RESUMEN
BACKGROUND: The diagnosis of T-cell lymphomas is typically established through a multiparameter approach that combines clinical, morphologic, immunophenotypic, and genetic features, utilizing a variety of histopathologic and molecular techniques. However, accurate diagnosis of such lymphomas and distinguishing them from reactive lymph nodes remains challenging due to their low prevalence and heterogeneous features, hence limiting the confidence of pathologists. We investigated the use of microRNA (miRNA) expression signatures as an adjunctive tool in the diagnosis and classification of T-cell lymphomas that involve lymph nodes. This study seeks to distinguish reactive lymph nodes (RLN) from two types of frequently occurring nodal T-cell lymphomas: nodal T-follicular helper (TFH) cell lymphomas (nTFHL) and peripheral T-cell lymphomas, not otherwise specified (nPTCL). METHODS: From the formalin-fixed paraffin-embedded (FFPE) samples from a cohort of 88 subjects, 246 miRNAs were quantified and analyzed by differential expression. Two-class logistic regression and random forest plot models were built to distinguish RLN from the nodal T-cell lymphomas. Gene set enrichment analysis was performed on the target genes of the miRNA to identify pathways and transcription factors that may be regulated by the differentially expressed miRNAs in each subtype. RESULTS: Using logistic regression analysis, we identified miRNA signatures that can distinguish RLN from nodal T-cell lymphomas (AUC of 0.92 ± 0.05), from nTFHL (AUC of 0.94 ± 0.05) and from nPTCL (AUC of 0.94 ± 0.08). Random forest plot modelling was also capable of distinguishing between RLN and nodal T-cell lymphomas, but performed worse than logistic regression. However, the miRNA signatures are not able to discriminate between nTFHL and nPTCL, owing to large similarity in miRNA expression patterns. Bioinformatic analysis of the gene targets of unique miRNA expression revealed the enrichment of both known and potentially understudied signalling pathways and genes in such lymphomas. CONCLUSION: This study suggests that miRNA biomarkers may serve as a promising, cost-effective tool to aid the diagnosis of nodal T-cell lymphomas, which can be challenging. Bioinformatic analysis of differentially expressed miRNAs revealed both relevant or understudied signalling pathways that may contribute to the progression and development of each T-cell lymphoma entity. This may help us gain further insight into the biology of T-cell lymphomagenesis.
RESUMEN
The 5th Workshop IRE on Translational Oncology was held in Rome (Italy) on 27-28 March at the IRCCS Regina Elena National Cancer Institute. This meeting entitled "The New World of RNA diagnostics and therapeutics" highlightes the significant progress in the RNA field made over the last years. Research moved from pure discovery towards the development of diagnostic biomarkers or RNA-base targeted therapies seeking validation in several clinical trials. Non-coding RNAs in particular have been the focus of this workshop due to their unique properties that make them attractive tools for the diagnosis and therapy of cancer.This report collected the presentations of many scientists from different institutions that discussed recent oncology research providing an excellent overview and representative examples for each possible application of RNA as biomarker, for therapy or to increase the number of patients that can benefit from precision oncology treatment.In particular, the meeting specifically emphasized two key features of RNA applications: RNA diagnostic (Blandino, Palcau, Sestito, Díaz Méndez, Cappelletto, Pulito, Monteonofrio, Calin, Sozzi, Cheong) and RNA therapeutics (Dinami, Marcia, Anastasiadou, Ryan, Fattore, Regazzo, Loria, Aharonov).
Asunto(s)
Neoplasias , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisión , Biomarcadores , Oncología Médica , ItaliaRESUMEN
Noncoding RNAs (ncRNAs), in the form of structural, catalytic or regulatory RNAs, have emerged to be critical effectors of many biological processes. With the advent of new technologies, we have begun to appreciate how intracellular and circulatory ncRNAs elegantly choreograph the regulation of gene expression and protein function(s) in the cell. Armed with this knowledge, the clinical utility of ncRNAs as biomarkers has been recently tested in a wide range of human diseases. In this review, we examine how critical factors govern the success of interrogating ncRNA biomarker expression in liquid biopsies and tissues to enhance our current clinical management of human diseases, particularly in the context of cancer. We also discuss strategies to overcome key challenges that preclude ncRNAs from becoming standard-of-care clinical biomarkers, including sample pre-analytics standardization, data cross-validation with closer attention to discordant findings, as well as correlation with clinical outcomes. Although harnessing multi-modal information from disease-associated noncoding RNome (ncRNome) in biofluids or in tissues using artificial intelligence or machine learning is at the nascent stage, it will undoubtedly fuel the community adoption of precision population health.
Asunto(s)
MicroARNs , ARN Largo no Codificante , Inteligencia Artificial , Biomarcadores , Humanos , MicroARNs/genética , Medicina de Precisión , ARN/genética , ARN Largo no Codificante/genética , ARN no Traducido/genética , ARN no Traducido/metabolismoRESUMEN
Oncogenic mutations in the RAS family of small GTPases are commonly found in human cancers and they promote tumorigenesis by altering gene expression networks. We previously demonstrated that Casein Kinase 1α (CK1α), a member of the CK1 family of serine/threonine kinases, is post-transcriptionally upregulated by oncogenic RAS signaling. Here, we report that the CK1α mRNA contains an exceptionally long 5'-untranslated region (UTR) harbouring several translational control elements, implicating its involvement in translational regulation. We demonstrate that the CK1α 5'-UTR functions as an IRES element in HCT-116 colon cancer cells to promote cap-independent translation. Using tobramycin-affinity RNA-pulldown assays coupled with identification via mass spectrometry, we identified several CK1α 5'-UTR-binding proteins, including SFPQ. We show that RNA interference targeting SFPQ reduced CK1α protein abundance and partially blocked RAS-mutant colon cancer cell growth. Importantly, transcript and protein levels of SFPQ and other CK1α 5'-UTR-associated RNA-binding proteins (RBPs) are found to be elevated in early stages of RAS-mutant cancers, including colorectal and lung adenocarcinoma. Taken together, our study uncovers a previously unappreciated role of RBPs in promoting RAS-mutant cancer cell growth and their potential to serve as promising biomarkers as well as tractable therapeutic targets in cancers driven by oncogenic RAS.
RESUMEN
Precision preventive healthcare aims to improve patient health by integrating preventive measures with early disease detection for timely intervention with precision medicine. Key to the delivery of preventive healthcare is the clinical adoption of novel assays that enable early disease detection. Such assays, typically based on biomarkers such as microRNAs (miRNAs) from liquid biopsy or excreta, are entering clinical practice after years of clinical development and validation. In this review, we discuss the clinical utility and validation of miRNA-based molecular diagnostics for early disease detection through large-cohort studies and key considerations for developing multi-analyte clinical assays. We also highlight recent advances in the ongoing development of integrated PCR-free miRNA detection systems for point-of-care testing.
Asunto(s)
MicroARNs , Biomarcadores , Humanos , MicroARNs/genética , Técnicas de Amplificación de Ácido Nucleico , Medicina de PrecisiónRESUMEN
Autophagy is a fundamental cellular homeostasis mechanism known to play multifaceted roles in the natural history of cancers over time. It has recently been shown that autophagy also mediates the crosstalk between the tumor and its microenvironment by promoting the export of molecular payloads such as non-coding RNA (ncRNAs) via LC3-dependent Extracellular Vesicle loading and secretion (LDELS). In turn, the dynamic exchange of exosomal ncRNAs regulate autophagic responses in the recipient cells within the tumor microenvironment (TME), for both tumor and stromal cells. Autophagy-dependent phenotypic changes in the recipient cells further enhance tumor growth and metastasis, through diverse biological processes, including nutrient supplementation, immune evasion, angiogenesis, and therapeutic resistance. In this review, we discuss how the feedforward autophagy-ncRNA axis orchestrates vital communications between various cell types within the TME ecosystem to promote cancer progression.
RESUMEN
MicroRNAs (miRNAs) are short single-stranded RNAs that have pivotal roles in disease pathophysiology through transcriptional and translational modulation of important genes. It has been implicated in the development of many diseases, such as stroke, cardiovascular conditions, cancers and inflammatory airway diseases. There is recent evidence that miRNAs play important roles in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD), and could help to distinguish between T2-low (non-eosinophilic, steroid-insensitive) versus T2-high (eosinophilic, steroid-sensitive) disease endotypes. As these are the two most prevalent chronic respiratory diseases globally, with rising disease burden, miRNA research might lead to the development of new diagnostic and therapeutic targets. Research involving miRNAs in airway disease is challenging because: (i) asthma and COPD are heterogeneous inflammatory airway diseases; there are overlapping but distinct inter- and intra-disease differences in the immunological pathophysiology, (ii) there exists more than 2000 known miRNAs and a single miRNA can regulate multiple targets, (iii) differential effects of miRNAs could be present in different cellular subtypes and tissues, and (iv) dysregulated miRNA expression might be a direct consequence of an indirect effect of airway disease onset or progression. As miRNAs are actively secreted in fluids and remain relatively stable, they have the potential for biomarker development and therapeutic targets. In this review, we summarize the preclinical data on potential miRNA biomarkers that mediate different pathophysiological mechanisms in airway disease. We discuss the framework for biomarker development using miRNA and highlight the need for careful patient characterization and endotyping in the screening and validation cohorts, profiling both airway and blood samples to determine the biological fluids of choice in different disease states or severity, and adopting an untargeted approach. Collaboration between the various stakeholders - pharmaceutical companies, laboratory professionals and clinician-scientists is crucial to reduce the difficulties and cost required to bring miRNA research into the translational stage for airway diseases.
Asunto(s)
MicroARNs/fisiología , Biosíntesis de Proteínas , Enfermedades Respiratorias/genética , Enfermedades Respiratorias/fisiopatología , Remodelación de las Vías Aéreas (Respiratorias)/genética , Animales , Asma/genética , Asma/fisiopatología , Marcadores Genéticos , Humanos , Inflamación/genética , Inflamación/fisiopatología , MicroARNs/análisis , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfermedades Respiratorias/diagnósticoRESUMEN
Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.
Asunto(s)
Diferenciación Celular , MicroARNs/metabolismo , ARN de Transferencia/metabolismo , Animales , Células Cultivadas , Células HCT116 , Humanos , Ratones , MicroARNs/genética , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Estabilidad del ARN , ARN de Transferencia/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Aberrant Wnt signaling has been widely accepted to be a key driver of a subset of human cancers and a heavily scrutinized molecular pathway for the development of personalized medicine. In a recently published issue of Science Translational Medicine, Rosenberg and coworkers reported that the delta isoform of the CK1 family of serine/threonine kinases (CK1δ), an important mediator of intracellular Wnt signaling, is amplified and overexpressed in human breast tumors. They further demonstrated that pharmacological inhibition of CK1δ is efficacious for these cancers and implicate ß-catenin signaling as a key target of CK1δ. In this perspective, we will discuss the salient features of this novel anti-cancer therapeutic approach and the challenges that lie ahead to translate it into a viable treatment option for cancer patients.
RESUMEN
Mutant RAS-driven cancer cells cope with proliferative stress by increasing basal autophagy to maintain protein/organelle and energy homeostasis. We recently demonstrated that casein kinase 1 alpha (CK1α), a therapeutically tractable enzyme, is critical for fine-tuning the transcriptional regulation of mutant RAS-induced autophagy and the development of mutant RAS-driven cancers.
RESUMEN
The RAS genes encode for members of a large superfamily of guanosine-5'-triphosphate (GTP)-binding proteins that control diverse intracellular signaling pathways to promote cell proliferation. Somatic mutations in the RAS oncogenes are the most common activating lesions found in human cancers. These mutations invariably result in the gain-of-function of RAS by impairing GTP hydrolysis and are frequently associated with poor responses to standard cancer therapies. In this review, we summarize key findings of past and present landmark studies that have deepened our understanding of the RAS biology in the context of oncogenesis. We also discuss how emerging areas of research could further bolster a renewed global effort to target the largely undruggable oncogenic RAS and/or its activated downstream effector signaling cascades to achieve better treatment outcomes for RAS-mutant cancer patients.
Asunto(s)
Neoplasias/patología , Proteínas ras/metabolismo , Animales , Autofagia , Transformación Celular Neoplásica , Guanosina Trifosfato/metabolismo , Humanos , Mutación , Neoplasias/metabolismo , Transducción de Señal , Proteínas ras/genéticaRESUMEN
The use of BCR-ABL1 tyrosine kinase inhibitors (TKI) has led to excellent clinical responses in patients with chronic phase chronic myeloid leukemia (CML). However these inhibitors have been less effective as single agents in the terminal blast phase (BP). We show that pyrvinium, a FDA-approved anthelminthic drug, selectively targets BP-CML CD34+ progenitor cells. Pyrvinium is effective in inducing apoptosis, inhibiting colony formation and self-renewal capacity of CD34+ cells from TKI-resistant BP-CML patients, while cord blood CD34+ are largely unaffected. The effects of pyrvinium are further enhanced upon combination with dasatinib, a second generation BCR-ABL1 TKI. In a CML xenograft model pyrvinium significantly inhibits tumor growth as a single agent, with complete inhibition in combination with dasatinib. While pyrvinium has been shown to inhibit the Wnt/ß-catenin signalling pathway via activation of casein kinase 1α , we find its activity in CML is not dependent on this pathway. Instead, we show that pyrvinium localizes to mitochondria and induces apoptosis by inhibiting mitochondrial respiration. Our study suggests that pyrvinium is a useful addition to the treatment armamentarium for BP-CML and that targeting mitochondrial respiration may be a potential therapeutic strategy in aggressive leukemia.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Mitocondrias/metabolismo , Compuestos de Pirvinio/administración & dosificación , Adenosina Trifosfato/química , Animales , Antígenos CD34/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis , Crisis Blástica/metabolismo , Quinasa de la Caseína I/metabolismo , Línea Celular Tumoral , Proliferación Celular , Dasatinib/administración & dosificación , Dasatinib/uso terapéutico , Humanos , Concentración 50 Inhibidora , Células K562 , Ratones , Ratones SCID , Trasplante de Neoplasias , Fosforilación , Compuestos de Pirvinio/uso terapéutico , Interferencia de ARN , beta Catenina/metabolismoRESUMEN
Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS-induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS-driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS-driven cancers.
Asunto(s)
Autofagia/fisiología , Quinasa de la Caseína I/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias/enzimología , Proteína Oncogénica p21(ras)/fisiología , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína I/antagonistas & inhibidores , Retroalimentación Fisiológica , Fibroblastos/enzimología , Fibroblastos/fisiología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/fisiología , ARN Interferente Pequeño/farmacología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina-Treonina Quinasas TOR/fisiología , Activación TranscripcionalRESUMEN
Obesity develops as a result of altered energy homeostasis favoring fat storage. Here we describe a new transcription co-regulator for adiposity and energy metabolism, SERTA domain containing 2 (TRIP-Br2, also called SERTAD2). TRIP-Br2-null mice are resistant to obesity and obesity-related insulin resistance. Adipocytes of these knockout mice showed greater stimulated lipolysis secondary to enhanced expression of hormone sensitive lipase (HSL) and ß3-adrenergic (Adrb3) receptors. The knockout mice also have higher energy expenditure because of increased adipocyte thermogenesis and oxidative metabolism caused by upregulating key enzymes in their respective processes. Our data show that a cell-cycle transcriptional co-regulator, TRIP-Br2, modulates fat storage through simultaneous regulation of lipolysis, thermogenesis and oxidative metabolism. These data, together with the observation that TRIP-Br2 expression is selectively elevated in visceral fat in obese humans, suggests that this transcriptional co-regulator is a new therapeutic target for counteracting the development of obesity, insulin resistance and hyperlipidemia.
Asunto(s)
Resistencia a la Insulina , Lipólisis , Obesidad/prevención & control , Termogénesis , Factores de Transcripción/fisiología , Células 3T3-L1 , Adipocitos/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Grasas de la Dieta/administración & dosificación , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Oxidación-Reducción , Receptores Adrenérgicos beta 3/fisiología , Esterol Esterasa/fisiologíaRESUMEN
Genome-wide association studies have identified genetic markers in kallikrein-related peptidase 3 (KLK3) associated with prostate cancer. However, some of these markers are also associated with prostate-specific antigen (PSA) levels, so it is unclear whether the polymorphisms are causal or if the association with risk is solely due to detection bias through PSA screening. PSA is a biologically active serine protease, cleaving insulin-like growth factor-binding protein. We examined the association of single-nucleotide polymorphisms (SNPs) in KLK3 with prostate cancer risk, disease-specific survival and pre-diagnostic PSA levels in a case-control study nested within the Physicians' Health Study, which began in 1982, with over 27 years of follow-up. We genotyped SNPs spanning the entire KLK3 locus to capture common variation at high resolution. Six polymorphisms were significantly associated with prostate cancer incidence (P < 0.05); the odds ratios per minor allele ranged from 0.88 to 0.73. For four of these, the odds ratios were lower when restricting to cases diagnosed in the pre-PSA screening era (before 1989). The four alleles significantly associated with lower PSA levels were also associated with lower prostate cancer risk. KLK3 variants were not significantly associated with stage at diagnosis, risk of lethal cancer or survival. Our results suggest that detection bias due to the association of KLK3 variants with PSA levels cannot completely explain the association with prostate cancer risk. Understanding the mechanism by which genetic variation in KLK3 affects prostate cancer risk has important implications for study of the biological role of PSA in prostate tumorigenesis.
Asunto(s)
Predisposición Genética a la Enfermedad , Calicreínas/genética , Polimorfismo de Nucleótido Simple/genética , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata/genética , Anciano , Estudios de Casos y Controles , Método Doble Ciego , Marcadores Genéticos , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Próstata/metabolismo , Factores de RiesgoRESUMEN
The CK1 family of serine/threonine kinases regulates diverse cellular processes, through binding to and phosphorylation a myriad of protein substrates. CK1 prefers substrates primed by prior phosphorylation, and works closely with other kinases in the Wnt pathway. CK1 is itself regulated by posttranslational modification, including autophosphorylation. We provide a brief overview of the fundamentals of CK1 biology with an emphasis on scaffold binding and kinase regulation in Wnt signaling and circadian rhythms.
Asunto(s)
Quinasa de la Caseína I/química , Quinasa de la Caseína I/metabolismo , Animales , Ritmo Circadiano , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Humanos , Neoplasias/enzimologíaRESUMEN
BACKGROUND: Members of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2). METHODS: Oncogenic potential of TRIP-Br2 was demonstrated by (1) inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target. RESULTS: Overexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro. CONCLUSION: This study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Transactivadores/genética , Factores de Transcripción/genética , Animales , Humanos , Ratones , Ratones DesnudosRESUMEN
TRIP-Br proteins are a novel family of transcriptional coregulators involved in E2F-mediated cell cycle progression. Three of the four mammalian members of TRIP-Br family, including TRIP-Br1, are known oncogenes. We now report the identification of the Balpha regulatory subunit of serine/threonine protein phosphatase 2A (PP2A) as a novel TRIP-Br1 interactor, based on an affinity binding assay coupled with mass spectrometry. A GST-TRIP-Br1 fusion protein associates with catalytically active PP2A-ABalphaC holoenzyme in vitro. Coimmunoprecipitation confirms this association in vivo. Immunofluorescence staining with a monoclonal antibody against TRIP-Br1 reveals that endogenous TRIP-Br1 and PP2A-Balpha colocalize mainly in the cytoplasm. Consistently, immunoprecipitation followed by immunodetection with anti-phosphoserine antibody suggest that TRIP-Br1 exists in a serine-phosphorylated form. Inhibition of PP2A activity by okadaic acid or transcriptional silencing of the PP2A catalytic subunit by small interfering RNA results in downregulation of total TRIP-Br1 protein levels but upregulation of serine-phosphorylated TRIP-Br1. Overexpression of PP2A catalytic subunit increases TRIP-Br1 protein levels and TRIP-Br1 co-activated E2F1/DP1 transcription. Our data support a model in which association between PP2A-ABalphaC holoenzyme and TRIP-Br1 in vivo in mammalian cells represents a novel mechanism for regulating the level of TRIP-Br1 protooncoprotein.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteína Fosfatasa 2/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Genes Reporteros , Humanos , Inmunoprecipitación , Mediciones Luminiscentes , Ratones , Ácido Ocadaico/farmacología , Fosforilación , Reacción en Cadena de la Polimerasa , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Carioferinas/fisiología , Complejo de la Endopetidasa Proteasomal/química , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/química , Transporte Activo de Núcleo Celular , Animales , Células COS , Ciclo Celular , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Carioferinas/química , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Proto-Oncogenes Mas , Receptores Citoplasmáticos y Nucleares/química , Activación Transcripcional , Ubiquitina/química , Proteína Exportina 1RESUMEN
TRIP-Br1 and TRIP-Br2 are potent cell growth promoting factors that function as components of the E2F1/DP1 transcription complex to integrate positive growth signals provided by PHD zinc finger- and/or bromodomain-containing transcription factors. TRIP-Br1 has been demonstrated to be an oncogene. We recently reported that antagonism of the TRIP-Br integrator function by synthetic decoy peptides that compete with TRIP-Br for binding to PHD zinc finger- and/or bromodomain-containing proteins elicit an anti-proliferative effect and induces caspase-3-independent sub-diploidization in cancer cells in vitro. We now demonstrate the chemotherapeutic potential of TRIP-Br decoy peptides for the treatment of cutaneous and intracavitary lesions in vitro as well as in vivo in representative human nasopharyngeal cancer (CNE2), cervical cancer (Ca Ski) and melanoma (MeWo) cancer cell lines. In vitro, BrdU incorporation, colony formation assays and cell cycle analysis confirmed that TRIP-Br decoy peptides possess strong anti-proliferative effects and induce nuclear sub-diploidization in cancer cells. In vivo, CNE2, Ca Ski and MeWo-derived chick embryo chorioallantoic membrane (CAM) tumor xenografts were used to evaluate the effect of topically applied TRIP-Br peptides. Confocal microscopy and flow cytometric analysis demonstrated that cells comprising the tumor xenografts efficiently internalized topically applied FITC-labeled peptides. Fifty muM of TRIP-Br1 decoy peptide significantly suppressed the growth of NPC2-derived human nasopharyngeal tumors, while 50 muM of TRIP-Br2 decoy peptide significantly inhibited tumor growth in all three CAM tumor xenograft models. Two hundred muM of TRIP-Br1 decoy peptide significantly inhibited MeWo-derived tumors. These results suggest that the TRIP-Br integrator function may represent a novel chemotherapeutic target for the treatment of human cutaneous and intracavitary proliferative lesions.