RESUMO
Sarcomas are cancers of mesenchymal origin with the potential to arise in diverse anatomic locations. With over 80 subtypes, which often demonstrate overlapping morphologies, sarcomas frequently require ancillary testing to enable accurate classification. Pathognomonic driver mutations can often be leveraged for diagnostic purposes and include fusion genes, amplification events, and recurrent point mutations. Until relatively recently, the major clinical molecular diagnostic tests have been karyotyping, fluorescence in situ hybridization, and polymerase chain reaction; however, these techniques have a number of limitations. Recent technological advances have led to the development of more comprehensive assays with higher throughput, thereby replacing the need for a suite of single gene tests. These approaches include next-generation sequencing, fluorescent bar code hybridization, and DNA methylation profiling, among others. Herein, we review the application of recently developed techniques relevant to the diagnosis of sarcomas, and emerging assays with the potential for future development and clinical implementation.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Hibridização in Situ Fluorescente , Patologia Molecular , Sarcoma/diagnóstico , Sarcoma/genética , Neoplasias de Tecidos Moles/genéticaRESUMO
AIMS: Clear cell sarcoma of the kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITDs) of the BCOR gene, whereas a minority have the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITDs nor YWHAE-NUTM2 fusion. The aim of this study was to characterise 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains) and genetically. METHODS AND RESULTS: By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITDs, with positive BCOR immunoreactivity being found in four (36%) or eight (72%) cases, depending on the antibody clone. By reverse transcription polymerase chain reaction, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative polymerase chain reaction showed markedly elevated BCOR expression in this case, whereas BCOR ITD cases had lower levels of elevated BCOR expression. CONCLUSIONS: The majority of the CCSKs in our cohort had BCOR ITDs, and none had the YWHAE-NUTM2 fusion. We verified the strong, diffuse cyclin D1 (CCND1) immunoreactivity in CCSKs described in recent reports. BCOR immunoreactivity was not consistently positive in all CCSKs with BCOR ITDs, and therefore cannot be used as a diagnostic immunohistochemical stain to identify BCOR ITD cases. The DN case was a BCOR-CCNB3 fusion sarcoma. BCOR-CCNB3 sarcoma is typically a primary bone sarcoma affecting male adolescents, and this is the first report of it presenting in a kidney of a young child as a CCSK. The full spectrum of DN CCSKs awaits more comprehensive characterisation.
Assuntos
Ciclina B/genética , Neoplasias Renais/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Sarcoma de Células Claras/genética , Criança , Pré-Escolar , Éxons , Feminino , Humanos , MasculinoRESUMO
Synovial sarcoma (SyS) is an aggressive soft-tissue malignancy characterized by a pathognomonic chromosomal translocation leading to the formation of the SS18::SSX fusion oncoprotein. SS18::SSX associates with mammalian BAF complexes suggesting deregulation of chromatin architecture as the oncogenic driver in this tumour type. To examine the epigenomic state of SyS we performed comprehensive multi-omics analysis on 52 primary pre-treatment human SyS tumours. Our analysis revealed a continuum of epigenomic states across the cohort at fusion target genes independent of rare somatic genetic lesions. We identify cell-of-origin signatures defined by enhancer states and reveal unexpected relationships between H2AK119Ub1 and active marks. The number of bivalent promoters, dually marked by the repressive H3K27me3 and activating H3K4me3 marks, has strong prognostic value and outperforms tumor grade in predicting patient outcome. Finally, we identify SyS defining epigenomic features including H3K4me3 expansion associated with striking promoter DNA hypomethylation in which SyS displays the lowest mean methylation level of any sarcoma subtype. We explore these distinctive features as potential vulnerabilities in SyS and identify H3K4me3 inhibition as a promising therapeutic strategy.
RESUMO
Sarcomas are a diverse group of tumors, with >70 subtypes in the current World Health Organization classification, each with distinct biological behavior requiring specific clinical management. A significant portion of sarcomas are molecularly defined by expression of a driver fusion gene; identification of such fusions is the basis of molecular diagnostics in sarcomas, which is of increasing complexity due to the ongoing discovery of new gene fusions. Recently, a multiplex NanoString platform-based assay was developed and clinically implemented, with fusion junction-spanning probes that detect the majority of sarcoma fusion types, with high sensitivity and specificity, and with lower cost and shorter turnaround time than those of targeted next-generation sequencing-based alternatives. Despite the effectiveness of this assay, there are several entities for which fusion-junction probes are not suitable due to multiple possible gene partners or excessive variability at the exon junctions. Here, the development and evaluation of a companion assay are described that uses NanoString-based gene expression analysis to detect aberrant 3'/5' exon expression imbalance and/or total gene overexpression as a surrogate marker for fusion gene rearrangement. This assay evaluates exon imbalance in 23 genes involved in over 25 mesenchymal tumor types and five genes specific to sarcomas with CIC rearrangements. Based on evaluation of 115 retrospectively and 91 prospectively collected cases, an assay sensitivity of 92.8% and specificity of 93.5% are demonstrated.
Assuntos
Sarcoma , Neoplasias de Tecidos Moles , Humanos , Estudos Retrospectivos , Sarcoma/diagnóstico , Sarcoma/genética , Sarcoma/patologia , Fusão Gênica , Éxons/genética , Neoplasias de Tecidos Moles/genética , Biomarcadores Tumorais/genética , Expressão Gênica , Proteínas de Fusão Oncogênica/genéticaRESUMO
PURPOSE: Predictive biomarkers for capecitabine benefit in triple-negative breast cancer (TNBC) have been recently proposed using samples from phase III clinical trials, including non-basal phenotype and biomarkers related to angiogenesis, stroma, and capecitabine activation genes. We aimed to validate these findings on the larger phase III GEICAM/CIBOMA clinical trial. EXPERIMENTAL DESIGN: Tumor tissues from patients with TNBC randomized to standard (neo)adjuvant chemotherapy followed by capecitabine versus observation were analyzed using a 164-gene NanoString custom nCounter codeset measuring mRNA expression. A prespecified statistical plan sought to verify the predictive capacity of PAM50 non-basal molecular subtype and tested the hypotheses that breast tumors with increased expression of (meta)genes for cytotoxic cells, mast cells, endothelial cells, PDL2, and 38 individual genes benefit from adjuvant capecitabine for distant recurrence-free survival (DRFS; primary endpoint) and overall survival. RESULTS: Of the 876 women enrolled in the GEICAM/CIBOMA trial, 658 (75%) were evaluable for analysis (337 with capecitabine and 321 without). Of these cases, 553 (84%) were profiled as PAM50 basal-like whereas 105 (16%) were PAM50 non-basal. Non-basal subtype was the most significant predictor for capecitabine benefit [HRcapecitabine, 0.19; 95% confidence interval (CI), 0.07-0.54; P < 0.001] when compared with PAM50 basal-like (HRcapecitabine, 0.9; 95% CI, 0.63-1.28; P = 0.55; Pinteraction<0.001, adjusted P value = 0.01). Analysis of biological processes related to PAM50 non-basal subtype revealed its enrichment for mast cells, extracellular matrix, angiogenesis, and features of mesenchymal stem-like TNBC subtype. CONCLUSIONS: In this prespecified correlative analysis of the GEICAM/CIBOMA trial, PAM50 non-basal status identified patients with early-stage TNBC most likely to benefit from capecitabine.
Assuntos
Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Humanos , Feminino , Capecitabina/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Células Endoteliais/patologia , Adjuvantes Imunológicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Quimioterapia Adjuvante , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversosRESUMO
The Golgi-specific zinc finger protein GODZ (palmitoyl acyltransferase/DHHC-3) mediates the palmitoylation and post-translational modification of many protein substrates that regulate membrane-protein interactions. Here, we show that GODZ also mediates Ca(2+) transport in expressing Xenopus laevis oocytes. Two-electrode voltage-clamp, fluorescence, and (45)Ca(2+) isotopic uptake determinations demonstrated voltage- and concentration-dependent, saturable, and substrate-inhibitable Ca(2+) transport in oocytes expressing GODZ cRNA but not in oocytes injected with water alone. Moreover, we show that GODZ-mediated Ca(2+) transport is regulated by palmitoylation, as the palmitoyl acyltransferase inhibitor 2-bromopalmitate or alteration of the acyltransferase DHHC motif (GODZ-DHHS) diminished GODZ-mediated Ca(2+) transport by approximately 80%. The GODZ mutation V61R abolished Ca(2+) transport but did not affect palmitoyl acyltransferase activity. Coexpression of GODZ-V61R with GODZ-DHHS restored GODZ-DHHS-mediated Ca(2+) uptake to values observed with wild-type GODZ, excluding an endogenous effect of palmitoylation. Coexpression of an independent palmitoyl acyltransferase (HIP14) with the GODZ-DHHS mutant also rescued Ca(2+) transport. HIP14 did not mediate Ca(2+) transport when expressed alone. Immunocytochemistry studies showed that GODZ and HIP14 co-localized to the Golgi and the same post-Golgi vesicles, suggesting that heteropalmitoylation might play a physiological role in addition to a biochemical function. We conclude that GODZ encodes a Ca(2+) transport protein in addition to its ability to palmitoylate protein substrates.
Assuntos
Aciltransferases/metabolismo , Cálcio/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Aciltransferases/química , Aciltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos , Transporte Proteico/genética , Transporte Proteico/fisiologia , Xenopus/genética , Xenopus/metabolismoRESUMO
Histologic examination neither reliably distinguishes benign lipomas from atypical lipomatous tumor/well-differentiated liposarcoma, nor dedifferentiated liposarcoma from other pleomorphic sarcomas, entities with different prognoses and management. Molecular confirmation of pathognomonic 12q13-15 amplifications leading to MDM2 overexpression is a diagnostic gold standard. Currently the most commonly used assay for this purpose is fluorescence in situ hybridization (FISH), but this is labor intensive. This study assessed whether newer NanoString-based technology could allow for more rapid and cost-efficient diagnosis of liposarcomas on standard formalin-fixed tissues through gene expression. Leveraging large-scale transcriptome data from The Cancer Genome Atlas, 20 genes were identified, most from the 12q13-15 amplicon, that distinguish dedifferentiated liposarcoma from other sarcomas and can be measured within a single NanoString assay. Using 21 cases of histologically ambiguous low-grade adipocytic tumors with available MDM2 amplification status, a machine learning-based analytical pipeline was built that assigns a given sample as negative or positive for liposarcoma based on quantitative gene expression. The effectiveness of the assay was validated on an independent set of 100 sarcoma samples (including 40 incident prospective cases), where histologic examination was considered insufficient for clinical diagnosis. The NanoString assay had a 93% technical success rate, and an accuracy of 97.8% versus an MDM2 amplification FISH gold standard. NanoString had a considerably faster turnaround time and was cheaper than FISH.
Assuntos
Biomarcadores Tumorais , Expressão Gênica , Testes Genéticos/métodos , Lipossarcoma/diagnóstico , Lipossarcoma/genética , Análise Custo-Benefício , Perfilação da Expressão Gênica , Testes Genéticos/economia , Testes Genéticos/normas , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Lipoma/diagnóstico , Lipoma/genética , Reprodutibilidade dos TestesRESUMO
NanoString nCounter is a multiplex nucleic acid hybridization technology that enables reliable and reproducible assessment of the expression of up to 800 genes or 228 gene fusions in 12 samples in a single assay. The technique works well with a variety of starting materials from fresh or formalin-fixed tissues, cell lysates or biological fluid samples. As low as 1 ng RNA input per sample is needed. In addition to gene expression, copy number variation, single nucleotide variation and chimeric RNAs can be analyzed. In this chapter, we discuss the design, setup, and performance of a NanoString-based RNA assay for gene expression or fusion transcript assessment.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Hibridização de Ácido Nucleico/métodos , RNA , Análise de Sequência de RNA , Biologia Computacional/métodos , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sondas Moleculares , RNA/química , RNA/genética , Análise de Sequência de RNA/métodos , NavegadorRESUMO
PURPOSE: Gene fusions involving neuregulin 1 (NRG1) have been noted in multiple cancer types and have potential therapeutic implications. Although varying results have been reported in other cancer types, the efficacy of the HER-family kinase inhibitor afatinib in the treatment of NRG1 fusion-positive pancreatic ductal adenocarcinoma is not fully understood. EXPERIMENTAL DESIGN: Forty-seven patients with pancreatic ductal adenocarcinoma received comprehensive whole-genome and transcriptome sequencing and analysis. Two patients with gene fusions involving NRG1 received afatinib treatment, with response measured by pretreatment and posttreatment PET/CT imaging. RESULTS: Three of 47 (6%) patients with advanced pancreatic ductal adenocarcinoma were identified as KRAS wild type by whole-genome sequencing. All KRAS wild-type tumors were positive for gene fusions involving the ERBB3 ligand NRG1. Two of 3 patients with NRG1 fusion-positive tumors were treated with afatinib and demonstrated a significant and rapid response while on therapy. CONCLUSIONS: This work adds to a growing body of evidence that NRG1 gene fusions are recurrent, therapeutically actionable genomic events in pancreatic cancers. Based on the clinical outcomes described here, patients with KRAS wild-type tumors harboring NRG1 gene fusions may benefit from treatment with afatinib.See related commentary by Aguirre, p. 4589.
Assuntos
Neoplasias Pulmonares/genética , Neoplasias Pancreáticas , Feminino , Fusão Gênica , Rearranjo Gênico , Humanos , Neuregulina-1 , Proteínas de Fusão Oncogênica/genética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Proteínas Proto-Oncogênicas p21(ras)RESUMO
The NanoString nCounter assay is a high-throughput hybridization technique using target-specific probes that can be customized to test for numerous fusion transcripts in a single assay using RNA from formalin-fixed, paraffin-embedded material. We designed a NanoString assay targeting 174 unique fusion junctions in 25 sarcoma types. The study cohort comprised 212 cases, 96 of which showed fusion gene expression by the NanoString assay, including all 20 Ewing sarcomas, 11 synovial sarcomas, and 5 myxoid liposarcomas tested. Among these 96 cases, 15 showed fusion expression not identified by standard clinical assay, including EWSR1-FLI1, EWSR1-ERG, BCOR-CCNB3, ZC3H7B-BCOR, HEY1-NCOA2, CIC-DUX4, COL1A1-PDGFB, MYH9-USP6, YAP1-TFE3, and IRF2BP2-CDX1 fusions. There were no false-positive results; however, four cases were false negative when compared with clinically available fluorescence in situ hybridization or RT-PCR testing. When batched as six cases, the per-sample reagent cost was less than conventional techniques, such as fluorescence in situ hybridization, with technologist hands-on time of 1.2 hours per case and assay time of 36 hours. In summary, the NanoString nCounter Sarcoma Fusion CodeSet reliably and cost-effectively identifies fusion genes in sarcomas using formalin-fixed, paraffin-embedded material, including many fusions missed by standard clinical assays, and can serve as a first-line clinical diagnostic test for sarcoma fusion gene identification, replacing multiple individual clinical assays.
Assuntos
Custos e Análise de Custo , Hibridização in Situ Fluorescente/economia , Hibridização in Situ Fluorescente/métodos , Proteínas de Fusão Oncogênica/genética , Sarcoma/genética , Estudos de Coortes , Humanos , Sondas RNA/metabolismo , Sensibilidade e EspecificidadeRESUMO
We describe a novel gene fusion, EWSR1-CREM, identified in 3 cases of clear cell carcinoma (CCC) using anchored multiplex polymerase chain reaction, a next-generation sequencing-based technique. CCC is a low-grade salivary tumor recently characterized to have EWSR1-ATF1 fusions in the majority of cases. Three cases of malignant tumor presenting in the base of tongue, lung, and nasopharynx were studied. All cases shared a clear cell morphology with hyalinized stroma, presence of mucin and p63 positivity and were initially diagnosed as mucoepidermoid carcinoma but were negative for evidence of any of the expected gene fusions. Anchored multiplex polymerase chain reaction demonstrated a EWSR1-CREM fusion in all 3 cases to confirm a diagnosis of CCC. This finding is biologically justified as CREM and ATF1 both belong to the CREB family of transcription factors. EWSR1-CREM fusions have not been previously reported in CCC and have only rarely been reported in other tumors. We show that the ability to discover novel gene variants with next-generation sequencing-based assays has clinical utility in the pathologic classification of fusion gene-associated tumors.
Assuntos
Adenocarcinoma de Células Claras/genética , Modulador de Elemento de Resposta do AMP Cíclico/genética , Neoplasias Pulmonares/genética , Neoplasias Nasofaríngeas/genética , Proteína EWS de Ligação a RNA/genética , Neoplasias da Língua/genética , Adenocarcinoma de Células Claras/patologia , Idoso , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Fusão Oncogênica , Neoplasias da Língua/patologia , TranscriptomaRESUMO
We have begun to identify and characterize genes that are differentially expressed with low magnesium. One of these sequences conformed to the ancient conserved domain protein, ACDP2. Real-time RT-PCR of mRNA isolated from distal epithelial cells cultured in low-magnesium media relative to normal media and in kidney cortex of mice maintained on low-magnesium diets compared with those animals consuming normal diets confirmed that the ACDP2 transcript is responsive to magnesium. Mouse ACDP2 was cloned from mouse distal convoluted tubule cells, expressed in Xenopus laevis oocytes, and studied with two-electrode voltage-clamp studies. When expressed in oocytes, ACDP2 mediates saturable Mg2+ uptake with a Michaelis constant of 0.56 +/- 0.05 mM. Transport of Mg2+ by ACDP2 is rheogenic, is voltage-dependent, and is not coupled to Na+ or Cl- ions. Expressed ACDP2 transports a range of divalent cations: Mg2+, Co2+, Mn2+, Sr2+, Ba2+, Cu2+, and Fe2+; accordingly, it is a divalent cation transporter with wide substrate selectivity. The cations Ca2+, Cd2+, Zn2+, and Ni2+ did not induce currents, and only Zn2+ effectively inhibited transport. The ACDP2 transcript is abundantly present in kidney, brain, and heart with lower amounts in liver, small intestine, and colon. Moreover, ACDP2 mRNA is upregulated with magnesium deficiency, particularly in the distal convoluted tubule cells, kidney, heart, and brain. These studies suggest that ACDP2 may provide a regulated transporter for Mg2+ and other divalent cations in epithelial cells.
Assuntos
Proteínas de Membrana Transportadoras/química , Sequência de Aminoácidos , Animais , Transporte Biológico , Proteínas de Transporte de Cátions , Cátions , Cátions Bivalentes , Eletrofisiologia , Células Epiteliais/metabolismo , Córtex Renal/metabolismo , Túbulos Renais/metabolismo , Cinética , Magnésio/química , Magnésio/metabolismo , Masculino , Proteínas de Membrana Transportadoras/fisiologia , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual , Regulação para Cima , Xenopus , Xenopus laevisRESUMO
We have begun to identify and characterize genes that are differentially expressed with low magnesium. One of these sequences conformed to the solute carrier SLC41A1. Real-time RT-PCR of RNA isolated from renal distal tubule epithelial [mouse distal convoluted tubule (MDCT)] cells cultured in low-magnesium media relative to normal media and in the kidney cortex of mice maintained on low-magnesium diets compared with those animals consuming normal diets confirmed that the SLC41A1 transcript is responsive to magnesium. Mouse SLC41A1 was cloned from MDCT cells, expressed in Xenopus laevis oocytes, and studied with two-electrode voltage-clamp studies. When expressed in oocytes, SLC41A1 mediates saturable Mg2+ uptake with a Michaelis constant of 0.67 mM. Transport of Mg2+ by SLC41A1 is rheogenic, voltage dependent, and not coupled to Na+ or Cl-. Expressed SLC41A1 transports a range of other divalent cations: Mg2+, Sr2+, Zn2+, Cu2+, Fe2+, Co2+, Ba2+, and Cd2+. The divalent cations Ca2+, Mn2+, and Ni2+ and the trivalent ion Gd3+ did not induce currents nor did they inhibit Mg2+ transport. The nonselective cation La3+ abolished Mg2+ uptake. The SLC41A1 transcript is present in many tissues, notably renal epithelial cells, and is upregulated in some tissues with magnesium deficiency. These studies suggest that SLC41A1 is a regulated Mg2+ transporter that might be involved in magnesium homeostasis in epithelial cells.
Assuntos
Antiporters/genética , Proteínas de Bactérias/genética , Magnésio/metabolismo , Proteínas de Membrana Transportadoras/genética , Animais , Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte de Cátions , Cátions Bivalentes/metabolismo , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Feminino , Vetores Genéticos , Humanos , Deficiência de Magnésio/genética , Oócitos/fisiologia , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Xenopus/fisiologiaRESUMO
BACKGROUND: Intracellular magnesium is abundant, highly regulated and plays an important role in biochemical functions. Despite the extensive evidence for unique mammalian Mg2+ transporters, few proteins have been biochemically identified to date that fulfill this role. We have shown that epithelial magnesium conservation is controlled, in part, by differential gene expression leading to regulation of Mg2+ transport. We used this knowledge to identify a novel gene that is regulated by magnesium. RESULTS: Oligonucleotide microarray analysis was used to identify a novel human gene that encodes a protein involved with Mg2+-evoked transport. We have designated this magnesium transporter (MagT1) protein. MagT1 is a novel protein with no amino acid sequence identity to other known transporters. The corresponding cDNA comprises an open reading frame of 1005 base pairs encoding a protein of 335 amino acids. It possesses five putative transmembrane (TM) regions with a cleavage site, a N-glycosylation site, and a number of phosphorylation sites. Based on Northern analysis of mouse tissues, a 2.4 kilobase transcript is present in many tissues. When expressed in Xenopus laevis oocytes, MagT1 mediates saturable Mg2+ uptake with a Michaelis constant of 0.23 mM. Transport of Mg2+ by MagT1 is rheogenic, voltage-dependent, does not display any time-dependent inactivation. Transport is very specific to Mg2+ as other divalent cations did not evoke currents. Large external concentrations of some cations inhibited Mg2+ transport (Ni2+, Zn2+, Mn2+) in MagT1-expressing oocytes. Ca2+and Fe2+ were without effect. Real-time reverse transcription polymerase chain reaction and Western blot analysis using a specific antibody demonstrated that MagT1 mRNA and protein is increased by about 2.1-fold and 32%, respectively, in kidney epithelial cells cultured in low magnesium media relative to normal media and in kidney cortex of mice maintained on low magnesium diets compared to those animals consuming normal diets. Accordingly, it is apparent that an increase in mRNA levels is translated into higher protein expression. CONCLUSION: These studies suggest that MagT1 may provide a selective and regulated pathway for Mg2+ transport in epithelial cells.
Assuntos
Proteínas de Transporte de Cátions/fisiologia , Magnésio/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico , Northern Blotting , Western Blotting , Cálcio/metabolismo , Proteínas de Transporte de Cátions/química , Células Cultivadas , Clonagem Molecular , DNA Complementar/metabolismo , Eletrofisiologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Genômica/métodos , Glicosilação , Humanos , Íons , Ferro/metabolismo , Rim/metabolismo , Córtex Renal/metabolismo , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Oócitos/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição Tecidual , Cromossomo X , Xenopus , Xenopus laevisRESUMO
A subset of endometrial stromal sarcoma harbors t(10;17)(q23;p13), which results in the genetic fusion between YWHAE and 1 of 2 highly homologous FAM22 family members-FAM22A or FAM22B. In contrast to classic low-grade endometrial stromal sarcoma with JAZF1-SUZ12 fusions, YWHAE-FAM22 endometrial stromal sarcoma displays high-grade histologic features and is associated with more aggressive disease course. Ancillary fluorescence in situ hybridization assay demonstrating the presence of YWHAE rearrangement can be used to support the diagnosis, but the detection of fusion transcript would be the most definitive test. We describe here an optimized reverse transcription-polymerase chain reaction assay for detection of YWHAE-FAM22 fusion transcript in formalin-fixed and paraffin-embedded tumor samples. We studied a series of 6 YWHAE-FAM22 endometrial stromal sarcomas, 7 JAZF-SUZ12 endometrial stromal sarcomas, 3 JAZF1-PHF1/EPC1-PHF1 endometrial stromal sarcomas, 6 undifferentiated endometrial sarcomas, 4 uterine leiomyosarcomas, and 4 uterine adenosarcomas. All 6 YWHAE-FAM22 endometrial stromal sarcomas were confirmed by fluorescence in situ hybridization assay, whereas all non-YWHAE-FAM22 tumors were confirmed to lack YWHAE rearrangement by fluorescence in situ hybridization assay. The reverse transcription-polymerase chain reaction assay optimized for formalin-fixed and paraffin-embedded samples detected YWHAE-FAM22 fusion transcripts in all 6 YWHAE-FAM22 endometrial stromal sarcomas and none of the 24 non-YWHAE-FAM22 uterine sarcomas. These findings show that this reverse transcription-polymerase chain reaction assay is sensitive and specific for detection of YWHAE-FAM22 fusion transcript and can serve as a useful adjunct diagnostic assay to confirm the diagnosis of YWHAE-FAM22 endometrial stromal sarcoma in formalin-fixed and paraffin-embedded tumor samples.
Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , Sarcoma do Estroma Endometrial/genética , Sarcoma do Estroma Endometrial/patologia , Feminino , Formaldeído , Humanos , Hibridização in Situ Fluorescente , Inclusão em Parafina , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Synovial sarcoma is a translocation-associated sarcoma where the underlying chromosomal event generates SS18-SSX fusion transcripts. In vitro and in vivo studies have shown that the SS18-SSX fusion oncoprotein is both necessary and sufficient to support tumorigenesis; however, its mechanism of action remains poorly defined. We have purified a core SS18-SSX complex and discovered that SS18-SSX serves as a bridge between activating transcription factor 2 (ATF2) and transducin-like enhancer of split 1 (TLE1), resulting in repression of ATF2 target genes. Disruption of these components by siRNA knockdown or treatment with HDAC inhibitors rescues target gene expression, leading to growth suppression and apoptosis. Together, these studies define a fundamental role for aberrant ATF2 transcriptional dysregulation in the etiology of synovial sarcoma.
Assuntos
Proteínas de Fusão Oncogênica/fisiologia , Sarcoma Sinovial/genética , Fator 2 Ativador da Transcrição/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas Correpressoras , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , RNA Interferente Pequeno , Proteínas Repressoras/metabolismo , Sarcoma Sinovial/metabolismo , Translocação GenéticaRESUMO
Magnesium is an essential metal, but few selective transporters have been identified at the molecular level. Microarray analysis was used to identify two similar transcripts that are upregulated with low extracellular Mg(2+). The corresponding cDNAs encode proteins of 131 and 123 amino acids with two predicted transmembrane domains. The two separate gene products comprise the family that we have termed "membrane Mg(2+) transporters" (MMgTs), because the proteins reside in the membrane and mediate Mg(2+) transport. When expressed in Xenopus laevis oocytes, MMgT1 and MMgT2 mediate Mg(2+) transport as determined with two-electrode voltage-clamp analysis and fluorescence measurements. Transport is saturable Mg(2+) uptake with Michaelis constants of 1.47 +/- 0.17 and 0.58 +/- 0.07 mM, respectively. Real-time RT-PCR demonstrated that MMgT mRNAs are present in a wide variety of cells. Subcellular localization with immunohistochemistry determined that the MMgT1-hemagglutinin (HA) and MMgT2-V5 fusion proteins reside in the Golgi complex and post-Golgi vesicles, including the early endosomes in COS-7 cells transfected with the respective tagged constructs. Interestingly, MMgT1-HA and MMgT2-V5 were found in separate populations of post-Golgi vesicles. MMgT1 and MMgT2 mRNA increased by about threefold, respectively, in kidney epithelial cells cultured in low-magnesium media relative to normal media and in the kidney cortex of mice maintained on low-magnesium diets compared with those animals consuming normal diets. With the increase in transcripts, there was an apparent increase in MMgT1 and MMgT2 protein in the Golgi and post-Golgi vesicles. These experiments suggest that MMgT proteins may provide regulated pathways for Mg(2+) transport in the Golgi and post-Golgi organelles of epithelium-derived cells.
Assuntos
Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Magnésio/metabolismo , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Linhagem Celular Transformada , Chlorocebus aethiops , Células Epiteliais/ultraestrutura , Feminino , Imunofluorescência , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/ultraestrutura , Túbulos Renais/metabolismo , Túbulos Renais/ultraestrutura , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Xenopus laevisRESUMO
Polyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14-like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 +/- 0.02 and 0.74 +/- 0.07 mm, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14DeltaDHHC, diminishes HIP14-mediated Mg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14DeltaDHHC mutant restored Mg2+ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg2+ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg2+ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes."
Assuntos
Aciltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Complexo de Golgi/enzimologia , Magnésio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Aciltransferases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Cães , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Transporte de Íons/fisiologia , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estrutura Terciária de Proteína/fisiologia , XenopusRESUMO
We used microarray analysis to identify renal cell transcripts that were upregulated with low magnesium. One transcript, identified as NIPA2 (nonimprinted in Prader-Willi/Angelman syndrome) subtype 2, was increased over twofold relative to cells cultured in normal magnesium. The deduced sequence comprises 129 amino acids with 8 predicted transmembrane regions. As the secondary structure of NIPA2 conformed to a membrane transport protein, we expressed it in Xenopus oocytes and determined that it mediated Mg(2+) uptake with two-electrode voltage-clamp and fluorescence studies. Mg(2+) transport was electrogenic, voltage dependent, and saturable, demonstrating a Michaelis affinity constant of 0.31 mM. Unlike other reported Mg(2+) transporters, NIPA2 was very selective for the Mg(2+) cation. NIPA2 mRNA is found in many tissues but particularly abundant in renal cells. With the use of immunofluorescence, it was shown that NIPA2 protein was normally localized to the early endosomes and plasma membrane and was recruited to the plasma membrane in response to low extracellular magnesium. We conclude that NIPA2 plays a role in magnesium metabolism and regulation of renal magnesium conservation.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/fisiologia , Proteínas de Transporte de Cátions/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Túbulos Renais Distais/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Filogenia , Análise Serial de Proteínas , XenopusRESUMO
Mutations in the NIPA1(SPG6) gene, named for "nonimprinted in Prader-Willi/Angelman" has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69+/-0.21 mM when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype.