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Background: Recent whole-genome sequencing identified four molecular subtypes of gastric cancer (GC), of which the subgroup of Epstein-Barr virus-associated GC (EBVaGC) showed a significant enrichment of PIK3CA mutations. We here aimed to validate independently the enrichment of PIK3CA mutations in EBVaGC of a Central European GC cohort, to correlate EBV status with clinico-pathological patient characteristics and to test for a major issue of GC, intratumoral heterogeneity. Patients and methods: In a first step, 484 GCs were screened for EBV and PIK3CA hot spot mutations of exon 9/20 using EBER in situ hybridization and pyrosequencing, respectively. Secondly, an extended sequencing of PIK3CA also utilizing next generation sequencing was carried out in all EBVaGCs and 96 corresponding lymph node metastases. Results: Twenty-two GCs were EBER-positive, all being of latency type I. Intratumoral heterogeneity of EBER-positivity was found in 18% of EBVaGCs. Twenty-three GCs held PIK3CA mutations in hot spot regions of exon 9 or 20, being significantly more common in EBVaGCs (P < 0.001). Subsequent extended sequencing of PIK3CA of EBVaGCs showed that 14% harvested three to five different PIK3CA genotypes (including wildtype) in the same primary tumor, albeit in histologically and spatially distinct tumor areas, and that intratumoral heterogeneity of PIK3CA was also present in the corresponding lymph node metastases. Conclusions: Our findings unravel issues of tumor heterogeneity and illustrate that the assessment of the EBV status in tissue biopsies might carry the risk of sampling errors, which may significantly hamper adequate molecular tumor classification in a more clinical setting. Moreover, this is the first report of intratumoral heterogeneity of PIK3CA mutations in GC, and our findings lead to the conclusion that PIK3CA mutant and -wildtype tumor subclones are skilled to metastasize independently to different regional lymph nodes.
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Adenocarcinoma/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Infecciones por Virus de Epstein-Barr/genética , Neoplasias Gástricas/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/secundario , Adenocarcinoma/virología , Anciano , Infecciones por Virus de Epstein-Barr/mortalidad , Infecciones por Virus de Epstein-Barr/patología , Infecciones por Virus de Epstein-Barr/virología , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Heterogeneidad Genética , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Técnicas de Diagnóstico Molecular , Mutación , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Neoplasias Gástricas/virologíaRESUMEN
The altered expression and/or activity of ion channels and transporters (transportome) have been associated with malignant behavior of cancer cells and were proposed to be a hallmark of cancer. However, the impact of altered transportome in epithelial cancers, such as pancreatic ductal adenocarcinoma (PDAC), as well as its pathophysiological consequences, still remains unclear. Here, we report the in silico analysis of 840 transportome genes in PDAC patients' tissues. Our study was focused on the transportome changes and their correlation with functional and behavioral responses in PDAC tumor and stromal compartments. The dysregulated gene expression datasets were filtered using a cut-off of fold-change values ≤-2 or ≥2 (adjusted p value ≤0.05). The dysregulated transportome genes were clearly associated with impaired physiological secretory mechanisms and/or pH regulation, control of cell volume, and cell polarity. Additionally, some down-regulated transportome genes were found to be closely linked to epithelial cell differentiation. Furthermore, the observed decrease in genes coding for calcium and chloride transport might be a mechanism for evasion of apoptosis. In conclusion, the current work provides a comprehensive overview of the altered transportome expression and its association with predicted PDAC malignancy with special focus on the epithelial compartment.
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Adenocarcinoma/metabolismo , Biología Computacional , Simulación por Computador , Perfilación de la Expresión Génica , Canales Iónicos/genética , Canales Iónicos/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Epitelio/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Microambiente TumoralRESUMEN
We have previously shown that the tumor necrosis factor family member a proliferation-inducing ligand (APRIL) enhances intestinal tumor growth in various preclinical tumor models. Here, we have investigated whether APRIL serum levels at time of surgery predict survival in a large cohort of colorectal cancer (CRC) patients. We measured circulating APRIL levels in a cohort of CRC patients (n=432) using a novel validated monoclonal APRIL antibody (hAPRIL.133) in an enzyme-linked immunosorbent assay (ELISA) setup. APRIL levels were correlated with clinicopathological features and outcome. Overall survival was examined with Kaplan-Meier survival analysis, and Cox proportional hazards ratios were calculated. We observed that circulating APRIL levels were normally distributed among CRC patients. High APRIL expression correlated significantly with poor outcome measures, such as higher stage at presentation and development of lymphatic and distant metastases. Within the group of rectal cancer patients, higher circulating APRIL levels at time of surgery were correlated with poor survival (log-rank analysis P-value 0.008). Univariate Cox regression analysis for overall survival in rectal cancer patients showed that patients with elevated circulating APRIL levels had an increased risk of poor outcome (hazard ratio (HR) 1.79; 95% confidence interval (CI) 1.16-2.76; P-value 0.009). Multivariate analysis in rectal cancer patients showed that APRIL as a prognostic factor was dependent on stage of disease (HR 1.25; 95% CI 0.79-1.99; P-value 0.340), which was related to the fact that stage IV rectal cancer patients had significantly higher levels of APRIL. Our results revealed that APRIL serum levels at time of surgery were associated with features of advanced disease and prognosis in rectal cancer patients, which strengthens the previously reported preclinical observation of increased APRIL levels correlating with disease progression.
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Localized in the plasma membrane, death domain-containing TNF-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induce apoptosis and non-apoptotic signaling when crosslinked by the ligand TRAIL or by agonistic receptor-specific antibodies. Recently, an increasing body of evidence has accumulated that TRAIL receptors are additionally found in noncanonical intracellular locations in a wide range of cell types, preferentially cancer cells. Thus, besides their canonical locations in the plasma membrane and in intracellular membranes of the secretory pathway as well as endosomes and lysosomes, TRAIL receptors may also exist in autophagosomes, in nonmembraneous cytosolic compartment as well as in the nucleus. Such intracellular locations have been mainly regarded as hide-outs for these receptors representing a strategy for cancer cells to resist TRAIL-mediated apoptosis. Recently, a novel function of intracellular TRAIL-R2 has been revealed. When present in the nuclei of tumor cells, TRAIL-R2 inhibits the processing of the primary let-7 miRNA (pri-let-7) via interaction with accessory proteins of the Microprocessor complex. The nuclear TRAIL-R2-driven decrease in mature let-7 enhances the malignancy of cancer cells. This finding represents a new example of nuclear activity of typically plasma membrane-located cytokine and growth factor receptors. Furthermore, this extends the list of nucleic acid targets of the cell surface receptors by pri-miRNA in addition to DNA and mRNA. Here we review the diverse functions of TRAIL-R2 depending on its intracellular localization and we particularly discuss the nuclear TRAIL-R2 (nTRAIL-R2) function in the context of known nuclear activities of other normally plasma membrane-localized receptors.
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Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Apoptosis/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Humanos , MicroARNs/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Improvements in the understanding of its molecular mechanism and the characterisation of CRC-specific biomarkers facilitating early detection are considered to increase overall survival. METHODS: A meta-analysis of microarray and Serial Analysis of Gene Expression (SAGE) has been performed to identify differentially regulated genes in CRC. Dipeptidase 1 (DPEP1/MDP/RDP) and Syntenin-2 (SDCBP2/SITAC18) were found to be differentially expressed in tumour tissue compared with normal mucosa. Expression of DPEP1 was assessed in a validation set of 87 normal mucosa samples, 20 hyperplastic polyps, 46 CR adenomas with low- and high-grade intraepithelial neoplasia (IEN) and 217 well-documented CRCs by immunohistochemistry and partially by immunoblotting and real-time PCR. RESULTS: Expression of DPEP1 was specifically increased in human CRC tissue samples compared with normal mucosa (P<0.0001, Mann-Whitney U-test), showing a striking upregulation in high-grade compared with low-grade IEN. Furthermore, high DPEP1 expression was found to strongly correlate with histological stage (P<0.0001, chi-square test) as well as localisation (P<0.0001, chi-square test) and has been recognised as an independent adverse prognostic factor, showing significant prognostic values with an ROC (receiver operating characteristic)-AUC of 0.9230. CONCLUSION: Dipeptidase 1 has been identified as an excellent marker of high-grade IEN and CRC, and may thus be applied for screening of early neoplastic lesions and for prognostic stratification.
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Biomarcadores de Tumor/biosíntesis , Carcinoma in Situ/enzimología , Carcinoma in Situ/patología , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Dipeptidasas/biosíntesis , Biomarcadores de Tumor/genética , Carcinoma in Situ/genética , Neoplasias Colorrectales/genética , Dipeptidasas/genética , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/genética , Humanos , Clasificación del Tumor , Pronóstico , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in Apc(Min) mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis.
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Transformación Celular Neoplásica , Neoplasias Colorrectales/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Modelos Animales de Enfermedad , Humanos , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones Transgénicos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Células Tumorales Cultivadas , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/antagonistas & inhibidores , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies, with an overall life expectancy of 6 months. Despite considerable advances in the understanding of the molecular mechanisms involved in the carcinogenesis of PDAC, the outcome of the disease was not significantly improved over the last 20 years. Although some achievements in molecular-targeted therapies have been made (that is, targeting the epidermal growth factor receptor by erlotinib), which already entered clinical settings, and despite the promising outcome of the FOLFIRINOX trial, there is an urgent need for improvement of the chemotherapy in this disease. A plethora of molecular alterations are thought to be responsible for the profound chemoresistance, including mutations in oncogenes and tumor suppressors. Besides these classical hallmarks of cancer, the constitutive or inducible activity of transcription factor pathways are characteristic changes in PDAC. Recently, three transcription factors-nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT) and nuclear factor-E2-related factor-2 (Nrf2)-have been shown to be crucial for tumor development and chemoresistance in pancreatic cancer. These transcription factors are key regulators of a variety of genes involved in nearly all aspects of tumorigenesis and resistance against chemotherapeutics and death receptor ligands. Furthermore, the pathways of NF-κB, NFAT and Nrf2 are functional, interacting on several regulatory steps, and, especially, natural compounds such as curcumin interfere with more than one pathway. Thus, targeting these pathways by established inhibitors or new drugs might have great potential to improve the outcome of PDAC patients, most likely in combination with established anticancer drugs. In this article, we summarize recent progress in the characterization of these transcription-factor pathways and their role in PDAC and therapy resistance. We also discuss future concepts for the treatment of PDAC relying on these pathways.
RESUMEN
BACKGROUND: Clinicopathologic stage is still the main parameter to evaluate the prognosis of newly diagnosed colorectal cancer (CRC) patients. Although molecular markers have been suggested for follow up of treated CRC patients, their complete clinical application is still under evaluation. MATERIALS AND METHODS: To evaluate the association of immune-related genes with CRC prognosis and survival, a total of 19 single nucleotide polymorphisms (SNPs) were genotyped in 614 German patients within the Kiel cohort (POPGEN). RESULTS: A promoter variant (rs1800872) in the Interleukin-10 (IL-10) gene was associated with an increased lymph node metastasis involvement [odds ratio (OR) = 2.1, 95% confidence interval (CI) = 1.03-4.2, for carriers of the TT genotype]. More importantly, among 582 followed up patients the SNP rs3775291 in the toll-like receptor 3 (TLR-3) gene was associated with CRC specific survival (150 events). Patients carrying the TT genotype had a 93% increased risk of death compared with the CC carriers [hazard ratio (HR) = 1.93, 95% CI 1.14-3.28]. The observed effect of the TLR-3 variant was restricted to stage II patients (HR = 4.14, 95% CI 1.24-13.84) and to patients who did not receive adjuvant therapy (HR = 3.2, 95% CI 1.4-7.7). CONCLUSIONS: Our results may provide additional candidates for risk assessment in stage II CRC patients for treatment decision. Further validation of the presented findings is warranted.
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Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Polimorfismo Genético , Receptor Toll-Like 3/genética , Anciano , Estudios de Cohortes , Femenino , Alemania , Humanos , Interleucina-10/genética , Metástasis Linfática , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo de Nucleótido Simple , Pronóstico , Regiones Promotoras GenéticasRESUMEN
Constitutively active PI3K catalytic subunit alpha (PIK3CA) interfered with apoptosis induction downstream of death receptor-signaling complex formation allowing robust caspase-8 activation without triggering the execution steps of apoptosis. In mutant PIK3CA-expressing cells, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95L stimulated nuclear factor kappaB (NFkappaB) activation, invasion, and transition to an amoeboid-like morphology. NFkappaB activation and adoption of amoeboid shape were inhibited by caspase-8 knockdown or FLIP-S expression, but only the cell morphology alterations required caspase-8 activity. Furthermore, we identified caspase-8-mediated, caspase-3-independent cleavage of the protein kinase rho-associated, coiled-coil containing protein kinase 1 as a novel mechanism for acquiring amoeboid shape and enhanced invasiveness in response to TRAIL and CD95L. Taken together, we provide evidence that mutated PIK3CA converts the 'tumor surveillance' activity of cancer cell-expressed death receptors and caspase-8 toward tumor promotion.
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Apoptosis/genética , Caspasa 8/metabolismo , Proteína Ligando Fas/farmacología , Mutación Missense/fisiología , Fosfatidilinositol 3-Quinasas/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Quinasas Asociadas a rho/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Apoptosis/efectos de los fármacos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 8/genética , Inhibidores de Caspasas , Forma de la Célula/efectos de los fármacos , Forma de la Célula/genética , Fosfatidilinositol 3-Quinasa Clase I , Inhibidores de Cisteína Proteinasa/farmacología , Células HCT116 , Humanos , Quinasa I-kappa B/metabolismo , Interleucina-8/metabolismo , Invasividad Neoplásica/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína X Asociada a bcl-2/genética , Receptor fas/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
An elevated proteasome activity contributes to tumorigenesis, particularly by providing cancer cells with antiapoptotic protection and efficient clearance from irregular proteins. Still, the underlying mechanisms are poorly known. In this study, we report that in colon cancer patients, higher proteasome activity was detected in tumoral tissue compared with surrounding normal tissue, and also that increased levels of proteasomal subunit proteins, such as S5a/PSMD4 and alpha-5/PSMA5, could be detected. Colon tumors showed higher nuclear levels of nuclear factor E2-related factor 2 (Nrf2), a transcription factor supposed to be involved in the control of proteasomal subunit protein expression. The induction or overexpression of Nrf2 led to stronger S5a and alpha-5 expression in the human colon cancer cell lines, Colo320 and Lovo, as well as in NCM460 colonocytes along with higher proteasome activity. The small interfering RNA (siRNA)-mediated Nrf2 knockdown decreased S5a and alpha-5 expression and reduced proteasome activity. Additionally, Nrf2-dependent S5a and alpha-5 expression conferred protection from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, an effect preceded by an increased nuclear factor (NF)-kappaB activation and higher expression of antiapoptotic NF-kappaB target genes. These findings point to an important role of Nrf2 in the gain of proteasome activity, thereby contributing to colorectal carcinogenesis. Nrf2 may therefore serve as a potential target in anticancer therapy.
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Neoplasias Colorrectales/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Western Blotting , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Células Epiteliales/citología , Humanos , Inmunohistoquímica , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/biosíntesis , Complejo de la Endopetidasa Proteasomal/genética , Transducción de Señal , UbiquitinaciónRESUMEN
Kallikreins play an important role in tumour microenvironment and as cancer biomarkers in different cancer entities. Previous studies suggested an upregulation of KLK10 and KLK6 in pancreatic ductal adenocarcinoma (PDAC). Therefore, we evaluated the clinicopathological role of these kallikreins and their value as biomarkers in PDAC.Differential expression was validated by DNA-microarrays and immunohistochemistry in normal and malignant pancreatic tissues. Sera concentrations of both kallikreins were evaluated using ELISA. In silico analysis of possible protein interactions and gene silencing of KLK10 in vitro using siRNAs gave further insights in the pathomechanisms.Gene expression analysis and immunohistochemistry demonstrated a strong expression for KLK10 and KLK6 in PDAC. Statistical analysis showed that co-expression of these kallikreins correlated with an R1-resection status (P=0.017) and worse outcome for overall survival (P=0.031). Multivariate analysis proofed that co-expression is an independent prognostic factor for survival (P=0.043). Importantly, KLK10 knockdown in AsPC-1 cells significantly reduced cell migration, whereas computational analysis suggested interaction of KLK6 with angiogenetic factors as an important mechanism.Co-expression of KLK10 and KLK6 plays an unfavourable role in PDAC. Our results suggest that this effect is likely mediated by an interaction with the factors of the extracellular matrix and enhancement of cancer cell motility.
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Adenocarcinoma/química , Carcinoma Ductal Pancreático/química , Calicreínas/análisis , Neoplasias Pancreáticas/química , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular , Proteínas del Ojo/fisiología , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Inmunohistoquímica , Calicreínas/genética , Calicreínas/fisiología , Factores de Crecimiento Nervioso/fisiología , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Pronóstico , Serpinas/fisiologíaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. AIMS: We analysed the expression levels of CK1 delta and epsilon (CK1delta/in) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. METHODS: CK1delta/in expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. RESULTS: We found that CK1delta/in are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1delta/in by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. CONCLUSIONS: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.
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Carcinoma Ductal Pancreático/patología , Caseína Cinasa 1 épsilon/antagonistas & inhibidores , Quinasa Idelta de la Caseína/antagonistas & inhibidores , Indoles/farmacología , Neoplasias Pancreáticas/patología , Floroglucinol/análogos & derivados , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/enzimología , Carcinoma Ductal Pancreático/secundario , Caseína Cinasa 1 épsilon/metabolismo , Caseína Cinasa 1 épsilon/fisiología , Quinasa Idelta de la Caseína/metabolismo , Quinasa Idelta de la Caseína/fisiología , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Indoles/uso terapéutico , Metástasis Linfática , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/enzimología , Floroglucinol/farmacología , Floroglucinol/uso terapéutico , Trasplante Heterólogo , Células Tumorales Cultivadas , Receptor fas/fisiología , GemcitabinaRESUMEN
Human gammadelta T cells expressing a V gamma 9V delta 2 T-cell receptor (TCR) kill various tumour cells including autologous tumours. In addition to TCR-dependent recognition, activation of NKG2D-positive gammadelta T cells by tumour cell-expressed NKG2D ligands can also trigger cytotoxic effector function. In this study, we investigated the involvement of TCR versus NKG2D in tumour cell recognition as a prerequisite to identify tumour types suitable for gammadelta T-cell-based immunotherapy. We have characterized epithelial tumour cells of different origin with respect to cell surface expression of the known NKG2D ligands MHC class I-chain-related antigens (MIC) A/B and UL16-binding proteins (ULBP), and susceptibility to gammadelta T-cell killing. Most tumour cells expressed comparable levels of MICA and MICB as well as ULBP with the exception of ULBP-1 which was absent or only weakly expressed. Most epithelial tumours were susceptible to allogeneic gammadelta T-cell lysis and in the case of an established ovarian carcinoma to autologous gammadelta T-cell killing. Lysis of resistant cells was enhanced by pre-treatment of tumour cells with aminobisphosphonates or pre-activation of gammadelta T cells with phosphoantigens. A potential involvement of TCR and/or NKG2D was investigated by antibody blockade. These experiments revealed three patterns of inhibition, i.e. preferential inhibition by anti-TCR antibody, preferential inhibition by anti-NKG2D antibody, or additive blockade by anti-TCR plus anti-NKG2D antibodies. Our results indicate for the first time that the NKG2D pathway is involved in the lysis of different melanomas, pancreatic adenocarcinomas, squamous cell carcinomas of the head and neck, and lung carcinoma.
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Citotoxicidad Inmunológica , Neoplasias Glandulares y Epiteliales/inmunología , Neoplasias Glandulares y Epiteliales/terapia , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Receptores Inmunológicos/fisiología , Subgrupos de Linfocitos T/inmunología , Adenocarcinoma/inmunología , Adenocarcinoma/terapia , Adulto , Células CACO-2 , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/terapia , Línea Celular , Femenino , Neoplasias de Cabeza y Cuello/inmunología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Ligandos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/terapia , Masculino , Melanoma/inmunología , Melanoma/terapia , Persona de Mediana Edad , Subfamilia K de Receptores Similares a Lectina de Células NK , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Células Asesinas Naturales , Subgrupos de Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
In the present study, we have analysed the effects of transforming growth factor-beta (TGF-beta) signaling on the growth behavior of pancreatic carcinoma cells in vitro and on their tumorigenicity in vivo. Ectopic expression of dominant-negative mutants of the TGF-beta type II receptor or type I receptor/activin receptor-like kinase 5 (ALK5) in TGF-beta-sensitive pancreatic ductal adenocarcinoma PANC-1 cells prevented the TGF-beta-induced activation of transfected Smad-responsive reporter genes and growth arrest. The growth-inhibitory effect was mimicked by stable expression of kinase-active ALK5 (ALK5-T204D), and was dependent on ALK5's ability to activate Smad signaling, as a ALK5-derived mutant with an intact kinase domain but deficient in its ability to activate Smads (RImL45) failed to suppress proliferation in the absence of added TGF-beta. Moreover, this mutant often displayed opposite effects to those of ALK5-TD and blocked various ligand-induced responses in vitro, indicating that it acts in a dominant-negative fashion to inhibit endogenous wild-type receptors. ALK5-TD-, but not RImL45-TD-transduced cells underwent epithelial-to-mesenchymal transition, exhibited a higher ratio of thrombospondin-1 to vascular endothelial growth factor-A expression and upregulated various metastasis-associated genes. Upon orthotopic transplantation of PANC-1 clones into immunodeficient mice, ALK5-TD, but not RImL45-TD, greatly reduced tumor size and induced the formation of liver metastases in otherwise non-metastatic PANC-1 cells. These results suggest a causal, dominant role for the endogenous Smad2/3 signaling pathway in the tumor suppressor and prometastatic activities of TGF-beta in pancreatic tumor cells.
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Receptores de Activinas Tipo I/fisiología , Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo I/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Ratones , Ratones SCID , Mutación , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas , Ratas , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Smad/metabolismo , Transfección , Factor de Crecimiento Transformador beta/farmacología , Carga TumoralRESUMEN
Pancreatic ductal adenocarcinoma is a devastating disease, characterized by a rapid progression and poor treatment response. Using gene expression profiling of pancreatic cancer tissues, we previously identified periostin as a potential diagnostic and therapeutic target. In this study, we report the overexpression of periostin in a larger set of pancreatic cancer tissues and show that although the periostin transcript is exclusively expressed in tumour cells, the protein product is only detected in the extracellular matrix adjacent to cancer cells. Using an enzyme-linked immunosorbent assay (ELISA) assay, we show significantly increased levels of periostin in the sera of pancreatic cancer patients compared to non-cancer controls. We demonstrate that periostin promotes the invasiveness of tumour cells by increasing the motility of cells without inducing expression of proteases, and enhances the survival of tumour cells exposed to hypoxic conditions. At the molecular level, we provide evidence that the alpha(6)beta(4) integrin complex acts as the cell receptor of periostin in pancreatic cancer cells and that interaction promotes phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT) though activation of the PI3 kinase pathway, but not the RAS/MEK/ERK pathway. These findings suggest an important role of periostin in pancreatic cancer and provide a rationale to study periostin for diagnostic and therapeutic applications.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Moléculas de Adhesión Celular/fisiología , Integrina beta4/metabolismo , Neoplasias Pancreáticas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Apoptosis , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/metabolismo , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Hipoxia/metabolismo , Integrina alfa6beta4/metabolismo , Invasividad Neoplásica , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease and one of the cancer entities with the lowest life expectancy. Beside surgical therapy, no effective therapeutic options are available yet. Here, we show that 4-phenylbutyrate (4-PB), a known and well-tolerable inhibitor of histone deacetylases (HDAC), induces up to 70% apoptosis in all cell lines tested (Panc 1, T4M-4, COLO 357, BxPc3). In contrast, it leads to cell cycle arrest in only half of the cell lines tested. This drug increases gap junction communication between adjacent T3M-4 cells in a concentration-dependent manner and efficiently inhibits cellular export mechanisms in Panc 1, T4M-4, COLO 357 and BxPc3 cells. Consequently, in combination with gemcitabine 4-PB shows an overadditive effect on induction of apoptosis in BxPc3 and T3M-4 cells (up to 4.5-fold compared to single drug treatment) with accompanied activation of Caspase 8, BH3 interacting domain death agonist (Bid) and poly (ADP-ribose) polymerase family, member 1 (PARP) cleavage. Although the inhibition of the mitogen-activated protein kinase-pathway has no influence on fulminant induction of apoptosis, the inhibition of the JNK-pathway by SP600125 completely abolishes the overadditive effect induced by the combined application of both drugs, firstly reported by this study.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Comunicación Celular/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Neoplasias Pancreáticas/tratamiento farmacológico , Fenilbutiratos/farmacología , Antracenos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 8/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Factores de Tiempo , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention for its potential use in tumor therapy, as some recombinant variants of this ligand induce apoptosis in tumor cells without harming most normal cells. Here, we show that TRAIL strongly induces the expression of the proinflammatory cytokines interleukin-8 and monocyte chemoattractant protein 1 and enhances the invasion of apoptosis-resistant pancreatic ductal adenocarcinoma cells in vitro by upregulation of the urokinase-type plasminogen activator expression. Most importantly, we also demonstrate for the first time that TRAIL treatment results in strongly increased distant metastasis of pancreatic tumors in vivo. We orthotopically transplanted human pancreatic ductal adenocarcinoma cells to the pancreata of severe combined immunodeficiency mice and observed a dramatic increase in metastatic spread including a sixfold increase in the volume and fourfold increase in the number of liver metastases upon TRAIL treatment. Our results point to the necessity to carefully evaluate in vivo side effects of TRAIL and to select therapy conditions that not only enhance apoptosis induction but in addition prevent proinvasive and proinflammatory non-apoptotic TRAIL signaling.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Metástasis de la Neoplasia , Neoplasias Pancreáticas/patología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Animales , Humanos , Ratones , Ratones SCIDRESUMEN
BACKGROUND/AIMS: The high rate of local recurrence after radical resection of pancreatic ductal adenocarcinoma fosters intensive efforts to develop new approaches for adjuvant treatment. Antisense and modified oligodeoxynucleotides (ODNs) have demonstrated significant tumour growth inhibitory effects in preclinical systems. Our aim was to evaluate the possible therapeutic potential of ODNs containing unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG motifs). METHODOLOGY: For in vitro analysis, [(3)H]thymidine incorporation for DNA synthesis, colorimetric cell vitality assay (EZ4U assay), and DNA fragmentation assay (JAM-[(3)H]thymidine incorporation assay) to test for apoptosis were performed. In vivo testing was done on an orthotopic pancreatic xenotransplantation model using severe combined immunodeficient (SCID) beige and nude mice. RESULTS: No significant differential effect of either control ODN or CpG-1826 ODN with regard to tumour cell proliferation or induction of apoptosis was observed in vitro. In vivo, ODN-1826 proved to have a significant inhibitory effect (up to 40% reduction of tumour weight) when compared with tumour-bearing animals treated with saline or control ODN. This was accompanied by sevenfold increase in splenomegaly and moderate hepatomegaly. The reduction of tumour weight by ODN-1826 was only slightly more pronounced in nude compared with SCID beige mice. CONCLUSION: CpG-1826 induces significant growth-inhibitory effects on orthotopic xenotransplanted pancreatic tumours in highly immunodeficient mice, which might be explained by innate immunity mechanisms and possibly a complex interaction of tumour and stroma cells.
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Carcinoma Ductal Pancreático/terapia , ADN/uso terapéutico , Neoplasias Pancreáticas/terapia , Animales , Carcinoma Ductal Pancreático/inmunología , Línea Celular Tumoral , Supervivencia Celular , Fragmentación del ADN , Modelos Animales de Enfermedad , Femenino , Hepatomegalia/patología , Huésped Inmunocomprometido , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Oligodesoxirribonucleótidos , Neoplasias Pancreáticas/inmunología , Esplenomegalia/patologíaRESUMEN
BACKGROUND: This study assesses the chemotherapeutic drug gemcitabine in the human non-small cell lung cancer (NSCLC) cell line KNS62 in relation to the CD95-induced apoptotic pathway, and the role of the anti-apoptotic protein Bcl-xL in vitro and in vivo. MATERIALS AND METHODS: Apoptosis was determined by JAM assay and DAPI staining analysis. Activation of key apoptotic proteins, including caspases 3, 8 and 9 and BID, as well as cytochrome c release and mitochondrial transmembrane potential (MTP), were measured. The impact of the caspase inhibitor zVAD on gemcitabine-induced apoptosis was quantified. The in vitro results were verified in vivo in an orthotopic murine xenotransplantation model. RESULTS: Gemcitabine treatment, as well as stimulation of CD95, resulted in cleavage of effector caspase 3 as well as its substrate PARP and caspase 9, followed by DNA fragmentation. Cleavage of caspase 8 was demonstrated after CD95 activation but not after the application of gemcitabine. In KNS62-Bcl-xL clones, release of cytochrome c and loss of mitochondrial transmembrane potential were suppressed. Consequently, apoptosis after gemcitabine therapy, as well as CD95-induced apoptosis, were significantly inhibited. Caspase inhibitor zVAD only partly reversed gemcitabine-induced DNA fragmentation. In vivo, there was a significant reduction in tumour volume under gemcitabine therapy. Bcl-xL over-expressing tumours were completely resistant to gemcitabine therapy. CONCLUSIONS: In NSCLC cell line KNS62 gemcitabine activated the mitochondrial apoptotic pathway downstream of mitochondria without activation of initiator caspases. Bcl-xL over-expression induced significant resistance to gemcitabine. In vivo, the anti-apoptotic effect of Bcl-xL was more pronounced than in vitro. Gemcitabine also induced caspase-independent DNA fragmentation in KNS62 cells.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Caspasas/metabolismo , Desoxicitidina/análogos & derivados , Neoplasias Pulmonares/tratamiento farmacológico , Proteína bcl-X/metabolismo , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Apoptosis/fisiología , Caspasa 8 , Línea Celular Tumoral , Fragmentación del ADN , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Femenino , Humanos , Immunoblotting , Ratones , Ratones SCID , Trasplante Heterólogo , Receptor fas/metabolismo , GemcitabinaRESUMEN
Mathematical modeling is required for understanding the complex behavior of large signal transduction networks. Previous attempts to model signal transduction pathways were often limited to small systems or based on qualitative data only. Here, we developed a mathematical modeling framework for understanding the complex signaling behavior of CD95(APO-1/Fas)-mediated apoptosis. Defects in the regulation of apoptosis result in serious diseases such as cancer, autoimmunity, and neurodegeneration. During the last decade many of the molecular mechanisms of apoptosis signaling have been examined and elucidated. A systemic understanding of apoptosis is, however, still missing. To address the complexity of apoptotic signaling we subdivided this system into subsystems of different information qualities. A new approach for sensitivity analysis within the mathematical model was key for the identification of critical system parameters and two essential system properties: modularity and robustness. Our model describes the regulation of apoptosis on a systems level and resolves the important question of a threshold mechanism for the regulation of apoptosis.