ABSTRACT
BACKGROUND: Pancreatic neuroendocrine tumors (PNETs) represent a distinct hypervascularized tumor entity, often diagnosed at metastatic stage. Therapeutic efficacy of anti-angiogenic multi-kinase inhibitors is frequently limited by primary or acquired resistance in vivo. This study aimed to characterize the molecular mode of action as well as resistance mechanisms to the anti-angiogenic multi-tyrosine kinase inhibitor (TKI) Regorafenib in vitro and in vivo. METHODS: In vitro, human and murine pancreatic neuroendocrine cell lines were comparatively treated with Regorafenib and other TKIs clinically used in PNETs. Effects on cell viability and proliferation were analyzed. In vivo, transgenic RIP1Tag2 mice were treated with Regorafenib at two different time periods during carcinogenesis and its impact on angiogenesis and tumor progression was evaluated. RESULTS: Compared to the established TKI therapies with Sunitinib and Everolimus, Regorafenib showed the strongest effects on cell viability and proliferation in vitro, but was unable to induce apoptosis. Unexpectedly and in contrast to these in vitro findings, Regorafenib enhanced proliferation during early tumor development in RIP1Tag2 mice and had no significant effect in late tumor progression. In addition, invasiveness was increased at both time points. Mechanistically, we could identify an upregulation of the pro-survival protein Bcl-2, the induction of the COX2-PGE2-pathway as well as the infiltration of CSF1R positive immune cells into the tumors as potential resistance mechanisms following Regorafenib treatment. DISCUSSION: Our data identify important tumor cell-autonomous and stroma-dependent mechanisms of resistance to antiangiogenic therapies.
Subject(s)
Neuroectodermal Tumors, Primitive , Mice , Humans , Animals , Mice, Transgenic , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Phenylurea Compounds/pharmacology , Phenylurea Compounds/therapeutic use , Carcinogenesis/genetics , Neuroectodermal Tumors, Primitive/drug therapy , Cell Line, TumorABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a dismal outcome. To improve understanding of sequential microbiome changes during PDAC development we analyzed mouse models of pancreatic carcinogenesis (KC mice recapitulating pre-invasive PanIN formation, as well as KPC mice recapitulating invasive PDAC) during early tumor development and subsequent tumor progression. Diversity and community composition were analyzed depending on genotype, age, and gender. Both mouse models demonstrated concordant abundance changes of several genera influenced by one or more of the investigated factors. Abundance was significantly impacted by gender, highlighting the need to further elucidate the impact of gender differences. The findings underline the importance of the microbiome in PDAC development and indicate that microbiological screening of patients at risk and targeting the microbiome in PDAC development may be feasible in future.
ABSTRACT
Neuroendocrine neoplasms (NENs) represent a heterogenous class of highly vascularized neoplasms that are increasing in prevalence and are predominantly diagnosed at a metastatic state. The molecular mechanisms leading to tumor initiation, metastasis, and chemoresistance are still under investigation. Hence, identification of novel therapeutic targets is of great interest. Here, we demonstrate that the RNA-binding Protein IGF2BP1 is a post-transcriptional regulator of components of the Polycomb repressive complex 2 (PRC2), an epigenic modifier affecting transcriptional regulation and proliferation: Comprehensive in silico analyses along with in vitro experiments showed that IGF2BP1 promotes neuroendocrine tumor cell proliferation by stabilizing the mRNA of Enhancer of Zeste 2 (EZH2), the catalytic subunit of PRC2, which represses gene expression by tri-methylation of histone H3 at lysine 27 (H3K27me3). The IGF2BP1-driven stabilization and protection of EZH2 mRNA is m6A-dependent and enhances EZH2 protein levels which stimulates cell cycle progression by silencing cell cycle arrest genes through enhanced H3K27 tri-methylation. Therapeutic inhibition of IGF2BP1 destabilizes EZH2 mRNA and results in a reduced cell proliferation, paralleled by an increase in G1 and sub-G1 phases. Combined targeting of IGF2BP1, EZH2, and Myc, a transcriptional activator of EZH2 and well-known target of IGF2BP1 cooperatively induces tumor cell apoptosis. Our data identify IGF2BP1 as an important driver of tumor progression in NEN, and indicate that disruption of the IGF2BP1-Myc-EZH2 axis represents a promising approach for targeted therapy of neuroendocrine neoplasms.
ABSTRACT
The transcription factor CUX1 has been implicated in either tumor suppression or progression, depending on the cancer entity and the prevalent CUX1 isoform. Previously, we could show that CUX1 acts as an important mediator of tumor cell proliferation and resistance to apoptosis in pancreatic cancer cell lines. However, in vivo evidence for its impact on pancreatic carcinogenesis, isoform-specific effects and downstream signaling cascades are missing. We crossbred two different CUX1 isoform mouse models (p200 CUX1 and p110 CUX1) with KC (KrasLSL-G12D/+; Ptf1aCre/+) mice, a genetic model for pancreatic precursor lesions (PanIN). In the context of oncogenic KRASs, both mice KCCux1p200 and KCCux1p110 led to increased PanIN formation and development of invasive pancreatic ductal adenocarcinomata (PDAC). In KCCux1p110 mice, tumor development was dramatically more accelerated, leading to formation of invasive PDAC within 4 weeks. In vitro and in vivo, we could show that CUX1 enhanced proliferation by activating MEK-ERK signaling via an upstream increase of ADAM17 protein, which in turn led to an activation of EGFR. Additionally, CUX1 further enhanced MEK-ERK activation through upregulation of the serine/threonine kinase MOS, phosphorylating MEK in a KRAS-independent manner. We identified p110 CUX1 as major driver of pancreatic cancer formation in the context of mutant KRAS. These results provide the first in vivo evidence for the importance of CUX1 in the development of pancreatic cancer, and highlight the importance of CUX1-dependent signaling pathways as potential therapeutic targets.
ABSTRACT
INTRODUCTION: Autophagic cell death in cancer cells can be mediated by inhibition of deacetylases. Although extensive studies have focused on the autophagic process in cancer, little is known about the role of autophagy in degrading cytosolic and nuclear components of pancreatic neuroendocrine neoplastic (pNEN) cells leading to cell death, thus improving the therapy of patients affected by pNEN. METHODS: 2D and 3D human pNEN and pancreatic stellate cells were treated with panobinostat and bafilomycin. Autophagy markers were detected by RT-qPCR, immunofluorescence, and Western blot. Autophagosomes were detected by electron microscopy and their maturation by real-time fluorescence of LC3B stable transfected cells. ChIP was performed at the cAMP responsive element. Immunofluorescence was performed in murine pancreatic tissue. RESULTS: We observed that pan-deacetylase inhibitor panobinostat treatment causes autophagic cell death in pNEN cells. We also found that although AMPK-α phosphorylation is counterbalanced by phosphorylated AKT, it is not capable to inhibiting autophagic cell death. However, the binding activity of the cAMP responsive element is prompted by panobinostat. Although autophagy inhibition prevented autophagosome synthesis, maturation, and cell death, panobinostat treatment induced the accumulation of mature autophagosomes in the cytosol and the nucleus, leading to disruption of the organelles, cellular digestion, and decay. Observation of autophagosome membrane proteins Beclin1 and LC3B aggregation in murine pancreatic islets indicates that autophagy restoration may also lead to autophagosome aggregation in murine insulinoma cells. A basal low expression of autophagy markers was detectable in patients affected by pNEN, and, interestingly, the expression of these markers was significantly lower in metastatic pNEN. DISCUSSION/CONCLUSION: Our study highlights that the autophagy functional restoration and prolongation of this catabolic process, mediated by inhibition of deacetylase, is responsible for the reduction of pNEN cells. Prompting of autophagy cell death could be a promising strategy for the therapy of pNEN.
Subject(s)
Autophagic Cell Death/drug effects , Enzyme Inhibitors/pharmacology , Neuroendocrine Tumors/drug therapy , Pancreatic Neoplasms/drug therapy , Cell Line, Tumor , Humans , Panobinostat/pharmacologyABSTRACT
BACKGROUND: The G-protein-coupled receptor Class C Group 6 Member A (GPRC6A) is activated by multiple ligands and is important for the regulation of calcium homeostasis. Extracellular calcium is capable to increase NLRP3 inflammasome activity of the innate immune system and deletion of this proinflammatory pathway mitigated pancreatitis severity in vivo. As such this pathway and the GPRC6A receptor is a reasonable candidate gene for pancreatitis. Here we investigated the prevalence of sequence variants in the GPRC6A locus in different pancreatitis aetiologies. METHODS: We selected 6 tagging SNPs with the SNPinfo LD TAG SNP Selection tool and the functional relevant SNP rs6907580 for genotyping. Cohorts from Germany, further European countries and China with up to 1,124 patients and 1,999 controls were screened for single SNPs with melting curve analysis. RESULTS: We identified an association of rs1606365(G) with alcoholic chronic pancreatitis in a German (odds ratio (OR) 0.76, 95% confidence interval (CI) 0.65-0.89, p = 8 × 10-5) and a Chinese cohort (OR 0.78, 95% CI 0.64-0.96, p = 0.02). However, this association was not replicated in a combined cohort of European patients (OR 1.18, 95% CI 0.99-1.41, p = 0.07). Finally, no association was found with acute and non-alcoholic chronic pancreatitis. CONCLUSIONS: Our results support a potential role of calcium sensing receptors and inflammasome activation in alcoholic chronic pancreatitis development. As the functional consequence of the associated variant is unclear, further investigations might elucidate the relevant mechanisms.
Subject(s)
Pancreatitis/genetics , Receptors, G-Protein-Coupled/genetics , Adult , Aged , Asian People , DNA/genetics , Europe , Female , Genetic Predisposition to Disease , Genetic Variation , Genome-Wide Association Study , Humans , Male , Middle Aged , Pancreatitis, Alcoholic/genetics , Polymorphism, Single Nucleotide , Receptors, Calcium-Sensing/genetics , Risk Factors , Signal Transduction/genetics , White PeopleABSTRACT
BACKGROUND: /Objectives: AGE and their receptors like RAGE and Galectin-3 can activate inflammatory pathways and have been associated with chronic inflammatory diseases. Several studies investigated the role of AGE, Galectin-3 and sRAGE in pancreatic diseases, whereas no comprehensive data for chronic pancreatitis (CP) are available. METHODS: Serum samples from CP patients without an active inflammatory process (85 ACP; 26 NACP patients) and 40 healthy controls were collected. Levels of AGE, sRAGE and Galectin-3 were measured by ELISA. To exclude potential influences of previously described RAGE SNPs on detected serum levels, we analyzed variants rs207128, rs207060, rs1800625, and rs1800624 by melting curve technique in 378 CP patients and 338 controls. RESULTS: AGE and Galectin-3 serum levels were significantly elevated in both ACP and NACP patients compared to controls (AGE: 56.61 ± 3.043 vs. 31.71 ± 2.308 ng/mL; p < 0.001; Galectin-3: 16.63 ± 0.6297 vs. 10.81 ± 0.4835 ng/mL; p < 0.001). In contrast, mean serum sRAGE levels were significantly reduced in CP patients compared to controls (sRAGE: 829.7 ± 37.10 vs. 1135 ± 55.74 ng/mL; p < 0.001). All results were consistent after correction for gender, age and diabetes mellitus. No genetic association with CP was found. CONCLUSIONS: Our extensive analysis demonstrated the importance of aging related pathways in the pathogenesis of CP. As the results were consistent in ACP and NACP, both entities most likely share common pathomechanisms. Most probably the involved pathways are a general hallmark of an inflammatory state in CP that is even present in symptom-free intervals.
Subject(s)
Antigens, Neoplasm/blood , Galectins/blood , Glycation End Products, Advanced/blood , Mitogen-Activated Protein Kinases/blood , Pancreatitis, Chronic/blood , Adult , Aged , Aged, 80 and over , Aging , Alcoholism/complications , Antigens, Neoplasm/genetics , Blood Proteins/genetics , Diabetes Complications/blood , Female , Galectins/genetics , Glycation End Products, Advanced/genetics , Humans , Inflammation/blood , Male , Middle Aged , Mitogen-Activated Protein Kinases/genetics , Pancreatitis, Chronic/complications , Pancreatitis, Chronic/genetics , Polymorphism, Single Nucleotide , Young AdultABSTRACT
INTRODUCTION: Chronic pancreatitis (CP) may be caused by oxidative stress. An important source of reactive oxygen species (ROS) is the methylglyoxal-derived formation of advanced glycation endproducts (AGE). Methylglyoxal is detoxified by Glyoxalase I (GLO1). A reduction in GLO1 activity results in increased ROS. Single nucleotide polymorphisms (SNPs) of GLO1 have been linked to various inflammatory diseases. Here, we analyzed whether common GLO1 variants are associated with alcoholic (ACP) and non-alcoholic CP (NACP). METHODS: Using melting curve analysis, we genotyped a screening cohort of 223 ACP, 218 NACP patients, and 328 controls for 11 tagging SNPs defined by the SNPinfo LD TAG SNP Selection tool and the functionally relevant variant rs4746. For selected variants the cohorts were extended to up to 1,441 patient samples. RESULTS: In the ACP cohort, comparison of genotypes for rs1937780 between patients and controls displayed an ambiguous result in the screening cohort (p = 0.08). However, in the extended cohort of 1,441 patients no statistically significant association was found for the comparison of genotypes (p = 0.11), nor in logistic regression analysis (p = 0.214, OR 1.072, 95% CI 0.961-1.196). In the NACP screening cohort SNPs rs937662, rs1699012, and rs4746 displayed an ambiguous result when patients were compared to controls in the recessive or dominant model (p = 0.08, 0.08, and 0.07, respectively). Again, these associations were not confirmed in the extended cohorts (rs937662, dominant model: p = 0.07, logistic regression: p = 0.07, OR 1.207, 95% CI 0.985-1.480) or in the replication cohorts for rs4746 (Germany, p = 0.42, OR 1.080, 95% CI 0.673-1.124; France, p = 0.19, OR 0.90, 95% CI 0.76-1.06; China, p = 0.24, OR 1.18, 95% CI 0.90-1.54) and rs1699012 (Germany, Munich; p = 0.279, OR 0.903, 95% CI 0.750-1.087). CONCLUSIONS: Common GLO1 variants do not increase chronic pancreatitis risk.
Subject(s)
Genetic Predisposition to Disease , Lactoylglutathione Lyase/genetics , Pancreatitis, Alcoholic/genetics , Pancreatitis, Chronic/genetics , Female , Genetic Association Studies , Genotype , Glycation End Products, Advanced/genetics , Humans , Male , Middle Aged , Oxidative Stress/genetics , Pancreatitis, Alcoholic/metabolism , Pancreatitis, Alcoholic/pathology , Pancreatitis, Chronic/metabolism , Pancreatitis, Chronic/pathology , Polymorphism, Single Nucleotide/genetics , Pyruvaldehyde/metabolism , Reactive Oxygen Species/metabolism , Risk FactorsABSTRACT
BACKGROUND/OBJECTIVES: Acute pancreatitis (AP) is one of the most common gastrointestinal disorders often requiring hospitalization. Frequent aetiologies are gallstones and alcohol abuse. In contrast to chronic pancreatitis (CP) few robust genetic associations have been described. Here we analysed whether common variants in the CLDN2-MORC4 and the PRSS1-PRSS2 locus that increase recurrent AP and CP risk associate with AP. METHODS: We screened 1462 AP patients and 3999 controls with melting curve analysis for SNPs rs10273639 (PRSS1-PRSS2), rs7057398 (RIPPLY), and rs12688220 (MORC4). Calculations were performed for the overall group, aetiology, and gender sub-groups. To examine genotype-phenotype relationships we performed several meta-analyses. RESULTS: Meta-analyses of all AP patients depicted significant (p-valueâ¯<â¯0.05) associations for rs10273639 (odds ratio (OR) 0.88, 95% confidence interval (CI) 0.81-0.97, p-value 0.01), rs7057398 (OR 1.27, 95% CI 1.07-1.5, p-value 0.005), and rs12688220 (OR 1.32, 95% CI 1.12-1.56, p-value 0.001). For the different aetiology groups a significant association was shown for rs10273639 (OR 0.76, 95% CI 0.63-0.92, p-value 0.005), rs7057398 (OR 1.43, 95% CI 1.07-1.92, p-value 0.02), and rs12688220 (OR 1.44, 95% CI 1.07-1.93, p-value 0.02) in the alcoholic sub-group only. CONCLUSIONS: The association of CP risk variants with different AP aetiologies, which is strongest in the alcoholic AP group, might implicate common pathomechanisms most likely between alcoholic AP and CP.
ABSTRACT
Pancreatic neuroendocrine tumors (PNETs) represent a heterogeneous group of neuroendocrine neoplasms with varying biological behavior and response to treatment. Although targeted therapies have been shown to improve the survival for patients at advanced stage, resistance to current therapies frequently occurs during the course of therapy. Previous reports indicate that the infiltration of tumor-associated macrophages (TAMs) in PNETs might correlate with tumor progression and metastasis formation. We aimed to evaluate the prognostic and functional impact of TAMs in human PNETs in vitro and in vivo and to investigate the effect of therapeutic targeting TAMs in a genetic PNET mouse model. TAM expression pattern was assessed immunohistochemically in human PNET tissue sections and a tissue-micro-array of PNET tumors with different functionality, stage, and grading. The effect of liposomal clodronate on TAM cell viability was analyzed in myeloid cell lines and isolated murine bone macrophages (mBMM). In vivo, RIP1Tag2 mice developing insulinomas were treated with liposomal clodronate or PBS-Liposomes. Tumor progression, angiogenesis and immune cell infiltration were assessed by immunohistochemistry. In human, insulinomas TAM density was correlated with invasiveness and malignant behavior. Moreover, TAM infiltration in liver metastases was significantly increased compared to primary tumors. In vitro, Liposomal clodronate selectively inhibited the viability of myeloid cells and murine bone macrophages, leaving PNET tumor cell lines largely unaffected. In vivo, repeated application of liposomal clodronate to RIP1Tag2 mice significantly diminished the malignant transformation of insulinomas, which was accompanied by a reduced infiltration of F4/80-positive TAM cells and simultaneously by a decreased microvessel density, suggesting a pronounced effect of clodronate-induced myeloid depletion on tumor angiogenesis. Concomitant treatment with the antiangiogenic TKI sunitinib, however, did not show any synergistic effects with liposomal clodronate. TAMs are crucial for malignant transformation in human PNET and correlate with metastatic behavior. Pharmacological targeting of TAMs via liposomal clodronate disrupts tumor progression in the RIP1Tag2 neuroendocrine tumor model and was associated with reduced tumor angiogenesis. Based on these results, using liposomal clodronate to target proangiogenic myeloid cells could be employed as novel therapeutic avenue in highly angiogenic tumors such as PNET.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Clodronic Acid/therapeutic use , Insulinoma/drug therapy , Macrophages/drug effects , Neovascularization, Pathologic/drug therapy , Neuroendocrine Tumors/drug therapy , Pancreatic Neoplasms/drug therapy , Sunitinib/therapeutic use , Adolescent , Adult , Aged , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Carriers/therapeutic use , Drug Synergism , Female , Humans , Liposomes/therapeutic use , Male , Mice , Mice, Transgenic , Middle Aged , Young AdultABSTRACT
OBJECTIVE: Alcohol-related pancreatitis is associated with a disproportionately large number of hospitalisations among GI disorders. Despite its clinical importance, genetic susceptibility to alcoholic chronic pancreatitis (CP) is poorly characterised. To identify risk genes for alcoholic CP and to evaluate their relevance in non-alcoholic CP, we performed a genome-wide association study and functional characterisation of a new pancreatitis locus. DESIGN: 1959 European alcoholic CP patients and population-based controls from the KORA, LIFE and INCIPE studies (n=4708) as well as chronic alcoholics from the GESGA consortium (n=1332) were screened with Illumina technology. For replication, three European cohorts comprising 1650 patients with non-alcoholic CP and 6695 controls originating from the same countries were used. RESULTS: We replicated previously reported risk loci CLDN2-MORC4, CTRC, PRSS1-PRSS2 and SPINK1 in alcoholic CP patients. We identified CTRB1-CTRB2 (chymotrypsin B1 and B2) as a new risk locus with lead single-nucleotide polymorphism (SNP) rs8055167 (OR 1.35, 95% CI 1.23 to 1.6). We found that a 16.6 kb inversion in the CTRB1-CTRB2 locus was in linkage disequilibrium with the CP-associated SNPs and was best tagged by rs8048956. The association was replicated in three independent European non-alcoholic CP cohorts of 1650 patients and 6695 controls (OR 1.62, 95% CI 1.42 to 1.86). The inversion changes the expression ratio of the CTRB1 and CTRB2 isoforms and thereby affects protective trypsinogen degradation and ultimately pancreatitis risk. CONCLUSION: An inversion in the CTRB1-CTRB2 locus modifies risk for alcoholic and non-alcoholic CP indicating that common pathomechanisms are involved in these inflammatory disorders.
Subject(s)
Chymotrypsin/genetics , Pancreatitis, Alcoholic , Adult , Aged , Europe/epidemiology , Female , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Pancreatitis, Alcoholic/epidemiology , Pancreatitis, Alcoholic/genetics , Polymorphism, Single NucleotideABSTRACT
BACKGROUND & AIMS: The ability of exocrine pancreatic cells to change the cellular phenotype is required for tissue regeneration upon injury, but also contributes to their malignant transformation and tumor progression. We investigated context-dependent signaling and transcription mechanisms that determine pancreatic cell fate decisions toward regeneration and malignancy. In particular, we studied the function and regulation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic cell plasticity and tissue adaptation. METHODS: We analyzed cell plasticity during pancreatic regeneration and transformation in mice with pancreas-specific expression of a constitutively active form of NFATC1, or depletion of enhancer of zeste 2 homologue 2 (EZH2), in the context of wild-type or constitutively activate Kras, respectively. Acute and chronic pancreatitis were induced by intraperitoneal injection of caerulein. EZH2-dependent regulation of NFATC1 expression was studied in mouse in human pancreatic tissue and cells by immunohistochemistry, immunoblotting, and quantitative reverse transcription polymerase chain reaction. We used genetic and pharmacologic approaches of EZH2 and NFATC1 inhibition to study the consequences of pathway disruption on pancreatic morphology and function. Epigenetic modifications on the NFATC1 gene were investigated by chromatin immunoprecipitation assays. RESULTS: NFATC1 was rapidly and transiently induced in early adaptation to acinar cell injury in human samples and in mice, where it promoted acinar cell transdifferentiation and blocked proliferation of metaplastic pancreatic cells. However, in late stages of regeneration, Nfatc1 was epigenetically silenced by EZH2-dependent histone methylation, to enable acinar cell redifferentiation and prevent organ atrophy and exocrine insufficiency. In contrast, oncogenic activation of KRAS signaling in pancreatic ductal adenocarcinoma cells reversed the EZH2-dependent effects on the NFATC1 gene and was required for EZH2-mediated transcriptional activation of NFATC1. CONCLUSIONS: In studies of human and mouse pancreatic cells and tissue, we identified context-specific epigenetic regulation of NFATc1 activity as an important mechanism of pancreatic cell plasticity. Inhibitors of EZH2 might therefore interfere with oncogenic activity of NFATC1 and be used in treatment of pancreatic ductal adenocarcinoma.
Subject(s)
Carcinoma, Pancreatic Ductal/genetics , Cell Plasticity/genetics , Cell Transformation, Neoplastic/genetics , Enhancer of Zeste Homolog 2 Protein/genetics , Gene Expression Regulation , NFATC Transcription Factors/genetics , Pancreatic Neoplasms/genetics , Regeneration/genetics , Acinar Cells/physiology , Animals , Carcinoma, Pancreatic Ductal/chemistry , Cell Proliferation/genetics , Cell Transdifferentiation/genetics , Ceruletide , Cyclin-Dependent Kinase Inhibitor p16/genetics , Enhancer of Zeste Homolog 2 Protein/analysis , Enhancer of Zeste Homolog 2 Protein/metabolism , Gene Silencing , Histones/metabolism , Humans , Methylation , Mice , NFATC Transcription Factors/analysis , NFATC Transcription Factors/metabolism , Pancreas/physiology , Pancreatic Neoplasms/chemistry , Pancreatitis, Chronic/chemically induced , Pancreatitis, Chronic/physiopathology , Promoter Regions, Genetic , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction/genetics , Transcription, GeneticABSTRACT
OBJECTIVES: Tumour-associated macrophages play an important role in mediating tumour progression. In pancreatic cancer, infiltrating macrophages are known to mediate tumour progression and have been identified in invasive tumours and in early preinvasive pancreatic intraepithelial precursor lesions. We aimed to study the impact of pharmacological macrophage depletion by liposomal clodronate in a genetic mouse model of pancreatic cancer. METHODS: KPC mice (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) were treated for 12â weeks with liposomal clodronate or control liposomes. Tumour and metastasis formation as well as alterations in local and circulating immune cells and cytokines were analysed. RESULTS: Treatment with liposomal clodronate effectively reduced CD11b-positive macrophages both in the pancreas and other organs such as liver, lung and spleen. While tumour incidence and growth were only slightly reduced, metastasis formation in the liver and lungs was significantly diminished after macrophage depletion. This antimetastatic effect was independent of the presence of an endogenous primary tumour, since reduced pulmonary colonisation was also detected in clodronate-pretreated mice after tail vein injection of syngeneic pancreatic cancer cell lines. Macrophage inhibition by liposomal clodronate was associated with significantly impaired angiogenesis, reduced circulating vascular endothelial growth factor levels and decreased circulating CD4+CD25+ T cells. These alterations could be confirmed in an independent macrophage depletion model using CD11b-diphtheria toxin receptor mice. CONCLUSIONS: Pharmacological depletion of macrophages in a genetic mouse model of pancreatic cancer markedly reduced metastasis formation and is associated with impaired angiogenesis and reduced CD4+CD25+ T cell levels. Pharmacological targeting of infiltrating macrophages represents a promising novel tool for antimetastatic therapeutic approaches.
Subject(s)
Antineoplastic Agents/pharmacology , Clodronic Acid/pharmacology , Macrophages/drug effects , Pancreatic Neoplasms/pathology , Animals , CD11b Antigen/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cell Proliferation/drug effects , Interleukin-2 Receptor alpha Subunit/metabolism , Liposomes , Liver Neoplasms/immunology , Liver Neoplasms/secondary , Lung Neoplasms/immunology , Lung Neoplasms/secondary , Macrophages/immunology , Mice , Models, Genetic , Neoplasms, Experimental , Neovascularization, Pathologic , Pancreatic Neoplasms/immunology , T-Lymphocytes/metabolism , Tumor Burden/drug effects , Vascular Endothelial Growth Factor A/bloodABSTRACT
Myeloid cells including tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) are known as important mediators of tumor progression in solid tumors such as pancreatic cancer. Infiltrating myeloid cells have been identified not only in invasive tumors, but also in early pre-invasive pancreatic intraepithelial precursor lesions (PanIN). The functional dynamics of myeloid cells during carcinogenesis is largely unknown. We aimed to systematically elucidate phenotypic and transcriptional changes in infiltrating myeloid cells during carcinogenesis and tumor progression in a genetic mouse model of pancreatic cancer. Using murine pancreatic myeloid cells isolated from the genetic mouse model at different time points during carcinogenesis, we examined both established markers of macrophage polarization using RT-PCR and FACS as well as transcriptional changes focusing on miRNA profiling. Myeloid cells isolated during carcinogenesis showed a simultaneous increase of established markers of M1 and M2 polarization during carcinogenesis, indicating that phenotypic changes of myeloid cells during carcinogenesis do not follow the established M1/M2 classification. MiRNA profiling revealed distinct regulations of several miRNAs already present in myeloid cells infiltrating pre-invasive PanIN lesions. Among them miRNA-21 was significantly increased in myeloid cells surrounding both PanIN lesions and invasive cancers. Functionally, miRNA-21-5p and -3p altered expression of the immune-modulating cytokines CXCL-10 and CCL-3 respectively. Our data indicate that miRNAs are dynamically regulated in infiltrating myeloid cells during carcinogenesis and mediate their functional phenotype by facilitating an immune-suppressive tumor-promoting micro-milieu.
ABSTRACT
Pancreatic neuroendocrine neoplasms (PNENs) constitute a rare tumour entity, and prognosis and treatment options depend on tumour-mediating hallmarks such as angiogenesis, proliferation rate and resistance to apoptosis. The molecular pathways that determine the malignant phenotype are still insufficiently understood and this has limited the use of effective combination therapies in the past. In this study, we aimed to characterise the effect of the oncogenic transcription factor Cut homeobox 1 (CUX1) on proliferation, resistance to apoptosis and angiogenesis in murine and human PNENs. The expression and function of CUX1 were analysed using knockdown and overexpression strategies in Ins-1 and Bon-1 cells, xenograft models and a genetically engineered mouse model of insulinoma (RIP1Tag2). Regulation of angiogenesis was assessed using RNA profiling and functional tube-formation assays in HMEC-1 cells. Finally, CUX1 expression was assessed in a tissue microarray of 59 human insulinomas and correlated with clinicopathological data. CUX1 expression was upregulated during tumour progression in a time- and stage-dependent manner in the RIP1Tag2 model, and associated with pro-invasive and metastatic features of human insulinomas. Endogenous and recombinant CUX1 expression increased tumour cell proliferation, tumour growth, resistance to apoptosis, and angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of CUX1 was mediated via upregulation of effectors such as HIF1α and MMP9. CUX1 mediates an invasive pro-angiogenic phenotype and is associated with malignant behaviour in human insulinomas.
Subject(s)
Apoptosis , Cell Movement , Homeodomain Proteins/metabolism , Insulinoma/pathology , Neuroendocrine Tumors/secondary , Nuclear Proteins/metabolism , Pancreatic Neoplasms/pathology , Repressor Proteins/metabolism , Animals , Blotting, Western , Cell Adhesion , Cell Proliferation , Female , Homeodomain Proteins/genetics , Humans , Immunoenzyme Techniques , Insulinoma/genetics , Insulinoma/metabolism , Lymphatic Metastasis , Male , Mice , Mice, Nude , Neoplasm Grading , Neoplasm Staging , Neuroendocrine Tumors/genetics , Neuroendocrine Tumors/metabolism , Nuclear Proteins/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
INTRODUCTION: WNT5A belongs to the Wnt family of secreted signaling molecules. Using transcriptional profiling, we previously identified WNT5A as target of the antiapoptotic transcription factor CUX1 and demonstrated high expression levels in pancreatic cancer. However, the impact of WNT5A on drug resistance and the signaling pathways employed by WNT5A remain to be elucidated. OBJECTIVES: This project aims to decipher the impact of WNT5A on resistance to apoptosis and the signaling pathways employed by WNT5A in pancreatic cancer. METHODS: The impact of WNT5A and its downstream effectors on tumor growth and drug resistance was studied in vitro and in xenograft models in vivo. Tissue microarrays of pancreatic cancer specimens were employed for immunohistochemical studies. RESULTS: Knockdown of WNT5A results in a significant increase in drug-induced apoptosis. In contrast, overexpression of WNT5A or addition of recombinant WNT5A mediates resistance to apoptosis in vitro. In our attempt to identify downstream effectors of WNT5A, we identified the transcription factor nuclear factor of activated T cells c2 (NFATc2) as transcriptional target of WNT5A signaling. NFATc2 confers a strong antiapoptotic phenotype mediating at least in part the effects of WNT5A on drug resistance and tumor cell survival. In vivo, WNT5A expression leads to resistance to gemcitabine-induced apoptosis in a xenograft model, which is paralleled by up-regulation of NFATc2. Both WNT5A and NFATc2 proteins are highly expressed in human pancreatic cancer tissues and their expression levels correlated significantly. CONCLUSION: We identified the WNT5A-NFATc2 axis as important mediator of drug resistance in pancreatic cancer.
Subject(s)
Apoptosis/physiology , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Pancreatic Neoplasms/metabolism , Proto-Oncogene Proteins/metabolism , Wnt Proteins/metabolism , Animals , Apoptosis/genetics , Cell Line, Tumor , Cell Survival , Drug Resistance, Neoplasm , Female , Humans , Mice , Mice, Nude , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , Random Allocation , Signal Transduction , TNF-Related Apoptosis-Inducing Ligand/genetics , Transcription, Genetic , Up-Regulation , Wnt Proteins/genetics , Wnt-5a Protein , beta Catenin/geneticsABSTRACT
Pancreatic cancer is characterized by a high degree of resistance to chemotherapy. Epidermal growth factor receptor (EGFR) inhibition using the small-molecule inhibitor erlotinib was shown to provide a small survival benefit in a subgroup of patients. To identify kinases whose inhibition acts synergistically with erlotinib, we employed a kinome-wide small-interfering RNA (siRNA)-based loss-of-function screen in the presence of erlotinib. Of 779 tested kinases, we identified several targets whose inhibition acted synergistically lethal with EGFR inhibition by erlotinib, among them the S6 kinase ribosomal protein S6 kinase 2 (RPS6KA2)/ribosomal S6 kinase 3. Activated RPS6KA2 was expressed in approximately 40% of 123 human pancreatic cancer tissues. RPS6KA2 was shown to act downstream of EGFR/RAS/mitogen-activated protein kinase kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling and was activated by EGF independently of the presence of KRAS mutations. Knockdown of RPS6KA2 by siRNA led to increased apoptosis only in the presence of erlotinib, whereas RPS6KA2 activation or overexpression rescued from erlotinib- and gemcitabine-induced apoptosis. This effect was at least in part mediated by downstream activation of ribosomal protein S6. Genetic as well as pharmacological inhibition of RPS6KA2 by the inhibitor BI-D1870 acted synergistically with erlotinib. By applying this synergistic lethality screen using a kinome-wide RNA interference-library approach, we identified RPS6KA2 as potential drug target whose inhibition synergistically enhanced the effect of erlotinib on tumor cell survival. This kinase therefore represents a promising drug candidate suitable for the development of novel inhibitors for pancreatic cancer therapy.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Drug Resistance, Neoplasm , ErbB Receptors/metabolism , Erlotinib Hydrochloride , Gene Knockdown Techniques , Genes, Lethal , Humans , MAP Kinase Signaling System , Mice , Pancreatic Neoplasms , Quinazolines/pharmacology , RNA, Small Interfering/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolismABSTRACT
OBJECTIVES: Novel imaging methods based on specific molecular targets to detect both established neoplasms and their precursor lesions are highly desirable in cancer medicine. Previously, we identified claudin-4, an integral constituent of tight junctions, as highly expressed in various gastrointestinal tumours including pancreatic cancer. Here, we investigate the potential of targeting claudin-4 with a naturally occurring ligand to visualise pancreatic cancer and its precursor lesions in vitro and in vivo by near-infrared imaging approaches. DESIGN: A non-toxic C-terminal fragment of the claudin-4 ligand Clostridium perfringens enterotoxin (C-CPE) was labelled with a cyanine dye (Cy5.5). Binding of the optical tracer was analysed on claudin-4 positive and negative cells in vitro, and tumour xenografts in vivo. In addition, two genetically engineered mouse models for pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer were used for in vivo validation. Optical imaging studies were conducted using 2D planar fluorescence reflectance imaging (FRI) technology and 3D fluorescence-mediated tomography (FMT). RESULTS: In vitro, the peptide-dye conjugate showed high binding affinity to claudin-4 positive CAPAN1 cells, while claudin-4 negative HT1080 cells revealed little or no fluorescence. In vivo, claudin-4 positive tumour xenografts, endogenous pancreatic tumours, hepatic metastases, as well as preinvasive PanIN lesions, were visualised by FRI and FMT up to 48 h after injection showing a significantly higher average of fluorochrome concentration as compared with claudin-4 negative xenografts and normal pancreatic tissue. CONCLUSIONS: C-CPE-Cy5.5 combined with novel optical imaging methods enables non-invasive visualisation of claudin-4 positive murine pancreatic tumours and their precursor lesions, representing a promising modality for early diagnostic imaging.
Subject(s)
Biomarkers, Tumor/metabolism , Claudin-4/metabolism , Pancreatic Neoplasms/diagnosis , Precancerous Conditions/diagnosis , Animals , Carcinoma, Pancreatic Ductal/diagnosis , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/secondary , Cell Line, Tumor , Enterotoxins , Female , Gene Expression Regulation, Neoplastic , Humans , Ligands , Liver Neoplasms/diagnosis , Liver Neoplasms/secondary , Mice , Mice, Transgenic , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Optical Imaging/methods , Pancreatic Neoplasms/metabolism , Precancerous Conditions/metabolism , Tomography, Optical/methods , Transplantation, Heterologous , Up-RegulationABSTRACT
Previously, we identified the transcription factor CUX1 as an important modulator of invasion and resistance to apoptosis. Expression profiles suggested that CUX1 regulates a complex transcriptional program mediating tumor progression. We aimed to identify functionally relevant targets of CUX1 by using RNA interference (RNAi)-based loss-of-function screens. Therefore, we generated an RNAi library containing putative transcriptional targets of CUX1 identified by microarrays and performed cell viability screens. Using this approach, several CUX1 targets with effect on tumor cell viability were identified, including the glutamate receptor GRIA3, which was validated in detail for its effects on proliferation, apoptosis, and cell migration using RNAi knock-down and overexpression strategies in vitro, as well as xenograft models in vivo. The expression of GRIA3 was evaluated in human pancreatic cancer tissues. We found that knock-down of GRIA3 significantly reduced proliferation and migration and enhanced apoptosis. In contrast, overexpression of GRIA3 significantly reduced apoptosis and enhanced both proliferation and tumor cell migration. GRIA3 could be confirmed as a downstream effector of CUX1 and was expressed in pancreatic cancer tissues. In vivo, GRIA3 significantly enhanced the growth of subcutaneous xenografts. Inhibitors of glutamate receptors such as GYKI52466 and SYM2206 significantly decreased survival of pancreatic cancer cells, suggesting the presence of glutamate signaling in pancreatic cancer. In conclusion, GRIA3 plays a role as a mediator of tumor progression in pancreatic cancer downstream CUX1. To our knowledge, this is the first report to identify a glutamate receptor as a modulator of tumor progression in a solid cancer outside the brain.
Subject(s)
Adenocarcinoma/genetics , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/physiology , Nuclear Proteins/physiology , Pancreatic Neoplasms/genetics , Receptors, AMPA/genetics , Receptors, AMPA/physiology , Repressor Proteins/physiology , Adenocarcinoma/pathology , Animals , Cell Movement/drug effects , Cell Movement/genetics , Cell Survival/drug effects , Cell Survival/genetics , Disease Progression , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Homeodomain Proteins/antagonists & inhibitors , Homeodomain Proteins/genetics , Humans , Mice , Mice, Nude , Neoplasm Invasiveness , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Oncogenes/genetics , Oncogenes/physiology , Pancreatic Neoplasms/pathology , RNA Interference/physiology , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/genetics , Transcription Factors , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
Induction of apoptosis by the tumor suppressor p53 is known to protect from Myc-driven lymphomagenesis. The p53 family member p73 is also a proapoptotic protein, which is activated in response to oncogenes like Myc. Here, we have investigated whether p73 provides a similar protection from Myc-driven lymphomas as p53. Confirming previous studies, the inactivation of a single p53 allele (p53+/-) strongly reduced the median survival of Emu-Myc transgenic mice from 103 to 39 days and was invariably associated with a loss of the wild-type p53 allele. In contrast, mutational inactivation of a p73 allele (p73+/-) reduced the median survival by only 12 days. Lymphomas that developed in the p73+/- background showed no loss of heterozygosity (LOH). Furthermore, gene expression profiling of p73+/+, p73+/- and p73-/- lymphomas indicated that p73+/- lymphomas retained p73 transcriptional activity. Subtle gene expression differences between p73+/+ and p73+/- lymphomas, however, suggest a haploinsufficient phenotype on some p73 target genes. This might help to explain why p73+/- animals succumbed to disease slightly earlier than their p73+/+ littermates (log-rank test p<0.0395) and why p73 often shows monoallelic inactivation in human lymphomas. Together these data demonstrate that in Myc-driven lymphomagenesis p73 has weak tumor suppressor activity compared with p53.