RESUMEN

BACKGROUND: Streptococcus pneumoniae (Spn) is a major causative agent of pneumonia, which can disseminate to the bloodstream and brain. Pneumonia remains a leading cause of death among children aged 1-59 months worldwide. This study aims to investigate the role of Kruppel-like factor 2 (KLF2) in lung injury caused by Spn in young mice. METHODS: Young mice were infected with Spn to induce pneumonia, and the bacterial load in the bronchoalveolar lavage fluid was quantified. KLF2 expression in lung tissues was analyzed using real-time quantitative polymerase chain reaction and Western blotting assays. Following KLF2 overexpression, lung tissues were assessed for lung wet-to-dry weight ratio and Myeloperoxidase activity. The effects of KLF2 on lung injury and inflammation were evaluated through hematoxylin and eosin staining and enzyme-linked immunosorbent assay. Chromatin immunoprecipitation and dual-luciferase assay were conducted to examine the binding of KLF2 to the promoter of microRNA (miR)-222-3p and cyclin-dependent kinase inhibitor 1B (CDKN1B), as well as the binding of miR-222-3p to CDKN1B. Levels of miR-222-3p and CDKN1B in lung tissues were also determined. RESULTS: In young mice with pneumonia, KLF2 and CDKN1B were downregulated, while miR-222-3p was upregulated in lung tissues. Overexpression of KLF2 reduced lung injury and inflammation, evidenced by decreased bacterial load, reduced lung injury, and lower levels of proinflammatory factors. Co-transfection of miR-222-3p-WT and oe-KLF2 significantly reduced luciferase activity, suggesting that KLF2 binds to the promoter of miR-222-3p and suppresses its expression. Transfection of CDKN1B-WT with miR-222-3p mimics significantly reduced luciferase activity, indicating that miR-222-3p binds to CDKN1B and downregulates its expression. Overexpression of miR-222-3p or downregulation of CDKN1B increased bacterial load in BALF, lung wet/dry weight ratio, MPO activity, and inflammation, thereby reversing the protective effect of KLF2 overexpression on lung injury in young mice with pneumonia. CONCLUSIONS: KLF2 alleviates lung injury in young mice with Spn-induced pneumonia by transcriptional regulation of the miR-222-3p/CDKN1B axis.

Asunto(s)

Modelos Animales de Enfermedad , Factores de Transcripción de Tipo Kruppel , Neumonía Neumocócica , Streptococcus pneumoniae , Animales , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Factores de Transcripción de Tipo Kruppel/biosíntesis , Ratones , Neumonía Neumocócica/metabolismo , Neumonía Neumocócica/microbiología , Pulmón/metabolismo , Pulmón/microbiología , MicroARNs/genética , MicroARNs/metabolismo , MicroARNs/biosíntesis , Ratones Endogámicos C57BL , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Masculino

RESUMEN

Lung cancer remains the leading cause of cancer-related deaths, with cigarette smoking being the most critical factor, linked to nearly 90% of lung cancer cases. NNK, a highly carcinogenic nitrosamine found in tobacco, is implicated in the lung cancer-causing effects of cigarette smoke. Although NNK is known to mutate or activate certain oncogenes, its potential interaction with p27 in modulating these carcinogenic effects is currently unexplored. Recent studies have identified specific downregulation of p27 in human squamous cell carcinoma, in contrast to adenocarcinoma. Additionally, exposure to NNK significantly suppresses p27 expression in human bronchial epithelial cells. Subsequent studies indicates that the downregulation of p27 is pivotal in NNK-induced cell transformation. Mechanistic investigations have shown that reduced p27 expression leads to increased level of ITCH, which facilitates the degradation of Jun B protein. This degradation in turn, augments miR-494 expression and its direct regulation of JAK1 mRNA stability and protein expression, ultimately activating STAT3 and driving cell transformation. In summary, our findings reveal that: (1) the downregulation of p27 increases Jun B expression by upregulating Jun B E3 ligase ITCH, which then boosts miR-494 transcription; (2) Elevated miR-494 directly binds to 3'-UTR of JAK1 mRNA, enhancing its stability and protein expression; and (3) The JAK1/STAT3 pathway is a downstream effector of p27, mediating the oncogenic effect of NNK in lung cancer. These findings provide significant insight into understanding the participation of mechanisms underlying p27 inhibition of NNK induced lung squamous cell carcinogenic effect.

Asunto(s)

Bronquios , Carcinoma de Células Escamosas , Transformación Celular Neoplásica , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Células Epiteliales , Neoplasias Pulmonares , Nitrosaminas , Humanos , Nitrosaminas/toxicidad , Bronquios/metabolismo , Bronquios/patología , Bronquios/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , MicroARNs/genética , MicroARNs/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Carcinógenos/toxicidad

RESUMEN

BACKGROUND: Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling. METHODS: In this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay. RESULTS: We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo. CONCLUSION: Together, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

Asunto(s)

Feto , Hematopoyesis , Células Madre Hematopoyéticas , Ratones Noqueados , Factor de Transcripción HES-1 , Animales , Factor de Transcripción HES-1/metabolismo , Factor de Transcripción HES-1/genética , Ratones , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Feto/citología , Feto/metabolismo , Diferenciación Celular , Apoptosis , Proliferación Celular , Fosfohidrolasa PTEN/metabolismo , Fosfohidrolasa PTEN/genética , Transducción de Señal , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética

RESUMEN

BACKGROUND: Extrachromosomal circular DNA (eccDNA), a kind of circular DNA that originates from chromosomes, carries complete gene information, particularly the oncogenic genes. This study aimed to examine the contributions of FAM84B induced by eccDNA to prostate cancer (PCa) development and the biomolecules involved. METHODS: The presence of eccDNA in PCa cells and the FAM84B transcripts that eccDNA carries were verified by outward and inward PCR. The effect of inhibition of eccDNA synthesis on FAM84B expression in PCa cells was analyzed by knocking down Lig3. The impact of FAM84B on the growth and metastases of PCa cells was verified by Cell Counting Kit-8 (CCK8), EdU, transwell assays, and a xenograft mouse model. Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and dual-luciferase reporter assays were carried out to examine the effect of FAM84B/MYC on WWP1 transcription, and a co-immunoprecipitation (Co-IP) assay was conducted to verify the modification of CDKN1B by WWP1. The function of this molecular axis in PCa was explored by rescue assays. RESULTS: The inhibited eccDNA synthesis significantly downregulated FAM84B in PCa cells, thereby attenuating the growth and metastasis of PCa. FAM84B promoted the transcription of WWP1 by MYC by activating the expression of MYC coterminous with the 8q24.21 gene desert in a beta catenin-dependent approach. WWP1 transcription promoted by MYC facilitated the ubiquitination and degradation of CDKN1B protein and inversely attenuated the repressive effect of CDKN1B on MYC expression. Exogenous overexpression of CDKN1B blocked FAM84B-activated MYC/WWP1 expression, thereby inhibiting PCa progression. CONCLUSIONS: FAM84B promoted by eccDNA mediates degradation of CDKN1B via MYC/WWP1, thereby accelerating PCa progression.

Asunto(s)

ADN Circular , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata , Proteínas Proto-Oncogénicas c-myc , Ubiquitina-Proteína Ligasas , Masculino , Humanos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Animales , ADN Circular/genética , ADN Circular/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ratones , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proliferación Celular/genética , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina

RESUMEN

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.

Asunto(s)

Carcinoma Hepatocelular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Neoplasias Hepáticas , Proteínas Quinasas Asociadas a Fase-S , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Animales , Ratones , Línea Celular Tumoral , Ácido Graso Sintasas/metabolismo , Acido Graso Sintasa Tipo I/metabolismo , Acido Graso Sintasa Tipo I/genética , Regulación hacia Abajo , Masculino

RESUMEN

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

Asunto(s)

Neoplasias de la Mama , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Hiperplasia , Células Madre Neoplásicas , Factor de Transcripción STAT3 , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Humanos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Animales , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Femenino , Fosforilación , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Hiperplasia/metabolismo , Ratones , Regulación Neoplásica de la Expresión Génica , Autorrenovación de las Células/genética , Línea Celular Tumoral , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/citología , Proteína Jagged-1/metabolismo , Proteína Jagged-1/genética

Asunto(s)

Proliferación Celular , Senescencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Neoplasia Intraepitelial Prostática , Neoplasias de la Próstata , Humanos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proliferación Celular/efectos de los fármacos , Masculino , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/tratamiento farmacológico , Senescencia Celular/efectos de los fármacos , Neoplasia Intraepitelial Prostática/patología , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasia Intraepitelial Prostática/genética , Animales , Progresión de la Enfermedad , Ratones , Línea Celular Tumoral , Puntos de Control del Ciclo Celular/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Proteína Potenciadora del Homólogo Zeste 2 , Proteínas Quinasas Asociadas a Fase-S , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Humanos , Transducción de Señal , Ciclo Celular/genética , Transición Epitelial-Mesenquimal/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Proliferación Celular

RESUMEN

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 Oncogénicas

RESUMEN

The anticancer potential and associated mechanisms of flavonoid fisetin are yet to be fully investigated on human head and neck squamous cell carcinoma (HNSCC). In the present study, fisetin (25-75 µM for 24-48 h) dose-dependently inhibited growth and induced death in HNSCC Cal33 and UM-SCC-22B cells, without showing any death in normal cells. Fisetin (25-50 µM) induced G2/M phase arrest via decrease in Cdc25C, CDK1, cyclin B1 expression, and an increase in p53(S15). A concentration-dependent increase in fisetin-induced DNA damage and apoptosis in HNSCC cells was authenticated by comet assay, gamma-H2A.X(S139) phosphorylation, and marked cleavage of PARP protein. Interestingly, fisetin-induced cell death occurred independently of p53 and reactive oxygen species production. The activation of JNK and inhibition of PI3K/Akt, ERK1/2, EGFR, and STAT-3 signaling were identified. Further, fisetin-induced apoptosis was mediated, in part, via p21Cip1 and p27Kip1 cleavage by caspase, which was reversed by z-VAD-FMK, a pan-caspase inhibitor. Subsequently, fisetin was also found to induce autophagy; nevertheless, autophagy attenuation exaggerated apoptosis. Oral fisetin (50 mg/kg body weight) treatment to establish Cal33 xenograft in mice for 19 days showed 73% inhibition in tumor volume (p < 0.01) along with a decrease in Ki67-positive cells and an increase in cleaved caspase-3 level in tumors. Consistent with the effect of 50 µM fisetin in vitro, the protein levels of p21Cip1 and P27Kip1 were also decreased by fisetin in tumors. Together, these findings showed strong anticancer efficacy of fisetin against HNSCC with downregulation of EGFR-Akt/ERK1/2-STAT-3 pathway and activation of JNK/c-Jun, caspases and caspase-mediated cleavage of p21Cip1 and p27Kip1.

Asunto(s)

Apoptosis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Flavonoides , Flavonoles , Puntos de Control de la Fase G2 del Ciclo Celular , Carcinoma de Células Escamosas de Cabeza y Cuello , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Flavonoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Ratones , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Flavonoides/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Línea Celular Tumoral , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Proliferación Celular/efectos de los fármacos , Ratones Desnudos , Caspasas/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Transducción de Señal/efectos de los fármacos , Daño del ADN/efectos de los fármacos

RESUMEN

Dysregulated nuclear-cytoplasmic trafficking has been shown to play a role in oncogenesis in several types of solid tumors and hematological malignancies. Exportin 1 (XPO1) is responsible for the nuclear export of several proteins and RNA species, mainly tumor suppressors. KPT-330, a small molecule inhibitor of XPO1, is approved for treating relapsed multiple myeloma and diffuse large B-cell lymphoma. Cutaneous T-cell lymphoma (CTCL) is an extranodal non-Hodgkin lymphoma with an adverse prognosis and limited treatment options in advanced stages. The effect of therapeutically targeting XPO1 with KPT-330 in CTCL has not been established. We report that XPO1 expression is upregulated in CTCL cells. KPT-330 reduces cell proliferation, induces G1 cell cycle arrest and apoptosis. RNA-sequencing was used to explore the underlying mechanisms. Genes associated with the cell cycle and the p53 pathway were significantly enriched with KPT-330 treatment. KPT-330 suppressed XPO1 expression, upregulated p53, p21WAF1/Cip1, and p27Kip1 and their nuclear localization, and downregulated anti-apoptotic protein (Survivin). The in vivo efficacy of KPT-330 was investigated using a bioluminescent xenograft mouse model of CTCL. KPT-330 blocked tumor growth and prolonged survival (p < 0.0002) compared to controls. These findings support investigating the use of KPT-330 and next-generation XPO1 inhibitors in CTCL.

Asunto(s)

Apoptosis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteína Exportina 1 , Carioferinas , Linfoma Cutáneo de Células T , Receptores Citoplasmáticos y Nucleares , Triazoles , Proteína p53 Supresora de Tumor , Humanos , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Linfoma Cutáneo de Células T/tratamiento farmacológico , Linfoma Cutáneo de Células T/patología , Linfoma Cutáneo de Células T/metabolismo , Linfoma Cutáneo de Células T/genética , Apoptosis/efectos de los fármacos , Animales , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Carioferinas/metabolismo , Carioferinas/antagonistas & inhibidores , Ratones , Línea Celular Tumoral , Triazoles/farmacología , Proliferación Celular/efectos de los fármacos , Hidrazinas/farmacología , Hidrazinas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Transducción de Señal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos

RESUMEN

Obesity is one of the significant health challenges in the world and is highly associated with abnormal adipogenesis. TG-interacting factor 1 (TGIF1) is essential for differentiating murine adipocytes and human adipose tissue-derived stem cells. However, the mode of action needs to be better elucidated. To investigate the roles of TGIF1 in differentiation in-depth, CRISPR/Cas9 knockout technology was performed to generate TGIF1-silenced preadipocytes. The absence of TGIF1 in 3 T3-F442A preadipocytes abolished lipid accumulation throughout the differentiation using Oil Red O staining. Conversely, we established 3 T3-F442A preadipocytes stably expressing TGIF1 and doxycycline-inducible TGIF1 in TGIF1-silenced 3 T3-F442A preadipocytes. Remarkably, the induction of TGIF1 by doxycycline during the initial differentiation phase successfully promoted lipid accumulation in TGIF1-silenced 3 T3-F442A cells. We further explored the mechanisms of TGIF1 in early differentiation. We demonstrated that TGIF1 promoted the mitotic clonal expansion via upregulation of CCAAT/enhancer-binding proteins ß expression, interruption with peroxisome proliferators activated receptor γ downstream regulation, and inhibition of p27kip1 expression. In conclusion, we strengthen the pivotal roles of TGIF1 in early differentiation, which might contribute to resolving obesity-associated metabolic syndromes.

Asunto(s)

Adipocitos , Adipogénesis , Diferenciación Celular , Proteínas de Homeodominio , Proteínas Represoras , Animales , Humanos , Ratones , Adipocitos/metabolismo , Adipocitos/citología , Adipogénesis/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mitosis/genética , PPAR gamma/metabolismo , PPAR gamma/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo

RESUMEN

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

Asunto(s)

Puntos de Control del Ciclo Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Enzimas Desubicuitinizantes , Interferón gamma , Ubiquitinación , Humanos , Interferón gamma/farmacología , Interferón gamma/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Puntos de Control del Ciclo Celular/genética , Enzimas Desubicuitinizantes/metabolismo , Cisteína Endopeptidasas/metabolismo , Cisteína Endopeptidasas/genética , Línea Celular Tumoral , Femenino , Proliferación Celular , Fosforilación , Transducción de Señal , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Estabilidad Proteica

RESUMEN

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Asunto(s)

Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Células Endoteliales , Pez Cebra , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Animales , Humanos , Células Endoteliales/metabolismo , Mecanotransducción Celular , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Ciclo Celular , Ratones , Células Cultivadas , Factores de Tiempo , Flujo Sanguíneo Regional , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proliferación Celular , Proteínas de Neoplasias

RESUMEN

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.

Asunto(s)

Sarcoma , Animales , Humanos , Ratones , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Sarcoma/genética , Sarcoma/patología , Ubiquitinación , Regulación hacia Arriba

RESUMEN

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/genética

RESUMEN

Epithelial ovarian cancer (EOC) is one of the most common malignant gynecological tumors with rapid growth potential and poor prognosis, however, the molecular mechanism underlying its outgrowth remained elusive. Germ cell-specific gene 2 (GSG2) was previously reported to be highly expressed in ovarian cancer and was essential for the growth of EOC. In this study, GSG2-knockdown cells and GSG2-overexpress cells were established through lentivirus-mediated transfection with Human ovarian cancer cells HO8910 and SKOV3. Knockdown of GSG2 inhibited cell proliferation and induced G2/M phase arrest in EOC. Interestingly, the expression of p27, a well-known regulator of the cell cycle showed a most significant increase after GSG2 knockdown. Further phosphorylation-protein array demonstrated the phosphorylation of GSK3αSer21 decreased in GSG2-knockdown cells to the most extent. Notably, inhibiting GSK3α activity effectively rescued GSG2 knockdown's suppression on cell cycle as well as p27 expression in EOC. Our study substantiates that GSG2 is able to phosphorylate GSK3α at Ser21 and then leads to the reduction of p27 expression, resulting in cell cycle acceleration and cell proliferation promotion. Thus, GSG2 may have the potential to become a promising target in EOC.

Asunto(s)

Carcinoma Epitelial de Ovario , Ciclo Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Glucógeno Sintasa Quinasa 3 , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Ováricas , Proteínas Serina-Treonina Quinasas , Femenino , Humanos , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Carcinoma Epitelial de Ovario/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Fosforilación , Transducción de Señal , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo

RESUMEN

TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.

Asunto(s)

Células 3T3-L1 , Adipocitos , Adipogénesis , Diferenciación Celular , Proteínas de Homeodominio , Insulina , Animales , Ratones , Fosforilación/efectos de los fármacos , Insulina/metabolismo , Adipocitos/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Humanos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Proliferación Celular/efectos de los fármacos , Butadienos/farmacología

RESUMEN

While loss of function (LOF) of retinoblastoma 1 (RB1) tumor suppressor is known to drive initiation of small-cell lung cancer and retinoblastoma, RB1 mutation is rarely observed in breast cancers at their initiation. In this study, we investigated the impact on untransformed mammary epithelial cells given by RB1 LOF. Depletion of RB1 in anon-tumorigenic MCF10A cells induced reversible growth arrest (quiescence) featured by downregulation of multiple cyclins and MYC, upregulation of p27KIP1, and lack of expression of markers which indicate cellular senescence or epithelial-mesenchymal transition (EMT). We observed a similar phenomenon in human mammary epithelial cells (HMEC) as well. Additionally, we found that RB1 depletion attenuated the activity of RAS and the downstream MAPK pathway in an RBL2/p130-dependent manner. The expression of farnesyltransferase ß, which is essential for RAS maturation, was found to be downregulated following RB1 depletion also in an RBL2/p130-dependent manner. These findings unveiled an unexpected mechanism whereby normal mammary epithelial cells resist to tumor initiation upon RB1 LOF.

Asunto(s)

Regulación hacia Abajo , Células Epiteliales , Proteínas de Unión a Retinoblastoma , Transducción de Señal , Proteínas ras , Humanos , Células Epiteliales/metabolismo , Femenino , Proteínas de Unión a Retinoblastoma/metabolismo , Proteínas de Unión a Retinoblastoma/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Glándulas Mamarias Humanas/citología , Línea Celular Tumoral , Proteína de Retinoblastoma/metabolismo , Proteína de Retinoblastoma/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética

RESUMEN

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin-conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized, and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293, and HELA cells. However, the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1 but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated the colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in the stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Asunto(s)

Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Humanos , Catálisis , Movimiento Celular/genética , Células HEK293 , Células HeLa , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/genética , Estabilidad Proteica , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo