RESUMO
Uveal melanoma (UM) is the deadliest form of eye cancer in adults. Inactivating mutations and/or loss of expression of the gene encoding BRCA1-associated protein 1 (BAP1) in UM tumors are associated with an increased risk of metastasis. To investigate the mechanisms underlying this risk, we explored the functional consequences of BAP1 deficiency. UM cell lines expressing mutant BAP1 grew more slowly than those expressing wild-type BAP1 in culture and in vivo. The ability of BAP1 reconstitution to restore cell proliferation in BAP1-deficient cells required its deubiquitylase activity. Proteomic analysis showed that BAP1-deficient cells had decreased phosphorylation of ribosomal S6 and its upstream regulator, p70S6K1, compared with both wild-type and BAP1 reconstituted cells. In turn, expression of p70S6K1 increased S6 phosphorylation and proliferation of BAP1-deficient UM cells. Consistent with these findings, BAP1 mutant primary UM tumors expressed lower amounts of p70S6K1 target genes, and S6 phosphorylation was decreased in BAP1 mutant patient-derived xenografts (PDXs), which grew more slowly than wild-type PDXs in the liver (the main metastatic site of UM) in mice. BAP1-deficient UM cells were also more resistant to amino acid starvation, which was associated with diminished phosphorylation of S6. These studies demonstrate that BAP1 deficiency slows the proliferation of UM cells through regulation of S6 phosphorylation. These characteristics may be associated with metastasis by ensuring survival during amino acid starvation.
Assuntos
Proliferação de Células , Melanoma , Transdução de Sinais , Proteínas Supressoras de Tumor , Ubiquitina Tiolesterase , Neoplasias Uveais , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Mutação , Fosforilação , Proteína S6 Ribossômica/metabolismo , Proteína S6 Ribossômica/genética , Estresse Fisiológico , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , FemininoRESUMO
Defective ribosome biogenesis (RiBi) underlies a group of clinically diverse human diseases collectively known as the ribosomopathies, core manifestations of which include cytopenias and developmental abnormalities that are believed to stem primarily from an inability to synthesize adequate numbers of ribosomes and concomitant activation of p53. The importance of a correctly functioning RiBi machinery for maintaining tissue homeostasis is illustrated by the observation that, despite having a paucity of certain cell types in early life, ribosomopathy patients have an increased risk for developing cancer later in life. This suggests that hypoproliferative states trigger adaptive responses that can, over time, become maladaptive and inadvertently drive unchecked hyperproliferation and predispose to cancer. Here we describe an experimentally induced ribosomopathy in the mouse and show that a normal level of hepatic ribosomal protein S6 (Rps6) is required for proper bile duct development and preservation of hepatocyte viability and that its insufficiency later promotes overgrowth and predisposes to liver cancer which is accelerated in the absence of the tumor-suppressor PTEN. We also show that the overexpression of c-Myc in the liver ameliorates, while expression of a mutant hyperstable form of p53 partially recapitulates specific aspects of the hepatopathies induced by Rps6 deletion. Surprisingly, co-deletion of p53 in the Rps6-deficient background fails to restore biliary development or significantly improve hepatic function. This study not only reveals a previously unappreciated dependence of the developing liver on adequate levels of Rps6 and exquisitely controlled p53 signaling, but suggests that the increased cancer risk in ribosomopathy patients may, in part, stem from an inability to preserve normal tissue homeostasis in the face of chronic injury and regeneration.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Animais , Camundongos , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Hepatócitos/metabolismo , Fenótipo , Ductos Biliares/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
In brief: Several factors affect the reprogramming efficiency of nuclear transfer embryos. This study shows that inhibiting 18S rRNA m6A methyltransferase METTL5 during nuclear transfer can improve the developmental rate of nuclear transfer embryos. Abstract: N6-methyladenosine (m6A) is one of the most important epigenetic modifications in eukaryotic RNAs, which regulates development and diseases. It is identified by several proteins. Methyltransferase-like 5 (METTL5), an enzyme that methylates 18S rRNA m6A, controls the translation of proteins and regulates pluripotency in embryonic stem cells. However, the functions of METTL5 in embryonic development have not been explored. Here, we found that Mettl5 was upregulated in somatic cell nuclear transfer (SCNT) embryos compared with normal fertilized embryos. Therefore, we hypothesized that METTL5 knockdown during the early stage of SCNT would improve the developmental rate of SCNT embryos. Notably, injection of Mettl5 siRNA (si-Mettl5) into enucleated oocytes during nuclear transfer increased the rate of development and the number of cells in blastocysts. Moreover, inhibition of METTL5 reduced the activity of phosphorylated ribosomal protein S6, decreased the levels of the repressive histone modification H3K27me3 and increased the expression of activating histone modifications H3K27ac and H3K4me3 and mRNA levels of some 2-cell-specific genes. These results expand our understanding of the role of METTL5 in early embryonic development and provide a novel idea for improving the efficiency of nuclear transfer cloning.
Assuntos
Reprogramação Celular , Histonas , Animais , Blastocisto/metabolismo , Desenvolvimento Embrionário , Feminino , Histonas/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Técnicas de Transferência Nuclear , Gravidez , RNA Mensageiro/metabolismo , RNA Ribossômico 18S/metabolismo , RNA Interferente Pequeno/genética , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismoRESUMO
Uveal melanoma is a rare form of melanoma that originates in the eye, exerts widespread therapeutic resistance, and displays an inherent propensity for hepatic metastases. Because metastatic disease is characterized by poor survival, there is an unmet clinical need to identify new therapeutic targets in uveal melanoma. Here, we show that the pleiotropic cytokine midkine is expressed in uveal melanoma. Midkine expression in primary uveal melanoma significantly correlates with poor survival and is elevated in patients that develop metastatic disease. Monosomy 3 and histopathologic staging parameters are associated with midkine expression. In addition, we demonstrate that midkine promotes survival, migration across a barrier of hepatic sinusoid endothelial cells and resistance to AKT/mTOR inhibition. Furthermore, midkine is secreted and mediates mTOR activation by maintaining phosphorylation of the mTOR target RPS6 in uveal melanoma cells. Therefore, midkine is identified as a uveal melanoma cell survival factor that drives metastasis and therapeutic resistance, and could be exploited as a biomarker as well as a new therapeutic target. IMPLICATIONS: Midkine is identified as a survival factor that drives liver metastasis and therapeutic resistance in melanoma of the eye.
Assuntos
Neoplasias Hepáticas , Melanoma , Midkina , Proteína S6 Ribossômica , Serina-Treonina Quinases TOR , Neoplasias Uveais , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Midkina/genética , Midkina/metabolismo , Metástase Neoplásica/patologia , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/genéticaRESUMO
BACKGROUND: Ovarian cancer typically is diagnosed late because insensitivity and lack of specificity of current biomarkers prior to its clinical detection. Ribosomal protein S6 (RPS6) is a ribosomal protein involved in the ribosomal 40S subunit, but its biological role in epithelial ovarian cancer (EOC) is still unknown. RESULTS: RPS6 was elevated in EOC compared to normal ovarian tissues and adenomas. Higher expression of RPS6 predicted worse prognosis in EOC. The level of RPS6 was correlated with clinical stage, histological type and pathological grade. Knockdown of RPS6 reduced the proliferation of ovarian cancer cell lines SKOV-3 and HO8910, and inhibit the migration and invasion ability. It revealed that cells arrested at G0G1 phase after knockdown of RPS6, and the expressions of CyclinD1, Cyclin E, CDK2, CDK4, CDK6 and pRb were also reduced. CONCLUSIONS: RPS6 is involved in EOC and knockdown of RPS6 could inhibit the proliferation, invasion and migration ability of EOC in vitro by inducing G0/G1 phase arrest. RPS6 is expected to be a novel biomarker and molecular target to the EOC.
Assuntos
Carcinoma Epitelial do Ovário/patologia , Neoplasias Ovarianas/patologia , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Regulação para Cima , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Prognóstico , Análise de SobrevidaRESUMO
OBJECTIVE: Esophageal cancer (EC) ranks as the sixth leading cause of cancer-related mortality worldwide. Circular RNAs (circRNAs) are involved in the pathogenesis of different cancers. However, the regulatory mechanism of circ_0006168 in EC progression is still unclear. MATERIALS AND METHODS: The expression of circ_0006168, microRNA (miR)-384, and retinoblastoma binding protein 7 (RBBP7) in tumors and cells was measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The stability of circ_0006168 was analyzed after RNase R treatment. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was conducted to evaluate cell viability. Transwell assay was applied to determine cell migration and invasion. Glucose consumption and lactate production were detected using glucose detection and lactic acid detection kits. The interaction between miR-384 and circ_0006168 or RBBP7 was certified by Dual-Luciferase reporter system. Protein expression of pyruvate kinase (PK), RBBP7, S6 ribosomal protein kinase (S6K), phosphorylated S6K (p-S6K), S6, phosphorylated S6 (p-S6) was analyzed by Western blot. RESULTS: Circ_0006168 and RBBP7 were over-expressed while miR-384 was low-expressed in EC tumors and cells. The repression of circ_0006168 attenuated cell proliferation, migration, invasion, and glycolysis in EC. Of note, circ_0006168 functioned as a sponge while RBBP7 acted as a target of miR-384 in EC. Rescue experiment revealed that miR-384 inhibitor abrogated circ_0006168 silencing-induced repression on cell proliferation, migration, and invasion in EC. Meanwhile, upregulation of RBBP7 restored the inhibition of miR-384 on EC cell progression. Moreover, circ_0006168 was able to improve RBBP7 level by interacting with miR-384. Also, circ_0006168 could activate S6K/S6 pathway by regulating RBBP7 expression. CONCLUSIONS: Abundance of circ_0006168 contributes to cell proliferation, migration, invasion, and glycolysis in EC by competitively sponging miR-384 to facilitate RBBP7 expression, representing prospective targets for EC therapy.
Assuntos
Neoplasias Esofágicas/metabolismo , MicroRNAs/metabolismo , RNA Circular/metabolismo , Proteína 7 de Ligação ao Retinoblastoma/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteína S6 Ribossômica/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Humanos , MicroRNAs/genética , RNA Circular/genética , Proteína 7 de Ligação ao Retinoblastoma/genética , Proteína S6 Ribossômica/genética , Proteínas Quinases S6 Ribossômicas/genéticaRESUMO
Postoperative distant metastasis dramatically affects rectal cancer patients who have undergone neoadjuvant chemoradiotherapy (NACRT). Here, we clarified the association between NACRT-mediated mammalian target of rapamycin (mTOR) signaling pathway activation and rectal cancer metastatic potential. We performed immunohistochemistry for phosphorylated mTOR (p-mTOR) and phosphorylated S6 (p-S6) on surgical specimen blocks from 98 rectal cancer patients after NACRT (cohort 1) and 80 colorectal cancer patients without NACRT (cohort 2). In addition, we investigated the association between mTOR pathway activity, affected by irradiation, and the migration ability of colorectal cancer cells in vitro. Based on the results of the clinical study, p-mTOR was significantly overexpressed in cohort 1 (with NACRT) as compared to levels in cohort 2 (without NACRT) (P < .001). High p-mTOR and p-S6 levels correlated with the development of distant metastasis only in cohort 1. Specifically, high p-S6 expression (HR 4.51, P = .002) and high pathological T-stage (HR 3.73, P = .020) after NACRT were independent predictors of the development of distant metastasis. In vitro, p-S6 levels and migration ability increased after irradiation in SW480 cells (TP53 mutation-type) but decreased in LoVo cells (TP53 wild-type), suggesting that irradiation modulates mTOR signaling and migration through cell type-dependent mechanisms. We next assessed the expression level of p53 by immunostaining in cohort 1 and demonstrated that p-S6 was overexpressed in samples with high p53 expression as compared to levels in samples with low p53 expression (P = .008). In conclusion, p-S6 levels after NACRT correlate with postoperative distant metastasis in rectal cancer patients, suggesting that chemoradiotherapy might modulate the mTOR signaling pathway, promoting metastasis.
Assuntos
Neoplasias Retais/tratamento farmacológico , Proteína S6 Ribossômica/genética , Serina-Treonina Quinases TOR/genética , Proteína Supressora de Tumor p53/genética , Idoso , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Quimiorradioterapia Adjuvante/efeitos adversos , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Retais/genética , Neoplasias Retais/patologia , Neoplasias Retais/radioterapia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
Tuberous sclerosis complex (TSC) is a dominant genetic neurocutaneous syndrome characterized by multiple organ hamartomas. Although rodent models bearing a germline mutation in either TSC1 or TSC2 gene have been generated, they do not develop pathogenic lesions matching those seen in patients with TSC because of the significant differences between mice and humans, highlighting the need for an improved large animal model of TSC. Here, we successfully generate monoallelic TSC1-modified Bama miniature pigs using the CRISPR/Cas9 system along with somatic cell nuclear transfer (SCNT) technology. The expression of phosphorylated target ribosomal protein S6 is significantly enhanced in the piglets, indicating that disruption of a TSC1 allele activate the mechanistic target of rapamycin (mTOR) signaling pathway. Notably, differing from the mouse TSC models reported previously, the TSC1+/- Bama miniature pig developed cardiac rhabdomyoma and subependymal nodules, resembling the major clinical features that occur in patients with TSC. These TSC1+/- Bama miniature pigs could serve as valuable large animal models for further elucidation of the pathogenesis of TSC and the development of therapeutic strategies for TSC disease.
Assuntos
Neoplasias Cardíacas/genética , Rabdomioma/genética , Proteína S6 Ribossômica/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Esclerose Tuberosa/genética , Alelos , Animais , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Neoplasias Cardíacas/complicações , Neoplasias Cardíacas/patologia , Humanos , Camundongos , Mutação/genética , Técnicas de Transferência Nuclear , Rabdomioma/complicações , Rabdomioma/patologia , Suínos/genética , Porco Miniatura/genética , Serina-Treonina Quinases TOR/genética , Esclerose Tuberosa/complicações , Esclerose Tuberosa/patologia , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
Maternal inflammation (MI) is associated with many adverse perinatal outcomes. The placenta plays a vital role in mediating maternal-fetal resource allocation. Studies have shown that MI contributes to placental dysfunction, which then leads to adverse birth outcomes and high health risks throughout childhood. Placental mammalian target of rapamycin complex 1 (mTORC1) signaling pathway links maternal nutrient availability to fetal growth; however, the impact of MI on mTORC1 signaling in the placenta remains unclear. In this study, we sought to explore the changes of mTORC1 signaling in the mouse placenta at late gestation by using two models of MI employing lipopolysaccharide (LPS) and interleukin-1ß (IL-1ß) to mimic acute (aMI) and sub-chronic (cMI) inflammatory states, respectively. We determined placental mTORC1 activity by measuring the activity of mTORC1 downstream molecules, including S6k, 4Ebp1, and rpS6. In the aMI model, we found that mTORC1 activity was significantly decreased in the placental decidual and junctional zone at 2 and 6 h after LPS surgery, respectively; however, mTORC1 activity was significantly increased in the placental labyrinth zone at 2, 6, and 24 h after LPS treatment, respectively. In the cMI model, we observed that mTORC1 activity was increased only in the placental labyrinth zone after consecutive IL-1ß exposure. Our study reveals that different parts of the mouse placenta react differently to MI, leading to variable mTORC1 activity throughout the placenta. This suggests that different downstream molecules of mTORC1 from different parts of the mouse placenta may be used in clinical research to monitor the fetal well-being during MI.
Assuntos
Inflamação/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Placenta/metabolismo , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Interleucina-1beta/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Fosforilação , Gravidez , Distribuição Aleatória , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismoRESUMO
Single germline or somatic activating mutations of mammalian target of rapamycin (mTOR) pathway genes are emerging as a major cause of type II focal cortical dysplasia (FCD), hemimegalencephaly (HME) and tuberous sclerosis complex (TSC). A double-hit mechanism, based on a primary germline mutation in one allele and a secondary somatic hit affecting the other allele of the same gene in a small number of cells, has been documented in some patients with TSC or FCD. In a patient with HME, severe intellectual disability, intractable seizures and hypochromic skin patches, we identified the ribosomal protein S6 (RPS6) p.R232H variant, present as somatic mosaicism at ~15.1% in dysplastic brain tissue and ~11% in blood, and the MTOR p.S2215F variant, detected as ~8.8% mosaicism in brain tissue, but not in blood. Overexpressing the two variants independently in animal models, we demonstrated that MTOR p.S2215F caused neuronal migration delay and cytomegaly, while RPS6 p.R232H prompted increased cell proliferation. Double mutants exhibited a more severe phenotype, with increased proliferation and migration defects at embryonic stage and, at postnatal stage, cytomegalic cells exhibiting eccentric nuclei and binucleation, which are typical features of balloon cells. These findings suggest a synergistic effect of the two variants. This study indicates that, in addition to single activating mutations and double-hit inactivating mutations in mTOR pathway genes, severe forms of cortical dysplasia can also result from activating mutations affecting different genes in this pathway. RPS6 is a potential novel disease-related gene.
Assuntos
Hemimegalencefalia/genética , Proteína S6 Ribossômica/genética , Serina-Treonina Quinases TOR/genética , Animais , Encéfalo/metabolismo , Criança , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/metabolismo , Epilepsia/genética , Feminino , Humanos , Malformações do Desenvolvimento Cortical/genética , Malformações do Desenvolvimento Cortical/metabolismo , Malformações do Desenvolvimento Cortical do Grupo I/genética , Camundongos , Mosaicismo , Mutação , Neurônios/metabolismo , Proteína S6 Ribossômica/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Progressive remodeling of the bone marrow microenvironment is recognized as an integral aspect of leukemogenesis. Expanding acute myeloid leukemia (AML) clones not only alter stroma composition, but also actively constrain hematopoiesis, representing a significant source of patient morbidity and mortality. Recent studies revealed the surprising resistance of long-term hematopoietic stem cells (LT-HSC) to elimination from the leukemic niche. Here, we examine the fate and function of residual LT-HSC in the BM of murine xenografts with emphasis on the role of AML-derived extracellular vesicles (EV). AML-EV rapidly enter HSC, and their trafficking elicits protein synthesis suppression and LT-HSC quiescence. Mechanistically, AML-EV transfer a panel of miRNA, including miR-1246, that target the mTOR subunit Raptor, causing ribosomal protein S6 hypo-phosphorylation, which in turn impairs protein synthesis in LT-HSC. While HSC functionally recover from quiescence upon transplantation to an AML-naive environment, they maintain relative gains in repopulation capacity. These phenotypic changes are accompanied by DNA double-strand breaks and evidence of a sustained DNA-damage response. In sum, AML-EV contribute to niche-dependent, reversible quiescence and elicit persisting DNA damage in LT-HSC.
Assuntos
Vesículas Extracelulares/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Nicho de Células-Tronco , Animais , Linhagem Celular Tumoral , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Feminino , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Proteína S6 Ribossômica/genéticaRESUMO
Studies have shown that the mTORC1/rpS6 signaling cascade regulates Sertoli cell blood-testis barrier (BTB) dynamics. For instance, specific inhibition of mTORC1 by treating Sertoli cells with rapamycin promotes the Sertoli cell barrier, making it "tighter." However, activation of mTORC1 by overexpressing a full-length rpS6 cDNA clone (i.e., rpS6-WT, wild type) in Sertoli cells promotes BTB remodeling, making the barrier "leaky." Also, there is an increase in rpS6 and p-rpS6 (phosphorylated and activated rpS6) expression at the BTB in testes at stages VIII-IX of the epithelial cycle, and it coincides with BTB remodeling to support the transport of preleptotene spermatocytes across the barrier, illustrating that rpS6 is a BTB-modifying signaling protein. Herein, we used a constitutively active, quadruple phosphomimetic mutant of rpS6, namely p-rpS6-MT of p-rpS6-S235E/S236E/S240E/S244E, wherein Ser (S) was converted to Glu (E) at amino acid residues 235, 236, 240, and 244 from the NH2 terminus by site-directed mutagenesis, for its overexpression in rat testes in vivo using the Polyplus in vivo jet-PEI transfection reagent with high transfection efficiency. Overexpression of this p-rpS6-MT was capable of inducing BTB remodeling, making the barrier "leaky." This thus promoted the entry of the nonhormonal male contraceptive adjudin into the adluminal compartment in the seminiferous epithelium to induce germ cell exfoliation. Combined overexpression of p-rpS6-MT with a male contraceptive (e.g., adjudin) potentiated the drug bioavailability by modifying the BTB. This approach thus lowers intrinsic drug toxicity due to a reduced drug dose, further characterizing the biology of BTB transport function.
Assuntos
Barreira Hematotesticular/metabolismo , Anticoncepcionais Masculinos/farmacologia , Hidrazinas/farmacologia , Indazóis/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteína S6 Ribossômica/metabolismo , Animais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Mutagênese Sítio-Dirigida , Ratos , Ratos Sprague-Dawley , Proteína S6 Ribossômica/genética , Epitélio Seminífero/metabolismo , Células de Sertoli/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermatócitos/metabolismo , Espermatogênese/efeitos dos fármacosRESUMO
The role of ribosomal protein S6 (rpS6) phosphorylation in mRNA translation remains poorly understood. Here, we reveal a potential role in modulating the translation rate of chemokine (C-X-C motif) ligand 8 (CXCL8 or Interleukin 8, IL8). We observed that more CXCL8 protein was being secreted from less CXCL8 mRNA in primary macrophages and macrophage-like HL-60 cells relative to other cell types. This correlated with an increase in CXCL8 polyribosome association, suggesting an increase in the rate of CXCL8 translation in macrophages. The cell type-specific expression levels were replicated by a CXCL8- UTR-reporter (Nanoluc reporter flanked by the 5' and 3' UTR of CXCL8). Mutations of the CXCL8-UTR-reporter revealed that cell type-specific expression required: 1) a 3' UTR of at least three hundred bases; and 2) an AU base content that exceeds fifty percent in the first hundred bases of the 3' UTR immediately after the stop codon, which we dub AU-rich proximal UTR sequences (APS). The 5' UTR of CXCL8 enhanced expression at the protein level and conferred cell type-specific expression when paired with a 3' UTR. A search for other APS-positive mRNAs uncovered TNF alpha induced protein 6 (TNFAIP6), another mRNA that was translationally upregulated in macrophages. The elevated translation of APS-positive mRNAs in macrophages coincided with elevated rpS6 S235/236 phosphorylation. Both were attenuated by the ERK1/2 signaling inhibitors, U0126 and AZD6244. In A549 cells, rpS6 S235/236 phosphorylation was induced by TAK1, Akt or PKA signaling. This enhanced the translation of the CXCL8-UTR-reporters. Thus, we propose that the induction of rpS6 S235/236 phosphorylation enhances the translation of mRNAs that contain APS motifs, such as CXCL8 and TNFAIP6. This may contribute to the role of macrophages as the primary producer of CXCL8, a cytokine that is essential for immune cell recruitment and activation.
Assuntos
Interleucina-8/biossíntese , Interleucina-8/genética , Proteína S6 Ribossômica/metabolismo , Células A549 , Elementos Ricos em Adenilato e Uridilato , Sequência de Bases , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/metabolismo , Células HL-60 , Humanos , Sistema de Sinalização das MAP Quinases , Macrófagos/imunologia , Macrófagos/metabolismo , Modelos Biológicos , Mutagênese , Fosforilação , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína S6 Ribossômica/química , Proteína S6 Ribossômica/genética , Regiões não TraduzidasRESUMO
Accumulation of senescent endothelial cells can contribute to endothelium dysfunction. Suppression of MTOR signaling has been shown to delay senescence but the mechanism that underpins this effect, particularly one that involves miRNAs, remains to be further defined. This study sought to identify miRNAs involved in MTORC1-mediated inhibition of replicative senescence in endothelial cells. Pre-senescent HUVECs were prolonged treated with low dose rapamycin (1â¯nM), an MTOR inhibitor. Rapamycin treatment down-regulated the phosphorylated MTOR, RPS6 and 4EBP1 expressions, which confirmed MTORC1 suppression. Prolonged low dose rapamycin treatment has significantly reduced the percentage of senescence-associated beta galactosidase (SA-ß gal) positively stained senescent cells and P16INK4A expression in these cells. On the contrary, the percentage of BrdU-labelled proliferating cells has significantly increased. RPTOR, a positive regulator of MTORC1 was knockdown using RPTOR siRNA to inhibit MTORC1 activation. RPTOR knockdown was evidenced by significant suppressions of RPTOR mRNA and protein expression levels. In these cells, the expression of miR-107 was down-regulated whereas miR-145-5p and miR-217 were up-regulated. Target gene prediction revealed PTEN as the target of miR-107 and this was confirmed by biotin pull-down assay. Over-expression of miR-107 has decreased PTEN expression, increased MTORC1 activity, induced cell cycle arrest at G0/G1 phase and up-regulated P16INK4A expression but mitigated tube formation. Collectively, our findings revealed that delayed endothelial replicative senescence caused by the inhibition of MTORC1 activation could be modulated by miR-107 via its influence on PTEN.
Assuntos
Senescência Celular/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , MicroRNAs/genética , PTEN Fosfo-Hidrolase/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína Regulatória Associada a mTOR/antagonistas & inibidores , Proteína Regulatória Associada a mTOR/genética , Proteína Regulatória Associada a mTOR/metabolismo , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Dietary anthocyanin compounds have multiple biological effects, including antioxidant, anti-inflammatory, and anti-atherosclerotic characteristics. The present study evaluated the anti-tumor capacity of mulberry anthocyanins (MA) in thyroid cancer cells. Our data showed that MA suppressed SW1736 and HTh-7 cell proliferation in a time- and dose-dependent manner. Meanwhile, flow cytometry results indicated that MA significantly increased SW1736 and HTh-7 cell apoptosis. We additionally observed that SW1736 and HTh-7 cell autophagy was markedly enhanced after MA treatment. Importantly, anthocyanin-induced cell death was largely abolished by 3-methyladenine (3-MA) or chloroquine diphosphate salt (CQ) treatment, suggesting that MA-induced SW1736 and HTh-7 cell death was partially dependent on autophagy. In addition, activation of protein kinase B (Akt), mammalian target of rapamycin (mTOR), and ribosomal protein S6 (S6) were significantly suppressed by anthocyanin exposure. In summary, MA may serve as an adjunctive therapy for thyroid cancer patients through induction of apoptosis and autophagy-dependent cell death.
Assuntos
Antocianinas/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Morus/química , Adenina/análogos & derivados , Adenina/farmacologia , Antocianinas/isolamento & purificação , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/genética , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/análogos & derivados , Cloroquina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/metabolismo , Glândula Tireoide/patologiaRESUMO
Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and Nε-methyl lysyl demethylations, have important cellular roles. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), which genetic studies reveal is essential in animal development, is reported as a histone Nε-methyl lysine demethylase (KDM). Here we report how extensive screening with peptides based on JMJD5 interacting proteins led to the finding that JMJD5 catalyses stereoselective C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6). High-resolution crystallographic analyses reveal overall fold, active site and substrate binding/product release features supporting the assignment of JMJD5 as an arginine hydroxylase rather than a KDM. The results will be useful in the development of selective oxygenase inhibitors for the treatment of cancer and genetic diseases.
Assuntos
Arginina/química , Proteínas de Transporte/química , Histona Desmetilases/química , Proteínas de Membrana/química , Proteína S6 Ribossômica/química , Arginina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Hidroxilação , Cinética , Lisina/química , Lisina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Estereoisomerismo , Especificidade por Substrato , TermodinâmicaRESUMO
The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.
Assuntos
Barreira Hematotesticular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Proteína S6 Ribossômica/fisiologia , Espermatogênese/fisiologia , Testículo/metabolismo , Animais , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Permeabilidade , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Proteína S6 Ribossômica/genética , Transdução de Sinais/genética , Espermatogênese/genética , Testículo/fisiologia , Regulação para Cima/genéticaRESUMO
BACKGROUND/AIMS: Trastuzumab is an important treatment used for patients with Her-2-positive breast cancer, but an increasing incidence of trastuzumab resistance has been observed clinically during the past decade. Aberrant microRNA (miR) expression levels are correlated with prognosis and response to trastuzumab in breast cancer. MiR-129-5p is downregulated in trastuzumab-resistant human breast cancer cells (JIMT-1), but its potential function and underlying mechanism remain unclear. METHODS: Quantitative RT-PCR (qRT-PCR) was used to determine the expression levels of miR-129-5p and its potential target genes. The effects of miR-129-5p on cell responses to trastuzumab were analyzed by CCK-8 and flow cytometry assays in Her-2-positive breast cancer cells (SKBR-3 and JIMT-1). Bio-informatics analyses were performed to predict target genes of miR-129-5p, and luciferase assays were carried out to confirm the binding of miR-129-5p and rpS6. RESULTS: MiR-129-5p, which was downregulated and predicted to target rpS6 in trastuzumab-resistant breast cancer cells, enhanced the sensitivity of breast cancer cells to trastuzumab by reducing the expression of rpS6. Moreover, the overexpression of rpS6 reversed the sensitivity of cells to trastuzumab induced by miR-129-5p. CONCLUSIONS: MiR-129-5p sensitized Her-2-positive breast cancer to trastuzumab by downregulating rpS6. These findings provide novel insights into the common role of rpS6 and its related molecular mechanisms in mediating trastuzumab-resistance in Her-2-positive breast cancers.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , MicroRNAs/genética , Receptor ErbB-2/análise , Proteína S6 Ribossômica/genética , Trastuzumab/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Trastuzumab/uso terapêuticoRESUMO
Gambogic acid (GA) has been shown to inhibit cancer cell proliferation, induce apoptosis, and enhance reactive oxygen species accumulation. However, whether GA could improve multidrug resistance through modulating autophagy has never been explored. We demonstrated that the combination of GA and cisplatin (CDDP) resulted in a stronger growth inhibition effect on A549 and NCI-H460 cells using the MTT assay. Furthermore, treatment with GA significantly increased autophagy in these cells. More importantly, GA-induced cell death could be largely abolished by 3-methyladenine (3-MA) or chloroquine (CQ) treatment, suggesting that GA-induced cell death was dependent on autophagy. Western blot analysis showed that GA treatment suppressed the activation of Akt, mTOR, and S6. In addition, using a GA and rapamycin combination induced more cell death compared to either GA or rapamycin alone. In summary, GA may have utility as an adjunct therapy for non-small cell lung cancer (NSCLC) patients through autophagy-dependent cell death, even when cancer cells have developed resistance to apoptosis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Garcinia/química , Regulação Neoplásica da Expressão Gênica , Xantonas/farmacologia , Células A549 , Adenina/análogos & derivados , Adenina/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Cisplatino/farmacologia , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/antagonistas & inibidores , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Xantonas/isolamento & purificaçãoRESUMO
Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.