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Primer pairs used for real-time quantitative RT-PCR.

Amalysis of Akt/Stat3 signaling induction following LIF stimulation. We measured the induction of STAT3 (A) and Akt (B) phosphorylation by western blot in sNF96.2 after incubation with LIF recombinant protein. We confirmed the specificity of induction triggered by LIF using LIFR blocking antibody or AG490 inhibitor.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive stroma and pathogenic modifications to the peripheral nervous system that elevate metastatic capacity. In this study, we show that the IL6-related stem cell–promoting factor LIF supports PDAC-associated neural remodeling (PANR). LIF was overexpressed in tumor tissue compared with healthy pancreas, but its receptors LIFR and gp130 were expressed only in intratumoral nerves. Cancer cells and stromal cells in PDAC tissues both expressed LIF, but only stromal cells could secrete it. Biological investigations showed that LIF promoted the differentiation of glial nerve sheath Schwann cells and induced their migration by activating JAK/STAT3/AKT signaling. LIF also induced neuronal plasticity in dorsal root ganglia neurons by increasing the number of neurites and the soma area. Notably, injection of LIF-blocking antibody into PDAC-bearing mice reduced intratumoral nerve density, supporting a critical role for LIF function in PANR. In serum from human PDAC patients and mouse models of PDAC, we found that LIF titers positively correlated with intratumoral nerve density. Taken together, our findings suggest LIF as a candidate serum biomarker and diagnostic tool and a possible therapeutic target for limiting the impact of PANR in PDAC pathophysiology and metastatic progression.Significance: This study suggests a target to limit neural remodeling in pancreatic cancer, which contributes to poorer quality of life and heightened metastatic progression in patients. Cancer Res; 78(4); 909–21. ©2017 AACR.

Supplementary methods and Figure legends

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions.Significance:This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

Supplemental Table 1. List of used Primary antibodies.

Supplementary Material & figure legends

In pancreatic ductal adenocarcinoma (PDAC), signaling from stromal cells is implicated in metastatic progression. Tumor-stroma cross-talk is often mediated through extracellular vesicles (EVs). We previously reported that EVs derived from cancer-associated stromal fibroblasts (CAFs) that are abundant in annexin A6 (ANXA6 + EVs) support tumor cell aggressiveness in PDAC. Here, we found that the cell surface glycoprotein and tetraspanin CD9 is a key component of CAF-derived ANXA6 + EVs for mediating this cross-talk. CD9 was abundant on the surface of ANXA6 + CAFs isolated from patient PDAC samples and from various mouse models of PDAC. CD9 colocalized with CAF markers in the tumor stroma, and CD9 abundance correlated with tumor stage. Blocking CD9 impaired the uptake of ANXA6 + EVs into cultured PDAC cells. Signaling pathway arrays and further analyses revealed that the uptake of CD9 + ANXA6 + EVs induced mitogen-activated protein kinase (MAPK) pathway activity, cell migration, and epithelial-to-mesenchymal transition (EMT). Blocking either CD9 or p38 MAPK signaling impaired CD9 + ANXA6 + EV–induced cell migration and EMT in PDAC cells. Analysis of bioinformatic datasets indicated that CD9 abundance was an independent marker of poor prognosis in patients with PDAC. Our findings suggest that CD9-mediated stromal cell signaling promotes PDAC progression.

Resume Les tumeurs reprogramment leur metabolisme pour repondre a leurs exigences energetiques et en biomasse essentielles a leur proliferation et dissemination. Ces changements metaboliques generent une dependance des tumeurs vis-a-vis des nutriments apportes par l’hote. Si le ciblage metabolique par des regimes alimentaires est une piste therapeutique attrayante de lutte contre le cancer, cette approche fait debat au sein de la communaute scientifique. Dans cette revue, nous apportons, sur la base d’experimentations pre-cliniques et/ou cliniques, des elements de reponses aux interrogations que les patients, les familles et le corps medical assurant leur prise en charge, se posent quant a l’interet clinique de ces interventions nutritionnelles. Nous discutons des consequences du ciblage de cette dependance aux nutriments, via des regimes alimentaires/inhibiteurs chimiques, sur l’evolution des cancers et des effets de ce ciblage lorsqu’il est associe aux therapies actuelles. L’efficacite de ces traitements, leurs consequences sur la qualite de vie des patients sont aussi discutees.

Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that β‐hydroxybutyrate (βOHB) is an alternative cell‐intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG‐CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage‐independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while βOHB stimulates metastatic dissemination to the liver. These findings suggest that βOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions. Significance: This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

Recent evidence has highlighted the presence of neuronal nerve processes in the tumor microenvironment. However, the origin of intra-tumoral nerves remains poorly known, in part because of technical difficulties in tracing nerve fibers via regular histological preparations. Here, we employed three-dimensional (3D) imaging of cleared tissues for a comprehensive analysis of sympathetic innervation in pancreatic ductal adenocarcinoma (PDAC). The results support two independent, but coexisting, mechanisms: passive engulfment of pre-existing nerves within tumors and active, localized sprouting of nerve terminals into non-neoplastic lesions and tumor periphery. Nerve ablation revealed an inverse correlation between sympathetic innervation and tumor growth and spread. Furthermore, sympathectomy increased CD163+macrophage levels, which contributed to worse outcomes. Altogether, our findings revealed protective properties of the sympathetic nervous system in PDAC immunity and progression that could pave the way for new treatments.

The malignant progression of pancreatic ductal adenocarcinoma (PDAC) is accompanied by a profound desmoplasia, which forces proliferating tumor cells to metabolically adapt to this new microenvironment. We established the PDAC metabolic signature to highlight the main activated tumor metabolic pathways. Comparative transcriptomic analysis identified lipid-related metabolic pathways as being the most highly enriched in PDAC, compared with a normal pancreas. Our study revealed that lipoprotein metabolic processes, in particular cholesterol uptake, are drastically activated in the tumor. This process results in an increase in the amount of cholesterol and an overexpression of the low-density lipoprotein receptor (LDLR) in pancreatic tumor cells. These findings identify LDLR as a novel metabolic target to limit PDAC progression. Here, we demonstrate that shRNA silencing of LDLR, in pancreatic tumor cells, profoundly reduces uptake of cholesterol and alters its distribution, decreases tumor cell proliferation, and limits activation of ERK1/2 survival pathway. Moreover, blocking cholesterol uptake sensitizes cells to chemotherapeutic drugs and potentiates the effect of chemotherapy on PDAC regression. Clinically, high PDAC Ldlr expression is not restricted to a specific tumor stage but is correlated to a higher risk of disease recurrence. This study provides a precise overview of lipid metabolic pathways that are disturbed in PDAC. We also highlight the high dependence of pancreatic cancer cells upon cholesterol uptake, and identify LDLR as a promising metabolic target for combined therapy, to limit PDAC progression and disease patient relapse.

SUMMARY Preclinical models based on patient-derived xenografts have remarkable specificity in distinguishing transformed human tumor cells from non-transformed murine stromal cells computationally. We obtained 29 pancreatic ductal adenocarcinoma (PDAC) xenografts from either resectable or non-resectable patients (surgery and endoscopic ultrasound-guided fine-needle aspirate, respectively). Extensive multiomic profiling revealed two subtypes with distinct clinical outcomes. These subtypes uncovered specific alterations in DNA methylation and transcription as well as in signaling pathways involved in tumor-stromal cross-talk. The analysis of these pathways indicates therapeutic opportunities for targeting both compartments and their interactions. In particular, we show that inhibiting NPC1L1 with Ezetimibe, a clinically available drug, might be an efficient approach for treating pancreatic cancers. These findings uncover the complex and diverse interplay between PDAC tumors and the stroma and demonstrate the pivotal role of xenografts for drug discovery and relevance to PDAC., In Brief Nicolle et al. present a genomic analysis of pancreatic cancer xenografts showing that tumor subtypes are defined by specific epigenetic, transcriptional, and stromal landscapes. They reveal potential therapeutic targets through analysis of signaling cross-talk between tumor and stromal cells.

> L’adenocarcinome canalaire pancreatique (ADKP) est un cancer de mauvais pronostic du fait de son diagnostic tardif et de sa resistance intrinseque aux therapies antitumorales existantes [1, 11]. A un stade avance de la maladie, les patients developpent un syndrome cachectique irreversible reduisant l’efficacite des therapies anticancereuses et degradant leur qualite de vie ; seulement 5 % survivent au-dela de 5 ans. Cette cachexie cancereuse, resultant d’un dialogue etroit et bidirectionnel entre les tissus metaboliques de l’hote et la tumeur pancreatique, engendre des troubles metaboliques se caracterisant principalement par une proteolyse musculaire importante et des anomalies affectant aussi le metabolisme lipidique (lipolyse) et glucidique (neoglucogenese) [2]. La tumeur pancreatique se caracterise par un stroma important, peu vascularise, encerclant les cellules cancereuses, les privant ainsi d’un apport suffisant en oxygene et en nutriments. Les cellules tumorales en expansion sont donc contraintes d’effectuer une reprogrammation metabolique pour pallier leurs besoins excessifs en energie et en nutriments.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive stroma and pathogenic modifications to the peripheral nervous system that elevate metastatic capacity. In this study, we show that the IL6-related stem cell–promoting factor LIF supports PDAC-associated neural remodeling (PANR). LIF was overexpressed in tumor tissue compared with healthy pancreas, but its receptors LIFR and gp130 were expressed only in intratumoral nerves. Cancer cells and stromal cells in PDAC tissues both expressed LIF, but only stromal cells could secrete it. Biological investigations showed that LIF promoted the differentiation of glial nerve sheath Schwann cells and induced their migration by activating JAK/STAT3/AKT signaling. LIF also induced neuronal plasticity in dorsal root ganglia neurons by increasing the number of neurites and the soma area. Notably, injection of LIF-blocking antibody into PDAC-bearing mice reduced intratumoral nerve density, supporting a critical role for LIF function in PANR. In serum from human PDAC patients and mouse models of PDAC, we found that LIF titers positively correlated with intratumoral nerve density. Taken together, our findings suggest LIF as a candidate serum biomarker and diagnostic tool and a possible therapeutic target for limiting the impact of PANR in PDAC pathophysiology and metastatic progression. Significance: This study suggests a target to limit neural remodeling in pancreatic cancer, which contributes to poorer quality of life and heightened metastatic progression in patients. Cancer Res; 78(4); 909–21. ©2017 AACR.

International audience; Although it is known to contain five cell types that synthesize and release hormones with a circadian pattern, the pituitary gland is poorly characterized as a circadian oscillator. By a differential microarray analysis, 252 genes were found to be differentially expressed in pituitaries from Bmal1(-/-) knockout versus wild-type mice. By integrative analyses of the data set with the Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources annotation analysis system, pituitary genes with altered expression in Bmal1(-/-) mice were dispatched among functional categories. Clusters of genes related to signaling and rhythmic processes as well as transcription regulators, in general, were found enriched in the data set, as were pathways such as circadian rhythm, transforming growth factor β (TGFβ) signaling, valine, leucine, and isoleucine degradation, and peroxisome proliferator-activated receptor (PPAR) signaling pathways. Gene Ontology term overrepresentation analyses revealed significant enrichment for genes involved in 10 key biological processes. To determine whether genes with altered expression in Bmal1(-/-) mice were actually circadian genes, we further characterized in the mouse pituitary gland the daily pattern of some of these genes, including core-clock genes. Core-clock genes and genes selected from three identified overrepresented biological processes, namely, hormone metabolic process, regulation of transcription from RNA polymerase II promoter, and cell adhesion, displayed a rhythmic pattern. Given the enrichment in genes dedicated to cell adhesion and their daily changes in the pituitary, it is hypothesized that cell-cell interactions could be involved in the transmission of information between endocrine cells, allowing rhythmic hormone outputs to be controlled in a temporally precise manner.

Daily rhythms in behavior and physiology are observed in most organisms. These rhythms are controlled by internal self-sustained circadian ( approximately 24 h) clocks, which are present in virtually all cells. The 24-h oscillations are generated by a molecular mechanism entrained by external or internal time cues and which, in turn, regulate rhythmic outputs. In mammals, the circadian system comprises a master clock located in the hypothalamus that is directly entrained by the light-dark cycle and which coordinates the phases of local clocks in the periphery in order to ensure optimal timing of the physiology. Nuclear receptors (NRs) form a large family of transcription factors that include both ligand-inducible and orphan receptors. These NRs are key regulators of major biological processes such as reproduction, development, cell growth and death, inflammation, immunity, and metabolic homeostasis. Recent observations indicate that several NR signaling pathways play a critical role in central and peripheral circadian clocks. The REV-ERB/retinoid-related orphan receptor orphan NR subfamily regulates the expression of core clock genes and contributes to the robustness of the clock mechanism. Glucocorticoid and retinoic acid receptors are involved in the resetting of peripheral clocks. Several other NRs such as peroxisome proliferator-activated receptor-alpha, short heterodimer partner, and constitutive androstane receptor act as molecular links between clock genes and specific rhythmic metabolic outputs. The expanding functional links between NRs and circadian clocks open novel perspectives for understanding the hormonal regulation of the mammalian circadian system as well as for exploring the role of circadian clocks in the pathogenesis of NR-related diseases such as cancer and metabolic syndrome.

The circadian system regulates rhythmically most of the mammalian physiology in synchrony with the environmental light/dark cycle. Alteration of circadian clock gene expression has been associated with tumour progression but the molecular links between the two mechanisms remain poorly defined. Here we show that Stra13 and Dec2, two circadian transcriptional regulators which play a crucial role in cell proliferation and apoptosis are overexpressed and no longer rhythmic in serum shocked fibroblasts treated with CoCl(2,) a substitute of hypoxia. This effect is associated with a loss of circadian expression of the clock genes Rev-erbalpha and Bmal1, and the clock-controlled gene Dbp. Consistently, cotransfection assays demonstrate that STRA13 and DEC2 both antagonize CLOCK:BMAL1 dependent transactivation of the Rev-erbalpha and Dbp promoters. Using a transplantable osteosarcoma tumour model, we show that hypoxia is associated with altered circadian expression of Stra13, Dec2, Rev-erbalpha, Bmal1 and Dbp in vivo. These observations collectively support the notion that overexpression of Stra13 and Dec2 links hypoxia signalling to altered circadian clock gene expression.

Many human diseases, including metabolic, immune and central nervous system disorders, as well as cancer, are the consequence of an alteration in lipid metabolic enzymes and their pathways. This illustrates the fundamental role played by lipids in maintaining membrane homeostasis and normal function in healthy cells. We reviewed the major lipid dysfunctions occurring during tumor development, as determined using systems biology approaches. In it, we provide detailed insight into the essential roles exerted by specific lipids in mediating intracellular oncogenic signaling, endoplasmic reticulum stress and bidirectional crosstalk between cells of the tumor microenvironment and cancer cells. Finally, we summarize the advances in ongoing research aimed at exploiting the dependency of cancer cells on lipids to abolish tumor progression.

International audience; Circadian rhythms in mammals are synchronized to the light (L)/dark (D) cycle through messages relaying in the master clock, the suprachiasmatic nucleus of the hypothalamus (SCN). Here, we provide evidence that the SCN undergoes rhythmic ultrastructural rearrangements over the 24-h cycle characterized by day/night changes of the glial, axon terminal, and/or somato-dendritic coverage of neurons expressing arginine vasopressin (AVP) or vasoactive intestinal peptide (VIP), the two main sources of SCN efferents. At nighttime, we noted an increase in the glial coverage of the dendrites of the VIP neurons (+29%) that was concomitant with a decrease in the mean coverage of the somata (-36%) and dendrites (-43%) of these neurons by axon terminals. Conversely, glial coverage of the dendrites of AVP neurons decreased (-19%) with parallel increase in the extent of somatal (+96%) and dendritic (+52%) membrane appositions involving these neurons. These plastic events were concomitant with daily fluctuations in quantitative expression of glial fibrillary acidic protein (GFAP), which were then used as an index of structural plasticity. The GFAP rhythm appeared to be strictly dependent on light entrainment, indicating that structural reorganization of the SCN may subserve synchronization of the clock to the L/D cycle. Other results presented reinforced this view while showing that circulating glucocorticoid hormones, which are known to modulate photic entrainment, were required to maintain amplitude of the GFAP rhythm to normal values.

Circadian rhythms result from feedback loops involving clock genes and their protein products. In mammals, 2 orphan nuclear receptors, REV-ERBalpha and RORalpha, play important roles in the transcription of the clock gene Bmal1. The authors now considerably extend these findings with the demonstration that all members of the REV-ERB (alpha and beta) and ROR (alpha, beta, and gamma) families repress and activate Bmal1 transcription, respectively. The authors further show that transcription of Bmal1 is the result of competition between REV-ERBs and RORs at their specific response elements (RORE). Moreover, they demonstrate that Reverb genes are similarly expressed in the thymus, skeletal muscle, and kidney, whereas Ror genes present distinct expression patterns. Thus, the results indicate that all members of the REV-ERB and ROR families are crucial components of the molecular circadian clock. Furthermore, their strikingly different patterns of expression in nervous and peripheral tissues provide important insights into functional differences between circadian clocks within the organism.

The circadian clock in the suprachiasmatic nucleus (SCN) controls day-to-day physiology and behavior by sending timing messages to multiple peripheral oscillators. In the pineal gland, a major SCN target, circadian events are believed to be driven exclusively by the rhythmic release of norepinephrine from superior cervical ganglia (SCG) neurons relaying clock messages through a polysynaptic pathway. Here we show in rat an SCN-driven daily rhythm of pineal MAPK activation that is not dependent on the SCG and whose maintenance requires vitamin A as a blood-borne factor. This finding challenges the dogma that SCG-released norepinephrine is an exclusive mediator of SCN-pineal communication and allows the assumption that humoral mechanisms are involved in pineal integration of temporal messages.

The main known function of the pineal gland in mammals is the temporal synchronization of physiological rhythms to seasonal changes of day length (photoperiod). In rat, the transcription factor activating protein-1 (AP-1) displays a circadian rhythm in its DNA binding in the pineal gland, which results from the rhythmic expression of Fra-2. We postulated that, if AP-1 is an important component of pineal gland functioning, then variations in photoperiodic conditions should lead to an adaptation of the AP-1 binding rhythm. Here we show that AP-1 binding patterns adapt to variations in lighting conditions, in the same way as the rhythm of arylalkylamine-N-acetyltransferase (AA-NAT) activity. This adaptation appeared to result from photoperiodic adaptation of the rhythmic fra-2 gene expression and was reflected by an adapted delay between the onset of night and the acrophase of the nocturnal peak. We further showed that photoperiodic adaptation of both the AP-1 binding and AA-NAT activity rhythms resulted from adapted changes in adrenergic inducibility of both variables at night onset. We finally provided evidence that AP-1 shared with the CREM gene encoding the transcriptional repressor protein inducible cAMP early repressor (ICER) the ability to be hypersensitive or subsensitive to adrenergic stimuli, depending on prior photoperiod.

The daily rhythm in circulating melatonin is driven by a circadian rhythm in the expression of the arylalkylamine N-acetyltransferase gene in the rat pineal gland. Turning off expression of this gene at the end of night is believed to involve inhibitory transcription factors, among which Fos-related antigen 2 (Fra-2) appears as a good candidate. Circadian rhythms in the expression of three proteins of activating protein-1 (AP-1) complexes, namely, Fra-2, c-Jun, and Jun-D, are shown here to account for circadian variations in AP-1 binding activity. Quantitative variations in the Fra-2 component over the circadian cycle were associated with qualitative variations in protein isoforms. Destruction of the suprachiasmatic nucleus resulted in decreased nocturnal AP-1 activity, showing that AP-1 circadian rhythm is driven by this nucleus. Exposure to light during subjective night and administration of a serotonin 5-HT1A/5-HT7 receptor agonist during subjective day, respectively, induced a 50% decrease and a 50% increase in both AP-1 and Fra-2 expression. These effects were impaired by suprachiasmatic nucleus lesions. These data show that pineal AP-1 binding activity, which results from Fra-2 expression, can be modulated by light and serotonin through the suprachiasmatic nucleus according to a “phase dependence” that is characteristic of the rhythm of clock sensitivity to both zeitgebers.

Patient-derived xenografts (PDX) are appearing as a prime approach for preclinical studies despite being insufficiently characterized as a model of the human disease and its diversity. In this work, 29 PDX were obtained from either surgery or endoscopic ultrasound-guided fine needle aspirate of pancreatic adenocarcinoma. The extensive genomic profiling of these pancreatic PDX, revealed two clinically-relevant subtypes having broad similarities with human primary tumors. These subtypes are defined by highly specific DNA methylation and transcriptomic profiles (mRNA, miRNA or lncRNA) but are not distinguishable by exonic mutations or copy number aberrations. Moreover, by specifically analyzing the stroma transcriptome, as defined by the expression of murine transcripts, we found that it is able to stratify the patients with the same efficiency than the analysis of grafted human tumor cells. This finding suggest that transformed pancreatic cells drive the composition of their own stroma. Finally, the multiomics analysis pinpoints novel therapeutic targets, one of which we demonstrate to be an efficient method for treating pancreatic cancer. Overall, we show that PDX are trustworthy pre-clinical models of pancreatic adenocarcinoma including of unresectable tumors. Their multiomics profiling allow the independent analysis of the uncontaminated cancer or stromal compartments and discloses several original therapeutics targets. Citation Format: Remy Nicolle, Yuna Blum, Laetitia Marisa, Celine Loncle, Odile Gayet, Vincent Moutardier, Olivier Turrini, Marc Giovannini, Benjamin Bian, Martin Bigonnet, Marion Rubis, Nabila Elarouci, Lucile Armenoult, Mira Ayadi, Pauline Duconseil, Mohamed Gasmi, Mehdi Ouaissi, Aurélie Maignan, Gwen Lomberk, Jean-Marie Boher, Jacques Ewald, Erwan Bories, Jonathan Garnier, Anthony Goncalves, Flora Poizat, Jean-Luc Raoul, Veronique Secq, Stephane Garcia, Philippe Grandval, Marine Barraud-Blanc, Emmanuelle Norguet, Marine Gilabert, Jean-Robert Delpero, Julie Roques, Ezequiel Calvo, Fabienne Guillaumond, Sophie Vasseur, Raul Urrutia, Aurélien de Reyniès, Nelson Dusetti, Juan Iovanna. Multiomics assessment of the cancer and stromal compartments of patient-derived pancreatic xenografts reveals clinically-relevant subtypes and novel targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4396. doi:10.1158/1538-7445.AM2017-4396

In mammals, photic entrainment of circadian rhythms likely involves light- and clock-dependent expression of immediate early genes, including fos-like and jun-like genes, in the rat suprachiasmatic nucleus. Using an electrophoretic mobility shift assay, we evaluated whether the photic regulation of DNA-binding activity and composition of activating protein-1 (AP-1) complexes in the suprachiasmatic nucleus is also dependent on circadian phase. Phase-dependent light inducibility in the expression of fra-2 and c-fos genes and in immunoreactive Fra-2 and c-Fos protein expression was also evaluated, by in situ hybridization and immunocytochemistry. Light's effects on AP-1 DNA-binding differed both qualitatively and quantitatively according to the circadian phase at which light was applied. This phase dependence accounted for by both compartmentalization of proteins involved in constitutive AP-1 complexes within the nucleus or cytoplasm and control of the extent to which the expression of specific complexes was induced. It was then shown that the mechanisms by which the circadian clock gates the photic induction of AP-1 components differed according to the nature of the protein.

Because of lack of effective treatment, pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of death by cancer in Western countries, with a very weak improvement of survival rate over the last 40years. Defeat of numerous conventional therapies to cure this cancer makes urgent to develop new tools usable by clinicians for a better management of the disease. Aggressiveness of pancreatic cancer relies on its own hallmarks: a low vascular network as well as a prominent stromal compartment (desmoplasia), which creates a severe hypoxic environment impeding correct oxygen and nutrients diffusion to the tumoral cells. To survive and proliferate in those conditions, pancreatic cancer cells set up specific metabolic pathways to meet their tremendous energetic and biomass demands. However, as PDAC is a heterogenous tumor, a complex reprogramming of metabolic processes is engaged by cancer cells according to their level of oxygenation and nutrients supply. In this review, we focus on the glycolytic activity of PDAC and the glucose-connected metabolic pathways which contribute to the progression and dissemination of this disease. We also discuss possible therapeutic strategies targeting these pathways in order to cure this disease which still until now is resistant to numerous conventional treatments.

International audience; Pancreatic ductal adenocarcinoma is one of the most intractable and fatal cancer. The decreased blood vessel density displayed by this tumor not only favors its resistance to chemotherapy but also participates in its aggressiveness due to the consequent high degree of hypoxia. It is indeed clear that hypoxia promotes selective pressure on malignant cells that must develop adaptive metabolic responses to reach their energetic and biosynthetic demands. Here, using a well-defined mouse model of pancreatic cancer, we report that hypoxic areas from pancreatic ductal adenocarcinoma are mainly composed of epithelial cells harboring epithelial-mesenchymal transition features and expressing glycolytic markers, two characteristics associated with tumor aggressiveness. We also show that hypoxia increases the "glycolytic" switch of pancreatic cancer cells from oxydative phosphorylation to lactate production and we demonstrate that increased lactate efflux from hypoxic cancer cells favors the growth of normoxic cancer cells. In addition, we show that glutamine metabolization by hypoxic pancreatic tumor cells is necessary for their survival. Metabolized glucose and glutamine converge toward a common pathway, termed hexosamine biosynthetic pathway, which allows O-linked N-acetylglucosamine modifications of proteins. Here, we report that hypoxia increases transcription of hexosamine biosynthetic pathway genes as well as levels of O-glycosylated proteins and that O-linked N-acetylglucosaminylation of proteins is a process required for hypoxic pancreatic cancer cell survival. Our results demonstrate that hypoxia-driven metabolic adaptive processes, such as high glycolytic rate and hexosamine biosynthetic pathway activation, favor hypoxic and normoxic cancer cell survival and correlate with pancreatic ductal adenocarcinoma aggressiveness.

For decades, the circadian system of animals was viewed as a single or a few centralized structures driving all overt physiological circadian rhythms. Recent data have questioned this view and have revealed a much more complex and dynamic picture. We now know that circadian oscillators are also present in many different tissues and constitute part of a hierarchical circadian system. In this article, we describe this multi-oscillator system that constitutes the basis for the variety of physiological rhythms.

International audience; Most clock-controlled genes (CCGs) lack the specific E-box response element necessary for direct circadian regulation. This is the case for the prolactin (Prl) gene, the expression of which oscillates in individual lactotrope pituitary cells. To characterize the processes underlying this oscillation, we used a lactotrope cell line (GH4C1 cells). In these cells, Prl gene expression fluctuated significantly during 24 h (P=0.0418). Circadian Prl transcription depended on an interaction between the pituitary-specific transcription factor, PIT-1, and the helicase-like transcription factor (HLTF), a SWI/SNF chromatin remodeler, shown here to bind the Prl promoter on an E-box that differs from the specific E-box preferentially bound by clock proteins. Circadian Prl transcription was further accompanied by marked daily chromatin transitions. While neither HLTF nor PIT-1 was rhythmically expressed, NONO and SFPQ, identified as HLTF-associated proteins by mass spectrometry, displayed a circadian pattern and bound rhythmically to the Prl promoter. Furthermore, NONO and SFPQ were functionally involved in circadian Prl transcription since overexpression of both proteins greatly reduced Prl promoter activity (P

p53 is a tumor suppressor that responds to various stress signals by initiating cell-cycle arrest, senescence and apoptosis. Mutations of the p53 gene are found in over 50% of human tumors, highlighting the importance of p53 in tumor suppression. Numerous studies have reported on the interactions between p53, IGF-1–AKT and mTOR pathways as potentially explaining some of the tumor suppressive activities of p53. To further understand the basis of these interactions, we analyzed the involvement of DJ-1, an oncogene known to drive AKT-mediated cell survival, in the p53–AKT axis. In this study, we show that DJ-1 and p53 are tightly ‘linked’: p53 prevents the accumulation of DJ-1 protein, whereas loss of p53 leads to stabilization and enhancement of DJ-1 expression. Interestingly, this increase in DJ-1 level is only observed when p53 loss is accompanied by transformation of cells. Moreover, DJ-1 seems to be required for the enhanced activation of AKT observed in p53-deficient cells. Such observation confers a new property to DJ-1 associated to transforming-process to its oncogenic ability to drive AKT activation. We also show that DJ-1 is necessary for p53 activation following oxidative stress, suggesting the existence of a finely regulated loop between these two proteins in transformed cells. Finally, we demonstrate that in the absence of p53, DJ-1 is stabilized by ROS accumulation, and surprisingly seems to be required for this high intracellular ROS production. These data offer new insights into the regulation of DJ-1 and suggest that DJ-1 is a target of p53. Importantly, our study highlights that during transformation, DJ-1 is having a key role in the p53-regulated AKT pathway and p53-driven oxidative-stress response.

International audience; The circadian timing system coordinates many aspects of mammalian physiology and behavior in synchrony with the external light/dark cycle. These rhythms are driven by endogenous molecular clocks present in most body cells. Many clock outputs are transcriptional regulators, suggesting that clock genes primarily control physiology through indirect pathways. Here, we show that Krüppel-like factor 10 (KLF10) displays a robust circadian expression pattern in wild-type mouse liver but not in clock-deficient Bmal1 knockout mice. Consistently, the Klf10 promoter recruited the BMAL1 core clock protein and was transactivated by the CLOCK-BMAL1 heterodimer through a conserved E-box response element. Profiling the liver transcriptome from Klf10(-/-) mice identified 158 regulated genes with significant enrichment for transcripts involved in lipid and carbohydrate metabolism. Importantly, approximately 56% of these metabolic genes are clock controlled. Male Klf10(-/-) mice displayed postprandial and fasting hyperglycemia, a phenotype accompanied by a significant time-of-day-dependent upregulation of the gluconeogenic gene Pepck and increased hepatic glucose production. Consistently, functional data showed that the proximal Pepck promoter is repressed directly by KLF10. Klf10(-/-) females were normoglycemic but displayed higher plasma triglycerides. Correspondingly, rhythmic gene expression of components of the lipogenic pathway, including Srebp1c, Fas, and Elovl6, was altered in females. Collectively, these data establish KLF10 as a required circadian transcriptional regulator that links the molecular clock to energy metabolism in the liver.

Most organisms adapt their behavior and physiology to the daily changes in their environment through internal ( approximately 24 h) circadian clocks. In mammals, this time-keeping system is organized hierarchically, with a master clock located in the suprachiasmatic nuclei of the hypothalamus that is reset by light, and that, in turn, coordinates the oscillation of local clocks found in all cells. Central and peripheral clocks control, in a highly tissue-specific manner, hundreds of target genes, resulting in the circadian regulation of most physiological processes. A great deal of knowledge has accumulated during the last decade regarding the molecular basis of mammalian circadian clocks. These studies have collectively demonstrated how a set of clock genes and their protein products interact together in complex feedback transcriptional/translational loops to generate 24-h oscillations at the molecular, cellular, and organism levels. In recent years, a number of nuclear receptors (NRs) have been implicated as important regulators of the mammalian clock mechanism. REV-ERB and retinoid-related orphan receptor NRs regulate directly the core feedback loop and increase its robustness. The glucocorticoid receptor mediates the synchronizing effect of glucocorticoid hormones on peripheral clocks. Other NR family members, including the orphan NR EAR2, peroxisome proliferator activated receptors-alpha/gamma, estrogen receptor-alpha, and retinoic acid receptors, are also linked to the clockwork mechanism. These findings together establish nuclear hormone receptor signaling as an integral part of the circadian timing system.

Daily rhythms in behavior and physiology are observed in most organisms. These rhythms are controlled by internal self-sustained circadian ( approximately 24 h) clocks, which are present in virtually all cells. The 24-h oscillations are generated by a molecular mechanism entrained by external or internal time cues and which, in turn, regulate rhythmic outputs. In mammals, the circadian system comprises a master clock located in the hypothalamus that is directly entrained by the light-dark cycle and which coordinates the phases of local clocks in the periphery in order to ensure optimal timing of the physiology. Nuclear receptors (NRs) form a large family of transcription factors that include both ligand-inducible and orphan receptors. These NRs are key regulators of major biological processes such as reproduction, development, cell growth and death, inflammation, immunity, and metabolic homeostasis. Recent observations indicate that several NR signaling pathways play a critical role in central and peripheral circadian clocks. The REV-ERB/retinoid-related orphan receptor orphan NR subfamily regulates the expression of core clock genes and contributes to the robustness of the clock mechanism. Glucocorticoid and retinoic acid receptors are involved in the resetting of peripheral clocks. Several other NRs such as peroxisome proliferator-activated receptor-alpha, short heterodimer partner, and constitutive androstane receptor act as molecular links between clock genes and specific rhythmic metabolic outputs. The expanding functional links between NRs and circadian clocks open novel perspectives for understanding the hormonal regulation of the mammalian circadian system as well as for exploring the role of circadian clocks in the pathogenesis of NR-related diseases such as cancer and metabolic syndrome.

Most living organisms show circadian (∼24 h) rhythms in physiology and behavior. These oscillations are generated by endogenous circadian clocks, present in virtually all cells where they control key biological processes. Although circadian gating of mitosis has been reported for many years in some peripheral tissues, the underlying molecular mechanisms have remained poorly understood. Here we show that the cell cycle inhibitor p21WAF1/CIP1 is rhythmically expressed in mouse peripheral organs. This rhythmic pattern of mRNA and protein expression was recapitulated in vitro in serum-shocked differentiated skeletal muscle cells. p21WAF1/CIP1 circadian expression is dramatically increased and no longer rhythmic in clock-deficient Bmal1–/– knock-out mice. Biochemical and genetic data show that oscillation of p21WAF1/CIP1 gene transcription is regulated by the antagonistic activities of the orphan nuclear receptors REV-ERBα/β and RORα4/γ, which are core clock regulators. Importantly, p21WAF1/CIP1 overexpressing Bmal1–/– primary hepatocytes exhibit a decreased proliferation rate. This phenotype could be reversed using small interfering RNA-mediated knockdown of p21WAF1/CIP1. These data establish a novel molecular link between clock and cell cycle genes and suggest that the G1 progression phase is a target of the circadian clock during liver cell proliferation.