RESUMO
Currently, as the largest family of E3 ubiquitin ligases, Skp1-Cullin 1-F-box (SCF) E3 ligase complexes have attracted extensive attention. Among SCF complexes, Skp2, ß-TrCP, and FBXW7 have undergone extensive research on their structures and functions. Previous studies suggest Skp2, ß-TrCP, and FBXW7 are overexpressed in numerous cancers. Thus, the SCF E3 ligase complex has become a significant target for the development of anti-cancer drugs. Over the past few decades, a variety of anti-tumor inhibitors targeting the SCF E3 ligase complex have been attempted. However, since almost none of the SCF E3 ligase inhibitors passed clinical trials, the design and synthesis of the new inhibitors are needed. Here, we will introduce the structure and function of Skp2, ß-TrCP, and FBXW7, their connections with cancer development, the relevant in vitro and in vivo activities, selectivity, structure-activity relationships, and the therapeutic or preventive application of small molecule inhibitors targeting these three F-box proteins reported in the patent (2010-present). This information will help develop drugs targeting the SCF E3 ubiquitin ligase, providing new strategies for future cancer treatments.
Assuntos
Antineoplásicos , Desenho de Fármacos , Neoplasias , Humanos , Relação Estrutura-Atividade , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Patentes como Assunto , Animais , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Estrutura Molecular , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/metabolismo , Proteína 7 com Repetições F-Box-WD/metabolismoRESUMO
Skp1-CUL1-ROC1-F-box E3 ubiquitin ligases' main component S-phase kinase-associated protein 2 (Skp2) is responsible for specifically recognizing ubiquitination-modified substrates to be degraded such as p27 and p21 in the case of binding with adaptor protein Cks1. Pharmacological inhibition of Skp2 has exhibited promising antitumor activity. Herein, we present the design and optimization of a series of [1,2,4]triazolo[1,5-a]pyrimidine-based small molecules targeting Skp2. Among them, E35 demonstrated excellent inhibitory activities against the binding of Skp2-Cks1. In addition, compound E35 significantly inhibited colony formation and migration, as well as arrested the cell cycle at the S-phase. Mechanistically, compound E35 markedly decreased the expression of Skp2, as well as increased the expression of its substrates p21 and p27. Furthermore, compound E35 showed an obvious inhibitory effect on MGC-803 xenograft mice without obvious toxicity. All of these results suggest that compound E35 might be a valuable lead compound for antitumor agents targeting Skp2.
Assuntos
Antineoplásicos , Pirimidinas , Proteínas Quinases Associadas a Fase S , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/metabolismo , Humanos , Animais , Pirimidinas/farmacologia , Pirimidinas/química , Pirimidinas/síntese química , Pirimidinas/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Antineoplásicos/uso terapêutico , Camundongos , Linhagem Celular Tumoral , Relação Estrutura-Atividade , Descoberta de Drogas , Triazóis/farmacologia , Triazóis/química , Triazóis/síntese química , Triazóis/uso terapêutico , Camundongos Nus , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/uso terapêutico , Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Quinases relacionadas a CDC2 e CDC28/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos BALB C , Proliferação de Células/efeitos dos fármacosRESUMO
The proliferative and migratory abilities of vascular smooth muscle cells (VSMCs) play a crucial role in neointima formation following vascular injury. Skp2 facilitates proliferation and migration in cells through cell cycle regulation, presenting an important therapeutic target for atherosclerosis, pulmonary hypertension, and vascular restenosis. This study aimed to identify a natural product capable of inhibiting neointima formation post vascular injury. Here, we demonstrate that troxerutin, a flavonoid, significantly reduced viability and downregulated Skp2 in VSMCs. Moreover, troxerutin exhibited anti-proliferative effects on VSMCs and mitigated neointima formation. These findings collectively elucidate the intrinsic mechanism of troxerutin in treating atherosclerosis, pulmonary hypertension, and vascular restenosis by targeting the E3-linked enzyme Skp2.
Assuntos
Proliferação de Células , Hidroxietilrutosídeo , Músculo Liso Vascular , Neointima , Proteínas Quinases Associadas a Fase S , Hidroxietilrutosídeo/análogos & derivados , Hidroxietilrutosídeo/farmacologia , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Neointima/tratamento farmacológico , Neointima/patologia , Neointima/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Proteólise/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Movimento Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , RatosRESUMO
INTRODUCTION: Psoriasis is a chronic immune-mediated disorder affecting over 2-3% of the population worldwide, significantly impacting quality of life. Despite the availability of various therapeutic interventions, concerns persist regarding lesion recurrence and potential alterations in immune surveillance promoting cancer progression. Recent advancements in understanding cellular and molecular pathways have unveiled key factors in psoriasis etiology, including IL-17, 22, 23, TNF-α, PDE-4, JAK-STAT inhibitors, and AhR agonists. This work explores the potential of S-phase kinase-associated protein 2 (Skp2) as a therapeutic target in psoriasis. AREA COVERED: This review covers the current understanding of psoriasis pathophysiology, including immune dysregulation, and the role of keratinocytes and ubiquitin. It also delves into Skp2 role in cell cycle regulation, and its correlation with angiogenesis and ubiquitin in psoriasis. The evolving therapeutic approaches targeting Skp2, including small molecule inhibitors, are also discussed. EXPERT OPINION: Targeting Skp2 holds promise for developing novel therapeutic approaches for psoriasis. By modulating Skp2 activity or expression, it may be possible to intervene in inflammatory and proliferative processes underlying the disease. Further research into Skp2 inhibitors and their efficacy in preclinical and clinical settings is warranted to harness the full potential of Skp2 as a therapeutic target in psoriasis management.
Assuntos
Terapia de Alvo Molecular , Psoríase , Proteínas Quinases Associadas a Fase S , Humanos , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/metabolismo , Psoríase/tratamento farmacológico , Psoríase/patologia , Animais , Qualidade de Vida , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Ubiquitina/metabolismo , Desenvolvimento de MedicamentosRESUMO
BACKGROUND: Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. The overall survival has not improved significantly over the last decades because no major therapeutic breakthroughs have been achieved for over 15 years. METHODS: We analyzed a genome-wide loss-of-function screening database to identify vulnerabilities in SCLC for the development of urgently needed novel therapies. RESULTS: We identified SKP2 (encoding S-phase kinase-associated protein 2) and CKS1B (encoding CDC28 protein kinase regulatory subunit 1B) as the two most essential genes in that order in SCLC. Notably, SKP2 and CKS1B comprise the p27 binding pocket of the E3 ubiquitin ligase SCFSKP2 complex. Immunohistochemistry on tissue microarrays revealed that SKP2 was expressed in >95% of samples at substantially higher levels than that observed for commonly used neuroendocrine markers. As expected, SCLC cell lines were sensitive to SKP2 inhibition. Furthermore, SKP2 or CKS1B knockdown induced apoptosis in RB1 mutant cells, whereas it induced senescence in RB1 wild-type cells. CONCLUSION: Although the mechanism underlying SKP2 knockdown-induced growth inhibition differs between RB1-wild-type and -mutant SCLC, SKP2 can be considered a novel therapeutic target for patients with SCLC regardless of the RB1 mutation status. Our findings indicate that SKP2 is a potential novel clinical diagnostic marker and therapeutic target in SCLC.
Assuntos
Biomarcadores Tumorais , Quinases relacionadas a CDC2 e CDC28 , Neoplasias Pulmonares , Proteínas Quinases Associadas a Fase S , Carcinoma de Pequenas Células do Pulmão , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Humanos , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/terapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Quinases relacionadas a CDC2 e CDC28/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Mutação , Terapia de Alvo Molecular , Apoptose/genética , Linhagem Celular Tumoral , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Ubiquitination, a post-translational modification, refers to the covalent attachment of ubiquitin molecules to substrates. This modification plays a critical role in diverse cellular processes such as protein degradation. The specificity of ubiquitination for substrates is regulated by E3 ubiquitin ligases. Dysregulation of ubiquitination has been associated with numerous diseases, including cancers. In our study, we first investigated the protein expression patterns of E3 ligases across 12 cancer types. Our findings indicated that E3 ligases tend to be up-regulated and exhibit reduced tissue specificity in tumors. Moreover, the correlation of protein expression between E3 ligases and substrates demonstrated significant changes in cancers, suggesting that E3-substrate specificity alters in tumors compared to normal tissues. By integrating transcriptome, proteome, and ubiquitylome data, we further characterized the E3-substrate regulatory patterns in lung squamous cell carcinoma. Our analysis revealed that the upregulation of the SKP2 E3 ligase leads to excessive degradation of BRCA2, potentially promoting tumor cell proliferation and metastasis. Furthermore, the upregulation of E3 ubiquitin-protein ligase TRIM33 was identified as a biomarker associated with a favorable prognosis by inhibiting the cell cycle. This work exemplifies how leveraging multi-omics data to analyze E3 ligases across various cancers can unveil prognosis biomarkers and facilitate the identification of potential drug targets for cancer therapy.
Assuntos
Neoplasias , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteômica/métodos , Transcriptoma , Proteoma/metabolismo , Prognóstico , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , MultiômicaRESUMO
Background and Objectives: Aberrant upregulation of fatty acid synthase (FASN), catalyzing de novo synthesis of fatty acids, occurs in various tumor types, including human hepatocellular carcinoma (HCC). Although FASN oncogenic activity seems to reside in its pro-lipogenic function, cumulating evidence suggests that FASN's tumor-supporting role might also be metabolic-independent. Materials and Methods: In the present study, we show that FASN inactivation by specific small interfering RNA (siRNA) promoted the downregulation of the S-phase kinase associated-protein kinase 2 (SKP2) and the consequent induction of p27KIP1 in HCC cell lines. Results: Expression levels of FASN and SKP2 directly correlated in human HCC specimens and predicted a dismal outcome. In addition, forced overexpression of SKP2 rendered HCC cells resistant to the treatment with the FASN inhibitor C75. Furthermore, FASN deletion was paralleled by SKP2 downregulation and p27KIP1 induction in the AKT-driven HCC preclinical mouse model. Moreover, forced overexpression of an SKP2 dominant negative form or a p27KIP1 non-phosphorylatable (p27KIP1-T187A) construct completely abolished AKT-dependent hepatocarcinogenesis in vitro and in vivo. Conclusions: In conclusion, the present data indicate that SKP2 is a critical downstream effector of FASN and AKT-dependent hepatocarcinogenesis in liver cancer, envisaging the possibility of effectively targeting FASN-positive liver tumors with SKP2 inhibitors or p27KIP1 activators.
Assuntos
Carcinoma Hepatocelular , Inibidor de Quinase Dependente de Ciclina p27 , Neoplasias Hepáticas , Proteínas Quinases Associadas a Fase S , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Ácido Graxo Sintases/metabolismo , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintase Tipo I/genética , Regulação para Baixo , MasculinoRESUMO
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The inflammatory cytokine storm causes systemic organ damage, especially acute lung injury in sepsis. In this study, we found that the expression of S-phase kinase-associated protein 2 (Skp2) was significantly decreased in sepsis-induced acute lung injury (ALI). Sepsis activated the MEK/ERK pathway and inhibited Skp2 expression in the pulmonary epithelium, resulting in a reduction of K48 ubiquitination of solute carrier family 3 member 2 (SLC3A2), thereby impairing its membrane localization and cystine/glutamate exchange function. Consequently, the dysregulated intracellular redox reactions induced ferroptosis in pulmonary epithelial cells, leading to lung injury. Finally, we demonstrated that intravenous administration of Skp2 mRNA-encapsulating lipid nanoparticles (LNPs) inhibited ferroptosis in the pulmonary epithelium and alleviated lung injury in septic mice. Taken together, these data provide an innovative understanding of the underlying mechanisms of sepsis-induced ALI and a promising therapeutic strategy for sepsis.
Assuntos
Lesão Pulmonar Aguda , Ferroptose , Camundongos Endogâmicos C57BL , Proteínas Quinases Associadas a Fase S , Sepse , Ubiquitinação , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/etiologia , Sepse/metabolismo , Sepse/complicações , Sepse/patologia , Animais , Camundongos , Humanos , Masculino , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Pulmão/patologia , Pulmão/metabolismo , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genéticaRESUMO
The development of new therapeutic uses for existing drugs is important for the treatment of some diseases. Cephalosporin antibiotics stand as the most extensively utilized antibiotics in clinical practice, effectively combating bacterial infections. Here, we found that the antimicrobial drug ceftazidime strongly upregulates p27 protein levels by inhibiting p27 ubiquitination. The p27 protein is a classic negative regulator of the cell cycle. Next, we demonstrated that ceftazidime can impede the cell cycle from G1 to S phase, thus inhibiting cell proliferation. Furthermore, we found that ceftazidime promotes p27 expression and inhibits cell proliferation by reducing Skp2, which is a substrate recognition component of the Skp2-Cullin-F-box (SCF) ubiquitin ligase. Moreover, ceftazidime downregulates transcriptional expression of Skp2. Importantly, we demonstrated that ceftazidime inhibited the proliferation of tumor cells in vivo. These findings reveal ceftazidime-mediated inhibition of cell proliferation through the Skp2-p27 axis, and could provide a potential strategy for anti-tumor therapy.
Assuntos
Antibacterianos , Ceftazidima , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27 , Proteínas Quinases Associadas a Fase S , Ceftazidima/farmacologia , Proliferação de Células/efeitos dos fármacos , Humanos , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Antibacterianos/farmacologia , Animais , Linhagem Celular Tumoral , Ubiquitinação/efeitos dos fármacos , Camundongos , Ciclo Celular/efeitos dos fármacos , Antineoplásicos/farmacologiaRESUMO
Glioblastoma is a malignant brain tumor with poor prognosis. Though several dysregulated pathways were found to mediate the tumor progression, hyperactivation of RAS-RAF-ERK pathway, enhanced glycolysis and SKP2 are associated with several glioblastomas. Recent findings on the role of USP10 in the transition from pro-neural to mesenchymal subtype of glioblastoma and, USP13 in the stabilization of RAF1 in mouse embryonic stem cells prompted us to examine their role in the mechanisms mediating the progression of glioblastoma. In the present study, we have examined the role of spautin-1, a pharmacological inhibitor of USP10 and USP13 in the mechanisms mediating glioblastoma. Our results indicate that spautin-1 as well as knockdown of its downstream targets, USP10 and USP13, reduced the proliferation and migration of glioblastoma cells. Also, spautin-1 mediated inhibition of RAF-ERK pathway or inhibition of RAF1 and MEK1 per se reduced the glycolytic function via PKM2/Glut-1 and inhibited the progression of glioblastoma. Further, the protooncogene, SKP2, which was shown to be a direct target of USP10 /USP13 was also reduced by spautin-1. While inhibition of SKP2 enhanced its downstream target p21, no apparent changes in the RAF-ERK levels or glycolytic function were evident. Also, inhibition of MEK1 did not affect SKP2 levels, indicating that these two pathways act independent of each other. Overall, our findings indicate that spautin-1 by virtue of its inhibitory effects on USP10/13 counteracts RAS-RAF-ERK mediated glycolysis and SKP2 that are critical in the progression of glioblastoma. Hence, further preclinical validation is warranted for taking the present observations forward.
Assuntos
Progressão da Doença , Glioblastoma , Glicólise , Proteínas Quinases Associadas a Fase S , Ubiquitina Tiolesterase , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Humanos , Linhagem Celular Tumoral , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/antagonistas & inibidores , Proliferação de Células , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Sistema de Sinalização das MAP Quinases , Movimento Celular , Animais , CamundongosRESUMO
S-phase kinase-associated protein 2 (Skp2) is an F-box protein overexpressed in human cancers and linked with poor prognosis. It triggers cancer pathogenesis, including stemness and drug resistance. In this study, we have explored the potential role of Skp2 targeting in restoring the expression of tumor suppressors in human cutaneous squamous cell carcinoma (cSCC) cells. Our results showed that genetic and pharmacological Skp2 targeting markedly suppressed cSCC cell proliferation, colony growth, spheroid formation, and enhanced sensitization to chemotherapeutic drugs. Further, western blot results demonstrated restoration of tumor suppressor (KLF4) and CDKI (p21) and suppression of vimentin and survivin in Skp2-knocked-down cSCC cells. Importantly, we also explored that Skp2 targeting potentiates apoptosis of cSCC cells through MAPK signaling. Moreover, co-targeting of Skp2 and PI3K/AKT resulted in increased cancer cell death. Interestingly, curcumin, a well-known naturally derived anticancer agent, also inhibits Skp2 expression with concomitant CDKI upregulation. In line, curcumin suppressed cSCC cell growth through ROS-mediated apoptosis, while the use of N-acetyl cysteine (NAC) reversed curcumin-induced cell death. Curcumin treatment also sensitized cSCC cells to conventional anticancer drugs, such as cisplatin and doxorubicin. Altogether, these data suggest that Skp2 targeting restores the functioning of tumor suppressors, inhibits the expression of genes associated with cell proliferation and stemness, and sensitizes cancer cells to anticancer drugs. Thus, genetic, and pharmacological ablation of Skp2 can be an important strategy for attenuating cancer pathogenesis and associated complications in skin squamous cell carcinoma.
Assuntos
Apoptose , Carcinoma de Células Escamosas , Fator 4 Semelhante a Kruppel , Proteínas Quinases Associadas a Fase S , Neoplasias Cutâneas , Humanos , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Apoptose/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Fator 4 Semelhante a Kruppel/metabolismo , Proliferação de Células/efeitos dos fármacos , Curcumina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
While EZH2 enzymatic activity is well-known, emerging evidence suggests that EZH2 can exert functions in a methyltransferase-independent manner. In this study, we have uncovered a novel mechanism by which EZH2 positively regulates the expression of SKP2, a critical protein involved in cell cycle progression. We demonstrate that depletion of EZH2 significantly reduces SKP2 protein levels in several cell types, while treatment with EPZ-6438, an EZH2 enzymatic inhibitor, has no effect on SKP2 protein levels. Consistently, EZH2 depletion leads to cell cycle arrest, accompanied by elevated expression of CIP/KIP family proteins, including p21, p27, and p57, whereas EPZ-6438 treatment does not modulate their levels. We also provide evidence that EZH2 knockdown, but not enzymatic inhibition, suppresses SKP2 mRNA expression, underscoring the transcriptional regulation of SKP2 by EZH2 in a methyltransferase-independent manner. Supporting this, analysis of the Cancer Genome Atlas database reveals a close association between EZH2 and SKP2 expression in human malignancies. Moreover, EZH2 depletion but not enzymatic inhibition positively regulates the expression of major epithelial-mesenchymal transition (EMT) regulators, such as ZEB1 and SNAIL1, in transformed cells. Our findings shed light on a novel mechanism by which EZH2 exerts regulatory effects on cell proliferation and differentiation through its methyltransferase-independent function, specifically by modulating SKP2 expression.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Proteínas Quinases Associadas a Fase S , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Humanos , Transdução de Sinais , Ciclo Celular/genética , Transição Epitelial-Mesenquimal/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de CélulasRESUMO
Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.
Assuntos
Proteínas Quinases Associadas a Fase S , Bibliotecas de Moléculas Pequenas , Humanos , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Proteólise/efeitos dos fármacos , Fenilpropionatos/química , Fenilpropionatos/farmacologia , Relação Estrutura-Atividade , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Molecular , Ligantes , Células HEK293 , DegronsRESUMO
Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.
Assuntos
Desoxicitidina , Gencitabina , Proteômica , Proteínas Quinases Associadas a Fase S , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Humanos , Proteômica/métodos , Linhagem Celular Tumoral , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Regulação para Cima/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Osteoarthritis (OA) is the main cause of cartilage damage and disability. This study explored the biological function of S-phase kinase-associated protein 2 (SKP2) and Kruppel-like factor 11 (KLF11) in OA progression and its underlying mechanisms. C28/I2 chondrocytes were stimulated with IL-1ß to mimic OA in vitro. We found that SKP2, Jumonji domain-containing protein D3 (JMJD3), and Notch receptor 1 (NOTCH1) were upregulated, while KLF11 was downregulated in IL-1ß-stimulated chondrocytes. SKP2/JMJD3 silencing or KLF11 overexpression repressed apoptosis and extracellular matrix (ECM) degradation in chondrocytes. Mechanistically, SKP2 triggered the ubiquitination and degradation of KLF11 to transcriptionally activate JMJD3, which resulted in activation of NOTCH1 through inhibiting H3K27me3. What's more, the in vivo study found that KLF11 overexpression delayed OA development in rats via restraining apoptosis and maintaining the balance of ECM metabolism. Taken together, ubiquitination and degradation of KLF11 regulated by SKP2 contributed to OA progression by activation of JMJD3/NOTCH1 pathway. Our findings provide promising therapeutic targets for OA.
Assuntos
Condrócitos , Histona Desmetilases com o Domínio Jumonji , Osteoartrite , Receptor Notch1 , Proteínas Quinases Associadas a Fase S , Ubiquitinação , Receptor Notch1/metabolismo , Receptor Notch1/genética , Animais , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Ratos , Condrócitos/metabolismo , Condrócitos/patologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Masculino , Transdução de Sinais , Ratos Sprague-Dawley , Humanos , Apoptose , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genéticaRESUMO
The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.
Assuntos
Cádmio , Ligação Proteica , Proteínas Ligases SKP Culina F-Box , Cádmio/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação , Domínios Proteicos , Humanos , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMO
Ovarian cancer is a common, highly lethal tumor. Herein, we reported that S-phase kinase-associated protein 2 (Skp2) is essential for the growth and aerobic glycolysis of ovarian cancer cells. Skp2 was upregulated in ovarian cancer tissues and associated with poor clinical outcomes. Using a customized natural product library screening, we found that xanthohumol inhibited aerobic glycolysis and cell viability of ovarian cancer cells. Xanthohumol facilitated the interaction between E3 ligase Cdh1 and Skp2 and promoted the Ub-K48-linked polyubiquitination of Skp2 and degradation. Cdh1 depletion reversed xanthohumol-induced Skp2 downregulation, enhancing HK2 expression and glycolysis in ovarian cancer cells. Finally, a xenograft tumor model was employed to examine the antitumor efficacy of xanthohumol in vivo. Collectively, we discovered that xanthohumol promotes the binding between Skp2 and Cdh1 to suppress the Skp2/AKT/HK2 signal pathway and exhibits potential antitumor activity for ovarian cancer cells.
Assuntos
Flavonoides , Glicólise , Neoplasias Ovarianas , Propiofenonas , Proteínas Quinases Associadas a Fase S , Ubiquitinação , Propiofenonas/farmacologia , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Flavonoides/farmacologia , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Glicólise/efeitos dos fármacos , Animais , Transdução de Sinais/efeitos dos fármacos , Caderinas/metabolismo , Carcinogênese/efeitos dos fármacos , Antígenos CD/metabolismo , Hexoquinase/metabolismo , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos , Fitoterapia , Camundongos Nus , Antineoplásicos Fitogênicos/farmacologiaRESUMO
BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. METHODS: Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. RESULTS: SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated ß-galactosidase (SA-ß-Gal) staining. CONCLUSIONS: Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.
Assuntos
Apoptose , Senescência Celular , Histona Desmetilases com o Domínio Jumonji , Neoplasias de Próstata Resistentes à Castração , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases Associadas a Fase S , Transdução de Sinais , Humanos , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/genética , Masculino , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Células PC-3 , Proteínas Nucleares , Proteínas RepressorasRESUMO
Soft-tissue sarcomas (STS) emerges as formidable challenges in clinics due to the complex genetic heterogeneity, high rates of local recurrence and metastasis. Exploring specific targets and biomarkers would benefit the prognosis and treatment of STS. Here, we identified RCC1, a guanine-nucleotide exchange factor for Ran, as an oncogene and a potential intervention target in STS. Bioinformatics analysis indicated that RCC1 is highly expressed and correlated with poor prognosis in STS. Functional studies showed that RCC1 knockdown significantly inhibited the cell cycle transition, proliferation and migration of STS cells in vitro, and the growth of STS xenografts in mice. Mechanistically, we identified Skp2 as a downstream target of RCC1 in STS. Loss of RCC1 substantially diminished Skp2 abundance by compromising its protein stability, resulting in the upregulation of p27Kip1 and G1/S transition arrest. Specifically, RCC1 might facilitate the nucleo-cytoplasmic trafficking of Skp2 via direct interaction. As a result, the cytoplasmic retention of Skp2 would further protect it from ubiquitination and degradation. Notably, recovery of Skp2 expression largely reversed the phenotypes induced by RCC1 knockdown in STS cells. Collectively, this study unveils a novel RCC1-Skp2-p27Kip1 axis in STS oncogenesis, which holds promise for improving prognosis and treatment of this formidable malignancy.
Assuntos
Sarcoma , Animais , Humanos , Camundongos , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Sarcoma/genética , Sarcoma/patologia , Ubiquitinação , Regulação para CimaRESUMO
FBXO43 is a member of the FBXO subfamily of F-box proteins, known to be a regulatory hub during meiosis. A body of data showed that FBXO43 is overexpressed in a number of human cancers. However, whether and how FBXO43 affects cell cycle progression and growth of cancer cells remain elusive. In this study, we provide first piece of evidence, showing a pivotal role of FBXO43 in cell cycle progression and growth of cancer cells. Specifically, FBXO43 acts as a positive cell cycle regulator with an oncogenic activity in variety types of human cancer, including non-small cell lung cancer, hepatocellular carcinoma and sarcoma. Mechanistically, FBXO43 interacts with phosphorylated SKP2 induced by AKT1, leading to reduced SKP2 auto-ubiquitylation and subsequent proteasome degradation. Taken together, our study demonstrates that FBXO43 promotes cell cycle progression by stabilizing SKP2, and FBXO43 could serve as a potential anti-cancer target.