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Toll-like receptors (TLRs) are key players in the innate immune system. Recent studies have suggested that they may affect the growth of pancreatic cancer, a disease with no cure. Among them, TLR7 shows promise for therapy but may also promotes tumor growth. Thus, we aimed to clarify the therapeutic potential of TLR7 ligands in experimental pancreatic cancer models, to open the door for clinical applications. In vitro, we found that TLR7 ligands strongly inhibit the proliferation of both human and murine pancreatic cancer cells, compared with TLR2 agonists. Hence, TLR7 treatment alters cancer cells' cell cycle and induces cell death by apoptosis. In vivo, TLR7 agonist therapy significantly delays the growth of murine pancreatic tumors engrafted in immunodeficient mice. Remarkably, TLR7 ligands administration instead increases tumor growth and accelerates animal death when tumors are engrafted in immunocompetent models. Further investigations revealed that TLR7 agonists modulate the intratumoral content and phenotype of macrophages and that depleting such tumor-associated macrophages strongly hampers TLR7 agonist-induced tumor growth. Collectively, our findings shine a light on the duality of action of TLR7 agonists in experimental cancer models and call into question their use for pancreatic cancer therapy.

Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow–derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti–PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell–dependent immune responses and overcome resistance to anti–PD-1.

TCGA dataset

Gating Strategies

This file contains supplementary table 1 and supplementary figures 1-7.

Pancreatic ductal adenocarcinoma (PDAC) offers an optimal model for discovering “druggable” molecular pathways that participate in inflammation-associated cancer development. Chronic pancreatitis, a common prolonged inflammatory disease, behaves as a well-known premalignant condition that contributes to PDAC development. Although the mechanisms underlying the pancreatitis-to-cancer transition remain to be fully elucidated, emerging evidence supports the hypothesis that the actions of proinflammatory mediators on cells harboring Kras mutations promote neoplastic transformation. Recent elegant studies demonstrated that the IL17 pathway mediates this phenomenon and can be targeted with antibodies, but the downstream mechanisms by which IL17 functions during this transition are currently unclear. In this study, we demonstrate that IL17 induces the expression of REG3β, a well-known mediator of pancreatitis, during acinar-to-ductal metaplasia and in early pancreatic intraepithelial neoplasia (PanIN) lesions. Furthermore, we found that REG3β promotes cell growth and decreases sensitivity to cell death through activation of the gp130-JAK2-STAT3-dependent pathway. Genetic inactivation of REG3β in the context of oncogenic Kras-driven PDAC resulted in reduced PanIN formation, an effect that could be rescued by administration of exogenous REG3β. Taken together, our findings provide mechanistic insight into the pathways underlying inflammation-associated pancreatic cancer, revealing a dual and contextual pathophysiologic role for REG3β during pancreatitis and PDAC initiation. Cancer Res; 75(22); 4852–62. ©2015 AACR.

Legend for Supplemental Figure 1.

Schematic representation of the IL-17A and REG3β signaling pathway and PDAC development.

PDF file - 43K, Correlation between the concentrations of GDF15 measured by ELISA in bone marrow (BM) plasma and in blood plasma in 24 patients with newly diagnosed MM

PDF file - 84K, Baseline phosphorylation of pT308 Akt in MOLP-6 and MM1.S cells

PDF file - 67K, Effect of GDF15 on T308 Akt phosphorylation of MOLP-6 stroma-dependent cells in culture condition with 10% fetal calf serum

PDF file - 90K, Effect of GDF15 on ERK1/2 phosphorylation in MOLP-6 and MM1.S cells

PDF file - 44K, Concentration of GDF15 measured by ELISA in 72 hours culture supernatants of primary BM-MSCs and MM cells from 3 patients, and MM cell lines MOLP-6 and MM1.S

PDF file - 67K, Transfection of BM-MSC with plasmids encoding shRNA targeting GDF15 and co-culture with MOLP-6 cells

Pancreatic ductal adenocarcinoma remains one of the greatest challenges in oncology for which therapeutic intervention is urgently needed. We demonstrated that the intratumoral gene transfer of somatostatin receptor 2, to combat tumor aggressiveness, or of deoxycytidine kinase and uridylate monophosphate kinase, to sensitize to gemcitabine chemotherapy, has antitumoral potential. Here, we describe the development of CYL-02 non-viral gene-therapy product, that comprises a DNA-plasmid encoding for the three aforementioned genes complexed with PolyEthylEnimine (22 kDa). In this work, we performed preclinical toxicology, biodistribution and therapeutic activity studies of CYL-02 in experimental models of pancreatic cancer. We demonstrated the safety of CYL-02 and defined the maximal tolerated dose in two animal species. CYL-02 co-administrated with gemcitabine did not increase gemcitabine toxicity. Biodistribution studies revealed that CYL-02 is rapidly cleared from blood following intravenous administration, and sequestered in tumors following intratumoral injection. CYL-02 drives the expression of therapeutic genes in cancer cells and strongly sensitizes tumor cells to gemcitabine, with significant inhibition of tumor cells dissemination. This study was instrumental for the later use of CYL-02 in patients with advanced pancreatic cancer, demonstrating that rigorous and thorough preclinical investigations are informative for the clinical transfer of gene therapy against pancreatic cancer.GRAPHICAL ABSTRACT

Despite tremendous efforts from the scientific community, pancreatic ductal adenocarcinoma (PDAC) is still a deadly disease and will soon become the second cause of death by cancer worldwide1. When surgery is not possible, therapeutic options are few and ineffective2. Patients are most often treated with Folfirinox or gemcitabine chemotherapy, that extends survival in weeks to months. Cytidine deaminase (CDA) catalyzes the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine to fuel RNA and DNA synthesis3. CDA also deaminates and neutralizes deoxycytidine-based therapies, and as such, has been identified as a major contributor of tumor chemoresistance, especially to gemcitabine in PDAC3. We previously identified that CDA is elevated in PDAC tumors at diagnosis and that CDA exerts an unexpected role on DNA replication that can be exploited for therapeutic intervention4. Very recently, CDA was associated with cellular metabolism5. Here, we show that CDA promotes mitochondrial biogenesis and oxidative phosphorylation independently of its deaminase activity. This uncloaks novel therapeutic vulnerabilities in primary cancer cells that overexpress this protein. This study shines a new light on the tumoral potential of CDA in PDAC.

Pancreatic cancer is an aggressive cancer with a dismal prognosis. This is due to the difficulty to detect the disease at an early and curable stage. In addition, only limited treatment options are available, and they are confronted by mechanisms of resistance. Monoclonal antibody (mAb) molecules are highly specific biologics that can be directly used as a blocking agent or modified to deliver a drug payload depending on the desired outcome. They are widely used to target extracellular proteins, but they can also be employed to inhibit intracellular proteins, such as oncoproteins. While mAbs are a class of therapeutics that have been successfully employed to treat many cancers, they have shown only limited efficacy in pancreatic cancer as a monotherapy so far. In this review, we will discuss the challenges, opportunities and hopes to use mAbs for pancreatic cancer treatment, diagnostics and imagery.

Chronic pancreatitis is one of the main risk factors for pancreatic cancer, but it is a rare event. Inflammation and oncogenes work hand in hand as key promoters of this disease. Tobacco is another co-factor. During alcoholic chronic pancreatitis, the cumulative risk of cancer is estimated at 4% after 15 to 20 years. This cumulative risk is higher in hereditary pancreatitis: 19 and 12% in the case of PRSS1 and SPINK1 mutations, respectively, at an age of 60 years. The diagnosis is difficult due to: (i) clinical symptoms of cancer shared with those of chronic pancreatitis; (ii) the parenchymal and ductal remodeling of chronic pancreatitis rendering imaging analysis difficult; and (iii) differential diagnoses, such as pseudo-tumorous chronic pancreatitis and paraduodenal pancreatitis. Nevertheless, the occurrence of cancer during chronic pancreatitis must be suspected in the case of back pain, weight loss, unbalanced diabetes, and jaundice, despite alcohol withdrawal. Imaging must be systematically reviewed. Endoscopic ultrasound-guided fine-needle biopsy can contribute by targeting suspicious tissue areas with the help of molecular biology (search for KRAS, TP53, CDKN2A, DPC4 mutations). Short-term follow-up of patients is necessary at the clinical and paraclinical levels to try to diagnose cancer at a surgically curable stage. Pancreatic surgery is sometimes necessary if there is any doubt.

The present work studies models of oncolytic virotherapy without space variable in which virus replication occurs at a faster time scale than tumor growth. We address the questions of the modeling of virus injection in this slow-fast system and of the optimal timing for different treatment strategies. To this aim, we first derive the asymptotic of a three-species slow-fast model and obtain a two-species dynamical system, where the variables are tumor cells and infected tumor cells. We fully characterize the behavior of this system depending on the various biological parameters. In the second part, we address the modeling of virus injection and its expression in the two-species system, where the amount of virus does not appear explicitly. We prove that the injection can be described by an instantaneous jump in the phase plane, where a certain amount of tumors cells are transformed instantly into infected tumor cells. This description allows discussing qualitatively the timing of different injections in the frame of successive treatment strategies. This work is illustrated by numerical simulations. The timing and amount of injected virus may have counterintuitive optimal values; nevertheless, the understanding is clear from the phase space analysis.

Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second most common cause of death within the next 10 years. The prognosis for this disease is poor despite diagnostic progress and new chemotherapeutic regimens. The oncogenic KRAS mutation is the major event in pancreatic cancer; it confers permanent activation of the KRAS protein, which acts as a molecular switch to activate various intracellular signalling pathways and transcription factors inducing cell proliferation, migration, transformation and survival. Several laboratory methods have been developed to detect KRAS mutations in biological samples, including digital droplet PCR (which displays high sensitivity). Clinical studies have revealed that a KRAS mutation assay in fine-needle aspiration material combined with cytopathology increases the sensitivity, accuracy and negative predictive value of cytopathology for a positive diagnosis of pancreatic cancer. In addition, the presence of KRAS mutations in serum and plasma (liquid biopsies) correlates with a worse prognosis. The presence of mutated KRAS can also have therapeutic implications, whether at the gene level per se, during its post-translational maturation, interaction with nucleotides and after activation of the various oncogenic signals. Further pharmacokinetic and toxicological studies on new molecules are required, especially small synthetic molecules, before they can be used in the therapeutic arsenal for pancreatic ductal adenocarcinoma. KRAS mutations are identified in >80% of patients with pancreatic ductal adenocarcinoma and tend to be associated with reduced overall survival. This Review discusses the role of oncogenic KRAS in the biology, diagnosis, prognosis and treatment of pancreatic cancer.

Genetic testing based on the analysis of circulating cell-free DNA (cfDNA) in body fluids, especially blood plasma, is raising interest for the management and follow-up of many diseases, including cancer. Because the concentration of cfDNA is low and its composition mostly degraded, this material can only be assayed with the most sensitive nucleic acid processing technologies. cfDNA analysis therefore constitutes a model target and a driving force for innovation in microfluidic biotechnologies. Here, we overview the main physico-chemical characteristics of cfDNA, and provide a critical review on the different methods for its processing out of blood samples. Then, we describe recent microfluidic developments for high sensitivity DNA analysis, evaluate their practical relevance for cfDNA analysis, and identify a few challenges for technologists in the near future. Keywords: Cell-free circulating DNA, Body fluid nucleic acid biomarkers, DNA separation, DNA concentration

International audience; In this work, we demonstrate that the analysis of spatially resolved nanofluidic-embedded biosensors permits the fast and direct discrimination of single-nucleotide difference (SND) within oligonucleotide sequences in a single step interaction. We design a sensor with a linear dimension much larger than the channel depth in order to ensure that the reaction over the whole sensor is limited by the convection rate. Thus, the targets are fully collected, inducing a nonuniform spatial hybridization profile. We also use the nanoscale height of the channel, which enables us to minimize the amount of labeled molecules flowing over the sensor and hence to reduce the fluorescence background, to carry out real-time hybridization detection by fluorescence microscopy. Taken together, these design rules allow us to show that the spatial hybridization profile depends on the duplex affinity, and we speculate that the on and off-rate constants can be inferred during target injection, which is not possible in local analysis where the dissociation step through rinsing must be conducted. We finally manage to discriminate a GT mismatch on a microRNA sequence by optimizing the interaction temperature and the probe design after a few minutes of interaction in a single step protocol. This work may be applied to any biosensing transduction scheme with spatial resolution, e.g., surface plasmon resonance imaging, integrated into nanofluidic channels for applications where high oligonucleotide sequence selectivity and short analysis times are required.

Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine within the pyrimidine salvage pathway for DNA and RNA synthesis. In this regard, loss of CDA provokes genomic instability in Bloom Syndrome, and CDA overexpression is associated with tumor resistance to chemotherapy with pyrimidine analogs. However, the precise role of CDA per se in cancer has been totally underexplored so far. Patient cohort analysis demonstrate that CDA is overexpressed in PDAC, a disease with no cure with increasing incidence, and associated with a worse prognosis in patient. Functional studies demonstrate that CDA is essential to PDAC cell proliferation and tumor growth. We found that CDA expression is associated with gene set enrichment in DNA replication signature, in both PDAC tumors and experimental models. Using enforced expression, genetic or pharmacologic targeting, we demonstrate that CDA promotes DNA replication, localizes to the replication fork and increases replication fork fitness. These effects are strictly dependent on CDA deaminase activity. Hence, we found that CDA controls replication stress as CDA expression is inversely correlated with the level of DNA breaks during S-phase, and that CDA targeting is associated with gene set enrichment in replication stress signature and CHK1 protein activation. Next, we demonstrate that CDA preserves genomic stability of PDAC cells, as CDA expression is inversely correlated with micronuclei formation and DNA damage transfer to daughter cells. We next explored PDAC cell lines from the Cancer Cell Lines Encyclopedia and found that CDA expression is associated with resistance to drug targeting DNA synthesis. Functional studies demonstrate that overexpressing CDA, and not CDA catalytically inactive mutant, protects PDAC cell lines from camptothecin, a topoisomerase inhibitor, while targeting CDA sensitizes PDAC cells to treatment by camptothecin and oxaliplatin, that forms platinum-DNA adducts. To gain further insights into the clinical potential of such finding, we targeted CDA expression in primary cells from patient with PDAC and found that CDA is essential to primary cell growth, and that CDA targeting sensitizes primary cells to treatment by oxaliplatin. Taken together, our results reveal for the first time that CDA controls DNA replication, replication stress level and genomic stability of PDAC cells, and that this new role of CDA is involved in tumor resistance to drugs inducing DNA damages. Thus, our work stems for new strategies based on CDA targeting to defeat PDAC resistance to treatment. Citation Format: Audrey Lumeau, Nicolas Bery, Cyril Ribeyre, Samad Elkaoutari, Guillaume Labrousse, Miguel Madrid-Mencia, Vera Pancaldi, Marie-Jeanne Pillaire, Valérie Bergoglio, Nelson Dusseti, Jean-Sébastien Hoffmann, Louis Buscail, Malik Lutzmann, Pierre Cordelier. Cytidine deaminase protects pancreatic cancer cells from replicative stress and drive response to DNA-targeting drugs [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-043.

Despite considerable promise and emerging clinical success, several challenges impede the broader implementation of novel immunotherapies such as oncolytic virus(OV)-based gene therapy, including for patients with pancreatic cancer (PDAC). One of such challenge is inter-patient variability that may impact on OV selectivity and killing efficacy for tumor cells. For this study, we selected the rat parvovirus H-1 (H-1PV) that is nonpathogenic in humans and has a natural oncolytic activity in several cancer models. The safety and tolerability of H-1PV was recently demonstrated in early clinical trials for glioma and PDAC. However, we report here that H-1PV infection, oncolytic and pro-apoptotic activity are limited in PDAC cells, including patient-derived primary cells. To address this concern, we applied a directed evolution strategy to generate H-1PV variants with specific activity towards PDAC cells. Following selection using patient primary cells, we managed to isolate clonal, PDAC-adapted H-1PVs that induce PDAC cells lysis as compared to parental H-1PV while infection of normal pancreatic cells remained negligeable. Genome sequencing of the tumor adapted virus reveals mutations in promoting and viral capsid sequences. In vivo, the tumor-adapted H1PV demonstrates greater anti-tumor effect than parental H-1PV, following intravenous administration in an experimental model of orthotopic pancreatic tumors engrafted in immunodeficient mice. To our knowledge, we report here for the first time the production of highly selective and potent OV using directed evolution to override PDAC resistance to virotherapy. While the molecular mechanisms involved are still under investigation, this project is a first step towards precision medicine strategies based on OV. Citation Format: Pierre Garcin, Monireh Kazemimanesh, Hubert Lulka, Nelson Dusetti, Guillaume Labrousse, Emilie Benuzzi, Louis Buscail, Pierre Cordelier. Directed evolution generates novel oncolytic H-1 parvoviruses with improved therapeutic efficacy in virus-resistant pancreatic cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-016.

International audience; The recycling activity of cytidine deaminase (CDA) within the pyrimidine salvage pathway is essential to DNA and RNA synthesis. As such, CDA deficiency can lead to replicative stress, notably in Bloom syndrome. Alternatively, CDA also can deaminate cytidine and deoxycytidine analog-based therapies, such as gemcitabine. Thus, CDA overexpression is often associated with lower systemic, chemotherapy-related, adverse effects but also with resistance to treatment. Considering the increasing interest of CDA in cancer chemoresistance, the aims of this review are to describe CDA structure, regulation of expression, and activity, and to report the therapeutic strategies based on CDA expression that recently emerged for tumor treatment.

In the era of precision medicine, the success of clinical trials, notably for patients diagnosed with cancer, strongly relies on biomarkers with pristine clinical value but also on robust and versatile analytical technologies to ensure proper patients' stratification and treatment. In this review, we will first address whether plasmatic and salivary microRNAs can be considered as a reliable source of biomarkers for cancer diagnosis and prognosis. We will then discuss the pre-analytical steps preceding miRNA quantification (from isolation to purification), and how such process could be biased and time-consuming. Next, we will review the most recent tools derived from micro- and nano-technologies for microRNA detection available to date and how they may compete with current standards. This review will prioritize publications using relevant biological samples. The significance of various physical transduction schemes (mechanical, optical, electrical, etc.) for biological detection will be compared, and pros and cons of each method will be widely discussed. Finally, we will debate on how micro and nanotechnologies could widespread the use of biomarkers in modern medicine, to help manage patients with serious diseases such as cancer.

International audience

Pancreatic ductal adenocarcinoma (PDAC) is a paradigmatic model of micrometastatic disease, characterized by both relapse of surgery and failure of long-term disease control with chemotherapies in resected, locally advanced unresectable and metastatic patients. Molecular characterization of large cohorts of PDAC patients failed to identify actionable pathways associated with mutations except for oncogenic Kras, found in more than 80% of all patients. Class I PI3K being a key regulator of both cell survival and cell migration, we questioned if nonmutated PI3K activity downstream oncogenic Kras is a clinically relevant target for this pathology. To achieve this goal, we devised a novel preclinical protocol in a genetically engineered mouse model (GEMM) including a noninvasive monitoring using circulating cell-free DNA (cfDNA) level as a novel biomarker of micrometastatic disease. We previously showed that, downstream Kras, nonmutant ubiquitous PI3Kalpha is crucial for PDAC development. Among all the mRNA expression-based hallmarks of biologic pathways, we now show that PI3K/Akt/mTOR hallmark is significantly increased in PDAC patients with advanced metastatic disease. High scoring of PI3Kalpha activation, based on expression levels of PI3Kalpha-regulated curated genes, is correlated to a poor prognosis, and is found in metastatic PDAC cell lines and in patient-derived ex vivo cultures of locally invasive PDAC cancer cells. When testing global or isoform-selective inhibitors in GEMM and human cell lines, we show that biologically efficient PI3K inhibitors annihilate PI3Kalpha strongly, but not necessarily specifically, among the four class I PI3K isoforms, resulting in Akt2 inactivation. Similar results are obtained with siRNA and in PIK3CA oncogenic cell lines. PI3Kalpha controls pancreatic tumor cell line motility and migration. In a GEMM model of PDAC, mice with high level of cfDNA present a shorter survival and develop a metastatic disease. Genetic or pharmacologic inhibition of PI3Kalpha in these mice decreases tumor volume, metastasis, ascitic fluid development, cfDNA quantity and M2 macrophage infiltration. Here, we used GEMMs to define a cfDNA-positive aggressive subtype of PDAC and identify a pharmacologically actionable prometastatic feature driven by PI3Kalpha. We propose PI3K as a therapeutic strategy to suppress PDAC progression and metastasis by scavenging peritumoral CD206-positive macrophages. Our data demonstrate that PI3K agents could be efficient on micrometastatic disease (measured by cfDNA), resulting in delay on macrometastatic dissemination. This abstract is also being presented as Poster B04. Citation Format: Benoit Thibault, Fernanda Ramos-Delgado, Elvire Pons-Tostivint, Nicole Therville, Gabriella Reyes-Castellanos, Marie Tosolini, Jean-Pierre Delord, Dieter Saur, Alice Carrier, Pierre Cordelier, Céline Basset, Julie Guillermet-Guibert. Pancreatic ductal adenocarcinoma requires PI3Kalpha activity to accelerate circulating DNA-positive metastatic disease [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr PR07.

Pancreatic ductal adenocarcinoma (PDAC) offers an optimal model for discovering >druggable> molecular pathways that participate in inflammation-associated cancer development. Chronic pancreatitis, a common prolonged inflammatory disease, behaves as a well-known premalignant condition that contributes to PDAC development. Although the mechanisms underlying the pancreatitis-to-cancer transition remain to be fully elucidated, emerging evidence supports the hypothesis that the actions of proinflammatory mediators on cells harboring Kras mutations promote neoplastic transformation. Recent elegant studies demonstrated that the IL17 pathway mediates this phenomenon and can be targeted with antibodies, but the downstream mechanisms by which IL17 functions during this transition are currently unclear. In this study, we demonstrate that IL17 induces the expression of REG3β, a wellknown mediator of pancreatitis, during acinar-to-ductal metaplasia and in early pancreatic intraepithelial neoplasia (PanIN) lesions. Furthermore, we found that REG3β promotes cell growth and decreases sensitivity to cell death through activation of the gp130-JAK2-STAT3-dependent pathway. Genetic inactivation of REG3β in the context of oncogenic Kras-driven PDAC resulted in reduced PanIN formation, an effect that could be rescued by administration of exogenous REG3β. Taken together, our findings provide mechanistic insight into the pathways underlying inflammation-associated pancreatic cancer, revealing a dual and contextual pathophysiologic role for REG3β during pancreatitis and PDAC initiation., This work was supported by La Ligue Contre le Cancer, INCa, Canceropole PACA, DGOS (labellisation SIRIC), and INSERM to J.L. Iovanna; NIH grants DK52913, the Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Foundation to R. Urrutia; by Fraternal Order of Eagles Cancer Award to G. Lomberk; and the FIS grant from Instituto de Salud Carlos III (PI13/01224) to E. Folch-Puy

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a mortality that is almost identical to incidence. Because early detected PDAC is potentially curable, blood-based biomarkers that could detect currently developing neoplasia would improve patient survival and management. PDAC develops from pancreatic intraepithelial neoplasia (PanIN) lesions, graded from low grade (PanIN1) to high grade (PanIN3). We made the hypothesis that specific proteomic signatures from each precancerous stage exist and are detectable in plasma. Methods: We explored the peptide profiles of microdissected PanIN cells and of plasma samples corresponding to the different PanIN grade from genetically engineered mouse models of PDAC using capillary electrophoresis coupled to mass spectrometry (CE-MS) and Chip-MS/MS. Results: We successfully characterised differential peptides profiles from PanIN microdissected cells. We found that plasma from tumor-bearing mice and age-matched controls exhibit discriminative peptide signatures. We also determined plasma peptide signatures corresponding to low- and high-grade precancerous step present in the mice pancreas using the two mass spectrometry technologies. Importantly, we identified biomarkers specific of PanIN3. Conclusions: We demonstrate that benign and advanced PanIN lesions display distinct plasma peptide patterns. This strongly supports the perspectives of developing a non-invasive screening test for prediction and early detection of PDAC.

The understanding of the biology of pancreatic carcinoma has greatly benefited from studies of genetic/epigenetic alterations and molecular expression in experimental models as well as precancerous and cancerous tissues by mean of molecular amplification and large-scale transcriptoma analysis. P16, TP53, DPC4/Smad4 tumor suppressor pathways are genetically inactivated in the majority of pancreatic carcinomas, whereas oncogenic k-ras is activated. The activating point mutation of the KRAS oncogene on codon 12 is the major event and occurs early in pancreatic carcinogenesis. At a late stage of tumor development, an increase of telomerase activity, an over expression of growth factors and/or their receptors (EGF, Nerve Growth Factor, gastrin), of pro-angiogenic factors (VEGF, FGF, PDGF), of invasiveness factors (metalloproteinases, tissue plasminogen activators) occurs. The microenvironment plays also a key role in the invasive and metastatic process of pancreatic carcinoma with a strong relationship between cancerous cells and pancreatic stellate cells as well as extracellular matrix. This microenvironment strongly participates to the tumor fibrosis, hypoxia and hypovascularization inducing an inaccessibility of drugs. Nowadays, the targeting of microenvironment takes a special place in the new therapeutic strategies of pancreatic cancer in combination with chemotherapy.

This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.

Pancreas transcription factor 1a (PTF1a) plays a crucial role in the early development of the pancreas and in the maintenance of the acinar cell phenotype. Several transcriptional mechanisms regulating expression of PTF1a have been identified. However, regulation of PTF1a protein stability and degradation is still unexplored. Here, we report that inhibition of proteasome leads to elevated levels of PTF1a and to the existence of polyubiquitinated forms of PTF1a. We used the Sos recruitment system, an alternative two-hybrid system method to detect protein-protein interactions in the cytoplasm and to map the interactome of PTF1a. We identified TRIP12 (thyroid hormone receptor-interacting protein 12), an E3 ubiquitin-protein ligase as a new partner of PTF1a. We confirmed PTF1a/TRIP12 interaction in acinar cell lines and in co-transfected HEK-293T cells. The protein stability of PTF1a is significantly increased upon decreased expression of TRIP12. It is reduced upon overexpression of TRIP12 but not a catalytically inactive TRIP12-C1959A mutant. We identified a region of TRIP12 required for interaction and identified lysine 312 of PTF1a as essential for proteasomal degradation. We also demonstrate that TRIP12 down-regulates PTF1a transcriptional and antiproliferative activities. Our data suggest that an increase in TRIP12 expression can play a part in PTF1a down-regulation and indicate that PTF1a/TRIP12 functional interaction may regulate pancreatic epithelial cell homeostasis.

International audience; Pancreatic cancer is a public health problem because of its increasing incidence, the absence of early diagnostic tools, and its aggressiveness. Despite recent progress in chemotherapy, the 5-year survival rate remains below 5%. Liquid biopsies are of particular interest from a clinical point of view because they are non-invasive biomarkers released by primary tumours and metastases, remotely reflecting disease burden. Pilot studies have been conducted in pancreatic cancer patients evaluating the detection of circulating tumour cells, cell-free circulating tumour DNA, exosomes, and tumour-educated platelets. There is heterogeneity between the methods used to isolate circulating tumour elements as well as the targets used for their identification. Performances for the diagnosis of pancreatic cancer vary depending of the technique but also the stage of the disease: 30-50% of resectable tumours are positive and 50-100% are positive in locally advanced and/or metastatic cases. A significant prognostic value is demonstrated in 50-70% of clinical studies, irrespective of the type of liquid biopsy. Large prospective studies of homogeneous cohorts of patients are lacking. One way to improve diagnostic and prognostic performances would be to use a combined technological approach for the detection of circulating tumour cells, exosomes, and DNA.

KLF6 is ubiquitously expressed in human tissues and regulates many pathways such as differentiation, development, cellular proliferation, growth-related signal transduction, and apoptosis. We previously demonstrated that KLF6 expression is altered during liver carcinogenesis. More importantly, KLF6 invalidation results in cell cycle progression inhibition and apoptosis of liver cancer cells. On the other hand, enforced expression of KLF6 variant 2 (SV2) induces cancer cell death by apoptosis. Thus, we and others demonstrated that KLF6 and its splicing variants play a critical role in liver cancer. However, little is known on the mechanisms governing KLF6 expression in HCC. In the present work, we asked whether the 3' untranslated region (3'UTR) of the KLF6 mRNA may be responsible for regulation of KLF6 expression in HCC. We found that KLF6 mRNA stability was altered in liver-derived cell lines as compared to cervical cancer-derived cell lines and human embryonic fibroblasts. Interestingly, KLF6 mRNA was highly unstable in liver cancer-derived cell lines as compared to normal hepatocytes. We next cloned the KLF6 mRNA 3'UTR into luciferase-expressing vectors and found that gene expression and activity were strongly impaired in all liver-derived cell lines tested. In addition, we found that most the KLF6 3'UTR destabilisation activity resides between nt 1,835 and nt 2,615 of the KLF6 gene. Taken together, we provide the first steps towards better understanding of the regulation of KLF6 expression in HCC. Further work is needed to identify the factors that bind to KLF6 3'UTR to regulate its expression in liver cancer-derived cell lines.

La mutation ponctuelle de l’oncogene Kras confere un gain de proliferation non controlee pour la cellule cancereuse qui acquiert ainsi un pouvoir oncogenique amplifie. Cette mutation est retrouvee dans 75 a 95 % des cancers du pancreas, mais aussi dans les lesions precancereuses de type PanIN (pancreatic intraepithelial neoplasia ) ou TIPMP (tumeur intracanalaire papillaire mucineuse du pancreas). Ces lesions, ainsi que le microenvironnement tumoral, ont ete reproduits dans des modeles transgeniques etablis chez la souris a partir de la mutation de Kras (souris Pdx1 [pancreatic and duodenal homeobox 1 ]-Cre ; KrasG12D ) associee ou non a l’inactivation de genes suppresseurs de tumeurs (TP53, DPC4, INK4A ). La recherche de la mutation de Kras en clinique humaine est facile et fiable a partir des milieux biologiques, en particulier sur le materiel de cytoponction de masses pancreatiques preleve sous echo-endoscopie. Cette recherche devrait etre utile dans un avenir proche pour l’aide au diagnostic positif de cancer, en cas de d’examen cytopathologique douteux ou non contributif, mais aussi pour le diagnostic differentiel avec la pancreatite chronique dans sa forme pseudotumorale.

DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results.

To date, pancreatic cancer (PDAC) can't be diagnosed early. Consequently, a majority of patient (80%) display an advanced disease that results in a low resection rate leading to an overall median survival of 4 to 6months. Accordingly, robust markers for the early diagnosis and prognosis of pancreatic cancer, or markers indicative of survival and/or metastatic disease are desperately needed to help alleviate the dismal prognosis of this disease. In this chapter, we review the translational studies demonstrating that miRNAs, alone or in combination, may soon translate into clinical applications as long-awaited screening tools for PDAC.

Despite tremendous efforts worldwide from clinicians and cancer scientists, pancreatic ductal adenocarcinoma (PDA) remains a deadly disease for which no cure is available. Recently, microRNAs (miRNAs) have emerged as key actors in carcinogenesis and we demonstrated that microRNA-21 (miR-21), oncomiR is expressed early during PDA. In the present study, we asked whether targeting miR-21 in human PDA-derived cell lines using lentiviral vectors (LVs) may impede tumor growth. We demonstrated that LVs-transduced human PDA efficiently downregulated miR-21 expression, both in vitro and in vivo. Consequently, cell proliferation was strongly inhibited and PDA-derived cell lines died by apoptosis through the mitochondrial pathway. In vivo, miR-21 depletion stopped the progression of a very aggressive model of PDA, to induce cell death by apoptosis; furthermore, combining miR-21 targeting and chemotherapeutic treatment provoked tumor regression. We demonstrate herein for the first time that targeting oncogenic miRNA strongly inhibit pancreatic cancer tumor growth both in vitro and in vivo. Because miR-21 is overexpressed in most human tumors; therapeutic delivery of miR-21 antagonists may still be beneficial for a large number of cancers for which no cure is available.

Since their discovery, microRNAs (miRNAs)20 have shown great promise in a wide array of clinical applications. In some cases, the use of miRNAs as new diagnostic markers might help answer diagnostic dilemmas that gene expression analyses or other types of analyses have not been able to satisfactorily address. In other instances, it is easy to perceive certain miRNAs as targets for novel therapies that downregulate an entire pathway via the targeting of a single miRNA. When and if this concept will come to fruition remain unclear. Scientists agree that this field of biology is exciting, offers much promise, and has numerous advantages, compared with experiences with other biomolecules. In this Q&A, 5 individuals with extensive miRNA experience share their vision for where the miRNA field is heading and whether we, in the clinical laboratory, will experience its implications soon. What are the most important characteristics of miRNAs that increase their potential as novel diagnostic or therapeutic targets? George Calin: One important characteristic of miRNAs that makes them an exciting potential biomarker for diagnosis and/or a target for therapy is the fact that specific miRNAs target multiple components from the same pathway. For example, the miR-15a/16–1 cluster targets genes from the apoptotic pathway: BCL2 21 (B-cell CLL/lymphoma 2) and MCL1 [myeloid cell leukemia sequence 1 ( BCL2 -related)]. Any given miRNA can regulate numerous genes, and each gene can be regulated by different miRNAs. Pierre Cordelier: In cancer research, miRNAs can differentiate normal from cancerous tissues and, more importantly, can discriminate different subtypes of cancer. The high stability of miRNAs in tissues and fluids is another key advantage that increases their potential as diagnostic markers over messenger RNA (mRNA). In addition, they can be quantified in very low amounts of material and in highly degraded samples. This is of prime importance …

The incidence of pancreatic cancer is increasing but the prognosis remains poor. The lack of clearly identified risk factors hinders screening and early diagnosis (when the disease is still curable). The sole curative treatment is surgical resection, which is feasible in only 15% of patients. Chemotherapy regimens are being developed in an attempt to prolong the survival of patients who do not quality for surgery. Recent advances include better knowledge and treatment of pre-neoplastic pancreatic lesions such as mucinous tumors. Basic and translational researchers are attempting to identify tumoral or circulating antigens that could serve as diagnostic markers and/or therapeutic targets.

Overexpression of growth differentiation factor 15 (GDF15) by bone marrow mesenchymal stem cells occurs widely in patients with multiple myeloma, but the pathophysiologic effects of GDF15 in this setting remain undefined. GDF15 has been described in numerous solid tumors but never in hematologic malignancies. In this study, we report that GDF15 significantly increases survival of stroma-dependent multiple myeloma cells including primary multiple myeloma cells. In particular, GDF15 conferred resistance to melphalan, bortezomib, and to a lesser extent, lenalidomide in both stroma-dependent and stroma-independent multiple myeloma cells. Akt-dependent signaling was critical to mediate the effects of GDF15, whereas Src and extracellular signal-regulated kinase 1/2 signaling pathways were not involved. Given these results, we tested the clinical significance of plasma concentrations of GDF15 (pGDF15) in 131 patients with multiple myeloma and found that it correlated with disease prognosis. Specifically, patients with high levels of pGDF15 had lower probabilities of event-free and overall survival 30 months after diagnosis than patients with low pGDF15 levels. Our findings suggest that tumor microenvironment-derived GDF15 is a key survival and chemoprotective factor for multiple myeloma cells, which is pathophysiologically linked to both initial parameters of the disease as well as patient survival. Cancer Res; 72(6); 1395–406. ©2012 AACR.

Endoscopic ultrasound-guided fine needle aspiration-biopsy is a safe and effective technique in diagnosing and staging of pancreatic ductal adenocarcinoma. However its predictive negative value does not exceed 50% to 60%. Unfortunately, the majority of pancreatic cancer patients have a metastatic and/or a locally advanced disease (i.e., not eligible for curative resection) which explains the limited access to pancreatic tissue specimens. Endoscopic ultrasound-guided fine needle aspiration-biopsy is the most widely used approach for cytological and histological material sampling in these situations used in up to two thirds of patients with pancreatic cancer. Based on this unique material, we and others developed strategies to improve the differential diagnosis between carcinoma and inflammatory pancreatic lesions by analysis of KRAS oncogene mutation, microRNA expression and methylation, as well as mRNA expression using both qRT-PCR and Low Density Array Taqman analysis. Indeed, differentiating pancreatic cancer from pseudotumoral chronic pancreatitis remains very difficult in current clinical practice, and endoscopic ultrasound-guided fine needle aspiration-biopsy analysis proved to be very helpful. In this review, we will compile the clinical and molecular advantages of using endoscopic ultrasound-guided fine needle aspiration-biopsy in managing pancreatic cancer.

In the paper untitled “high-throughput creation and functional profiling of DNA sequence variant libraries using CRISP-Cas9 in yeast” (1) published online in May 2018 in Nature Biotechnology, George M. Church, an eminent expert in CRISPR-Cas9 biology and technology, makes, yet another, giant step in the field of genomics.