RESUMO
UNLABELLED: We present rCGH, a comprehensive array-based comparative genomic hybridization analysis workflow, integrating computational improvements and functionalities specifically designed for precision medicine. rCGH supports the major microarray platforms, ensures a full traceability and facilitates profiles interpretation and decision-making through sharable interactive visualizations. AVAILABILITY AND IMPLEMENTATION: The rCGH R package is available on bioconductor (under Artistic-2.0). The aCGH-viewer is available at https://fredcommo.shinyapps.io/aCGH_viewer, and the application implementation is freely available for installation at https://github.com/fredcommo/aCGH_viewer CONTACT: frederic.commo@gustaveroussy.fr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Hibridização Genômica Comparativa , Genômica , Medicina de Precisão , Humanos , SoftwareRESUMO
BACKGROUND: Major advances have been achieved in the characterization of early breast cancer (eBC) genomic profiles. Metastatic breast cancer (mBC) is associated with poor outcomes, yet limited information is available on the genomic profile of this disease. This study aims to decipher mutational profiles of mBC using next-generation sequencing. METHODS AND FINDINGS: Whole-exome sequencing was performed on 216 tumor-blood pairs from mBC patients who underwent a biopsy in the context of the SAFIR01, SAFIR02, SHIVA, or Molecular Screening for Cancer Treatment Optimization (MOSCATO) prospective trials. Mutational profiles from 772 primary breast tumors from The Cancer Genome Atlas (TCGA) were used as a reference for comparing primary and mBC mutational profiles. Twelve genes (TP53, PIK3CA, GATA3, ESR1, MAP3K1, CDH1, AKT1, MAP2K4, RB1, PTEN, CBFB, and CDKN2A) were identified as significantly mutated in mBC (false discovery rate [FDR] < 0.1). Eight genes (ESR1, FSIP2, FRAS1, OSBPL3, EDC4, PALB2, IGFN1, and AGRN) were more frequently mutated in mBC as compared to eBC (FDR < 0.01). ESR1 was identified both as a driver and as a metastatic gene (n = 22, odds ratio = 29, 95% CI [9-155], p = 1.2e-12) and also presented with focal amplification (n = 9) for a total of 31 mBCs with either ESR1 mutation or amplification, including 27 hormone receptor positive (HR+) and HER2 negative (HER2-) mBCs (19%). HR+/HER2- mBC presented a high prevalence of mutations on genes located on the mechanistic target of rapamycin (mTOR) pathway (TSC1 and TSC2) as compared to HR+/HER2- eBC (respectively 6% and 0.7%, p = 0.0004). Other actionable genes were more frequently mutated in HR+ mBC, including ERBB4 (n = 8), NOTCH3 (n = 7), and ALK (n = 7). Analysis of mutational signatures revealed a significant increase in APOBEC-mediated mutagenesis in HR+/HER2- metastatic tumors as compared to primary TCGA samples (p < 2e-16). The main limitations of this study include the absence of bone metastases and the size of the cohort, which might not have allowed the identification of rare mutations and their effect on survival. CONCLUSIONS: This work reports the results of the analysis of the first large-scale study on mutation profiles of mBC. This study revealed genomic alterations and mutational signatures involved in the resistance to therapies, including actionable mutations.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Exoma , Mutação , Feminino , Humanos , Metástase Neoplásica , Estudos Retrospectivos , Análise de Sequência de DNARESUMO
BACKGROUND: The excision repair cross-complementation group 1 (ERCC1) protein is a potential prognostic biomarker of the efficacy of cisplatin-based chemotherapy in non-small-cell lung cancer (NSCLC). Although several ongoing trials are evaluating the level of expression of ERCC1, no consensus has been reached regarding a method for evaluation. METHODS: We used the 8F1 antibody to measure the level of expression of ERCC1 protein by means of immunohistochemical analysis in a validation set of samples obtained from 494 patients in two independent phase 3 trials (the National Cancer Institute of Canada Clinical Trials Group JBR.10 and the Cancer and Leukemia Group B 9633 trial from the Lung Adjuvant Cisplatin Evaluation Biology project). We compared the results of repeated staining of the entire original set of samples obtained from 589 patients in the International Adjuvant Lung Cancer Trial Biology study, which had led to the initial correlation between the absence of ERCC1 expression and platinum response, with our previous results in the same tumors. We mapped the epitope recognized by 16 commercially available ERCC1 antibodies and investigated the capacity of the different ERCC1 isoforms to repair platinum-induced DNA damage. RESULTS: We were unable to validate the predictive effect of immunostaining for ERCC1 protein. The discordance in the results of staining for ERCC1 suggested a change in the performance of the 8F1 antibody since 2006. We found that none of the 16 antibodies could distinguish among the four ERCC1 protein isoforms, whereas only one isoform produced a protein that had full capacities for nucleotide excision repair and cisplatin resistance. CONCLUSIONS: Immunohistochemical analysis with the use of currently available ERCC1 antibodies did not specifically detect the unique functional ERCC1 isoform. As a result, its usefulness in guiding therapeutic decision making is limited. (Funded by Eli Lilly and others.).
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/uso terapêutico , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Quimioterapia Adjuvante , DNA de Neoplasias , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Resistencia a Medicamentos Antineoplásicos/genética , Endonucleases/genética , Endonucleases/imunologia , Mapeamento de Epitopos , Epitopos , Humanos , Imunoglobulina G , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
BACKGROUND: Breast cancer is characterised by genomic alterations. We did a multicentre molecular screening study to identify abnormalities in individual patients with the aim of providing targeted therapy matched to individuals' genomic alterations. METHODS: From June 16, 2011, to July 30, 2012, we recruited patients who had breast cancer with a metastasis accessible for biopsy in 18 centres in France. Comparative genomic hybridisation (CGH) array and Sanger sequencing on PIK3CA (exon 10 and 21) and AKT1 (exon 4) were used to assess metastatic biopsy samples in five centres. Therapeutic targets were decided on the basis of identified genomic alterations. The primary objective was to include 30% of patients in clinical trials testing a targeted therapy and, therefore, the primary outcome was the proportion of patients to whom a targeted therapy could be offered. For the primary endpoint, the analyses were done on the overall population registered for the trial. This trial is registered with ClinicalTrials.gov, number NCT01414933. FINDINGS: 423 patients were included, and biopsy samples were obtained from 407 (metastatic breast cancer was not found in four). CGH array and Sanger sequencing were feasible in 283 (67%) and 297 (70%) patients, respectively. A targetable genomic alteration was identified in 195 (46%) patients, most frequently in PIK3CA (74 [25%] of 297 identified genomic alterations), CCND1 (53 [19%]), and FGFR1 (36 [13%]). 117 (39%) of 297 patients with genomic tests available presented with rare genomic alterations (defined as occurring in less than 5% of the general population), including AKT1 mutations, and EGFR, MDM2, FGFR2, AKT2, IGF1R, and MET high-level amplifications. Therapy could be personalised in 55 (13%) of 423 patients. Of the 43 patients who were assessable and received targeted therapy, four (9%) had an objective response, and nine others (21%) had stable disease for more than 16 weeks. Serious (grade 3 or higher) adverse events related to biopsy were reported in four (1%) of enrolled patients, including pneumothorax (grade 3, one patient), pain (grade 3, one patient), haematoma (grade 3, one patient), and haemorrhagic shock (grade 3, one patient). INTERPRETATION: Personalisation of medicine for metastatic breast cancer is feasible, including for rare genomic alterations. FUNDING: French National Cancer Institute, Breast Cancer Research Foundation, Odyssea, Operation Parrains Chercheurs.
Assuntos
Neoplasias da Mama/genética , Hibridização Genômica Comparativa/métodos , Análise de Sequência de DNA/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Feminino , Genes erbB-2 , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Estudos ProspectivosRESUMO
PURPOSE: Targeted therapies that use the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets for overcoming chemoresistance.Experimental Design: We established 4 chemoresistant prostate cancer cell lines and used image-based high-content siRNA functional screening, based on gene-expression signature, to explore mechanisms of chemoresistance and identify new potential targets with potential roles in taxane resistance. The functional role of a new target was assessed by in vitro and in vivo silencing, and mass spectrometry analysis was used to identify its downstream effectors. RESULTS: We identified FKBP7, a prolyl-peptidyl isomerase overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. This is the first study to characterize the function of human FKBP7 and explore its role in cancer. We discovered that FKBP7 was upregulated in human prostate cancers and its expression correlated with the recurrence observed in patients receiving docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with eIF4G, a component of the eIF4F translation initiation complex, to mediate the survival of chemoresistant cells. Using small-molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to kill docetaxel- and cabazitaxel-resistant cells. CONCLUSIONS: Targeting FKBP7 or the eIF4G-containing eIF4F translation initiation complex could be novel therapeutic strategies to eradicate taxane-resistant prostate cancer cells.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fator de Iniciação 4F em Eucariotos/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Taxoides/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Biologia Computacional , Modelos Animais de Doenças , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Ligação Proteica , RNA Interferente Pequeno/genética , Transcriptoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play key roles in tumorigenesis, but little is known of their expression according to mucinous or serous type. This study aimed to evaluate the immunohistochemical expression of MMP-2, -7, -9, MT1-MMP, TIMP-1 and -2 in these tumors. A tissue microarray was set up including 99 serous (25 benign, 27 borderline, 47 malignant) and 79 mucinous (25 benign, 44 borderline, 10 malignant) ovarian tumors. Immunostaining results were scored by using the HSCORE and assessed by univariate, unsupervised hierarchical clustering and multidimensional scaling analyses. Epithelial expression of MMP-2, -7, -9, MT1-MMP, TIMP-2, but not TIMP-1, was higher in serous than mucinous tumors. Stromal expression of MMP-7 was higher in serous tumors. Alterations in MT1-MMP, MMP-7 and -9 were found in malignant serous tumors, while benign and borderline tumors shared similar expressions. By unsupervised hierarchical clustering analysis, mucinous and serous tumors were better differentiated by epithelial than stromal MMP and TIMP immunolabelling. By multidimensional scaling analysis, the expressions of MMPs and TIMPs were scattered in serous tumors and homogeneous for mucinous tumors. In conclusion, our results support the differential expression in MMPs and TIMPs of ovarian tumors according to serous or mucinous histology.
Assuntos
Cistadenoma Mucinoso/química , Cistadenoma Seroso/química , Metaloproteinases da Matriz/análise , Neoplasias Ovarianas/química , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-2/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise por Conglomerados , Cistadenoma Mucinoso/enzimologia , Cistadenoma Mucinoso/patologia , Cistadenoma Seroso/enzimologia , Cistadenoma Seroso/patologia , Feminino , Humanos , Imuno-Histoquímica , Metaloproteinase 14 da Matriz/análise , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 7 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , Pessoa de Meia-Idade , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Análise Serial de Tecidos , Adulto JovemRESUMO
The duration and magnitude of clinical response are unpredictable in ALK-rearranged non-small cell lung cancer (NSCLC) patients treated with crizotinib, although all patients invariably develop resistance. Here, we evaluated whether circulating tumor cells (CTC) with aberrant ALK-FISH patterns [ALK-rearrangement, ALK-copy number gain (ALK-CNG)] monitored on crizotinib could predict progression-free survival (PFS) in a cohort of ALK-rearranged patients. Thirty-nine ALK-rearranged NSCLC patients treated with crizotinib as first ALK inhibitor were recruited prospectively. Blood samples were collected at baseline and at an early time-point (2 months) on crizotinib. Aberrant ALK-FISH patterns were examined in CTCs using immunofluorescence staining combined with filter-adapted FISH after filtration enrichment. CTCs were classified into distinct subsets according to the presence of ALK-rearrangement and/or ALK-CNG signals. No significant association between baseline numbers of ALK-rearranged or ALK-CNG CTCs and PFS was observed. However, we observed a significant association between the decrease in CTC number with ALK-CNG on crizotinib and a longer PFS (likelihood ratio test, P = 0.025). In multivariate analysis, the dynamic change of CTC with ALK-CNG was the strongest factor associated with PFS (HR, 4.485; 95% confidence interval, 1.543-13.030, P = 0.006). Although not dominant, ALK-CNG has been reported to be one of the mechanisms of acquired resistance to crizotinib in tumor biopsies. Our results suggest that the dynamic change in the numbers of CTCs with ALK-CNG may be a predictive biomarker for crizotinib efficacy in ALK-rearranged NSCLC patients. Serial molecular analysis of CTC shows promise for real-time patient monitoring and clinical outcome prediction in this population. Cancer Res; 77(9); 2222-30. ©2017 AACR.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Resistencia a Medicamentos Antineoplásicos/genética , Receptores Proteína Tirosina Quinases/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Quinase do Linfoma Anaplásico , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Crizotinibe , Variações do Número de Cópias de DNA/genética , Intervalo Livre de Doença , Feminino , Rearranjo Gênico/efeitos dos fármacos , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/patologia , Prognóstico , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Receptores Proteína Tirosina Quinases/genéticaRESUMO
The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.
Assuntos
Antígenos CD57/metabolismo , Necrose Tubular Aguda/metabolismo , Rim/metabolismo , Animais , Biomarcadores/metabolismo , Epitopos , Humanos , Imuno-Histoquímica , Imunoprecipitação , Rim/embriologia , Rim/patologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Necrose Tubular Aguda/patologia , Alça do Néfron/embriologia , Alça do Néfron/metabolismo , Alça do Néfron/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Proteoglicanas/metabolismo , Coelhos , Ratos , Ratos WistarRESUMO
PURPOSE: Expression of erythropoietin (Epo) and its receptor (Epo-R) has been shown in various normal and neoplastic nonhematopoietic tissues. This study, in non-small cell lung carcinoma, was designed to investigate the previously unreported expression of Epo and Epo-R as well as hypoxia-inducible factor-1alpha (HIF-1alpha), which is known to control Epo expression. EXPERIMENTAL DESIGN: Samples from lung squamous cell carcinomas (n = 17) and adenocarcinomas (n = 12) were obtained from patients undergoing curative surgery. mRNA transcripts of Epo, Epo-R, soluble Epo-R (sEpo-R), HIF-1alpha, and factor inhibiting HIF-1 (FIH-1) were evaluated by reverse transcription-PCR, whereas localization of Epo, Epo-R, and HIF-1alpha was assessed by immunohistochemistry. RESULTS: Epo, Epo-R, sEpo-R, HIF-1alpha, and FIH-1 transcripts were detected by reverse transcription-PCR in all samples tested, but with heterogeneous levels of expression for Epo, Epo-R, and sEpo-R. Coordinated levels of mRNA were observed for HIF-1alpha and FIH-1.Epo was detected in carcinomatous cells by immunohistochemistry in 50% of samples and Epo-R was detected in 96% of samples. Co-expression of Epo and Epo-R was observed on contiguous sections from 50% of tumors. HIF-1alpha was immunolocalized in 80% of non-small cell lung carcinomas. CONCLUSION: Epo-R was expressed in almost all samples and Epo was expressed in one half of samples on immunohistochemistry and in 100% of samples by mRNA detection, suggesting a potential paracrine and/or autocrine role of endogenous Epo in non-small cell lung carcinoma. The detection of stabilized HIF-1alpha suggests a possible role in Epo expression. Moreover, in the light of these results, the potential interactions between therapeutic recombinant Epo and the putative neoplastic Epo/Epo-R signaling pathways must be considered.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Eritropoetina/genética , Neoplasias Pulmonares/patologia , Receptores da Eritropoetina/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Eritropoetina/análise , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica , Antígeno Ki-67/análise , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Oxigenases de Função Mista , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores da Eritropoetina/análise , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Transcrição/análise , Fatores de Transcrição/genéticaRESUMO
Cubilin and megalin are multiligand epithelial endocytic receptors well characterized in the adult kidney and ileum where they form a complex essential for protein, lipid and vitamin uptake. Although inactivation of the megalin gene leads to holoprosencephaly and administration of anti-cubilin antibodies induces fetal resorptions or cranio-facial malformations their function in the developing embryo remains unclear. We recently showed that both proteins are strongly expressed by the maternal-fetal interfaces and the neuroepithelium of the early rodent embryo where they co-localize and form a complex important for nutrient uptake. The aim of the present study was the further investigation of cubilin expression at later developmental stages of the rodent embryo and its correlation to that of megalin. Immunohistochemical and in situ hybridization analysis showed striking similarities in the spatial and temporal expression patterns of cubilin and megalin. The electrophoretic mobility of both proteins was identical to that of the adult as revealed by Western blot analysis. Cubilin and megalin were strongly expressed in the sensory organs, the central nervous system, the respiratory and urogenital tracts as well as in the thymus, parathyroids and thyroid. In each site, the expression mainly concerned epithelial structures and correlated with the onset of epithelial induction. Depending on the site, a decreased or restricted expression was observed by the end of the gestation for both proteins.
Assuntos
Sistema Nervoso Central/embriologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Ratos/embriologia , Receptores de Superfície Celular/metabolismo , Órgãos dos Sentidos/embriologia , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Sistema Nervoso Central/química , Sistema Nervoso Central/metabolismo , Orelha/embriologia , Embrião de Mamíferos/química , Embrião de Mamíferos/metabolismo , Endocitose , Epitélio/embriologia , Epitélio/metabolismo , Olho/química , Olho/embriologia , Olho/metabolismo , Feminino , Trato Gastrointestinal/química , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Imunoquímica , Hibridização In Situ , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/análise , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Mucosa Nasal/metabolismo , Nariz/química , Nariz/embriologia , Glândulas Paratireoides/química , Glândulas Paratireoides/embriologia , Glândulas Paratireoides/metabolismo , Ratos/genética , Ratos/metabolismo , Ratos Wistar , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/genética , Sistema Respiratório/química , Sistema Respiratório/embriologia , Sistema Respiratório/metabolismo , Órgãos dos Sentidos/química , Órgãos dos Sentidos/metabolismo , Medula Espinal/química , Medula Espinal/embriologia , Medula Espinal/metabolismo , Timo/química , Timo/embriologia , Timo/metabolismo , Glândula Tireoide/química , Glândula Tireoide/embriologia , Glândula Tireoide/metabolismo , Distribuição Tecidual , Sistema Urogenital/química , Sistema Urogenital/embriologiaRESUMO
Type 1 plasminogen activator inhibitor (PAI-1) is the primary inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Whereas PAI-1 is not expressed in normal kidneys, it is strongly induced in glomerular diseases and thus could promote the local accumulation of fibrin. To study the role of PAI-1 in the development of inflammatory glomerular injury, passive antiglomerular basement membrane (GBM) glomerulonephritis (GN) was induced in PAI-1 knockout mice and in wild-type mice of the same genetic background. Unexpectedly, PAI-1 deficiency was associated with an early and severe exacerbation of glomerular injury: Infiltration by CD4 T cells, proportion of fibrinous crescents, and renal function impairment were significantly more pronounced in PAI-1 -/- mice. Interestingly, activation of transforming growth factor (TGF)- beta, which is known to be dependent on the PA/plasmin system in vitro, was dramatically enhanced in the kidneys in the absence of PAI-1. Moreover, administration of neutralizing antibodies against TGF-beta significantly attenuated the disease in PAI-1 -/- mice. This suggests that the poor outcome of GN in PAI-1 -/- mice is consecutive to an uncontrolled activation of TGF-beta and confers PAI-1 with a new, immunomodulatory role.
Assuntos
Glomerulonefrite/etiologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Rim/metabolismo , Rim/fisiopatologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Inibidor 1 de Ativador de Plasminogênio/genética , Ativadores de Plasminogênio/metabolismo , Trombina/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
PURPOSE: Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. EXPERIMENTAL DESIGN: p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. RESULTS: Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. CONCLUSIONS: The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.
Assuntos
Análise Mutacional de DNA/métodos , Genes p53 , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutação , Oligonucleotídeos/genética , Idoso , Alelos , Biópsia , Estudos de Coortes , DNA/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Ácidos Nucleicos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Estudos Prospectivos , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/metabolismoRESUMO
The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification.
Assuntos
Antígenos B7/metabolismo , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Neuroblastoma/metabolismo , Adolescente , Adulto , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Criança , Pré-Escolar , Intervalo Livre de Doença , Humanos , Lactente , Células Jurkat , Ligantes , Metástase Neoplásica , Neuroblastoma/mortalidade , Fenótipo , Prognóstico , Estudos Prospectivos , Ligação Proteica , Fatores de Risco , Adulto JovemRESUMO
Docetaxel is used as a standard treatment in patients with metastatic castration-resistant prostate cancer. However, a large subset of patients develops resistance. Understanding resistance mechanisms, which are largely unknown, will allow identification of predictive biomarkers and therapeutic targets. We established resistant IGR-CaP1 prostate cancer cell lines for different doses of Docetaxel. We investigated gene expression profiles by microarray analyses in these cell lines and generated a signature of 99 highly differentially expressed genes potentially implicated in chemoresistance. We focused on the role of the cell cycle regulator LZTS1, which was under-expressed in the Docetaxel-resistant cell lines, its inhibition resulting from the promoter methylation. Knockdown of LZTS1 in parental cells with siRNA showed that LZTS1 plays a role in the acquisition of the resistant phenotype. Furthermore, we observed that targeting CDC25C, a partner of LZTS1, with the NSC663284 inhibitor specifically killed the Docetaxel-resistant cells. To further investigate the role of CDC25C, we used inhibitors of the mitotic kinases that regulate CDC25C. Inhibition of CHEK1 and PLK1 induced growth arrest and cell death in the resistant cells. Our findings identify an important role of LZTS1 through its regulation of CDC25C in Docetaxel resistance in prostate cancer and suggest that CDC25C, or the mitotic kinases CHEK1 and PLK1, could be efficient therapeutic targets to overcome Docetaxel resistance.
Assuntos
Proteínas de Ligação a DNA/deficiência , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Proteínas Quinases/metabolismo , Taxoides/farmacologia , Proteínas Supressoras de Tumor/deficiência , Fosfatases cdc25/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Terapia de Alvo Molecular , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Análise Serial de Tecidos , Transcriptoma , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fosfatases cdc25/metabolismo , Quinase 1 Polo-LikeRESUMO
BACKGROUND: In lung adenocarcinoma, inactivation of the tumor suppressor p53 may abrogate a safeguard mechanism preventing the development of tumors with activating mutations in EGFR or KRAS. To assess this hypothesis, we analyzed TP53 mutations and downregulation of p14(arf), a negative regulator of p53 activated by oncogenic signals, in a retrospective series of 96 patients with primary adenocarcinoma of the lung. PATIENTS AND METHODS: Mutations in TP53 (exons 4-9), KRAS (exon 1), and EGFR (exons 18-21) were identified by direct sequencing of DNA from formalin-fixed, paraffin-embedded resected tumors. Expression of p14(arf) was semiquantitatively evaluated by immunohistochemical analysis. RESULTS: TP53, KRAS, and EGFR mutations were detected in 42 of 93 (45.2%), 15 of 95 (15.8%), and 31 of 90 (34.4%) cases, respectively. Low p14(arf) expression was observed in 19 of 91 cases (20.9%). Disruption of the p53/p14(arf) pathway (defined as TP53 mutation or decreased p14(arf) expression, or both) was observed in 18 of 31 EGFR-mutated (58.1%) tumors and in 9 of 13 KRAS-mutated (69.2%) tumors. CONCLUSION: Inactivation of the p53/p14(arf) pathway is common but not systematic in EGFR- or KRAS-mutated lung adenocarcinomas. Our work highlights the need to better investigate the association between EGFR and KRAS mutations and alterations in tumor suppressor pathways.
Assuntos
Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Proto-Oncogênicas p21(ras) , Estudos Retrospectivos , Transdução de SinaisRESUMO
BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare tumor that is caused by activating mutations in the proto-oncogene RET. Vandetanib, a tyrosine-kinase inhibitor, has been recently approved to treat adult patients with metastatic MTC. The aim of this study was to investigate changes in signaling pathways induced by vandetanib treatment in preclinical MTC models, using the reverse-phase protein array method (RPPA). METHODS: The human TT cell line was used to assess in vitro and in vivo activity of vandetanib. Protein extracts from TT cells or TT xenografted mice, treated by increasing concentrations of vandetanib for different periods of time, were probed with a set of 12 antibodies representing major signaling pathways, using RPPA. Results were validated using two distinct protein detection methods: Western immunoblotting and immunohistochemistry. RESULTS: Vandetanib displays antiproliferative and antiangiogenic activities and inhibits RET autophosphorylation. The MAPK and AKT pathways were the two major signaling pathways inhibited by vandetanib. Interestingly, phosphorylated levels of NFκB-p65 were significantly increased by vandetanib. Comparable results were obtained in both the in vitro and in vivo approaches, as well as for the protein detection methods. However, some discrepancies were observed between RPPA and Western immunoblotting, possibly due to lack of specificity of the primary antibodies used. CONCLUSIONS: Overall, our results confirmed the interest of RPPA for screening global changes induced in signaling pathways by kinase inhibitors. MAPK and AKT were identified as the main pathways involved in vandetanib response in MTC models. Our results also suggest alternative routes for controlling the disease, and provide a rationale for the development of therapeutic combinations based on the comprehensive identification of molecular events induced by inhibitors.
Assuntos
Piperidinas/uso terapêutico , Quinazolinas/uso terapêutico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Adulto , Animais , Carcinoma Neuroendócrino , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVES: Bioinformatics analyses of pathways and genes differentially expressed between malignant and benign lesions could allow discovering new therapeutic targets. Here, we identified Checkpoint kinase 1 (Chk1) as a potent therapeutic target in triple-negative breast cancer (TNBC). MATERIALS AND METHODS: Differential gene expression between TNBC, other malignant and benign lesions was performed on two breast cancer datasets. Chk1 was targeted using RNA interference or chemical inhibitor in several TNBC cell lines. RESULTS: DNA repair pathway was identified as one mostly deregulated pathway in TNBC as compared to benign lesions. Chk1 was identified as candidate target among the 35 genes included in this pathway. Gene expression analysis revealed that Chk1 gene was significantly overexpressed in TNBC as compared to non-TNBC and benign lesions. Depletion of Chk1 protein expression induced a marked reduction of cell viability and led to mitotic catastrophe in TNBC cells. Chemical Chk1 inhibitor decreased survival in TNBC cells, and transcriptome analyze revealed a modulation of gene expression profile in response to Chk1 treatment. CONCLUSION: These findings suggest that Chk1 may represent a therapeutic target in TNBC, and provide a rationale to evaluate Chk1 inhibitors in breast cancer patients.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 1 do Ponto de Checagem , Reparo do DNA/efeitos dos fármacos , Reposicionamento de Medicamentos , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Humanos , Terapia de Alvo Molecular , Interferência de RNA , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/terapiaRESUMO
Natural killer (NK) cells play a prominent role at the intersection between innate and cognate immunity, thus influencing the development of multiple pathological conditions including HIV-1-induced AIDS. Not only NK cells directly kill HIV-1-infected cells, but also control the maturation and/or elimination of dendritic cells (DCs). These functions are regulated by the delicate balance between activating and inhibiting receptors expressed at the NK-cell surface. Among the former, NKp30 has raised significant interest since the alternative splicing of its intracellular domain leads to differential effector functions, dictating the prognosis of patients bearing gastrointestinal sarcoma, and B7-H6 has recently been identified as its main ligand. Since NKp30 is downregulated in CD56-/CD16+ NK cells expanded in viremic, chronically infected HIV-1+ patients, we decided to investigate the predictive value of NKp30 splice variants for spontaneous disease progression in 89 therapy-naïve HIV-1-infected individuals enrolled in an historical cohort of patients followed since diagnosis (ANRS SEROCO cohort). We found no difference in the representation of NK-cell subsets (CD56bright, CD56dim, CD56neg) in HIV-1-infected patients as compared with healthy subjects. NKp30 downregulation was detected in CD56dim and CD56neg NK-cell subsets, yet this did not convey any prognostic value. None of the NKp30 isoforms did affect disease progression, as measured in terms of time-to-loss of circulating CD4+ T cells, time-to-AIDS-defining events and overall survival. NKp30 isoforms do not seem to play a major role in the outcome of HIV-1 infection, but the heterogeneity of the immuno-virological status of patients at enrollment could have to be taken into account.
RESUMO
The progressive introduction of high-throughput molecular techniques in the clinic allows for the extensive and systematic exploration of multiple biologic layers of tumors. Molecular profiles and classifiers generated from these assays represent the foundation of what the National Academy describes as the future of "precision medicine". However, the analysis of such complex data requires the implementation of sophisticated bioinformatic and statistical procedures. It is critical that oncology practitioners be aware of the advantages and limitations of the methods used to generate classifiers to usher them into the clinic. This article uses publicly available expression data from patients with non-small cell lung cancer to first illustrate the challenges of experimental design and preprocessing of data before clinical application and highlights the challenges of high-dimensional statistical analysis. It provides a roadmap for the translation of such classifiers to clinical practice and makes key recommendations for good practice.
Assuntos
Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Controle de Qualidade , Projetos de Pesquisa , Pesquisa Translacional BiomédicaRESUMO
BACKGROUND: Medullary thyroid carcinoma (MTC) is a rare tumor that is due to activating mutations in the proto-oncogene RET. Vandetanib, a tyrosine-kinase inhibitor, has been recently approved to treat adult patients with metastatic MTC. The aim of this study was to investigate changes in signaling pathways induced by vandetanib treatment in preclinical MTC models, using the reverse-phase protein array method (RPPA). METHODS: The human TT cell line was used to assess in vitro and in vivo activity of vandetanib. Protein extracts from TT cells or TT xenografted mice, treated by increasing concentrations of vandetanib for different periods of time, were probed with a set of 12 antibodies representing major signaling pathways, using RPPA. Results were validated using two distinct protein detection methods, western-immunoblotting and immunohistochemistry. RESULTS: Vandetanib displays antiproliferative and antiangiogenic activities and inhibits RET auto-phosphorylation. MAPK and AKT pathways were the two major signaling pathways inhibited by vandetanib. Interestingly, phosphorylated levels of NFκB-p65 were significantly increased by vandetanib. Comparable results were obtained in both the in vitro and in vivo approaches as well as for the protein detection methods, although some discrepancies were observed between RPPA and western-immunoblotting. CONCLUSIONS: Results confirmed the reliability and the utility of RPPA for screening global changes induced in signaling pathways by kinase inhibitors. MAPK and AKT were identified as the main pathways involved in vandetanib response in MTC models. Our results also suggest alternative routes for controlling the disease and provide a rationale for the development of therapeutic combinations based on the comprehensive identification of molecular events induced by inhibitors.