RESUMEN
BACKGROUND: Dihydropyrimidinase-like 3 (DPYSL3) is a cytosolic phosphoprotein expressed in the nervous system and is crucial for neurogenesis. A previous study showed that increased DPYSL3 expression promotes tumour aggressiveness in pancreatic ductal adenocarcinoma, gastric cancer, and colon cancer. However, the role of DPYSL3 in affecting the biological behaviour of urothelial carcinoma (UC) is not yet understood. METHODS: A UC transcriptomic dataset from the Gene Expression Omnibus and the Urothelial Bladder Cancer (BLCA) dataset from The Cancer Genome Atlas were used for the in silico study. We collected 340 upper urinary tract urothelial carcinoma (UTUC) and 295 urinary bladder urothelial carcinoma (UBUC) samples for the immunohistochemical study. Fresh tumour tissue from 50 patients was used to examine the DPYSL3 mRNA level. In addition, urothelial cell lines with and without DPYSL3 knockdown were used for the functional study. RESULTS: The in silico study revealed that DPYSL3 correlated with advanced tumour stage and metastasis development while functioning primarily in the nucleobase-containing compound metabolic process (GO:0006139). DPYSL3 mRNA expression is significantly upregulated in advanced UC. Furthermore, overexpression of the DPYSL3 protein is significantly associated with the aggressive behaviour of UTUC and UBUC. DPYSL3 expression independently predicts disease-specific survival (DSS) and metastatic-free survival (MFS) in patients with UC. In non-muscle-invasive UBUC, DPYSL3 expression predicts local recurrence-free survival. UC cell lines with DPYSL3 knockdown exhibited decreased proliferation, migration, invasion, and human umbilical vein endothelial cells (HUVECs) tube formation but increased apoptosis and G1 arrest. Gene ontology enrichment analysis revealed that the enriched processes related to DPYSL3 overexpression in UC were tissue morphogenesis, cell mesenchyme migration, smooth muscle regulation, metabolic processes, and RNA processing. In vivo study revealed DPYSL3 knockdown in UC tumours significantly suppressed the growth of tumours and decreased MYC and GLUT1 protein expression. CONCLUSIONS: DPYSL3 promotes the aggressiveness of UC cells by changing their biological behaviours and is likely associated with cytoskeletal and metabolic process modifications. Furthermore, DPYSL3 protein overexpression in UC was associated with aggressive clinicopathological characteristics and independently predicted poor clinical outcomes. Therefore, DPYSL3 can be used as a novel therapeutic target for UC.
Asunto(s)
Carcinoma de Células Transicionales , Neoplasias Pancreáticas , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/genética , Regulación hacia Arriba , Células Endoteliales , Pronóstico , Proteínas Musculares/genéticaRESUMEN
Ribosomal protein S6 (rpS6) phosphorylation mediates the hypertrophic growth of kidney proximal tubule cells. However, the role of rpS6 phosphorylation in podocyte hypertrophy and podocyte loss during the pathogenesis of focal segmental glomerulosclerosis (FSGS) remains undefined. Here, we examined rpS6 phosphorylation levels in kidney biopsy specimens from patients with FSGS and in podocytes from mouse kidneys with Adriamycin-induced FSGS. Using genetic and pharmacologic approaches in the mouse model of FSGS, we investigated the role of rpS6 phosphorylation in podocyte hypertrophy and loss during development and progression of FSGS. Phosphorylated rpS6 was found to be markedly increased in the podocytes of patients with FSGS and Adriamycin-induced FSGS mice. Genetic deletion of the Tuberous sclerosis 1 gene in kidney glomerular podocytes activated mammalian target of rapamycin complex 1 signaling to rpS6 phosphorylation, resulting in podocyte hypertrophy and pathologic features similar to those of patients with FSGS including podocyte loss, leading to segmental glomerulosclerosis. Since protein phosphatase 1 is known to negatively regulate rpS6 phosphorylation, treatment with an inhibitor increased phospho-rpS6 levels, promoted podocyte hypertrophy and exacerbated formation of FSGS lesions. Importantly, blocking rpS6 phosphorylation (either by generating congenic rpS6 knock-in mice expressing non-phosphorylatable rpS6 or by inhibiting ribosomal protein S6 kinase 1-mediated rpS6 phosphorylation with an inhibitor) significantly blunted podocyte hypertrophy, inhibited podocyte loss, and attenuated formation of FSGS lesions. Thus, our study provides genetic and pharmacologic evidence indicating that specifically targeting rpS6 phosphorylation can attenuate the development of FSGS lesions by inhibiting podocyte hypertrophy and associated podocyte depletion.
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Glomeruloesclerosis Focal y Segmentaria , Podocitos , Animales , Doxorrubicina , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Humanos , Hipertrofia , Mamíferos/metabolismo , Ratones , Fosforilación , Podocitos/patología , Proteínas Serina-Treonina Quinasas , Proteína S6 Ribosómica/metabolismoRESUMEN
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.
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Carcinoma Epitelial de Ovario/patología , Neoplasias Ováricas/patología , Proteína S6 Ribosómica/genética , Proteína S6 Ribosómica/metabolismo , Regulación hacia Arriba , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Pronóstico , Análisis de SupervivenciaRESUMEN
Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreERT2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreERT2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway.
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Fosfatidilinositol 3-Quinasas Clase III/genética , Túbulos Renales Proximales/crecimiento & desarrollo , Riñón/efectos de los fármacos , Nefronas/crecimiento & desarrollo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Animales , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Integrasas/genética , Riñón/crecimiento & desarrollo , Riñón/patología , Riñón/cirugía , Túbulos Renales Proximales/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Nefrectomía , Nefronas/metabolismo , Fosforilación/genética , Proteína-Lisina 6-Oxidasa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
Amplification and overexpression of the myeloid cell leukemia differentiation protein MCL1 and the murine double minute protein MDM2 have been reported in various human tumors as well as hematological malignancies including acute myeloid leukemia (AML). While MCL1 is an anti-apoptotic member of the BCL-2 family proteins, MDM2 is an important cellular inhibitor of the p53 tumor suppressor. The key oncogene in AML is the FLT3 growth factor receptor gene. FLT3 signaling pathways including the MAPK cascade (RAS-RAF-MEK-ERK) are highly active in AML cells, leading to induced protein translation and cell proliferation as well as reduced apoptosis. Consequently, combined administration of MCL1-, MDM2-, and MEK-inhibitors may present a promising anti-leukemic treatment strategy. Here, we assessed the MCL1-antagonist S63845, the MDM2-inhibitor HDM201, and the MEK1/2-inhibitor trametinib as single agents and in combination in a variety of AML cell lines and mononuclear cells isolated from patients with hematological malignancies centered on myeloid leukemia, some lymphatic leukemia, as well as some lymphomas, for their ability to induce apoptosis and cell death. We observed a considerably varying anti-leukemic efficacy of the MCL1-inhibitor S63845 and the MEK1/2-inhibitor trametinib. Hematological cells with susceptibility to the single compounds as well as to the combined treatment were defined by elevated MCL1- and MEK-protein levels, independent of the mutational status of FLT3 and TP53. Our data indicate that hematological cells with elevated MCL1- and MEK-protein levels are most sensitive to the combined treatment with S63845 and trametinib. MCL1- and MEK1/2-protein expression may be valid biomarkers for treatment response to S63845 and trametinib, respectively.
RESUMEN
BACKGROUND: The 5'-nucleotidase, cytosolic II gene (NT5C2, cN-II) is associated with disorders characterized by psychiatric and psychomotor disturbances. Common psychiatric risk alleles at the NT5C2 locus reduce expression of this gene in the fetal and adult brain, but downstream biological risk mechanisms remain elusive. METHODS: Distribution of the NT5C2 protein in the human dorsolateral prefrontal cortex and cortical human neural progenitor cells (hNPCs) was determined using immunostaining, publicly available expression data, and reverse transcriptase quantitative polymerase chain reaction. Phosphorylation quantification of adenosine monophosphate-activated protein kinase (AMPK) alpha (Thr172) and ribosomal protein S6 (Ser235/Ser236) was performed using Western blotting to infer the degree of activation of AMPK signaling and the rate of protein translation. Knockdowns were induced in hNPCs and Drosophila melanogaster using RNA interference. Transcriptomic profiling of hNPCs was performed using microarrays, and motility behavior was assessed in flies using the climbing assay. RESULTS: Expression of NT5C2 was higher during neurodevelopment and was neuronally enriched in the adult human cortex. Knockdown in hNPCs affected AMPK signaling, a major nutrient-sensing mechanism involved in energy homeostasis, and protein translation. Transcriptional changes implicated in protein translation were observed in knockdown hNPCs, and expression changes to genes related to AMPK signaling and protein translation were confirmed using reverse transcriptase quantitative polymerase chain reaction. The knockdown in Drosophila was associated with drastic climbing impairment. CONCLUSIONS: We provide an extensive neurobiological characterization of the psychiatric risk gene NT5C2, describing its previously unknown role in the regulation of AMPK signaling and protein translation in neural stem cells and its association with Drosophila melanogaster motility behavior.
Asunto(s)
5'-Nucleotidasa/genética , Proteínas Quinasas Activadas por AMP/genética , Trastornos Mentales/genética , Células-Madre Neurales/metabolismo , Biosíntesis de Proteínas/genética , Transducción de Señal/genética , Adulto , Animales , Drosophila melanogaster , Técnicas de Silenciamiento del Gen , Humanos , Actividad Motora/genética , Trastornos del Movimiento/genética , Trastornos del Movimiento/psicología , Fosforilación , Interferencia de ARNRESUMEN
PURPOSE: Failure of retinal detachment surgery is most commonly due to the development of proliferative vitreoretinopathy (PVR). Everolimus is an inhibitor of mammalian target of rapamycin (mTOR), and is available as oral tablets. In this study, we investigated the effect of everolimus on retinal pigment epithelial cells and modification of the severity of experimental PVR. METHODS: In our in vitro studies, primary culture of retinal pigment epithelium (RPE) cells was obtained from pigmented Rex rabbits. Cell proliferation was assayed with the tetrazolium dye cytotoxicity test, and cell migration assay was performed in 24-well transwell units with 8-µm filters. In the in vivo study, pigmented Rex rabbits weighing between 2 and 2.5 kg were used. Each rabbit eye underwent gas compression; one week later, 5 × 104 RPE cells were injected into the vitreous cavity to induce PVR, and each eye was graded with indirect ophthalmoscopy on days 1, 3, 7, 14, 21, and 28. The rabbits were administered everolimus (0.5 mg/day orally) from the day of PVR induction. Total proteins extracted from RPE cells and dissected retinal samples were processed for Western blotting analysis of mTOR and ribosomal protein S6 (RPS6). RESULTS: The in vitro studies showed that everolimus significantly inhibited the proliferation of RPE cells at 0.1 µg/ml; additionally, at 10 µg/ml, it suppressed the migration of RPE cells and significantly suppressed the expression of mTOR and RPS6 in RPE cells. The in vivo study did not show any benefit of oral everolimus (0.5 mg/day) in suppressing experimental PVR. Thus, everolimus significantly suppressed the expression of mTOR and RPS6 in PVR. CONCLUSIONS: Everolimus suppressed the proliferation and migration of RPE cells in vitro. Oral everolimus (0.5 mg/day) suppressed the expression of mTOR and RPS6 in the retina, but showed no effect in suppressing experimental PVR.
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Everolimus/farmacología , Epitelio Pigmentado de la Retina/patología , Vitreorretinopatía Proliferativa/tratamiento farmacológico , Animales , Western Blotting , Movimiento Celular , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Inmunosupresores/farmacología , Conejos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Proteína S6 Ribosómica/biosíntesis , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patologíaRESUMEN
The 90 kDa ribosomal S6 kinases (RSK) are effectors of the Ras-ERK1/2 signaling pathway. RSK signaling controls proliferation and protein synthesis, and is altered in several types of tumors. BI-D1870 and SL0101 are two widely used inhibitors of RSK. After revision of the literature, discrepancies in the effects of the inhibitors were identified. Herein we report that while SL0101 inhibited mTORC1-p70S6K signaling, BI-D1870 increased p70S6K activation. Both effects were independent of ERK1/2 and RSK, and thus nonspecific. We also demonstrated how these opposite nonspecific effects mislead the identification of the RSK-dependent phosphorylation of rpS6 (S235/236), a known RSK and p70S6K substrate. Phosphorylation of tuberin at S1798 by RSK was proposed to mediate ERK1/2-dependent activation of mTORC1-p70S6K signaling. In glioblastoma-derived cells, phosphorylation of tuberin was abolished after RSK depletion or ERK1/2 inhibition, suggesting that RSK is its main kinase. However, RSK depletion did not reduce PMA-dependent p70S6K phosphorylation, which suggests that tuberin phosphorylation at S1798 is not the main mediator of ERK1/2-dependent activation of mTORC1. Remarkably, tuberin phosphorylation (S1798) followed the activation status of RSK in different cells and experimental conditions, suggesting that phosphorylation of that residue could be used as readout for RSK activation in cells. We confirmed the difference in the effects of SL0101 and BI-D1870 in cellular proliferation assays. Rapamycin potentiated the inhibition of proliferation induced by BI-D1870, but not by SL0101. We thus conclude that SL0101 and BI-D1870 induce distinct off-target effects in mTORC1-p70S6K signaling, and thus, the functions previously ascribed to RSK based on these inhibitors should be reassessed.
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Benzopiranos/farmacología , Monosacáridos/farmacología , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pteridinas/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Minería de Datos , Relación Dosis-Respuesta a Droga , Glioblastoma/enzimología , Glioblastoma/genética , Glioblastoma/patología , Células HEK293 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/metabolismo , Fosforilación , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Especificidad por Sustrato , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Transfección , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
Ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit, has been found to be associated with multiple physiological and pathophysiological functions. However, its effects and mechanisms in non-small cell lung cancer (NSCLC) still remain unknown. Here, we showed that expressions of total rpS6 and phosphorylation rpS6 (p-rpS6) were both significantly overexpressed in NSCLC. Further survival analysis revealed the shortened overall survival (OS) and relapse-free survival (RFS) in p-rpS6 overexpressed patients and confirmed it as an independent adverse predictor. Stable downregulation of rpS6 in lung adenocarcinoma A549 and squamous cell carcinoma H520 cell lines was then achieved by two specific small hairpin RNA (shRNA) lentiviruses separately. Subsequent experiments showed that downregulation of rpS6 dramatically inhibited cell proliferation in vitro and tumorigenicity in vivo. Moreover, loss of rpS6 promoted cells arrested in G0-G1 phase and reduced in G2-M phase, along with the expression alterations of relative proteins. However, no notable change in apoptosis was observed. Collectively, these results suggested that rpS6 is overactivated in NSCLC and its downregulation suppresses the growth of NSCLC mainly by inducing G0-G1 cell cycle arrest rather than apoptosis.