RESUMO
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.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27 , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Humanos , Catálise , Movimento Celular/genética , Células HEK293 , Células HeLa , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Estabilidade Proteica , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismoRESUMO
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.
Assuntos
Brônquios , Carcinoma de Células Escamosas , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p27 , Células Epiteliais , Neoplasias Pulmonares , Nitrosaminas , Humanos , Nitrosaminas/toxicidade , Brônquios/metabolismo , Brônquios/patologia , Brônquios/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação para Baixo/efeitos dos fármacos , Carcinógenos/toxicidadeRESUMO
Noncoding RNAs, including miRNAs (microRNAs) and circRNAs (circular RNA), are crucial regulators of myoblast proliferation and differentiation during muscle development. However, the specific roles and molecular mechanisms of circRNAs in muscle development remain poorly understood. Based on the existing circRNA-miRNA-mRNA network, our study focuses on circUBE3C, exploring its differential expression in fetal and adult muscle tissue of the cattle and investigating its impact on myoblast proliferation, apoptosis, and differentiation. The functional analysis of overexpression plasmids and siRNAs (small interfering RNAs) targeting circUBE3C was comprehensively evaluated by employing an array of advanced assays, encompassing CCK-8 (cell counting kit-8), EdU (5-ethynyl-20-deoxyuridine), flow cytometry, western blot analysis, and RT-qPCR. In vivo investigations indicated that overexpression of circUBE3C impedes the process of skeletal muscle regeneration. Mechanistically, we demonstrated that circUBE3C interacts with miR-191 and alleviates the suppression of p27 through cytoplasmic separation, bioinformatics prediction, dual-luciferase reporter assay, and RIP (RNA immunoprecipitation). Our findings indicate that the novel circRNA circUBE3C competitively binds to miR-191, thereby inhibiting proliferation and promoting apoptosis in bovine primary myoblasts and unveiling a regulatory pathway in bovine skeletal muscle development. These findings expand our understanding of circRNA functions in mammals and provide a basis for further exploration of their role in myogenesis and muscle diseases.
Assuntos
MicroRNAs , RNA Circular , Animais , Bovinos , Diferenciação Celular/genética , Proliferação de Células/genética , Mamíferos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , RNA Interferente Pequeno/metabolismo , Células Cultivadas , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Ubiquitination factor E4B (UBE4B) has a tumor-promoting effect, demonstrated by its aberrant expression in various types of cancers, and in vitro studies have shown that the retardation of cancer cell proliferation can be induced by targeting UBE4B. However, the molecular pathways through which UBE4B exerts its oncogenic activities have not yet been clearly identified and existing knowledge is limited to p53 and its subsequent downstream targets. In this study, we demonstrated that UBE4B regulates p27 expression in A549 cells via the cap-independent translation pathway following treatment with rapamycin and cycloheximide (CHX). Subsequently, we identified that UBE4B regulates p27 translation by regulating the interaction between human antigen R (HuR) and the p27 internal ribosomal entry site (IRES). First, UBE4B interacts with HuR, which inhibits p27 translation through the IRES. Secondly, the interaction between HuR and the p27 IRES was diminished by UBE4B depletion and enhanced by UBE4B overexpression. Finally, HuR depletion-induced growth retardation, accompanied by p27 accumulation, was restored by UBE4B overexpression. Collectively, these results suggest that the oncogenic properties of UBE4B in A549 cells are mediated by HuR, suggesting the potential of targeting the UBE4B-HuR-p27 axis as a therapeutic strategy for lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Inibidor de Quinase Dependente de Ciclina p27 , Neoplasias Pulmonares , Ubiquitina-Proteína Ligases , Humanos , Regiões 5' não Traduzidas , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Semelhante a ELAV 1/metabolismoRESUMO
Respiratory syncytial virus (RSV) fusion protein (F) is highly conserved between subtypes A and B (RSV/A and RSV/B). To become fully active, F precursor undergoes enzymatic cleavage to yield F1 and F2 subunits and releases a 27-amino-acid peptide (p27). Virus-cell fusion occurs when RSV F undergoes a conformational change from pre-F to post-F. Previous data show that p27 is detected on RSV F, but questions remain regarding if and how p27 affects the conformation of mature RSV F. Monoclonal antibodies against p27, site Ø (pre-F specific), and site II were used to monitor RSV F conformation by enzyme-linked immunosorbent assay (ELISA) and imaging flow cytometry. Pre-F to post-F conformational change was induced by a temperature stress test. We found that p27 cleavage efficiency was lower on sucrose-purified RSV/A (spRSV/A) than on spRSV/B. In addition, cleavage of RSV F was cell line dependent: HEp-2 cells had higher retention of p27 than did A549 cells when infected with RSV. Higher levels of p27 were also found on RSV/A-infected cells than on RSV/B-infected cells. We observed that RSV/A F with higher p27 levels could better sustain the pre-F conformation during the temperature stress challenge in both spRSV- and RSV-infected cell lines. Our findings suggest that despite F sequence similarity, p27 of RSV subtypes was cleaved with different efficiencies, which were also dependent on the cell lines used for infection. Importantly, the presence of p27 was associated with greater stability of the pre-F conformation, supporting the possibility that RSV has more than one mechanism for fusion to the host cell. IMPORTANCE RSV fusion protein (F) plays an important role in entry and viral fusion to the host cell. The F undergoes proteolytic cleavages releasing a 27-amino-acid peptide (p27) to become fully functional. The role of p27 in viral entry and the function of the partially cleaved F containing p27 has been overlooked. p27 is thought to destabilize the F trimers, and thus, there is need for a fully cleaved F. In this study, we detected p27 on purified RSV virions and on the surface of virus-infected HEp-2 and A549 cells for circulating RSV strains of both subtypes. Higher levels of partially cleaved F containing p27 better sustained the pre-F conformation during the temperature stress challenge. Our findings highlight that the cleavage efficiency of p27 is different between RSV subtypes and among cell lines and that the presence of p27 contributes to the stability of the pre-F conformation.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vacinas contra Vírus Sincicial Respiratório , Vírus Sincicial Respiratório Humano , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , Linhagem Celular , Proteínas Virais de Fusão/metabolismoRESUMO
BACKGROUND: PD-L1 intrinsically promotes tumor progression through multiple mechanisms, which potentially leads to resistance to anti-PD-1/PD-L1 therapies. The intrinsic effect of PD-L1 on breast cancer (BC) cell proliferation has not been fully elucidated. METHODS: we used proteomics, gene expression knockdown (KD), quantitative immunofluorescence (qIF), western blots, functional assays including colony-forming assay (CFA) and real-time cell analyzer (RTCA), and in vivo data using immunohistochemistry in breast cancer patients. RESULTS: PD-L1 promoted BC cell proliferation by accelerating cell cycle entry at the G1-to-S phase transition. Global proteomic analysis of the differentially expressed nuclear proteins indicated the involvement of several proliferation-related molecules, including p21CIP1/WAF1. Western blotting and qIF demonstrated the higher expression of SKP2 and the lower expression of p21CIP1/WAF1 and p27Kip1 in PD-L1 expressing (PD-L1pos) cells as compared to PD-L1 KD (PD-L1KD) cells. Xenograft-derived cells and the TCGA BC dataset confirmed this relationship in vivo. Functionally, CFA and RTCA demonstrated the central role of SKP2 in promoting PD-L1-mediated proliferation. Finally, immunohistochemistry in 74 breast cancer patients confirmed PD-L1 and SKP-p21/p27 axis relationship, as it showed a highly statistically significant correlation between SKP2 and PD-L1 expression (p < 0.001), and both correlated significantly with the proliferation marker Ki-67 (p < 0.001). On the other hand, there was a statistically significant inverse relationship between PD-L1 and p21CIP1/WAF1 expression (p = 0.005). Importantly, double negativity for p21CIP1/WAF1 and p27Kip1 correlated significantly with PD-L1 (p < 0.001), SKP2 (p = 0.002), and Ki-67 (p = 0.002). CONCLUSIONS: we have demonstrated the role of the SKP2-p27/p21 axis in intrinsic PD-L1-enhanced cell cycle progression. Inhibitors of SKP2 expression can alleviate resistance to ICPIs.
RESUMO
Thioacetamide (TAA) within the liver generates hepatotoxic metabolites that can be induce hepatic fibrosis, similar to the clinical pathological features of chronic human liver disease. The potential protective effect of Albiflorin (ALB), a monoterpenoid glycoside found in Paeonia lactiflora Pall, against hepatic fibrosis was investigated. The mouse hepatic fibrosis model was induced with an intraperitoneal injection of TAA. Hepatic stellate cells (HSCs) were subjected to treatment with transforming growth factor-beta (TGF-ß), while lipopolysaccharide/adenosine triphosphate (LPS/ATP) was added to stimulate mouse peritoneal macrophages (MPMs), leading to the acquisition of conditioned medium. For TAA-treated mice, ALB reduced ALT, AST, HYP levels in serum or liver. The administration of ALB reduced histopathological abnormalities, and significantly regulated the expressions of nuclear receptor-related 1 protein (NURR1) and the P2X purinoceptor 7 receptor (P2×7r) in liver. ALB could suppress HSCs epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) deposition, and pro-inflammatory factor level. ALB also remarkably up-regulated NURR1, inhibited P2×7r signaling pathway, and worked as working as C-DIM12, a NURR1 agonist. Moreover, deficiency of NURR1 in activated HSCs and Kupffer cells weakened the regulatory effect of ALB on P2×7r inhibition. NURR1-mediated inhibition of inflammatory contributed to the regulation of ALB ameliorates TAA-induced hepatic fibrosis, especially based on involving in the crosstalk of HSCs-macrophage. Therefore, ALB plays a significant part in the mitigation of TAA-induced hepatotoxicity this highlights the potential of ALB as a protective intervention for hepatic fibrosis.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Transdução de Sinais , Tioacetamida , Animais , Tioacetamida/toxicidade , Células Estreladas do Fígado/efeitos dos fármacos , Camundongos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Masculino , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Camundongos Endogâmicos C57BL , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacosRESUMO
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
Placentas control the maternal-fetal transport of nutrients and gases. Placental reactions to adverse intrauterine conditions affect fetal development. Such adverse conditions occur in pregnancies complicated by diabetes, leading to alterations in placental anatomy and physiology. In this study, streptozocin (STZ) injection produced sustained hyperglycemia during pregnancy in rats. Hyperglycemic pregnant rats had gained significantly less weight than normal pregnant rats on embryonic day 15.5. We investigated the influence of diabetes on placental anatomy and physiology. Compared with controls, the diabetic group had a markedly thicker junctional zone at embryonic day 15.5. To explore a mechanism for this abnormality, we examined Nodal expression in the junctional zone of control and diabetic groups. We found lower expression of Nodal in the diabetic group. We then investigated the expression of its target gene p27Kip1 (p27), which is related to cell proliferation. In vitro, Nodal overexpression up-regulated p27 protein levels while interfered EBAF up-regulated p27. In vivo, the expression of p27 was lower in diabetic compared with normal rats, and localization was similar between the two groups. In contrast, a higher expression of PCNA was found in diabetic versus normal placenta. Endometrial bleeding associated factor (EBAF), an up-stream molecular regulator of Nodal, was expressed at higher levels in placenta from diabetic versus normal rats. Based on these results, we speculate that the EBAF/Nodal/p27 signaling pathway plays a role in morphological change of diabetic placenta.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Determinação Direita-Esquerda/metabolismo , Proteína Nodal/metabolismo , Placenta/metabolismo , Gravidez em Diabéticas/metabolismo , Transdução de Sinais , Animais , Feminino , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Gastrointestinal epithelial cells respond to milk-born molecules throughout breastfeeding, influencing growth, and development. The rapid renewal of the small intestine depends on the proliferation in the crypt that drives cell fates. We used early weaning model to investigate immediate and late effects of breastfeeding on proliferation, differentiation of jejunal epithelial cells. Wistar rats were either allowed to suckle (S) until 21 postnatal days or submitted to early weaning (EW) at 15 days. By comparing ages (18, 60, and 120 days), we found that EW decreased Ki67 indices and villi height at 18 and 60 days (p < 0.05), and at 120 days they were similar between diets. Proliferative reduction and augmented expression of Cdkn1b (p27 gene) were parallel. In the stem cell niche, EW increased the number and activity (Defa24) of Paneth cells at 18 and 60 days (p < 0.05), and Lgr5 and Ascl2 genes showed inverted responses between ages. Among target cells, EW decreased goblet cell number at 18 and 60 days (p < 0.05) and increased it at 120 days (p < 0.05), whereas enteroendocrine marker genes were differentially altered. EW reduced enterocytes density at 18 days (p < 0.05), and at 120 days this population was decreased (vs. 60 days). Among cell fate crypt-controlling genes, Notch and Atoh1 were the main targets of EW. Metabolically, intraperitoneal glucose tolerance was immediately reduced (18 days), being reverted until 120 days (p < 0.05). Currently, we showed that breastfeeding has a lifespan influence on intestinal mucosa and on its stem cell compartment. We suggest that, although jejunum absorptive function is granted after early weaning, the long lasting changes in gene expression might prime the mucosa with a different sensitivity to gut disorders that still have to be further explored.
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Pulmonary arterial hypertension (PAH) is a disease characterized by increased vasoconstriction and vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) highly express the transcription factor hypoxia-inducible factor-1α (HIF-1α), yet the role of PASMC HIF-1α in the development of PAH remains controversial. To study the role of SMC HIF-1α in the pulmonary vascular response to acute and chronic hypoxia, we used a gain-of-function strategy to stabilize HIF-1α in PASMC by generating mice lacking prolyl hydroxylase domain (PHD) 1 and 2 in SM22α-expressing cells. This strategy increased HIF-1α expression and transcriptional activity under conditions of normoxia and hypoxia. Acute hypoxia increased right ventricular systolic pressure (RVSP) in control, but not in SM22α-PHD1/2-/- mice. Chronic hypoxia increased RVSP and vascular remodeling more in control SM22α-PHD1/2+/+ than in SM22α-PHD1/2-/- mice. In vitro studies demonstrated increased contractility and myosin light chain phosphorylation in isolated PHD1/2+/+ compared with PHD1/2-/- PASMC under both normoxic and hypoxic conditions. After chronic hypoxia, there was more p27 and less vascular remodeling in SM22α-PHD1/2-/- compared with SM22α-PHD1/2+/+ mice. Hypoxia increased p27 in PASMC isolated from control patients, but not in cells from patients with idiopathic pulmonary arterial hypertension (IPAH). These findings highlight an SM22α-expressing cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling. Modulating HIF-1α expression in PASMC may represent a promising preventative and therapeutic strategy for patients with PAH.NEW & NOTEWORTHY In a mouse model wherein hypoxia-inducible factor 1 alpha (HIF-1α) is stabilized in vascular smooth muscle cells, we found that HIF-1α regulates vasoconstriction by limiting phosphorylation of myosin light chain and regulates vascular remodeling through p27 induction. These findings highlight a cell-specific role for HIF-1α in the inhibition of pulmonary vasoconstriction and vascular remodeling.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Humanos , Camundongos , Hipertensão Pulmonar Primária Familiar/metabolismo , Hipertensão Pulmonar/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Prolil Hidroxilases/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Remodelação VascularRESUMO
Accumulating evidence suggests an association between iron metabolism and lung cancer progression. In biological systems, iron is present in either reduced (Fe2+ ; ferrous) or oxidized (Fe3+ ; ferric) states. However, ferrous and ferric iron exhibit distinct chemical and biological properties, the role of ferrous and ferric iron in lung cancer cell growth has not been clearly distinguished. In this study, we manipulated the balance between cellular ferrous and ferric iron status by inducing gene mutations involving the FBXL5-IRP2 axis, a ubiquitin-dependent regulatory system for cellular iron homeostasis, and determined its effects on lung cancer cell growth. FBXL5 depletion (ferrous iron accumulation) was found to suppress lung cancer cell growth, whereas IRP2 depletion (ferric iron accumulation) did not suppress such growth, suggesting that ferrous iron but not ferric iron plays a suppressive role in cell growth. Mechanistically, the depletion of FBXL5 impaired the degradation of the cyclin-dependent kinase inhibitor, p27, resulting in a delay in the cell cycle at the G1/S phase. FBXL5 depletion in lung cancer cells also improved the survival of tumor-bearing mice. Overall, this study highlights the important function of ferrous iron in cell cycle progression and lung cancer cell growth.
Assuntos
Proteínas F-Box , Neoplasias Pulmonares , Animais , Camundongos , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Neoplasias Pulmonares/genética , Ferro/metabolismo , Ubiquitina/metabolismo , Compostos Férricos , Proteínas F-Box/genética , Proteínas F-Box/metabolismoRESUMO
Recent comprehensive analyses of mtDNA and orthogonal RNA-sequencing data revealed that in numerous human cancers, mtDNA copy numbers and mtRNA amounts are significantly reduced, followed by low respiratory gene expression. Under such conditions (called mt-Low), cells encounter severe cell proliferation defects; therefore, they must acquire countermeasures against this fatal disadvantage during malignant transformation. This study elucidated a countermeasure against the mt-Low condition-induced antiproliferative effects in hepatocellular carcinoma (HCC) cells. The mechanism relied on the architectural transcriptional regulator HMGA2, which was preferably expressed in HCC cells of the mt-Low type in vitro and in vivo. Detailed in vitro analyses suggest that HMGA2 regulates insulin-like growth factor binding protein 1 (IGFBP1) expression, leading to AKT activation, which then phosphorylates the cyclin-dependent kinase inhibitor (CKI), P27KIP1, and facilitates its ubiquitin-mediated degradation. Accordingly, intervention in the HMGA2 function by RNAi resulted in an increase in P27KIP1 levels and an induction of senescence-like cell proliferation inhibition in mt-Low-type HCC cells. Conclusively, the HMGA2/IGFBP1/AKT axis has emerged as a countermeasure against P27KIP1 CKI upregulation under mt-Low conditions, thereby circumventing cell proliferation inhibition and supporting the tumorigenic state. Notably, similar to in vitro cell lines, HMGA2 was likely to regulate IGFBP1 expression in HCC in vivo, thereby contributing to poor patient prognosis. Considering the significant number of cases under mt-Low or the threat of CKI upregulation cancer-wide, the axis is noteworthy as a vulnerability of cancer cells or target for tumor-agnostic therapy inducing irreversible cell proliferation inhibition via CKI upregulation in a large population with cancer.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , RNA , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Hepáticas/patologia , DNA Mitocondrial , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Proliferação de Células/genética , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão GênicaRESUMO
Renal cell carcinoma (RCC) is the most lethal of the urologic malignancies. We previously discovered that DAB2IP, a novel Ras GTPase-activating protein, was frequently epigenetically silenced in RCC, and DAB2IP loss was correlated with the overall survival of RCC patients. In this study, we determined the biological functions of DAB2IP in clear cell RCC (ccRCC) and its potential mechanisms of action. Correlations between DAB2IP expression level and ccRCC tumor size and patient survival were analyzed, and the results showed that ccRCC patients with high DAB2IP mRNA level exhibited smaller tumor size and better survival than the patients with low DAB2IP. Compared to control, DAB2IP knockdown significantly increased cell proliferation, promoted cell cycle progression in G1/S phase, and decreased p27 expression. Mechanism studies demonstrated that loss of DAB2IP promoted p27 protein phosphorylation, cytosolic sequestration, and subsequently ubiquitination-mediated degradation in ccRCC cells. Further studies confirmed that the proline-rich domain in C terminal (CPR) of DAB2IP suppressed AKT phosphorylation and p27 phosphorylation on S10. Hence, DAB2IP is essential for p27 protein stabilization in ccRCC, which is at less partly mediated by PI3K/AKT signaling pathway.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Proliferação de Células/genética , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Linhagem Celular Tumoral , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismoRESUMO
p27Kip1 is known as a major cyclin-dependent kinase inhibitor and a tumor suppressor, and often functionally hampered at protein level. p27 protein expression levels are frequently low in various cancers and negatively correlated with malignancy of cancer. However, in our previous study, we discovered that p27 overexpression does not inhibit the proliferation of two cancer cell lines due to a functional suppression of p27 by nucleophosmin isoform 1 (NPM1); that is, a qualitative, not quantitative, suppression of p27 function occurs in these cancer cell lines. To clarify the regulation of p27 in several types of cancer, we investigated p27 function in other cancer cell lines, based on proliferation assays in those cell lines carrying doxycycline-inducible p27, and found that MDAH041 cells which express p14ARF, an antagonist of NPM1, show growth inhibition depending on p27 induction. Moreover, to investigate p27 function under anchorage-independent culture conditions, we performed soft agar colony formation assay and observed that the colony formation of some cell lines carrying wild-type p53, a major tumor suppressor, was inhibited depending on p27 induction. These results suggest that p27 function is regulated differentially among cancer cell types under anchorage-dependent and anchorage-independent culture conditions.
Assuntos
Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53 , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The asymptomatic nature of cholangiocarcinoma (CCA), particularly during its early stages, in combination with its high aggressiveness and chemoresistance, significantly compromises the efficacy of current therapeutic options, contributing to a dismal prognosis. As a tumor suppressor that inhibits the cell cycle, abnormal cytoplasmic p27kip1 localization is related to chemotherapy resistance and often occurs in various cancers, including CCA. Nevertheless, the underlying mechanism is unclear. SUMOylation, which is involved in regulating subcellular localization and the cell cycle, is a posttranslational modification that regulates p27kip1 activity. Here, we confirmed that UBE2I, as the only key enzyme for SUMOylation, was highly expressed and p27kip1 was downregulated in CCA tissues, which were associated with poor outcomes in CCA. Moreover, UBE2I silencing inhibited CCA cell proliferation, delayed xenograft tumor growth in vivo, and sensitized CCA cells to the chemotherapeutics, which may be due to cell cycle arrest induced by p27kip1 nuclear accumulation. According to the immunoprecipitation result, we found that UBE2I could bind p27kip1, and the binding amount of p27kip1 and SUMO-1 decreased after UBE2I silencing. Moreover, nuclear retention of p27kip1 was induced by UBE2I knockdown and SUMOylation or CRM1 inhibition, further suggesting that UBE2I could cooperate with CRM1 in the nuclear export of p27kip1. These data indicate that UBE2I-mediated SUMOylation is a novel regulatory mechanism that underlies p27kip1 export and controls CCA tumorigenesis, providing a therapeutic option for CCA treatment.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Transporte Ativo do Núcleo Celular , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , AnimaisRESUMO
BACKGROUND AND AIMS: Accumulating circular RNAs (circRNAs) play important roles in tissue repair and organ regeneration. However, the biological effects of circRNAs on liver regeneration remain largely unknown. This study aims to systematically elucidate the functions and mechanisms of circRNAs derived from lipopolysaccharide-responsive beige-like anchor protein (LRBA) in regulating liver regeneration. METHODS: CircRNAs derived from mouse LRBA gene were identified using CircBase. In vivo and in vitro experiments were conducted to confirm the effects of circLRBA on liver regeneration. RNA pull-down and RNA immunoprecipitation assays were used to investigate the underlying mechanisms. Clinical samples and cirrhotic mouse models were used to evaluate the clinical significance and transitional value of circLRBA. RESULTS: Eight circRNAs derived from LRBA were registered in CircBase. The circRNA mmu_circ_0018031 (circLRBA) was significantly upregulated in the liver tissues after 2/3 partial hepatectomy (PHx). Adeno-associated virus serotype 8 (AAV8)-mediated knockdown of circLRBA markedly inhibited mouse liver regeneration after 2/3 PHx. In vitro experiments confirmed that circLRBA exerted its growth-promoting function mainly through liver parenchymal cells. Mechanistically, circLRBA acted as a scaffold for the interaction between E3 ubiquitin-protein ligase ring finger protein 123 and p27, facilitating the ubiquitination degradation of p27. Clinically, circLRBA was lowly expressed in cirrhotic liver tissues and negatively correlated with perioperative levels of total bilirubin. Furthermore, overexpression of circLRBA enhanced cirrhotic mouse liver regeneration after 2/3 PHx. CONCLUSIONS: We conclude that circLRBA is a novel growth promoter in liver regeneration and a potential therapeutic target related to deficiency of cirrhotic liver regeneration.
Assuntos
MicroRNAs , RNA Circular , Animais , Camundongos , Cirrose Hepática , Regeneração Hepática , MicroRNAs/genética , RNA/genética , RNA Circular/genética , RNA Circular/metabolismo , UbiquitinaçãoRESUMO
Among the hallmarks of cholangiocarcinoma (CCA) progression and unresponsiveness to therapy is impaired ubiquitin-dependent degradation of nuclear tumor suppressor protein. In the previous stage, our research group found that as a key tumor suppressor, nuclear dysfunction of p27kip1 is closely related to chemotherapy resistance of CCA, but the specific mechanism is unclear. It was recently shown that p27kip1-driven tumors were strongly dependent on the SUMO pathway. RNF4, as the SUMO-targeted ubiquitin ligase (STUbL), identifies SUMOylated proteins as a substrate through sumo-interacting motifs (SIM) and causes its degradation via the ubiquitin proteasome pathway. Here we described that the expression of RNF4 was upregulated in CCA tissues and related to malignant features. Silencing RNF4 arrested human CCA cells at the G1 phase, which was associated with the upregulation of p27kip1 and the downregulation of its downstream cycle-related proteins. Silencing RNF4 inhibited cell proliferation and migration, increased cell apoptosis, and sensitized CCA cells to treatment of chemotherapeutic drugs in vitro. Immunofluorescence showed that p27kip1 and RNF4 were mainly co-located in the nucleus. Immunoprecipitation and Western blot showed that p27kip1 was a target protein for SUMOylation and high expression of RNF4 decreased the levels of nuclear p27kip1, enhanced the levels of ubiquitinated and SUMOylated p27kip1, indicating that RNF4 could regulate cell cycle progression via recognizing SUMOylated p27kip1 and facilitating its ubiquitination degradation. These data indicate that RNF4-mediated ubiquitination degradation of SUMOylated proteins is a novel regulatory mechanism of p27kip1 dysfunction and CCA tumorigenesis, which provides a potential option for therapeutic intervention of CCA.
Assuntos
Colangiocarcinoma , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Carcinogênese , Ciclo Celular , Humanos , Sumoilação , Ubiquitina , UbiquitinaçãoRESUMO
PURPOSE: The foremost drawback of ovarian tissue cryopreservation and re-transplantation (OTCT) technique is the rapid loss of the primordial follicle (PF) pool. In recent studies, we have demonstrated that post-transplantation burnout of the PFs occurs due to the altered expression of the activatory and inhibitory proteins that control PF reserve, and rapamycin prevented it. METHODS: Here, we investigated whether anti-Mullerian hormone administration in the bilateral oophorectomy and transplantation group and internal AMH in the unilateral oophorectomy and transplantation group protect follicle reserve by regulating the expression of the molecules that control follicle growth after OTCT in mice. RESULTS: After 14 days of OTCT, PF reserve is significantly reduced in both unilateral oophorectomy and transplantation and bilateral oophorectomy and transplantation groups, while anti-Mullerian hormone treatment attenuates PF loss after bilateral oophorectomy and transplantation. The expression of KitL, Bmp-15, and p27 decreased after unilateral oophorectomy and transplantation and bilateral oophorectomy and transplantation, yet recombinant anti-Mullerian hormone treatment did not restore the expression of these proteins in the BLO-T group. CONCLUSION: Exogenous recombinant anti-Mullerian hormone administration in the BLO-T group preserved the expressions of Tsc1 and Gdf-9 in PF and p-s6k and Gdf-9 in growing follicles after OTCT. Nonetheless, recombinant anti-Mullerian hormone administration did not affect granulosa cell proliferation and death rates in the growing follicles. These findings suggest a novel hormonal replacement strategy for fertility preservation by restoring anti-Mullerian hormone to regulate Tsc1 and p-s6k, thereby linking this hormone with the mTOR pathway and Gdf-9 signaling.
Assuntos
Hormônio Antimülleriano , Fator 9 de Diferenciação de Crescimento , Feminino , Camundongos , Animais , Hormônio Antimülleriano/metabolismo , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Folículo Ovariano , Ovário/metabolismo , CriopreservaçãoRESUMO
The aim of this work was to investigate the effect of the whole-body deletion of p27 on the activity of brown adipose tissue and the susceptibility to develop obesity and glucose homeostasis disturbances in mice, especially when subjected to a high fat diet. p27 knockout (p27-/-) and wild type (WT) mice were fed a normal chow diet or a high fat diet (HFD) for 10-weeks. Body weight and composition were assessed. Insulin and glucose tolerance tests and indirect calorimetry assays were performed. Histological analysis of interscapular BAT (iBAT) was carried out, and expression of key genes/proteins involved in BAT function were characterized by qPCR and Western blot. iBAT activity was estimated by 18F-fluorodeoxyglucose (18FDG) uptake with microPET. p27-/- mice were more prone to develop obesity and insulin resistance, exhibiting increased size of all fat depots. p27-/- mice displayed a higher respiratory exchange ratio. iBAT presented larger adipocytes in p27-/- HFD mice, accompanied by downregulation of both Glut1 and uncoupling protein 1 (UCP1) in parallel with defective insulin signalling. Moreover, p27-/- HFD mice exhibited impaired response to cold exposure, characterized by a reduced iBAT 18FDG uptake and difficulty to maintain body temperature when exposed to cold compared to WT HFD mice, suggesting reduced thermogenic capacity. These data suggest that p27 could play a role in BAT activation and in the susceptibility to develop obesity and insulin resistance.