RESUMEN
KDM6A, an X chromosome-linked histone lysine demethylase, was reported to be frequently mutated in many tumor types including breast and bladder cancer. However, the functional role of KDM6A is not fully understood. Using MCF10A as a model of non-tumorigenic epithelial breast cells, we found that silencing KDM6A promoted cell migration and transformation demonstrated by the formation of tumor-like acini in three-dimensional culture. KDM6A loss reduced the sensitivity of MCF10A cells to therapeutic agents commonly used to treat patients with triple-negative breast cancer and also induced TGFß extracellular secretion leading to suppressed expression of cytotoxic genes in normal human CD8+ T cells in vitro. Interestingly, when cells were treated with TGFß, de novo synthesis of KDM6A protein was suppressed while TGFB1 transcription was enhanced, indicating a TGFß/KDM6A-negative regulatory axis. Furthermore, both KDM6A deficiency and TGFß treatment promoted disorganized acinar structures in three-dimensional culture, as well as transcriptional profiles associated with epithelial-to-mesenchymal transition and metastasis, suggesting KDM6A depletion and TGFß drive tumor progression. IMPLICATIONS: Our study provides the preclinical rationale for evaluating KDM6A and TGFß in breast tumor samples as predictors for response to chemo and immunotherapy, informing personalized therapy based on these findings.
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Antineoplásicos , Neoplasias de la Mama , Neoplasias de la Vejiga Urinaria , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Células Epiteliales/patología , Femenino , Histona Demetilasas/genética , Humanos , Factor de Crecimiento Transformador beta , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Bladder cancer is the 10th most commonly diagnosed cancer in the world, accounting for around 573,000 new cases and 213,000 deaths in 2020. The current standard treatment for locally advanced bladder cancer is neoadjuvant cisplatin (NAC)-based chemotherapy followed by cystectomy. The significant progress being made in the genomic and molecular understandings of bladder cancer has uncovered the genetic alterations and signaling pathways that drive bladder cancer progression. These developments have led to a dramatic increase in the evaluation of molecular agents targeting at these alterations. One example is Erdafitinib, a first-in-class FGFR inhibitor being approved as second-line treatment for locally advanced or metastatic urothelial carcinoma with FGFR mutations. Immunotherapy has also been approved as second-line treatment for advanced and metastatic bladder cancer. Preclinical studies suggest targeted therapy combined with immunotherapy has the potential to markedly improve patient outcome. Given the prevalence of FGFR alternations in bladder cancer, here we review recent preclinical and clinical studies on FGFR inhibitors and analyze possible drug resistance mechanisms to these agents. We also discuss FGFR inhibitors in combination with other therapies and its potential to improve outcome.
RESUMEN
The field of research in bladder cancer has seen significant advances in recent years. Next-generation sequencing has identified the genes most mutated in bladder cancer. This wealth of information allowed the definition of driver mutations, and identification of actionable therapeutic targets, as well as a clearer picture of patient prognosis and therapeutic direction. In a similar vein, our understanding of the cellular aspects of bladder cancer has grown. The identification of the cellular geography and the populations of different cell types and quantifications of normal and abnormal cell types in tumours provide a better prediction of therapeutic response. Non-invasive methods of diagnosis, including liquid biopsies, have seen major advances as well. These methods will likely find considerable utility in assessing minimal residual disease following treatment and for early-stage diagnosis. A significant therapeutic impact on patients with bladder cancer is found in the use of immune checkpoint inhibitor therapeutics. These therapeutics have been shown to cure some patients with bladder cancer and significantly decrease adverse events. These developments provide patients with better monitoring opportunities, unique therapeutic options and greater hope for prolonged survival.
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Carcinoma de Células Transicionales/genética , Neoplasias de la Vejiga Urinaria/genética , Animales , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Células Transicionales/patología , Carcinoma de Células Transicionales/terapia , Modelos Animales de Enfermedad , Factores Epidemiológicos , Epigénesis Genética/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones , Mutación , Estadificación de Neoplasias , Pronóstico , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/terapiaRESUMEN
Immunotherapies targeting the PD-1/PD-L1 axis are now a mainstay in the clinical management of multiple cancer types, however, many tumors still fail to respond. CCL2 is highly expressed in various cancer types and has been shown to be associated with poor prognosis. Inhibition or blockade of the CCL2/CCR2 signaling axis has thus been an area of interest for cancer therapy. Here we show across multiple murine tumor and metastasis models that CCR2 antagonism in combination with anti-PD-1 therapy leads to sensitization and enhanced tumor response over anti-PD-1 monotherapy. We show that enhanced treatment response correlates with enhanced CD8+ T cell recruitment and activation and a concomitant decrease in CD4+ regulatory T cell. These results provide strong preclinical rationale for further clinical exploration of combining CCR2 antagonism with PD-1/PD-L1-directed immunotherapies across multiple tumor types especially given the availability of small molecule CCR2 inhibitors and antibodies.
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Antineoplásicos/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/terapia , Receptores CCR2/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Terapia Combinada , Femenino , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , RNA-Seq , Neoplasias de la Vejiga Urinaria/terapiaRESUMEN
While a fraction of cancer patients treated with anti-PD-1 show durable therapeutic responses, most remain unresponsive, highlighting the need to better understand and improve these therapies. Using an in vivo screening approach with a customized shRNA pooled library, we identified DDR2 as a leading target for the enhancement of response to anti-PD-1 immunotherapy. Using isogenic in vivo murine models across five different tumor histologies-bladder, breast, colon, sarcoma, and melanoma-we show that DDR2 depletion increases sensitivity to anti-PD-1 treatment compared to monotherapy. Combination treatment of tumor-bearing mice with anti-PD-1 and dasatinib, a tyrosine kinase inhibitor of DDR2, led to tumor load reduction. RNA-seq and CyTOF analysis revealed higher CD8+ T cell populations in tumors with DDR2 depletion and those treated with dasatinib when either was combined with anti-PD-1 treatment. Our work provides strong scientific rationale for targeting DDR2 in combination with PD-1 inhibitors.
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Linfocitos T CD8-positivos/inmunología , Dasatinib/farmacología , Receptor con Dominio Discoidina 2/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos , Inmunidad Celular , Inmunoterapia , Neoplasias Experimentales/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Receptor con Dominio Discoidina 2/inmunología , Femenino , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Urothelial carcinoma accounts for most of the bladder cancer cases. Using next-generation sequencing (NGS) technology, we found that a significant percentage (83%) of tumors had mutations in chromatin-remodeling genes. Here, we examined the functional relevance of mutations in two chromatin-remodeling genes, EP300 and its paralog, CREBBP, which are mutated in almost one-third of patients. Interestingly, almost half of missense mutations cluster in the histone-acetyltransferase (HAT) domain of EP300/CREBBP. This domain catalyzes the transfer of an acetyl group to target molecules such as histones, thereby regulating chromatin dynamics. Thus, patients with EP300 or CREBBP mutations may have alterations in the ability of the corresponding proteins to modify histone proteins and control transcriptional profiles. In fact, it was determined that many of the missense HAT mutations in EP300 (64%) and CREBBP (78%) were HAT-inactivating. These inactivating mutations also correlated with invasive disease in patients. Strikingly, the prediction software Mutation Assessor accurately predicted the functional consequences of each HAT missense mutation. Finally, a gene expression signature was developed that associated with loss of HAT activity and that this signature was associated with more aggressive cancer in four patient datasets. Further supporting the notion that this score accurately reflects HAT activity, we found it is responsive to treatment of cancer cells to mocetinostat, a histone deacetylase (HDAC) inhibitor.Implication: This study provides a rationale for targeted sequencing of EP300 and CREBBP and use of a gene profiling signature for predicting therapeutic response in patients. Mol Cancer Res; 16(1); 69-77. ©2017 AACR.
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Ensamble y Desensamble de Cromatina/genética , Neoplasias de la Vejiga Urinaria/genética , Línea Celular Tumoral , Humanos , Mutación Missense , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Elevated tumor expression of the cell surface GPI-linked CD24 protein signals poor patient prognosis in many tumor types. However, some cancer cells selected to be negative for surface CD24 (surCD24-) still retain aggressive phenotypes in vitro and in vivo Here, we resolve this apparent paradox with the discovery of biologically active, nuclear CD24 (nucCD24) and finding that its levels are unchanged in surCD24- cells. Using the complementary techniques of biochemical cellular fractionation and immunofluorescence, we demonstrate a signal for CD24 in the nucleus in cells from various histologic types of cancer. Nuclear-specific expression of CD24 (NLS-CD24) increased anchorage-independent growth in vitro and tumor formation in vivo Immunohistochemistry of patient tumor samples revealed the presence of nucCD24, whose signal intensity correlated positively with the presence of metastatic disease. Analysis of gene expression between cells expressing CD24 and NLS-CD24 revealed a unique nucCD24 transcriptional signature. The median score derived from this signature was able to stratify overall survival in four patient datasets from bladder cancer and five patient datasets from colorectal cancer. Patients with high scores (more nucCD24-like) had reduced survival. These findings define a novel and functionally important intracellular location of CD24; they explain why surCD24- cells can remain aggressive, and they highlight the need to consider nucCD24 in both fundamental research and therapeutic development. Cancer Res; 77(18); 4858-67. ©2017 AACR.
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Biomarcadores de Tumor/metabolismo , Antígeno CD24/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Neoplasias de la Vejiga Urinaria/patología , Animales , Apoptosis , Humanos , Metástasis Linfática , Ratones , Ratones Desnudos , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In principle, the inhibition of candidate gain-of-function genes defined through genomic analyses of large patient cohorts offers an attractive therapeutic strategy. In this study, we focused on changes in expression of CD24, a well-validated clinical biomarker of poor prognosis and a driver of tumor growth and metastasis, as a benchmark to assess functional relevance. Through this approach, we identified GON4L as a regulator of CD24 from screening a pooled shRNA library of 176 candidate gain-of-function genes. GON4L depletion reduced CD24 expression in human bladder cancer cells and blocked cell proliferation in vitro and tumor xenograft growth in vivo Mechanistically, GON4L interacted with transcription factor YY1, promoting its association with the androgen receptor to drive CD24 expression and cell growth. In clinical bladder cancer specimens, expression of GON4L, YY1, and CD24 was elevated compared with normal bladder urothelium. This pathway is biologically relevant in other cancer types as well, where CD24 and the androgen receptor are clinically prognostic, given that silencing of GON4L and YY1 suppressed CD24 expression and growth of human lung, prostate, and breast cancer cells. Overall, our results define GON4L as a novel driver of cancer growth, offering new biomarker and therapeutic opportunities. Cancer Res; 76(17); 5175-85. ©2016 AACR.
Asunto(s)
Antígeno CD24/metabolismo , Carcinoma de Células Transicionales/patología , Receptores Androgénicos/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Factor de Transcripción YY1/metabolismo , Animales , Western Blotting , Carcinoma de Células Transicionales/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Proteínas Co-Represoras , Proteínas de Unión al ADN , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
UNLABELLED: Next-generation sequencing (NGS) of human bladder cancer has revealed many gene alterations compared with normal tissue, with most being predicted to be "loss of function." However, given the high number of alterations, evaluating the functional impact of each is impractical. Here, we develop and use a high-throughput, in vivo strategy to determine which alterations are loss of function in tumor growth suppressors. Genes reported as altered by NGS in bladder cancer patients were bioinformatically processed by MutationTaster and MutationAssessor, with 283 predicted as loss of function. An shRNA lentiviral library targeting these genes was transduced into T24 cells, a nontumorigenic human bladder cancer cell line, followed by injection into mice. Tumors that arose were sequenced and the dominant shRNA constructs were found to target IQGAP1, SAMD9L, PCIF1, MED1, and KATNAL1 genes. In vitro validation experiments revealed that shRNA molecules directed at IQGAP1 showed the most profound increase in anchorage-independent growth of T24 cells. The clinical relevance of IQGAP1 as a tumor growth suppressor is supported by the finding that its expression is lower in bladder cancer compared with benign patient urothelium in multiple independent datasets. Lower IQGAP1 protein expression associated with higher tumor grade and decreased patient survival. Finally, depletion of IQGAP1 leads to increased TGFBR2 with TGFß signaling, explaining in part how reduced IQGAP1 promotes tumor growth. These findings suggest IQGAP1 is a bladder tumor growth suppressor that works via modulating TGFß signaling and is a potentially clinically useful biomarker. IMPLICATIONS: This study used gene mutation information from patient-derived bladder tumor specimens to inform the development of a screen used to identify novel tumor growth suppressors. This included identification of the protein IQGAP1 as a potent bladder cancer growth suppressor.
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Genes Supresores de Tumor , Pruebas Genéticas/métodos , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Proteínas Activadoras de ras GTPasa/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Computadores Moleculares , Supervivencia sin Enfermedad , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Invasividad Neoplásica , Pronóstico , ARN Interferente Pequeño/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/metabolismo , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
BACKGROUND: Bladder cancer is the most common malignancy of the urinary system, yet our molecular understanding of this disease is incomplete, hampering therapeutic advances. METHODS: Here we used a genome-wide functional short-hairpin RNA (shRNA) screen to identify suppressors of in vivo bladder tumor xenograft growth (n = 50) using bladder cancer UMUC3 cells. Next-generation sequencing was used to identify the most frequently occurring shRNAs in tumors. Genes so identified were studied in 561 patients with bladder cancer for their association with stratification of clinical outcome by Kaplan-Meier analysis. The best prognostic marker was studied to determine its mechanism in tumor suppression using anchorage-dependent and -independent growth, xenograft (n = 20), and metabolomic assays. Statistical significance was determined using two-sided Student t test and repeated-measures statistical analysis. RESULTS: We identified the glycogen debranching enzyme AGL as a prognostic indicator of patient survival (P = .04) and as a novel regulator of bladder cancer anchorage-dependent (P < .001), anchorage-independent (mean ± standard deviation, 180 ± 23.1 colonies vs 20±9.5 in control, P < .001), and xenograft growth (P < .001). Rescue experiments using catalytically dead AGL variants revealed that this effect is independent of AGL enzymatic functions. We demonstrated that reduced AGL enhances tumor growth by increasing glycine synthesis through increased expression of serine hydroxymethyltransferase 2. CONCLUSIONS: Using an in vivo RNA interference screen, we discovered that AGL, a glycogen debranching enzyme, has a biologically and statistically significant role in suppressing human cancer growth.
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Sistema de la Enzima Desramificadora del Glucógeno/deficiencia , Neoplasias de la Vejiga Urinaria/enzimología , Neoplasias de la Vejiga Urinaria/patología , Animales , Línea Celular Tumoral , Estudio de Asociación del Genoma Completo , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno/enzimología , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , ARN Interferente Pequeño/genética , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Overexpression of CD24, a glycosyl phosphatidylinositol-linked sialoglycoprotein, is associated with poor outcome in urothelial carcinoma and contributes to experimental tumor growth and metastasis. However, the requirement for CD24 (Cd24a in mice) in tumorigenesis and spontaneous metastasis from the orthotopic site remains uncharacterized. Using N-butyl-N-(4-hydroxybutyl) nitrosamine induction of invasive and metastatic bladder cancer, we show that Cd24a-deficient male mice developed fewer bladder tumors than C57BL/6 control male mice. Evaluating only mice with evidence of primary tumors, we observed that Cd24a-deficient male mice also had fewer metastases than wild-type counterparts. In parallel observations, stratification of patients based on CD24 immunohistochemical expression in their tumors revealed that high levels of CD24 are associated with poor prognosis in males. In female patients and mice the above observations were not present. Given the significant role of CD24 in males, we sought to assess the relationship between androgen and CD24 regulation. We discovered that androgen receptor knockdown in UM-UC-3 and TCCSUP human urothelial carcinoma cell lines resulted in suppression of CD24 expression and cell proliferation. Androgen treatment also led to increased CD24 promoter activity, dependent on the presence of androgen receptor. In vivo, androgen deprivation resulted in reduced growth and CD24 expression of UM-UC-3 xenografts, and the latter was rescued by exogenous CD24 overexpression. These findings demonstrate an important role for CD24 in urothelial tumorigenesis and metastasis in male mice and indicate that CD24 is androgen regulated, providing the foundation for urothelial bladder cancer therapy with antiandrogens.
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Andrógenos/metabolismo , Antígeno CD24/biosíntesis , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Urotelio/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Trasplante de Neoplasias , Regiones Promotoras Genéticas , Receptores Androgénicos/metabolismo , Factores SexualesRESUMEN
Epidermal growth factor receptor (EGFR) is involved in development and progression of many human cancers. We have previously demonstrated that the ubiquitin-specific peptidase Usp18 (Ubp43) is a potent regulator of EGFR protein expression. Here we report that the 3'-untranslated region (3'-UTR) of the EGFR message modulates RNA translation following cell treatment with Usp18 siRNA, suggesting microRNA as a possible mediator. Given earlier evidence of EGFR regulation by the microRNA miR-7, we assessed whether miR-7 mediates Usp18 siRNA effects. We found that Usp18 depletion elevates miR-7 levels in several cancer cell lines because of a transcriptional activation and/or mRNA stabilization of miR-7 host genes and that miR-7 acts downstream of Usp18 to regulate EGFR mRNA translation via the 3'-UTR. Also, depletion of Usp18 led to a decrease in protein levels of other known oncogenic targets of miR-7, reduced cell proliferation and soft agar colony formation, and increased apoptosis. Notably, all of these phenotypes were reversed by a specific inhibitor of miR-7. Thus, our findings support a model in which Usp18 inhibition promotes up-regulation of miR-7, which in turn inhibits EGFR expression and the tumorigenic activity of cancer cells.
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Regiones no Traducidas 3' , Endopeptidasas/metabolismo , Receptores ErbB/biosíntesis , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , ARN Neoplásico/metabolismo , Apoptosis/genética , Proliferación Celular , Endopeptidasas/genética , Receptores ErbB/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , MicroARNs/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Estabilidad del ARN/genética , ARN Neoplásico/genética , Ubiquitina TiolesterasaRESUMEN
Endocytosis of signaling receptors, such as epidermal growth factor receptor (EGFR), tightly controls the signal transduction process triggered by ligand activation of these receptors. To identify new regulators of the endocytic trafficking of EGFR an RNA interference screen was performed for genes involved in ubiquitin conjugation and down-regulation of EGFR. The screen revealed that small interfering RNAs (siRNAs) that target the conserved ubiquitin-binding domain Uev1 increased down-regulation of EGFR. Since these siRNAs simultaneously targeted multiple genes containing a Uev1 domain, we analyzed the role of these gene products by overexpressing individual Uev1-related proteins. This analysis revealed that overexpression of Uev1A (UBE2V1) has no effect on the degradation of EGFR:EGF complexes. In contrast, overexpression of Uev1B (TMEM189-UBE2V1 isoform 2) slowed the degradation of EGF:receptor complexes. The Uev1B protein was found to strongly colocalize and associate with ubiquitin and Hrs in endosomes. Moreover, overexpression of Uev1B abrogated the ability of Hrs to colocalize with EGFR. The B-domain of Uev1B, and not the UEV-domain, was mainly responsible for the observed phenotypes suggesting the presence of a novel endosomal targeting sequence within the B-domain. Together, the data show that elevated levels of Uev1B protein in cells lead to decreased efficiency of endosomal sorting by associating with ubiquitinated proteins and Hrs.
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Endocitosis/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/metabolismo , Receptores ErbB/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Células COS , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Células Cultivadas , Chlorocebus aethiops , Regulación hacia Abajo , Receptores ErbB/genética , Células HeLa , Humanos , Isoformas de Proteínas , Transporte de Proteínas , ARN Interferente Pequeño/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) initiates signal transduction, ultimately leading to altered gene expression. Ligand-activated EGFR is also rapidly internalized and then targeted to lysosomes for degradation or recycled back to the plasma membrane. Endocytosis is a major regulator of EGFR signaling. Therefore, elucidation of the mechanisms of EGFR endocytosis is essential for a better understanding of EGFR biology. In order to achieve a comprehensive analysis of these mechanisms, reliable methods for measuring the rates of EGFR protein turnover and the rate parameters for individual steps of EGFR endocytic trafficking must be employed. The protocols in this unit describe methodologies to measure the rates of EGFR synthesis and degradation, to monitor EGF-induced down-regulation of surface EGFR, to measure the kinetic rate parameters of internalization, recycling, and degradation of radiolabeled EGF, and to perform radioiodination of EGF by the chloramine T method.
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Bioquímica/métodos , Endocitosis , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Animales , Regulación hacia Abajo , Receptores ErbB/biosíntesis , Radioisótopos de Yodo , Ratones , Células 3T3 NIH , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Coloración y Etiquetado , Sus scrofaRESUMEN
Elevated expression of epidermal growth factor receptor (EGFR) contributes to the progression of many types of cancer. Therefore, we developed a high-throughput screen to identify proteins that regulate the levels of EGFR in squamous cell carcinoma. Knocking down various ubiquitination-related genes with small interfering RNAs led to the identification of several novel genes involved in this process. One of these genes, Usp18, is a member of the ubiquitin-specific protease family. We found that knockdown of Usp18 in several cell lines reduced expression levels of EGFR by 50-80%, whereas the levels of other receptor tyrosine kinases remained unchanged. Overexpression of Usp18 elevated EGFR levels in a manner requiring the catalytic cysteine of Usp18. Analysis of metabolically radiolabeled cells showed that the rate of EGFR protein synthesis was reduced up to fourfold in the absence of Usp18. Interestingly, this dramatic reduction occurred despite no change in the levels of EGFR mRNA. This suggests that depletion of Usp18 inhibited EGFR mRNA translation. In fact, this inhibition required the presence of native 5' and 3' untranslated region sequences on EGFR mRNA. Together, our data provide evidence for the novel mechanism of EGFR regulation at the translational step of receptor synthesis.
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Endopeptidasas/metabolismo , Receptores ErbB/biosíntesis , Interferencia de ARN , Animales , Línea Celular , Chlorocebus aethiops , Regulación hacia Abajo , Endopeptidasas/genética , Receptores ErbB/genética , Eliminación de Gen , Genes Reporteros/genética , ARN Mensajero/genética , Regulación hacia ArribaRESUMEN
The signalling lipid PI(3,5)P(2) is generated on endosomes and regulates retrograde traffic to the trans-Golgi network. Physiological signals regulate rapid, transient changes in PI(3,5)P(2) levels. Mutations that lower PI(3,5)P(2) cause neurodegeneration in human patients and mice. The function of Vac14 in the regulation of PI(3,5)P(2) was uncharacterized previously. Here, we predict that yeast and mammalian Vac14 are composed entirely of HEAT repeats and demonstrate that Vac14 exerts an effect as a scaffold for the PI(3,5)P(2) regulatory complex by direct contact with the known regulators of PI(3,5)P(2): Fig4, Fab1, Vac7 and Atg18. We also report that the mouse mutant ingls (infantile gliosis) results from a missense mutation in Vac14 that prevents the association of Vac14 with Fab1, generating a partial complex. Analysis of ingls and two additional mutants provides insight into the organization of the PI(3,5)P(2) regulatory complex and indicates that Vac14 mediates three distinct mechanisms for the rapid interconversion of PI3P and PI(3,5)P(2). Moreover, these studies show that the association of Fab1 with the complex is essential for viability in the mouse.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositoles/metabolismo , Saccharomyces cerevisiae/metabolismo , Sustitución de Aminoácidos/genética , Animales , Proteínas Relacionadas con la Autofagia , Viabilidad Fetal , Flavoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Modelos Biológicos , Mutación Missense , Monoéster Fosfórico Hidrolasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Secuencias Repetitivas de Aminoácido , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Phosphoinositide lipids regulate complex events via the recruitment of proteins to a specialized region of the membrane at a specific time. Precise control of both the synthesis and turnover of phosphoinositide lipids is integral to membrane trafficking, signal transduction, and cytoskeletal rearrangements. Little is known about the acute regulation of the levels of these signaling lipids. When Saccharomyces cerevisiae cells are treated with hyperosmotic medium the levels of phosphatidylinositol 3,5-bisphosphate (PI3,5P(2)) increase 20-fold. Here we show that this 20-fold increase is rapid and occurs within 5 min. Surprisingly, these elevated levels are transient. Fifteen minutes following hyperosmotic shock they decrease at a rapid rate, even though the cells remain in hyperosmotic medium. In parallel with the rapid increase in the levels of PI3,5P(2), vacuole volume decreases rapidly. Furthermore, concomitant with a return to basal levels of PI3,5P(2) vacuole volume is restored. We show that Fig 4p, consistent with its proposed role as a PI3,5P(2) 5-phosphatase, is required in vivo for this rapid return to basal levels of PI3,5P(2). Surprisingly, we find that Fig 4p is also required for the hyperosmotic shock-induced increase in PI3,5P(2) levels. These findings demonstrate that following hyperosmotic shock, large, transient changes occur in the levels of PI3,5P(2) and further suggest that Fig 4p is important in regulating both the acute rise and subsequent fall in PI3,5P(2) levels.
Asunto(s)
Flavoproteínas/fisiología , Presión Osmótica , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Membrana Celular/química , Monoéster Fosfórico Hidrolasas , Proteínas de Saccharomyces cerevisiae/análisis , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Phosphoinositide-signaling lipids function in diverse cellular pathways. Dynamic changes in the levels of these signaling lipids regulate multiple processes. In particular, when Saccharomyces cerevisiae cells are exposed to hyperosmotic shock, PI3,5P2 (phosphatidylinositol [PI] 3,5-bisphosphate) levels transiently increase 20-fold. This causes the vacuole to undergo multiple acute changes. Control of PI3,5P2 levels occurs through regulation of both its synthesis and turnover. Synthesis is catalyzed by the PI3P 5-kinase Fab1p, and turnover is catalyzed by the PI3,5P2 5-phosphatase Fig4p. In this study, we show that two putative Fab1p activators, Vac7p and Vac14p, independently regulate Fab1p activity. Although Vac7p only regulates Fab1p, surprisingly, we find that Vac14 regulates both Fab1p and Fig4p. Moreover, Fig4p itself functions in both PI3,5P2 synthesis and turnover. In both the absence and presence of Vac7p, the Vac14p-Fig4p complex controls the hyperosmotic shock-induced increase in PI3,5P2 levels. These findings suggest that the dynamic changes in PI3,5P2 are controlled through a tight coupling of synthesis and turnover.
Asunto(s)
Flavoproteínas/fisiología , Proteínas de la Membrana/fisiología , Fosfatos de Fosfatidilinositol/biosíntesis , Proteínas de Saccharomyces cerevisiae/fisiología , Proteínas de la Membrana/genética , Mutagénesis , Presión Osmótica , Fenotipo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P(2)) was first identified as a non-abundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P(2) via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P(2) synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P(2) levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P(2) are 18-28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P(2). After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P(2) rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P(2) plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P(2) continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P(2) and provide insight into why PtdIns(3,5)P(2) levels rise in response to osmotic stress.