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1.
Clin Exp Gastroenterol ; 12: 219-229, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190949

RESUMO

Purpose: The incidence of esophageal adenocarcinoma (EAC) has increased by 700% in Western countries over the last 30 years. Although clinical guidelines call for endoscopic surveillance for EAC among high-risk populations, fewer than 5% of new EAC patients are under surveillance at the time of diagnosis. We studied the accuracy of combined cytopathology and MUC2 immunohistochemistry (IHC) for screening of Intestinal Metaplasia (IM), dysplasia and EAC, using specimens collected from the EsophaCap swallowable encapsulated cytology sponge from Canada and United States. Patients and methods: By comparing the EsophaCap cytological diagnosis with concurrent endoscopic biopsies performed on the same patients in 28 cases, we first built up the cytology diagnostic categories and criteria. Based on these criteria, 136 cases were evaluated by both cytology and MUC2 IHC with blinded to patient biopsy diagnosis. Results: We first set up categories and criteria for cytological diagnosis of EscophaCap samples. Based on these, we divided our evaluated cytological samples into two groups: non-IM group and IM or dysplasia or adenocarcinoma group. Using the biopsy as our gold standard to screen IM, dysplasia and EAC by combined cytology and MUC2 IHC, the sensitivity and specificity were 68% and 91%, respectively, which is in the range of clinically useful cytological screening tests such as the cervical Pap smear. Conclusions: Combined EsophaCap cytology and MUC2 IHC could be a good screening test for IM and Beyond.

2.
Ann Thorac Surg ; 108(2): 343-349, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31059681

RESUMO

BACKGROUND: Recent literature has demonstrated the potential of "liquid biopsy" and detection of circulating tumor (ct)DNA as a cancer biomarker. However, to date there is a lack of data specific to esophageal adenocarcinoma (EAC). This study was conducted to determine how detection and quantification of ctDNA changes with disease burden in patients with EAC and evaluate its potential as a biomarker in this population. METHODS: Blood samples were obtained from patients with stage I to IV EAC. Longitudinal blood samples were collected from a subset of patients. Imaging studies and pathology reports were reviewed to determine disease course. Tumor samples were sequenced to identify mutations. Mutations in plasma DNA were detected using custom, barcoded, patient-specific sequencing libraries. Mutations in plasma were quantified, and associations with disease stage and response to therapy were explored. RESULTS: Plasma samples from a final cohort of 38 patients were evaluated. Baseline plasma samples were ctDNA positive for 18 patients (47%) overall, with tumor allele frequencies ranging from 0.05% to 5.30%. Detection frequency of ctDNA and quantity of ctDNA increased with stage. Data from longitudinal samples indicate that ctDNA levels correlate with and precede evidence of response to therapy or recurrence. CONCLUSIONS: ctDNA can be detected in plasma of EAC patients and correlates with disease burden. Detection of ctDNA in early-stage EAC is challenging and may limit diagnostic applications. However, our data demonstrate the potential of ctDNA as a dynamic biomarker to monitor treatment response and disease recurrence in patients with EAC.


Assuntos
Adenocarcinoma/diagnóstico , DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Neoplasias Esofágicas/diagnóstico , Mutação , Estadiamento de Neoplasias/métodos , Adenocarcinoma/sangue , Adenocarcinoma/genética , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/sangue , Progressão da Doença , Neoplasias Esofágicas/sangue , Neoplasias Esofágicas/genética , Feminino , Humanos , Biópsia Líquida , Masculino
3.
Head Neck ; 41(5): 1351-1358, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30554450

RESUMO

BACKGROUND: Recommendations for perioperative therapy in head and neck cancer are not explicit and recurrence occurs frequently. Circulating tumor DNA is an emerging cancer biomarker, but has not been extensively explored for detection of recurrence in head and neck cancer. METHODS: Patients diagnosed with head and neck squamous cell carcinoma were recruited into the study protocol. Tumors were sequenced to identify patient-specific mutations. Mutations were then identified in plasma circulating tumor DNA from pre-treatment blood samples and longitudinally during standard follow-up. Circulating tumor DNA status during follow-up was correlated to disease recurrence. RESULTS: Samples were taken from eight patients. Tumor mutations were verified in seven patients. Baseline circulating tumor DNA was positive in six patients. Recurrence occurred in four patients, two of whom had detectable circulating tumor DNA prior to recurrence. CONCLUSION: Circulating tumor DNA is a potential tool for disease and recurrence monitoring following curative therapy in head and neck cancer, allowing for better prognostication, and/or modification of treatment strategies.

4.
BMC Mol Biol ; 18(1): 19, 2017 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-28728573

RESUMO

BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.


Assuntos
Precursores de RNA/genética , Processamento de RNA , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Linhagem Celular , Análise por Conglomerados , Biologia Computacional/métodos , Éxons , Fibroblastos , Perfilação da Expressão Gênica , Humanos , Reprodutibilidade dos Testes , Transdução de Sinais , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteínas ras/metabolismo
5.
Genome Med ; 9(1): 59, 2017 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-28659176

RESUMO

BACKGROUND: A key step in cancer genome analysis is the identification of somatic mutations in the tumor. This is typically done by comparing the genome of the tumor to the reference genome sequence derived from a normal tissue taken from the same donor. However, there are a variety of common scenarios in which matched normal tissue is not available for comparison. RESULTS: In this work, we describe an algorithm to distinguish somatic single nucleotide variants (SNVs) in next-generation sequencing data from germline polymorphisms in the absence of normal samples using a machine learning approach. Our algorithm was evaluated using a family of supervised learning classifications across six different cancer types and ~1600 samples, including cell lines, fresh frozen tissues, and formalin-fixed paraffin-embedded tissues; we tested our algorithm with both deep targeted and whole-exome sequencing data. Our algorithm correctly classified between 95 and 98% of somatic mutations with F1-measure ranges from 75.9 to 98.6% depending on the tumor type. We have released the algorithm as a software package called ISOWN (Identification of SOmatic mutations Without matching Normal tissues). CONCLUSIONS: In this work, we describe the development, implementation, and validation of ISOWN, an accurate algorithm for predicting somatic mutations in cancer tissues in the absence of matching normal tissues. ISOWN is available as Open Source under Apache License 2.0 from https://github.com/ikalatskaya/ISOWN .


Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/genética , Aprendizado de Máquina Supervisionado , Humanos
6.
Ann N Y Acad Sci ; 1381(1): 74-91, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27415609

RESUMO

Esophageal adenocarcinoma (EAC) develops in the sequential transformation of normal epithelium into metaplastic epithelium, called Barrett's esophagus (BE), then to dysplasia, and finally cancer. BE is a common condition in which normal stratified squamous epithelium of the esophagus is replaced with an intestine-like columnar epithelium, and it is the most prominent risk factor for EAC. This review aims to impartially systemize the knowledge from a large number of publications that describe the molecular and biochemical alterations occurring over this progression sequence. In order to provide an unbiased extraction of the knowledge from the literature, a text-mining methodology was used to select genes that are involved in the BE progression, with the top candidate genes found to be TP53, CDKN2A, CTNNB1, CDH1, GPX3, and NOX5. In addition, sample frequencies across analyzed patient cohorts at each stage of disease progression are summarized. All six genes are altered in the majority of EAC patients, and accumulation of alterations correlates well with the sequential progression of BE to cancer, indicating that the text-mining method is a valid approach for gene prioritization. This review discusses how, besides being cancer drivers, these genes are functionally interconnected and might collectively be considered a central hub of BE progression.


Assuntos
Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Esôfago de Barrett/diagnóstico , Esôfago de Barrett/genética , Biomarcadores Tumorais/genética , Progressão da Doença , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Adenocarcinoma/metabolismo , Animais , Esôfago de Barrett/metabolismo , Biomarcadores Tumorais/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Neoplasias Esofágicas/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NADPH Oxidase 5 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
7.
Breast Cancer Res ; 18(1): 16, 2016 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-26852132

RESUMO

BACKGROUND: Drug resistance in breast cancer is the major obstacle to effective treatment with chemotherapy. While upregulation of multidrug resistance genes is an important component of drug resistance mechanisms in vitro, their clinical relevance remains to be determined. Therefore, identifying pathways that could be targeted in the clinic to eliminate anthracycline-resistant breast cancer remains a major challenge. METHODS: We generated paired native and epirubicin-resistant MDA-MB-231, MCF7, SKBR3 and ZR-75-1 epirubicin-resistant breast cancer cell lines to identify pathways contributing to anthracycline resistance. Native cell lines were exposed to increasing concentrations of epirubicin until resistant cells were generated. To identify mechanisms driving epirubicin resistance, we used a complementary approach including gene expression analyses to identify molecular pathways involved in resistance, and small-molecule inhibitors to reverse resistance. In addition, we tested its clinical relevance in a BR9601 adjuvant clinical trial. RESULTS: Characterisation of epirubicin-resistant cells revealed that they were cross-resistant to doxorubicin and SN-38 and had alterations in apoptosis and cell-cycle profiles. Gene expression analysis identified deregulation of histone H2A and H2B genes in all four cell lines. Histone deacetylase small-molecule inhibitors reversed resistance and were cytotoxic for epirubicin-resistant cell lines, confirming that histone pathways are associated with epirubicin resistance. Gene expression of a novel 18-gene histone pathway module analysis of the BR9601 adjuvant clinical trial revealed that patients with low expression of the 18-gene histone module benefited from anthracycline treatment more than those with high expression (hazard ratio 0.35, 95 % confidence interval 0.13-0.96, p = 0.042). CONCLUSIONS: This study revealed a key pathway that contributes to anthracycline resistance and established model systems for investigating drug resistance in all four major breast cancer subtypes. As the histone modification can be targeted with small-molecule inhibitors, it represents a possible means of reversing clinical anthracycline resistance. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00003012 . Registered on 1 November 1999.


Assuntos
Antraciclinas/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Histonas/biossíntese , Adulto , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Doxorrubicina/administração & dosagem , Epirubicina/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/administração & dosagem , Histonas/genética , Humanos , Irinotecano , Células MCF-7 , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Adulto Jovem
8.
Genome Med ; 5(7): 68, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23890051

RESUMO

New strategies to combat complex human disease require systems approaches to biology that integrate experiments from cell lines, primary tissues and model organisms. We have developed Pathprint, a functional approach that compares gene expression profiles in a set of pathways, networks and transcriptionally regulated targets. It can be applied universally to gene expression profiles across species. Integration of large-scale profiling methods and curation of the public repository overcomes platform, species and batch effects to yield a standard measure of functional distance between experiments. We show that pathprints combine mouse and human blood developmental lineage, and can be used to identify new prognostic indicators in acute myeloid leukemia. The code and resources are available at http://compbio.sph.harvard.edu/hidelab/pathprint.

9.
J Pharmacol Exp Ther ; 344(1): 85-95, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23086229

RESUMO

The DRY motif with the highly conserved R3.50 is a hallmark of family A G protein-coupled receptors (GPCRs). The crystal structure of rhodopsin revealed a salt bridge between R135(3.50) and another conserved residue, E247(6.30), in helix 6. This ionic lock was shown to maintain rhodopsin in its inactive state. Thus far, little information is available on how interruption of this ionic bond affects signaling properties of nonrhodopsin GPCRs, because the focus has been on mutations of R3.50, although this residue is indispensable for G protein activation. To investigate the importance of an ionic lock for overall receptor activity in a nonrhodopsin GPCR, we mutated R128(3.50) and E238(6.30) in the bradykinin (BK) B(2) receptor (B(2)R) and stably expressed the constructs in HEK293 cells. As expected, mutation of R3.50 resulted in lack of G protein activation. In addition, this mutation led to considerable constitutive receptor internalization. Mutation of E6.30 (mutants E6.30A and E6.30R) also caused strong constitutive internalization. Most intriguingly, however, although the two E6.30 mutants displayed no increased basal phosphatidylinositol hydrolysis, they gave a response to three different B(2)R antagonists that was almost comparable to that obtained with BK. In contrast, swapping of R3.50 and E6.30, thus allowing the formation of an inverse ionic bond, resulted in rescue of the wild type phenotype. These findings demonstrate for the first time, to our knowledge, that interruption of the ionic lock in a family A GPCR can have distinctly different effects on receptor internalization and G protein stimulation, shedding new light on its role in the activation process.


Assuntos
Receptor B2 da Bradicinina/efeitos dos fármacos , Aminoácidos/metabolismo , Biotinilação , Bradicinina/metabolismo , Antagonistas de Receptor B2 da Bradicinina , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Hidrólise , Fosfatos de Inositol/metabolismo , Íons/metabolismo , Fosforilação , Mutação Puntual , Piridonas/farmacologia , Quinolinas/farmacologia , Receptor B2 da Bradicinina/agonistas , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Temperatura Ambiente
10.
Mol Cell Proteomics ; 11(12): 1870-84, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22986220

RESUMO

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.


Assuntos
Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Secretadas pela Próstata/análise , Proteínas Secretadas pela Próstata/urina , Proteínas 14-3-3/análise , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Exonucleases/análise , Exorribonucleases , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Marcação por Isótopo , Masculino , Proteínas Oncogênicas/análise , Antígeno Prostático Específico/metabolismo , Análise Serial de Proteínas , Proteína Desglicase DJ-1 , Proteoma/análise , Inibidor Tecidual de Metaloproteinase-1/análise , Transglutaminases/análise
11.
Mol Cell Biol ; 32(19): 3913-24, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22851698

RESUMO

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.


Assuntos
Neoplasias da Mama/genética , Glândulas Mamárias Humanas/citologia , Receptor ErbB-2/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/metabolismo , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Prognóstico , Estrutura Terciária de Proteína , Receptor ErbB-2/genética , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/química , Transcriptoma , Regulação para Cima
12.
Ann Surg ; 255(6): 1113-20, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22498892

RESUMO

OBJECTIVE: This study aimed to identify pathways and cellular processes that are modulated by exposure of normal esophageal cells to bile and acid. BACKGROUND: Barrett's esophagus most likely develops as a response of esophageal stem cells to the abnormal reflux environment. Although insights into the underlying molecular mechanisms are slowly emerging, much of the metaplastic process remains unknown. METHODS: We performed a global analysis of gene expression in normal squamous esophageal cells in response to bile or acid exposure. Differentially expressed genes were classified into major biological functions using pathway analysis and interaction network software. Array data were verified by quantitative PCR and western blot both in vitro and in human esophageal biopsies. RESULTS: Bile modulated expression of 202 genes, and acid modulated expression of 103 genes. Genes involved in squamous differentiation formed the largest functional group (n = 45) all of which were downregulated by bile exposure. This included genes such as involucrin (IVL), keratinocyte differentiation-associated protein (KRTDAP), grainyhead-like 1 (GRHL1), and desmoglein1 (DSG1) the downregulation of which was confirmed by quantitative PCR and western blot. Bile also caused expression changes in genes involved in cell adhesion, DNA repair, oxidative stress, cell cycle, Wnt signaling, and lipid metabolism. Analysis of human esophageal biopsies demonstrated greatly reduced expression of IVL, KRTDAP, DSG1, and GRHL1 in metaplastic compared to squamous epithelia. CONCLUSIONS: We report for the first time that bile inhibits the squamous differentiation program of esophageal epithelial cells. This, coordinated with induction of genes driving intestinal differentiation, may be required for the development of Barrett's esophagus.


Assuntos
Bile/fisiologia , Diferenciação Celular/genética , Células Epiteliais/citologia , Esôfago/patologia , Esôfago/fisiopatologia , Ácido Gástrico/fisiologia , Biópsia , Linhagem Celular , Células Epiteliais/fisiologia , Esôfago/citologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
13.
Breast Cancer Res ; 14(1): R2, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22225778

RESUMO

INTRODUCTION: The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance. METHODS: MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR). RESULTS: MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways. CONCLUSIONS: We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Paclitaxel/farmacologia , Transdução de Sinais , Taxoides/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias da Mama , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Docetaxel , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Neoplasias Ovarianas , Inibidores da Síntese de Proteínas/farmacologia , Proteólise , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Ativação Transcricional/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética
14.
Clin Cancer Res ; 17(13): 4513-22, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21593195

RESUMO

PURPOSE: Chromosomal gain at 7q21 is a frequent event in esophageal adenocarcinoma (EAC). However, this event has not been mapped with fine resolution in a large EAC cohort, and its association with clinical endpoints and functional relevance are unclear. EXPERIMENTAL DESIGN: We used a cohort of 116 patients to fine map the 7q21 amplification using SNP microarrays. Prognostic significance and functional role of 7q21 amplification and its gene expression were explored. RESULTS: Amplification of the 7q21 region was observed in 35% of tumors with a focal, minimal amplicon containing six genes. 7q21 amplification was associated with poor survival and analysis of gene expression identified cyclin-dependent kinase 6 (CDK6) as the only gene in the minimal amplicon whose expression was also associated with poor survival. A low-level amplification (10%) was observed at the 12q13 region containing the CDK6 homologue cyclin-dependent kinase 4 (CDK4). Both amplification and expression of CDK4 correlated with poor survival. A combined model of both CDK6 and CDK4 expressions is a superior predictor of survival than either alone. Specific knockdown of CDK4 and/or CDK6 by siRNAs shows that they are required for proliferation of EAC cells and that their function is additive. PD-0332991 targets the kinase activity of both molecules and suppresses proliferation and anchorage independence of EAC cells through activation of the pRB pathway. CONCLUSIONS: We suggest that CDK6 is the driver of 7q21 amplification and that both CDK4 and CDK6 are prognostic markers and bona fide oncogenes in EAC. Targeting these molecules may constitute a viable new therapy for this disease.


Assuntos
Adenocarcinoma/diagnóstico , Adenocarcinoma/enzimologia , Biomarcadores Tumorais/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/enzimologia , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromossomos Humanos Par 7/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Variações do Número de Cópias de DNA/genética , Neoplasias Esofágicas/mortalidade , Feminino , Amplificação de Genes/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Piperazinas/farmacologia , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Análise de Sobrevida
15.
Nucleic Acids Res ; 39(Database issue): D691-7, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21067998

RESUMO

Reactome (http://www.reactome.org) is a collaboration among groups at the Ontario Institute for Cancer Research, Cold Spring Harbor Laboratory, New York University School of Medicine and The European Bioinformatics Institute, to develop an open source curated bioinformatics database of human pathways and reactions. Recently, we developed a new web site with improved tools for pathway browsing and data analysis. The Pathway Browser is an Systems Biology Graphical Notation (SBGN)-based visualization system that supports zooming, scrolling and event highlighting. It exploits PSIQUIC web services to overlay our curated pathways with molecular interaction data from the Reactome Functional Interaction Network and external interaction databases such as IntAct, BioGRID, ChEMBL, iRefIndex, MINT and STRING. Our Pathway and Expression Analysis tools enable ID mapping, pathway assignment and overrepresentation analysis of user-supplied data sets. To support pathway annotation and analysis in other species, we continue to make orthology-based inferences of pathways in non-human species, applying Ensembl Compara to identify orthologs of curated human proteins in each of 20 other species. The resulting inferred pathway sets can be browsed and analyzed with our Species Comparison tool. Collaborations are also underway to create manually curated data sets on the Reactome framework for chicken, Drosophila and rice.


Assuntos
Bases de Dados Factuais , Modelos Biológicos , Fenômenos Biológicos , Gráficos por Computador , Bases de Dados Genéticas , Bases de Dados de Proteínas , Regulação da Expressão Gênica , Humanos , Internet , Redes e Vias Metabólicas , Transdução de Sinais
16.
Mol Pharmacol ; 75(5): 1240-7, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19255243

RESUMO

The bicyclam AMD3100 is known as a small synthetic inhibitor of the CXCL12-binding chemokine receptor CXCR4. Here, we show that AMD3100 also binds to the alternative CXCL12 receptor CXCR7. CXCL12 or AMD3100 alone activate beta-arrestin recruitment to CXCR7, which we identify as a previously unreported signaling pathway of CXCR7. In addition, AMD3100 increases CXCL12 binding to CXCR7 and CXCL12-induced conformational rearrangements in the receptor dimer as measured by bioluminescence resonance energy transfer. Moreover, small but reproducible increases in the potency of CXCL12-induced arrestin recruitment to CXCR7 by AMD3100 are observed. Taken together, our data suggest that AMD3100 is an allosteric agonist of CXCR7. The finding that AMD3100 not only binds CXCR4, but also to CXCR7, with opposite effects on the two receptors, calls for caution in the use of the compound as a tool to dissect CXCL12 effects on the respective receptors in vitro and in vivo.


Assuntos
Compostos Heterocíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR/agonistas , Regulação Alostérica , Arrestinas/metabolismo , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacologia , Dimerização , Humanos , Luminescência , Receptores CXCR/química , beta-Arrestinas
17.
Biol Chem ; 387(5): 603-10, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16740132

RESUMO

A functional comparison was made between the wild-type bradykinin B2 receptor (B2wt) and the chimera B2eGFP (enhanced green-fluorescent protein fused to the C-terminus of B2wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 microM bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B2wt, but not in B2eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B2wt(low) to less than 20% within 1 h, whereas the chimera B2eGFP(low) still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B2wt(low) after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B2eGFP(low) levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B2wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation.


Assuntos
Proteínas de Fluorescência Verde/metabolismo , Receptor B2 da Bradicinina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Bradicinina/metabolismo , Bradicinina/farmacologia , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Immunoblotting , Fosfatos de Inositol/metabolismo , Cinética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Transporte Proteico/efeitos dos fármacos , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/genética , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodos
18.
FEBS J ; 272(1): 129-40, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15634338

RESUMO

Determinants for desensitization and sequestration of G protein-coupled receptors often contain serine or threonine residues located in their C-termini. The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown. Mutagenesis studies with the B(2) bradykinin receptor (B(2)wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including I347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist-inducible internalization and the prevention of receptor hypersensitivity. Chimeras of the noninternalizing B(1) bradykinin receptor (B(1)wt) containing these B(2)wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required. In fact, further substitution of the B(1)wt C-terminus with corresponding B(2)wt regions either at C330(7.71) following putative helix 8 (B(1)CB(2)) or at the preceding Y312(7.53) in the NPXXY sequence (B(1)YB(2)) resulted in chimeras displaying rapid internalization. Intriguingly, however, exchange performed at K322(7.63) within putative helix 8 generated a slowly internalizing chimera (B(1)KB(2)). Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B(1)wt to serine (as in B(2)wt) as being responsible for this effect. The slowly internalizing chimera as well as a B(1)wt point-mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B(1)wt or the other chimeras. The slow internalization of B(1)KB(2) was also accompanied by a lack of agonist-induced phosphorylation, that in contrast was observed for B(1)YB(2) and B(1)CB(2), suggesting that putative helix 8 is either directly or indirectly (e.g. via G protein activation) involved in the interaction between the receptor and receptor kinases.


Assuntos
Citosol/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Linhagem Celular , Endocitose , Humanos , Dados de Sequência Molecular , Fosforilação , Mutação Puntual , Receptor B1 da Bradicinina/química , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/química , Receptor B2 da Bradicinina/genética
19.
J Biol Chem ; 279(30): 31268-76, 2004 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-15161928

RESUMO

Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPXXY motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/beta-adrenergic-like receptors. Exchange of Tyr(305) in the corresponding NPLVY sequence of the bradykinin B(2) receptor (B(2)R) for Ala resulted in a mutant, termed Y305A, that internalized [(3)H]bradykinin (BK) almost as rapidly as the wild-type (wt) B(2)R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B(2)R. Confocal fluorescence microscopy revealed that, in contrast to the B(2)R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B(2)R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G(q/11) without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B(2)R. We conclude, therefore, that the Y305A mutation of B(2)R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G(q/11), thereby limiting the extent of ligand-independent receptor internalization.


Assuntos
Receptor B2 da Bradicinina/genética , Receptor B2 da Bradicinina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Sequência Conservada , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Técnicas In Vitro , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Fosforilação , Conformação Proteica , Receptor B2 da Bradicinina/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tirosina/química , Tirosina/genética
20.
Am J Physiol Heart Circ Physiol ; 284(6): H1892-8, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12742821

RESUMO

Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B(2) kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 microM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [(3)H]BK internalization (internalized [(3)H]BK in percentage of total bound [(3)H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B(2) kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.


Assuntos
Receptores da Bradicinina/biossíntese , Receptores da Bradicinina/genética , Animais , Bradicinina/metabolismo , Células CHO , Células COS , Linhagem Celular , Cricetinae , Eletroporação , Vetores Genéticos , Humanos , Ligantes , Mutação/genética , Proteínas/metabolismo , Receptor B2 da Bradicinina , Transfecção
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