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1.
Elife ; 102021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34545812

RESUMO

Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model, we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem/progenitor cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks' level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Dinaminas/metabolismo , Dinâmica Mitocondrial , Células-Tronco/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Ciclina E/genética , Ciclina E/metabolismo , Dinaminas/genética , Células HaCaT , Humanos , Queratina-15/genética , Queratina-15/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Fosforilação , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
2.
Sci Rep ; 11(1): 14736, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34282211

RESUMO

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Fase G1/fisiologia , Divisão Celular , Células Cultivadas , Ciclina D/metabolismo , Ciclina E/metabolismo , Humanos , Proteínas Oncogênicas/metabolismo , Fosforilação , Proteoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína do Retinoblastoma/metabolismo
3.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070839

RESUMO

BACKGROUND: Ovarian clear cell carcinoma (OCCC) is resistant to platinum chemotherapy and is characterized by poor prognosis. Today, the use of poly (ADP-ribose) polymerase (PARP) inhibitor, which is based on synthetic lethality strategy and characterized by cancer selectivity, is widely used for new types of molecular-targeted treatment of relapsed platinum-sensitive ovarian cancer. However, it is less effective against OCCC. METHODS: We conducted siRNA screening to identify synthetic lethal candidates for the ARID1A mutation; as a result, we identified Cyclin-E1 (CCNE1) as a potential target that affects cell viability. To further clarify the effects of CCNE1, human OCCC cell lines, namely TOV-21G and KOC7c (ARID1A mutant lines), and RMG-I and ES2 (ARID1A wild type lines) were transfected with siRNA targeting CCNE1 or a control vector. RESULTS: Loss of CCNE1 reduced proliferation of the TOV-21G and KOC7c cells but not of the RMG-I and ES2 cells. Furthermore, in vivo interference of CCNE1 effectively inhibited tumor cell proliferation in a xenograft mouse model. CONCLUSION: This study showed for the first time that CCNE1 is a synthetic lethal target gene to ARID1A-mutated OCCC. Targeting this gene may represent a putative, novel, anticancer strategy in OCCC treatment.


Assuntos
Adenocarcinoma de Células Claras/genética , Ciclina E/genética , Proteínas de Ligação a DNA/genética , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/genética , Mutações Sintéticas Letais , Fatores de Transcrição/genética , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Ciclina E/antagonistas & inibidores , Ciclina E/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Anticancer Res ; 41(5): 2239-2245, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33952450

RESUMO

BACKGROUND/AIM: This study was designed to investigate the effect of IL-39 on T24 bladder cancer (BC) cell line survival and growth. MATERIALS AND METHODS: In order to assess the direct effect of IL-39 on survival, proliferation, and apoptosis of T24 BC cells, we utilized a clonogenic survival assay, a cell proliferation assay, and caspase-3 activity kits. Potential proliferative and apoptotic molecular mechanisms were evaluated by RT-PCR. RESULTS: Treatment of T24 BC cells with IL-39 resulted in a significant reduction in the percentage of colonies. The anti-tumor effect of IL-39 on T24 bladder cancer cells correlated strongly with a decrease in cyclin E, in combination with an increase in the mRNA levels of Fas. CONCLUSION: IL-39 impedes the growth and survival of T24 BC cells by inhibiting growth and promoting apoptosis. This ability to modulate gene transcription in neoplastic cells shows promise and warrants further research in immunotherapy.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina E/metabolismo , Interleucinas/farmacologia , Receptor fas/metabolismo , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ciclina D/genética , Ciclina D/metabolismo , Ciclina E/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor fas/genética
5.
Bioorg Chem ; 112: 104940, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33965780

RESUMO

A series of novel substituted bisurea 1,4-Diisocyanatobenzene compounds were designed, synthesized and introduced as potent anticancer compounds and screened for their in vitro anti-proliferative activities in human cancer cell lines. The structures of all titled compounds were characterized using Fourier-transform infrared mass spectra, nuclear magnetic resonance spectroscopy, elemental analysis and evaluated their sustainability using biological experiments. A selected group of ten derivatives were apprised for their anti-proliferative activity. The compounds 3d and 3e displayed potent anticancer activity with low IC50 value of 5.40, and 5.89 µM against HeLa cancer cell lines. The observed apoptosis data has demonstrated that compounds 3d and 3e induce the activaties of caspase-9 and caspase-3, the compounds 3d and 3e regulated fungal zone inhibition. Due to promising growth inhibitions, the all synthesized compounds were allowed to campaign includes quantum-polarized-ligand, quantum mechanical and molecular mechanical, docking experiments. The compounds 3d and 3e have exhibited a higher affinity for ERK/MAP kinase and CDK2 proteins. The molecular docking interactions have demonstrated two stage inhibition of cancer cells by binding with ERK/MAP kinase and CDK2 leads to inactivation of cell proliferation,cell cycle progression,cell divisionanddifferentiation, and hypo-phosphorylation of ribosome leading cells to restricts at point boundary of the G1/S phase. The long-range molecular dynamics, 150 ns, simulations were also revealed more consistency by 3d. Our study conclude good binding propensity for active-tunnel of ERK/MAP kinase and CDK2 proteins, by 3d (1,1'-(1,4-phenylene) bis(3-(2-chlorobenzyl)urea)), to suggest that the designed and synthesized 3d is to use as selective novel nuclei in anti-cancer chemotherapeutics.


Assuntos
Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Derivados de Benzeno/farmacologia , Isocianatos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ureia/farmacologia , Animais , Antifúngicos/síntese química , Antifúngicos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Derivados de Benzeno/síntese química , Derivados de Benzeno/química , Proliferação de Células/efeitos dos fármacos , Ciclina E/antagonistas & inibidores , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/deficiência , Quinase 2 Dependente de Ciclina/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Isocianatos/síntese química , Isocianatos/química , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Estrutura Molecular , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Ureia/análogos & derivados , Ureia/química
6.
Biol Pharm Bull ; 44(4): 507-514, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790102

RESUMO

Preeclampsia (PE) is a severe pregnancy-specific complication responsible for a majority of maternal and fetal mortality. The dysfunction of trophoblast cells is known to be associated with the etiology of PE. Moreover, elevated expression of hsa_circ_0001326 was found in patients with PE without elucidating specific mechanisms. Thus, this study aimed to investigate the role of hsa_circ_0001326 in the dysfunction of trophoblast cells in vitro. Human trophoblast SWAN71 cells were used in this study. Cell proliferation, apoptosis and cell cycle were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, cell counting kit-8 assay, Annexin V/propidium iodide (PI) staining and flow cytometry, respectively. Dual luciferase assay was performed to validate the predicted targets. Additionally, Western blot was conducted for protein detection. The results indicated overexpression (OE) of hsa_circ_0001326 remarkably decreased the viability and proliferation of SWAN71 cells. MiR-186-5p was identified as the target of hsa_circ_0001326. Meanwhile, p27 Kip1 was validated as the target of hsa_miR-186-5p. Moreover, the increased apoptosis and decreased migration induced by hsa_circ_0001326 OE were inhibited by p27 Kip1 knockdown. Hsa_circ_0001326 OE upregulated p27 Kip1 and cleaved caspase3 and downregulated CDK2 and cyclin E1 in cells, while these phenomena were reversed by p27 Kip1 knockdown. In addition, hsa_circ_0001326 OE induced G0/G1 cell cycle arrest was also attenuated in the presence of p27 Kip1 knockdown. Taken together, hsa_circ_0001326 OE contributed to the decreased viability of SWAN71 cells by targeting miR-186-5p via upregulation of p27 Kip1. Our findings were helpful to uncover the pathophysiological process of PE, as well as inspire the development of novel targeted therapy against PE.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , MicroRNAs , RNA Circular , Linhagem Celular , Fenômenos Fisiológicos Celulares , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas Oncogênicas/metabolismo , Regulação para Cima
7.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33669811

RESUMO

Although the lignan compound fargesin is a major ingredient in Shin-Yi, the roles of fargesin in carcinogenesis and cancer cell growth have not been elucidated. In this study, we observed that fargesin inhibited cell proliferation and transformation by suppression of epidermal growth factor (EGF)-stimulated G1/S-phase cell cycle transition in premalignant JB6 Cl41 and HaCaT cells. Unexpectedly, we found that signaling pathway analyses showed different regulation patterns in which fargesin inhibited phosphatidylinositol 3-kinase/AKT signaling without an alteration of or increase in mitogen activated protein kinase (MAPK) in JB6 Cl41 and HaCaT cells, while both signaling pathways were abrogated by fargesin treatment in colon cancer cells. We further found that fargesin-induced colony growth inhibition of colon cancer cells was mediated by suppression of the cyclin dependent kinase 2 (CDK2)/cyclin E signaling axis by upregulation of p21WAF1/Cip1, resulting in G1-phase cell cycle accumulation in a dose-dependent manner. Simultaneously, the suppression of CDK2/cyclin E and induction of p21WAF1/Cip1 were correlated with Rb phosphorylation and c-Myc suppression. Taken together, we conclude that fargesin-mediated c-Myc suppression inhibits EGF-induced cell transformation and colon cancer cell colony growth by the suppression of retinoblastoma (Rb)-E2F and CDK/cyclin signaling pathways, which are mainly regulated by MAPK and PKB signaling pathways.


Assuntos
Benzodioxóis/farmacologia , Transformação Celular Neoplásica/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Fator de Crescimento Epidérmico/efeitos adversos , Lignanas/farmacologia , Transdução de Sinais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
8.
Genetics ; 217(1): 1-15, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33683365

RESUMO

Polyploidy is an integral part of development and is associated with cellular stress, aging, and pathological conditions. The endocycle, comprised of successive rounds of G and S phases without mitosis, is widely employed to produce polyploid cells in plants and animals. In Drosophila, maintenance of the endocycle is dependent on E2F-governed oscillations of Cyclin E (CycE)-Cdk2 activity, which is known to be largely regulated at the level of transcription. In this study, we report an additional level of E2F-dependent control of CycE-Cdk2 activity during the endocycle. Genetic experiments revealed that an alternative isoform of Drosophila de2f1, dE2F1b, regulates the expression of the p27CIP/KIP-like Cdk inhibitor Dacapo (Dap). We provide evidence showing that dE2F1b-dependent Dap expression in endocycling tissues is necessary for setting proper CycE-Cdk2 activity. Furthermore, we demonstrate that dE2F1b is required for proliferating cell nuclear antigen expression that establishes a negative feedback loop in S phase. Overall, our study reveals previously unappreciated E2F-dependent regulatory networks that are critical for the periodic transition between G and S phases during the endocycle.


Assuntos
Ciclo Celular , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Retroalimentação Fisiológica , Proteínas Nucleares/genética , Poliploidia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição/genética
9.
Mol Cell ; 81(9): 1951-1969.e6, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33761311

RESUMO

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Replicação do DNA , Proteínas Intrinsicamente Desordenadas/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Domínio AAA , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Fase G1 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Nucleares/genética , Complexo de Reconhecimento de Origem/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Estabilidade Proteica , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo
10.
Nature ; 590(7847): 671-676, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33536622

RESUMO

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies3-7. Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.


Assuntos
Proteínas F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Regulação Alostérica , Biocatálise , Microscopia Crioeletrônica , Ciclina E/metabolismo , Humanos , Fosforilação , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
11.
Med Oncol ; 38(3): 25, 2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33586074

RESUMO

Skin cancers are the most common cancers in the world and among the different types of skin cancers, melanoma is the deadliest and incidence is rising. Previous studies have shown promising in vitro and human evidence of kiwifruit exhibiting anti-cancer effects. This study was designed to investigate if kiwifruit extract (KE) has any effect on CRL-11147 melanoma cancer cells and to investigate the possible mechanisms behind the results. The effects of KE on CRL-11147 melanoma cell survival, proliferation, and apoptosis was investigated using clonogenic survival assay, cell proliferation, and caspase-3 activity kits. Potential anti-tumor molecular mechanisms were elucidated using RT-PCR and IHC. Addition of KE decreased CRL-11147 cell colonies percentages indicated by a decreased optical density value of cancer cells when compared to control. Furthermore, treatment with KE increased relative caspase-3 activity in cancer cells, which indicated increased apoptosis of cancer cells. The anti-proliferative effect of KE on cancer cells corresponded with decreased expression of the pro-proliferative molecule Cyclin E and CDK4, while increased expression of the pro-apoptotic molecule TRAILR1 corresponded with the pro-apoptotic effect. KE decreases CRL-11147 melanoma cell growth via downregulation of Cyclin E and CDK4 and upregulation in TRAILR1. Our study suggests a potential use for KE in treatment of melanoma.


Assuntos
Actinidia/química , Ciclina E/metabolismo , Frutas/química , Melanoma/tratamento farmacológico , Proteínas Oncogênicas/metabolismo , Extratos Vegetais/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
12.
J Exp Clin Cancer Res ; 40(1): 48, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33516252

RESUMO

BACKGROUND: As a novel type of non-coding RNA, circular RNAs (circRNAs) play a critical role in the initiation and development of various diseases, including cancer. However, the exact function of circRNAs in human cervical cancer remains largely unknown. METHODS: We identified the circRNA signature of upregulated circRNAs between cervical cancer and paired adjacent normal tissues. Using two different cohorts and GEO database, a total of six upregulated circRNAs were identified with a fold change > 2, and P < 0.05. Among these six circRNAs, hsa_circ_0072088 (circZFR) was the only exonic circRNA significantly overexpressed in cervical cancer. Functional experiments were performed to investigate the biological function of circZFR. CircRNA pull-down, circRNA immunoprecipitation (circRIP) and Co-immunoprecipitation (Co-IP) assays were executed to investigate the molecular mechanism underlying the function of circZFR. RESULTS: Functionally, circZFR knockdown represses the proliferation, invasion, and tumor growth. Furthermore, circRNA pull-down experiments combined with mass spectrometry unveil the interactions of circZFR with Single-Stranded DNA Binding Protein 1 (SSBP1). Mechanistically, circZFR bound with SSBP1, thereby promoting the assembly of CDK2/cyclin E1 complexes. The activation of CDK2/cyclin E1 complexes induced p-Rb phosphorylation, thus releasing activated E2F1 leading to cell cycle progression and cell proliferation. CONCLUSION: Our findings provide the first evidence that circZFR is a novel onco-circRNA and might be a potential biomarker and therapeutic target for cervical cancer patients.


Assuntos
RNA Circular , RNA não Traduzido/genética , Proteínas de Ligação a RNA/genética , Proteína do Retinoblastoma/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Biologia Computacional , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Neoplasias do Colo do Útero/patologia
13.
J Mol Biol ; 433(5): 166795, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33422522

RESUMO

The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.


Assuntos
Ciclina A/química , Quinase 2 Dependente de Ciclina/química , Inibidor de Quinase Dependente de Ciclina p27/química , Pontos de Checagem da Fase G1 do Ciclo Celular , Proteínas Quinases Associadas a Fase S/química , Sítios de Ligação , Quinases relacionadas a CDC2 e CDC28/química , Quinases relacionadas a CDC2 e CDC28/genética , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ciclina A/genética , Ciclina A/metabolismo , Ciclina E/química , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
14.
Mol Biol Rep ; 48(1): 915-925, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33409716

RESUMO

Cyclin-dependent kinase (CDK) 4/6 inhibitors have emerged in the treatment of metastatic hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. However, most patients will eventually present disease progression, highlighting the inevitable resistance of cancer cells to CDK4/6 inhibition. Several studies have suggested that resistance mechanisms involve aberrations of the molecules that regulate the cell cycle, and the re-wiring of the cell to escape CDK4/6 dependence and turn to alternative pathways. Loss of retinoblastoma function, overexpression of CDK 6, upregulation of cyclin E, overexpression of CDK 7, and dysregulation of several signaling pathways, notably the PI3/AKT/mTOR pathway, have been implicated in the development of resistance to CDK4/6 inhibitors. Overlap with endocrine resistance mechanisms might be possible. Combinational therapeutic strategies should be explored in order to prevent resistance and optimize the management of patients after progression under CDK 4/6 inhibition.


Assuntos
Neoplasias da Mama/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Ciclina E/genética , Ciclina E/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Terapia de Alvo Molecular/métodos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
15.
BMC Cancer ; 21(1): 39, 2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33413211

RESUMO

BACKGROUND: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer. METHODS: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit. RESULTS: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%). CONCLUSIONS: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample.


Assuntos
Ciclina E/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Biópsia Líquida/métodos , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Lesões Pré-Cancerosas/patologia , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasia Intraepitelial Cervical/metabolismo , Neoplasia Intraepitelial Cervical/patologia , Neoplasia Intraepitelial Cervical/cirurgia , Neoplasia Intraepitelial Cervical/virologia , Citodiagnóstico/métodos , Progressão da Doença , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/cirurgia , Lesões Pré-Cancerosas/virologia , Prognóstico , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/cirurgia , Neoplasias do Colo do Útero/virologia
16.
Biomed Pharmacother ; 133: 111030, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378944

RESUMO

Drug resistance has always been an important problem affecting the therapeutic effect of hepatocellular carcinoma (HCC). To investigate the potential role of lncRNA TTN-AS1 in HCC cells with sorafenib (SOR) resistance, and explore the underlying pathways, quantitative real time polymerase chain reaction (qRT-PCR) was used to test the expression of TTN-AS1 in HCC tissues and cells. Then, the expression of TTN-AS1 was down-regulated by shRNA, the activity changes, apoptosis and related protein expression in HCC cells with/without SOR treatment were observed in succession. Expression levels of the downstream target of TTN-AS1, miR-16-5p were studied by dual-luciferase binding assay, cell proliferation, and western blotting analysis. Nude mice models of human HCC with TTN-AS1 gene knockdown were established to observe the tumor growth. As the results revealed, TTN-AS1 silencing in HCC cells induced apoptosis by enhancing the sensitivity of cells to SOR, and the tumor in nude mice became smaller. The mechanism study showed that miR-16-5p was affected by TTN-AS1 sponge, up-regulated cyclin E1 expression, and regulated PTEN/Akt signaling pathway, thereby significantly alleviating the inhibition of apoptosis of HCC cells induced by TTN-AS1 gene. Collectively, our results provided TTN-AS1 as a potential therapeutic target for sorafenib resistance in HCC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Ciclina E/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/metabolismo , Proteínas Oncogênicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Longo não Codificante/metabolismo , Sorafenibe/farmacologia , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Ciclina E/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Proteínas Oncogênicas/genética , RNA Longo não Codificante/genética , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Mol Med Rep ; 23(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33215214

RESUMO

Long non­coding RNAs (lncRNAs) serve important roles in the tumorigenesis of a diverse range of cancer types. The lung cancer­associated transcript 1 (LUCAT1), has been reported to promote the proliferation, migration and invasion of oral squamous cell carcinoma cells. However, the exact role of LUCAT1 in laryngeal squamous cell carcinoma (LSCC) remains to fully understood. The present study aimed to interrogate the role and modulatory mechanism of LUCAT1 in LSCC. Reverse transcription­quantitative PCR and western blotting were used to investigate the expression of LUCAT1 and miR­493, as well as the protein expression of cyclin­dependent kinase 2, cyclin E1, p21, matrix metalloproteinase (MMP)2, MMP9, vascular endothelial growth factor­C, Bcl­2, Bax, cleaved caspase­3 and procaspase­3. Cell Counting Kit­8, flow cytometry, wound healing and Transwell assays were performed to analyze the proliferation, cell cycle, apoptosis levels, and the migratory and invasive abilities, respectively, of the LSCC AMC­HN­8 cell line. In addition, dual­luciferase reporter and ribonucleoprotein immunoprecipitation assays were used to investigate the binding between LUCAT1 and microRNA (miR)­493. The results of the present study revealed that the expression levels of LUCAT1 were upregulated in AMC­HN­8 cells. The genetic knockdown of LUCAT1 expression levels significantly suppressed the cell proliferation, alongside downregulating the expression levels of CDK2 and cyclin E1 and upregulating p21 expression levels. In addition, the knockdown of LUCAT1 inhibited cell migration and invasion, as demonstrated using the wound healing and Transwell assays, respectively. Moreover, LUCAT1 knockdown promoted cell apoptosis and upregulated the expression levels of Bax and cleaved caspase­3, whilst downregulating the expression levels of Bcl­2. Furthermore, LUCAT1 was discovered to directly bind to and inhibit the well­known tumor suppressor, miR­493. Notably, the specific inhibition of miR­493 partly blocked the anticancer effects of LUCAT1 knockdown in AMC­HN­8 cells. In conclusion, these results suggested that LUCAT1 may facilitate tumorigenesis in LSCC through the targeted inhibition of miR­493, which provides evidence for a novel target for the treatment of LSCC.


Assuntos
Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , MicroRNAs/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Apoptose/genética , Caspase 3/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Longo não Codificante/fisiologia , Regulação para Cima , Fator C de Crescimento do Endotélio Vascular , Proteína X Associada a bcl-2/metabolismo
18.
Ann Surg ; 274(2): e150-e159, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31436549

RESUMO

BACKGROUND: Pathologic complete response (pCR) has been shown to be associated with favorable outcomes in breast cancer. Predictors of pCR could be useful in guiding treatment decisions regarding neoadjuvant therapy. The objective of this study was to evaluate cyclin E as a predictor of response to neoadjuvant chemotherapy in breast cancer. METHODS: Patients (n = 285) with stage II-III breast cancer were enrolled in a prospective study and received neoadjuvant chemotherapy with anthracyclines, taxanes, or combination of the two. Pretreatment biopsies from 190 patients and surgical specimens following chemotherapy from 192 patients were available for immunohistochemical analysis. Clinical and pathologic responses were recorded and associated with presence of tumor infiltrating lymphocytes, cyclin E, adipophilin, programmed cell death-ligand 1, and elastase staining and other patient, tumor and treatment characteristics. RESULTS: The pCR rate was significantly lower in patients with cytoplasmic cyclin E staining compared with those who had no cyclin E expression (16.1% vs 38.9%, P = 0.0005). In multivariable logistic regression analysis, the odds of pCR for patients who had cytoplasmic negative tumors was 9.35 times (P value < 0.0001) that compared with patients with cytoplasmic positive tumors after adjusting for ER, PR, and HER2 status. Cytoplasmic cyclin E expression also predicts long-term outcome and is associated with reduced disease free, recurrence free, and overall survival rates, independent of increased pretreatment tumor infiltrating lymphocytes. CONCLUSIONS: Cyclin E independently predicted response to neoadjuvant chemotherapy. Hence, its routine immunohistochemical analysis could be used clinically to identify those breast cancer patients expected to have a poor response to anthracycline/taxane-based chemotherapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Ciclina E/metabolismo , Adulto , Idoso , Antraciclinas/administração & dosagem , Biomarcadores Tumorais/metabolismo , Biópsia , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Quimioterapia Adjuvante , Feminino , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Estudos Prospectivos , Taxa de Sobrevida , Taxoides/administração & dosagem
19.
Pathol Res Pract ; 216(12): 153231, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33059240

RESUMO

Thyroid cancer (TC) is the most common endocrine cancer in the world and about 80-85 % patients with TC belong to papillary thyroid cancer (PTC). MicroRNAs(MiRNAs) are a class of non-coding RNAs that can negatively modulate gene expression post-transcriptionally and play a role in tumorigenesis and development. The purpose of this study was to explore the biological function of miR-506-3p in PTC. We found that miR-506-3p suppressed cell proliferation disrupted the cell cycle of PTC cells in vitro. Mechanistically, we found that YAP1 was a direct target gene of miR-506-3p. Western Blot and RT-qPCR results indicated that miR-506-3p could down-regulate the expression of YAP1 at both mRNA and protein levels. Furthermore, miR-506-3p could also suppress the expression of CDK2/Cyclin E1 compound which could be affected by YAP1 gene. Therefore miR-506-3p might have proliferation-suppressive function in PTC by inhibiting YAP1 expression and regulating YAP1-CDK2/Cy clin E1 cell cycle pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , MicroRNAs/metabolismo , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Ciclo Celular , Linhagem Celular Tumoral , Ciclina E/genética , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transdução de Sinais , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição/genética
20.
Cancer Biol Ther ; 21(11): 994-1004, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33054513

RESUMO

The efficacy of trastuzumab, a treatment for HER2+ breast cancer, can be limited by the development of resistance. Cyclin E (CCNE) overexpression has been implicated in trastuzumab resistance. We sought to uncover a potential mechanism for this trastuzumab resistance and focused on a model of CCNE overexpressing HER2+ breast cancer and noncanonical phosphorylation of the TGF-ß signaling protein, SMAD3. Network analysis of transcriptional activity in a HER2+, CCNE overexpressing, trastuzumab-resistant cell line (BT474R2) identified decreased SMAD3 activity was associated with treatment resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p < .01), and noncanonical phosphorylation of SMAD3 was increased in these CCNE-overexpressing cells. Also, in response to CDK2 inhibition, expression patterns linked to restored canonical SMAD3 signaling, including decreased cMyc and increased cyclin-dependent inhibitor, p15, were identified. The BT474R2 cell line was modified through overexpression of SMAD3 (BT474R2-SMAD3), a mutant construct resistant to CCNE-mediated noncanonical phosphorylation of SMAD3 (BT474R2-5M), and a control (BT474R2-Blank). In vitro studies examining the response to trastuzumab showed increased sensitivity to treatment for BT474R2-5M cells. These findings were then validated in NSG mice inoculated with BT474R2-5M cells or BT474R2 control cells. After treatment with trastuzumab, the NSG mice inoculated with BT474R2-5M cells developed significantly lower tumor volumes (p < .001), when compared to mice inoculated with BT474R2 cells. Taken together, these results indicate that for patients with HER2+ breast cancer, a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, could be treated with CDK2 inhibition to help enhance the efficacy of trastuzumab therapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Ciclina E/metabolismo , Proteína Smad3/metabolismo , Trastuzumab/uso terapêutico , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Fosforilação , Trastuzumab/farmacologia
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