RESUMEN
Melanoma clinical outcomes emerge from incompletely understood genetic mechanisms operating within the tumor and its microenvironment. Here, we used single-cell RNA-based spatial molecular imaging (RNA-SMI) in patient-derived archival tumors to reveal clinically relevant markers of malignancy progression and prognosis. We examined spatial gene expression of 203,472 cells inside benign and malignant melanocytic neoplasms, including melanocytic nevi and primary invasive and metastatic melanomas. Algorithmic cell clustering paired with intratumoral comparative two-dimensional analyses visualized synergistic, spatial gene signatures linking cellular proliferation, metabolism, and malignancy, validated by protein expression. Metastatic niches included up-regulation of CDK2 and FABP5, which independently predicted poor clinical outcome in 473 patients with melanoma via Cox regression analysis. More generally, our work demonstrates a framework for applying single-cell RNA-SMI technology toward identifying gene regulatory landscapes pertinent to cancer progression and patient survival.
Asunto(s)
Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Melanoma , Análisis de la Célula Individual , Humanos , Melanoma/patología , Melanoma/genética , Melanoma/metabolismo , Melanoma/mortalidad , Pronóstico , Análisis de la Célula Individual/métodos , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Microambiente Tumoral , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Masculino , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/mortalidad , Perfilación de la Expresión GénicaRESUMEN
Vitamin D signals through the vitamin D receptor (VDR) to induce its end-organ effects. Hepatic stellate cells control development of liver fibrosis in response to stressors and vitamin D signaling decreases fibrogenesis. VDR expression in hepatocytes is low in healthy liver, and the role of VDR in hepatocyte proliferation is unclear. Hepatocyte-VDR null mice (hVDR) were used to assess the role of VDR and vitamin D signaling in hepatic regeneration. hVDR mice have impaired liver regeneration and impaired hepatocyte proliferation associated with significant differential changes in bile salts. Notably, mice lacking hepatocyte VDR had significant increases in expression of conjugated bile acids after partial hepatectomy, consistent with failure to normalize hepatic function by the 14-day time point tested. Real-time PCR of hVDR and control livers showed significant changes in expression of cell-cycle genes including cyclins D1 and E1 and cyclin-dependent kinase 2. Gene expression profiling of hepatocytes treated with vitamin D or control showed regulation of groups of genes involved in liver proliferation, hepatitis, liver hyperplasia/hyperproliferation, and liver necrosis/cell death. Together, these studies demonstrate an important functional role for VDR in hepatocytes during liver regeneration. Combined with the known profibrotic effects of impaired VDR signaling in stellate cells, the studies provide a mechanism whereby vitamin D deficiency would both reduce hepatocyte proliferation and permit fibrosis, leading to significant liver compromise.
Asunto(s)
Ácidos y Sales Biliares , Proliferación Celular , Hepatectomía , Hepatocitos , Regeneración Hepática , Ratones Noqueados , Receptores de Calcitriol , Animales , Regeneración Hepática/efectos de los fármacos , Regeneración Hepática/fisiología , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Masculino , Ratones , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ácidos y Sales Biliares/metabolismo , Hígado/metabolismo , Ciclina D1/metabolismo , Ciclina D1/genética , Ciclina E/metabolismo , Ciclina E/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Ratones Endogámicos C57BL , Vitamina D/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas OncogénicasRESUMEN
Merkel Cell Carcinoma (MCC) is a rare neuroendocrine skin cancer. In our previous work, we decoded genes specifically deregulated by MCPyV early genes as opposed to other polyomaviruses and established functional importance of NDRG1 in inhibiting cellular proliferation and migration in MCC. In the present work, we found the SET protein, (I2PP2A, intrinsic inhibitor of PP2A) upstream of NDRG1 which was modulated by MCPyV early genes, both in hTERT-HK-MCPyV and MCPyV-positive (+) MCC cell lines. Additionally, MCC dermal tumour nodule tissues showed strong SET expression. Inhibition of the SET-PP2A interaction in hTERT-HK-MCPyV using the small molecule inhibitor, FTY720, increased NDRG1 expression and inhibited cell cycle regulators, cyclinD1 and CDK2. SET inhibition by shRNA and FTY720 also decreased cell proliferation and colony formation in MCPyV(+) MCC cells. Overall, these results pave a path for use of drugs targeting SET protein for the treatment of MCC.
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Carcinoma de Células de Merkel , Movimiento Celular , Proliferación Celular , Poliomavirus de Células de Merkel , Proteína Fosfatasa 2 , Humanos , Poliomavirus de Células de Merkel/fisiología , Poliomavirus de Células de Merkel/genética , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Carcinoma de Células de Merkel/virología , Carcinoma de Células de Merkel/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Clorhidrato de Fingolimod/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Línea Celular Tumoral , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Infecciones por Polyomavirus/virología , Neoplasias Cutáneas/virología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genéticaRESUMEN
Oncogene activation through DNA amplification or overexpression is a crucial driver of cancer initiation and progression. The FOXK2 gene, located on chromosome 17q25, encodes a transcription factor with a forkhead DNA-binding domain. Analysis of genomic datasets reveals that FOXK2 is frequently amplified and overexpressed in breast cancer, correlating with poor patient survival. Knockdown of FOXK2 significantly inhibited breast cancer cell proliferation, migration, anchorage-independent growth, and delayed tumor growth in a xenograft mouse model. Additionally, inhibiting FOXK2 sensitized breast cancer cells to chemotherapy. Co-overexpression of FOXK2 and mutant PI3KCA transformed non-tumorigenic MCF-10A cells, suggesting a role for FOXK2 in PI3KCA-driven tumorigenesis. CCNE2, PDK1, and ESR1 were identified as transcriptional targets of FOXK2 in MCF-7 cells. Small-molecule inhibitors of CCNE2/CDK2 (dinaciclib) and PDK1 (dichloroacetate) exhibited synergistic anti-tumor effects with PI3KCA inhibitor (alpelisib) in vitro. Inhibition of FOXK2 by dinaciclib synergistically enhanced the anti-tumor effects of alpelisib in a xenograft mouse model. Collectively, these findings highlight the oncogenic function of FOXK2 and suggest that FOXK2 and its downstream genes represent potential therapeutic targets in breast cancer.
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Neoplasias de la Mama , Proliferación Celular , Resistencia a Antineoplásicos , Factores de Transcripción Forkhead , Animales , Femenino , Humanos , Ratones , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido/genética , Antineoplásicos/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células MCF-7 , Ratones Desnudos , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Tiazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dysregulation of cyclin-dependent kinases (CDKs) impacts cell proliferation, driving cancer. Here, we ask why the cyclin-D/CDK4 complex governs cell cycle progression through the longer G1 phase, whereas cyclin-E/CDK2 regulates the shorter G1/S phase transition. We consider available experimental cellular and structural data including cyclin-E's high-level burst, sustained duration of elevated cyclin-D expression, and explicit solvent molecular dynamics simulations of the inactive monomeric and complexed states, to establish the conformational tendencies along the landscape of the distinct activation scenarios of cyclin-D/CDK4 and cyclin-E/CDK2 in the G1 phase and G1/S transition of the cell cycle, respectively. These lead us to propose slower activation of cyclin-D/CDK4 and rapid activation of cyclin-E/CDK2. We provide the mechanisms through which this occurs, offering innovative CDK4 drug design considerations. Our insightful mechanistic work addresses a compelling cell cycle regulation question and illuminates the distinct activation speeds between the G1 and the G1/S phases, which are crucial for function.
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Ciclo Celular , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Simulación de Dinámica Molecular , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/química , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/química , Humanos , Unión Proteica , Ciclina E/metabolismo , Ciclina E/química , Ciclina E/genética , Ciclina D/metabolismo , Ciclina D/química , Ciclina D/genética , Sitios de Unión , Activación EnzimáticaRESUMEN
Cyclin-dependent kinase 2 (CDK2) is a key cell cycle regulator, with essential roles during G1/S transition. The clinicopathological significance of CDK2 in ductal carcinomas in situ (DCIS) and early-stage invasive breast cancers (BCs) remains largely unknown. Here, we evaluated CDK2's protein expression in 479 BC samples and 216 DCIS specimens. Analysis of CDK2 transcripts was completed in the METABRIC cohort (n = 1980) and TCGA cohort (n = 1090), respectively. A high nuclear CDK2 protein expression was significantly associated with aggressive phenotypes, including a high tumour grade, lymph vascular invasion, a poor Nottingham prognostic index (all p-values < 0.0001), and shorter survival (p = 0.006), especially in luminal BC (p = 0.009). In p53-mutant BC, high nuclear CDK2 remained linked with worse survival (p = 0.01). In DCIS, high nuclear/low cytoplasmic co-expression showed significant association with a high tumour grade (p = 0.043), triple-negative and HER2-enriched molecular subtypes (p = 0.01), Comedo necrosis (p = 0.024), negative ER status (p = 0.004), negative PR status (p < 0.0001), and a high proliferation index (p < 0.0001). Tumours with high CDK2 transcripts were more likely to have higher expressions of genes involved in the cell cycle, homologous recombination, and p53 signaling. We provide compelling evidence that high CDK2 is a feature of aggressive breast cancers. The clinical evaluation of CDK2 inhibitors in early-stage BC patients will have a clinical impact.
Asunto(s)
Neoplasias de la Mama , Carcinoma Intraductal no Infiltrante , Quinasa 2 Dependiente de la Ciclina , Humanos , Femenino , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Carcinoma Intraductal no Infiltrante/patología , Carcinoma Intraductal no Infiltrante/genética , Carcinoma Intraductal no Infiltrante/metabolismo , Pronóstico , Persona de Mediana Edad , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Estadificación de Neoplasias , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/mortalidad , Anciano , Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
This study explored the effects of 4-hydroxy-2(3H)-benzoxazolone(HBOA) on the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. The L3.6 cells cultured in vitro were treated with HBOA of 0-1.0 mmol·L~(-1). The cell viability was detected by the cell counting kit-8(CCK-8) method, and the half inhibitory concentration(IC_(50)) was analyzed to determine the drug concentration and time. The cell morphology was observed under an inverted microscope and by acridine orange(AO) staining. The ability of proliferation and self-renewal were evaluated through live cell counting and colony formation experiments. The cell cycle progression and cell apoptosis rate were detected by flow cytometry. The morphology of cell apoptosis was observed by scanning electron microscopy. The mRNA expression of proliferating cell nuclear antigen(PCNA), cyclinA1, cyclinA2, cyclin dependent kinase 2(CDK2), and cyclin dependent kinase inhibitor 1A(P21) were determined by qPCR. The level of reactive oxygen species(ROS), lipid peroxide, and mitochondrial membrane potential were measured by flow cytometry. The activity of protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway was detected by Western blot. Compared with the control group, the cells treated with HBOA exhibited a significant decrease in viability. Then the optimal concentration and intervention time of HBOA were determined to be 0.4 mmol·L~(-1), 0.6 mmol·L~(-1), and 48 h. Compared with the control group, groups with HBOA of 0.4 mmol·L~(-1 )and 0.6 mmol·L~(-1) showed a significant suppression in cell proliferation and colony formation ability, down-regulated mRNA of PCNA, cyclinA1, cyclinA2, and CDK2, up-regulated P21 mRNA, S-phase cell cycle arrest, and increased cell apoptosis rate. There was an appearance of apoptotic bodies, increased ROS and lipid peroxide, decreased mitochondrial membrane potential(with a significant decrease in 0.6 mmol·L~(-1) group), and down-regulated p-Akt and p-mTOR proteins. The results show that HBOA inhibits the proliferation of pancreatic cancer L3.6 cells and induces cell apoptosis, which may be related to the increase in reactive oxygen species and the inhibition of the Akt/mTOR pathway.
Asunto(s)
Apoptosis , Proliferación Celular , Neoplasias Pancreáticas , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Humanos , Línea Celular Tumoral , Benzoxazoles/farmacología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ciclo Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Supervivencia Celular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Ubiquitin like with PHD and ring finger domains 2 (UHRF2) regulates the cell cycle and epigenetics as a multi-domain protein sharing homology with UHRF1. UHRF1 functions with DNMT1 to coordinate daughter strand methylation during DNA replication, but UHRF2 can't perform this function, and its roles during cell cycle progression are not well defined. UHRF2 role as an oncogene vs. tumor suppressor differs in distinct cell types. UHRF2 interacts with E2F1 to control Cyclin E1 (CCNE1) transcription. UHRF2 also functions in a reciprocal loop with Cyclin E/CDK2 during G1, first as a direct target of CDK2 phosphorylation, but also as an E3-ligase with direct activity toward both Cyclin E and Cyclin D. In this study, we demonstrate that UHRF2 is expressed in early G1 following either serum stimulation out of quiescence or in cells transiting directly out of M-phase, where UHRF2 protein is lost. Further, UHRF2 depletion in G2/M is reversed with a CDK1 specific inhibitor. UHRF2 controls expression levels of cyclins and CDK inhibitors and controls its own transcription in a negative-feedback loop. Deletion of UHRF2 using CRISPR/Cas9 caused a delay in passage through each cell cycle phase. UHRF2 loss culminated in elevated levels of cyclins but also the CDK inhibitor p27KIP1, which regulates G1 passage, to reduce retinoblastoma phosphorylation and increase the amount of time required to reach G1/S passage. Our data indicate that UHRF2 is a central regulator of cell-cycle pacing through its complex regulation of cell cycle gene expression and protein stability.
Asunto(s)
Ciclina E , Fase G1 , Mitosis , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Humanos , Ciclina E/metabolismo , Ciclina E/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Ciclo Celular/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Fosforilación , Proteínas OncogénicasRESUMEN
Lung adenocarcinoma (LUAD), a type of non-small cell lung cancer (NSCLC), originates from not only bronchial epithelial cells but also alveolar type 2 (AT2) cells, which could differentiate into AT2-like cells. AT2-like cells function as cancer stem cells (CSCs) of LUAD tumorigenesis to give rise to adenocarcinoma. However, the mechanism underlying AT2 cell differentiation into AT2-like cells in LUAD remains unknown. We analyze genes differentially expressed and genes with significantly different survival curves in LUAD, and the combination of these two analyses yields 147 differential genes, in which 14 differentially expressed genes were enriched in cell cycle pathway. We next analyze the protein levels of these genes in LUAD and find that Cyclin-A2 (CCNA2) is closely associated with LUAD tumorigenesis. Unexpectedly, high CCNA2 expression in LUAD is restrictedly associated with smoking and independent of other driver mutations. Single-cell sequencing analyses reveal that CCNA2 is predominantly involved in AT2-like cell differentiation, while inhibition of CCNA2 significantly reverses smoking-induced AT2-like cell differentiation. Mechanistically, CCNA2 binding to CDK2 phosphorylates the AXIN1 complex, which in turn induces ubiquitination-dependent degradation of ß-catenin and inhibits the WNT signaling pathway, thereby failing AT2 cell maintenance. These results uncover smoking-induced CCNA2 overexpression and subsequent WNT/ß-catenin signaling inactivation as a hitherto uncharacterized mechanism controlling AT2 cell differentiation and LUAD tumorigenesis.
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Adenocarcinoma del Pulmón , Carcinogénesis , Diferenciación Celular , Ciclina A2 , Neoplasias Pulmonares , Fumar , Animales , Femenino , Humanos , Masculino , Ratones , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , beta Catenina/metabolismo , beta Catenina/genética , Carcinogénesis/genética , Línea Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismo , Fumar/efectos adversos , Vía de Señalización Wnt/genética , RatasRESUMEN
BACKGROUND: Reduced numbers and dysfunction of thymic epithelial cells (TECs) are important factors of thymic degeneration. Previous studies have found that umbilical cord mesenchymal stem cells (UCMSCs) reverse the structure and function of the senescent thymus in vivo. However, the transcriptomic regulation mechanism is unclear. METHODS: TECs were cultured with H2O2 for 72 hours to induce senescence. UCMSCs were cocultured with senescent TECs for 48 hours to detect SA-ß-gal, P16 and Ki67. The cocultured TECs were collected for lncRNA, mRNA and miRNA sequencing to establish a competitive endogenous regulatory network (ceRNA). And RT-qPCR, immunofluorescence staining, and western blot were used to identified key genes. RESULTS: Our results showed that H2O2 induced TEC aging and that UCMSCs reversed these changes. Compared with those in aged TECs, 2260 DE mRNAs, 1033 DE lncRNAs and 67 DE miRNAs were differentially expressed, and these changes were reversed by coculturing the cells with UCMSCs. Differential mRNA enrichment analysis of ceRNA regulation revealed that the PI3K-AKT pathway was a significant signaling pathway. UCMSC coculture upregulated VEGFA, which is the upstream factor of the PI3K-AKT signaling pathway, and the expression of the key proteins PI3K and AKT. Thus, the expression of the cell cycle suppressor P27, which is downstream of the PI3K-AKT signaling pathway, was downregulated, while the expression of the cell cycle regulators CDK2 and CCNE was upregulated. CONCLUSION: UCMSC coculture upregulated the expression of VEGFA, activated the PI3K-AKT signaling pathway, increased the expression of CDK2 and CCNE, decreased the expression of P27, and promoted the proliferation of TECs.
Asunto(s)
Senescencia Celular , Técnicas de Cocultivo , Células Epiteliales , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas , MicroARNs , Proteínas Oncogénicas , Timo , Cordón Umbilical , Células Madre Mesenquimatosas/metabolismo , Humanos , Células Epiteliales/metabolismo , Cordón Umbilical/citología , Timo/citología , Timo/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Ciclina E/metabolismo , Ciclina E/genética , Biomarcadores/metabolismo , Peróxido de Hidrógeno/toxicidad , Peróxido de Hidrógeno/farmacología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Células Cultivadas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transcriptoma , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genéticaRESUMEN
We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.
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Proliferación Celular , Citocinas , Proteína con Dominio Pirina 3 de la Familia NLR , Psoriasis , Humanos , Ciclo Celular/genética , Proliferación Celular/genética , Ciclina B1/metabolismo , Ciclina B1/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Citocinas/metabolismo , Silenciador del Gen , Células HaCaT , Interleucina-17/metabolismo , Interleucina-17/genética , Interleucina-23/metabolismo , Interleucina-23/genética , Interleucina-6/metabolismo , Interleucina-6/genética , Antígeno Ki-67/metabolismo , Antígeno Ki-67/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Psoriasis/genética , Psoriasis/metabolismo , Psoriasis/patología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.
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Neoplasias Ováricas , Neoplasias de la Retina , Retinoblastoma , Femenino , Humanos , Carcinoma Epitelial de Ovario , Ciclina D1/genética , Neoplasias Ováricas/genética , Quinasa 2 Dependiente de la Ciclina/genética , Ubiquitina-Proteína Ligasas , Proteínas de Unión a Retinoblastoma/genéticaRESUMEN
BACKGROUND: Circular RNAs (circRNAs), which are covalently closed non-coding RNAs, are frequently dysregulated in cancer. However, their precise role in bladder cancer (BCa) remains largely unknown. METHODS: Expression of hsa_circ_0005320 in tissues and cell lines was detected using quantitative real-time PCR. Proliferation and colony forming capacity of BCa cells were assessed using Cell Counting Kit-8, ethynyl-labeled deoxyuridine, and colony formation assays. The cell cycle was analyzed using flow cytometry. Protein expression of insulin-like growth factor II mRNA-binding protein 3 (IGF2BP3) and cyclin dependent kinase 2 (CDK2) was examined using western blots. The binding of RNA and protein was validated using RNA immunoprecipitation. Additionally, xenograft tumor models were established to validate the function of hsa_circ_0005320 in vivo. RESULTS: We screened hsa_circ_0005320 from previous high-throughput sequencing and found that it was highly expressed in BCa tissues and associated with tumor differentiation and depth of invasion in BCa patients. Through functional experiments, we demonstrated that hsa_circ_0005320 promoted cell proliferation and regulated the cell cycle. Mechanistically, hsa_circ_0005320 interacted with and upregulated the expression of IGF2BP3, which binds to and enhances the stability of CDK2 mRNA. Furthermore, knockdown of hsa_circ_0005320 resulted in a reduction in tumor burden in vivo. CONCLUSIONS: Collectively, these findings highlight the pro-oncogenic role of hsa_circ_0005320 in BCa through the IGF2BP3/CDK2 axis, providing valuable insights into the mechanism of circRNAs in tumor progression.
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Ciclo Celular , Proliferación Celular , Quinasa 2 Dependiente de la Ciclina , ARN Circular , Proteínas de Unión al ARN , Neoplasias de la Vejiga Urinaria , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Circular/metabolismo , ARN Circular/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Breast cancer, the most common cancer, presents a significant challenge to the health and longevity of women. Aspongopus chinensis Dallas is an insect with known anti-breast cancer properties. However, the anti-breast cancer effects and underlying mechanisms have not been elucidated. Exogenous microRNAs (miRNAs), which are derived from plants and animals, have been revealed to have notable capacities for controlling the proliferation of cancerous cells. To elucidate the inhibitory effects of miRNAs derived from A. chinensis and the regulatory mechanism involved in the growth of breast cancer cells, miRNA sequencing was initially employed to screen for miRNAs both in A. chinensis hemolymph and decoction and in mouse serum and tumor tissue after decoction gavage. Subsequently, the experiments were performed to assess the suppressive effect of ach-miR-276a-3p, the miRNA screened out from a previous study, on the proliferation of MDA-MB-231 and MDA-MB-468 breast cancer cell lines in vitro and in vivo. Finally, the regulatory mechanism of ach-miR-276a-3p in MDA-MB-231 and MDA-MB-468 breast cancer cells was elucidated. The results demonstrated that ach-miR-276a-3p notably inhibited breast cancer cell proliferation, migration, colony formation, and invasion and induced cell cycle arrest at the G0/G1 phase. Moreover, the ach-miR-276a-3p mimics significantly reduced the tumor volume and weight in xenograft tumor mice. Furthermore, ach-miR-276a-3p could induce cell cycle arrest by targeting APPL2 and regulating the CDK2-Rb-E2F1 signaling pathway. In summary, ach-miR-276a-3p, derived from A. chinensis, has anti-breast cancer activity by targeting APPL2 and regulating the CDK2-Rb-E2F1 signaling pathway and can serve as a promising candidate anticancer agent.
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Neoplasias de la Mama , MicroARNs , Humanos , Femenino , Animales , Ratones , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , MicroARNs/genética , MicroARNs/metabolismo , Puntos de Control del Ciclo Celular , Transducción de Señal , Regulación Neoplásica de la Expresión Génica , Quinasa 2 Dependiente de la Ciclina/genética , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
SUMMARY: In this issue, Dietrich, Trub, and colleagues describe and characterize a novel selective CDK2 inhibitor: INX-315. This agent shows promise in CCNE1-amplified cancers and in CDK4/6 inhibitor-resistant breast cancers. See related article by Dietrich et al., p. 446 (8).
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Neoplasias de la Mama , Humanos , Femenino , Quinasa 2 Dependiente de la Ciclina/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Quinasa 4 Dependiente de la Ciclina/genéticaRESUMEN
Posttranslational modifications (PTMs) of proteins can be investigated by Nuclear Magnetic Resonance (NMR) spectroscopy as a powerful analytical tool to define modification sites, their relative stoichiometry, and crosstalk between modifications. As a Structural Biology method, NMR provides important additional information on changes in protein conformation and dynamics upon modification as well as a mapping of binding sites upon biomolecular interactions. Indeed, PTMs not only mediate functional modulation in protein-protein interactions, but can also induce diverse structural responses with different biological outcomes. Here we present protocols that have been developed for the production and phosphorylation of the neuronal tau protein. Under its aggregated form, tau is a hallmark of Alzheimer's disease and other neurodegenerative diseases named tauopathies involving tau dysfunction and/or mutations. As a common feature shared by various tauopathies, tau aggregates are found into a form displaying an increased, abnormal phosphorylation, also referred to hyperphosphorylation. We have used NMR to investigate the phosphorylation patterns of tau induced by several kinases or cell extracts, how phosphorylation affects the local and overall conformation of tau, its interactions with partners (proteins, DNA, small-molecules, etc.) including tubulin and microtubules, and its capacity to form insoluble fibrillar aggregates. We present here detailed protocols for in vitro phosphorylation of tau by the recombinant kinases CDK2/cyclin A and GSK3ß, the production of the recombinant kinases thereof, as well as the analytical characterization of phosphorylated tau by NMR spectroscopy.
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Enfermedad de Alzheimer , Proteínas tau , Humanos , Proteínas tau/metabolismo , Fosforilación , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ciclina A/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Enfermedad de Alzheimer/metabolismo , Espectroscopía de Resonancia Magnética , Quinasa 2 Dependiente de la Ciclina/genéticaRESUMEN
BACKGROUND: Uveal melanoma (UVM) is an aggressive malignant tumor originating from melanocytes in the eye. Here, we screened the possible genes involved in the development and prognosis of UVM, and identified that FOXM1 and MET were associated with the prognosis of UVM patients. Forkhead box protein M1 (FOXM1) is a transcription factor that regulates the expression of cell cycle-related genes that are necessary for DNA duplication. However, the regulatory mechanism of FOXM1 in UVM was still not clear. Here, we investigated the regulation of FOXM1 in the malignant phenotype of UVM cells and its effect on the prognosis of UVM patients. METHODS: UVM gene expression profiles were obtained using GSE22138 data from the gene expression omnibus (GEO). Weighted gene co-expression network analysis (WGCNA) was used to construct a key module gene for metastasis, which was strongly correlated with UVM prognosis. The latent biological pathways were identified through gene ontology analysis. Protein-protein interaction (PPI) networks and hub shared gene authentication were performed. GEPIA and UALCAN databases were used for the analysis of relationship between candidate genes (FOXM1 or MET) and the prognosis of UVM patients. The abundance of FOXM1 was examined by quantitative real time polymerase chain reaction (qRT-PCR) and western blot. Colony formation and cell counting kit-8 (CCK-8) assays for cell proliferation, wound healing assay for migration, and transwell invasion analysis for invasion were performed. RESULTS: GEO database showed the differentially expressed genes between UVM samples with or without metastasis, and a key module gene for metastasis was constructed by WGCNA. The PPI network revealed that seven candidate genes (VEGFA, KRAS, MET, SRC, EZR, FOXM1, and CCNB1) were closely associated with UVM metastasis. GEPIA and UALCAN analyzes suggested that FOXM1 and MET are related to the prognosis of patients with UVM. These experimental results suggested that FOXM1 was highly expressed in UVM cells. FOXM1 deficiency represses the proliferative, migratory, and invasive abilities of UVM cells. CONCLUSIONS: FOXM1 silencing may hinder UVM cell progression, providing a novel theoretical basis and new insights for UVM treatment.
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Melanoma , Neoplasias de la Úvea , Humanos , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Melanoma/metabolismo , Neoplasias de la Úvea/genética , Proliferación Celular , Quinasa 2 Dependiente de la Ciclina/genéticaRESUMEN
Diabetic retinopathy (DR) is a leading cause of irreversible vision loss in working-age populations. Fat mass and obesity-associated protein (FTO) is an N6-methyladenosine (m6A) demethylase that demethylates RNAs involved in energy homeostasis, though its influence on DR is not well studied. Herein, we detected elevated FTO expression in vitreous fibrovascular membranes of patients with proliferative DR. FTO promoted cell cycle progression and tip cell formation of endothelial cells (ECs) to facilitate angiogenesis in vitro, in mice, and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetic microvascular leakage, and mediated EC-microglia interactions to induce retinal inflammation and neurodegeneration in vivo and in vitro. Mechanistically, FTO affected EC features via modulating CDK2 mRNA stability in an m6A-YTHDF2-dependent manner. FTO up-regulation under diabetic conditions was driven by lactate-mediated histone lactylation. FB23-2, an inhibitor to FTO's m6A demethylase activity, suppressed angiogenic phenotypes in vitro. To allow for systemic administration, we developed a nanoplatform encapsulating FB23-2 and confirmed its targeting and therapeutic efficiency in mice. Collectively, our study demonstrates that FTO is important for EC function and retinal homeostasis in DR, and warrants further investigation as a therapeutic target for DR patients.
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Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Quinasa 2 Dependiente de la Ciclina , Diabetes Mellitus , Retinopatía Diabética , Animales , Ratones , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Células Endoteliales/metabolismo , Retina/metabolismo , ARN , Pez Cebra/genéticaRESUMEN
Cyclin-dependent kinase 2 (CDK2) is thought to play an important role in driving proliferation of certain cancers, including those harboring CCNE1 amplification and breast cancers that have acquired resistance to CDK4/6 inhibitors (CDK4/6i). The precise impact of pharmacologic inhibition of CDK2 is not known due to the lack of selective CDK2 inhibitors. Here we describe INX-315, a novel and potent CDK2 inhibitor with high selectivity over other CDK family members. Using cell-based assays, patient-derived xenografts (PDX), and transgenic mouse models, we show that INX-315 (i) promotes retinoblastoma protein hypophosphorylation and therapy-induced senescence (TIS) in CCNE1-amplified tumors, leading to durable control of tumor growth; (ii) overcomes breast cancer resistance to CDK4/6i, restoring cell cycle control while reinstating the chromatin architecture of CDK4/6i-induced TIS; and (iii) delays the onset of CDK4/6i resistance in breast cancer by driving deeper suppression of E2F targets. Our results support the clinical development of selective CDK2 inhibitors. SIGNIFICANCE: INX-315 is a novel, selective inhibitor of CDK2. Our preclinical studies demonstrate activity for INX-315 in both CCNE1-amplified cancers and CDK4/6i-resistant breast cancer. In each case, CDK2 inhibition induces cell cycle arrest and a phenotype resembling cellular senescence. Our data support the development of selective CDK2 inhibitors in clinical trials. See related commentary by Watts and Spencer, p. 386. This article is featured in Selected Articles from This Issue, p. 384.
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Neoplasias de la Mama , Animales , Ratones , Humanos , Femenino , Quinasa 2 Dependiente de la Ciclina/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Puntos de Control del Ciclo Celular , Senescencia Celular , Cromatina , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Ratones TransgénicosRESUMEN
With the significant increase in the availability of microbial genome sequences in recent years, resistance gene-guided genome mining has emerged as a powerful approach for identifying natural products with specific bioactivities. Here, we present the use of this approach to reveal the roseopurpurins as potent inhibitors of cyclin-dependent kinases (CDKs), a class of cell cycle regulators implicated in multiple cancers. We identified a biosynthetic gene cluster (BGC) with a putative resistance gene with homology to human CDK2. Using targeted gene disruption and transcription factor overexpression in Aspergillus uvarum, and heterologous expression of the BGC in Aspergillus nidulans, we demonstrated that roseopurpurin C (1) is produced by this cluster and characterized its biosynthesis. We determined the potency, specificity, and mechanism of action of 1 as well as multiple intermediates and shunt products produced from the BGC. We show that 1 inhibits human CDK2 with a Kiapp of 44 nM, demonstrates selectivity for clinically relevant members of the CDK family, and induces G1 cell cycle arrest in HCT116 cells. Structural analysis of 1 complexed with CDK2 revealed the molecular basis of ATP-competitive inhibition.