ABSTRACT
The xenobiotic transporter ABCC4/MRP4 is highly expressed in pancreatic ductal adenocarcinoma (PDAC) and correlates with a more aggressive phenotype and metastatic propensity. Here, we show that ABCC4 promotes epithelial-mesenchymal transition (EMT) in PDAC, a hallmark process involving the acquisition of mesenchymal traits by epithelial cells, enhanced cell motility, and chemoresistance. Modulation of ABCC4 levels in PANC-1 and BxPC-3 cell lines resulted in the dysregulation of genes present in the EMT signature. Bioinformatic analysis on several cohorts including tumor samples, primary patient-derived cultured cells, patient-derived xenografts, and cell lines, revealed a positive correlation between ABCC4 expression and EMT markers. We also characterized the ABCC4 cistrome and identified four candidate clusters in the distal promoter and intron one that showed differential binding of pro-epithelial FOXA1 and pro-mesenchymal GATA2 transcription factors in low ABCC4-expressing HPAF-II and high ABCC4-expressing PANC-1 xenografts. HPAF-II xenografts showed exclusive binding of FOXA1, and PANC-1 xenografts exclusive binding of GATA2, at ABCC4 clusters, consistent with their low and high EMT phenotype respectively. Our results underscore ABCC4/MRP4 as a valuable prognostic marker and a potential therapeutic target to treat PDAC subtypes with prominent EMT features, such as the basal-like/squamous subtype, characterized by worse prognosis and no effective therapies.
ABSTRACT
BACKGROUND: After surgical resection of pancreatic ductal adenocarcinoma (PDAC), patients are predominantly treated with adjuvant chemotherapy, commonly consisting of gemcitabine (GEM)-based regimens or the modified FOLFIRINOX (mFFX) regimen. While mFFX regimen has been shown to be more effective than GEM-based regimens, it is also associated with higher toxicity. Current treatment decisions are based on patient performance status rather than on the molecular characteristics of the tumor. To address this gap, the goal of this study was to develop drug-specific transcriptomic signatures for personalized chemotherapy treatment. PATIENTS AND METHODS: We used PDAC datasets from preclinical models, encompassing chemotherapy response profiles for the mFFX regimen components. From them we identified specific gene transcripts associated with chemotherapy response. Three transcriptomic artificial intelligence signatures were obtained by combining independent component analysis and the least absolute shrinkage and selection operator-random forest approach. We integrated a previously developed GEM signature with three newly developed ones. The machine learning strategy employed to enhance these signatures incorporates transcriptomic features from the tumor microenvironment, leading to the development of the 'Pancreas-View' tool ultimately clinically validated in a cohort of 343 patients from the PRODIGE-24/CCTG PA6 trial. RESULTS: Patients who were predicted to be sensitive to the administered drugs (n = 164; 47.8%) had longer disease-free survival (DFS) than the other patients. The median DFS in the mFFX-sensitive group treated with mFFX was 50.0 months [stratified hazard ratio (HR) 0.31, 95% confidence interval (CI) 0.21-0.44, P < 0.001] and 33.7 months (stratified HR 0.40, 95% CI 0.17-0.59, P < 0.001) in the GEM-sensitive group when treated with GEM. Comparatively patients with signature predictions unmatched with the treatments (n = 86; 25.1%) or those resistant to all drugs (n = 93; 27.1%) had shorter DFS (10.6 and 10.8 months, respectively). CONCLUSIONS: This study presents a transcriptome-based tool that was developed using preclinical models and machine learning to accurately predict sensitivity to mFFX and GEM.
ABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) patients are frequently treated by chemotherapy. Even if personalized therapy based on molecular analysis can be performed for some tumors, PDAC regimens selection is still mainly based on patients' performance status and expected efficacy. Therefore, the establishment of molecular predictors of chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. We have recently developed an RNA-based signature that predicts the efficacy of adjuvant gemcitabine using 38 PDAC primary cell cultures. While demonstrated its efficiency, a significant association with the classical/basal-like PDAC spectrum was observed. We hypothesized that this flaw was due to the basal-like biased phenotype of cellular models used in our strategy. To overcome this limitation, we generated a prospective cohort of 27 consecutive biopsied derived pancreatic organoids (BDPO) and include them in the signature identification strategy. As BDPO's do not have the same biased phenotype as primary cell cultures we expect they can compensate one with each other and cover a broader range of molecular phenotypes. We then obtained an improved signature predicting gemcitabine sensibility that was validated in a cohort of 300 resected PDAC patients that have or have not received adjuvant gemcitabine. We demonstrated a significant association between the improved signature and the overall and disease-free survival in patients predicted as sensitive and treated with adjuvant gemcitabine. We propose then that including BDPO along primary cell cultures represent a powerful strategy that helps to overcome primary cell cultures limitations producing unbiased RNA-based signatures predictive of adjuvant treatments in PDAC.
ABSTRACT
BACKGROUND: Chemotherapy is the only systemic treatment approved for pancreatic ductal adenocarcinoma (PDAC), with a selection of regimens based on patients' performance status and expected efficacy. The establishment of a potent stratification associated with chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments. PATIENTS AND METHODS: Concomitant chemosensitivity and genome-wide RNA profiles were carried out on preclinical models (primary cell cultures and patient-derived xenografts) derived from patients with PDAC included in the PaCaOmics program (NCT01692873). The RNA-based stratification was tested in a monocentric cohort and validated in a multicentric cohort, both retrospectively collected from resected PDAC samples (67 and 368 patients, respectively). Forty-three (65%) and 203 (55%) patients received adjuvant gemcitabine in the monocentric and the multicentric cohorts, respectively. The relationships between predicted gemcitabine sensitivity and patients' overall survival (OS) and disease-free survival were investigated. RESULTS: The GemPred RNA signature was derived from preclinical models, defining gemcitabine sensitive PDAC as GemPred+. Among the patients who received gemcitabine in the test and validation cohorts, the GemPred+ patients had a higher OS than GemPred- (P = 0.046 and P = 0.00216). In both cohorts, the GemPred stratification was not associated with OS among patients who did not receive gemcitabine. Among gemcitabine-treated patients, GemPred+ patients had significantly higher OS than the GemPred-: 91.3 months [95% confidence interval (CI): 61.2-not reached] versus 33 months (95% CI: 24-35.2); hazard ratio 0.403 (95% CI: 0.221-0.735, P = 0.00216). The interaction test for gemcitabine and GemPred+ stratification was significant (P = 0.0245). Multivariate analysis in the gemcitabine-treated population retained an independent predictive value. CONCLUSION: The RNA-based GemPred stratification predicts the benefit of adjuvant gemcitabine in PDAC patients.
Subject(s)
Adenocarcinoma , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Chemotherapy, Adjuvant , Deoxycytidine/analogs & derivatives , Humans , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Retrospective Studies , Transcriptome , GemcitabineABSTRACT
The original version of this Article omitted the author Dr Mathias Chamaillard from the l'Institut de Pasteur, Lille, France. This has been corrected in both the PDF and HTML versions of the Article.
ABSTRACT
Upon oral infection with Toxoplasma gondii cysts (76 K strain) tachyzoites are released into the intestinal lumen and cross the epithelial barrier causing damage and acute intestinal inflammation in C57BL/6 (B6) mice. Here we investigated the role of microbiota and IL-22 in T.gondii-induced small intestinal inflammation. Oral T.gondii infection in B6 mice causes inflammation with IFNγ and IL-22 production. In IL-22-deficient mice, T.gondii infection augments the Th1 driven inflammation. Deficiency in either IL-22bp, the soluble IL-22 receptor or Reg3γ, an IL-22-dependent antimicrobial lectin/peptide, did not reduce inflammation. Under germ-free conditions, T.gondii-induced inflammation was reduced in correlation with parasite load. But intestinal inflammation is still present in germ-free mice, at low level, in the lamina propria, independently of IL-22 expression. Exacerbated intestinal inflammation driven by absence of IL-22 appears to be independent of IL-22 deficiency associated-dysbiosis as similar inflammation was observed after fecal transplantation of IL-22-/- or WT microbiota to germ-free-WT mice. Our results suggest cooperation between parasite and intestinal microbiota in small intestine inflammation development and endogenous IL-22 seems to exert a protective role independently of its effect on the microbiota. In conclusion, IL-22 participates in T.gondii induced acute small intestinal inflammation independently of microbiota and Reg3γ.
Subject(s)
Gastrointestinal Microbiome/immunology , Inflammation/immunology , Interleukins/metabolism , Intestines/immunology , Toxoplasma/physiology , Toxoplasmosis/immunology , Animals , Cells, Cultured , Disease Progression , Interleukins/genetics , Intestines/parasitology , Mice , Mice, Inbred C57BL , Mice, Knockout , Pancreatitis-Associated Proteins/genetics , Pancreatitis-Associated Proteins/metabolism , Parasite Load , Receptors, Interleukin/genetics , Receptors, Interleukin/metabolism , Interleukin-22ABSTRACT
Flavones represent a class of polyphenols that are found in many plant-derived food sources. Herein, we provide evidence that the anti-inflammatory and antiproliferative effect of the flavone apigenin relies on the regulation of the gut microbiota by the NOD-like receptor family pyrin domain containing 6 (Nlrp6). When challenged by dextran sulfate sodium (DSS) in drinking water, mice were protected against colitis upon cohousing with apigenin-treated animals. In contrast, the protective effect was lost in the absence of Nlrp6. Sequencing of the 16S ribosomal RNA gene revealed a shift in the composition of the gut microbiota in apigenin-treated mice that was not observed in the absence of Nlrp6. Equally important, we find that the antiproliferative effect of apigenin was dominantly transmitted after cohousing, while being compromised in Nlrp6-deficient mice. In contrast, the symptoms of colitis were alleviated upon apigenin administration even in the absence of either caspase-1/11 or Asc. Collectively, these data indicate that apigenin modulated an inflammasome-independent mechanism by which Nlrp6 reprograms the gut microbiota for protecting mice against colitis. Our study highlights a modulation of the Nlrp6 signaling pathway by a prominent constituent of the human diet that may point toward improved ways to treat inflammatory bowel diseases.
Subject(s)
Apigenin/administration & dosage , Colitis/prevention & control , Diet , Flavones/administration & dosage , Gastrointestinal Microbiome/physiology , Inflammatory Bowel Diseases/diet therapy , Receptors, Cell Surface/metabolism , Animals , Colitis/chemically induced , Dextran Sulfate , Housing, Animal , Humans , Inflammasomes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Ribosomal, 16S/genetics , Receptors, Cell Surface/genetics , Signal TransductionABSTRACT
PURPOSE: To assess the K-ras gene mutation in the histologically negative venous margin of a pancreaticoduodenectomy (PD) specimen and its impact on survival. METHOD: From 2007 to 2010, 22 patients underwent R0 PD for resecable pancreatic adenocarcinoma. All specimens were stained and the portal vein (PV) bed was identified by blue ink; a 2mm3 sample (including the blue ink) was cut from a microscopic free-tumor block. DNA was extracted and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation. Twelve specimens (55%) (kras+ group) were identified with a K-ras mutation in the venous margin resection, and 10 specimens (kras- group) did not have K-ras mutation detected in the venous margin resection. RESULTS: The two groups were comparable. Overall 3years survival of patients of kras+ group versus patients of kras- group was 0 and 17% (P=0.03), respectively. Median survival time of patients of kras+ group versus patients of kras- group was 16months vs 25months (P=0.04; 95% confidence interval [1,11-1,88]), respectively. CONCLUSION: Genetic evaluation of venous resection margin affirmed unrecognized disease with strong impact on survival in more than 50% of patients with histologically R0 resection.
Subject(s)
Adenocarcinoma/surgery , Gene Expression Regulation , Margins of Excision , Pancreatic Neoplasms/surgery , Proto-Oncogene Proteins p21(ras)/genetics , Adenocarcinoma/genetics , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Adult , Aged , Cohort Studies , Disease-Free Survival , Female , Humans , Male , Mesenteric Veins/surgery , Middle Aged , Mutation/genetics , Neoplasm Grading , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Pancreaticoduodenectomy/methods , Pancreaticoduodenectomy/mortality , Portal Vein/surgery , Prognosis , Retrospective Studies , Risk Assessment , Survival Analysis , Treatment OutcomeABSTRACT
Despite the recent description of the mucosal vaccine-induced reduction of Helicobacter pylori natural infection in a phase 3 clinical trial, the absence of immune correlates of protection slows the final development of the vaccine. In this study, we evaluated the role of interleukin (IL)-22 in mucosal vaccine-induced protection. Gastric IL-22 levels were increased in mice intranasally immunized with urease+cholera toxin and challenged with H. felis, as compared with controls. Flow cytometry analysis showed that a peak of CD4+IL-22+IL-17+ T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice. The inhibition of the IL-22 biological activity prevented the vaccine-induced reduction of H. pylori infection. Remarkably, anti-microbial peptides (AMPs) extracted from the stomachs of vaccinated mice, but not from the stomachs of non-immunized or immunized mice, injected with anti-IL-22 antibodies efficiently killed H. pylori in vitro. Finally, H. pylori infection in vaccinated RegIIIß-deficient mice was not reduced as efficiently as in wild-type mice. These results demonstrate that IL-22 has a critical role in vaccine-induced protection, by promoting the expression of AMPs, such as RegIIIß, capable of killing Helicobacter. Therefore, it can be concluded that urease-specific memory Th17/Th22 cells could constitute immune correlates of vaccine protection in humans.
Subject(s)
Antigens, Bacterial/immunology , Bacterial Vaccines/immunology , Helicobacter Infections/immunology , Helicobacter pylori/immunology , Interleukins/metabolism , Mucous Membrane/immunology , Th17 Cells/immunology , Urease/immunology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Cells, Cultured , Disease Models, Animal , Female , Gene Expression Regulation , Helicobacter Infections/prevention & control , Humans , Interleukins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mucous Membrane/microbiology , Pancreatitis-Associated Proteins , Proteins/genetics , Proteins/metabolism , Interleukin-22ABSTRACT
Both clinical and experimental evidence have firmly established that chronic pancreatitis, in particular in the context of Kras oncogenic mutations, predisposes to pancreatic ductal adenocarcinoma (PDAC). However, the repertoire of molecular mediators of pancreatitis involved in Kras-mediated initiation of pancreatic carcinogenesis remains to be fully defined. In this study we demonstrate a novel role for vacuole membrane protein 1 (VMP1), a pancreatitis-associated protein critical for inducible autophagy, in the regulation of Kras-induced PDAC initiation. Using a newly developed genetically engineered model, we demonstrate that VMP1 increases the ability of Kras to give rise to preneoplastic lesions, pancreatic intraepithelial neoplasias (PanINs). This promoting effect of VMP1 on PanIN formation is due, at least in part, by an increase in cell proliferation combined with a decrease in apoptosis. Using chloroquine, an inhibitor of autophagy, we show that this drug antagonizes the effect of VMP1 on PanIN formation. Thus, we conclude that VMP1-mediated autophagy cooperate with Kras to promote PDAC initiation. These findings are of significant medical relevance, molecules targeting autophagy are currently being tested along chemotherapeutic agents to treat PDAC and other tumors in human trials.
Subject(s)
Carcinoma, Ductal/genetics , Gene Expression Regulation, Neoplastic , Membrane Proteins/genetics , Pancreatic Neoplasms/genetics , Pancreatitis/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Autophagy/drug effects , Carcinoma, Ductal/etiology , Carcinoma, Ductal/metabolism , Carcinoma, Ductal/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Chloroquine/pharmacology , Genes, Reporter , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Pancreas/metabolism , Pancreas/pathology , Pancreatic Neoplasms/etiology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatitis/complications , Pancreatitis/metabolism , Pancreatitis/pathology , Pancreatitis-Associated Proteins , Proto-Oncogene Proteins p21(ras)/metabolism , Signal TransductionABSTRACT
Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits are modest, and as its mechanisms of action remain elusive, a better understanding of its anticancer effects is needed. Based on our previous study results, we investigated here the implication of the nuclear protein 1 (NUPR1) in HCC and its role in sorafenib treatment. NUPR1 is a stress-inducible protein that is overexpressed in various malignancies, but its role in HCC is not yet fully understood. We found that NUPR1 expression was significantly higher in primary human HCC samples than in the normal liver. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibited the cell growth, migration and invasion of HCC cells, both in vitro and in vivo. Moreover, NUPR1 silencing influenced the expression of RELB and IER3 genes. Unsurprisingly, RELB and IER3 knockdown also inhibited HCC cell viability, growth and migration. Using gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as downregulated gene nodes, and several HCC-related oncogenes were also suppressed. We identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1-regulated gene and demonstrated that RUNX2 gene silencing inhibits HCC cell viability, growth, migration and increased cell sensitivity to sorafenib. We propose that the NUPR1/RELB/IER3/RUNX2 pathway has a pivotal role in hepatocarcinogenesis. The identification of the NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Movement/drug effects , Drug Resistance, Neoplasm/drug effects , Liver Neoplasms/pathology , Molecular Targeted Therapy , Neoplasm Proteins/metabolism , Niacinamide/analogs & derivatives , Phenylurea Compounds/pharmacology , Aged , Aged, 80 and over , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Computational Biology , Core Binding Factor Alpha 1 Subunit/metabolism , Down-Regulation/drug effects , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Gene Silencing/drug effects , Humans , Liver Neoplasms/genetics , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Middle Aged , Neoplasm Invasiveness , Neoplasm Proteins/genetics , Niacinamide/pharmacology , RNA, Small Interfering/metabolism , Sorafenib , Transcription Factor RelB/genetics , Transcription Factor RelB/metabolism , Transcriptome/genetics , Young AdultABSTRACT
Advances made in pancreatic cancer therapy have been far from sufficient and have allowed only a slight improvement in global survival of patients with pancreatic ductal adenocarcinoma (PDA). Recent progresses in chemotherapy have offered some hope for an otherwise gloomy outlook, however, only a limited number of patients are eligible because of important cytotoxicity. In this context, enhancing our knowledge on PDA initiation and evolution is crucial to highlight certain weaknesses on which to specifically target therapy. We found that loss of transcriptionally active p73 (TAp73), a p53 family member, impacted PDA development. In two relevant and specific engineered pancreatic cancer mouse models, we observed that TAp73 deficiency reduced survival and enhanced epithelial-to-mesenchymal transition (EMT). Through proteomic analysis of conditioned media from TAp73 wild-type (WT) and deficient pancreatic tumor cells, we identified a secreted protein, biglycan (BGN), which is necessary and sufficient to mediate this pro-EMT effect. Interestingly, BGN is modulated by and modulates the transforming growth factor-ß (TGF-ß) pathway, a key regulator of the EMT process. We further examined this link and revealed that TAp73 impacts the TGF-ß pathway by direct regulation of BGN expression and Sma and Mad-related proteins (SMADs) expression/activity. Absence of TAp73 leads to activation of TGF-ß signaling through a SMAD-independent pathway, favoring oncogenic TGF-ß effects and EMT. Altogether, our data highlight the implication of TAp73 in the aggressiveness of pancreatic carcinogenesis through modulation of the TGF-ß signaling. By suggesting TAp73 as a predictive marker for response to TGF-ß inhibitors, our study could improve the classification of PDA patients with a view to offering combined therapy involving TGF-ß inhibitors.
Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Nuclear Proteins/metabolism , Pancreatic Neoplasms/pathology , Signal Transduction , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolism , Animals , Biglycan/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/mortality , Cell Movement/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Epithelial-Mesenchymal Transition , Humans , Male , Mice , Mice, Knockout , Mice, Nude , Mice, Transgenic , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/mortality , RNA Interference , Signal Transduction/physiology , Survival Rate , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/pharmacology , Tumor Cells, CulturedABSTRACT
We recently reported that deletion of the stress-regulated nuclear protein 1 (Nupr1) protected against obesity-associated metabolic alterations due to increased beta cell mass, but complete Nupr1 ablation was not advantageous since it led to insulin resistance on a normal diet. The current study used Nupr1 haplodeficient mice to investigate whether a partial reduction in Nupr1 expression conferred beneficial effects on glucose homeostasis. Islet number, morphology and area, assessed by immunofluorescence and morphometric analyses, were not altered in Nupr1 haplodeficient mice under normal diet conditions and nor was beta cell BrdU incorporation. Glucose and insulin tolerance tests indicated that there were no significant changes in in vivo insulin secretion and glucose clearance in Nupr1 haplodeficient mice, and beta cell function in vitro was normal. However, reduced Nupr1 expression decreased visceral fat deposition and significantly increased insulin sensitivity in vivo. In contrast to wild type animals, high fat diet-fed Nupr1 haplodeficient mice were not hyperinsulinaemic or glucose intolerant, and their sustained insulin sensitivity was demonstrated by appropriate insulin-induced Akt phosphorylation, as determined by Western blotting. At the molecular level, measurements of gene expression levels and promoter activities identified Nupr1-dependent inhibition of heat shock factor-1-induced heat shock protein 70 (Hsp70) expression as a mechanism through which Nupr1 regulates insulin sensitivity. We have shown for the first time that Nupr1 plays a central role in inhibiting Hsp70 expression in tissues regulating glucose homeostasis, and reductions in Nupr1 expression could be used to protect against the metabolic defects associated with obesity-induced insulin resistance.
Subject(s)
DNA-Binding Proteins/genetics , Glucose Intolerance/genetics , HSP70 Heat-Shock Proteins/genetics , Insulin Resistance/genetics , Neoplasm Proteins/genetics , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation/genetics , DNA-Binding Proteins/metabolism , Diet, High-Fat/adverse effects , Gene Expression , Glucose Intolerance/etiology , Glucose Intolerance/metabolism , HSP70 Heat-Shock Proteins/metabolism , Immunohistochemistry , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/metabolism , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Proteins/metabolism , Phosphorylation , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-akt/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Up-RegulationABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is a critical health issue in the field of cancer, with few therapeutic options. Evidence supports an implication of the intratumoral microenvironment (stroma) on PDA progression. However, its contribution to the role of neuroplastic changes within the pathophysiology and clinical course of PDA, through tumor recurrence and neuropathic pain, remains unknown, neglecting a putative, therapeutic window. Here, we report that the intratumoral microenvironment is a mediator of PDA-associated neural remodeling (PANR), and we highlight factors such as 'SLIT2' (an axon guidance molecule), which is expressed by cancer-associated fibroblasts (CAFs), that impact on neuroplastic changes in human PDA. We showed that 'CAF-secreted SLIT2' increases neurite outgrowth from dorsal root ganglia neurons as well as from Schwann cell migration/proliferation by modulating N-cadherin/ß-catenin signaling. Importantly, SLIT2/ROBO signaling inhibition disrupts this stromal/neural connection. Finally, we revealed that SLIT2 expression and CAFs are correlated with neural remodeling within human and mouse PDA. All together, our data demonstrate the implication of CAFs, through the secretion of axon guidance molecule, in PANR. Furthermore, it provides rationale to investigate the disruption of the stromal/neural compartment connection with SLIT2/ROBO inhibitors for the treatment of pancreatic cancer recurrence and pain.
Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Animals , Axons/drug effects , Axons/metabolism , Cadherins/metabolism , Cell Communication/drug effects , Cell Compartmentation/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Culture Media/pharmacology , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice, Nude , Models, Biological , Neurons/drug effects , Neurons/metabolism , Pancreatic Neoplasms/genetics , Schwann Cells/drug effects , Schwann Cells/metabolism , Schwann Cells/pathology , Signal Transduction/drug effects , Stromal Cells/drug effects , Stromal Cells/metabolism , Stromal Cells/pathology , Transcriptome/genetics , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , beta Catenin/metabolism , Pancreatic NeoplasmsABSTRACT
Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis.
Subject(s)
Cell Cycle Proteins/metabolism , Forkhead Transcription Factors/metabolism , Neoplasms/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Repressor Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Line, Tumor , Forkhead Box Protein O3 , Forkhead Transcription Factors/genetics , Humans , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Knockout , Mice, Nude , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation/genetics , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Repressor Proteins/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Nuclear protein 1 (Nupr1), a small chromatin protein, has a critical role in cancer development, progression and resistance to therapy. Previously, we had demonstrated that Nupr1 cooperates with Kras(G12D) to induce pancreas intraepithelial neoplasias (PanIN) formation and pancreatic ductal adenocarcinoma development in mice. However, the molecular mechanisms by which Nupr1 influences Kras-mediated preneoplastic growth remain to be fully characterized. In the current study, we report evidence supporting a role for Nupr1 as a gene modifier of Kras(G12D)-induced senescence, which must be overcome to promote PanIN formation. We found that genetic inactivation of Nupr1 in mice impairs Kras-induced PanIN, leading to an increase in ß-galactosidase-positive cells and an upregulation of surrogate marker genes for senescence. More importantly, both of these cellular and molecular changes are recapitulated by the results of mechanistic experiments using RNAi-based inactivation of Nupr1 in human pancreatic cancer cell models. In addition, the senescent phenotype, which results from Nupr1 inactivation, is accompanied by activation of the FoxO3a-Skp2-p27(Kip1)-pRb-E2F pathway in vivo and in vitro. Thus, combined, these results show, for the first time, that Nupr1 aids oncogenic Kras to bypass senescence in a manner that cooperatively promotes PanIN formation. Besides its mechanistic importance, this new knowledge bears medical relevance as it delineates early pathobiological events that may be targeted in the future as a means to interfere with the formation of preneoplastic lesions early during pancreatic carcinogenesis.
Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Cellular Senescence/genetics , DNA-Binding Proteins/genetics , Neoplasm Proteins/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Cell Line , Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase Inhibitor p27/biosynthesis , Forkhead Box Protein O3 , Forkhead Transcription Factors/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Knockout , Proto-Oncogene Proteins p21(ras)/biosynthesis , RNA Interference , RNA, Small Interfering , S-Phase Kinase-Associated Proteins/biosynthesis , Up-Regulation , beta-Galactosidase/biosynthesisABSTRACT
Tumor Protein p53-Induced Nuclear Protein 1 (TP53INP1) is a tumor suppressor that modulates the p53 response to stress. TP53INP1 is one of the key mediators of p53 antioxidant function by promoting the p53 transcriptional activity on its target genes. TP53INP1 expression is deregulated in many types of cancers including pancreatic ductal adenocarcinoma in which its decrease occurs early during the preneoplastic development. In this work, we report that redox-dependent induction of p53 transcriptional activity is enhanced by the oxidative stress-induced SUMOylation of TP53INP1 at lysine 113. This SUMOylation is mediated by PIAS3 and CBX4, two SUMO ligases especially related to the p53 activation upon DNA damage. Importantly, this modification is reversed by three SUMO1-specific proteases SENP1, 2 and 6. Moreover, TP53INP1 SUMOylation induces its binding to p53 in the nucleus under oxidative stress conditions. TP53INP1 mutation at lysine 113 prevents the pro-apoptotic, antiproliferative and antioxidant effects of TP53INP1 by impairing the p53 response on its target genes p21, Bax and PUMA. We conclude that TP53INP1 SUMOylation is essential for the regulation of p53 activity induced by oxidative stress.
Subject(s)
Carrier Proteins/metabolism , Heat-Shock Proteins/metabolism , Oxidative Stress , Sumoylation , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , Autophagy , Cell Proliferation , HEK293 Cells , Humans , Ligases , MCF-7 Cells , Molecular Chaperones/metabolism , Oxidation-Reduction , Polycomb-Group Proteins/metabolism , Protein Inhibitors of Activated STAT/metabolism , SUMO-1 Protein/metabolism , Serine Endopeptidases/metabolism , Transcriptional Activation , Ubiquitin-Protein Ligases/metabolismABSTRACT
BACKGROUND: The amplification of epidermal growth factor receptor (EGFR) in triple negative breast carcinomas (TNBC) suggests its potential therapeutic application, as for HER-2, using standardised methods of measurement. In this regard, we aimed to compare several methods for evaluating EGFR amplification along with potential mutations for suitability in clinical practice. METHODS: Tissue sections of 138 TNBCs were used (1) to compare EGFR amplification and expression by silver in situ hybridisation (SISH) to qPCR and immunohistochemistry (IHC) and (2) to search for EGFR mutations, along with Kras, PI3K, Braf and HER-2 mutations and echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) translocation. RESULTS: (1) Amplification of EGFR was observed in well-characterised TNBCs (up to 92%); (2) qPCR correlated with SISH with 94% specificity and 75.6% sensitivity; (3) IHC correlated with SISH with 97% sensitivity and 78% specificity; (4) no EGFR, Kras mutations or EML4-ALK translocations were found, but PI3K and Braf mutations were observed in 26% of cases; and (5) small, acentric circular extrachromosomal DNA similar to 'double minutes' in glioblastomas was observed in 18% of SISH sections. CONCLUSIONS: SISH and IHC are methods that are suitable in clinical practice to screen for EGFR amplification and overexpression, which are frequently observed in TNBC. Patients with TNBC are potential candidates for EGFR-targeted therapy combined with PI3K and Braf inhibitors.
Subject(s)
ErbB Receptors/genetics , Gene Amplification , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Triple Negative Breast Neoplasms/genetics , ras Proteins/genetics , Anaplastic Lymphoma Kinase , Cell Cycle Proteins/genetics , DNA, Circular/genetics , DNA, Circular/isolation & purification , ErbB Receptors/metabolism , Female , Humans , Microtubule-Associated Proteins/genetics , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras) , Receptor, ErbB-2/genetics , Serine Endopeptidases/genetics , Translocation, Genetic , Triple Negative Breast Neoplasms/metabolismABSTRACT
The multiple isoforms of p73, a member of the p53 family, share the ability to modulate p53 activities but also have unique properties, leading to a complex and poorly understood functional network. In vivo, p73 isoforms have been implicated in tumor suppression (TAp73(-/-) mice), DNA damage (ΔNp73(-/-) mice) and development (p73(-/-) mice). In this study, we investigated whether TAp73 contributes to innate immunity and septic shock. In response to a lethal lipopolysaccharide (LPS) challenge, TAp73(-/-) mice showed higher blood levels of proinflammatory cytokines and greater mortality than their wild-type littermates. In vitro, TAp73(-/-) macrophages exhibited elevated production of tumor necrosis factor alpha , interleukin-6 and macrophage inflammatory protein-2 as well as prolonged survival, decreased phagocytosis and increased major histocompatibility complex class II expression. Mice depleted of endogenous macrophages and reconstituted with TAp73(-/-) macrophages showed increased sensitivity to LPS challenge. These results suggest that macrophage polarization is altered in the absence of TAp73 such that maintenance of the M1 effector phenotype is prolonged at the expense of the M2 phenotype, thus impairing resolution of the inflammatory response. Our data indicate that TAp73 has a role in macrophage polarization and innate immunity, enhancing the action field of this important regulatory molecule.
Subject(s)
DNA-Binding Proteins/metabolism , Immunity, Innate , Macrophages/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cells, Cultured , Chemokine CXCL2/metabolism , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Gene Expression Regulation/drug effects , Interferon-beta/metabolism , Interleukin-6/metabolism , Lipopolysaccharides/toxicity , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Phenotype , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Protein p73 , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/geneticsABSTRACT
TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy.