RESUMEN
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy. Differentiation from chronic pancreatitis (CP) is currently inaccurate in about one-third of cases. Misdiagnoses in both directions, however, have severe consequences for patients. We set out to identify molecular markers for a clear distinction between PDAC and CP. DESIGN: Genome-wide variations of DNA-methylation, messenger RNA and microRNA level as well as combinations thereof were analysed in 345 tissue samples for marker identification. To improve diagnostic performance, we established a random-forest machine-learning approach. Results were validated on another 48 samples and further corroborated in 16 liquid biopsy samples. RESULTS: Machine-learning succeeded in defining markers to differentiate between patients with PDAC and CP, while low-dimensional embedding and cluster analysis failed to do so. DNA-methylation yielded the best diagnostic accuracy by far, dwarfing the importance of transcript levels. Identified changes were confirmed with data taken from public repositories and validated in independent sample sets. A signature of six DNA-methylation sites in a CpG-island of the protein kinase C beta type gene achieved a validated diagnostic accuracy of 100% in tissue and in circulating free DNA isolated from patient plasma. CONCLUSION: The success of machine-learning to identify an effective marker signature documents the power of this approach. The high diagnostic accuracy of discriminating PDAC from CP could have tremendous consequences for treatment success, once the result from still a limited number of liquid biopsy samples would be confirmed in a larger cohort of patients with suspected pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatitis Crónica , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pancreatitis Crónica/diagnóstico , Pancreatitis Crónica/genética , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Metilación de ADN , ADN , Biomarcadores de Tumor/genética , Neoplasias PancreáticasRESUMEN
Binding of transcription factors to mutated DNA sequences is a likely regulator of cancer progression. Noncoding regulatory mutations such as those on the core promoter of the gene encoding human telomerase reverse transcriptase have been shown to affect gene expression in cancer. Using a protein microarray of 667 transcription factor DNA-binding domains and subsequent functional assays, we looked for transcription factors that preferentially bind the mutant hTERT promoter and characterized their downstream effects. One of them, friend leukemia integration 1 (FLI1), which belongs to the E26 transforming-specific family of transcription factors, exhibited particularly strong effects with respect to regulating hTERT expression, while the even better binding ELK3 did not. Depletion of FLI1 decreased expression of the genes for cyclin D1 (CCND1) and E2F transcription factor 2 (E2F2) resulting in a G1/S cell cycle arrest and in consequence a reduction of cell proliferation. FLI1 also affected CMTM7, another gene involved in G1/S transition, although by another process that suggests a balanced regulation of the tumor suppressor gene's activity via opposing regulation processes. FLI1 expression was found upregulated and correlated with an increase in CCND1 expression in pancreatic cancer and brain tumors. In non-neoplastic lung cells, however, FLI1 depletion led to rapid progression through the cell cycle. This coincides with the fact that FLI1 is downregulated in lung tumors. Taken together, our data indicate a cell cycle regulatory hub involving FLI1, hTERT, CCND1 and E2F2 in a tissue- and context-dependent manner.
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Neoplasias/genética , Neoplasias/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclina D1/biosíntesis , Ciclina D1/genética , Ciclina D1/metabolismo , Progresión de la Enfermedad , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F2/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Neoplasias/patología , Regiones Promotoras Genéticas , Análisis por Matrices de Proteínas , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Early onset pancreatic cancer (EOPC) is a rare disease with a very high mortality rate. Almost nothing is known on the genetic susceptibility of EOPC, therefore, we performed a genome-wide association study (GWAS) to identify novel genetic variants specific for patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) at younger ages. In the first phase, conducted on 821 cases with age of onset ≤60 years, of whom 198 with age of onset ≤50, and 3227 controls from PanScan I-II, we observed four SNPs (rs7155613, rs2328991, rs4891017 and rs12610094) showing an association with EOPC risk (P < 1 × 10-4 ). We replicated these SNPs in the PANcreatic Disease ReseArch (PANDoRA) consortium and used additional in silico data from PanScan III and PanC4. Among these four variants rs2328991 was significant in an independent set of 855 cases with age of onset ≤60 years, of whom 265 with age of onset ≤50, and 4142 controls from the PANDoRA consortium while in the in silico data, we observed no statistically significant association. However, the resulting meta-analysis supported the association (P = 1.15 × 10-4 ). In conclusion, we propose a novel variant rs2328991 to be involved in EOPC risk. Even though it was not possible to find a mechanistic link between the variant and the function, the association is supported by a solid statistical significance obtained in the largest study on EOPC genetics present so far in the literature.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Estudios de Casos y Controles , Femenino , Estudio de Asociación del Genoma Completo/métodos , Humanos , Masculino , Persona de Mediana Edad , Páncreas/patología , Neoplasias Pancreáticas/patología , Polimorfismo de Nucleótido Simple/genética , Factores de Riesgo , Neoplasias PancreáticasRESUMEN
Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples. Pancreatic ductal adenocarcinomas (PDAC) and cystic tumours were most different in these non-tumorous tissues surrounding them, whereas the actual tumours exhibited rather similar transcript patterns. The environment of cystic tumours was transcriptionally nearly identical to normal pancreas tissue. In contrast, the tissue around PDAC behaved a lot like the tumour, indicating some kind of field defect, while showing far less molecular resemblance to both chronic pancreatitis and healthy tissue. This suggests that the major pathogenic difference between cystic and ductal tumours may be due to their cellular environment rather than the few variations between the tumours. Lack of correlation between DNA methylation and transcript levels makes it unlikely that the observed field defect in the peritumoral tissue of PDAC is controlled to a large extent by such epigenetic regulation. Functionally, a strikingly large number of autophagy-related transcripts was changed in both PDAC and its peritumoral tissue, but not in other pancreatic tumours. A transcription signature of 15 autophagy-related genes was established that permits a prognosis of survival with high accuracy and indicates the role of autophagy in tumour biology.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Regulación Neoplásica de la Expresión Génica , Quiste Pancreático/genética , Neoplasias Pancreáticas/genética , Pancreatitis Crónica/genética , Microambiente Tumoral/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal Pancreático/patología , Metilación de ADN , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Persona de Mediana Edad , Quiste Pancreático/patología , Neoplasias Pancreáticas/patología , Pancreatitis Crónica/patología , Pronóstico , Tasa de Supervivencia , Adulto JovenRESUMEN
BACKGROUND & AIMS: Incidence of and mortality from pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, are almost equivalent, so better treatments are needed. We studied gene expression profiles of PDACs and the functions of genes with altered expression to identify new therapeutic targets. METHODS: We performed microarray analysis to analyze gene expression profiles of 195 PDAC and 41 non-tumor pancreatic tissue samples. We undertook an extensive analysis of the PDAC transcriptome by superimposing interaction networks of proteins encoded by aberrantly expressed genes over signaling pathways associated with PDAC development to identify factors that might alter regulation of these pathways during tumor progression. We performed tissue microarray analysis to verify changes in expression of candidate protein using an independent set of 152 samples (40 nontumor pancreatic tissues, 63 PDAC sections, and 49 chronic pancreatitis samples). We validated the functional relevance of the candidate molecule using RNA interference or pharmacologic inhibitors in pancreatic cancer cell lines and analyses of xenograft tumors in mice. RESULTS: In an analysis of 38,276 human genes and loci, we identified 1676 genes that were significantly up-regulated and 1166 genes that were significantly down-regulated in PDAC compared with nontumor pancreatic tissues. One gene that was up-regulated and associated with multiple signaling pathways that are dysregulated in PDAC was G protein subunit αi2, which has not been previously associated with PDAC. G protein subunit αi2 mediates the effects of dopamine receptor D2 (DRD2) on cyclic adenosine monophosphate signaling; PDAC tissues had a slight but significant increase in DRD2 messenger RNA. Levels of DRD2 protein were substantially increased in PDACs, compared with non-tumor tissues, in tissue microarray analyses. RNA interference knockdown of DRD2 or inhibition with pharmacologic antagonists (pimozide and haloperidol) reduced proliferation of pancreatic cancer cells, induced endoplasmic reticulum stress and apoptosis, and reduced cell migration. RNA interference knockdown of DRD2 in pancreatic tumor cells reduced growth of xenograft tumors in mice, and administration of the DRD2 inhibitor haloperidol to mice with orthotopic xenograft tumors reduced final tumor size and metastasis. CONCLUSIONS: In gene expression profile analysis of PDAC samples, we found the DRD2 signaling pathway to be activated. Inhibition of DRD2 in pancreatic cancer cells reduced proliferation and migration, and slowed growth of xenograft tumors in mice. DRD2 antagonists routinely used for management of schizophrenia might be tested in patients with pancreatic cancer.
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Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptores de Dopamina D2/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/secundario , Estudios de Casos y Controles , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Antagonistas de los Receptores de Dopamina D2/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Haloperidol/farmacología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Neoplasias Pancreáticas/patología , Fosforilación/efectos de los fármacos , Pimozida/farmacología , ARN Interferente Pequeño , Receptores de Dopamina D2/metabolismo , Transducción de Señal , Transcriptoma , Respuesta de Proteína Desplegada/efectos de los fármacos , Regulación hacia Arriba , eIF-2 Quinasa/metabolismoRESUMEN
Germline genetic variability might contribute, at least partially, to the survival of pancreatic ductal adenocarcinoma (PDAC) patients. Two recently performed genome-wide association studies (GWAS) on PDAC overall survival (OS) suggested (P < 10(-5)) the association between 30 genomic regions and PDAC OS. With the aim to highlight the true associations within these regions, we analyzed 44 single-nucleotide polymorphisms (SNPs) in the 30 candidate regions in 1722 PDAC patients within the PANcreatic Disease ReseArch (PANDoRA) consortium. We observed statistically significant associations for five of the selected regions. One association in the CTNNA2 gene on chromosome 2p12 [rs1567532, hazard ratio (HR) = 1.75, 95% confidence interval (CI) 1.19-2.58, P = 0.005 for homozygotes for the minor allele] and one in the last intron of the RUNX2 gene on chromosome 6p21 (rs12209785, HR = 0.88, 95% CI 0.80-0.98, P = 0.014 for heterozygotes) are of particular relevance. These loci do not coincide with those that showed the strongest associations in the previous GWAS. In silico analysis strongly suggested a possible mechanistic link between these two SNPs and pancreatic cancer survival. Functional studies are warranted to confirm the link between these genes (or other genes mapping in those regions) and PDAC prognosis in order to understand whether these variants may have the potential to impact treatment decisions and design of clinical trials.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Estudio de Asociación del Genoma Completo , alfa Catenina/genética , Adenocarcinoma/patología , Anciano , Carcinoma Ductal Pancreático/patología , Supervivencia sin Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Polimorfismo de Nucleótido Simple , PronósticoRESUMEN
Biomarkers for early diagnosis of patients with pancreatic cancer (PC) are needed. Our aim was to identify panels of miRNAs in serum in combination with CA 19-9 for use in the diagnosis of PC. Four hundred seventeen patients with PC were included prospectively from Denmark (n = 306) and Germany (n = 111). Controls included 59 patients with chronic pancreatitis (CP) and 248 healthy subjects (HS). MiRNAs were analyzed in pretreatment serum samples from 3 cohorts: discovery cohort (754 human miRNAs, TaqMan(®) Human MicroRNA assay, Applied Biosystem; PC n = 133, controls n = 72); training cohort (34 miRNAs, real-time qPCR using the Fluidigm BioMark™ System; PC n = 198, controls n = 184); validation cohort (13 miRNAs, real-time qPCR using the Fluidigm BioMark™ System; PC n = 86, controls n = 51). We found that 34 miRNAs in serum from PC patients in the discovery cohort were expressed differently than in controls. These miRNAs were tested in the training cohort, and four diagnostic panels were constructed that included 5 or 12 miRNAs (miR-16, -18a, -20a, -24, -25, -27a, -29c, -30a.5p, -191, -323.3p, -345 and -483.5p). Diagnostic accuracy of detecting PC in the training cohort was AUC (Index I 0.85; II 0.87; III 0.85; IV 0.95; CA 19-9 0.93); specificity (I 0.71; II 0.76; III 0.66; IV 0.90 (fixed sensitivity at 0.85); CA 19-9 0.93). Combining serum CA 19-9 and Index II best discriminated Stages I and II PC from HS [AUC 0.93 (0.90-0.96), sensitivity 0.77 (0.69-0.84), specificity 0.94 (0.90-0.96) and accuracy 0.88 (0.84-0.91)]. In conclusion, we identified four diagnostic panels based on 5 or 12 miRNAs in serum that could distinguish patients with PC from HS and CP.
Asunto(s)
Carcinoma Ductal Pancreático/genética , MicroARNs/sangre , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/sangre , Estudios de Casos y Controles , Humanos , MicroARNs/genética , Neoplasias Pancreáticas/sangre , Pancreatitis Crónica/sangre , Pancreatitis Crónica/genética , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. PURPOSE: (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. METHODS: PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. RESULTS: Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. CONCLUSIONS: Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/fisiología , Pancreatitis Crónica/patología , Técnicas de Cultivo de Célula , Separación Celular , Supervivencia Celular , HumanosRESUMEN
Late diagnosis contributes to pancreatic cancer (PaCa) dismal prognosis, urging for reliable, early detection. Serum-exosome protein and/or miRNA markers might be suitable candidates, which we controlled for patients with PaCa. Protein markers were selected according to expression in exosomes of PaCa cell line culture supernatants, but not healthy donors' serum-exosomes. miRNA was selected according to abundant recovery in microarrays of patients with PaCa, but not healthy donors' serum-exosomes and exosome-depleted serum. According to these preselections, serum-exosomes were tested by flow cytometry for the PaCa-initiating cell (PaCIC) markers CD44v6, Tspan8, EpCAM, MET and CD104. Serum-exosomes and exosome-depleted serum was tested for miR-1246, miR-4644, miR-3976 and miR-4306 recovery by qRT-PCR. The majority (95%) of patients with PaCa (131) and patients with nonPa-malignancies reacted with a panel of anti-CD44v6, -Tspan8, -EpCAM and -CD104. Serum-exosomes of healthy donors' and patients with nonmalignant diseases were not reactive. Recovery was tumor grading and staging independent including early stages. The selected miR-1246, miR-4644, miR-3976 and miR-4306 were significantly upregulated in 83% of PaCa serum-exosomes, but rarely in control groups. These miRNA were also elevated in exosome-depleted serum of patients with PaCa, but at a low level. Concomitant evaluation of PaCIC and miRNA serum-exosome marker panels significantly improved sensitivity (1.00, CI: 0.95-1) with a specificity of 0.80 (CI: 0.67-0.90) for PaCa versus all others groups and of 0.93 (CI: 0.81-0.98) excluding nonPa-malignancies. Thus, the concomitant evaluation of PaCIC and PaCa-related miRNA marker panels awaits retrospective analyses of larger cohorts, as it should allow for a highly sensitive, minimally-invasive PaCa diagnostics.
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Biomarcadores de Tumor/sangre , Exosomas/metabolismo , MicroARNs/sangre , Técnicas de Diagnóstico Molecular/métodos , Neoplasias Pancreáticas/sangre , Biomarcadores de Tumor/genética , Exosomas/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Sensibilidad y EspecificidadRESUMEN
A small number of common susceptibility loci have been identified for pancreatic cancer, one of which is marked by rs401681 in the TERT-CLPTM1L gene region on chromosome 5p15.33. Because this region is characterized by low linkage disequilibrium, we sought to identify whether additional single nucleotide polymorphisms (SNPs) could be related to pancreatic cancer risk, independently of rs401681. We performed an in-depth analysis of genetic variability of the telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC) genes, in 5,550 subjects with pancreatic cancer and 7,585 controls from the PANcreatic Disease ReseArch (PANDoRA) and the PanScan consortia. We identified a significant association between a variant in TERT and pancreatic cancer risk (rs2853677, odds ratio = 0.85; 95% confidence interval = 0.80-0.90, p = 8.3 × 10(-8)). Additional analysis adjusting rs2853677 for rs401681 indicated that the two SNPs are independently associated with pancreatic cancer risk, as suggested by the low linkage disequilibrium between them (r(2) = 0.07, D' = 0.28). Three additional SNPs in TERT reached statistical significance after correction for multiple testing: rs2736100 (p = 3.0 × 10(-5) ), rs4583925 (p = 4.0 × 10(-5) ) and rs2735948 (p = 5.0 × 10(-5) ). In conclusion, we confirmed that the TERT locus is associated with pancreatic cancer risk, possibly through several independent variants.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Telomerasa/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Humanos , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Mutación , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
UNLABELLED: Novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires induction of immunogenic cell death (ICD), coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study shows that in human pancreatic cancer cells (pancreatic ductal adenocarcinoma [PDAC] cells n=4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0±0.5 times (58%±9% of total content; up to 100 ng/ml) in all infected cultures, whether nondying, necrotic, or apoptotic. An alternative secretory route allowed H-1PV to overcome the failure of gemcitabine to trigger HMGB1 release, without impeding cytotoxicity or other ICD activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided with but was uncoupled from an autocrine interleukin-1ß (IL-1ß) loop. That and the pattern of viral determinants maintained in gemcitabine-treated cells suggested the activation of an inflammasome/caspase 1 (CASP1) platform alongside DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signaled through secretion of the alarmin HMGB1 and, besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD process. A transient arrest of cells in the cyclin A1-rich S phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV, which is not pathogenic in humans, into multimodal anticancer treatments. IMPORTANCE: The current therapeutic concepts targeting aggressive malignancies require an induction of immunogenic cell death characterized by exposure of calreticulin (CRT) as well as release of ATP and HMGB1 from dying cells. In pancreatic tumor cells (PDAC cells) infected with the oncolytic parvovirus H-1PV, only HMGB1 was released by all infected cells, whether nondying, necrotic, or succumbing to one of the programmed death pathways, including contraproductive apoptosis. Our data suggest that active secretion of HMGB1 from PDAC cells is a sentinel reaction emerging during early stages of the viral life cycle, irrespective of cell death, that is compatible with and complements cytotoxic regimens. Consistent induction of HMGB1 secretion raised the possibility that this reaction might be a general "alarming" phenomenon characteristic of H-1PV's interaction with the host cell; release of IL-1ß points to the possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.
Asunto(s)
Antineoplásicos/metabolismo , Muerte Celular , Desoxicitidina/análogos & derivados , Células Epiteliales/fisiología , Virus Oncolíticos/crecimiento & desarrollo , Parvovirus/crecimiento & desarrollo , Línea Celular Tumoral , Desoxicitidina/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Proteína HMGB1/metabolismo , Humanos , Transducción de Señal , GemcitabinaRESUMEN
BACKGROUND/OBJECTIVES: Meaningful profiling of pancreatic cancer samples is particularly challenging due to their complex cellular composition. Beyond tumor cells, surgical biopsies contain desmoplastic stroma with infiltrating inflammatory cells, adjacent normal parenchyma, and "non-pancreatic tissues". The risk of misinterpretation rises when the heterogeneous cancer tissues are sub-divided into smaller fragments for multiple analytic procedures. Pre-analytic histological evaluation is the best option to characterize pancreatic tissue samples. Our aim was to develop a complement or alternative procedure to determine the cellular composition of pancreatic cancerous biopsies, basing on intra-analytic molecular annotation. A standard process for sample stratification at a molecular level does not yet exist. Particularly in the case of retrospective or data depository-based studies, when hematoxylin-eosin stained sections are not available, it supports the correct interpretation of expression profiles. METHODS: A five-gene transcriptional signature (RNACellStrat) was defined that allows cell type-specific stratification of pancreatic tissues. Testing biopsy material from biobanks with this procedure demonstrated high correspondence of molecular (qRT-PCR and microarray) and histologic (hematoxylin-eosin stain) evaluations. RESULTS: Notably, about a quarter of randomly selected samples (tissue fragments) were exposed as inappropriate for subsequent clinico-pathological interpretation. CONCLUSIONS: Via immediate intra-analytical procedure, our RNA-based stratification RNACellStrat increases the accuracy and reliability of the conclusions drawn from diagnostic and prognostic molecular information.
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Páncreas/patología , ARN Mensajero/genética , Anciano , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Biopsia , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Anotación de Secuencia Molecular , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa , Pronóstico , ARN Mensajero/biosíntesis , Reproducibilidad de los Resultados , Estudios Retrospectivos , Análisis de Supervivencia , Bancos de Tejidos , Transcripción Genética/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that Notch and janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathways are both important for the initiation and progression of PDAC. The purpose of this study was to determine the outcome of targeting these two tumor signaling pathways simultaneously both in vitro and in vivo. We assessed the combinational effects of the γ-secretase inhibitor IX (GSI IX) and JAK2 inhibitor (AG-490) on growth and epithelial plasticity of human pancreatic cancer cell lines, and in a genetically engineered mouse model (Pdx1-Cre, LSL-KrasG12D, p53(lox/+)) of PDAC. Dual treatment with GSI IX and AG-490 significantly impaired cell proliferation, migration, invasion, soft agar growth and apoptosis when compared with monotherapies. Most importantly, combinational treatment significantly attenuates tumor progression in vivo and suppresses conversion from acinar-ductal-metaplasia to PDAC. Our results suggest that targeting Notch and JAK2/STAT3 signaling pathways simultaneously is superior to single inhibitions, supporting combined treatment by GSI IX and AG-490 as a potential therapeutic approach for PDAC. However, the study design limits the direct transfer into the clinic and the impact of dual treatment in patients with PDAC remains still to be determined.
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Neoplasias/patología , Proteínas Quinasas/metabolismo , Receptores Notch/antagonistas & inhibidores , Factores de Transcripción STAT/antagonistas & inhibidores , Línea Celular Tumoral , Progresión de la Enfermedad , HumanosRESUMEN
Pancreatic stellate cells are key mediators in chronic pancreatitis and play a central role in the development of pancreatic fibrosis, stromal formation, and progression of pancreatic cancer. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely TNF-α, FGF2, IL6, and chemokine (C-C motif) ligand 4 (CCL4). They were added individually to cells growing in serum-free medium next to controls in medium supplemented with serum, thus containing a mixture of them all, or in serum-free medium alone. Variations were detected by means of a microarray of 810 antibodies targeting relevant proteins. All tested factors triggered increased proliferation and migration. Further analysis showed that TNF-α is the prime factor responsible for the activation of pancreatic stellate cells. CCL4 is associated with cellular neovascularization, whereas FGF2 and IL6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells. The identified direct effects of individual cytokines on human pancreatic stellate cells provide new insights about their contribution to pancreatic cancer promotion.
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Citocinas/farmacología , Células Estrelladas Hepáticas/metabolismo , Mediadores de Inflamación/farmacología , Proteoma/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Mediadores de Inflamación/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.
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Enfermedad/genética , MicroARNs/sangre , MicroARNs/genética , Perfilación de la Expresión Génica , Variación Genética/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Exocrine and endocrine pancreas are interconnected anatomically and functionally, with vasculature facilitating bidirectional communication. Our understanding of this network remains limited, largely due to two-dimensional histology and missing combination with three-dimensional imaging. In this study, a multiscale 3D-imaging process was used to analyze a porcine pancreas. Clinical computed tomography, digital volume tomography, micro-computed tomography and Synchrotron-based propagation-based imaging were applied consecutively. Fields of view correlated inversely with attainable resolution from a whole organism level down to capillary structures with a voxel edge length of 2.0 µm. Segmented vascular networks from 3D-imaging data were correlated with tissue sections stained by immunohistochemistry and revealed highly vascularized regions to be intra-islet capillaries of islets of Langerhans. Generated 3D-datasets allowed for three-dimensional qualitative and quantitative organ and vessel structure analysis. Beyond this study, the method shows potential for application across a wide range of patho-morphology analyses and might possibly provide microstructural blueprints for biotissue engineering.
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Imagenología Tridimensional , Imagen Multimodal , Páncreas , Animales , Imagenología Tridimensional/métodos , Páncreas/diagnóstico por imagen , Páncreas/irrigación sanguínea , Porcinos , Imagen Multimodal/métodos , Microtomografía por Rayos X/métodos , Islotes Pancreáticos/diagnóstico por imagen , Islotes Pancreáticos/irrigación sanguínea , Tomografía Computarizada por Rayos X/métodosRESUMEN
Pancreatic adenocarcinoma (PaCa) being the deadliest cancer is partly due to early metastatic spread. Thus, we searched for PaCa-initiating cell (PaCIC) markers with emphasis on markers contributing to metastatic progression. PaCIC were enriched from long-term and freshly established lines by repeated selection for spheroid or holoclone growth in advance of evaluating PaCIC markers. Sphere and holoclone formation steeply increased by recloning and remained stable thereafter. Cells not forming spheres or holoclones died on recloning. PaCIC enrichment in spheres and holoclones was accompanied by increased motility, anchorage independence and upregulated CXCR4 expression. After subcutaneous injection in NOD/SCID mice tumorigenicity and, impressively, recovery of metastasizing tumor cells in peripheral blood, spleen, bone marrow, lung and pancreas was strongly increased in spheres and holoclones. PaCIC enrichment in spheres and holoclones was accompanied, besides CXCR4, by upregulated CD44v6, alpha6beta4, weakly CD133 and tetraspanin Tspan8 expression. Notably, CD44v6, alpha6beta4, CXCR4 and Tspan8 expressing PaCa cells had a growth advantage in vivo and became dominating in migrating and in distant organs settled tumor cells. This is the first report showing that CD44v6, alpha6beta4, Tspan8 and CXCR4 are biomarkers in PaCIC allowing for long-term survival, expansion and migration in immunocompromised mice. The stability of the percentage of PaCIC in long-term and freshly established lines after a roughly 8-fold enrichment by cloning indicates PaCIC, though required for long-term survival, concomitantly depending on support by non-CIC.
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Adenocarcinoma/metabolismo , Biomarcadores de Tumor/sangre , Receptores de Hialuranos/sangre , Integrina alfa6beta4/sangre , Neoplasias Pancreáticas/metabolismo , Tetraspaninas/sangre , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Exosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Trasplante de NeoplasiasRESUMEN
Meningiomas are among the most frequent intracranial tumors. The secretory variant of meningioma is characterized by glandular differentiation, formation of intracellular lumina and pseudopsammoma bodies, expression of a distinct pattern of cytokeratins and clinically by pronounced perifocal brain edema. Here we describe whole-exome sequencing analysis of DNA from 16 secretory meningiomas and corresponding constitutional tissues. All secretory meningiomas invariably harbored a mutation in both KLF4 and TRAF7. Validation in an independent cohort of 14 secretory meningiomas by Sanger sequencing or derived cleaved amplified polymorphic sequence (dCAPS) assay detected the same pattern, with KLF4 mutations observed in a total of 30/30 and TRAF7 mutations in 29/30 of these tumors. All KLF4 mutations were identical, affected codon 409 and resulted in a lysine to glutamine exchange (K409Q). KLF4 mutations were not found in 89 non-secretory meningiomas, 267 other intracranial tumors including gliomas, glioneuronal tumors, pituitary adenomas and metastases, 59 peripheral nerve sheath tumors and 52 pancreatic tumors. TRAF7 mutations were restricted to the WD40 domains. While KLF4 mutations were exclusively seen in secretory meningiomas, TRAF7 mutations were also observed in 7/89 (8 %) of non-secretory meningiomas. KLF4 and TRAF7 mutations were mutually exclusive with NF2 mutations. In conclusion, our findings suggest an essential contribution of combined KLF4 K409Q and TRAF7 mutations in the genesis of secretory meningioma and demonstrate a role for TRAF7 alterations in other non-NF2 meningiomas.
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Factores de Transcripción de Tipo Kruppel/genética , Neoplasias Meníngeas/genética , Meningioma/genética , Mutación/genética , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/genética , Análisis Mutacional de ADN , Genómica , Glutamina/genética , Humanos , Factor 4 Similar a Kruppel , Lisina/genética , Meningioma/patología , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: The glycoprotein MFG-E8 mediates phagocytic clearance of apoptotic cells and influences the pathogenesis and progression of inflammatory diseases. MFG-E8 was shown to attenuate the progression of inflammation and to improve survival in septic rats. Accumulating evidence suggests an immunomodulatory link between MFG-E8 and the pro-inflammatory chemokine fractalkine, which may determine the severity of pain, fibrosis, and inflammation in chronic pancreatitis (CP). METHODS: The expression and localization of MFG-E8 was investigated in CP (n=62), and normal pancreas (NP; n=34) by QRT-PCR, Western-blot and immunohistochemistry analyses. Results were correlated with mRNA expression of fractalkine, CX3CR1, and with the presence and degree of pain and fibrosis. Human pancreatic stellate cells (hPSCs) were isolated from CP tissues and evaluated for MFG-E8 mRNA expression after fractalkine stimulation. RESULTS: MFG-E8-mRNA was significantly overexpressed in CP and isolated hPSCs when compared to NP. Western-blot and immunohistochemistry analysis confirmed accumulation of MFG-E8 in CP, with noticeably increased MFG-E8 immunoreactivity in tubular complexes. MFG-E8 expression correlated significantly with fractalkine expression, severe fibrosis, and the presence of pain in CP patients. Stimulation of hPSCs with fractalkine led to a significant increase in MFG-E8 expression. CONCLUSIONS: In the present study, we demonstrated for the first time that MFG-E8 is significantly up-regulated in CP patients and together with fractalkine correlated noticeably with severe fibrosis and the presence of pain. hPSCs overexpress MFG-E8 upon fractalkine stimulation in vitro, which underlines the suggested immunmodulatory link in CP and may be a key mechanism in CP fibrogenesis and pain generation. Taken together, these novel findings suggest that MFG-E8 blockade may be a promising tool for future immunotherapy in CP to attenuate both fibrosis and pain sensation.
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Antígenos de Superficie/metabolismo , Inmunoterapia , Proteínas de la Leche/metabolismo , Páncreas/metabolismo , Pancreatitis Crónica/metabolismo , Adulto , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CX3CL1/metabolismo , Quimiocina CX3CL1/farmacología , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Dimensión del Dolor , Páncreas/patología , Células Estrelladas Pancreáticas/efectos de los fármacos , Células Estrelladas Pancreáticas/metabolismo , Células Estrelladas Pancreáticas/patología , Pancreatitis Crónica/patología , Pancreatitis Crónica/terapia , Índice de Severidad de la EnfermedadRESUMEN
Autoantibodies, a hallmark of both autoimmunity and cancer, represent an easily accessible surrogate for measuring adaptive immune responses to cancer. Sera can now be assayed for reactivity against thousands of proteins using microarrays, but there is no agreed-upon standard to analyze results. We developed a set of tailored quality control and normalization procedures based on ELISA validation to allow patient comparisons and determination of individual cutoffs for specificity and sensitivity. Sera from 60 patients with pancreatic cancer, 51 patients with ovarian cancer, and 53 age-matched healthy donors were used to assess the binding of IgG antibodies against a panel of >8000 human antigens using protein microarrays and fluorescence detection. The resulting data interpretation led to the definition and ranking of proteins with preferred recognition by the sera from cancer patients in comparison with healthy donors, both by frequency and strength of signal. We found that 202 proteins were preferentially immunogenic in ovarian cancer sera compared to 29 in pancreatic cancer, with few overlaps. Correlates of autoantibody signatures with known tumor expression of corresponding antigens, functional pathways, clinical stage, and outcome were examined. Serological analysis of arrays displaying the complete human proteome (seromics) represents a new era in cancer immunology, opening the way to defining the repertoire of the humoral immune response to cancer.