RESUMEN
OBJECTIVE: Gastric cancer patients generally have a poor outcome, particularly those with advanced-stage disease which is defined by the increased invasion of cancer locally and is associated with higher metastatic potential. This study aimed to identify genes that were functional in the most fundamental hallmark of cancer, namely invasion. We then wanted to assess their value as biomarkers of gastric cancer progression and recurrence. DESIGN: Data from a cohort of patients profiled on cDNA expression arrays was interrogated using K-means analysis. This genomic approach classified the data based on patterns of gene expression allowing the identification of the genes most correlated with the invasion of GC. We evaluated the functional role of a key protein from this analysis in invasion and as a biomarker of recurrence after curative resection. RESULTS: Expression of secreted frizzled-related protein 4 (SFRP4) was identified as directly proportional to gastric cancer invasion. This finding was validated in multiple, independent datasets and its functional role in invasion was also confirmed using invasion assays. A change in serum levels of SFRP4 after curative resection, when coupled with AJCC stage, can accurately predict the risk of disease recurrence after curative therapy in an assay we termed PredictR. CONCLUSIONS: This simple ELISA-based assay can help predict recurrence of disease after curative gastric cancer surgery irrespective of adjuvant therapy. The results require further evaluation in a prospective trial but would help in the rational prescription of cancer therapies and surveillance to prevent under or over treatment of patients after curative resection.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Recurrencia Local de Neoplasia/cirugía , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias Gástricas/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Femenino , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Valor Predictivo de las Pruebas , Neoplasias Gástricas/patologíaRESUMEN
We investigated the molecular basis for the remarkably different survival outcomes of mice expressing different alloforms of the pro-apoptotic serine protease granzyme B to mouse cytomegalovirus infection. Whereas C57BL/6 mice homozygous for granzyme BP (GzmBP/P) raise cytotoxic T lymphocytes that efficiently kill infected cells, those of C57BL/6 mice congenic for the outbred allele (GzmBW/W) fail to kill MCMV-infected cells and died from uncontrolled hepatocyte infection and acute liver failure. We identified subtle differences in how GzmBP and GzmBW activate cell death signalling - both alloforms predominantly activated pro-caspases directly, and cleaved pro-apoptotic Bid poorly. Consequently, neither alloform initiated mitochondrial outer membrane permeabilization, or was blocked by Bcl-2, Bcl-XL or co-expression of MCMV proteins M38.5/M41.1, which together stabilize mitochondria by sequestering Bak/Bax. Remarkably, mass spectrometric analysis of proteins from MCMV-infected primary mouse embryonic fibroblasts identified 13 cleavage sites in nine viral proteins (M18, M25, M28, M45, M80, M98, M102, M155, M164) that were cleaved >20-fold more efficiently by either GzmBP or GzmBW. Notably, M18, M28, M45, M80, M98, M102 and M164 were cleaved 20- >100-fold more efficiently by GzmBW, and so, would persist in infected cells targeted by CTLs from GzmBP/P mice. Conversely, M155 was cleaved >100-fold more efficiently by GzmBP, and would persist in cells targeted by CTLs of GzmBW/W mice. M25 was cleaved efficiently by both proteases, but at different sites. We conclude that different susceptibility to MCMV does not result from skewed endogenous cell death pathways, but rather, to as yet uncharacterised MCMV-intrinsic pathways that ultimately inhibit granzyme B-induced cell death.
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Granzimas/química , Granzimas/metabolismo , Muromegalovirus/inmunología , Péptidos/metabolismo , Animales , Apoptosis , Caspasas/metabolismo , Muerte Celular , Línea Celular , Modelos Animales de Enfermedad , Femenino , Granzimas/genética , Infecciones por Herpesviridae/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Péptidos/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especificidad por Sustrato , Linfocitos T Citotóxicos/inmunología , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Proteína bcl-X/metabolismoRESUMEN
The Hippo pathway is an evolutionarily conserved signaling network that regulates organ size, cell fate, and tumorigenesis. In the context of organ size control, the pathway incorporates a large variety of cellular cues, such as cell polarity and adhesion, into an integrated transcriptional response. The central Hippo signaling effector is the transcriptional coactivator Yorkie, which controls gene expression in partnership with different transcription factors, most notably Scalloped. When it is not activated by Yorkie, Scalloped can act as a repressor of transcription, at least in part due to its interaction with the corepressor protein Tgi. The mechanism by which Tgi represses transcription is incompletely understood, and therefore we sought to identify proteins that potentially operate together with Tgi. Using an affinity purification and mass-spectrometry approach we identified Pits and CtBP as Tgi-interacting proteins, both of which have been linked to transcriptional repression. Both Pits and CtBP were required for Tgi to suppress the growth of the Drosophila melanogaster eye and CtBP loss suppressed the undergrowth of yorkie mutant eye tissue. Furthermore, as reported previously for Tgi, overexpression of Pits repressed transcription of Hippo pathway target genes. These findings suggest that Tgi might operate together with Pits and CtBP to repress transcription of genes that normally promote tissue growth. The human orthologs of Tgi, CtBP, and Pits (VGLL4, CTBP2, and IRF2BP2) have previously been shown to physically and functionally interact to control transcription, implying that the mechanism by which these proteins control transcriptional repression is conserved throughout evolution.
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Oxidorreductasas de Alcohol/metabolismo , Proteínas Portadoras/metabolismo , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/metabolismo , Oxidorreductasas de Alcohol/genética , Animales , Proteínas Portadoras/genética , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Nucleares/genética , Unión Proteica , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Perforin is a highly cytotoxic pore-forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca2+-rich endoplasmic reticulum, remains unknown. Here, we show that N-linked glycosylation of the perforin C-terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C-terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C-terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post-translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule.
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Sinapsis Inmunológicas , Perforina/química , Perforina/metabolismo , Animales , Gránulos Citoplasmáticos/metabolismo , Retículo Endoplásmico/metabolismo , Glicosilación , Humanos , Interleucina-2/inmunología , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana , Ratones , Perforina/genética , Procesamiento Proteico-Postraduccional , ProteolisisRESUMEN
TP53, a critical tumour suppressor gene, is mutated in over half of all cancers resulting in mutant-p53 protein accumulation and poor patient survival. Therapeutic strategies to target mutant-p53 cancers are urgently needed. We show that accumulated mutant-p53 protein suppresses the expression of SLC7A11, a component of the cystine/glutamate antiporter, system xC-, through binding to the master antioxidant transcription factor NRF2. This diminishes glutathione synthesis, rendering mutant-p53 tumours susceptible to oxidative damage. System xC- inhibitors specifically exploit this vulnerability to preferentially kill cancer cells with stabilized mutant-p53 protein. Moreover, we demonstrate that SLC7A11 expression is a novel and robust predictive biomarker for APR-246, a first-in-class mutant-p53 reactivator that also binds and depletes glutathione in tumours, triggering lipid peroxidative cell death. Importantly, system xC- antagonism strongly synergizes with APR-246 to induce apoptosis in mutant-p53 tumours. We propose a new paradigm for targeting cancers that accumulate mutant-p53 protein by inhibiting the SLC7A11-glutathione axis.
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Sistema de Transporte de Aminoácidos y+/metabolismo , Glutatión/metabolismo , Mutación/genética , Proteína p53 Supresora de Tumor/genética , Sistema de Transporte de Aminoácidos y+/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos y+/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Peroxidación de Lípido/efectos de los fármacos , Modelos Biológicos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Quinuclidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
Perforin is a key protein of the vertebrate immune system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10-20â nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptotic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane-bound prepore intermediate state, typically consisting of up to approximately eight loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subsequently recruit additional prepore oligomers to grow the pore size.
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Membrana Eritrocítica , Nanoporos/ultraestructura , Proteínas Citotóxicas Formadoras de Poros , Animales , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestructura , Ratones , Microscopía de Fuerza Atómica , Microscopía Electrónica , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , OvinosRESUMEN
Histone deacetylase (HDAC) inhibitors (HDACis) have demonstrated activity in hematological and solid malignancies. Vorinostat, romidepsin, belinostat, and panobinostat are Food and Drug Administration-approved for hematological malignancies and inhibit class II and/or class I HDACs, including HDAC1, 2, 3, and 6. We combined genetic and pharmacological approaches to investigate whether suppression of individual or multiple Hdacs phenocopied broad-acting HDACis in 3 genetically distinct leukemias and lymphomas. Individual Hdacs were depleted in murine acute myeloid leukemias (MLL-AF9;Nras(G12D); PML-RARα acute promyelocytic leukemia [APL] cells) and Eµ-Myc lymphoma in vitro and in vivo. Strikingly, Hdac3-depleted cells were selected against in competitive assays for all 3 tumor types. Decreased proliferation following Hdac3 knockdown was not prevented by BCL-2 overexpression, caspase inhibition, or knockout of Cdkn1a in Eµ-Myc lymphoma, and depletion of Hdac3 in vivo significantly reduced tumor burden. Interestingly, APL cells depleted of Hdac3 demonstrated a more differentiated phenotype. Consistent with these genetic studies, the HDAC3 inhibitor RGFP966 reduced proliferation of Eµ-Myc lymphoma and induced differentiation in APL. Genetic codepletion of Hdac1 with Hdac2 was pro-apoptotic in Eµ-Myc lymphoma in vitro and in vivo and was phenocopied by the HDAC1/2-specific agent RGFP233. This study demonstrates the importance of HDAC3 for the proliferation of leukemia and lymphoma cells, suggesting that HDAC3-selective inhibitors could prove useful for the treatment of hematological malignancies. Moreover, our results demonstrate that codepletion of Hdac1 with Hdac2 mediates a robust pro-apoptotic response. Our integrated genetic and pharmacological approach provides important insights into the individual or combinations of HDACs that could be prioritized for targeting in a range of hematological malignancies.
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Histona Desacetilasas/metabolismo , Leucemia Promielocítica Aguda/enzimología , Leucemia Promielocítica Aguda/genética , Linfoma/enzimología , Linfoma/genética , Animales , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/patología , Linfoma/tratamiento farmacológico , Linfoma/patología , Ratones , Células 3T3 NIH , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Elucidating the mechanisms that underlie metastasis is of paramount importance to understanding tumor progression and to the development of novel therapeutics. Epithelial to Mesenchymal Transition (EMT) plays a vital role in tumor cell dissemination and is regulated by a core cassette of transcription factors. Despite recent advances, the molecular pathways that regulate the EMT program have not yet been fully delineated. We show that Siah ubiquitin ligases regulate Zeb1 protein, a key EMT transcription factor. The induction of EMT in breast cancer cells leads to the down-regulation of Siah, while the loss of Siah induces a mesenchymal phenotype, concurrent with an up-regulation of Zeb1. Overexpression of Siah in vitro mediates Zeb1 degradation, which can be blocked with a Siah peptide inhibitor. Thus, this work demonstrates that Siah is a novel regulator of EMT. This work is the first to identify a mechanism of post-translational regulation of the key Epithelial to Mesenchymal Transition transcription factor Zeb1.
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Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Células MCF-7 , Ratones , Microscopía Fluorescente , Modelos Genéticos , Proteínas Nucleares/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador alfa/farmacología , Factor de Crecimiento Transformador beta/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings. We detected Foxp3 protein in macrophages infiltrating mouse renal cancer tumors injected subcutaneously or in the kidney. Expression was demonstrated using flow cytometry and Western blot with two individual monoclonal antibodies. Further analyses confirmed Foxp3 expression in macrophages by RT PCR, and studies using ribonucleic acid-sequencing (RNAseq) demonstrated a previously unknown Foxp3 messenger (m)RNA transcript in tumor-associated macrophages. In addition, depletion of Foxp3+ cells using diphtheria toxin in Foxp3DTR mice reduced the frequency of type-2 macrophages (M2) in kidney tumors. Collectively, these results indicate that tumor-associated macrophages could express Foxp3.
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Factores de Transcripción Forkhead/biosíntesis , Neoplasias Renales/inmunología , Macrófagos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Factores de Transcripción Forkhead/genética , Ratones , Ratones Endogámicos BALB C , Ratones SCID , ARN Mensajero/biosíntesisRESUMEN
Tumor hypoxia is associated with resistance to antiangiogenic therapy and poor prognosis. The Siah E3 ubiquitin ligases regulate the hypoxic response pathway by modulating the turnover of the master proangiogenic transcription factor hypoxia-inducible factor-1α (Hif-1α). In this study, we show that genetic deficiency in the Siah family member Siah2 results in vascular normalization and delayed tumor growth in an established transgenic model of aggressive breast cancer. Tumors arising in a Siah2(-/-) genetic background showed increased perfusion and pericyte-associated vasculature, similar to that occurring with antiangiogenic therapy. In support of the role of Siah2 in regulating levels of Hif-1α, expression of angiogenic factors was decreased in Siah2(-/-) tumors. Blood vessel normalization in Siah2(-/-) tumors resulted in an increased response to chemotherapy and prolonged survival. Together, our findings offer a preclinical proof of concept that targeting Siah2 is sufficient to attenuate Hif-1α-mediated angiogenesis and hypoxia signaling, thereby improving responses to chemotherapy.
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Neoplasias Mamarias Experimentales/irrigación sanguínea , Neovascularización Patológica/prevención & control , Ubiquitina-Proteína Ligasas/fisiología , Animales , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is â¼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.
RESUMEN
Molecular subtypes of serous ovarian cancer have been recently described. Using data from independent datasets including over 900 primary tumour samples, we show that deregulation of the Let-7 pathway is specifically associated with the C5 molecular subtype of serous ovarian cancer. DNA copy number and gene expression of HMGA2, alleles of Let-7, LIN28, LIN28B, MYC, MYCN, DICER1, and RNASEN were measured using microarray and quantitative reverse transcriptase PCR. Immunohistochemistry was performed on 127 samples using tissue microarrays and anti-HMGA2 antibodies. Fluorescence in situ hybridisation of bacterial artificial chromosomes hybridized to 239 ovarian tumours was used to measure translocation at the LIN28B locus. Short interfering RNA knockdown in ovarian cell lines was used to test the functionality of associations observed. Four molecular subtypes (C1, C2, C4, C5) of high-grade serous ovarian cancers were robustly represented in each dataset and showed similar pattern of patient survival. We found highly specific activation of a pathway involving MYCN, LIN28B, Let-7 and HMGA2 in the C5 molecular subtype defined by MYCN amplification and over-expression, over-expression of MYCN targets including the Let-7 repressor LIN28B, loss of Let-7 expression and HMGA2 amplification and over-expression. DICER1, a known Let-7 target, and RNASEN were over-expressed in C5 tumours. We saw no evidence of translocation at the LIN28B locus in C5 tumours. The reported interaction between LIN28B and Let-7 was recapitulated by siRNA knockdown in ovarian cancer cell lines. Our results associate deregulation of MYCN and downstream targets, including Let-7 and oncofetal genes, with serous ovarian cancer. We define for the first time how elements of an oncogenic pathway, involving multiple genes that contribute to stem cell renewal, is specifically altered in a molecular subtype of serous ovarian cancer. By defining the drivers of a molecular subtype of serous ovarian cancers we provide a novel strategy for targeted therapeutic intervention.
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Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Neoplasias Ováricas/genética , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteína Proto-Oncogénica N-Myc , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70(S6K1) isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85(S6K1) isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85(S6K1) substrates. Four novel putative p85(S6K1) substrates, GRP75, CCTß, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTß was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.
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Proteínas/metabolismo , Proteómica , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Anexina A4/metabolismo , Procesos de Crecimiento Celular , Línea Celular , Chaperonina con TCP-1/química , Chaperonina con TCP-1/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos , Neuropéptidos/metabolismo , Fosfoglicerato Quinasa/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/fisiología , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Cinasa C Activada , Proteínas Quinasas S6 Ribosómicas 70-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Especificidad por Sustrato , Serina-Treonina Quinasas TORRESUMEN
PURPOSE: Ovarian clear cell adenocarcinoma (OCCA) is an uncommon histotype that is generally refractory to platinum-based chemotherapy. We analyze here the most comprehensive gene expression and copy number data sets, to date, to identify potential therapeutic targets of OCCA. EXPERIMENTAL DESIGN: Gene expression and DNA copy number were carried out using primary human OCCA tumor samples, and findings were confirmed by immunohistochemistry on tissue microarrays. Circulating interleukin (IL) 6 levels were measured in serum from patients with OCCA or high-grade serous cancers and related to progression-free and overall survival. Two patients were treated with sunitinib, and their therapeutic responses were measured clinically and by positron emission tomography. RESULTS: We find specific overexpression of the IL6-STAT3-HIF (interleukin 6-signal transducer and activator of transcription 3-hypoxia induced factor) pathway in OCCA tumors compared with high-grade serous cancers. Expression of PTHLH and high levels of circulating IL6 in OCCA patients may explain the frequent occurrence of hypercalcemia of malignancy and thromboembolic events in OCCA. We describe amplification of several receptor tyrosine kinases, most notably MET, suggesting other potential therapeutic targets. We report sustained clinical and functional imaging responses in two OCCA patients with chemotherapy-resistant disease who were treated with sunitinib, thus showing significant parallels with renal clear cell cancer. CONCLUSIONS: Our findings highlight important therapeutic targets in OCCA, suggest that more extensive clinical trials with sunitinib in OCCA are warranted, and provide significant impetus to the growing realization that OCCA is molecularly and clinically distinct to other forms of ovarian cancer.
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Adenocarcinoma de Células Claras/tratamiento farmacológico , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Indoles/uso terapéutico , Interleucina-6/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Pirroles/uso terapéutico , Factor de Transcripción STAT3/metabolismo , Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/metabolismo , Adulto , Anciano , Inhibidores de la Angiogénesis/uso terapéutico , Línea Celular Tumoral , Análisis por Conglomerados , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inmunohistoquímica , Interleucina-6/sangre , Interleucina-6/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Sunitinib , Análisis de Matrices Tisulares , Resultado del TratamientoRESUMEN
The Siah (seven in absentia homolog) family of RING-domain proteins are components of ubiquitin ligase complexes, targeting proteins for proteasomal degradation. Siah family members have been reported to function in Ras, estrogen, DNA-damage, and hypoxia response pathways. Although earlier reports implicated Siah proteins as tumor suppressors, recent studies in mouse models have shown that Siah inhibition impairs tumor growth and metastasis. Given their central role in oncogenic and angiogenic pathways, Siah proteins are attractive novel therapeutic targets in cancer.
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Antineoplásicos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas ras/metabolismo , Animales , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Sistemas de Liberación de Medicamentos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
Perforin (PRF), a pore-forming protein expressed in cytotoxic lymphocytes, plays a key role in immune surveillance and immune homeostasis. The A91V substitution has a prevalence of 8% to 9% in population studies. While this variant has been suspected of predisposing to various disorders of immune homeostasis, its effect on perforin's function has not been elucidated. Here we complemented, for the first time, the cytotoxic function of perforin-deficient primary cytotoxic T lymphocytes (CTLs) with wild-type (hPRF-WT) and A91V mutant (hPRF-A91V) perforin. The cytotoxicity of hPRF-A91V-expressing cells was about half that of hPRF-WT-expressing counterparts and coincided with a moderate reduction in hPRF-A91V expression. By contrast, the reduction in cytotoxic function was far more pronounced (more than 10-fold) when purified proteins were tested directly on target cells. The A91V substitution can therefore be manifested by abnormalities at both the lymphocyte (presynaptic) and target cell (postsynaptic) levels. However, the severe intrinsic defect in activity can be partly rescued by expression in the physiological setting of an intact CTL. These findings provide the first direct evidence that hPRF-A91V is functionally abnormal and provides a rationale for why it may be responsible for disordered immune homeostasis if inherited with another dysfunctional perforin allele.
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Citotoxicidad Inmunológica/inmunología , Polimorfismo Genético/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/fisiología , Transmisión Sináptica , Linfocitos T Citotóxicos/patología , Animales , Western Blotting , Cromatografía en Gel , Eritrocitos/inmunología , Eritrocitos/metabolismo , Eritrocitos/patología , Humanos , Ratones , Ratones Noqueados , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Ovinos , Linfocitos T Citotóxicos/inmunología , Timoma/inmunología , Timoma/metabolismo , Timoma/patologíaRESUMEN
The Siah family of RING proteins function as ubiquitin ligase components, contributing to the degradation of multiple targets involved in cell growth, differentiation, angiogenesis, oncogenesis, and inflammation. Previously, a binding motif (degron) was recognized in many of the Siah degradation targets, suggesting that Siah itself may facilitate substrate recognition. We report the crystal structure of the Siah in complex with a peptide containing the degron motif. Binding is within a groove formed in part by the zinc fingers and the first two beta strands of the TRAF-C domain of Siah. We show that residues in the degron, previously described to facilitate binding to Siah, interact with the protein. Mutagenesis of Siah at sites of interaction also abrogates both in vitro peptide binding and destabilization of a known Siah target.
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Proteínas Nucleares/química , Proteínas/química , Ubiquitina-Proteína Ligasas/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Diferenciación Celular , Línea Celular , Clonación Molecular , Cristalografía por Rayos X , Factores de Transcripción de la Respuesta de Crecimiento Precoz/química , Glutatión Transferasa/metabolismo , Humanos , Inflamación , Factores de Transcripción de Tipo Kruppel/química , Ratones , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación , Neovascularización Patológica , Proteínas Nucleares/metabolismo , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/metabolismo , Factores de Tiempo , Transfección , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The Drosophila SINA (seven in absentia) protein and its mammalian orthologs (Siah, seven in absentia homolog) are RING domain proteins that function in E3 ubiquitin ligase complexes and facilitate ubiquitination and degradation of a wide range of cellular proteins, including beta-catenin. Despite these diverse targets, the means by which SINASiah recognize substrates or binding proteins has remained unknown. Here we identify a peptide motif (RPVAxVxPxxR) that mediates the interaction of Siah protein with a range of protein partners. Sequence alignment and mutagenesis scanning revealed residues that are important to this interaction. This consensus sequence correctly predicted a high-affinity interaction with a peptide from the cytoskeletal protein plectin-1 (residues 95-117). The unusually high-affinity binding obtained with a 23-residue peptide (K(Dapp) = 29 nM with SINA) suggests that it may serve as a useful dominant negative reagent for SINASiah proteins.
Asunto(s)
Proteínas de Unión al Calcio , Ligasas/química , Ligasas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Secuencia Conservada , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Choque Térmico/metabolismo , Técnicas In Vitro , Ligasas/genética , Ratones , Datos de Secuencia Molecular , Mutagénesis , Proteínas Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Ubiquitina-Proteína LigasasRESUMEN
The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.
Asunto(s)
Ligasas/química , Meiosis , Proteínas Nucleares/metabolismo , Proteínas , Animales , Western Blotting , Femenino , Células Germinativas/metabolismo , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/mortalidad , Trastornos del Crecimiento/patología , Hibridación in Situ , Infertilidad/genética , Ligasas/deficiencia , Ligasas/genética , Masculino , Metafase , Ratones , Mutación/genética , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Profase , ARN Mensajero/metabolismo , Túbulos Seminíferos/patología , Espermatogénesis/genética , Ubiquitina-Proteína LigasasRESUMEN
Members of the Siah (seven in absentia homolog) family of RING domain proteins are components of E3 ubiquitin ligase complexes that catalyze ubiquitination of proteins. We have determined the crystal structure of the substrate-binding domain (SBD) of murine Siah1a to 2.6 A resolution. The structure reveals that Siah is a dimeric protein and that the SBD adopts an eight-stranded beta-sandwich fold that is highly similar to the TRAF-C region of TRAF (TNF-receptor associated factor) proteins. The TRAF-C region interacts with TNF-alpha receptors and TNF-receptor associated death-domain (TRADD) proteins; however, our findings indicate that these interactions are unlikely to be mimicked by Siah. The Siah structure also reveals two novel zinc fingers in a region with sequence similarity to TRAF. We find that the Siah1a SBD potentiates TNF-alpha-mediated NF-kappa B activation. Therefore, Siah proteins share important similarities with the TRAF family of proteins, including their overall domain architecture, three-dimensional structure and functional activity.