Odile Gayet

Cholesterol uptake disruption, in association with chemotherapy, is a promising combined metabolic therapy for pancreatic adenocarcinoma

Significance Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second deadliest cancer by 2030. Advances in therapeutic treatments are urgently required to fight against this fatal disease. Here, elucidation of the metabolic signature of PDAC has identified the low-density lipoprotein receptor (LDLR), which facilitates cholesterol uptake, as a promising therapeutic target. Blocking of LDLR reduces the proliferative and clonogenic potential of PDAC cells and decreases activation of the ERK1/2 survival pathway. Moreover, LDLR silencing sensitizes PDAC cells to chemotherapeutic drugs and potentiates the tumoral regression promoted by chemotherapy. Finally, Ldlr is highly expressed at all stages of human PDAC and expression is associated with an increased risk of PDAC recurrence. 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.

Juxtaposition of CNR protocadherins and reelin expression in the developing spinal cord.

The CNR (cadherin-related neuronal receptors) family of protocadherins is of great interest because of their potential roles as molecular tags in the formation of specific synaptic connections, and as receptors for reelin, during neuronal migration, and cell body positioning. In order to know more about potential functions of CNRs we have mapped their expression during mouse nervous system development and compared their expression with that of reelin and its intracellular effector Dab1 in several tissues. In spinal cord, CNRs and Dab1 are expressed in motoneurons, while reelin is located in adjacent cells. In the hindbrain, there is a differential expression of CNRs and Dab1 in various motor nuclei. In the retina and olfactory system, we observe CNR and reelin expression but not that of Dab1. These results provide new insights into the potential functions of CNRs and their possible integration in the reelin pathway during development.

Identification of lynx2, a novel member of the ly-6/neurotoxin superfamily, expressed in neuronal subpopulations during mouse development.

We isolated a new gene which shares all the features of the Ly-6/neurotoxin superfamily, from gene organization to predicted 3D structure. As it is preferentially expressed in the nervous system, we called this gene lynx2, by analogy with lynx1, a nAChR modulator. In embryonic and postnatal mouse, lynx2 is expressed in postmitotic central and peripheral neurons. These include subpopulations of motor neurons, sensory neurons, interneurons and neurons of the autonomous nervous system. In addition, lynx2 is transiently expressed around the growing nerves in the limb bud. Comparison of its spatio-temporal expression pattern with that of two other members of this family, lynx1 and ly-6h, shows that these genes are detected both in distinct and overlapping neuron populations.

The Functional Landscape of Hsp27 Reveals New Cellular Processes such as DNA Repair and Alternative Splicing and Proposes Novel Anticancer Targets

Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells.

Changes in subcellular distribution of protocadherin gamma proteins accompany maturation of spinal neurons

Protocadherins gamma (Pcdhγ) are a family of transmembrane proteins in which variable extracellular domains are associated with an invariant cytoplasmic domain, potentially allowing these proteins to trigger common cellular responses through diverse extracellular signals. We studied the expression of the family by in situ hybridisation and immunohistochemistry for the conserved portion of the mRNA or protein. During mouse development, Pcdhγ expression is highest in neural tissues, but is also present in some nonneural tissues. In the adult, Pcdhγ expression is maintained at high levels in brain, in particular in hippocampus and in the Purkinje cells of the cerebellum, whereas it is downregulated in spinal cord. Using antibodies against the conserved cytoplasmic domain, we show that in cultured embryonic spinal cord neurons, Pcdhγ protein is present initially in both axonal and dendritic growth cones. At later stages of differentiation in vitro, Pcdhγ distribution becomes polarised to the somatodendritic compartment. We propose that members of the Pcdhγ family may play roles in neuronal growth and maturation.

Sorting nexin-14, a gene expressed in motoneurons trapped by an in vitro preselection method

A gene‐trap strategy was set up in embryonic stem (ES) cells with the aim of trapping genes expressed in restricted neuronal lineages. The vector used trap genes irrespective of their activity in undifferentiated totipotent ES cells. Clones were subjected individually to differentiation in a system in which ES cells differentiated into neurons. Two ES clones in which the trapped gene was expressed in ES‐derived neurons were studied in detail. The corresponding cDNAs were cloned, sequenced, and analysed by in situ hybridisation on wild‐type embryo sections. Both genes are expressed in the nervous system. One gene, YR‐23, encodes a large intracellular protein of unknown function. The second clone, YR‐14, represents a sorting nexin (SNX14) gene whose expression in vivo coincides with that of LIM‐homeodomain Islet‐1 in several tissues. Sorting nexins are proteins associated with the endoplasmic reticulum (ER) and may play a role in receptor trafficking. Gene trapping followed by screening based on in vitro preselection of differentiated ES recombinant clones, therefore, has the potential to identify integration events in subsets of genes before generation of mouse mutants.

The b1 isoform of protocadherin-gamma (Pcdhγ) interacts with the microtubule-destabilizing protein SCG10

Due to their structural characteristics and their diversity, the 22 members of the protocadherin‐gamma (Pcdhγ) family have been suggested to contribute to the establishment of specific connections in the nervous system. Here, we focus on a single isoform, Pcdhγ‐b1. Its expression is found in different brain regions and in developing spinal cord it is restricted to scattered cells, whereas all cells are labeled using an antibody that recognizes all Pcdhγ isoforms. As a first step to understanding the signaling mechanisms downstream of Pcdhγ, we identify the microtubule‐destabilizing protein SCG10 as a cytoplasmic interactor for Pcdhγ‐b1 and other isoforms of the Pcdhγ‐b subfamily, and show that SCG10 and Pcdhγ‐b1 are found together in certain neuronal growth cones.

Expression of Deltex1 during mouse embryogenesis: comparison with Notch1, 2 and 3 expression.

The Notch signalling pathway defines a phylogenetically conserved cell-cell communication process that enables cell-fate specification in multicellular organisms. Deltex is a component of the Notch signalling network that physically interacts with the ankyrin repeats of Notch. Here, we report on the expression pattern of the Deltex1 gene during mouse embryonic development and, furthermore, we compare its expression with that of the Notch1, 2 and 3 genes. Complementary and combinatorial expression patterns between Deltex1 and the three Notch genes were observed throughout embryogenesis since Deltex1 expression was related either to cytodifferentiation (i.e. neuronal tissues) or to cell proliferation events (i.e. eye, vascular structures, hematopoiesis).

Enhanced expression of P‐selectin (CD62P) by endothelial cells seeded onto synthetic arterial prostheses (PET, Dacron®) is correlated with leukocyte interactions

This study evaluated the expression by seeded endothelial cells (S-EC) of P-selectin (CD62P/GMP-140/PADGEM), an adhesion molecule implicated in endothelial-leukocyte interactions. Endothelial cells were seeded onto knitted polyethylene terephthalate (PET, Dacron(R)) prostheses and compared with control endothelial cells (C-EC) cultured in flasks to the same stage. Using flow cytometry techniques, we observed that CD62P expression by PET S-EC was significantly increased (p<0.05) compared to C-EC. Moreover, RT PCR techniques showed that the CD62P RNA level was higher on S-ECs compared to C-ECs. Following adhesion assays, increased polymorphonuclear neutrophil (PMN) attachment to the PET-seeded prostheses as compared to control cultures (p<0.001) was observed. PMN adherence was enhanced by TNFalpha activation. PMN adhesion was decreased significantly (p<0.001) after the incubation of resting EC or TNFalpha-activated EC-seeded prostheses with a blocking monoclonal antibody (LYP20) directed against the P-selectin. Such results suggest that: (1) PET prosthetic material may induce the expression of P-selectin by S-EC; (2) seeding conditions provoke an increase in PMN adhesion; (3) increased PMN interactions with seeded PET material is partially dependent upon P-selectin expression by the S-EC.

A pancreatic ductal adenocarcinoma subpopulation is sensitive to FK866, an inhibitor of NAMPT

Treating pancreatic cancer is extremely challenging due to multiple factors, including chemoresistance and poor disease prognosis. Chemoresistance can be explained by: the presence of a dense stromal barrier leading to a lower vascularized condition, therefore limiting drug delivery; the huge intra-tumoral heterogeneity; and the status of epithelial-to-mesenchymal transition. These factors are highly variable between patients making it difficult to predict responses to chemotherapy. Nicotinamide phosphoribosyl transferase (NAMPT) is the main enzyme responsible for recycling cytosolic NAD+ in hypoxic conditions. FK866 is a noncompetitive specific inhibitor of NAMPT, which has proven anti-tumoral effects, although a clinical advantage has still not been demonstrated. Here, we tested the effect of FK866 on pancreatic cancer-derived primary cell cultures (PCCs), both alone and in combination with three different drugs typically used against this cancer: gemcitabine, 5-Fluorouracil (5FU) and oxaliplatin. The aims of this study were to evaluate the benefit of drug combinations, define groups of sensitivity, and identify a potential biomarker for predicting treatment sensitivity. We performed cell viability tests in the presence of either FK866 alone or in combination with the drugs above-mentioned. We confirmed both inter- and intra-tumoral heterogeneity. Interestingly, only the in vitro effect of gemcitabine was influenced by the addition of FK866. We also found that NAMPT mRNA expression levels can predict the sensitivity of cells to FK866. Overall, our results suggest that patients with tumors sensitive to FK866 can be identified using NAMPT mRNA levels as a biomarker and could therefore benefit from a co-treatment of gemcitabine plus FK866.