RESUMEN
BACKGROUND: Mesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype. METHODS: We conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1) in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination. RESULTS: Knockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only) and tumor cells (all three genes). No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene. CONCLUSIONS: We provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma.
Asunto(s)
Mesotelioma/diagnóstico , Mesotelioma/genética , Neoplasias Pleurales/diagnóstico , Neoplasias Pleurales/genética , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Técnicas de Silenciamiento del Gen , Inhibidores de Disociación de Guanina Nucleótido/genética , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Humanos , Mesotelioma/patología , Mesotelioma/cirugía , Mitosis/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenotipo , Neoplasias Pleurales/patología , Neoplasias Pleurales/cirugía , Pronóstico , Interferencia de ARNRESUMEN
PURPOSE: Malignant pleural mesothelioma (MPM) is an aggressive disease associated with median survival between 9 and 12 months. The correct diagnosis of MPM is sometimes challenging and usually requires solid tissue biopsies rather than fine-needle aspiration biopsies (FNA). We postulated that the accuracy of FNA-based diagnosis might be improved by the addition of molecular tests using a gene expression ratio-based algorithm and that prognostic tests could be similarly performed. EXPERIMENTAL DESIGN: Two MPM and 2 lung cancer cell lines were used to establish the minimal quantity of RNA required to perform the gene ratio test. On the basis of these results, 276 ex vivo FNA biopsies from 63 MPM patients and 250 ex vivo FNA samples from 92 lung cancer patients were analyzed using previously described diagnostic and prognostic tests based on gene expression ratios. RESULTS: We found that the sensitivity of the diagnostic test for MPM was 100% [95% confidence interval (CI): 95%-100%] and the specificity in primary lung adenocarcinoma was 90% (95% CI: 81%-95%). The FNA-based prognostic classification was concordant among 76% (95% CI: 65%-87%) of patients with the risk assignment in a subset of the matched surgical specimens previously analyzed by the prognostic test. CONCLUSIONS: Sufficient RNA can be extracted from most FNA biopsies to perform gene expression molecular tests. In particular, we show that the gene expression ratio algorithms performed well when applied to diagnosis and prognosis in MPM. This study provides support for the development of additional RNA molecular tests that may enhance the utility of FNA in the management of other solid cancers.
Asunto(s)
Biopsia con Aguja Fina , Mesotelioma/genética , Neoplasias Pleurales/genética , Línea Celular Tumoral , Expresión Génica , Humanos , Neoplasias Pulmonares/diagnóstico , Mesotelioma/diagnóstico , Técnicas de Diagnóstico Molecular , Neoplasias Pleurales/diagnóstico , Pronóstico , ARN Neoplásico/análisis , Sensibilidad y EspecificidadRESUMEN
The current paradigm for elucidating the molecular etiology of cancers relies on the interrogation of small numbers of genes, which limits the scope of investigation. Emerging second-generation massively parallel DNA sequencing technologies have enabled more precise definition of the cancer genome on a global scale. We examined the genome of a human primary malignant pleural mesothelioma (MPM) tumor and matched normal tissue by using a combination of sequencing-by-synthesis and pyrosequencing methodologies to a 9.6X depth of coverage. Read density analysis uncovered significant aneuploidy and numerous rearrangements. Method-dependent informatics rules, which combined the results of different sequencing platforms, were developed to identify and validate candidate mutations of multiple types. Many more tumor-specific rearrangements than point mutations were uncovered at this depth of sequencing, resulting in novel, large-scale, inter- and intra-chromosomal deletions, inversions, and translocations. Nearly all candidate point mutations appeared to be previously unknown SNPs. Thirty tumor-specific fusions/translocations were independently validated with PCR and Sanger sequencing. Of these, 15 represented disrupted gene-encoding regions, including kinases, transcription factors, and growth factors. One large deletion in DPP10 resulted in altered transcription and expression of DPP10 transcripts in a set of 53 additional MPM tumors correlated with survival. Additionally, three point mutations were observed in the coding regions of NKX6-2, a transcription regulator, and NFRKB, a DNA-binding protein involved in modulating NFKB1. Several regions containing genes such as PCBD2 and DHFR, which are involved in growth factor signaling and nucleotide synthesis, respectively, were selectively amplified in the tumor. Second-generation sequencing uncovered all types of mutations in this MPM tumor, with DNA rearrangements representing the dominant type.
Asunto(s)
Genoma Humano/genética , Mesotelioma/genética , Neoplasias Pleurales/genética , Análisis de Secuencia de ADN/métodos , Aberraciones Cromosómicas , Mapeo Cromosómico , Cromosomas Humanos Par 21/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico/genética , Genes Relacionados con las Neoplasias/genética , Humanos , Mutación INDEL/genética , Cariotipificación , Mutación Puntual/genética , Polimorfismo de Nucleótido Simple/genética , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Malignant pleural mesothelioma has few effective treatments, one being cytoreductive surgery. We previously developed a gene ratio test to predict outcome of malignant pleural mesothelioma patients undergoing surgery. In this study, we investigated the predictive value and technical assay performance of this test in patients with malignant pleural mesothelioma. METHODS: Clinical data were obtained prospectively from 120 consecutive patients with malignant pleural mesothelioma who were scheduled for debulking surgery at one institution. Specimens were obtained at surgery or by pleural biopsy examination. Expression data for four genes were collected from tumor specimens, and three ratios of gene expression (TM4SF1/PKM2, TM4SF1/ARHGDIA, and COBLL1/ARHGDIA) were determined by quantitative reverse transcriptase-polymerase chain reaction. Patients were assigned to good or poor outcome groups by the gene ratio test. Survival was estimated by the Kaplan-Meier method and the log-rank test in univariate analyses. A multivariable Cox proportional hazards model was used to control for prognostic factors. Technical robustness was determined by using up to 30 specimens per patient, two biopsy techniques, and two performance sites. All statistical tests were two-sided. RESULTS: The test predicted overall survival (P < .001) and cancer-specific survival (P = .007) in univariate analysis and overall survival in multivariable analysis (hazard ratio for death = 2.09, 95% confidence interval [CI] = 1.27 to 3.45, P = .004). The test was reproducible within patients and repeatable between two determinations for specimens with widely varying tumor cell contents. Repeatability between two determinations was 88.5% (95% CI = 84.0% to 92.2%) or, when technically unacceptable test values were excluded, 91.9% (95% CI = 87.4% to 95.1%). Reproducibility between two determinations was 96.1% (95% CI = 86.5% to 99.5%). Combining the gene ratio test and other prognostic factors allowed prospective discrimination between patients at high risk (median survival = 6.9 months, 95% CI = 2.6 to 8.9 months; 3-year survival = 0%) and low risk (median survival = 31.9 months, 95% CI = 21.9 to 41.7 months; 3-year survival = 42%). CONCLUSION: The gene ratio test for survival of patients with malignant pleural mesothelioma has robust predictive value and technical assay performance.
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Biomarcadores de Tumor/análisis , Perfilación de la Expresión Génica , Mesotelioma/química , Mesotelioma/mortalidad , Neoplasias Pleurales/química , Neoplasias Pleurales/mortalidad , Adulto , Anciano , Análisis de Varianza , Antígenos de Superficie/análisis , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Mesotelioma/patología , Mesotelioma/cirugía , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Estadificación de Neoplasias , Oportunidad Relativa , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pleurales/patología , Neoplasias Pleurales/cirugía , Valor Predictivo de las Pruebas , Pronóstico , Estudios Prospectivos , Piruvato Quinasa/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples. METHODS: We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens. RESULTS: We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively). CONCLUSION: Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.
Asunto(s)
Empalme Alternativo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Mesotelioma/genética , Neoplasias Pleurales/genética , Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Precursores del ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARNRESUMEN
PURPOSE: Bladder cancer is relatively common but early detection techniques such as cystoscopy and cytology are somewhat limited. We developed a broadly applicable, platform-independent and clinically relevant method based on simple ratios of gene expression to diagnose human cancers. In this study, we sought to determine whether this technique could be applied to the diagnosis of bladder cancer. EXPERIMENTAL DESIGN: We developed a model for the diagnosis of bladder cancer using expression profiling data from 80 normal and tumor bladder tissues to identify statistically significant discriminating genes with reciprocal average expression levels in each tissue type. The expression levels of select genes were used to calculate individual gene pair expression ratios in order to assign diagnosis. The optimal model was examined in two additional published microarray data sets and using quantitative RT-PCR in a cohort of 13 frozen benign bladder urothelium samples and 13 bladder cancer samples from our institution. RESULTS: A five-ratio test utilizing six genes proved to be 100% accurate (26 of 26 samples) for distinguishing benign from malignant bladder tissue samples (P < 10(-6)). CONCLUSIONS: : We have provided a proof of principle study for the use of gene expression ratios in the diagnosis of bladder cancer. This technique may ultimately prove to be a useful adjunct to cytopathology in screening urine specimens for bladder cancer.
RESUMEN
Solid tumors such as mesothelioma exhibit a stubborn resistance to apoptosis that may derive from survival pathways, such as PI3K/Akt/mTOR, that are activated in many tumors, including mesothelioma. To address the role of PI3K/Akt/mTOR, we used a novel approach to study mesothelioma ex vivo as tumor fragment spheroids. Freshly resected mesothelioma tissue from 15 different patients was grown in vitro as 1- to 2-mm-diameter fragments, exposed to apoptotic agents for 48 hours with or without PI3K/Akt/mTOR inhibitors, and doubly stained for cytokeratin and cleaved caspase 3 to identify apoptotic mesothelioma cells. Mesothelioma cells within the tumor spheroids exhibited striking resistance to apoptotic agents such as TRAIL plus gemcitabine that were highly effective against monolayers. In a majority of tumors (67%; 10 of 15), apoptotic resistance could be reduced by more than 50% by rapamycin, an mTOR inhibitor, but not by LY294002, a PI3K inhibitor. Responsiveness to rapamycin correlated with staining for the mTOR target, p-S6K, in the original tumor, but not for p-Akt. As confirmation of the role of mTOR, siRNA knockdown of S6K reproduced the effect of rapamycin in three rapamycin-responsive tumors. Finally, in 37 mesotheliomas on tissue microarray, p-S6K correlated only weakly with p-Akt, suggesting the existence of Akt-independent regulation of mTOR. We propose that mTOR mediates survival signals in many mesothelioma tumors. Inhibition of mTOR may provide a nontoxic adjunct to therapy directed against malignant mesothelioma, especially in those with high baseline expression of p-S6K.
Asunto(s)
Mesotelioma/metabolismo , Mesotelioma/patología , Proteínas Quinasas/metabolismo , Transducción de Señal , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromonas/farmacología , Cicloheximida/farmacología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Mesotelioma/genética , Morfolinas/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Esferoides Celulares , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Serina-Treonina Quinasas TOR , GemcitabinaRESUMEN
Cancers arise by the gradual accumulation of mutations in multiple genes. We now use shotgun pyrosequencing to characterize RNA mutations and expression levels unique to malignant pleural mesotheliomas (MPMs) and not present in control tissues. On average, 266 Mb of cDNA were sequenced from each of four MPMs, from a control pulmonary adenocarcinoma (ADCA), and from normal lung tissue. Previously observed differences in MPM RNA expression levels were confirmed. Point mutations were identified by using criteria that require the presence of the mutation in at least four reads and in both cDNA strands and the absence of the mutation from sequence databases, normal adjacent tissues, and other controls. In the four MPMs, 15 nonsynonymous mutations were discovered: 7 were point mutations, 3 were deletions, 4 were exclusively expressed as a consequence of imputed epigenetic silencing, and 1 was putatively expressed as a consequence of RNA editing. Notably, each MPM had a different mutation profile, and no mutated gene was previously implicated in MPM. Of the seven point mutations, three were observed in at least one tumor from 49 other MPM patients. The mutations were in genes that could be causally related to cancer and included XRCC6, PDZK1IP1, ACTR1A, and AVEN.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Mesotelioma/genética , Mutación , Proteínas de Neoplasias/genética , Neoplasias Pleurales/genética , Receptores de Activinas Tipo I/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Antígenos Nucleares/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Autoantígeno Ku , Proteínas de la Membrana/genética , Mutación Puntual , Edición de ARN , ARN Neoplásico , Eliminación de SecuenciaRESUMEN
Lung cancer is characterized by abnormal cell growth and invasion, and the actin cytoskeleton plays a major role in these processes. The focal adhesion protein paxillin is a target of a number of oncogenes involved in key signal transduction and important in cell motility and migration. In lung cancer tissues, we have found that paxillin was highly expressed (compared with normal lung), amplified (12.1%, 8 of 66) and correlated with increased MET and epidermal growth factor receptor (EGFR) gene copy numbers, or mutated (somatic mutation rate of 9.4%, 18 of 191). Paxillin mutations (19 of 21) were clustered between LD motifs 1 and 2 and the LIM domains. The most frequent point mutation (A127T) enhanced lung cancer cell growth, colony formation, focal adhesion formation, and colocalized with Bcl-2 in vitro. Gene silencing from RNA interference of mutant paxillin led to reduction of cell viability. A murine in vivo xenograft model of A127T paxillin showed an increase in tumor growth, cell proliferation, and invasion. These results establish an important role for paxillin in lung cancer.
Asunto(s)
Neoplasias Pulmonares/patología , Paxillin/metabolismo , Animales , Proliferación Celular , Dosificación de Gen , Genes erbB-1 , Humanos , Neoplasias Pulmonares/etnología , Neoplasias Pulmonares/genética , Ratones , Ratones Endogámicos , Mutación , Invasividad Neoplásica , Paxillin/análisis , Paxillin/genética , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Interferencia de ARNRESUMEN
Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm that is resistant to chemotherapy. Bortezomib is an FDA-approved proteasome inhibitor that is currently under clinical investigation in multiple neoplasms but has not been studied extensively in MPM. In this report, we determine the biological and molecular response of cultured MPM cells to bortezomib alone and in combination with cisplatin or pemetrexed. We used four MPM cell lines (MS589, H28, H2052, JMN), a normal mesothelial cell line (HM3), and a lung cancer cell line (H23) in survival studies utilizing bortezomib, cisplatin, and pemetrexed alone and in combination by administering concurrently or by varying the order of administration. We determined the effect of bortezomib on the cell cycle, apoptosis, and on the expression of cell cycle proteins p21/WAF1 and p27/KIP1 and on apoptosis-related proteins IAP-1, IAP-2, survivin, and XIAP. Bortezomib was highly cytotoxic to MPM cells and induced both G(2)/M and G(1)/S cell cycle arrest. Apoptosis increased in a concentration- and time-dependent manner in 3 of 4 MPM cell lines. Bortezomib stabilized or increased protein levels of p21/WAF1 and IAP-1 and to a lesser degree p27/KIP1, IAP-2, XIAP, and survivin. In combination studies with cisplatin, bortezomib was generally synergistic at high concentrations and antagonistic at low concentrations. Bortezomib increased the cytotoxicity of cisplatin and pemetrexed in a concentration-dependent manner when administered prior to either. Bortezomib may improve outcome in MPM patients alone or in combination with standard chemotherapy but the order of administration is likely to be important. This study justifies further evaluation of bortezomib in MPM.
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Antineoplásicos/uso terapéutico , Ácidos Borónicos/uso terapéutico , Mesotelioma/tratamiento farmacológico , Neoplasias Pleurales/tratamiento farmacológico , Inhibidores de Proteasoma , Pirazinas/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Bortezomib , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Evaluación Preclínica de Medicamentos , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Citometría de Flujo , Genes de Partícula A Intracisternal/efectos de los fármacos , Glutamatos/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Mesotelioma/patología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Pemetrexed , Neoplasias Pleurales/patología , Análisis de SupervivenciaRESUMEN
OBJECTIVE: We have developed a new technique that uses the ratios of select gene expression levels to translate complex genomic data into simple clinically relevant tests for the diagnosis and prognosis of cancer. We determined whether select gene pair ratio combinations can be used to detect and diagnose lung cancer with high accuracy and sensitivity. METHODS: We used gene expression profiling data to train a ratio-based predictor model to discriminate among a set of samples (n = 145 total) composed of normal lung, small cell lung cancer, adenocarcinoma, squamous cell carcinoma, and pulmonary carcinoid (the training set). We then examined the optimal test in an independent set of samples (the test set, n = 122). Finally, we used one aspect of the test to determine whether the gene ratio technique was capable of detecting cancer in specimens from fine-needle aspirations performed ex vivo with normal lung (n = 14) and suspected tumor nodules (n = 15) acquired at our institution. RESULTS: We found that a ratio-based test with 23 genes could be used to classify training set samples with 90% accuracy. This same test was similarly accurate (88%) when applied to the test set of samples. We also found that this test was 87% and 100% accurate at detecting cancer in normal and tumorous fine-needle aspiration specimens, respectively. CONCLUSION: The gene expression ratio diagnostic technique is likely to aid in the differential diagnosis of solitary lung nodules in patients with suspected cancer and may also prove useful in developing lung cancer screening strategies that incorporate analysis of fine-needle aspiration specimens.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Expresión Génica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Nódulo Pulmonar Solitario/diagnóstico , Nódulo Pulmonar Solitario/genética , Biopsia con Aguja , Diagnóstico Diferencial , HumanosRESUMEN
c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal-regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC50 < 2.5 micromol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC50 > 10 micromol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurales/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Activación Enzimática , Factor de Crecimiento Epidérmico/sangre , Factor de Crecimiento de Hepatocito/sangre , Humanos , Indoles/farmacología , Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Mesotelioma/patología , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Piperazinas/farmacología , Neoplasias Pleurales/tratamiento farmacológico , Neoplasias Pleurales/genética , Neoplasias Pleurales/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal , Sulfonamidas/farmacologíaRESUMEN
Integrins are adhesion receptors that transmit signals bidirectionally across the plasma membrane. In our previous report we have shown that the squamous lung cancer cell line, Calu-1, binds to collagen type IV (Coll IV) through beta1-integrin and results in phosphorylation of focal adhesion kinase (FAK) (Ann Thorac Surg 2004; 78:450-457). Considering the critical role of FAK in cell migration, proliferation, and survival, here we investigated potential mechanisms of its activation and regulation in Calu-1 cells. We observed the phosphorylation of Tyr397 of FAK (the autophosphorylation site of FAK) and paxillin, the immediate downstream substrate of FAK following the adhesion of Calu-1 cells to Coll IV. FAK remains phosphorylated during proliferation either on Coll IV or on uncoated plates for 72 h, as determined by peroxivanadate treatment. Exposure of Calu-1 cells with 60 microM genistein, reduces FAK phosphorylation (7.6 fold) and cell proliferation. Extracellular signal regulated kinases (ERKs) were also phosphorylated after Coll IV attachment. Disruption of Calu-1 cell cytoskeleton integrity by 1-5 muM Cytochalasin D resulted in the inhibition of cell adhesion (50% to 75%, p<0.19 - 6.6 x 10(7)) and ERKs phosphorylation (2 fold) without any effect on FAK phosphorylation. Protein Kinase C inhibitor, Calphostin C at 100 and 250 nM concentrations did not block Coll IV induced FAK phosphorylation but activated the ERKs in a dose dependent manner. beta1-integrin is essential for Coll IV induced FAK activation, but it is not physically associated with FAK as determined by immunodetection assay. Collectively, this report defines the existence of multiple and potentially parallel Coll IV/beta1-integrin mediated signaling events in Calu-1 cells, which involve FAK, ERKs, and PKC.
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Neoplasias Pulmonares/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Adhesión Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Colágeno Tipo IV/metabolismo , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Integrina beta1/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Naftalenos/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Tirosina/metabolismoRESUMEN
Mesothelioma is an asbestos-related neoplasm of the thoracic pleura about which little is known and for which effective therapy is lacking. Large-scale transcriptional profiling using microarrays is frequently a part of studies to explore gene expression patterns in cancer and other diseases. In general, microarray based experiments can facilitate the identification of tumor molecular markers, provide clues relating to mechanisms carcinogenesis, as well as aid in the discovery of candidate targets for therapy. Relatively few studies of this sort have been attempted for mesothelioma, likely due to its relatively rare incidence and by extension the difficulty in acquiring suitable tissues for analysis. Microarray analysis of mesothelioma will likely lead to a better understanding of a highly lethal malignancy and result in the identification of potential therapeutic targets to ultimately affect better treatment options and patient clinical outcome. This mini-review will address general issues pertaining to all expression profiling experiments (e.g., data interpretation) and summarize similar studies that have been attempted for mesothelioma.
Asunto(s)
Mesotelioma/genética , Neoplasias Pleurales/genética , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
PURPOSE: Malignant pleural mesothelioma (MPM) is a highly lethal neoplasm with limited pretreatment prognostication strategies. In this report, we examine the accuracy of a previously proposed prognostic test in an independent cohort of MPM patients. This test uses simple ratios of gene expression levels to provide a novel prognostication scheme. EXPERIMENTAL DESIGN: Gene expression data using high-density oligonucleotide microarrays (approximately 22,000 genes) were obtained for a new cohort of human MPM tumors from patients undergoing similar treatments (n = 39). The relative expression levels for specific genes were also determined using real-time quantitative reverse transcription-PCR. We also used a subset of these tumors associated with widely divergent patient survival (n = 23) as a training set to identify new treatment-specific candidate prognostic molecular markers and gene ratio-based prognostic tests. The predictive nature of these newly discovered markers and gene ratio-based prognostic tests were then examined in an independent group of tumors (n = 52) using microarray data and quantitative reverse transcription-PCR. RESULTS: Previously described MPM prognostic genes and gene ratio-based prognostic tests predicted clinical outcome in 39 independent MPM tumor specimens in a statistically significant manner. Newly discovered treatment-specific prognostic genes and gene ratio-based prognostic tests were highly accurate and statistically significant when examined in an independent group of 52 tumors from patients undergoing similar treatment. CONCLUSIONS: The data support the use of gene ratios in translating gene expression data into easily reproducible, statistically validated clinical tests for the prediction of outcome in MPM.
Asunto(s)
Genómica/métodos , Mesotelioma/patología , Neoplasias Pleurales/patología , Adulto , Anciano , Estudios de Cohortes , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Mesotelioma/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Neoplasias Pleurales/genética , Pronóstico , Reproducibilidad de los Resultados , Análisis de SupervivenciaRESUMEN
Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n = 40), normal lung specimens (n = 4), normal pleura specimens (n = 5), and MPM and SV40-immortalized mesothelial cell lines (n = 5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma that correlated loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (ie, software) to create and explore complex physiological pathways. Combined, all of these data may advance our understanding of mesothelioma tumorigenesis, pathobiology, or both.
Asunto(s)
Perfilación de la Expresión Génica , Genes Supresores de Tumor , Mesotelioma/genética , Oncogenes , Neoplasias Pleurales/genética , Western Blotting , Línea Celular Tumoral , Humanos , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
MPM is a poorly understood lethal malignancy. Although the pathobiology of MPM is not completely elucidated, new genomic technology is likely to help shed light on the mechanisms of carcinogenesis through genome-wide screening of tumor-specific gene expression. Related efforts to identify the molecular markers of mesothelioma are pursued with the aim of refining current diagnostic capabilities, predicting prognosis, and designing appropriate trimodality programs. These new genomic tools also will assist efforts to tailor current adjuvant and neoadjuvant therapies, optimizing their effect and furthering research that may lead to new therapeutic options.
Asunto(s)
Perfilación de la Expresión Génica , Mesotelioma/genética , Neoplasias Pleurales/genética , Apoptosis/genética , Biomarcadores de Tumor , Aberraciones Cromosómicas , Diagnóstico Diferencial , Genes Supresores de Tumor , Humanos , Mesotelioma/patología , Estadificación de Neoplasias , Neoplasias Pleurales/patología , Proteínas Proto-Oncogénicas/genéticaRESUMEN
BACKGROUND: The clinical phenomenon of lung cancer metastasis to specific target organs is believed to be a direct interaction between tumor cells and extracellular matrix components. During invasion, tumor cells attach to the basement membrane protein, collagen type IV, degrade it, migrate through blood vessels, and adhere to extracellular matrix proteins. METHODS: Four nonsmall-cell lung cancer cells were tested for adhesion, proliferation, migration and morphologic alterations on collagen type IV matrix by immunoprecipitation, Western blotting, phase contrast and time lapse video microscopy. RESULTS: Collagen type IV promoted Calu-1 cell adhesion (< 15 minutes) and motility (< 6 hours) without any significant effect on proliferation. beta(1)-integrin is essential for collagen type IV adhesion and 8 to 10 fold higher expression of beta1-integrin on the surface of Calu-1 cells was identified. A protein tyrosine phosphatase inhibitor, peroxyvanadate, caused 50% inhibition in the adhesion process within 5 minutes but exposure to 60 micromol/L genistein for 72 hours, a protein tyrosine kinase inhibitor, drastically inhibits Calu-1 cell proliferation (> 70%). We examined the influence of beta1-integrin, peroxyvanadate and genistein on the spreading morphogenesis of Calu-1 cells on Collagen type IV. Use of either 1 microg of anti beta1-integrin inhibitory antibody (P5D2), 100 micromol/L peroxyvanadate or 60 micromol/L genistein had dramatic influence on the spreading of Calu-1 cells. Finally, Focal adhesion kinase was identified as a phosphoprotein target. CONCLUSIONS: We have characterized an in vitro model of matrix-specific binding of a lung cancer cell line, Calu-1 to Coll IV. Calu-1 cells use primarily a beta1-integrin mediated intracellular tyrosine phosphorylation phenomenon as the key regulatory mechanism for its binding advantage to Coll IV matrix.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Colágeno Tipo IV/metabolismo , Integrina beta1/fisiología , Neoplasias Pulmonares/patología , Invasividad Neoplásica/fisiopatología , Proteínas de Neoplasias/fisiología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular Tumoral/citología , Línea Celular Tumoral/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Tamaño de la Célula/efectos de los fármacos , Medio de Cultivo Libre de Suero , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Genisteína/farmacología , Humanos , Microscopía por Video , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/fisiología , Seudópodos/efectos de los fármacos , Vanadatos/farmacologíaRESUMEN
PURPOSE: Multiple recent studies show excellent classification accuracy using bioinformatics tools applied to expression profiling data on various tumors. However, the clinical applicability of these techniques remains unfulfilled because of difficulty in translating complex multigene mathematical algorithms into reproducible, platform independent tests. We recently developed a broadly applicable platform independent method based on simple ratios of gene expression to diagnose and predict outcome in cancer. In the current study we applied this technique to the diagnosis of prostate cancer. MATERIALS AND METHODS: We developed a ratio based predictive model using a training set of 32 samples with previously published gene profiling data. We then tested and refined the model using additional independent samples with previously published microarray data from another source (that is the test set of 34 samples). Finally, the optimal ratio based test was examined with quantitative reverse transcriptase-polymerase chain reaction for data acquisition in a third cohort of samples consisting of 10 frozen normal and 10 tumor prostate tissues. RESULTS: A 3-ratio test using 4 genes was 90% accurate (18 of 20 samples) for distinguishing normal prostate and prostate cancer samples obtained at surgery (Fisher's exact test p = 0.0007). This test did not result in any false-negative findings. CONCLUSIONS: We describe and validate a new gene ratio based test for the diagnosis of prostate cancer, which was developed from the analysis of extensive gene profiling data for the diagnosis of prostate cancer. This test can be easily adapted to the clinical arena without the need for complex computer software or hardware. We anticipate that the gene ratio based diagnosis of prostate cancer using fine needle aspirations could serve as a useful adjunct to standard histopathological techniques.