarian cancer and enhances our knowledge of the ovarian cancer immune microenvironment and immune exhaustion. We have revealed the heightened susceptibility of the C2 TOP2A+ TCs subgroup to neoadjuvant chemotherapy and emphasized the role of MYBL2 within the C2 subgroup in promoting the occurrence and progression of ovarian cancer. These insights provide valuable guidance for the management of ovarian cancer treatment.


Asunto(s)
Progresión de la Enfermedad , Células Epiteliales , Neoplasias Ováricas , Análisis de la Célula Individual , Microambiente Tumoral , Femenino , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/tratamiento farmacológico , Microambiente Tumoral/inmunología , Microambiente Tumoral/genética , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Transactivadores/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Biomarcadores de Tumor/genética , Análisis de Secuencia de ARN , Pronóstico , Proteínas de Unión al ADN/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , RNA-Seq , Persona de Mediana Edad , ADN-Topoisomerasas de Tipo II
10.
Proc Natl Acad Sci U S A ; 121(33): e2405177121, 2024 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-39110738

RESUMEN

The ring-shaped Cohesin complex, consisting of core subunits Smc1, Smc3, Scc1, and SA2 (or its paralog SA1), topologically entraps two duplicated sister DNA molecules to establish sister chromatid cohesion in S-phase. It remains largely elusive how the Cohesin release factor Wapl binds the Cohesin complex, thereby inducing Cohesin disassociation from mitotic chromosomes to allow proper resolution and separation of sister chromatids. Here, we show that Wapl uses two structural modules containing the FGF motif and the YNARHWN motif, respectively, to simultaneously bind distinct pockets in the extensive composite interface between Scc1 and SA2. Strikingly, only when both docking modules are mutated, Wapl completely loses the ability to bind the Scc1-SA2 interface and release Cohesin, leading to erroneous chromosome segregation in mitosis. Surprisingly, Sororin, which contains a conserved FGF motif and functions as a master antagonist of Wapl in S-phase and G2-phase, does not bind the Scc1-SA2 interface. Moreover, Sgo1, the major protector of Cohesin at mitotic centromeres, can only compete with the FGF motif but not the YNARHWN motif of Wapl for binding Scc1-SA2 interface. Our data uncover the molecular mechanism by which Wapl binds Cohesin to ensure precise chromosome segregation.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Segregación Cromosómica , Cohesinas , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Unión Proteica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Secuencias de Aminoácidos , Mitosis , Cromátides/metabolismo , Proteínas Portadoras , Proteínas Proto-Oncogénicas
11.
Sci Rep ; 14(1): 19026, 2024 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-39152186

RESUMEN

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA.


Asunto(s)
Adenosina Trifosfatasas , Proteínas Bacterianas , Replicación del ADN , Proteínas de Unión al ADN , Complejos Multiproteicos , Mycobacterium smegmatis , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Adenosina Trifosfatasas/metabolismo , Complejos Multiproteicos/metabolismo , Cromosomas Bacterianos/metabolismo , Cromosomas Bacterianos/genética , ADN Bacteriano/metabolismo , ADN Bacteriano/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética
12.
Nat Commun ; 15(1): 6953, 2024 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-39138205

RESUMEN

Filovirus-host interactions play important roles in all stages of the virus lifecycle. Here, we identify LATS1/2 kinases and YAP, key components of the Hippo pathway, as critical regulators of EBOV transcription and egress. Specifically, we find that when YAP is phosphorylated by LATS1/2, it localizes to the cytoplasm (Hippo "ON") where it sequesters VP40 to prevent egress. In contrast, when the Hippo pathway is "OFF", unphosphorylated YAP translocates to the nucleus where it transcriptionally activates host genes and promotes viral egress. Our data reveal that LATS2 indirectly modulates filoviral VP40-mediated egress through phosphorylation of AMOTp130, a positive regulator of viral egress, but more surprisingly that LATS1/2 kinases directly modulate EBOV transcription by phosphorylating VP30, an essential regulator of viral transcription. In sum, our findings highlight the potential to exploit the Hippo pathway/filovirus axis for the development of host-oriented countermeasures targeting EBOV and related filoviruses.


Asunto(s)
Ebolavirus , Vía de Señalización Hippo , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Factores de Transcripción , Transcripción Genética , Liberación del Virus , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Fosforilación , Ebolavirus/fisiología , Ebolavirus/genética , Ebolavirus/metabolismo , Células HEK293 , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Señalizadoras YAP/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Matriz Viral/genética , Fiebre Hemorrágica Ebola/virología , Fiebre Hemorrágica Ebola/metabolismo , Interacciones Huésped-Patógeno , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética
13.
Cell Chem Biol ; 31(8): 1490-1502.e42, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-39116881

RESUMEN

We describe a protein proximity inducing therapeutic modality called Regulated Induced Proximity Targeting Chimeras or RIPTACs: heterobifunctional small molecules that elicit a stable ternary complex between a target protein (TP) selectively expressed in tumor cells and a pan-expressed protein essential for cell survival. The resulting co-operative protein-protein interaction (PPI) abrogates the function of the essential protein, thus leading to death selectively in cells expressing the TP. This approach leverages differentially expressed intracellular proteins as novel cancer targets, with the advantage of not requiring the target to be a disease driver. In this chemical biology study, we design RIPTACs that incorporate a ligand against a model TP connected via a linker to effector ligands such as JQ1 (BRD4) or BI2536 (PLK1) or CDK inhibitors such as TMX3013 or dinaciclib. RIPTACs accumulate selectively in cells expressing the HaloTag-FKBP target, form co-operative intracellular ternary complexes, and induce an anti-proliferative response in target-expressing cells.


Asunto(s)
Antineoplásicos , Proteínas de Ciclo Celular , Bibliotecas de Moléculas Pequeñas , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Proliferación Celular/efectos de los fármacos , Triazoles/química , Triazoles/farmacología , Quinasa Tipo Polo 1 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Azepinas/farmacología , Azepinas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Indolizinas/química , Indolizinas/farmacología , Línea Celular Tumoral , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Ligandos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Compuestos Heterocíclicos con 2 Anillos/farmacología , Compuestos Heterocíclicos con 2 Anillos/química , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas que Contienen Bromodominio , Óxidos N-Cíclicos , Compuestos de Piridinio
14.
Nat Commun ; 15(1): 7154, 2024 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-39168984

RESUMEN

Roberts syndrome (RBS) is an autosomal recessive disorder with profound growth deficiency and limb reduction caused by ESCO2 loss-of-function variants. Here, we elucidate the pathogenesis of limb reduction in an Esco2fl/fl;Prrx1-CreTg/0 mouse model using bulk- and single-cell-RNA-seq and gene co-expression network analyses during embryogenesis. Our results reveal morphological and vascular defects culminating in hemorrhage of mutant limbs at E12.5. Underlying this abnormal developmental progression is a pre-apoptotic, mesenchymal cell population specific to mutant limb buds enriched for p53-related signaling beginning at E9.5. We then characterize these p53-related processes of cell cycle arrest, DNA damage, cell death, and the inflammatory leukotriene signaling pathway in vivo. In utero treatment with pifithrin-α, a p53 inhibitor, rescued the hemorrhage in mutant limbs. Lastly, significant enrichments were identified among genes associated with RBS, thalidomide embryopathy, and other genetic limb reduction disorders, suggesting a common vascular etiology among these conditions.


Asunto(s)
Apoptosis , Proteínas Cromosómicas no Histona , Cohesinas , Modelos Animales de Enfermedad , Deformidades Congénitas de las Extremidades , Proteína p53 Supresora de Tumor , Animales , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Apoptosis/genética , Ratones , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/genética , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/patología , Deformidades Congénitas de las Extremidades/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Femenino , Tolueno/análogos & derivados , Tolueno/farmacología , Ectromelia/genética , Ectromelia/metabolismo , Ectromelia/patología , Benzotiazoles/farmacología , Transducción de Señal , Masculino , Daño del ADN , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Esbozos de los Miembros/metabolismo , Hemorragia/patología , Hemorragia/genética , Hipertelorismo , Proteínas de Homeodominio , Anomalías Craneofaciales
15.
Nat Commun ; 15(1): 7132, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-39164253

RESUMEN

Although the E3 ligase Mdm2 and its homologue and binding partner MdmX are the major regulators of the p53 tumor suppressor protein, it is now evident that Mdm2 and MdmX have multiple functions that do not involve p53. As one example, it is known that Mdm2 can regulate cell migration, although mechanistic insight into this function is still lacking. Here we show in cells lacking p53 expression that knockdown of Mdm2 or MdmX, as well as pharmacological inhibition of the Mdm2/MdmX complex, not only reduces cell migration and invasion, but also impairs cell spreading and focal adhesion formation. In addition, Mdm2 knockdown decreases metastasis in vivo. Interestingly, Mdm2 downregulates the expression of Sprouty4, which is required for the Mdm2 mediated effects on cell migration, focal adhesion formation and metastasis. Further, our findings indicate that Mdm2 dampening of Sprouty4 is a prerequisite for maintaining RhoA levels in the cancer cells that we have studied. Taken together we describe a molecular mechanism whereby the Mdm2/MdmX complex through Sprouty4 regulates cellular processes leading to increase metastatic capability independently of p53.


Asunto(s)
Movimiento Celular , Adhesiones Focales , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p53 Supresora de Tumor , Proteína de Unión al GTP rhoA , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/genética , Adhesiones Focales/metabolismo , Adhesiones Focales/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Humanos , Animales , Movimiento Celular/genética , Línea Celular Tumoral , Ratones , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Regulación Neoplásica de la Expresión Génica
16.
Cell Death Dis ; 15(8): 603, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-39164278

RESUMEN

Triple negative breast cancer (TNBC) is an aggressive disease which currently has no effective therapeutic targets and prominent biomarkers. The Sperm Associated antigen 5 (SPAG5) is a mitotic spindle associated protein with oncogenic function in several human cancers. In TNBC, increased SPAG5 expression has been associated with tumor progression, chemoresistance, relapse, and poor clinical outcome. Here we show that high SPAG5 expression in TNBC is regulated by coordinated activity of YAP, mutant p53 and MYC. Depletion of YAP or mutant p53 proteins reduced SPAG5 expression and the recruitment of MYC onto SPAG5 promoter. Targeting of MYC also reduced SPAG5 expression and concomitantly tumorigenicity of TNBC cells. These effects of MYC targeting were synergized with cytotoxic chemotherapy and markedly reduced TNBC oncogenicity in SPAG5-expression dependent manner. These results suggest that mutant p53-MYC-SPAG5 expression can be considered as bona fide predictors of patient's outcome, and reliable biomarkers for effective anticancer therapies.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas Proto-Oncogénicas c-myc , Neoplasias de la Mama Triple Negativas , Proteína p53 Supresora de Tumor , Humanos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Línea Celular Tumoral , Femenino , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Animales , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Mutación/genética , Ratones , Proteínas Señalizadoras YAP/metabolismo , Ratones Desnudos
17.
Cell Rep ; 43(8): 114610, 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39116201

RESUMEN

The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation.


Asunto(s)
Empalme Alternativo , Exones , Precursores del ARN , Proteínas Ribosómicas , Proteína p53 Supresora de Tumor , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Humanos , Exones/genética , Precursores del ARN/metabolismo , Precursores del ARN/genética , Empalme Alternativo/genética , Nucléolo Celular/metabolismo , Proliferación Celular , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Unión Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ribosomas/metabolismo , Estrés Fisiológico/genética , Proteínas de Unión al ARN
18.
Cell Rep ; 43(8): 114606, 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-39120974

RESUMEN

Patients with small-cell lung cancer (SCLC) are in dire need of more effective therapeutic options. Frequent disruption of the G1 checkpoint in SCLC cells creates a dependency on the G2/M checkpoint to maintain genomic integrity. Indeed, in pre-clinical models, inhibiting the G2/M checkpoint kinase WEE1 shows promise in inhibiting SCLC growth. However, toxicity and acquired resistance limit the clinical effectiveness of this strategy. Here, using CRISPR-Cas9 knockout screens in vitro and in vivo, we identified multiple factors influencing the response of SCLC cells to the WEE1 kinase inhibitor AZD1775, including the GCN2 kinase and other members of its signaling pathway. Rapid activation of GCN2 upon AZD1775 treatment triggers a stress response in SCLC cells. Pharmacological or genetic activation of the GCN2 pathway enhances cancer cell killing by AZD1775. Thus, activation of the GCN2 pathway represents a promising strategy to increase the efficacy of WEE1 inhibitors in SCLC.


Asunto(s)
Proteínas de Ciclo Celular , Neoplasias Pulmonares , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Pirimidinonas , Carcinoma Pulmonar de Células Pequeñas , Humanos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Línea Celular Tumoral , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Pirazoles/farmacología , Ratones , Transducción de Señal/efectos de los fármacos , Ratones Desnudos
19.
Proc Natl Acad Sci U S A ; 121(36): e2405543121, 2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-39190349

RESUMEN

Higher levels of aneuploidy, characterized by imbalanced chromosome numbers, are associated with lethal progression in prostate cancer. However, how aneuploidy contributes to prostate cancer aggressiveness remains poorly understood. In this study, we assessed in patients which genes on chromosome 8q, one of the most frequently gained chromosome arms in prostate tumors, were most strongly associated with long-term risk of cancer progression to metastases and death from prostate cancer (lethal disease) in 403 patients and found the strongest candidate was cohesin subunit gene, RAD21, with an odds ratio of 3.7 (95% CI 1.8, 7.6) comparing the highest vs. lowest tertiles of mRNA expression and adjusting for overall aneuploidy burden and Gleason score, both strong prognostic factors in primary prostate cancer. Studying prostate cancer driven by the TMPRSS2-ERG oncogenic fusion, found in about half of all prostate tumors, we found that increased RAD21 alleviated toxic oncogenic stress and DNA damage caused by oncogene expression. Data from both organoids and patients indicate that increased RAD21 thereby enables aggressive tumors to sustain tumor proliferation, and more broadly suggests one path through which tumors benefit from aneuploidy.


Asunto(s)
Aneuploidia , Carcinogénesis , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Progresión de la Enfermedad , Neoplasias de la Próstata , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Carcinogénesis/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Cromosomas Humanos Par 8/genética , Regulación Neoplásica de la Expresión Génica , Daño del ADN
20.
Sci Adv ; 10(34): eadr0036, 2024 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-39178265

RESUMEN

CDCA7, encoding a protein with a carboxyl-terminal cysteine-rich domain (CRD), is mutated in immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a disease related to hypomethylation of juxtacentromeric satellite DNA. How CDCA7 directs DNA methylation to juxtacentromeric regions is unknown. Here, we show that the CDCA7 CRD adopts a unique zinc-binding structure that recognizes a CpG dyad in a non-B DNA formed by two sequence motifs. CDCA7, but not ICF mutants, preferentially binds the non-B DNA with strand-specific CpG hemi-methylation. The unmethylated sequence motif is highly enriched at centromeres of human chromosomes, whereas the methylated motif is distributed throughout the genome. At S phase, CDCA7, but not ICF mutants, is concentrated in constitutive heterochromatin foci, and the formation of such foci can be inhibited by exogenous hemi-methylated non-B DNA bound by the CRD. Binding of the non-B DNA formed in juxtacentromeric regions during DNA replication provides a mechanism by which CDCA7 controls the specificity of DNA methylation.


Asunto(s)
Centrómero , Islas de CpG , Metilación de ADN , Síndromes de Inmunodeficiencia , Enfermedades de Inmunodeficiencia Primaria , Unión Proteica , Humanos , Enfermedades de Inmunodeficiencia Primaria/metabolismo , Enfermedades de Inmunodeficiencia Primaria/genética , Síndromes de Inmunodeficiencia/metabolismo , Síndromes de Inmunodeficiencia/genética , Centrómero/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Dominios Proteicos , ADN/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Mutación , Heterocromatina/metabolismo , Heterocromatina/genética , Cara/anomalías , Proteínas Nucleares
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